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SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL ACTIVITIES OF ISOCOUMARINS, TRIAZOLES,
THIADIAZOLES AND INDOLINONES
A DISSERTATION SUBMITTED TO THE QUAID-I-AZAM UNIVERSITY ISLAMABAD
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY IN
ORGANIC CHEMISTRY
BY
GHULAM QADEER
Department of Chemistry, Quaid-i-Azam University,
Islamabad, Pakistan. 2008
SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL ACTIVITIES OF
ISOCOUMARINS, TRIAZOLES, THIADIAZOLES AND INDOLINONES
BY
GHULAM QADEER
Department of Chemistry, Quaid-i-Azam University,
Islamabad, Pakistan. 2008
Allah will exalt those who believe among you,
and those who have knowledge to high ranks.
Saying of Holy Prophet Hazrat Muhammad
“Do you know who is the most beneficent? Allah is the
most beneficent, than of the children of the man; I am the most
beneficent and after me the most beneficent among them is the
man who acquires knowledge and spreads it, he will come on the
day of resurrection as a chief by himself”.
My parents
ACKNOWLEDGEMENT First of All I bow my head before “Almighty Allah” Who bestowed me the
opportunity and potential to make material contribution to the already existing
ocean of knowledge and all respects are for the Holy Prophet, Hazrat
Muhammad who enabled us to recognize our Creator.
It is a matter of great pleasure to express my obligations and sincere thanks to
my teacher and research supervisor Prof. Dr. Nasim Hasan Rama, Department
of Chemistry, Q. A. U. Islamabad, for his constant encouragement, keen interest
and thought provoking guidance through out my research work. I am thankful to
Prof. Dr S. Sakhawat shah, Chairman, Department of Chemistry, for providing
necessary research facilities. Thanks to all the teachers of organic section for
being a source of inspiration and enlightenment for me during my course work
and stay in the department.
I obliged to express my heartfelt thanks to Prof. Dr. Erik De Clercq, (Belgium) Dr.
Lieve Naesens, (Belgium), Prof. Dr. Bob Hill (Scotland), Dr. Dra. Maria Luisa
Garduño, (New Maxico), Dr. Wai Yeung-Wong (Hong Kong), Dr. Ales Ruzicka,
(Czec republic) Dr. Yong Hong Li, (China) Dr. Fan Zhijin, (China), Dr. Sauli Vuoti,
(Finland) Dr. M. Azad Malik, (U.K) James Rafetry (U.K) and Dr. Abdul Malik, H.
E. J. Research institute, University of Karachi, Pakistan for their co-operation and
generous help especially in the form of spectroscopic analysis, X-ray
crystallography and bioassay.
I ought to submit my thanks to my dear friends, who remember me in their
prayers and hearts. I wish to acknowledge the support, co-operation and
encouragement provided by Syed Jabbar Hussain Shah, Mazhar Ali Kalyar,
Shahid Ashraf, Javeed Akhter (U.K), I do remember the company of my research
fellows Dr. Muhammad Zareef, M. Arfan, Tasfeen Akhter, K. Ansar Yasin and
Muhammad Sher. My acknowledgement remains incomplete if I don’t mention
the help, encouragement and companionship of my lab fellows, Gul S. Khan,
Naveed Umer, Taj-ud-din, Ahsan Farid, Obaid-ur-Rehman Abid, Tariq Mehmood
Baber, Muhammad Hanif, Furrukh Iftikhar Ali, Muhammad Shahzad & Hakim
Luqman.
I am also heartily thankful to all the members of non-teaching staff of the
department especially M. Sharif Chohan, Shamas Pervaiz Qureshi, Ali Zaman,
Shamas Tabrez Qureshi, Muhammad Ilyas, M. Rashid, Muhammad Raza and
Jumma Khan, for their help during the entire period of research work.
My sincere thanks are due to my wife. Without her encouragement and
excessive generosity I would not been able to complete the task. Lastly my
special thanks are due to my parents, brothers, sisters and all of other relatives.
It is due to their prayers that I have been successful in my educational career.
May Almighty Allah shower his choicest blessings and prosperity on all those
who assisted me in any way during completion of my thesis
Thank you all,
(GHULAM QADEER)
CERTIFICATE
This is to certify that this thesis submitted by Mr. GHULAM QADEER is
accepted in its present form by the Department of Chemistry, Quaid-i-Azam
University, Islamabad as satisfying the requirements for the Degree of Doctor of
Philosophy in Organic Chemistry.
SUPERVISOR ---------------------------
(Prof. Dr. Nasim Hasan Rama)
Departmet of Chemistry,
Quaid-i-Azam University,
Islamabad
CHAIRMAN ---------------------------------
(Prof. Dr. S. Sakhawat shah)
Department of Chemistry,
Quaid-i-Azam University,
Islamabad
Date:
ABSTRACT
The work presented in this thesis consists of the synthesis, characterization, and
biological screening of heterocyclic compounds. For convenience, the work has
been divided into two parts, part one is related to the compounds containing
oxygen atom in the heterocyclic ring whereas part two refers to the compounds
containing nitrogen and/ or sulphur atoms in the heterocyclic ring.
Part one of this thesis is related to the synthesis, characterization and biological
activity of some heterocyclic compounds containing oxygen in the ring. These
compounds include some naturally and unnaturally occurring substituted
isocoumarins and 3,4-dihydroisocoumarins. The synthesis of naturally occurring
isocoumarin Thunberginol B is reported, in which 3,5-dimethoxyhomophthalic
acid is a key intermediate for the synthesis of Thunberginol B. It was
synthesized efficiently in five steps from 3,5-dimethoxybenzaldehyde via a series
of reactions including synthesis of 3,5-dimethoxycinnamic acid, 3-(3′,5′-
dimethoxyphenyl)propionic acid, cyclization of 3-(3′,5′-dimethoxyphenyl)propionic
acid to 5,7-dimethoxy-1-indanone and oxidative decomposition of methyl 2-
hydroxy-2-(5,7-dimethyoxy-1-oxo-1H-inden-2(3H)-ylidene)acetate to 3,5-
dimethoxyhomophthalic acid. 3,4-Dimethoxybenzoylchloride was prepared
from 3,4-dimethoxybenzoic acid on reaction with thionyl chloride which on
condensation with 3,5-dimethoxyhomophthalic acid afforded 3-(3',4'-
dimethoxyphenyl)-6,8-dimethoxyisocoumarin. Complete demethylation of 3-(3',4'-
dimethoxyphenyl)-6,8-dimethoxyisocoumarin with hydrobromic acid in acetic acid
gave 3-(3',4'-dihydroxyphenyl)-6,8-dihydroxyisocoumarin (Thunberginol B). In
addition to above, some unnaturally occurring halogenated isocoumarins and
their 3,4-dihydrodrivatives were also synthesized. The difluorophenyl- and
dichlorophenylisocoumarins by condensation of homophthalic acid with an
appropriate acid chloride. Alkaline hydrolysis of the isocoumarins yielded
corresponding keto-acids, which on reduction give the corresponding racemic
hydroxy-acids. 3,4-Dihydroisocoumarins were obtained from these racemic
hydroxy-acids by cyclodehydration using acetic anhydride.
All the synthesised compounds were identified using their IR, 1H NMR and mass
spectral data. In many cases 13C NMR and elemental analysis data were
employed to support the characterization. In each case, a plasible mass
fragmentation pattern is suggested. The synthesized compounds were screened
for their antifungal, antibacterial, herbicidal, insecticidal, fungicidal, anti-
metastatic, brine shrimp lethality, antioxidant, anti -inflammatory, antiviral, anti-
HIV, anti-HBV and anticancer activities and in some cases, very fascinating
results were obtained which were then published in different international
journals. These synthetic schemes have tremendous potential for further
synthesis of novel biological active compounds.
Part two of the thesis describes the synthesis and biological screening of some,
hitherto unreported, isatin derivatives (Indolinones), 1,4-disubstituted
thiosemicarbazides and their related 2,5-disubstituted-1,3,4-thiadiazoles and 4,5-
disubstituted-3H-1,2,4-triazole-3-thiones. Indolinones were formed by the direct
condensation of hydrazides with halogenated isatins. Triazoles were formed by
intramolecular dehydrative cyclization of thiosemicarbazides in basic media while
thiadiazoles were formed in acidic media, which is an intermediate during the
synthesis of various heterocyclic compounds. Thiosemicarbazides were formed
by aldol type condensation reaction of acid hydrazides and isothiocyanates.
Isothiocyanates were formed by the reaction of anilines with carbon disulphide in
ammonium hydroxide solution to yield ammonium dithiocarbamate, an
intermediate which on oxidation with lead nitrate yield isothiocyanate. Acid
hydrazides were formed by the reaction of esters of carboxylic acid with
hydrazine hydrate and esters were formed by refluxing carboxylic acid in
methanol in catalytic amount of carboxylic acids. As a result of these synthetic
schemes, thirty new indolinones, ten disubstituted-1,3,4-thiadiazoles and twenty
disubstituted 1,2,4-triazole-3-thiones were synthesized. The characterization of
these synthesized compounds was carried out by IR, 1H NMR, 13C NMR,
elemental analysis, Mass spectral data and XRD analysis. The synthesized
compounds were screened for their antifungal, antibacterial, herbicidal,
insecticidal, fungicidal, plant growth regulatory activity and antiviral activities.
CONTENTS Acknowledgement I
Abstract III
Part one CHAPTER 1 INTRODUCTION
1.1 Nomenclature and structural types 1
1.2 Physical properties 5
1.3 Biological activities 5
1.4 Behavior towards human beings 12 1.5 Biosynthesis 14
1.6 Synthesis of isocoumarins and 3,4-dihydro- 18
isocoumarins
1.6.1 Synthesis involving metals 19
1.6.2 Oxidation of Indenes, Indanones and Indones 24
1.6.3 Oxidation of isochromans 26
1.6.4 Aldol type condensation between homophthalic 27
acids, esters or anhydrides and carbonyl compounds
1.6.5 Synthesis of isocoumarins via iodocyclization 32
1.6.6 Synthesis of isocoumarin by the use of new 33
technology
1.7 Interconverision of isocoumarins and 3,4-dihydro- 34
isocumarins 1.7.1 Conversion of 3,4-dihydroisocoumarins to 34
isocoumarins 1.7.2 Conversion of isocoumarins to 3,4-dihydro- 35
isocoumarins
1.8 Reactions of isocoumarins and 3,4-dihydtoisocoumarins 36
1.8.1 Hydrolysis 36
1.8.2 Reaction with ammonia and amines 36
1.8.3 Reaction with phosphorus pentasulphide 37
1.8.4 Nitration 38
1.8.5 Reaction with Grignard reagents 38
1.8.6 Oxidation 39
1.8.7 Reduction 39
1.9 Plan of work 39
1.9.1 Naturally occurring isocoumarin 40
1.9.2 Unnaturally occurring isocoumarins and 3,4- 40
dihydroisocoumarins
CHAPTER 2 SYNTHESIS OF NATRUAL ISOCOUMARIN 6.1 Synthesis of Naturally Occurring Thunberginol B 41
2.1.1 Introduction 41 2.1.2 Plan of work 42 2.1.3 Results and discussions 43 2.1.4 Experimental 56
CHAPTER 3 SYNTHESIS OF UNNATRUAL ISOCOUMARINS
AND 3,4-DIHYDROISOCOUMARINS 3.1. Synthesis of Dihalophenylisocouamrins 62
3.1.1 Introduction 62
3.1.2 Plan of Work 63
3.1.3 Results and discussion 63
3.1.4 Experimental 75
3.2. Synthesis of 3H-Furo[3,4-c]-isochromene-1,5-dione 85
(an unusal isocoumarin) and 3-(3′,4′,5′-trimethoxyphenyl)
isocoumarin 3.2.1 Synthetic scheme 85
3.2.2 Results and Discussion 86
3.2.3 Experimental 96
CHAPTER 4 BIOLOGICAL ACTIVITIES
4.1. Antioxidant Studies 98 4.2. Anti-inflammatory Studies 102
4.3. Herbicide studies 104 4.4. Fungicide studies 108
4.5. Insecticide Studies 111
4.6. Antifungal studies 112
4.7. Antibacterial studies 116
4.8. Brine shrimp lethality (Artemia salina) studies 118 4.9. Antiviral studies against different Cel lCulture 120 4.10. Anti-HIV Studies 124
4.11. Anti-HBV studies 127 4.12. Anti-cancer studies 129
4.13. Antimetastatic studies 131
REFERENCES (PART ONE) 133
Part two CHAPTER 5 INTRODUCTION 5.1 1,2,4-Triazole 146
5.2 Chemistry of 1,2,4-triazole 147
5.2.1 Aromaticity and stability 147
5.2.2 Amphoteric nature 148
5.2.3 Tautomerism in triazoles 148
5.3 Spectroscopy of 1,2,4-triazoles 150
5.3.1 Ultraviolet spectroscopy 150
5.3.2 Infrared spectroscopy 151
5.3.3 NMR and mass spectrometry 151
5.4 Applications and biological activities 152
5.4.1 Agricultural applications 152
5.4.2 Pharmacological applications 153
5.4.3 Industrial applications 154
5.5 Synthetic approaches towards 1,2,4-triazoles 155
5.5.1 From semicarbazides 155
5.5.2 From triazine 156
5.5.3 From thiosemicarbazides 156
5.5.4 From benzalsemicarbazones with ferric chloride 157
5.5.5 From carboxylic acid hydrazides 158
5.5.6 From 1,3,4-oxadiazol-5-thiones 158
5.5.7 From thiosemicarbazides and carbonyl compounds 159
5.5.8 From thiocarbohydrazides and carbohydrazides 159
5.5.9 From thiosemicarbazides with benzoyl chloride 160
5.5.10 From phenylthiosemicarbazide with ethylphenylimidate
hydrochloride 160
5.5.11 From condensation of a nitrile and a hydrazide 160
5.5.12 From isothiocyanates 161
5.5.13 From aromatic nitriles 161
5.5.14 Solid phase synthesis of triazoles 162
5.5.15 Synthesis of 1,2,4-triazoles under microwave irradiation 163
5.6 1,3,4-Thiadiazoles 165
5.7 Applications 166 5.8 Synthetic approaches towards 1,3,4-thiadiazoles 170
5.8.1 From thiosemicarbazides 170
5.8.2 From diacylhydrazides 171
5.8.3 From dithiocarbazinic acid derivatives 172
5.8.4 From Fluorous Lawesson’s reagent 172
5.9 Isatin derivatives-Indolinones 173 5.9.1 Isatin 173
5.9.2 Indolinones 174
5.9.3 Synthetic approaches towards indolinones 175
5.10 Plan of work 177 CHAPTER 6 RESULTS AND DISCUSSION
6.1 Synthesis of methyl / ethyl esters 180 6.1.1 Crystal structure of Ethyl 2-(3-methoxyphenyl)acetate 182
6.1.2 Crystal structure of Methyl 2,6-dimethoxybenzoate 185
6.2 Synthesis of hydrazides 187 6.2.1 Crystal structure of 3,5-difluorobenzohydrazide 189
6.2.2 Crystal structure of 2,6-dimethoxybenzohydrazide 192
6.2.3 Crystal structure of 3,4-dimethoxybenzohydrazide 193
6.2.4 Crystal structure of 2-(2′,4′-dichlorophenylsulfanyl)-
acetohydrazide 196
6.2.5 Crystal structure of (E)-3-(4′-methoxyphenyl)acrylo-
hydrazide 198
6.2.6 Crystal structure of 3-(3′,4′,5′-trimethoxyphenyl)-
propanehydrazide 200
6.2.7 Crystal structure of 3-(4′-methoxyphenyl)-
propanohydrazide 203
6.3 Synthesis of isothiocyanates 205
6.4 Synthesis of thiosemicarbazides 207 6.4.1 Crystal structure of 1-(3,5-Dimethoxybenzoyl)-4-(2-
methoxyphenyl) thiosemicarbazide 212
6.4.2 Crystal structure of 1-[2-(2,4-Dichlorophenoxy)-
acetyl]-4-cyclohexylthiosemicarbazide 214
6.4.3 Crystal structure of 1-[3-(4-methoxyphenyl)
propanoyl]-4-(2- methoxyphenyl)thiosemicarbazide 216
6.4.4 Crystal structure of 1-(3,5-Difluorobenzoyl)-4-
Cyclohexylthiosemicarbazide 217
6.5 Synthesis of substituted 1,2,4-triazol-3-thiones 219
6.5.1 Crystal structure of 4-(2-methoxyphenyl)-5-(3,5-
dimethoxyphenyl)-2H-1,2,4-triazole-3(4H)-thione 224
6.5.2 Crystal Structure of 5-(3,4,5-trimethoxyphenylethyl)-
4-(2-methoxyphenyl)-2H-1,2,4-triazole-3(4H)-thione 227
6.5.3 Crystal Structure of 3-(4-Methoxyphenethyl)-4-(2-
methoxyphenyl)-1H-1,2,4-triazole-5(4H)-thione 229
6.5.4 Crystal Structure of 3-(4-bromophenoxymethyl)-
4-(4-methoxyphenyl)-1H-1,2,4-triazole-5(4H)-thione 231
6.5.5 Crystal Structure of 3-(2,4-dichlorophenoxymethyl)-
4-(4-methoxyphenyl)-1H-1,2,4-triazole-5(4H)-thione 233
6.6 Synthesis of 1,3,4-thiadiazoles 235 6.7 Synthesis of indolinones 239
6.7.1 Crystal Structure of (Z)-N'-(5-chloro-2-oxoindolin-
3-ylidene)-3,5 difluorobenzohydrazide 243
CHAPTER 7 EXPERIMENTAL
7.1 Substrates and reagents 245 7.2 Purification of solvents 245 7.3 Instrumentation 246 7.4 General procedure for the synthesis of esters 247 7.5 General procedure for the synthesis of hydrazides 250 7.6 General procedure for the synthesis of isothiocyanate 256 7.7 General procedure for the synthesis of
Thiosemicarbazides 257 7.8 General procedure for the synthesis of 1, 2, 4-
triazol-3-thiones 267 7.9 General procedure for the Synthesis of 1,3,4-
Thiadiazoles 276 7.10 General procedure for the synthesis of Indolinones 280
CHAPTER 8 BIOLOGICAL ACTIVITIES
8.1 Herbicide studies 294 8.2 Fungicide studies 296 8.3 Insecticide studies 298 8.4 Plant growth regulating studies 300 8.5 Antiviral studies against different cell cultures 302 8.6 Antifungal studies 310 8.7 Antibacterial studies 314
REFERENCES (PART TWO) 316
Chapter-1 INTRODUCTION
Isocoumarins and 3,4-dihydroisocoumarins are the secondary metabolites1.
These are found in wide varieties of fungi, lichens, molds, bacteria, higher plants
and insects. Majority of isocoumarins have been isolated from various species of
fungal genera Artemisia, Aspergillus, Ceratocystis, Fusarium, Penicillum, and
Streptomyces etc. A number of them are constituents of a few higher plant
families e.g. Bignoniaceae, Compositae, Leguminoseae, Myricaceae,
Saxifragaceae, Myristicaceae and Liliaceae families. A number of reviews have
been published about isocoumarins. These include the review by Barry2 (1964),
Turner and Aldridge3 (1983), Yamato4 (1983), Hill5 (1986), Napolitano6 (1997)
and Bin7 et al. (2000) on isocoumarins and 3,4-dihydroisocoumarins.
1.1 Nomenclature and structural types
The name isocoumarin (1) is derived from the fact that these compounds
are isomeric to coumarins (2). Coumarin8 was isolated (1820) from tonka tree
formerly known as Coumarouna odorata. In an isocoumarin, a lactonic pyran ring
is fused to a benzene ring. The IUPAC and Chemical Abstract name for
isocoumarin is 1H-2-benzopyran-1-one, numbered as shown and its 3,4-dihydro-
analogue (3) is named as 3,4-dihydroisocoumarin rather than isochroman-1-one.
O
O
R1
R2R3R4
R5R6
12
3456
78
4a
8a
O
O
R1
R2R3R4
R5R6
1 2
3
456
7 8
4a
8aO O
(1) (2) (3)
As in case of other classes of the natural products (alkaloids, flavonoids
etc.) no systematic nomenclature exists for isocoumarins. Majority of naturally
1
occurring isocoumarins and 3,4-dihydroisocoumarins have been assigned trivial
names9, which are derived from generic or specific names of source plant and
fungi. Examples of the names derived from those of parent genera are
agrimonolide (Agrimonia pilosa), fusamarin (Fusarium spp.), alternariol
(Alternaria spp.), artemidin (Artemisia glauca), peniolactol (Peniophora
sanguinea), cladosporin (Cladosporium spp.), homalicine (Homalium
zeylancum), oosponol (Oospora astringes) etc. Those names derived from
species are found in mellein (Aspergillus melleus), ustic acid (A. ustus),
duclauxin (P. duclauxi), ochratoxin A, B and C (A. ochraceus), capillarin
(Artemisia capillaris), viridotoxin (A. virinutans), moncerin (H. monoceros) etc.
Trivial names of a large number of isocoumarins end in the suffix "-in" for
example artemidin, bergenin, bactobolin A, B and C, actinobolin, baciphelacin,
coriandrin, asperentin, canescin, fusamarin, mellein, stellatin etc. However
isocoumarin names ending in other suffixes like "-ol, -one, -ide, -oic acid,
anhydride" indicating their chemical class are also common. Example are
altenuisol, hydrangenol, oosponol, oospoglycol, peniolactol, reticulol,
oospolactone, agrimonolide, feralolide, monocerolide, ustic acid, β-callatolic
acid, β-alectoronic acid, ardisic acid B, chebulic acid, lamellicolic anhydride,
naphthalic anhydride, etc.
Isocoumarin (1) itself (R1-R6=H) had never been found to occur naturally
however its simple derivatives are found in nature. Isocoumarin may be
substituted either on lactone ring or the aromatic ring or on both. Thus R1-R6 in
(1) or (3) may be alkyl, aryl, heterocyclyl, halo, nitro or any other substituent. A
number of naturally occurring isocoumarins possess a C-3 carbon substituent
and all isocoumarins, biogenetically derived from acetate have C-8 oxygenation
and some have retained the C-6 oxygen.
Hydrangenol, phyllodulcin, chebulic acid, dihydrohomalicine and
blepherigenin are isocoumarins found in plants, lack C-6 oxygenation and are
2
not acetate derived. Isocoumarins having a C-4, C-5 or C-7 substituents are
relatively uncommon in nature nevertheless C-7 oxygenation is fairly uncommon.
Mellein (4), the 3,4-dihydro-8-hydroxy-3-methylisocoumarin has been
taken as the parent compound for simple isocoumarins. Thus 3,4-dihydro-8-
hydroxy-6-methoxy-3-methylisocoumarin (5) is known as 6-methoxymellein.
Similarly the compounds (6, R1=H, R2=COOH) and (6, R1=CHO, R2=H) are
called as 7-carboxymellein and 5-formylmellein respectively.
O
OOH
O
OOH
O
OOH
OR1
R2
(4) (5) (6)
Peniolactol (7) and 3-alkyl-3-hydroxy-3,4-dihydroisocoumarins such as
ustic acid (8) and its derivatives exist in tautomeric equilibrium between their
keto acid forms (7a & 8a) and lactol forms (7b & 8b), respectively.
O
OH
C15H31
O
OOH
C15H31HO
(7a) (7b)
COOH
HOHO
O
OOH
HO
(8b)
OH
O
OH(8a)
COOH
HOOHO OHO
The lactam analogue of isocoumarin, 1-(2H)-isoquinolinone (9a) trivially
known as isocarbostyril exists in equilibrium with its tautomeric form (9b). A large
3
number of variously substituted isocarbostyrils10 and tetrahydroisoquinolinones
(10), which can also exist as its other tautomer, have been prepared.
NHR
O
NR
OH
NHR
O(9a) (9b) (10)
Sulphur analogues have also been known since times and a number of
substituted 1-thio- (11, Z=S), 1-hydrazino-(11, Z=NNH2), 1-phenylhydrazino- (11, Z=NNHC6H5), 2-thio- (12), and 1,2-dithioisocoumarins11 (13), have been
prepared.
O
Z
S
O
S
S
(11) (12) (13)
OR
In 1980, a three-step synthesis of 2-seleno- and 2-telluroisocoumarins
was reported12. Regiospesific nucleophilic β-addition of methaneselenolate or
methanetellurolate anion to the triple bond of ethyl-2-ethenylbenzoate (14) afforded the chalcogenated esters (14a). Saponification afforded the
corresponding acids (14b) which were electrophilically cyclized via the acid
chlorides to 1H-2-seleno- (15) and 1H-2-telluro- (16)-3-benzopyran-1-ones.
`
OR
Y
O
X
O
14a) R = C2H5, Y = Se or Te14b) R = H, Y = Se or Te
15) X = Se, m.p. 80°C16) X = Te, m.p. 83° C
4
1.2 Physical properties
Isocoumarins are usually solid crystalline compounds having melting points
ranging from 49-50°C (trans-artemidin) to 350°C (alternariol). Some
isocoumarins like 3-pentylisocoumarin and 3-propylisocoumarin are oils. It is
observed that melting points of isocoumarins are invariably higher than those of
corresponding dihydroisocoumarins.
1.3 Biological activities
In 1964 when the review by R. D. Barry2 appeared there were only a few
valid reports of the biological activities of isocoumarins. A literature survey of the
later period shows that isocoumarins display a wide range of biological activities.
A large number of 3-phenylisocoumarin13 has been tested for various
pharmacological activities. Some of these are useful sweeteners, anti-corrosives,
fluorescent agents and laxatives, whereas others possess anti-inflammatory,
anti-allergic, anti-malarial activities and have proved to be useful in the treatment
of asthma14-15.
Mellein (17) has been found in several insects. The defensive secretion of
termites16, Australian onerine ants17, the mandibular gland secretion of
Camponotus herculeanus, C. lighiperda and C. pensylvanicus18 (carpenter ants)
and the male hair pencil of the oriental fruit moth19, all contain mellein. Mellein
and its dihydroderivatives20 are found in the defensive secretions of tenebrionid
beetle, Apsena pubescencs.
O
OOH
(17) Many fungal isocoumarins exhibit antifungal activities21 particularly
oospolactone22 (18), chladosporin23 (19), 6-methoxymellein24 (20), 3-phenyl-3,4-
5
dihydroisocoumarin-4-carboxylic acid25 (21) and 3-phenyl-4-(hydroxyacetyl)-3,4-
dihydroisocoumarin (22).
Coriandrin (23), one of the two naturally occurring furoisocoumarins
known to date, was isolated in 1988 from dry coriander leaves26. In addition to
the expected psoralen activity, it shows in vitro anti-HIV activity27.
O
OOH
O
O
(20)
OH
OHO O
O
OOH
(18) (19)
O
COOH
O
O
O
OHO
(21) (22) (23)
O
O
O
6-[2-Chloro-4-(trifluoromethyl)phenoxy]-3,4-dihydroisocoumarin (24) has
shown herbicidal activity28 and its application of 1kg/ha almost totally controlled
Schinochloa crus-galli, Sinapis alba and other weeds.
Twenty isocoumarins derivatives29 were tested for biological activities
towards rice, radish, barnyard grass and A. niger. At 100 ppm, 4-carboxy-6-
chloro- (25, R=R1=H, R2=6-Cl), 4-carboxy-7-chloro- (25, R=R1=H, R2=7-Cl), and
6-chloro-4-ethoxycarbonyl-3-methylisocoumarin (25, R=Me, R1=Et, R2=6-Cl), were phytotoxic to radish and rice plants while 4-ethoxycarbonyl-6,7-dimethoxy-
3-methylisocoumarin (25, R=Me, R1=Et, R2=6,7-(OMe)2), was phytotoxic to
radish. 3-Methyl-, 3,7-dimethyl- and 6-methoxy-3-methylisocoumarins inhibited
the growth of A. niger.
6
O
O(24)
OCl
F3CO
RCOOR1
O
R2
(25)
Several isocoumarins30 (26) (R=H, alkyl, alkenyl, alkoxy, nitro) are useful
as antihypertensives, antiarrhythmics and β-sympatholytics. These were
prepared starting from 3-hydroxyhomophthalic acid. Antiarrhythmic activity of
(26) (R=H) was comparable to that of pindolol (standard).
Isocoumarins 27(a-b) with different substituents, isolated from the fungus
Ceratocysistis fimbriata coffea, were found to have toxic activity on coffee tree
leaves31 and horse radish peroxidase. Compound (27b) also exhibits antiviral
activity as well as a distinct inhibiting activity on 3α-hydroxysteroid
dehydrogenase (3α -HSD) 32.
Cytogenin (28) shows antitumour activity33 against Ehrlich carcinoma at
6.3 to 100mg/ Kg/ day.
(26)
O
O
RRR
R
ROHO
NH
O O
O O
R1R2
OH OH
OOH
(28)27a) R1 = CH3, R2 = OCH327b) R1 = CH3, R2 = OH
Some derivatives34 of isocoumarin and thioisocoumarin (29) (R=H, OH,
NO2, NH2, halo, alkyl; Y=H, halo, OCH3, CF3; X=O, S; Z=H, halo, C1-6 alkyl,
7
alkylphenyl etc.) are serine protease inhibitors and useful in treatment of
emphysema.
Five isocoumarins35 (-)-S-5-methyl, (-)-S-5-carboxy-, (-)-5-hydroxyethyl-
mellein, cis- and trans-4-hydroxy-5-methylmellein (30) isolated from pathogenic
fungus of apple canker, Valsa ceratosperma showed phytotoxicity in apple
shoots and the lettuce seedlings.
XR
Z
O
(30)
Y
O
O
RR
OH
(29)
3-Alkoxy-7-amino-4-chloroisocoumarin derivatives (31) were synthesized
as new beta-amyloid peptide production inhibitors and found very active against
various classes of proteases36-38.
The isocoumarinyl penicillin derivatives39 (32) were quite effective
bacteriocides at 3.15-100 mg/mL.
O
O
OR
H2N
(32)
O
O
O
NH
N
S
ONaO
O
(31)
Cl
A new isocoumarin 2-(8-hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-
yl)propionic acid (NM-3)40-41 (33), which is an analogue of the natural product
cytogenin, induces the lethality of human carcinoma cells by generation of
reactive oxygen species42 and inhibits angiogenesis43-44. It increases the
antitumour effects of radiotherapy with toxicity45 and Potentiates
Dexamethasone-Induced Apoptosis of Human Multiple Myeloma Cells46. It also
8
increases the antitumor effects of various existing chemotherapeutic drugs in
breast and prostate tumor model systems, as measured by TGI47.
Dihydroisocoumarin PF1223 (34) from the culture of Neosartorya
quadricincta inhibit the specific binding of the non competitive antagonist [3H]
EBOB to housefly head membranes. This compound at 2.2 µM inhibited [3H]
EBOB binding by 65%48.
(33)
O
OH
O
O
O
OH O
OOH
O
O
OH
O
OH
(34)
Phytochemical investigation of the aqueous extract of the roots of
Agrimonia pilosa Ledeb (Rosaceae), as guided by hepatoprotective activity in
vitro, furnished two isocoumarins, agrimonolide (35a) and agrimonolide 6-O-β-D-
glucoside (35b). Compound (35a) showed hepatoprotective effects on both
tacrine-induced cytotoxicity in human liver-derived Hep G2 cells and tert-butyl
hydroperoxide-induced cytotoxicity in rat primary hepatocytes with EC50 values
of 88.2 ± 2.8 and 37.7 ± 1.6 µM, respectively49.
Reticulol (36), produced from a strain of Streptoverticillium, was found to
be active against Topo I mediated DNA relaxation in vitro50. The treatment with
45 µM reticulol afforded inhibitory activity, but with 0.45 and 4.5 µM reticulol,
relaxation of DNA was not significantly reduced. The potency of 45 µM reticulol
in inhibiting relaxation was almost the same as that of 0.1 mM camptothecin
(molecular weight 348.34). Consequently, reticulol exhibited Topo I-inhibitory
efficacy similar to a positive control, camptothecin. This result demonstrated that
reticulol blocked the relaxation of DNA with the formation of supercoiled DNA by
inducing the inactivation of Topo I.
9
O
OOH
RO
O
35a) R = H35b) R = Glc
O
HO
OOH O
(36)
Seven new naturally occurring 3-butylisocoumarins (37) were isolated and
identified from the lipophilic extracts of aerial as well as underground parts of
Asteraceae-Anthemideae51. The antifungal activities of all naturally occurring
derivatives were determined in a germ-tube inhibition test against a susceptible
strain of rice blast fungus Pyricularia grisea. The 3-butyl side chain is
prerequisite for high activity.
Eleven new guanidino-, amino alkoxy- and isothiureido alkoxy substituted
isocoumarins52 (38) are potent mechanism based inhibitors for blood coagulation
serine proteases and other trypsin-like enzymes. In many cases, the inhibited
enzymes are very stable. These isocoumarins are effective anticoagulants in
human plasma.
A substituted isocoumarin53 (39) had a dose dependent reproducible
anticoagulant effect in rabbit. Its effect however ceased shortly after stopping
administration, indicating that its half-life is very short.
(37)
O
O O
OH2NHN
Cl
O
O
OH2N
ClS NH2
NH
(38) (39)
10
Isocoumarins substituted with chloro and basic groups such as guanidino
and isothiureido alkoxy are inhibitors of complement serine proteases. 3,4-
Dichloroisocoumarins are general serine protease inhibitor and 4-chloro-3-(3-
isothiureiodopropxy)isocoumarin (40) is found to be the best inhibitor54 .
Several 6,8-dihydroxyisocoumarins and 6,8-dihydroxy-3,4-dihydro-
isocoumarins are the metabolites of phytopathogenic fungi. The 3-methyl-3-
hydroxy-6,8-dihydroxy-3,4-dihydroisocoumarins (41) and 3,4,6,8-tetrahydroxy-3-
methyl-3,4-dihydroisocoumarin (42) implicated as possible phytotoxins produced
by ceratocystis ulmi, the fungus responsible for Dutch Elm disease55-56. The
dihydroisocoumarin (42) causes lesion on the leaves of pear trees and inhibit the
growth of rice seedlings57.
O
O
Cl
Oisothiuredine
(40)
O
OH
HO OH
O
O
OH
HO OH
O
OH
(41) (42)
8-Hydroxy-3,4-dihydroisocoumarins have been found to constitute a new
class of ant pheromone58. The insects use trail pheromones for making trail
between the feeding place and the nest. Mellein and other 8-
hydroxydihydroisocoumarins 43(a-d) and 3-ethyl-7-methyl-8-hydroxy-3,4-
dihydroisocoumarin (44) have been isolated as trail pheromones in the hindgut
of ants of various species of the genera Formica and Lasius found in Germany.
11
O
OH
R1
O
O
OH O
R
R2
43a) R = R1 = R2 = H 43b) R =R1 = Me, R2 = H43c) R1 = R2 = Me, R = H 43d) R = R2 = Me, R1 = H
(44)
Sclerin (45) and sclerotinins A (46) and sclerotinins B (47), metabolites of
sclerotinia sclerotiorum have plant growth regularity effect59-60.
O
OH
O
O
O
OH O
HOR
46) R = CH347) R = H
(45)
The mode of action of these isocoumarins in phytopathogenic organisms
is not clear although they are observed to build up in the trees and plants after
infection by the fungi61.
1.4 Behaviour towards human beings
Some isocoumarins have beneficial activities while others are harmful
towards human beings. Some show diuretic and antihypertensive activities62 (48) (R=Phenyl, 3,4-dichlorophenyl, 4-methoxyphenyl, R1=H, methyl, NR2R3=NMe2,
N-diallyl, 4-methylpiperazino, morpholino , etc.). Some have been used in the
treatment of lymphedema63. The antitumour activity of duclauxin (49) has been
demonstrated64. Analogues of bactobolin A (50) have antileukemic activity65
whereas bactobolin A and its related metabolites are active against bacteria and
viruses66. Among the most important isocoumarins, the AI-77s (51), endowed of
12
gastroprotective properties67-68 but free of effects on the central nervous system,
Phyllodulcin, a lead compound in the discovery of novel low calorie sweeteners,
coriandrin, active against HIV69. Agrimonolide possesses strong anthelmintic
activity70. The observations of scanning electron microscope have shown that
anthelmintic effects of agrimonolide are due to its destructive effects on the body
wall of tapeworms. A number of isocoumarin-3-carboxylic acids71 (52) (R, R1, R2
= H, methyl, methoxy, bromo, etc.) were active in the passive cutaneous
anaphylaxis test.
O
OOAcO
HO
O
OH
O
O
O
O
N R3
R R1 R2
O
O
NH
OH
O
OOH
O
O
COOH
O
R2
R1
R
O
OOH
HOCHCl2
HNH
OHNH2
O
(48)
(49)
(50)
(51) (52)
Some isocoumarins and dihydroisocoumarins are harmful to man, like
ochratoxin A (53) and ochratoxin B (54) which are nephratoxic and hepatotoxic
metabolites of several Aspergillus and Penicillium species72-73. Ochratoxin A (53)
inhibits protein synthesis74. Oosponol inhibits dopamine β-hydroxylase and
causes severe skin rash, bronchitis and pneumonia75 and reticulol (36) inhibits
cyclic AMPase76.
O
OH O
HN
COOHO
O
OH O
HN
COOHO
Cl
(53) (54)
13
1.5 Biosynthesis
J. N. Collie (1907) made the first suggestion of the biosynthesis of certain
classes of aromatic compounds, by the head to tail condensation of acetate
units. These considerations were based upon the reactivity of synthetic linear β-
polyketones (55; n ≥2) which underwent aldol type condensations to form
aromatic phenolic compounds.
R
OO
n
(55) Biogenetic definitions of the aromatic polyketides are due to the work of
Birch (1951), by whom a detailed theoretical analysis of carbon skeletons and
oxygenation patterns of known compounds was first combined with an extensive
series of tracer incorporation experiments. The elaboration of the β-ketide chain
in metabolites derived from poly-β-ketide precursors, normally proceeds by
condensation of terminal unit of acetyl Co-enzyme A with chain building units of
malonyl Co-enzyme A (Scheme 1.1).
SCoA
O
HSCoA + CO2
+ HO SEnz
O O
SEnz
O O
Scheme 1.1 The orsellinic acid may be derived from a β-tetraketone, which might undergo
the aldol or claisen type condensation (Scheme 1.2).
SEnz
O OO O
OHHO
OH
OHHO
OH
O
O
a
b
Scheme 1.2
14
T. Money and F. W. Comer et al.77 using pyrones as masked β-
polycarbonyls provided the experimental verification of the acetate-malonate
pathway for the synthesis of isocoumarins. Thus hydrolysis of trypyrone (57)
which is a protected β-pentaketide (56) afforded eight crystalline compounds, six
of which were obtained by internal aldol condensations of (57) at positions 2 and
7 (Scheme 1.3). The products (58) and (59) appeared due to prior degradation
of (56 or 57). The remaining products represented conversion of the intact C10
chain and are variants of the 2,7-aldol condensation of (57). The most significant
of these are 6,8-dihydroxy-3-methylisocoumarin (60, R = H) and 7-
carbomethoxy-6,8-dihydroxy-3-methylisocoumarin (62, R = COOCH3). The
structure of (62) was confirmed by spectroscopy and its conversion to dimethyl
ester (63, R=COOCH3) which is another product of the reaction. The methyl
ether (64) has been isolated from Endothia parasitica, the lactol viz. 3,4-dihydro-
3,6-dimethoxy-8-hydroxy-3-methylisocoumarin (65) has also been isolated.
O OO O
OR
O
COORCOOR2378
(56)
Scheme 1.3
O
O
O
O
O
OHO
23
78
OO
O
OH
O
O
COOCH3
O
O
OH
O
HO OH
OO
OH
HO
O O
O
HO
OH OR COOCH3
O
HO
OHR
60) R = H62) R = COOCH3
61) R = H63) R = COOCH3
(57)
(58)
(59)
(65)
(64)
15
This biogenetic type synthesis of isocoumarins and related compounds
confirmed the acetate-malonate pathway for biosynthesis of such compounds. 14C Labeled malonate in these metabolites yields a product in which each of the
chain building unit carries a label but the terminal unit of the chain is inactive. On
the basis of structure analysis and tracer work many fungal metabolites appear
to be derived biogenetically from the acetate and polymalonate pathway78. For
example mellein is formed from acetate and malonate as given in (Scheme 1.4).
O
O
COOH
CH3
OO
O
HO
COOHOOCH3COOH 4H2C
COOH
COOH
OCOOH
OHO
O
O
OH
Scheme 1.4
The early reduction of two carbonyl groups in the polyketide chain
followed by the loss of oxygen function at C-6 and then aldol type condensation
result in the aromatization, to give mellein. Loss of the oxygen function at C-6 of
an isocoumarins is quite common but loss of the hydroxyl group at C-8 never
occurs in those isocoumarins derived from acetate presumably a consequence
of the cyclization mechanism79.
In 1970 Y. Suzuki80 isolated fusamarin (66) from a strain of fusarium sp.
first synthesis was completed in 1978 under the supervision of Prof. W. B.
Whalley 81. In the biosynthesis of fusamarin the main chain may be derived from
poly-β-ketide pathway and is biosynthesised from seven acetate units.
16
O
OH
HOH
O
(66) The butyl chain at C-5 is supposed to be derived from isopentyl
pyrophosphate in which one carbon atom is missing presumably may involve an
electrophilic attack on the poly-β-ketide chain. This would be a unique example
of the deviation from the "biogenetic isoprene rule". 6,8-Dihydroxy-3-undecyl-3,4-
dihydroisocoumarin82 (67) isolated from Ononis natrix and peniolactol83 (7a, 7b)
isolated from Peniophora sanguinea Bres belong to poly-β-ketide pathway and
can be biosynthesized from ten and twelve acetate units and their synthesis
have been completed under the supervision of Prof. N. H. Rama84-85.
O
O
OH
OHHO
O
OH
HO
COOH
(7a)
(7b)
Biosynthesized from twelve acetate units
O
O
OH
HO
(67)
Biosynthesized from ten acetate units
17
The biosynthesis of plant derived isocoumarins has been studied to a
lesser extent than fungal isocoumarins. Phyllodulcin (69), the sweet principle of
Hydrangea macrophylla and hydrangenol (68), has been shown to be formed
from phenylalanine via cinnamic acid and p-coumaric acid with the addition of
three acetate units86-89 (Scheme 1.5).
OHNH2
O
OH
O
OH
O
HO
O
O
OH
OH
O
O
O
OH
OH
(68)(69)Scheme 1.5
Bergenin (70) is derived from C-glucosylation of gallic acid and
subsequent lactone formation90 (Scheme 1.6).
Scheme 1.6
O
OO
O OHCH2OH
OHH
HO
O
OH
OHHO
HO
Glucose
(70)
1.6 Synthesis of isocoumarins and 3,4-dihydro- isocoumarins
A wide spectrum of synthetic methods have been used towards the
synthesis of isocoumarins and 3,4-dihydroisocoumarins91. A number of new
18
methods 92-102 are being developed and reported each year. Some of these
methods provide the isocoumarins directly whereas others lead to the 3,4-
dihydroisocoumarins. Some of the most important, high yield methods applicable
to the synthesis of a large number of these compounds are mentioned below.
1.6.1 Synthesis Involving Metals
Literature reveals that isocoumarins and 3,4-dihydroisocoumarins have
been extensively prepared by such methods, involving metallation at specific
position like lithiation, silylation and thallation etc.
a. Lithiation Reaction
Benzoic acid derivatives are important precursors of isocoumarins.
Among the methods available for introducing a β-functionalized carbon
substituent ortho to the carboxyl group, those involving ortho-metallation of the
benzene ring have enjoyed a great popularity. This approach has been
thoroughly reviewed103-105. Summarizing the general concepts, carboxylic acid
derivatives suitable for promoting ortho lithiation106-107 are tertiary amides (4,4-
dimethyl)oxazolin-2-yl group and secondary amides. Lithiated tertiary amides are
readily and generaly ortho-lithiated using s-butyllithium and tetramethylethylene-
diamine, but their reaction with alkylating agents other than methyl iodide gives
low yields because of a poor nucleophilicity. Allylation of lithiated tertiary
benzamides has however been accomplished in high yields by previous trans-
metallation to a magnesium or (better) to a copper derivative; the allyl group thus
introduced has been converted to the β-hydroxyalkyl group required to complete
the lactone ring in the conditions of the acid hydrolysis of the benzamide, leading
to racemic 3,4-dihydroisocoumarins directly, apparently without the possibility of
isolating the intermediate allylbenzoic acids; alternatively, asymmetric
hydroxylation of the double bond followed by treatment with acids has been used
to obtain 3,4-dihydroisocoumarins with a high degree of enantiomeric purity, as
19
demonstrated by the enantioselective synthesis of the isocoumarin portion of
AI77B (71) (Scheme 1.7)108-111.
O
OOO
N
O
a,b,c
O
N
O
d,e
OH
O
O
NH2
a) BuLi, TMEDA b) CuCN(LiCl2) c) (E)-1-bromo-5-methyl-2-hexene
d) Sharples AD e) aq. NaOH and then HCl
Scheme 1.7
(71)81% 69.6% OH
Enantiomerically pure natural 3,4-dihydroisocoumarins have been
obtained from lithiated secondary benzamides and homochiral epoxides.
Coupling between lithiated secondary benzamides and epoxides belongs to the
beginning of the anionic chemistry of aromatic compounds; unfortunately, yields
are generally modest and N-alkylation can complicate the reaction112. Good
yields have occasionally been reported though, as in the synthesis of the
allergenic principle of gingko biloba (72) (Scheme 1.8)113 and of a variety of
mellein derivatives114.
O
C12H25
OOO
LiN
O
a
O
HN
O
bO
O
a) (R)-1,2-epoxytetradecane b) OH, then neutralization CuCN(LiCl2) c) BBr3
Li
C12H25 OH
OH
C12H25
70%98%
c
_
Scheme 1.8
(72)
Lateral lithiation of (S)-4-isopropyl-2-(o-tolyl)oxazoline in diethyl ether
followed by the reaction with aldehydes in the presence of TMEDA produced the
addition products with stereoselectivities up to 84% de115. Utilization of TMEDA
20
as a ligand is essential for the good selectivity. Rationale for the stereoselectivity
is proposed based on ab-initio calculation of the lateral lithio species. The major
(S,S)-products lactonized faster than the minor (S,R)-products to the
corresponding 3,4-dihydroisocoumarins under acidic conditions. Thus, (3S)-3,4-
dihydroisocoumarins were obtained in good optical purities up to 97% ee by
sequential application of these matched stereo-selective reactions (Scheme 1.9)
LiN
ON
O
RHO*
O
O
R*
RCHO H3O+
Scheme 1.9
b. Silylation Method
Closely related to lithiation is the desilylation of 2-(trimethylsilylmethyl)-
benzamides, which generates carbanions suitable for additions to aldehydes116.
2-(Trimethylsilylmethyl)benzoyl chlorides also undergo desilylation and addition
to aldehydes to give dihydroisocoumarins through a concerted mechanism
involving ortho-quinodimethanes rather than carbanions as reactive
intermediates (Scheme 1.10)117.
Si(CH3)3Cl
OOO
OH
O
a
O
OH
O
bO
Ar
O
a) n-BuLi, (CH3)3SiCl b)SOCl2 c) CsF, ArCHO
O
cSi(CH3)3
50-53%
Scheme 1.10
OO
To this class of reactive intermediates belong the products of UV
irradiation of ortho-toluyl cyanides which add to aliphatic and aromatic acyl
cyanides to give 3-cyano-3-phenyl-8-methoxy-3,4-dihydroisocoumarins which
are converted to isocoumarins by treatment with strong bases (Scheme 1.11)118.
21
O O
a,b
O
CN
O
cO
Ph
O
a) (CH3)3SiCN b)PCC c) hv, PhCOCN
OOHO
CNCN
Scheme 1.11
c. Thallation-olefination of Arenes
Isocoumarins and 3,4-dihydroisocoumarins were prepared in a single pot
reaction119, by reacting a benzoic acid with an electrophilic thallium salt in the
presence of an organic solvent to give O-thalliated benzoic acid followed by
reaction with an organic compound e. g. an alkene in the presence of PdCl2
(Scheme 1.12).
Tl
COOH
O
O
RRH2C=CH2
PdCl2
Scheme 1.12
d. Palladium catalyzed method
Aryl iodides with a nucleophilic substituent at the ortho position react with
1,2-dienes in the presence of a palladium catalyst120 and a chiral bisoxazoline
ligand to afford isocoumarin (73) in good yield and with 46-86% enantiomeric
excess (Scheme 1.13).
22
I
COOHO
O
n-C8H17
+ n-C8H17-CH=C=CH2
5mol% Pd(dba)25mol% Ligand
1.2 equiv Ag3PO4
Scheme 1.13
(73)
e. Iridium catalyzed method
Two new cyclizations of ketoaldehydes121 have been developed using
an Ir-ligand bifunctional catalyst. Oxidative lactonization of δ-ketoaldehydes
proceeded smoothly at room temperature to give coumarin derivatives in
excellent yields. Intramolecular Tishchenko reaction of δ-ketoaldehydes
afforded 3,4-dihydroisocoumarins (74a-b) in good yields (Scheme 1.14).
R
O
O
R
OH
O
R
O
O+
OIr
HN
PhPh
(5mol%)cooxidantbast, rt, 16 h
R= CH3R= Ph
R
O
O
R
O
O+
Ir cat (5mol%)
t-BuOH, reflux
R
O
O
74a) R= CH3, 70%74b) R= Ph, 45%
28%47%
Scheme 1.14
f. Rhodium-Catalyzed Oxidative Coupling of Benzoic Acids with
Alkynes via Regioselective C-H Bond Cleavage
The oxidative coupling of benzoic acids with internal alkynes effectively
proceeds in the presence of [Cp*RhCl2]2 and Cu (OAc)2. H2O as catalyst122 and
oxidant respectively to produce the corresponding isocoumarin derivatives. The
copper salt can be reduced to a catalytic quantity under air (Scheme 1.15).
23
COOHO
OScheme 1.15
H+ R R
Rh-Cat.
Cu-Salt
RR
g. Mercury catalyzed method
Sulphuric acid–catalyzed chloralhydrate condensation with different m-
substituted benzoic acids formed trichlorophthalides, from which Zn+AcOH
reduction afforded various dichloro derivatives. These derivatives on treatment
with alkaline Hg(OAc)2 + I2 furnished different substituted isocoumarins123
(Scheme 1.16).
COOHRRO
O
CCl3Zn-AcOH
Cl
Cl RO
O
ClHg(OAc)2NaHCO3, I2
DMSO70-80°C3-4h 75a) R= OCH3
75b) R= OH75c) R= OCH2PhScheme 1.16
1.6.2 Oxidation of Indenes, Indanones and Indones
Isocoumarins and 3,4-dihydroisocoumarins have been prepared in high
yield by ozonization of indene (76) in ethyl alcohol, followed by decomposition of
the intermediate cyclic perester124 (77). Treatment of 2-
carboxyphenylacetaldehyde (78) with mineral acid (or copper powder) 125 lead to
isocoumarin and sodium borohydride give 3,4-dihydroisocoumarin (Scheme 1.17).
24
O3
OO
HO
OC2H5OH
OO
O
O
O
O
O
O
OH
O
ONaBH4
AlkAlk
H+
(76) (77)
(78)
Scheme 1.17 Oxidative cleavage of indanone has been used for synthesis of 6,8-
dimethoxy-3-methylisocoumarin126-127and related isocoumarins. Thus 2-
methylindan-1-one (79) was converted into silyl ether (80) which on ozonolysis
afforded the 2-hydroxyindanone (81). The latter on periodate cleavage afforded
the isocoumarin (82) presumably via the keto acid (82a). Alternatively, the
conversion of indanone (79) into enol (81) followed by ozonolysis gave the
desired isocoumarin (83) (Scheme 1.18).
O
R
OOSi(CH3)3
R
O OSi(CH3)3
R
OO
OCOCF3
R
OO
R
OOH
R
O
R
OCOOH
OO
O
Scheme 1.18
(79) (80)
(81)
(82a)
(82)
(83)
25
Indanone epoxide (84) prepared from indanone by epoxidation with H2O2/
(C2H5)3N in acetone was submitted to flash vacuum pyrolysis128 (FVP)
(450oC/0.1 mm).The epoxide undergoes rearrangement during FVP to afford
isocoumarin129 (85) (Scheme 1.19).
OO OO
(C2H5)3N, H2O2
OO
O
O
OO
R
O
OO O
O
FVPπ4a+π2acycloreversion
(84)
(85)Scheme 1.19
1.6.3 Oxidation of Isochromans
Oxidation of isochromans with a variety of reagents e.g. selenium dioxide,
chromium trioxide, potassium permanganate, nitric acid or air yields the
corresponding 3,4-dihydroisocoumarins (Scheme 1.20).
OR O
R
OScheme 1.20
Isochroman formed from 2-arylethanol130 is also converted to 3,4-
dihydroisocoumarin by oxidation with PCC in boiling CH2Cl2129, which give
isocoumarin (86) (R=H, 7-Me, 5-CF3, 5,6-C4H4) on treatment with NBS and Et3N
(Scheme 1.21).
26
OR O
R
O
OR'R
(R' = H, MEM)
a bO
R
O
c
b) PCC c) NBS, (C2H5)3N(86)
Scheme 1.21
a) TiCl4
1.6.4 Aldol-type Condensation between Homophthalic Acids, Esters or Anhydrides and Carbonyl Compounds
This type of condensation is mostly used in the synthesis of isocoumarins
and 3,4-dihydroisocoumarins. The most important methods of aldol type
condensation are discussed in four main groups.
a. Stobbe Condensation of Homophthalates with Aldehydes and Ketones
Stobbe condensation is used for synthesis of a number of 3,4-
dihydroisocoumarins131-135. Synthesis of (dl)-agrimonolide136 provides a good
example of application of Stobbe condensation.
Thus, diethyl 3,4-dibenzyloxyhomophthalate (87) on condensation with 4-
methoxybenzaldehyde in presence of sodium hydride afforded 2,4-dibenzyloxy-
6-[1-ethoxycarbonyl-4-(4'-methoxyphenyl)buten-1-yl]benzoic acid (88a,
R=COOEt). Hydrolysis and decarboxylation gave 2,4-dibenzyloxy-6-[4-(4'-
methoxyphenyl)buten-1-yl]benzoic acid (88b, R=H) which on cyclization with
bromine gave the 4-bromo-3,4-dihydroisocoumarin (89). Reductive
debromination and debenzylation was simultaneously effected by adding triethyl
amine to the catalytic reduction medium to furnish the (dl)-agrimonolide (90) (Scheme 1.22)
27
BzO
OBz
OMeBr
O
O
COOC2H5
COOC2H5OBz
BzO
OBz
BzO
COOH
R O
O
O
NaOH
88a) R= COOC2H5
OBz
BzO
COOH
O
Br2/CHCl3
HO
OH
OMe
O
O
H2/Pd-C/(C2H5)3N
(87)
88b) R = H (89)
(90)
Scheme 1.22
b. Claisen Condensation of Homophthalates with Formates
Diethyl homophthalate (91) condenses with methyl formate in the
presence of sodium ethoxide imparting a 66% yield of isocoumarin-4-carboxylic
acid (92). Decarboxylation with phosphoric acid furnishes isocoumarin (93)137
(Scheme 1.23).
28
COOC2H5
COOC2H5
HCOOCH3
C2H5ONa O
O
COOH
H3PO4
-CO2O
O(91) (92) (93)
Scheme 1.23
6,7-Dimethoxyisocoumarin and 5,7-dimethoxyisocoumarin were also
prepared by the above procedure. Ethyl 5,6,7-trimethoxyisocoumarin-4-
carboxylate was prepared from corresponding homophthalate and ethyl formate
in the presence of potassium ethoxide in good yield138.
c. Claisen Condensations of Homophthalates with Oxalates
Metallic sodium in ether, or better without a solvent, effects ready
condensation between diethyl homophthalate (94) and diethyl oxalate, giving a
67% yield of triester (95). This triester loses ethanol when heated yielding diethyl
isocoumarin-3,4-dicarboxylate (96). Under different hydrolysis conditions
different products are formed. Thus heating (96) at 68-72°C for 3hr. gives ethyl
isocoumarin-3-(carboxylic acid)-4-carboxylate (97) and prolonged heating yields
isocoumarin-3-carboxylic acid (98). Boiling hydrochloric acid or heating in a
sealed tube at 180-190°C converts (96) to isocoumarin-3-carboxylic acid in 84%
yield139. These results indicate that the ester at position 3 in (96) is hydrolyzed
first, but the acid at position 4 is more easily decarboxylated (Scheme 1.24).
29
COOC2H5
COOC2H5COCOOC2H5
OC2H5NaOO
OO+
O
O O
O
COOC2H5
COOC2H5
O
O
O
COOH
O
O
COOHCOOC2H5
(94)(95)
(96)
(97)
(98)
Scheme 1.24
d. Condensation of malonyl heterocycles with diphenylcarbonate
Reaction of diphenylcarbonate (100) with enolized phenylmalonyl
heterocyclic compounds as (99 a-d) yields the condensed isocoumarins140 like
(101 a-d) (Scheme 1.25).
X
OH
O X
O
O
O
C6H5O OC6H5
O
a) X= Hb) X= NHc) X= NCH3d) X= NC6H5
(99) (101)
(100)
Scheme 1.25
30
e. Condensation of Acid chlorides, Phenols, Phenol acids with homophthalic acids and Anhydrides
Tirodkar and R. N. Usgaonkar141-142 carried out two or three step
synthesis of various 3-alkyl/aryl isocoumarins. The synthesis involved pyridine
catalysed acylation of homophtalic acids with acid chlorides or anhydrides to
give isochroman-1,3-dione (102). Treatment of (102) with conc. sulphuric acid at
room temperature gave the 3-alkyl/aryl isocoumarin-3-carboxylic acid whereas
on treatment with 90% sulphuric acid at 90°C directly gave the isocoumarins
(Scheme 1.26).
COOH
COOHR (R'CO)2O/Py
90-100°C RO
R'COOH
Or.t
RO
OCOR'
O
RO
R'
O
conc H2SO4
90% H2SO4
(102)Scheme 1.26
S. Nakajima et. al. synthesized various 3-arylisocoumarins (104, Ar =Ph,
p-anisyl, p-(OH)phenyl etc.) and later on 3-alkylisocoumarins in high yields
(80%) by heating directly the homophthalic acids (103, R, R1, R2=H, OH, OMe,
Cl) with aryl or acyl chlorides at 190°C. These isocoumarins were converted into
corresponding 3,4-dihydroisocoumarins by reducing with NaBH4(Scheme 1.27).
COOH
COOH
Ar/ RCOCl190°C O
Ar/ R
O
RR1
R2
RR1
R2(103) (104)
Scheme 1.27
31
A. Rose143 and later on H. Yoshikawa144 prepared a large number of 3-
(hydroxyphenyl)isocoumarins by condensation of various phenols with
substituted homophthalic acids in moderate yields in presence of polyphosphoric
acid (PPA) or anhydrous stannic chloride e.g. 7-methyl-3-(2′-hydroxy-4′-
methylphenyl)isocoumarin (105) was obtained from 7-methylhomophthalic acid
(Scheme 1.28).
Scheme 1.28
COOH
COOHO
O
OH
(105)
PPA
anh. SnCl4
3-(2',4'-Dimethoxyphenyl)-, 3-(2'-methyl-4'-hydroxyphenyl)isocoumarins
etc. were prepared145 by condensation of homophthalic anhydride with
appropriate phenols. 3-(4'-Methoxyphenyl)isocoumarin was prepared by
condensation of homophthalic acid with anisole (Scheme 1.29).
COOH
COOH O
O
OO
/PPA
Scheme 1.29
1.6.5 Synthesis of isocoumarins via iodocyclization
A variety of 3-substituted 4-iodoisocoumarins and 6-substituted 5-iodo-
2(2H)-pyranones are readily prepared in excellent yields under mild reaction
conditions by the reaction of o-(1-alkynyl)benzoates and (Z)-2-alken-4-ynoates
with ICl146-148 (Scheme 1.30).
32
Scheme 1.30
OR1
R2
O
ICI O
O
IR2
OR1
R2
O
ICI O
O
IR2
1.6.6 Synthesis of isocoumarin by the use of new technology Microreactor technology has been studied as a suitable process to
produce chemicals via multicomponent reactions. Efforts were made to produce
3,4-diamino-1H-isochromen-1-ones. Based on a known reaction procedure,
using in situ generated HCN, a safe reaction setup was created to avoid the
release of the hazardous gas during the process. The 3,4-diamino-1H-
isochromen-1-ones149 were produced continuously in moderate to good yields
(Scheme 1.31).
NH
O
O
Scheme 1.31
OHH
O
O
OHH
N
O
R
RNH2KCN
HOAc O.
NH
O
R
N
HOAc
HNR
NH2
O
O
HNR
33
1.7 Interconverision of isocoumarins and 3,4-dihydroisocumarins
We have seen so far that some methods directly afford the isocoumarins
while others produce dihydroisocoumarins. Their interconversion is carried out
depending upon whether the synthesis of isocoumarin is easier or that of its
dihydro derivative.
1.7.1 Conversion of 3,4-Dihydroisocoumarins to Isocoumarins
There are two routs mainly used for conversion of 3,4-
dihydroisocoumarins to isocoumarins are as follows.
1.7.1.a Alkaline Hydrolysis Followed by Oxidation and Recyclization
Alkaline hydrolysis of 3,4-dihydroisocoumarins150 yields the hydroxy acids
which could be oxidized to corresponding keto-acids. Since the hydroxy acids on
standing recyclize to parent dihydroisocoumarins, the oxidation should be carried
out immediately. The keto-acids are readily cyclized e.g. by heating with acetic
anhydride to corresponding isocoumarins (Scheme 1.32).
O
O
RR
OH
RR
COOH
CrO3
O
RR
COOHO
O
RR
Scheme 1.32
1.7.1.b Benzylic Bromination Followed by Dehydrobromination
Isocoumarins can be prepared from 3,4-dihydroisocoumarin151-152 via
benzylic bromination with N-bromosuccinimide (NBS), followed by
dehydrohalogenation with triethylamine (Scheme 1.33)
34
O
O
OO
O
O
OO
O
O
OO
Br
NBSUV
N(C2H5)3
Scheme 1.33
1.7.2 Conversion of Isocoumarins to 3,4-Dihydroisocoumarins
Two different methods of reduction mainly used for conversion of
isocoumarins to 3,4-dihydroisocoumarins.
1.7.2.a Alkaline Hydrolysis Followed by Reduction and Recyclization
Alkaline hydrolysis of isocoumarins with dilute aqueous alkali affords the
keto-acids, which upon reduction with sodium borohydride are converted into
corresponding hydroxy-acids. Cyclodehydration of the latter affords the
dihydroisocoumarins (Scheme 1.34)
O
O
Ar
OH
Ar
COOHO
Ar
COOH
O
O
Ar
O OO O
KOHCH3OH
NaBH4 -H20
Scheme 1.34
1.7.2.b Catalytic Reduction
Hydrogenation153-154 in the presence of palladium charcoal or some
other catalyst has been used to reduce the 3,4-double bond of isocoumarins
thereby converting them directly into 3,4-dihydroisocoumarins.
35
1.8 Reactions of isocoumarins and 3,4-dihydro- isocoumarin
1.8.1 Hydrolysis
Isocoumarins are lactones and undergo ring opening on alkaline
hydrolysis to give homophthaldehydic acids or ketones (106, R=H) or hydroxy
acid (107, R=alkyl, aryl, etc.). Similar treatment of 3,4-dihydroisocoumarins
yields the corresponding β-(2-carboxyphenyl)ethyl alcohol (107). In most of the
cases isolation of the free acids due to spontaneous recyclization to lactonic ring
is not possible. In some cases e.g. during the hydrolysis of cis-3-phenyl-4-
hydroxy-3,4-dihydroisocoumarin (108), the glycol produced recyclizes to the
more stable erythro-γ-lactone (109) under acid treatment155-156(Scheme 1.35).
OH
R'R
COOHO
R'R
COOH
O
O
OH
Ph
HH
COOOH
OHH
-
PhO
O
H H
OHPh
(106) (107)
(109)(108)Scheme 1.35
1.8.4 Reaction with Ammonia and Amines Ammonia and amines add to isocoumarins furnishing isocarbostyrils157
(110), a reaction typical of esters (Scheme 1.36).
O
O
NR
O
NH3orNH2R
(110)Scheme 1.36
36
For example isocoumarin and 3-carboxylic acid have been condensed
with tryptamine, and the product subsequently converted to yobyrine (111) and
other derivatives158 (Scheme 1.37). .
Scheme 1.37
O
ON
NH2
N
ON
N
O
HN
+
(111)
It is reported that 3,4-dihydroisocoumarin with ammonia gives the
corresponding tetrahydroisoquinolinones e.g. heating agrimonolide with
ammonia at 100°C gave the isoquinolinone analogue (112) (Scheme 1.38).
.
O
OOH
HO
O
NH
OOH
HO
O
NH3
100°C
Scheme 1.38(112)
1.8.3 Reaction with Phosphorus Pentasulfide
Isocoumarin can be converted to 1-thioisocoumarin (113) with
phosphorus pentasulfide, and treatment of 1-thioisocoumarin with ammonium
sulfide or aniline yields isoquinolins (Scheme 1.39). Analogously, 3-
phenylisocoumarin has been converted to 1-thio-3-phenylisocoumarin, and
treatment with aniline produced (114)159.
37
O
O
O
S
P2S5
N N
O
Ph
Ph
(113) (114)
Scheme 1.39
1.8.4 Nitration
The only report of the nitration of an isocoumarin is that of 3-phenyl-3,4-
dihydroisocoumarin (115), in which nitric acid in sulfuric acid gives 3-(4-
nitrophenyl)-7-nitro-3,4-dihydroisocoumarin (116)160 (Scheme 1.40).
O
O
Ph
O
O
NO2
HNO3
O2N
(115) (116)Scheme 1.40
1.8.5 Reaction with Grignard Reagents
Addition of phenylmagnesium bromide161-166 to 3-phenylisocoumarin (117) followed by perchloric acid, anhydrous hydrochloric acid, ferric chloride or ferric
bromide yields the isobenzopyrilium salt (118) (Scheme 1.41).
O
O
Ph
O
Ph
O
Ph
Ph
PhHO
C6H5MgBr HY Y-
+
(117) (118)Scheme 1.41
Y= perchlorate, chloride, ferric chloride and ferric bromide, respectively.
38
1.8.6 Oxidation
Chromium trioxide oxidation of 3,4,6,7-tetraphenylisocoumarin (119) produce 2-benzoyl-4,5-diphenylbenzoic acid161 (120) (Scheme 1.42).
O
Ph
Ph
PhPh
COOH
Ph
Ph
O
Ph
O
CrO3
(119) (120)Scheme 1.42
1.8.7 Reduction
The 3,4-double bond of isocoumarin is readily reduced with hydrogen and
palladium on charcoal or with other catalyst167-168. Catalytic reduction also has
been used to remove the halogen from cis- and trans-3-phenyl-4-halo-3,4-
dihydroisocoumarin169-170.
1.9 Plan of work More than thirty years elapsed, still increasing number of new
isocoumarins has been found in nature exhibiting a wide structural diversity in
their natural source and biosynthetic pathways. These findings are a constant
stimulant for synthetic work, which have been undertaken either to confirm novel
structures or to provide substantial amounts of material for biological and
pharmaceutical studies in those cases in which an isocoumarin exhibiting
interesting properties or was suspected of being responsible for the significant
properties associated with its natural source. In view of biological activity of
isocoumarin and 3,4-dihydroisocoumarin, it was planned to synthesize some of
outlined compounds. The work is divided in two groups.
i. Naturally occurring isocoumarin.
ii. Non naturally occurring isocoumarins and 3,4-dihydroisocoumarins.
39
1.9.1 Naturally occurring isocoumarin In order to achieve the objective of the present work, a plan was made for
the synthesis of naturally occurring isocoumarin, Thunberginol B.
1.9.2 Unnaturally occurring isocoumarins and 3,4- dihydro isocoumarins
The plan for the preparation of unnatural isocoumarin and 3,4-
dihydroisocoumarin is to synthesize 3-dihalophenylisocoumarins and 3,4-
dihydroisocoumarins. Synthesis of all these compounds was based on the
following known principles, which are illustrated in (Scheme 1.43). i. Direct condensation of homophthalic acid with corresponding aryl
chlorides to obtain 3-substituted isocoumarins.
ii. Alkaline hydrolysis of the isocoumarin to obtain corresponding keto-acid.
iii. Reformation of the isocoumarin by refluxing of the keto-acid with acetic
anhydride.
iv. Preparation of recemic 3,4-dihydroisocoumarins by reduction of keto-
acids using sodium borohydride to yield corresponding hydroxy-acids
followed by cyclodehydration with acetic anhydride.
OHO
R
O
OHO
OH
O
200°C, 6h
OO
R
5% KOH/ C2H5OH
NaBH4
OHO
R
OH
OO
R
R Cl
O+
Ac2O
Ac2O
reflux
Scheme 1.43 General synthetic scheme for the synthesis of isocoumarins
and 3,4-dihydroisocoumarins
40
42
Chapter-2 TOTAL SYNTHESIS OF THUNBERGINOL B
(A NATURAL ISOCOUMARIN)
Various naturally occurring isocoumarins have been isolated and synthesized
so far. In the present work, synthesis of thunberginol B has been carried out.
The detail of this work is as follows.
2.1 Synthesis of 6,8-dihydroxy-3-(3′,4′-dihydroxy-
phenyl)isocoumarin (Thunberginol B)
2.1.1 Introduction Thunberginols A (121), B (122), and F (125) were isolated from
Hydrangeae Dulcis Folium, the fermented leaves of Hydrangea macrophylla
SERINGE var. thunbergii MAKINO, as antiallergic constituents171-174.
Compounds 121, 122, and 125 inhibited histamine release from rat peritoneal
mast cells stimulated by compound 48/80, calcium ionophore A23187, or
antigen173-175 and oral administration of compound 121 inhibited passive
cutaneous anaphylaxis reactions in rats 176-177. However, their inhibitory
effects on the antigen-induced degranulation and release of cytokines in
basophils have not been reported to date.
Wang et al178 examined the effects of the isolated compounds
[thunberginols A (121), B (122), C (123), E (124), and F (125), phyllodulcin
(127), hydrangenol (128)] from Hydrangeae Dulicis Folium, thunberginol G
(126) and 3′-hydroxyhydrangeaic acid (129) derived from 127 on the
degranulations and/or releases of TNF-α and IL-4 via FcεRI signaling in rat
basophilic leukemia (RBL-2H3) cells. In addition, effects of the active
constituents on increase in [Ca2+]i were examined to get some information for
their mechanism of action. In conclusion, the 3-phenylisocoumarins
thunberginols A (121) and B (122)] and a benzylidene-phthalide thunberginol
F (125)] from the processed leaves of Hydrangea macrophylla var. thunbergii
(Hydrangeae Dulcis Folium) substantially inhibited the degranulation by
43
antigen and calcium ionophore A23187, and the releases of TNF-α and IL-4
by antigen in RBL-2H3 cells. With regard to structural requirements of the 3-
phenylisocoumarins for the activity, the 3,4-double bond was essential for the
strong activity and the 6-hydroxyl group and lactone ring enhanced the
activity. The active compounds 121, 122 and 125 inhibited increase in [Ca2]i in
RBL-2H3 cells induced by antigen, but not by calcium ionophore A23187.
OOH
OH
OH O
OOH
OH
OH O
HO
O
OH
OH O
HO
OOH
O
OH O
HOH
OOH
OH
OH O
OOH
O
OH O
H
O
OH
OH O
OHOH
OH
OH O
OH
OH
OH
O
O
Thunberginol A (121) Thunberginol B (122) Thunberginol C (123)
Thunberginol G (126)Thunberginol F (125)Thunberginol E (124)
Phyllodulcin (127) hydrangenol (128) 3'-Hydroxyhydrangeaic acid (129)
Due to their biological importance, various routes were adopted to
synthesize thurnberginol B 179-180. We wish to report here a convenient route
for the synthesis of Thurnberginol B.
2.1.2 Plan of work
Keeping in mind the importance of Thunberginol B, It was planned to
synthesize it by the short and convenient route established M. Arfan et al. 3,5-
Dimethoxyhomophthalic acid is a key intermediate for the synthesis of highly
biological active naturally and unnaturally occurring isocoumarins and 3,4-
dihydroisocoumarins. It was synthesized efficiently in five steps from 3,5-
44
dimethoxybenzaldehyde (130) via a series of reactions including synthesis of
3,5-dimethoxycinnamic acid181-182 (131) and 3-(3′,5′-dimethoxyphenyl)-
propionic acid183-184 (132). Cyclization of 3-(3′,5′-dimethoxyphenyl)propionic
acid (132) to 5,7-dimethoxy-1-indanone185-186 (133) and oxidative
decomposition of methyl 2-hydroxy-2-(5,7-dimethyoxy-1-oxo-1H-inden-2(3H)-
ylidene)acetate187(134) to 3,5-dimethoxyhomo-phthalic acid187-188 (135). 3,4-
Dimethoxybenzoylchloride (137) was prepared from 3,4-dimethoxybenzoic
acid (136) on reaction with thionyl chloride. Direct condensation of 3,4-
Dimethoxybenzoylchloride (137) with 3,5-dimethoxy- homophthalic acid (135) at 200oC afforded 3-(3',4'-dimethoxyphenyl)-6,8-dimethoxyisocoumarin (138). This isocoumarin (138) was purified by HPLC. Complete demethylation of
isocoumarin (138) with hydrobromic acid (48%) in acetic acid gave 3-(3',4'-
dihydroxyphenyl)-6,8-dihydroxyisocoumarin (139). Synthetic scheme is given
below (Scheme 2.1). 2.1.3 Results and discussion
Condensation of 3,5-dimethoxybenzaldehyde (130) with malonic acid
in the presence of pyridine (dry) and piperidine (dry) afforded 3,5-
dimethoxycinnamic acid (131), which showed a characteristic broad singlet at
δ 11.7 ppm for -OH in 1H NMR and C=C absorption at 1600 cm-1 in IR
spectrum. The detailed 1H NMR data is illustrated in (Table 2.1). The mass
spectrum of this compound showed molecular ion peak at m/z 208, which
agreed with its molecular weight. HREIMS of its molecular ion is in good
agreement with the calculated value. The fragmentation pattern of 3,5-
dimethoxycinnamic acid (131) is shown in scheme (Scheme 2.2). The
physical constants, IR and elemental analysis of all the synthesized
compounds is given in the experimental section.
45
Na/Hg
MeONa / benzene
Diethyl oxalate
CH2(COOH)2
Pyridine(dry)/Piperidine(dry)
PPA
SOCl230min, 90°C
reflux6 hrs200°C
HBracetic acid
KOHH2O2
Thunberginol B
O
O
OO
O
COOH O
O
COOH
O
O O
O
O O
OH
COOCH3
O
O
COOH
COOH
OO
COOH
OO
COCl
O
O
O O
O
O
O
HO
OH O
OH
OH
(130) (131) (132)
(134)(133)
(136)
(135)
(137)
(138)(139)
[Yield: 86%][Yield: 83%]
[Yield: 77%][Yield: 91.5%]
[Yield: 72%]
[Yield: 60%]
[Yield: 53%]
Overall Yield = 0.86 × 0.83 × 0.77 × 0.915 × 0.72 × 0.60 × 0.53 ×100 = 11.5% Scheme. 2.1
This Part of the chapter has been published: Ghulam Qadeer. Nasim Hasan
Rama and Syed Jabbar Hussain Shah, “A new total synthesis of natural
isocoumarin, Thunberginol B”. ARKIVOC, 2007, (xiv), 12-19.
46
O
O
COOHH
H
1'
2'1
2
34
56
(131) (132)
O
O
COOH1'
2'1
2
34
56
Table 2.1: 1H NMR Data of (131) & (132)
Carbon (131) (132) 2,6 6.66 (2H, dd, J = 1.9, 2.1 Hz) 6.36 (2H, dd, J = 2.1, 2.3 Hz)
4 6.5 (1H, t, J = 2.2 Hz) 6.32 (1H , t, J = 2.1 Hz)
1′ 7.69 (1H, d, J = 15.5 Hz) 2.89 (2H, t, J = 8.0 Hz)
2′ 6.4 (1H, d, J = 15.8 Hz) 2.66 (2H, t, J = 7.5 Hz)
OCH3 3.8 (6H, s) 3.76 (6H, s)
COOH 11.7 br s exchangeable with D2O 11.66 br s exchangeable with D2O
O
O
COOHO
O
OO COOH
O
O
O
O
O
O
O
-OH
-CO
-C2H2
-CH2O
-OCH3
-COOH
-C2H2
-CH2O
.
-OCH3
(m/z = 222, 8.58 % )
+
+
(m/z = 208, 100 % )
(m/z = 163, 15.17 % )
(m/z = 137, 6.98 % )
(m/z = 177, 9.87 % )
+
(m/z = 107, 3.78 % )(m/z = 77, 24.43 % )
(m/z = 106, 2.07 % )
(m/z = 132, 5.21 % )
(m/z = 191, 8.99 % )
+
+
-C2H2
(131)
++ .
..
.
-COOH.
+ .
.+
+
(m/z = 75, 3.81% )
+
Scheme 2.2: Mass Spectral Data and Fragmentation Pattern of (131)
47
Reduction of cinnamic acid (131) with sodium amaglum yielded 3-
(3',5'-dimethoxyphenyl )propionic acid (132) which exhibited a broad singlet
at δ 11.66 ppm for COOH in 1H NMR which is exchangeable with D2O. The
carboxylic carbonyl absorption in IR spectrum was observed at 1722 cm-1.
The mass spectrum of the propionic acid (132) showed a molecular ion peak
at m/z 210 and a characteristic peak at m/z 165 [M+-COOH]. The
fragmentation pattern of propionic acid is illustrated in scheme (Scheme 2.3) and 1H NMR data is shown in (Table 2.1)
O
O
COOHO COOH-H2CO -COOH
+
+
(m/z = 210, 71 % )
(m/z = 150, 11.0 % )
(m/z = 105, 30.49 % )
(m/z = 165, 100 % )
(m/z = 139, 3.27 % )
(m/z = 180, 1.52 % )
O
O
O
O
-C2H4COOH-H2CO
+
+
(m/z = 65, 28.80 % )
(m/z = 91, 39.59 % )
+
(132)
+ .+ .
+ .
.
-CH2COOH.
-C2H2-COOH.
Scheme 2.3: Mass Spectral Data and Fragmentation Pattern of (132)
3-(3′,5′-Dimethoxyphenyl)propionic acid (132) on cyclization with poly-
phosphoric acid furnished the 5,7-dimethoxy-1-indanone (133) and its
structure was confirmed by the disappearance of broad singlet due to OH
proton (Exchangeable) and removal of one aromatic proton in 1H NMR while
carbonyl absorption in IR spectrum appeared at 1685 cm-1. Mass spectrum of
the indanone (133) showed molecular ion peak at m/z 192. The fragmentation
pattern of indanone is illustrated in scheme (Scheme 2.4) and 1H NMR data is
shown in (Table 2.2).
48
O
O
O
OO
-OCH3
.
OO
O
O
O
O
O
O
O
OOO
++
+
+
+
+
++
(m/z = 134, 38.19 % )
(m/z = 106, 41.71% )
(m/z = 162, 36.01 % )
(m/z = 75, 16.68 % )(m/z = 103, 26.96 % )
(m/z = 131, 31.04 % )
(m/z = 163, 100 % )
(m/z = 105, 33.86 % )
(m/z = 192, 96.62 % ) (m/z = 150, 3.27 % )
(m/z = 119, 25.62 % )(m/z = 161, 90.11 % )(m/z = 133, 28.90 % )
-HCO.
-CH2CO
-CH2CO
-OCH3
.
-CO
-C2H4
-CO
-C2H4
-OCH3
.
-CO
-C2H4
+
(133)
. + .
+ . + .
+ .
-CH2O
Scheme 2.4: Mass Spectral Data and Fragmentation Pattern of (133)
5,7-Dimethoxy-1-indanone (133) on reaction with diethyl oxalate in
the presence of sodium methoxide in dry benzene gave methyl 2-hydroxy-2-
(5,7-dimethyoxy-1-oxo-1H-inden-2(3H)-ylidene)acetate (134) and its structure
was confirmed by 3H singlet for -COOMe at 3.95ppm in 1H NMR. IR spectrum
of this ester showed two additional bands at 3600-3000 cm-1 for OH and 1744
cm-1 for carbonyl of ester. Mass spectrum of (134) showed molecular ion peak
at m/z 278. The fragmentation pattern of indanone is illustrated in scheme
(Scheme 2.5) and 1H NMR data is shown in (Table 2.2).
49
O
O
+
OO
OO
O
OH C2H3O2OO
O
OH
(m/z = 278, 18.90 % )
+
(134)
-CO
(m/z = 219, 96.33 % )
HO
(m/z = 191, 08.47 % )O
O
OO
O
OH
HO
O
O-
OO
O
(m/z = 191, 08.47 % )
O
O
(m/z = 278, 18.90 % )
-CO-CH2O
(m/z =133, 04.54% )
O
OH
HO
O
+ +
(m/z = 83, 100 % ) (m/z = 83, 100 % )
O
O HO
-CO
O
O
(m/z = 250, 02.10 % )
+
HOO
O
(m/z = 190, 03.17 % )
+ . .+ .
+ .
+ .
+ .
Scheme 2.5: Mass Spectral Data & Fragmentation Pattern of Compound (134)
Methyl-2-hydroxy-2-(5,7-dimethyoxy-1-oxo-1H-inden-2(3H)-ylidene)
acetate (134) on oxidation with hydrogen peroxide in the presence of
potassium hydroxide furnished 3,5-dimethoxyhomophthalic acid (135), which
showed a characteristic broad singlet at δ 11.2 ppm exchangeable with D2O
for -OH (Table 2.2). The two absorption bands of carbonyl groups appeared
at 1748 and 1697 cm-1 in IR spectrum. The mass spectrum of this compound
showed molecular ion peak at m/z 240, which agreed with its molecular
weight. The mass spectrum also showed two characteristic peaks at m/z =
50
222, 195 [M+-H2O, -COOH] (Scheme 2.6). The structure of 3,5-
dimethoxyhomophthalic acid was also confirmed by X-ray crystallography.
1
2
34
56
7O
O
O
1
234
56
7O
O
O
OH
COOCH3
6
54
3
O
O
COOH
COOH
1'1
2
(133) (134) (135)
Table 2.2: 1H NMR Data of (133), (134) & (135)
Carbon (133) (134) (135)
2 2.9 (2H, t, J = 6.2 Hz) -- --
3 2.53 (2H, t, J = 6.1 Hz) 3.93 (2H, s) --
4 6.37 (1H, d, J = 1.4 Hz) 6.80 (1H, d, J = 3.0 Hz) 6.55 (1H , d, J = 1.2 Hz)
6 6.18 (1H, d, J = 1.6 Hz) 6.38 (1H,d, J = 3.05 Hz) 6.84 (1H , d, J = 1.4 Hz)
1′ -- -- 4.2 (2H, s)
OCH3 3.77 (3H, s)
3.80 (3H, s)
4.05 (6H, s)
3.65(3H, s )
3.67(3H, s)
COOCH3 -- 4.01 (3H, s) --
OH -- 13.57 (s) --
COOH -- -- 11.2 br. s exchangeable
with D2O
51
OHO
OHO
O
O
-H2O
OHO
O
O-CO2 O
O
O
O
O
O
OO
OOHO
O
O
-CO
O
O
O
OO
-CO
(m/z = 194, 57.79 % )(m/z = 195 , 12.77 % )
m/z = 196 ( 56.89 % ) (m/z = 222, 8.58 % )
(m/z = 151, 14.85 % )(m/z = 178, 100 % )
(m/z = 150, 2.51 % )
(m/z = 120, 3.55 % )
CO O
O
.
-COOH.
.
+
+
+
-CO2-COOH.
-OCH3
(m/z = 240, 21.69 % )(135)
+ . + .
+ .
+ .
+ .
+ .
Scheme 2.6: Mass Fragmentation Pattern of Compound (135)
The X-ray crystal structure of (135) is shown in Fig. 2.1. The C9/O3/O4
carboxyl group is tilted by 30.34 (9) with respect to the plane of the benzene
This Part of the chapter has been published: Ghulam Qadeer, Nasim Hasan
Rama and Fan Zhijin, “Synthesis, mass fragmentation studies and bioloigical
evaluation of 3,5-dimethoxyhomophthalic acid and related compounds”. Chem.
Res. Chinese U., 2007, 23(5), 533-537.
52
ring, possibly as a result of the steric hindrance of the ortho substituents. The
O3—C9—C2—C3 and O3—C9—C2—C1 torsion angles are 149.68 (14) and
29.6 (2), respectively.
Figure 2.1: View of the 3,5-dimethoxyhomophthalic acid, with displacement
ellipsoids drawn at the 30% probability level
.
It is noteworthy that the C9 and O3 carbonyl group derivatives from the
phenyl plane, with the mean deviation from the plane of 0.1750\%A. The
torsion angles of O3-C9-C2-C3 (149.68(14)\%) and O3-C9-C2-C1 (-
21.6(2)\%) have also indicated this carbonyl group π system is not coplanar
with the phenyl plane. These results display that some repulsions among
substitutes may exist. It is also noted that this compound forms an infinite 2D
network structure by the intermolecular hydrogen bonds between C7-
H7A...O3 (2.7829 (19)\%A), C10-H10C...O2 (3.4144 (19)\%A), O4-H4A...O3
(2.6606 (14)\%A) and O1-H1...O2 (2.6532 (14)\ %A) (Fig. 2.2). In the crystal
packing, intermolecular C—H….O hydrogen-bond interactions link the
molecules, forming a two-dimensional network (Fig. 2.2).
53
Figure 2.2: The crystal packing of the 3,5-dimethoxyhomophthalic acid,
viewed approximately along a axis. Hydrogen bonds are shown as dashed
lines
[symmetry codes: (a)1x, 1y, 2z (b) x, 2y, 2z (c) x, 1y, 1z]
Crystal data
Mr = 240.21 Triclinic, P1 a = 7.2886 (10) Å b = 7.9631 (11) Å c = 10.2758 (14) Å α = 105.937 (2)º β = 103.444 (2)º γ = 93.211 (2)º
V = 553.17 (13) Å3 Z = 2 Mo Kα µ = 0.12 mm−1 T = 292 (2) K 0.24 × 0.20 × 0.20 mm
This Part of the chapter has been published: Ghulam Qadeer. Nasim Hasan
Rama and Qing-Shan Li. “Crystal structure of 2-(Carboxymethyl)-4,6-
dimethoxybenzoic acid”. Acta Cryst. 2006, E62, 906–907.
Geometric parameters (°A, °)
Selected bond lengths Selected bond angles
C1—C6 1.3743 (19) C1—C2 1.4128 (18) C1—C7 1.5131 (18) C9—O3 1.2346 (17) C2—C3 1.4085 (18) C9—O4 1.2979 (17) C2—C9 1.4822 (18) C11—O6 1.4243 (18) C5—O6 1.3625 (16) C5—C6 1.3888 (19
Hydrogen-bond geometry (Å, °)
D—H···A D—H H···A C7—H7B···O3 0.97 2.43 C10—H10C···O2i 0.96 2.56 O4—H4A···O3ii 0.850 (10) 1.821 (O1—H1···O2iii 0.853 (10) 1.801 (
Symmetry codes: (i) −x, −y+1,−y+1, −
3,4-Dimethoxybenzoylchloride (dimethoxybenzoic acid (136) on rea
condensation of 3,4-dimethoxybenz
dimethoxyhomophthalic acid (135) at 2
dimethoxyphenyl)isocoumarin (138), absoption at 1716 and C=C absorbtion
characteristic C4-H singlet at δ 7.0 ppm
data is illustrated in (Table 2.3). The
showed molecular ion peak at m/z 34
weight. The mass spectrum also showe
254, 164 (Scheme 2.7). This isocoumari
53
C6—C1—C2 120.35 (12) C6—C1—C7 116.69 (12) O2—C8—O1 122.97 (13) C2—C1—C7 122.77 (12) O2—C8—C7 124.33 (12) C1—C2—C9 119.88 (12) C4—C5—C6 120.42 (12) C8—C7—C1 114.89 (11) C8—C7—H7A 108.5 C8—O1—H1 108.8 (16)
D···A D—H···A
2.7829 (19) 101 3.4144 (19) 148 11) 2.6606 (14) 169 (2) 10) 2.6532 (14) 178 (2)
−z+1; (ii) −x, −y+2, −z+2; (iii) −x+1, z+2
137) was prepared from 3,4-
ction with thionyl chloride. Direct
oylchloride (137) with 3,5-
00oC afforded 6,8-dimethoxy-3-(3',4'-
which showed lactonic carbonyl
at 1599 cm-1 in IR spectrum and
in 1H NMR. The detailed 1H NMR
mass spectrum of this compound
2, which agreed with its molecular
d two characteristic peaks at m/z =
n (138) was purified by HPLC.
O
O
O O
O
O
12
345
67
8
1' 2'
3'
4'5'
6'
(138) (139)
O
HO
OH O
OH
OH
12
345
67
8
1' 2'
3'
4'5'
6'
Table 2.3: 1H NMR and 13 C NMR data of (138) and (139)
(138)
(139)
Carbon 1H NMR 13 C NMR 1H NMR 13 C NMR
1 --- 154.9 --- 151.9 3 --- 149.2 --- 147.2 4 7.0(1H,s) 111.3 6.91(1H,s) 111.3 4a --- 148.7 --- 148.7
5 7.55 (1H, d, J = 1.8 Hz) 112.2 7.47 (1H, d, J = 1.8
Hz) 115.2
6 --- 167.6 167.6
7 7.44(1H, d, J = 1.8 Hz) 111.8 7.41(1H, d, J = 1.8
Hz) 113.8
8 --- 162.8 --- 162.8 8a --- 112.6 --- 112.6 1′ --- 125.4 --- 125.4
2′ 7.57(1H, d, J = 1.8 Hz) 120.5 7.52(1H, d, J = 1.8
Hz) 122.5
3′ --- 154.9 --- 154.9 4′ --- 154.9 --- 154.9
5′ 7.16(1H, d, J = 8.7 Hz) 123.4 7.11(1H, d, J = 8.7
Hz) 125.4
6′ 7.76(1H, dd, J = 8.4, 1.2Hz) 123.6 7.72 (1H, dd, J =
8.4,1.2 Hz) 124.6
OCH3 3.79-3.88 (12H, 4×s) 55.8-56.3 --- --- OH --- --- 5.22(s) ---
54
O
O
O
O
O
OO
O
O
O
O
O
O
O
O
O
OO
O
O
-CO
-CO
-CO2-OCH3
O-
.
-HCO
(m/z = 138, 21 %)
(m/z = 166, 7.0 %)(m/z = 342, 100 %)
(m/z = 300, 2.2 %)
(m/z = 284, 30 %)
(m/z = 314, 30 %)
(m/z = 178, 01 %)(m/z = 206, 32 %)
OO
O
O
O
O
O
O
O
O
O
-.O
O
(138)
+
+ .++ .
+ .
++
.+
Scheme 2.7: Mass Fragmentation Pattern of Compound (138)
Complete demethylation of this 6,8-dimethoxy-3-(3',4'-dimethoxy-
phenyl)isocoumarin (138) with hydrobromic acid (48%) in acetic acid gave 3-
(3′,4′-dihydroxyphenyl)-6,8-dihydroxyisocoumarin (139). Its IR spectrum
showed a broad absoption at 3386-3360cm-1 for phenolic OH group. In 1H
NMR, the signal for methoxy groups in (138) at δ 3.83 ppm disappeared. The
detailed 1H NMR data is illustrated in (Table 2.3). The mass spectrum of this
compound showed molecular ion peak at m/z 286, which agreed with its
molecular weight. The mass spectrum also showed two characteristic peaks
at m/z = 227, 136 (Scheme 2.8). This isocoumarin (139) is also purified by
HPLC.
55
O
O
OH
HO
OH
OH
OH
OH
OH
HO
OH
OH
OOH
OH
OH
OHO
OH
OH
O
O
HO
OH
O
OH
HO
HO
OH
OOH
HO
OH
-CO
-CO
-CO2-OH
-.
O-
.
-HCO
.
-.OH
OH
(m/z = 110, 32 %)
(m/z = 138, 7.0 %)(m/z = 286, 7.0 %)
(m/z = 258, 2.2 %)
(m/z = 136, 100 %)
(m/z = 243, 27 %)
(m/z = 259, 52 %)
(m/z = 150, 01 %)(m/z = 178, 31 %)
+
+
(139)
+
+ .
+
+ .
.+ .
+
+
Scheme 2.8: Mass Fragmentation Pattern of Compound (139)
2.1.4 Experimental
All reagents and solvents were commercially available and used
as supplied. The melting points of the compounds were determined in open
capillaries using a Gallenkemp melting point apparatus and are uncorrected.
The infrared spectra were recorded on a Hitachi model 270-50
spectrophotometer as KBr disks or as neat liquids. 1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were recorded on a Bruker AM-300 in CDCl3
56
solution using TMS as an internal standard. HPLC-MS analyses were
performed HPLC-Anlage Waters 2695 Alliance, Säule Phenomenex Luna 3µ
Phenyl-Hexyl (2 × 150 mm), mobile Phase Water/Acetonitrile (0:100 bis
50:50), Fluss: 0.2 ml/min, photodiode array detector Waters 996, MS-Detector
Waters ZQ2000. In the mass detector, the fragmenter operated at 70eV.
Elemental analysis was performed on a LECO CHNS-932 instrument. XRD
data collection was carried out by, Data collection: SMART (Bruker, 1998);
cell refinement: SAINT (Bruker, 1999); data reduction: SAINT; program(s)
used to solve structure: SHELXS97 (Sheldrick, 1997); program(s) used to
refine structure: SHELXL97 (Sheldrick, 1997); molecular graphics: SHELXTL
(Bruker, 1999); software used to prepare material; SHELXTL.
3, 5-Dimethoxycinnamic acid (131)
3,5-Dimethoxybenzaldehyde (130) (17g, 102 mmol), malonic acid
(51g), dry pyridine (183.6mL) and dry piperidine (40.2mL) were heated on
boiling water bath for 4h. The reaction mixture was cooled and poured into
cold water (500mL) and carefully acidified with dilute hydrochloric acid (1:1)
with stirring. On filtration white ppt of 3,5-dimethoxycinnamic acid (131)(18.3g,
88 mmol) was obtained. Yield: 86%; m.p: 170°C; IR (KBr, νmax, cm-1): 3354,
1685,1600; 1H NMR (CDCl3, δ ppm): 7.69 (1H, d, J =15.5 Hz, H-1′), 6.66 (2H,
d, J = 2.1 Hz, H-2,6), 6.50 (1H, t, J = 2.2 Hz, H-4), 6.41 (1H, d, J = 15.8 Hz, H-
2′), 3.84 (6H, s, OCH3), 11.70 (bs COOH); EIMS (DMF, m/z, %): 208 (100,
M), 191 (9), 177 (10), 163 (15), 137 (7), 132 (5), 107 (4), 106 (2), 77 (24), 75
(4), 51 (24); Anal. Cald for C11H12O4 : C, 63.45; H, 5.81; Found: C, 63.01; H,
5.54.
3-(3′,5′-Dimethoxyphenyl)propionic acid (132)
Cinnamic acid (131) (17.85g, 86 mmol) was dissolved in 93mL of IM
solution of sodium hydroxide contained in a 500ml two necked flask equipped
with a mechanical stirrer and added sodium amalgam (222g) gradually during
1 hour while the reaction mixture was well stirred. When hydrogen was not
57
evolved, the mercury was separated and washed with water, washings was
added to the solution and acidified the reaction mixture with dilute
hydrochloric acid (1:1). Propionic acid was precipitated at first in the form of
an oil, which solidified on cooling and rubbing with a glass rod. Filtered at the
pump and recrystalized with pet. ether to get 3-(3′,5′-Dimethoxyphenyl)-
propionic acid (132) (15g, 71mmol). Yield: 83%; m.p: 58°C; IR (KBr, νmax, cm-
1): 3390, 1722, 1207; 1H NMR (CDCl3, δ ppm): 6.36 (2H, d, J = 2.3 Hz, H-2,6),
6.32 (1H, t, J = 2.1 Hz, H-4), 2.89 (2H, t, J = 8.0 Hz, H-1′), 2.66 (2H, t, J = 7.5
Hz, H-2′), 3.76(6H, s, OCH3), 11.66 (bs COOH); EIMS (DMF, m/z, %): 210
(71, M), 180 (2), 165 (100), 150 (11), 139 (3), 105 (30), 91 (40), 65 (29); Anal.
Cald for C11H14O4 : C, 62.85; H, 6.71; Found: C, 62.39; H, 6.14.
5,7-Dimethoxy-1-indanone (133) 3-(3,5-Dimethoxyphenyl)propionic acid (132) was dissolved in 250g of hot
poly phosphoric acid (90°C). The resulting yellow solution was heated on oil
bath with stirring for 2 hours. The cooled solution was added to 500ml of ice
water. Extracted the mixture with three 150ml portions of ethyl acetate and
washed the combined extracts with 5% sodium hydroxide solution and then
with water until the washings were neutral. Dried the ethyl acetate solution
over anhydrous magnesium sulphate. The organic layer was concentrated
and chromatographed on silica gel using pet. ether (40-80°C) as eluent to
afford 5,7-dimethoxy-1-indanone (133) (8.8g, 6 mmol). Yield: 77%; m.p: 97-
98°C; IR (KBr, νmax, cm-1): 1685, 1209,1057; 1H NMR (CDCl3, δ ppm): 6.18
(1H, d, J = 1.6 Hz, H-6), 6.37 (1H, d, J =1.4 Hz, H-4), 2.9 (2H, t, J = 6.2 Hz, H-
2), 2.53 (2H, t, J = 6.1 Hz, H-3), 3.80 (3H, s, OCH3), 3.77 (3H, s, OCH3);
EIMS (DMF, m/z, %): 192 (97, M), 163 (100), 162 (36), 161 (90), 150 (3) 134
(38), 133 (29), 131(31), 119 (25), 105 (34), 106 (42), 103 (30), 75 (17); Anal.
Cald for C11H12O3: C, 68.74; H, 6.29; Found: C, 68.11; H, 6.94.
58
Methyl 2-hydroxy-2-(5′,7′-dimethyoxy-1-oxo-1H-inden-2(3H)-ylidene)-
acetate (134)
Sodium (15.2g) was dissolved in methanol (27.5g) and benzene (440
ml) was added to this solution. Indanone (133) (20g, 100 mmol) was added in
portions with good stirring, then diethyl oxalate (38.5mL) was added as single
aliquot and the reaction mixture was stirred for over night at room
temperature. Hydrochloric acid (5%) was added under ice cooling to the
residue obtained after removal of the solvent, to give a final pH of 1.0. The
precipitate that deposited was collected on a filter, washed with hydrochloric
acid (5%), then with water, and dried. The solid thus obtained was crystallized
from methanol to give methyl 2-hydroxy-2-(5,7-dimethyoxy-1-oxo-1H-inden-
2(3H)-ylidene)acetate (134) (26.5g, 90 mmol) as yellow needles. Yield:
91.5%; m.p: 234-237 °C; IR (KBr, νmax, cm-1): 3400-2400, 1744 ,1692, 1633,
1605, 1365; 1H NMR (CDCl3, δ ppm): 13.57 (s, OH), 6.80 (1H, d, J = 3.0 Hz,
Ar-H), 6.38 (1H,d, J = 3.05 Hz, Ar-H), 4.05 (6H, s, 2 × OMe), 4.01 (3H, s,
COOMe), 3.93 (2H, s, CH2); EIMS (DMF, m/z, %): 278 (19, M), 219(95),
191(8), 175(6), 85(65), 83(100), 42 (25). Anal. Cald for C14H14O6: C, 60.43; H,
5.07; Found: C, 60.08; H, 5.43.
3,5-Dimethoxyhomophthalic acid (135)
A solution of methyl 2-hydroxy-2-(5,7-dimethyoxy-1-oxo-1H-inden-
2(3H)-ylidene)acetate (134) (26.5g, 100 mmol) and potassium hydroxide
(20.8g) in water (442mL) was cooled to 5-10°C, and hydrogen peroxide
(90.0g, 35%) was added. The reaction mixture was stirred at this temperature
for 6 hours. When the temperature was gradually raised to 50°C, vigorous
frothing occurred. After stirring for further 2h at this temperature, the reaction
mixture was filtered and the filtrate was acidified with concentrated
hydrochloric acid. The crystals that precipitated were collected by filtration
and the filtrate was extracted with ethyl acetate (4 × 30mL). The solvent was
evaporated to give solid residue, which was purified by recrystalization from
dichloromethane to give 3,5-dimethoxyhomophthalic acid (135) (19g, 72
59
mmol) as light yellow color micro crystals. Yield: 72%; m.p: 170°C; IR (KBr,
νmax, cm-1): 2580-3300, 1714, 1659, 1226; 1H NMR (CDCl3, δ ppm): 11.7 (bs
COOH), 6.84 (1H, d, J = 1.4 Hz, Ar-H), 6.55 (1H, d, J = 1.2 Hz, Ar-H), 4.2 (2H,
s, H-1′), 3.67 (3H, s, OCH3), 3.65 (3H, s, OCH3); EIMS (DMF, m/z, %): 240
(38, M), 222 (17), 196 (66), 195 (40), 194 (85), 178 (100), 151 (28), 150 (15),
120 (41), 119(12). Anal. Cald for C11H12O6: C, 55.00; H, 5.04; Found: C,
54.52; H, 5.41.
3-(3′,4′-Dimethoxyphenyl)-6,8-dimethoxyisocoumarin (138)
Mixture of 3,4-dimethoxybenzoic acid (135) (2g, 10.9 mmol) and thionyl
chloride (1.57g, 1mL, 13.2 mmol) was heated for 30 min in the presence of a
drop of dimethylformamide at 90°C. Completion of the reaction was indicated
by the stoppage of gas evolution. Removal of excess of thionyl chloride was
carried out under reduced pressure to afford 3,4-dimethoxybenzoyl chloride
(137) (1.8g, 9mmol). 3,5-Dimethoxyhomophthalic acid (136) (0.63g, 2.6
mmol) and 3,4-dimethoxybenzoyl chloride (137) (1.8g, 9 mmol) were refluxed
at 200°C for six hours. Residue, after concentration, was purified by HPLC to
give the 3-(3′,4′-dimethoxyphenyl)-6,8-dimethoxyisocoumarin (138). Yield:
60%; m.p: 175-177°C; IR (KBr, νmax, cm-1): 1716 (C=O, lactonic), 1599 (C=C); 1H NMR (CDCl3, δ ppm): 7.76(1H, dd, J = 1.2, 8.4 Hz, H-6′), 7.57(1H, d, J =
1.8 Hz, H-2′), 7.55 (1H, d, J= 1.8 Hz, H-5′), 7.44 (1H, d, J= 1.8 Hz, H-7),
7.16(1H, d, J= 8.7 Hz, H-5′),7.0(1H, s, H-4), 3.88(3H, s), 3.85(3H, s),
3.82(3H, s), 3.79 (3H, s); 13C NMR (CDCl3, δ ppm): (167.6, 162.8, 154.9,
149.2, 148.7, 125.4 , 123.6, 123.4, 112.6, 112.2, 111.8)(Ar-C), 55.8-56.3 (4×
OCH3); EIMS (DMF, m/z, %): 342(6.0, M), 314(30), 300(2), 284(30), 254(54),
206(32), 178(1), 166(7), 164(100), 138(21), 94(13); Anal. Cald for C19H18O6:
C, 66.66; H, 5.30; Found: C, 66.20; H, 5.05.
60
3-(3′,4′-Dihydroxyphenyl)-6,8-dihydroxyisocoumarin (139)
Freshly distilled hydrobromic acid (55%, 16 mL) was added to stirred
solution of isocoumarin (138) (0.3g, 1.1 mmol) in glacial acetic acid (16mL).
Reaction mixture was then refluxed for four hours, cooled and poured on
crushed ice and then treated with solid sodium carbonate till PH-7 and
extracted with diethyl ether (2 × 30 mL), dried over anhydrous sodium
sulphate and concentrated to give crude solid which was purified by HPLC to
get 3-(3′,4′-dihydroxyphenyl)-6,8-dihydroxyisocoumarin (139) Yield: 53%;
m.p: 247–249°C(lit. mp 244°C); IR (KBr, νmax, cm-1): 1716 (C=O, lactonic),
1599 (C=C); 1H NMR (CDCl3, δ ppm): 7.69(1H, dd, J = 1.2, 8.4 Hz, H-6′),
7.52(1H, d, J = 1.8 Hz, H-2′), 7.49 (1H, d, J = 1.8 Hz, H-5′), 7.38(1H, d, J =
1.8 Hz, H-7), 7.19(1H, d, J = 8.7 Hz, H-5′), 6.94(1H, s, H-4); 13C NMR
(CDCl3, δ ppm): 166.9, 164.8, 157.1, 148.2, 147.7, 128.4, 124.6, 122.4, 119.5,
111.6, 110.2, 109.8; EIMS (DMF, m/z, %): 286 (52), 269 (100), 259 (4), 241
(13), 213 (11), 177 (23), 149 (3), 137 (4), 121 (7); Anal. Cald for C15H10O6: C,
62.94; H, 3.52; Found: C, 62.71; H, 3.84.
61
Chapter-3 SYNTHESIS OF UNNATURAL ISOCOUMARINS
3.1 Synthesis of dihalophenylisocoumarins
3.1.1 Introduction Naturally occurring isocoumarins containing halogens have been
seldom reported. Examples of naturally occurring isocoumarins containing
fluorine are not known yet. However, a few examples of naturally occurring
chlorine- and bromine-containing isocoumarins have been reported.
Laresenb189 has isolate chlorine-containing metabolite dichlorodiaportin (140) from the cheese-associated cultures of Penicillium nalgiovense. 4-Chloro-3-
(4′-fluorobenzyloxy)isocoumarin (141) has been found190 to be quite an
effective inhibitor for human Q31 granzyme A, murine and human granzyme
A, isolated from cytotoxic T lymphocytes. This isocoumarin derivative has also
been found191, to be useful in the treatment of emphysema as serine protease
inhibitor.6-(2′-Chloro-4′-trifluoromethylphenoxy)-3,4-dihydroisocoumarin (142)
has been used192, as a herbicide, which almost totally controlled the growth of
Schinochloa crusgall, Sinapis alba and other weeds. 7-Amino-4-chloro-3-(2′-
bromoethoxy)isocoumarin (143) has been synthesized,193 and evaluated as a
potent inhibitor of human leuko elastase and several blood coagulation
enzymes194. 7-Amino-3-(2′-bromopropoxy)-4-chloroisocoumarin and 7-Amino-
3-(3′-bromopropoxy)-4-chloroisocoumarin have been patented195, as an
ascapain inhibitor in the inhibition and treatment of neurodegeneration.
O
O
ClO
F
O
O
OCl
F3CO
O
OBr
Cl
H2N
O
Cl
ClO
OH O
OH
(140)(141)
(142)(143)
62
3.1.2 Plan of work
As a continuation of our previous studies196-197 and biological activities
associated with halo substituted isocoumarins, prompted us to synthesize
some new 3-(dihalophenyl)isocoumarins 146(a-g) and their conversion to the
corresponding 3,4-dihydro-derivatives 149(a-g) in order to check their
antimicrobial, anti-inflammatory, antioxidant, antiviral, anti-HIV, anti-HBV,
pesticide and plant disease control activities. Synthetic scheme is shown as
follows (Scheme 3.1)
OHO
Ar
O
OHO
OH
O
Ar OH
O
SOCl2Ar Cl
O
200°C, 6h
OO
Ar
5% KOH/ C2H5OH
NaBH4 /H+
OHO
Ar
OH
OO
Ar
FF
F
F
F
F
b ) Ar =a) Ar = d) Ar =c) Ar =F
F
Cl
Cl ClCl
Cl
Cl
e) Ar = f) Ar = g) Ar =
Ar Cl
O+
Ac2O
Ac2O
30 minreflux
reflux
148(a-g) 147(a-g)149(a-g)
146(a-g)
144(a-g) 145(a-g)
145(a-g)[Yield = 77- 81%]
[Yield = 69 - 81%]
[Yield = 69 - 79%][Yield = 69- 80%]
Scheme 3.1
3.1.3 Results and Discussion
Difluorobenzoic acids 144(a-d) and dichlorobenzoic acids 144(e-g) were converted into their respective acid chlorides 145(a-g) by reaction with
thionyl chloride in the presence of a drop of DMF. Direct condensation of acid
63
chlorides 145(a-g) with unsubtituted homophthalic acid at 200oC afforded 3-
(dichlorophenyl)isocoumarins 146(a-d) and 3-(difluorophenyl)isocoumarins 146(e-g) These isocoumarins 146(a-g) were purified by column
chromatography and showed a single spot on TLC.These isocoumarins
146(a-g) exhibited characteristic 1H singlet at δ 6.81-7.19 ppm for C4-H in 1H
NMR (Table 3.1). The aromatic protons appeared in the acceptable region i.e.
7.50-8.31 ppm. In IR spectra of isocoumarins 146(a-g) lactonic carbonyl
absorptions were observed at 1702-1709 cm-1. The molecular ion peaks in
the mass spectrum of 3-(3′,5′-difluorophenyl)isocoumarin 146a and for 3-
(3′,5′-dichlorophenyl)-isocoumarin 146e were obtained at m/z 258 and 292
respectively (Scheme 3.2 & 3.3)
O
O1
2
3456
78
1' 2'
3'4'5'
6'
146a
F
F
Table 3.1: 1H NMR Data of 3-(3′.5′-Difluorophenyl)isocoumarin (146a)
1H NMR δ (ppm) Carbon
No 3-(3′.5′-Difluorophenyl)isocoumarin
146a
4 6.94 (s)
5 7.54 (d, J = 7.40 Hz)
6 7.74 (1H, m)
7 7.50 (dd, J = 7.88 Hz)
8 8.31 ( d, J = 7.92 Hz
2′ 7.40 ( dd, J = 2.0, 8.30 Hz)
3′ ---
4′ 6.81 ( m)
5′ ---
6′ 7.40 ( dd, J = 2.0, 8.30 Hz)
64
OO
O
-CO
-CO
-F-CO2
-.
O-
.
+
+
+
OO F
F
F
F
F
F
O
F
F
OF
FF
.
O
F
F
F
F
-HCO
(m/z = 258, 56.72 %) (m/z = 230, 30.88 %)(m/z = 141, 7.74 %)
(m/z = 113, 32.74 %)(m/z = 216, 2.7 %) (m/z = 212, 24.83 %)
(m/z = 139, 100%)
(m/z = 117, 2.61 %)(m/z = 145, 34.75 %)
+
(146a)
+ .+ .
+
+ + ..
Scheme 3.2: Mass Fragmentation Pattern of 3-(3′,5′ Difluorophenyl)-
isocoumarin 146a
65
O
O1
2
3456
78
1' 2'
3'4'5'
6'
146e
Cl
Cl
Table 3.2: 1H NMR Data of 3-(3′.5′-Dichlorophenyl)isocoumarin (146e)
1H NMR δ (ppm)
Carbon No
3-(3′.5′-Dichlorophenyl)isocoumarin
146e
4 6.95 (s)
5 7.55 (1H, dd, J 2.2, 7.6 Hz)
6 7.53 (m)
7 7.50 (m)
8 8.31 (dd, J = 1.9, 7.9 Hz)
2′ 7.75 (t, J = 1.2 Hz)
3′ ----
4′ 7.39 (t, J = 1.2 Hz)
5′ ----
6′ 7.75 (t, J = 1.2 Hz)
66
OO
O
-CO
-CO
-Cl-CO2
-.
O-
.O
O Cl
Cl
Cl
Cl
Cl
Cl
O
Cl
Cl
O
Cl
ClCl
.
O
Cl
Cl
Cl
Cl
-HCO
m/z = 290 (56.72 %, 2 Cl35) 292 (38.18 %, Cl35Cl37) 294 (6.52 %, 2 Cl37)
m/z = 262 (30.88 %, 2 Cl35) 264(20.28 %, Cl35Cl37) 266 (3.59 %, 2 Cl37)
m/z = 174 (7.74 %, 2 Cl35) 176 (4.84 %, Cl35Cl37) 178 (0.75 %, 2 Cl37)
m/z = 145 (32.74 %, 2 Cl35) 147 (20.85 %, Cl35Cl37) 149 (3.85 %, 2 Cl37)
m/z = 246 (2.7 %, 2 Cl35) 248 (1.6 % Cl35Cl37) 250 (0.3 % 2 Cl37)
m/z = 227 (24.83 %, 2 Cl35) 229 (8.23 %, Cl35Cl37)
m/z = 173(100%, 2 Cl35) 175(64.21 %, Cl35Cl37) 177(10.32 %, 2 Cl37)
m/z = 117 (0.61 %)m/z = 145 (32.74 %)
(146e)
++
+
+ .+ .
+
+ .
+
+
.
Scheme 3.3: Mass Fragmentation Pattern of 3-(3′,5′-Dichlorophenyl)-
isocoumarin 146e
Alkaline hydrolysis of isocoumarins 146(a-g) afforded 2-(difluoro-
benzoylmethyl)benzoic acids 147(a-d) and 2-(dichlorobenzoylmethyl)-
benzoic acids 147(e-g). In the IR spectra of keto-acids 147(a-g), the
absorptions for the ketonic carbonyl were observed at 1708-1713cm-1.The
keto-acids 147(a-g) showed 2H singlets at δ 4.60-4.65 ppm for methylene
protons at C-1′ (Table 3.3 & 3.4). The molecular ion peak for the keto-acids
147(a-d) and for 147(e-g) were observed at m/z 276 (M+) and 308(M+)
67
(Scheme 3.4 & 3.5). Isocoumarins 146(a-g) can also be obtained on
refluxing keto-acids 147(a-g) with acetic anhydride (reversible reaction).
COOHO
2'1'34
56
1'' 2''3''
4''5''6''
1
2
147a
F
F
Table 3.3: 1H NMR Data of 2′-(3′′,5′′-Difluorobenzoylmethyl)benzoic
acid (147a)
1H NMR δ (ppm) Carbon
No 2′-(3′′,5′′-difluorobenzoylmethyl)benzoic acid
147a
3 7.23 (d, J = 7.4Hz)
4 7.37 (dd, J = 7.6 Hz)
5 7.77 (m)
6 8.08 (d, J = 7.23 Hz)
1′ 4.61 (s)
2′′ 7.51 (m)
3′′ ----
4′′ 6.98 ( dd, J = 8.25, 16.6 Hz)
5′′ ----
6′′ 7.51 (m)
COOH 11.31 (s) Exchangeable with D2O
68
OO
F
-H2O
O OH
OF
O OH
O
OF
F
F
OO
OF
O
2(-F)
-CO
-C2H2
-CO2
F
-
F.-CO.-
-C2H2
F
(m/z = 290, 22 %)
(m/z = 164, 4.20 %)
(m/z = 145, 63.99 %)
(m/z = 101, 4.22 %)
(m/z = 75, 22.71 %)(m/z = 224, 22 %)
(m/z = 276, 2.5 %)
(m/z = 158, 17.39 %)
(m/z = 262, 1 %)
(m/z = 109, 100 %)(m/z = 113, 63.99%)F
F
F
F
F
F
(147a)
+ + +
+ .+ .
+ .
+ . + .+ .
+.
+ .
Scheme 3.4: Mass Fragmentation Pattern of 2′-(3′′,5′′-
Difluorobenzoylmethyl) benzoic acid 147a
69
COOHO
2'1'3
4
56
1'' 2''
3''
4''5''6''
1
2
147e
Cl
Cl
Table 3.4: 1H NMR Data of 2′-(3′′,5′′-Dichlorobenzoylmethyl)benzoic
acid (147e)
1H NMR δ (ppm) Carbon
No 2′-(3′′,5′′-dichlorobenzoylmethyl)benzoic acids
147e
3 7.18 (dd, J = 1.56, 7.99 Hz )
4 7.24 (m)
5 7.52 (m)
6 8.15 (dd, J = 1.2, 7.9 Hz
1′ 4.62 (s)
2′′ 7.94 (d, J = 1.76 Hz
3′′ ----
4′′ 7.56 (dd, J= 2.41 Hz
5′′ ----
6′′ 7.94 (d, J = 1.76 Hz
COOH 10.31 (s) Exchangeable with D2O
70
OO
Cl
-H2O
O OH
OCl
O OH
O
OCl
Cl
Cl
OO
OCl
O
2(-Cl)
-CO
-C2H2
-CO2
Cl
-
Cl.
-CO.-
-C2H2
++
+
Cl
(m/z = 290, 22 %, 2Cl35)
(m/z = 164, 4.20 %)
(m/z = 145, 63.99 %) (m/z = 292, 11 %, 2Cl35Cl37)(m/z = 294, 2.2 %, 2Cl37)
(m/z = 101, 4.22 %)
(m/z = 75, 22.71 %)
(m/z = 292, 22 %)
(m/z = 308, 2.5 %, 2Cl35)(m/z = 310, 1.1 %, 2Cl35Cl37)
(m/z = 312, 2.2 %, 2Cl37)
(m/z = 174, 17.39 %, 2Cl35)(m/z = 176, 6.12 %, 2Cl35Cl37)(m/z = 178, 1.5 %, 2Cl37)
(m/z = 262, 1 %, 2Cl35
(m/z = 264, 0.7 %, 2Cl35Cl37)(m/z = 266, o.15 %, 2Cl37)
(m/z = 173, 100 %, 2Cl35)(m/z = 175, 64.21 %, 2Cl35Cl37)(m/z = 177, 10.32 %, 2Cl37)
(m/z = 145, 63.99%, 2Cl35)(m/z = 147, 41.25 %, 2Cl35Cl37)
(m/z = 149, 11.58 %, 2Cl37)
Cl
Cl
Cl
Cl
Cl
Cl (147e)
+ .+ . + .
+ .
+ .
+ .
+.
+ .
Scheme 3.5: Mass Fragmentation Pattern of 2′-(3′′,5′′-Dichloro-
benzoylmethyl) benzoic acids 147e
Sodium borohydride reduction of keto-acids 147(a-g) afforded the
corresponding racemic hydroxyl-acids 148(a-g), which were cyclodehydrated
with acetic anhydride to produce (dl)-3-difluorophenyl-3,4-dihydro-
isocoumarins 149(a-d) and (dl)-3-dichlorophenyl-3,4-dihydroisocoumarins
149(e-g) which exhibited the carbonyl absorptions at 1703-1710 cm-1 in IR
spectra. The typical AB pattern for C3-H and ABX pattern for C4-H protons
were observed in 1H NMR spectrum of the compound 149a. Thus, each of the
C4-H showed a doublet of doublet at δ 3.46 & 3.81 ppm and other doublet of
doublet observed at δ 5.92 ppm due to C3-H. The mass spectrum of 149a
71
and 149e showed molecular ion peak at m/z 260 (4%)(M+) and 294(M+)
respectively. Almost the same is the case with dihydroisocoumarins 149(b-d) and 149(f-g).
O
O1
2
34
56
78
1' 2'
3'
4'5'6'
149a
HH
F
F
Table 3.5: 1H NMR Data of 3-(3′,5′-Difluorophenyl)-3,4-
dihydroisocoumarin (149a)
1H NMR δ (ppm) Carbon
No 3-(3,5-difluorophenyl)-3,4-dihydroisocoumarin
(149a)
3 5.92 (dd, J = 2.95,13.10 Hz)
4a 3.81 (dd, J = 4.09, 13.04 Hz)
4b 3.46 (dd, J = 2.92, 16.37 Hz)
5 7.51 (dd, J = 2.15, 7.68 Hz)
6 7.67(dd, J = 3.37,5.67 Hz)
7 7.28 (m)
8 8.14 (d, J = 7.58 Hz)
2′ 7.12 (m)
3′ ----
4′ 6.90 (m)
5′ ----
6′ 7.12 (m)
72
OO
O
O
O
F
F
O
-CO
-CO
-F
F.
O-
.
OO F
-F
O
CO
O
F
-CO
-
F
-
-CO2
+
++
+
+
+
(m/z = 147, 4 %) (m/z = 119, 23.1 %)
F
F F
F
F
F
(m/z = 118, 100 %)
-HCO
(m/z = 104, 58.3 %)(m/z = 90, 38.21 %)
(m/z = 192, 11.41 %)
F
(m/z = 142, 1.41 %)
(m/z = 260, 5.21 %)
(m/z = 114, 21.7 %)
(m/z = 211, 9.17 %)
(m/z = 141, 12.22 %)
(149a)
.
++ .
+ .
+
.
+ .
.
Scheme 3.6: Mass Fragmentation Pattern of 3-(3′,5′-Difluorophenyl)-
3,4-dihydroisocoumarin 149a
73
O
O1
2
34
56
78
1' 2'
3'
4'5'6'
149e
HH
Cl
Cl
Table 3.6: 1H NMR Data of 3-(3′,5′-Dichlorophenyl)-3,4-
dihydroisocoumarin (149e)
1H NMR δ (ppm) Carbon
No 3-(3′,5′-dichlorophenyl)-3,4-dihydroisocoumarin
(149e)
3 5.93 (dd, J = 2.90, 12.03 Hz)
4a 3.29 (dd, J = 2.90,16.36 Hz)
4b 3.09 (dd, J = 12.10, 16.25 Hz)
5 7.58 (dd, J = 2.24, 7.60 Hz)
6 7.66 (m)
7 7.48 (m)
8 8.15 (dd, J = 1.34, 7.6 Hz)
2′ 7.30 (d, J = 2.6 Hz)
3′ ----
4′ 7.44 (d, J = 2.3 Hz)
5′ ----
6′ 7.30 (d, J = 2.6 Hz)
74
OO
O
O
O
Cl
Cl
O
-CO
-CO
-Cl
Cl.
O-
.
OO Cl
-Cl
O
CO
O
Cl
-CO
-
Cl
-
-CO2
+
++
+
+
+
(m/z = 147, 4 %) (m/z = 119, 23.1 %)
(m/z = 292, 32.55 %, 2Cl35)(m/z = 294, 21.41 %, 2Cl35Cl37)
(m/z = 296, 10.15 %, 2Cl37)
Cl
Cl Cl
Cl
Cl Cl
(m/z = 227, 5.5 %, Cl35)(m/z = 229, 2.41 %, Cl37)
(m/z = 173,5.5.%, 2Cl35)(m/z = 175, 4.9%, 2Cl35Cl37)
(m/z = 177, 5.2 %, 2Cl37)
(m/z = 174, 8.0 %, 2Cl35)(m/z = 176 1.41 %, 2Cl35Cl37)
(m/z = 178,8.8%, 2Cl37)
(m/z = 145, 3.55 %, 2Cl35)(m/z = 147, 2.41 %, 2Cl35Cl37)
(m/z = 149, 1.15 %, 2Cl37) (m/z = 118, 100 %)
-HCO
(m/z = 104, 1.41 %)
(m/z = 90, 38.41 %)
(m/z = 192, 11.41 %)
Cl
(149e)
.
.+ .
+ .+
+ .
+ .
Scheme 3.7: Mass Fragmentation Pattern of 3-(3′,5′-Dichlorophenyl)-
3,4-dihydroisocoumarin 149e
3.1.4 Experimental
The difluoro- and dichlorobenzoic acids were purchased from Aldrich
and used without further purification. All reagents and solvents were
commercially available and used as supplied. The petroleum ether used
corresponds to the fraction with a boiling range of 40-80 °C. The melting
points of the compounds were determined in open capillaries using a
Gallenkemp melting point apparatus and are uncorrected. The infrared
75
spectra were recorded on a Hitachi model 270-50 spectrophotometer as KBr
disks or as neat liquids. 1H NMR (300 MHz) spectra were recorded on a
Bruker AM-400 as a CDCl3 solution using TMS as an internal standard, while
the EI MS were recorded on a MAT-112-S machine.
General procedure for 3-(Difluorophenyl)- 146(a-d) and (Dichlorophenyl)- isocoumarins 146(a-g)
A mixture of difluorobenzoic acid 144(a-d) (63.3 mmol) / dichlorobenzoic
acid 144(e-g) (53 mmol) and thionyl chloride (76.0 mmol & 63 mmol) was
heated for 30 min in the presence of a drop of DMF under reflux. Completion of
the reaction was determined by the stoppage of gas evolution. Removal of
excess of thionyl chloride was carried out under reduced pressure to afford
difluoro- 145(a-d) (62.0 mmol) and dichlorobenzoyl chlorides (52.0 mmole)
145(e-g)
A mixture of unsubstituted homophthalic acid (11.3 mmol/ 9.48 mmol)
and difluoro- (62.0 mmol) 145(a-d) / dichlorobenzoyl chloride (52.0 mmol)
145(e-g) was heated at 200 oC under reflux for four hours. The mixture was
dissolved in ethyl acetate and aqueous solution of sodium carbonate was
added in order to remove the unreacted homophthalic acid. The organic layer
was separated, concentrated and chromatographed on silica gel using pet
ether (40-80 oC fraction) as eluent to afford 3-(difluorophenyl)- 146(a-d) and 3-
(dichlorophenyl)isocoumarins 146(e-g) as solids, which were further purified by
recrystallization from methanol.
3-(3′,5′-Difluorophenyl)isocoumarin (146a): Yield: 80%; m.p.: 149–151°C;
IR (KBr, νmax, cm-1): 2920(C-H),, 1704(C=O), 1569, 1515(C=C arom), 1241(C-
O), 1042(C-F); 1H NMR (CDCl3, δ ppm): 8.31 (1H, d, J = 7.92 Hz, H-8), 7.74
(1H, m, H-6), 7.54 (1H, d, J = 7.40 Hz, H-5), 7.50 (1H, dd, J = 7.88 Hz, H-7),
7.40(2H, dd, J = 1.98, 8.30 Hz, H-2′, 6′), 6.94 (1H, s, H-4), 6.81(1H, m, H-4′);
EIMS (DMF, m/z, %): 258(56.72, M); Anal. Calcd. (%) for C15H8F2O2: C,
69.77; H, 3.12; Found: C, 69.73; H, 3.10.
76
3-(2′,3′-Difluorophenyl)isocoumarin (146b): Yield: 77%; m.p.: 156–158°C;
IR (KBr, νmax, cm-1): 2935(C-H),, 1707(C=O), 1566, 1508 (C=C arom),
1242(C-O), 1141(C-F); 1H NMR (CDCl3, δ ppm): 8.31(1H, d, J = 7.90 Hz, H-
8), 7.92(1H, m, H-6), 7.74(1H, m, H-6′), 7.68(1H, d, J = 7.36 Hz, H 5),
7.54(1H, dd, J = 8.2 Hz, H-7), 7.44(1H, m, H-5′), 7.36(1H, m, H-4′), 7.19(1H,
s, H-4); EIMS (DMF, m/z, %): 258(43.60, M). Anal. Calcd. (%) for C15H8F2O2:
C, 69.77; H, 3.12; Found: C, 69.79; H, 3.13.
3-(2′,4′-Difluorophenyl)isocoumarin (146c): Yield; 78%: m.p.: 131–133°C;
IR (KBr, νmax, cm-1): 2927 (C-H), 1709 (C=O), 1562, 1491 (C=C arom), 1248
(C-O), 1141 (C-F); 1H NMR (CDCl3, δ ppm): 8.30 (1H, J = 7.89 Hz, H-8), 7.99
(1H, m, H-6′), 7.73 (1H, m, H-6), 7.53 (1H, d, J = 7.56 Hz, H-5), 7.50 (1H, dd,
J = 3.0, 7.8 Hz, H-7), 7.12 (1H, s, H-4), 6.99 (1H, m, H-5′), 6.93 (1H, m, H-3′);
EIMS (DMF, m/z, %): 258(83, M); Anal. Calcd. (%) for C15H8F2O2: C, 69.77;
H, 3.12; Found: C, 69.72; H, 3.11.
3-(3′,4′-Difluorophenyl)isocoumarin (146d): Yield: 79%; m.p.: 156–159°C;
IR (KBr, νmax, cm-1): 2923 (C-H), 1703 (C=O), 1565, 1511 (C=C arom), 1247
(C-O), 1189 (C-F); 1H NMR (CDCl3, δ ppm): 8.30 (1H, dd, J = 8.0 Hz, H-8),
7.75 (1H, d, J = 1.24, 7.8, 11.2 Hz, H-6′), 7.74 (1H, m, H-6), 7.72 (1H, m, H-
5′), 7.53(1H, dd, J = 1.0, 7.8 Hz, H-5), 7.51 (1H, m, H-2′), 7.23 (1H, m), 6.88
(1H, s, H-4); EIMS (DMF, m/z, %): 258 (77, M). Anal. Calcd. (%) for
C15H8Cl2O2: C, 69.77; H, 3.12; Found: C, 69.71; H, 3.13.
3-(3′,5′-Dichlorophenyl)isocoumarin (146e): Yield: 81%: m.p: 208-210°C;
IR (KBr, νmax, cm-1): 3156 (C-H), 2935 (C-H), 1708 (C=O), 1590, 1516 (C=C
arom), 1099 (C-Cl); 1H NMR (CDCl3, δ ppm): 8.31 (1H, dd, J = 1.9, 7.9 Hz, H-
8), 7.75 (2H, t, J = 1.2 Hz, H-2′, 6′), 7.55 (1H, dd, J = 2.2, 7.6 Hz, H-5), 7.53
(1H, m, H-6), 7.50 (1H, m, H-7), 7.39 (1H, t, J = 1.2 Hz, H-4′), 6.95 (1H , s ,
H-4); EIMS (DMF, m/z, %): 290 (100, M), 292 (67.5, M+2), 294 (13, M+4);
Anal. Calcd. (%) for C15H8Cl2O2: C, 62.07; H, 2.76; Found: C, 61.83; H, 2.73.
77
3-(2′, 3′-Dichlorophenyl)isocoumarin (146f): Yield: 80%; m.p: 180-182°C;
IR (KBr, νmax, cm-1): 3152 (C-H), 2931 (Ar-H), 1710 (C=O), 1593, 1490 (C=C
arom), 1096 (C-Cl); 1H NMR (CDCl3, δ ppm): 8.33 (1H, dd, J = 2.1, 7.9 Hz, H-
8), 7.74 (1H, m, H-6), 7.56 (1H, dd, J = 2.2, 8.0 Hz, H-5), 7.54 (1H, m, H-7),
7.50 (2H, t, J = 7.8 Hz, H-6′), 7.33 (2H, t, J = 7.8 Hz, H-5′), 7.29(1H, dd, J =
1.9, 7.9 Hz, H-4′), 6.89(1H, s, H-4); EIMS (DMF, m/z, %): 290 (28, M), 292
(17, M+2), 294 (4, M+4); Anal. Calcd. (%) for C15H8F2O2: C, 62.07; H, 2.76;
Found; C, 61.99; H, 2.69.
3-(2′, 5′-Dichlorophenyl)isocoumarin (146g): Yield: 81%; m.p:187-188°C;
IR (KBr, νmax, cm-1): 3155 (C-H), 2935 (Ar-H), 1710 (C=O), 1593 (C=C arom),
1093 (C-Cl); 1H NMR (CDCl3, δ ppm): 8.32 (1H, dd, J = 2.7, 7.9 Hz, H-8), 7.75
(1H, m, H-6), 7.72 (1H, d, J = 2.0 Hz, H-6’), 7.56 (1H, m, H-7), 7.50 (1H, dd, J
= 2.1, 7.8 Hz, H-5), 7.41 (1H, d, J = 8.5 Hz, H-3’), 7.32 (1H, dd, J = 2.4, 8.5
Hz, H-4'), 7.01 (1H, s, H-4); EIMS (DMF, m/z, %): 290 (100, M), 292 (70,
M+2), 294 (13, M+4); Anal. Calcd. (%) for C15H8F2O2: C, 62.07; H, 2.76;
Found; C, 61.89; H, 2.71.
General procedure for 2-(Difluoro/Dichlorobenzoylmethyl)benzoic acid 147(a-g)
A solution of isocoumarins 146(a-g) in ethanol (50 mL) and 5%
potassium hydroxide (100 mL) were refluxed for 4 h. Ethanol was removed
from the reaction mixture by distillation. Ice cold water (20 mL) was added
and the reaction mixture was acidified with hydrochloric acid. The reaction
mixtures were then extracted with dichloromethane (3 × 20 mL). The extracts
were dried (Na2SO4) and evaporated to yield crude solids 147(a-c), which
were recrystallized from methanol.
2′-(3′′,5′′-Difluorobenzoylmethyl)benzoic acid (147a): Yield, 80%; m.p.:
124–126°C; IR (KBr, νmax, cm-1): 3150 (sp2 CH), 2920 (sp3 C-H) , 1708(C=O),
1470 (C-H), 1142 (C-F); 1H NMR (D2O, δ ppm): 8.08 (1H, d, J = 7.23 Hz, H-6),
78
7.77 (1H, m, H-5), 7.51 (2H, m, H-2′′,6′′), 7.37 (1H, dd, J = 7.6 Hz, H-4), 7.23
(1H, d, J = 7.4 Hz, H-3), 6.98 (1H, dd, J = 8.25, 16.6 Hz, H-4′′), 4.61 (1H, s,
H-1′); EIMS (DMF, m/z, %): 276 (2, M). Anal. Calcd. for C15H10F2O3: C, 65.22;
H, 3.65; Found: C, 65.24; H, 3.66.
2′-(2′′,3′′-Difluorobenzoylmethyl)benzoic acid (147b): Yield, 70%; m.p.:
151–154°C; IR (KBr, νmax, cm-1): 3300–3250 (-OH), 2920, 2835 (sp3 C-H),
1704 (C=O), 1471 (C-H), 1145 (C-F); 1H NMR (D2O, δ ppm): 8.05 (1H, d, J =
7.2 Hz, H-6), 7.73 (1H, m, H-4), 7.51 (2H, m, H-6′′), 7.31 (1H, dd, J = 7.6 Hz,
H-5), 7.27 (1H, d, J = 7.1 Hz, H-3), 6.98 (1H, dd, J = 8.0, 16.6 Hz, H- 4′′,5′′),
4.65 (1H, s, H-1′); EIMS (DMF, m/z, %): 276 (1.5, M). Anal. Calcd. for
C15H10F2O3: C, 65.22; H, 3.65; Found: C, 65.29; H, 3.62.
2′-(2′′,4′′-Difluorobenzoylmethyl)benzoic acid (147c): Yield, 77%; m.p.:
174–177°C; IR (KBr, νmax, cm-1): 3300–3250 (-OH), 2924, 2831(sp3 C-H),
1708 (C=O), 1477 (C-H), 1140 (C-F); 1H NMR(D2O, δ ppm): 8.10 (1H, d, J =
7.25 Hz, H-6), 7.71 (1H, m, H-4), 7.51 (1H, m, H-6′′), 7.35 (1H, dd, J = 7.6 Hz,
H-5), 7.23 (1H, d, J = 7.4 Hz, H-3), 6.97 (1H, dd, J = 8.23, 16.6 Hz, H-5′′),
6.79 (1H, m, H-3′′), 4.62 (1H, s, H-1′); EIMS (DMF, m/z, %): 276 (3.0, M).
Anal. Calcd. for C15H10F2O3: C, 65.22; H, 3.65; Found: C, 65.19; H, 3.67.
2′-(3′′,4′′-Difluorobenzoylmethyl)benzoic acid (147d): Yield, 69%; m.p.:
132–135°C; IR (KBr, νmax, cm-1): 3300–3250 (-OH), 2927, 2837 (sp3 C-H),
1710 (C=O), 1473 (C-H), 1141 (C-F); 1H NMR (D2O, δ ppm): 8.03 (1H, d, J =
7.21 Hz, H-6), 7.73 (1H, m, H-4), 7.55 (2H, m, H-2′′,6′′), 7.33 (1H, dd, J = 7.1
Hz, H-5), 7.24 (1H, d, J = 7.1 Hz, H-3), 6.89 (1H, dd, J = 8.22, 16.1 Hz, H-5′′),
4.62 (1H, s, H-1′); EIMS (DMF, m/z, %): 276 (2.1, M). Anal. Calcd. for
C15H10F2O3: C, 65.22; H, 3.65; Found: C, 65.22; H, 3.69.
2′-(3′′,5′′-Dichlorobenzoylmethyl) benzoic acid (147e): Yield: 81%;
m.p.:187-188°C; IR (KBr, νmax, cm-1): 3300-3250 (-OH), 3155 (C-H), 2935 (Ar-
H), 1710 (C=O), 1593 (C=C arom), 1093 (C-Cl); 1H NMR (D2O, δ ppm): 8.15
79
(1H, dd, J = 1.2, 7.9 Hz, H-6), 7.94 (2H, d, J = 1.7 Hz, H-2′′and 6′′), 7.56 (1H,
dd, J = 2.4 Hz, H-4′′ ), 7.52 (1H, m, H-5), 7.24 (1H, m, H-4), 7.18 (1H, dd, J
1.5, 7.9 Hz, H-3), 4.62 (2H, s, H-1′); EIMS (DMF, m/z, %): 308 (2.1, M), 310
(1.0, M+2), 312 (1.2, M+4); Anal. Calcd. (%) for C15H10Cl2O3: C, 58.44; H,
3.25; Cl, 25.26; Found; C, 58.23; H, 3.28.
2′-(2′′,3′′-Dichlorobenzoylmethyl)benzoic acid (147f): Yield: 77%; m.p.;
160-162°C; IR (KBr, νmax, cm-1): 3300-3250 (-OH), 3151 (C-H), 2935 (Ar-H),
1712 (C=O), 1590 (C=C arom), 1091 (C-Cl); 1H NMR (D2O, δ ppm): 8.02 (1H,
dd, J = 2.2, 8.0 Hz, H-6), 7.81 (1H, dd, J = 1.4, 7.7 Hz, H-6′′), 7.63 (1H, m,
H-5), 7.42 (1H, t, J = 8.1 Hz, H-4′′), 7.29 (1H, m, H-4), 7.20 (1H, dd, J = 1.5,
8.0 Hz, H-3), 7.06 (1H, t, J = 7.9 Hz, H-5′′), 4.64 (1H, s, H-1’); EIMS (DMF,
m/z, %): 308 (1.7, M), 310 (5.5, M+2), 312 (1.3, M+4); Anal. Calcd. (%) for
C15H10Cl2O3: C, 58.44; H, 3.25; Found: C, 58.30; H, 3.23.
2′-(2′′,5′′-Dichlorobenzoylmethyl) benzoic acid (147g):Yield: 71%;
m.p.:140-142°C; IR (KBr, νmax, cm-1): 3300-3250 (-OH), 3157 (C-H), 2933 (Ar-
H), 1711 (C=O), 1590 (C=C arom), 1094 (C-Cl); 1H NMR (D2O, δ ppm): 8.17
(1H, dd, J = 1.7, 7.9 Hz, H-6), 7.97 (1H, d, J = 1.8 Hz, H-6′′), 7.58 ( 1H, m, H-
5), 7.44 (1H, d, J = 8.8 Hz, H-4′′), 7.41 (1H, d, J = 9.0 Hz, H-3′′), 7.34 (1H,
dd, J = 2.0, 8.0 Hz, H-3), 7.32 (1H, m, H-4), 4.63 (1H, s, H-1’); EIMS (DMF,
m/z, %): 308 (3, M), 310 (2, M+2), 312 (3, M+4); Anal. Calcd. (%) for
C15H10Cl2O3: C, 58.44; H, 3.25; Found: C, 58.33; H, 3.20.
General procedure for 3-(Difluoro/Dichlorophenyl)-3,4-dihydro- isocoumarin 149(a–g)
To a solution of the keto-acids 147(a–d) (2.07mmol) dissolved in 1%
potassium hydroxide solution (25mL), sodium borohydride (0.25g) was added
and the reaction mixture was stirred for 1 h at room temperature. After
acidification with hydrochloric acid, the reaction mixture was extracted with
ethyl acetate (2 × 50mL). The usual workup gave the crude hydroxy-acids
148(a–d), which were dissolved in acetic anhydride (1mL) and heated under
80
reflux for 2 h. The reaction mixture was cooled, water (25mL) was added and
the reaction mixture was stirred overnight. The crystals that deposited were
collected by filtration and the filtrate was extracted with dichloromethane (2 ×
20mL). The solvent was removed under reduced pressure. The crude
dihydroisocoumarin 149(a–d) was purified by column chromatography on
silica gel using petroleum ether as an eluent.
2-[2′-Hydroxy-2′-(3′′,5′′-difluorophenyl)ethyl]benzoic acid (148a): Yield:
69%; m.p.: 132-133°C; IR (KBr, νmax, cm-1): 3300-3250 (O-H), 2920 (C-H),
1708 (C=O), 1470 (C-H), 1142 (C-F); 1H NMR (D2O, δ ppm): 8.08 (1H, dd, J
= 7.23 Hz, H-6), 7.37 (1H, dd, J = 7.6 Hz,H-5), 7.23 (1H, d, J = 7.4 Hz, H-3),
6.91 (1H, m , J = 8.2 , 16.6 Hz, H-2′′, 4′′,6′′), 4.49 (1H, dd, J = 7.1, 14.2 Hz,
H-2′), 2.67 (1H, dd, J = 6.2, 15.6 Hz, H-1′a), 2.33 (1H, dd, J = 8.2, 15.6 Hz, H-
1′b); EIMS (DMF, m/z, %): 278 (4, M). Anal. Calcd. for C15H12F2O3: C, 64.75;
H, 4.35 ; Found: C, 64.24; H, 4.66.
2-[2′-Hydroxy-2′-(2′′,3′′-difluorophenyl)ethyl]benzoic acid (148b): Yield:
75%; m.p.: 144-146°C; IR (KBr, νmax, cm-1): 3300-3250 (-OH), 2922 (C-H),
1701 (C=O), 1475 (C-H), 1141 (C-F); 1H NMR (D2O, δ ppm): 8.03 (1H, dd, J
= 7.2 Hz, H-6), 7.67 (m, J = 1.6, 7.9 Hz, H-4), 7.39(1H, dd, J = 7.6 Hz, H-5),
7.43 (1H, d, J = 7.4 Hz, H-3), 6.99 (1H, m , J = 8.2 , 16.6 Hz, H- H-5′′,6′′),
6.69 (1H, ddd , J = 8.2 , 16.0 Hz, H- H-4′′), 4.62 (1H, dd, J = 7.1, 14.2 Hz, H-
2′), 3.01 (1H, dd, J = 6.1, 15.0 Hz, H-1′a), 2.53 (1H, dd, J = 8.2, 15.1 Hz, H-
1′b); EIMS (DMF, m/z, %): 278 (2, M). Anal. Calcd. for C15H12F2O3: C, 64.75;
H, 4.35 ; Found: C, 64.77; H, 4.46.
2-[2′-Hydroxy-2′-(2′′,4′′-difluorophenyl)ethyl]benzoic acid (148c): Yield,
79%; m.p.: 129-130°C. IR (KBr, νmax, cm-1): 3300-3250 (-OH), 2923 (C-H),
1705 (C=O), 1477 (C-H), 1145 (C-F).1H NMR (D2O, δ ppm): 8.10 (1H, dd, J =
7.23 Hz, H-6), 7.69 (m, J = 1.6, 7.92 Hz, H-4), 7.45 (1H, dd, J = 7.6 Hz,H-5),
7.23 (1H, d, J = 7.4 Hz, H-3), 7.01 (1H, dd , J = 8.25 , 16.66 Hz, H-,6′′), 6.55
(1H, ddd , J = 8.21 , 16.61 Hz, H- H-3′′,5′′), 4.75 (1H, dd, J = 7.10, 14.25 Hz,
81
H-2′), 2.99 (1H, dd, J = 6.21, 15.56 Hz, H-1′a), 2.57 (1H, dd, J = 8.20, 15.56
Hz, H-1′b); EI MS (DMF, m/z, %): 278.00(1.1. M+); Anal. Calcd. for
C15H12F2O3: C, 64.75; H, 4.35 ; Found: C, 64.67; H, 4.26.
2-[2′-Hydroxy-2′-(3′′, 4′′-difluorophenyl)ethyl]benzoic acid (148d): Yield,
75.25 % ; m.p.: 121-123°C. IR (KBr, νmax, cm-1): 3300-3250 (-OH), 2933 (C-
H), 1703 (C=O), 1467 (C-H), 1143 (C-F).1H NMR (D2O, δ ppm): 8.09 (1H, dd,
J = 7.23 Hz, H-6), 7.61 (m, J = 1.6, 7.90 Hz, H-4), 7.55 (1H, dd, J = 7.6Hz,H-
5), 7.43 (1H, d, J = 7.4 Hz, H-3), 6.87 (1H, dd , J = 8.20 , 16.6 Hz, H-6′′), 6.71
(1H, ddd , J = 8.15 , 16.76 Hz, H- H-2′′,5′′), 4.85 (1H, dd, J = 7.01, 14.22 Hz,
H-2′), 2.89 (1H, dd, J = 6.12, 15.6 Hz, H-1′a), 2.55 (1H, dd, J = 8.02, 15.36
Hz, H-1′b); EI MS (DMF, m/z, %): 278(1.6, M+). Anal. Calcd. for C15H12F2O3:
C, 64.75; H, 4.35 ; Found: C, 64.60; H, 4.28.
2-[2′-Hydroxy-2′-(3′′, 5′′-dichlorophenyl)ethyl]benzoic acid (148e): Yield:
77%; m.p.: 138-140°C; IR (KBr, νmax, cm-1): 3300-3250 (-OH), 3157 (C-H),
2933 (Ar-H), 1711 (C=O), 1590 (C=C arom), 1094 (C-Cl):; 1H NMR (D2O, δ
ppm): 8.16 (1H, dd, J 1.77, 7.9 Hz, H-6), 7.73 (1H, dd, J 1.78, 8.9 Hz, H-3),
7.78 (1H, m, H-4), 7.57 (1H, m, H-5), 7.05 (3H, t, J 1.76 Hz, H-2′′, 4′′ and 6′′),
4.10(1H, dd, J 7.12, 14.20 Hz, H-2’), 2.60 (1H, dd, J 6.24, 15.6 Hz, H-1′a),
2.25 (1H, dd, J 8.22, 15.6 Hz, H-1′b); EI MS (DMF, m/z, %): 310 (2.1, M+),
312 (1.2, M++2), 314 (1.9, M++4) Anal. Calcd. (%) for C15H12Cl2O3: C, 58.06;
H, 3.87; Found: C, 57.91; H, 3.88.
2-[2′-Hydroxy-2′-(2′′, 3′′-dichlorophenyl)ethyl]benzoic acid (148f): Yield:
70 %; m.p.: 150-152°C; IR (KBr, νmax, cm-1): 3300-3250 (-OH), 3159 (C-H),
2936 (Ar-H), 1713 (C=O), 1593 (C=C arom), 1094 (C-Cl): 1H NMR (D2O, δ
ppm): 8.13 (1H, dd, J 2.1, 7.9 Hz, H-6), 7.78 (1H, m, H-4), 7.73 (1H, dd, J
1.87, 8.9 Hz, H-3), 7.57 (1H, m, H-5), 7.63 (1H, dd, J 1.76, 7.78 Hz, H-6′′),
7.59 (1H, t, J 7.72 Hz, H-5′′), 7.55 (1H, dd, J 1.76, 8.1 Hz, H-4′′), 4.17(1H,
dd, J 7.12, 14.20 Hz, H-2’), 2.63 (1H, dd, J 6.24, 15.6 Hz, H-1’a), 2.22 (1H,
dd, J 8.22, 15.6 Hz, H-1’b); EI MS (DMF, m/z, %): 310 (8.6, M+), 312 (6.5,
82
M++2), 314 (1.4, M++4); Anal. Calcd. (%) for C15H12Cl2O3: C, 58.06; H, 3.87;
Found; C, 58.12; H, 3.77.
2-[2′-Hydroxy-2′-(2′′, 5′′-dichlorophenyl)ethyl]benzoic acid (148g): Yield:
72 %; m.p.: 172-174°C; IR (KBr, νmax, cm-1): 3300-3250 (-OH), 3153 (C-H),
2936 (Ar-H), 1716 (C=O), 1593 (C=C arom), 1099 (C-Cl): 1H NMR (D2O, δ
ppm): 8.15 (1H, dd, J 1.87, 7.79 Hz, H-6), 7.78 (1H, m, H-4), 7.73 (1H, dd, J
2.3, 8.9 Hz, H-3), 7.42 (1H, m, H-5), 7.27 (1H, d, J 1.0 Hz, H-6′′), 7.24 (1H,
dd, J 1.56, 7.8 Hz, H-4′′), 7.23 (1H, d, J 8.9 Hz, H-3′′), 4.14(1H, dd, J7.12,
14.20 Hz, H-2’), 2.61 (1H, dd, J 6.24, 15.6 Hz, H-1’a), 2.21 (1H, dd, J 8.22,
15.6 Hz, H-1’b); EI MS (DMF, m/z, %): 310 (2.3, M+), 312 (5.5, M++2), 314
(1.8, M++4); Anal. Calcd. (%) for C15H12Cl2O3: C, 58.06; H, 3.87; Found: C,
57.99; H, 3.58.
(dl)-3-(3′, 5′-Difluorophenyl)-3,4-dihydroisocoumarin (149a): Yield: 69% ;
m.p.: 121-122°C; IR (KBr, νmax, cm-1): 2920 (C-H), 1704, 1244, 1142 (C-F); 1H
NMR (CDCl3, δ ppm): 8.14 (1H, d, J = 7.5 Hz, H-8), 7.67(1H, dd, J = 3.3, 5.6
Hz, H-6), 7.51 (1H, dd, J = 2.1, 7.6 Hz, H-5), 7.28 (1H, m, J=1.7, 6.7 Hz, H-7),
7.12 (1H, m, H-2′, 6′), 6.90 (1H, m, H-4′), 5.92 (1H, dd, J = 2.9, 13.1 Hz, H-3),
3.81 (1H, dd, J = 4.0, 13.0 Hz, H-4a), 3.46 (1H, dd, J = 2.9, 16.3 Hz, H-4b);
EIMS (DMF, m/z, %): 261 (4, M++1); Anal. Calcd. For C15H10F2O2: C, 69.23;
H, 3.87; Found: C, 69.23; H, 3.03.
(dl)-3-(2′,3′-Difluorophenyl)-3,4-dihydroisocoumarin (149b): Yield: 80%;
m.p.: 118-121°C; IR (KBr, νmax, cm-1): 2935 (C-H), 1707, 1244, 1141(C-F); 1H
NMR (CDCl3, δ ppm): 8.13 (1H, d, J = 7.2 Hz, H-8), 7.68(1H, dd, J = 3.2,
5.7Hz, H-6), 7.62 (1H, dd, J = 2.5, 7.5 Hz, H-5), 7.54 (1H, dd, J = 2.2, 7.9 Hz,
H-7), 7.46 (1H, m, J = 5.0, 8.6 Hz, H-5′), 6.71 (m, J = 2.3, 8.5 Hz, H-6′), 6.61
(1H, m, H-4′), 5.76 (1H, dd, J = 2.9, 12.1 Hz, H-3), 3.31 (1H, dd, J = 4.0, 16.3
Hz, H-4a), 3.09 (1H, dd, J = 2.9, 16.3 Hz, H-4b); EIMS (DMF, m/z, %): 260
(5.1, M+). Anal. Calcd. For C15H10F2O2: C, 69.23; H, 3.87; Found: C, 69.23;
H, 3.03.
83
(dl)-3-(2′,4′-Difluorophenyl)-3,4-dihydroisocoumarin (149c): Yield: 80%;
m.p.: 121-122°C; IR (KBr, νmax, cm-1): 2935(C-H), 1707, 1244, 1141(C-F); 1H
NMR (CDCl3, δ ppm): 8.13 (1H, d, J = 7.2 Hz, H-8), 7.68(1H, dd, J = 3.2, 5.7
Hz, H-6), 7.62 (1H, dd, J = 2.5, 7.5 Hz, H-5), 7.54 (1H, dd, J = 2.2, 7.9Hz, H-
7), 7.46 (1H, m, J = 5.0, 8.6 Hz, H-5’), 6.71 (m, J = 2.3, 8.5 Hz, H-6′), 6.61
(1H, m, H-3′), 5.76(1H, dd, J = 2.9,12.1 Hz, H-3), 3.31(1H, dd, J = 4.0, 16.3
Hz, H-4a), 3.09 (1H, dd, J = 2.9,16.3Hz, H-4b); EIMS (DMF, m/z, %): 260 (11,
M+). Anal. Calcd. For C15H10F2O2: C, 69.23; H, 3.87; Found: C, 69.23; H,
3.03.
(dl)-3-(3′,4′-Difluorophenyl)-3,4-dihydroisocoumarin (149d): Yield: 69%;
m.p.: 121-122°C; IR (KBr, νmax, cm-1): 2923, 1703, 1243, 1149 (C-F); 1H NMR
(CDCl3, δ ppm): 3.08(1H, dd, J= 3.1, 16.3Hz, H-4b), 3.29(1H, dd, J = 4.4,16.3
Hz, H-4a), 5.46(1H, dd, J = 3.0, 11.9 Hz, H-3), 7.15 (2H, m, J = 7.8, 11.3 Hz,
H-2’, 5’), 7.21(1H, m, J = 3.8, 5.1, 8.2 Hz, H-6’), 7.41 (1H, m, J = 1.1, 8.6 Hz,
H-7), 7.55 (1H, dd, J = 1.2, 7.4 Hz, H-5), 7.88 (1H, m, J = 1.8, 6.9 Hz, H-6),
8.13(1H, d, J = 7.7 Hz, H-8); EIMS (DMF, m/z, %): 260 (9, M+); Anal. Calcd.
For C15H10F2O2: C, 69.23; H, 3.87; Found: C, 69.23; H, 3.03.
(dl)-3-(3′,5′-Dichlorophenyl)-3,4-dihydroisocoumarin (149e): Yield: 73%;
m.p.: 184-186°C; IR (KBr, νmax, cm-1): 3153 (C-H), 2935 (Ar-H), 1708 (C=O),
1593 (C=C arom), 1099 (C-Cl); 1H NMR (CDCl3, δ ppm): 8.15 (1H, dd, J =
1.3, 7.6 Hz, H-8), 7.66 (1H, m, H-6), 7.58 (1H, dd, J = 2.2, 7.6 Hz, H-5), 7.48
(1H, m, H-7), 7.44 (1H, d, J = 2.3 Hz, H-4’), 7.30 (2H, d, J = 2.6 Hz, H-2', 6’),
5.93 (1H, dd, J = 2.9, 12.0 Hz, H-3), 3.29 (1H, dd, J = 2.9, 16.3 Hz, H-4a),
3.09 (1H, dd, J = 12.1, 16.2 Hz, H-4b); EIMS (DMF, m/z, %): 292 (11, M), 294
(10, M+2), 296 (3, M+4); Anal. Calcd. (%) for C15H10Cl2O2: C, 61.64; H, 3.08;
Found: C, 61.44; H, 3.44.
(dl)-3-(2',3'-Dichlorophenyl)-3,4-dihydroisocoumarin (149f): Yield: 77 %;
m.p.: 136-138°C; IR (KBr, νmax, cm-1): 3151 (C-H), 2939 (Ar-H), 1710 (C=O),
1597 (C=C arom), 1097 (C-Cl); 1H NMR (CDCl3, δ ppm): 8.15 (1H, dd, J =
2.6, 7.6 Hz, H-8), 7.66 (1H, dd, J = 2.6, 7.7 Hz, H-5), 7.45 (1H, m, H-7), 7.58
84
(1H, m, H-6), 7.32 (1H, dd, J = 1.7, 7.9Hz, H-4’), 7.30 (1H, t, J = 5.9 Hz, H-
5’), 7.29 (1H, dd, J = 2.6, 7.4 Hz, H-6’), 5.93 (1H, dd, J = 2.8, 12.0 Hz, H-3),
3.27 (1H, dd, J = 2.9, 16.3 Hz, H-4a), 3.10 (1H, dd, J = 12.1, 16.2 Hz, H-4b);
EIMS (DMF, m/z, %): 292 (58, M), 294 (18, M+2), 296 (13, M+4); Anal. Calcd.
(%) for C15H10Cl2O2: C, 61.64; H, 3.08; Found: C, 61.54; H, 3.04.
(dl)-3-(2',5'-Dichlorophenyl)-3,4-dihydroisocoumarin (149g): Yield: 77 %;
m.p.: 121-124°C; IR (KBr, νmax, cm-1): 3154 (C-H), 2933 (Ar-H), 1712 (C=O),
1598 (C=C arom), 1097 (C-Cl); 1H NMR (CDCl3, δ ppm): 8.15 (1H, J = 1.2,
7.6 Hz, H-8), 7.66 (1H, dd, J = 1.2, 7.7 Hz, H-5), 7.48 (1H, m, H-7), 7.58 (1H,
m, H-6), 7.44 (1H, dd, J = 2.3, 8.1 Hz, H-4’), 7.30 (1H, dd, J = 2.6 Hz, H-6’),
7.24 (1H, dd, J = 7.6 Hz, H-3' ), 5.93 (1H, dd, J = 2.9, 12.0 Hz, H-3), 3.29 (1H,
dd, J = 2.9, 16.3 Hz, H-4a), 3.09 (1H, dd, J = 12.1, 16.2 Hz, H-4b); EIMS
(DMF, m/z, %): 292 (35, M), 294 (21, M+2), 296 (11, M+4); Anal. Calcd. (%)
for C15H10Cl2O2: C, 61.64; H, 3.08; Found: C, 61.44; H, 3.44.
3.2 Synthesis of 3H-Furo[3,4-c]-isochromene-1,5-
dione (152) (an unusal isocoumarin) and 3-(3′,4′,5′-
trimethoxyphenyl)isocoumarin (153)
3.2.1 Synthetic scheme
As a continuation of our previous studies196-197 and biological activities
associated with 3-substituted isocoumarins, prompted us to synthesize some
new 3-substituted isocoumarins. Synthetic scheme is shown as follows
(Scheme 3.8)
85
O
OH
O
R OH
O
SOCl2R Cl
O
200°C, 6hCl
O+
R
HO
150a) R = CH2Cl
O
O
O
150b) R =
where
O
OO
O
OO
O
O
O
(152)
(153)
150(a-b) 151(a-b)
O
O
Cl[Yield = 66%]
[Yield = 66%]
[Yield = 71%]
Scheme: 3.8
3.2.2 Results and discussion Chloroacetic acid (150) was converted into their respective acid
chlorides (151a) by reaction with thionyl chloride in the presence of a drop of
DMF and direct condensation of acid chloride (151a) with homophthalic acid
at 200oC afforded 3,4-substituted isocoumarins (152). Unfortunately, the
desired 3-chloromethylisocoumarin was not detected and 3H-Furo[3,4-
c]isochromene-1,5-dione (152) formed instead of 3-chloromethylisocoumarin which was characterized by spectral data and X-ray crystallography.
3H-Furo[3,4-c]isochromene-1,5-dione (152) was purified by column
chromatography and showed a single spot on TLC. This isocoumarin (152)
did not exhibit characteristic 1H singlet at δ 6.81-7.19 ppm for C4-H in 1H NMR
which indicates the absence of C4-H but a singlet of two protons in aliphatic
86
region was observed. The aromatic protons appeared in the acceptable
region i.e. 7.50-8.31 ppm (Table). In IR spectra of isocoumarins (152) lactonic
carbonyl absorptions were observed at 1702 - 1709 cm-1. The molecular ion
peak in the mass spectrum was obtained at m/z 202. The structure was finally
confirmed by X-ray crystallography.
The tentative mechanism for the formation of 3H-Furo[3,4-
c]isochromene-1,5-dione (152) is outlined in scheme 3.9. The reaction
presumably occurs by the initial attack of acid chloride (151a), formed under
the reaction conditions, at the active methylene group of homophthalic acid to
form condensation product (Scheme 3.9) Intermediate A undergoes
cyclization to produce isocoumarin derivative B. Further cyclization of B
followed by water elimination results in the formation of 3H-Furo[3,4-
c]isochromene-1,5-dione (152).
O
OH
O
OH
O
SOCl2Cl
O
200°C, 6hCl
O+
HO
O
OO
O
(152)
Cl Cl
Cl
90°C, 30min
OH
OCOOH
ClO
O
HO O
Cl
OOH....
-H+
O
O OHO-
Cl
O
H+
O
O OHOH
Cl
O
-HCl
-H2O
(A)
(B)
Scheme 3.9: Proposed mechanism for the syntesis of 3H-Furo[3,4-c]-
isochromene-1,5-dione
The detail of X-ray crystallographic data is as follows.
87
a. X-ray crystal structure of 3H-Furo[3,4-c]-isochromene-1,5-dione
The molecular structure of the title compound is shown in Fig. 3.1. In
the title compound, all bond lengths and angles are within normal ranges. The
five member ring is planar to the isocoumarin ring. The bonds lengths within
phenyl ring lie between 1.386 (4)°A and 1.411 (4)°A which highlights the
aromatic character (Fig. 3.1). In case of five membered ring attached to the
isocoumarin ring, a certain variations are found in bond lengths and bond
angles. These variations are due to presence of O atom on C10 and due to
C10---C11. There is no hydrogen bonding below 3.0°A found in the crystal
lattice.
Fig 3.1: Molecular Structure of the Compound 3H-Furo[3,4-c]-isochromene-
1,5-dione 152
This Part of the chapter has been published: Ghulam Qadeer, Nasim
Hasan Rama, Muhammad Tahir Hussain and Wai-Yeung Wong;
Crystal structure of 3H-Furo [3, 4-c] isochromene-1, 5-dione, Acta
Cryst, 2006, E62, o4022–o4023.
88
Crystal data C11H6O4Mr = 202.16 Triclinic P1 a = 7.9892 (13) °A b = 8.0899 (13) °A c = 8.2075 (13) °A α = 67.089 (3)° β = 83.871 (3)° γ = 62.738 (3)° V = 432.68 (12) °A3
Z = 2 Dx = 1.552 Mg m−3
Dm not measured Mo K_ radiation λ = 0.71073 °A µ = 0.120 mm−1
T = 293 (2) K Block Pale yellow 0.32 × 0.24 × 0.20 mm
Geometric parameters (°A, °) Selected bond lengths C1—C2 1.375 (3) C1—C6 1.389 (2) C5—C6 1.405 (2) C5—C9 1.443 (2) C6—C7 1.476 (2) C7—O1 1.196 (2)
C7—O2 1.405 (2) C8—C9 1.330 (2) C8—O2 1.348 (2) C8—C11 1.484 (3) C9—C10 1.458 (2) C10—O3 1.207 (2)
Selected bond angles
C2—C1—C6 119.60 (15) C3—C4—C5 119.49 (15) C4—C5—C9 125.06 (14) C6—C5—C9 115.88 (14)
C1—C6—C5 120.36 (15) O2—C8—C11 122.09 (15) C8—C9—C10 106.99 (15) C5—C9—C10 132.42 (14)
Selected torsional angles H11A—C11—H11B 109.2 C6—C1—C2—C3 0.1 (3) C3—C4—C5—C6 −0.3 (2)
C6—C5—C9—C8 0.6 (2) C4—C5—C9—C10 0.0 (3)
3,4,5-Trimethoxybenzoic acid was purified by recrystalization before
use and its crystal structure is also reported. The X-ray crystallographic data
of 3,4,5-trimethoxybenzoic acid is as follows.
89
b. X-ray crystal structure of 3,4,5-Trimethoxybenzoic acid
Fig. 3.2: Molecular Structure of Compound 150b
In the title crystal structure, the bonds lengths within phenyl ring lie
between 1.374(4) %A and 1.399(4) %A which highlights the aromatic
character (Fig. 3.2). The valence angle C3--C2--C7 [121.5(3)%] is larger than
the standard value of 120%. The opening of this angle is due to the presence
of the methoxy and carboxyl groups on C2 and C6, respectively, which
involves a decrease of the ring angles of C7 [119.0(3)\%] and C5 [119.7(2)%].
The C1---O1 [1.264(3) %A] bond distance are compatible with
respective distances in related structures198 and smaller than those usually
observed in carboxylic acids (1.365%A). The three methoxy groups are nearly
coplanar with the benzene ring (C3-C4-O3-C8 = 7.2, C4-C5-O4-C9 = 7.1 and
C7-C6-O5-C10 = 4.5). As observed in 2,6-dimethoxybenzoic acid or 2,6-
dimethoxy-3-nitrobenzoic acid, the hydrogen interaction from the hydroxyl O7
of one molecule to the remote carbonyl O2 of a neighbour results in
90
catemers. The torsion angle between the plane of the acid group and the
benzene ring (C3-C2-C1-O2) is 176.0 (3). The crystal structure is stablized by
inter and intra-molecular hydrogen bonding (Fig. 3.3)
Fig 3.3: A partial packing diagram of 150b, hydrogen bonds are shown as
dashed lines
Crystal data C10H12O5 F000 = 448 Mr = 212.20 Dx = 1.389 Mg m−3
Monoclinic, Pc Mo Kα radiation λ = 0.71073 Å Hall symbol: P -2yc Cell parameters from 1520 reflections a = 7.3384 (3) Å θ = 2.8–22.6º b = 8.8325 (3) Å µ = 0.11 mm−1
c = 15.7560 (5) Å T = 298 (2) K β = 96.576 (2)º Monoclinic, colorless V = 1014.53 (6) Å 3 0.25 × 0.15 × 0.13 mm Z = 4
This Part of the chapter has been published: Ghulam Qadeer, Nasim
Hasan Rama, Murat Tas¸, Okan Zafer Yesilel and Wai-Yeung Wong;
Crystal structure of 3,4,5-Trimethoxybenzoic acid; Acta Cryst.; 2007,
E63 ,o3456.
91
Geometric parameters (°A, °) Selected bond lengths C1—O1 1.243 (4) C12—C17 1.372 (4) C2—C3 1.373 C16—C17 1.385 (4)
C6—C7 1.392 (4) C17—H17 0.9 C7—H7 0.99 (3) C18—O8 1.420 (4)
Selected bond angles O1—C1—O2 123.1(3) C17—C12—C11 118.7(2) O1—C1—C2 119.8(3) C13—C12—C11 119.4(3) C4—C5—C6 120.4(3) C12—C17—C16 119.4(3) O5—C6—C7 123.9(3) C12—C17—H17 120.3 O5—C6—C5 116.0(2) C16—C17—H17 120.3 Selected torsional angles C3—C4—C5—O4 −177.4 (3) C14—C15—C16—C17 −0.9 (5) O3—C4—C5—C6 179.1 (3) C13—C12—C17—C1 6 0.8 (5) C3—C4—C5—C6 −0.5 (5) O4—C5—C6—O5 −2.0(4) O10—C16—C17—C12 178.8 (3) C15—C16—C17—C12 −0.3 (5) Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A C9—H9A···O9i 0.96 2.53 3.406 (4) 152 C9—H9C···O7ii 0.96 2.49 3.405 (4) 159 C18—H18A···O5iii 0.96 2.51 3.321 (4) 142 C19—H19C···O1iv 0.96 2.50 3.404 (5) 158 O2—H2···O7v 0.82 1.83 2.641 (3) 172 O6—H6···O1vi 0.96 (2) 1.71 (2) 2.656 (3) 170 (5)
Symmetry codes: (i) x, −y, z+1/2; (ii) x−1, y, z; (iii) x, −y+1, z−1/2;
(iv) x+1, y, z; (v) x−1, −y+1, z−1/2; (vi) x+1, −y+1, z+1/2.
3,4,5-Trimethoxybenzoic acid (150b) was converted into their
respective acid chloride (151b) by reaction with thionyl chloride in the
presence of a drop of DMF. Direct condensation of acid chloride (151b) with
simple homophthalic acid at 200oC afforded 3-(3,4,5-trimethoxyphenyl)-
isocoumarin (153). This isocoumarin (153) was purified by column
chromatography and showed a single spot on TLC.
92
This isocoumarin (153) exhibited characteristic 1H singlet at δ 7.26
ppm for C4-H in 1H NMR. The aromatic proton appeared in the acceptable
region i.e. 7.44-8.30 ppm (Table. 3.7). In IR spectra of isocoumarins (153),
lactonic carbonyl absorptions were observed at 1702 cm-1. The molecular ion
peaks in the mass spectrum of 3-(3,4,5-trimethoxyphenyl)isocoumarin (153)
were obtained at m/z 312. The mass fragmentation pattern is shown in
scheme 3.10.
OO
O
O
O
O
OO
O1
23
4 56
78
9
1011
1 23
456
7
81'
2' 3'
4'5'6'
Table 3.7: 1H NMR spectral data of compounds 152 and 153
Carbon 152 153
4 --- 7.26 (1H, s, H-4)
5 7.54 (1H, d, J = 7.54 Hz) 7.78 (1H, d, J = 7.56 Hz)
6 7.74 (1H, m) 7.69-7.74 (1H, m)
7 7.50 (1H, dd, J = 7.88 Hz) 7.44 (1H, m)
8 8.31 (1H, d, J = 7.82 Hz) 8.30 (1H, J = 7.89 Hz)
9 5.01(2H, s) ---
2′ --- 7.62 (2H, d, J = 1.8 Hz)
6′ --- 7.62 (2H, d, J = 1.8 Hz)
OCH3 --- 3.91-3.94 (9H, s, 3 × OCH3)
The structure of the 3-(3,4,5-trimethoxyphenyl)isocoumarin (153) was
also confirmed by X-ray crystallography. The X-ray crystallographic data is as
follows.
93
OO
O
O
O
OO
O
O
OO
O
O
O
O
O
O
O
OO
O
OO
O
OO
O(m/z = 312, 100 %) (m/z = 284, 68.8 %)
(m/z = 253, 54.6 %)(m/z = 89, 15.3 %)(m/z = 117, 21.0 %)
(m/z =145, 7.8 %)
(m/z = 167, 19.6 %) (m/z = 195, 19.5 %)
-.
-CO
-.
-CO
-CO
-OCH3.
+
+++
+
+ +
-. -CO
. + .
(153)
Scheme 3.10: Mass Fragmentation Pattern of 3-(3′,4′,5′-trimethoxy-
phenyl)isocoumarin (153) c. X-ray crystal structure of 3-(3,4,5-trimethoxyphenyl)isocoumarin
A perspective view of (I) is shown in Fig 3.4. Bond lengths and angles
can be regarded as normal. The molecules are essentially planar (r.m.s.
deviation for all non H atoms = 0.033 A %). No hydrogen bonding is found
within the crystal lattice.
Crystal data: C18H16O5 F000 = 656 Mr = 312.31 Dx = 1.416 Mg m−3
Monoclinic, P 2(1)/n Melting point: 437(1) K Hall symbol: -P2yn Mo Kα radiation λ = 0.71073
94
a = 9.1496 (11) Å Cell parameters from 2460 reflections b = 12.8426 (15) Å θ = 2.3–26.3º c = 12.6835 (14) Å µ = 0.10 mm−1
β = 100.592 (2)º T = 100 (2) K V = 1465.0 (3) Å3 Irregular, colourless Z = 4 0.60 × 0.50 × 0.40 mm
Fig 3.4: Molecular Structure of the Compound 153
Geometric parameters (°A, °)
Selected bond lengths C7—C8 1.392 (2) O1—C9 1.391 (2) C7—H7 0.9500 O2—C9 1.209 (2) C12—C13 1.393 (2) O5—C18 1.426 (2)
C2—H2 0.9500 C3—C4 1.405 (2) C3—C8 1.408 (2) C4—H4 0.9500 C5—C6 1.410 (2) C5—H5 0.9500
This Part of the chapter has been published: Ghulam Qadeer, Nasim Hasan
Rama, Mohammad Azad Malik, Javeed Akhtar and Madeleine Helliwell;
Crystal structure of 3-(3,4,5-Trimethoxyphenyl)-1H-isochromen-1-one; Acta
Cryst. 2007, E63, o3447.
95
Selected bond angles C13—O4—C17 113.61 (13) C10—C11—H11 120.0 C14—O5—C18 117.34 (13) C12—C13—C14 119.54 (15) C5—C4—H4 119.9
C10—C15—H15 120.1 C3—C4—H4 119.9 C4—C5—C6 120.63 (16) C4—C5—H5 119.7
Selected torsional angles
C9—O1—C1—C2 −1.0 (2) C2—C1—C10—C15 2.5 (3) C4—C5—C6—C7 −0.9 (3) C17—O4—C13—C14 −75.9 (2) C5—C6—C7—C8 −0.9 (3) O3—C12—C13—O4 −4.2 (2) C6—C7—C8—C3 2.3 (3) C2—C3—C8—C9 −3.0(2) 3.2.3 Experimental
The chloroacetic acid and 3,4,5-trimethoxybenzoic acid were
purchased from Aldrich and used with further purification by recrystallization.
All reagents and solvents were commercially available and used as supplied.
The petroleum ether used corresponds to the fraction with a boiling range of
40-80 °C. The melting points of the compounds were determined in open
capillaries using a Gallenkemp melting point apparatus and are uncorrected.
The infrared spectra were recorded on a Hitachi model 270-50
spectrophotometer as KBr disks or as neat liquids. 1H-NMR (300 MHz)
spectra were recorded on a Bruker AM-400 as aCDCl3 solution using TMS as
an internal standard, while the EIMS were recorded on a MAT-112-S
machine. XRD data collection was carried out by Data collection: SMART
(Bruker, 1998); cell refinement: SAINT (Bruker, 1999); data reduction: SAINT;
program(s) used to solve structure: SHELXS97 (Sheldrick, 1997); program(s)
used to refine structure: SHELXL97 (Sheldrick, 1997); molecular graphics:
SHELXTL (Bruker, 1999); software used to prepare material; SHELXTL.
General procedure for 3H-Furo[3,4-c]isochromene-1,5-dione (152) and
3-(3′,4′,5′-Trimethoxyphenyl)isocoumarins (153).
A mixture of chloroacetic acid (150a) (47 mmol) / 3,4,5-
trimethoxybenzoic acid (150b) (28 mmol) and thionyl chloride (39 mmol & 34
96
mmol) was heated for 30 min in the presence of a drop of DMF under reflux.
Completion of the reaction was determined by the stoppage of gas evolution.
Removal of excess of thionyl chloride was carried out under reduced pressure
to afford chloroacetyl (151a) (46 mmol) and 3,4,5-trimethoxybenzoyl chlorides
(27 mmole) (151b)
A mixture of homophthalic acid (11 mmol/ 7.2 mmol) and chloroacetyl-
(46 mmol) (151a) / 3,4,5-trimethoxybenzoyl chloride (27 mmol) (151b) was
heated at 200 oC under reflux for four hours. The mixture was dissolved in ethyl
acetate and aqueous solution of sodium carbonate was added in order to
remove the unreacted homophthalic acid. The organic layer was separated,
concentrated and chromatographed on silica gel using pet ether (40-80 oC
fraction) as eluent to afford 3H-Furo[3,4-c]isochromene-1,5-dione (152) and 3-
(3′,4′,5′-trimethoxyphenyl) isocoumarin (153) as white crystalline solids, which
were further purified by recrystallization from methanol.
3H-Furo[3,4-c]isochromene-1,5-dione (152): Yield: 66 %; m.p.: 131-132°C;
IR (KBr, νmax, cm-1): 2920 (C-H), 1704 (C=O), 1569(C=C), 1241(C-O),
1142(C-F); 1H NMR (CDCl3, δ ppm ): 8.31 (1H, d, J = 7.92 Hz, H-8), 7.74 (1H,
m, H-6), 7.54 (1H, d, J = 7.4 Hz, H-5), 7.50 (1H, dd, J = 7.8 Hz, H-7), 5.01
(2H, s, H-9); EIMS (DMF, m/z, %): 202 (100, M). Anal. Calcd. (%) for
C11H6O4: C, 65.35; H, 2.99; Found: C, 65.29; H, 3.01.
3-(3′,4′,5′-Trimethoxyphenyl)isocoumarin (153). Yield: 71 %; m.p.: 164-
165°C; (KBr, νmax, cm-1): 2926 (C-H), 1720 (C=O), 1603 (C=C), 1561 (C=C),
1245 (C-O); 1H NMR (CDCl3, δ ppm ): 8.30 (1H, J = 7.8 Hz, H-8), 7.78 (1H, d,
J = 7.5 Hz, H-5), 7.69-7.74 (1H, m, H-6), 7.62 (2H, d, J = 1.8 Hz, H-2′,6′), 7.44
(1H, m, H-7), 7.26 (1H, s, H-4), 3.91 (9H, s, 3 × OCH3); EIMS (DMF, m/z, %):
312 (100, M+); Anal. Calcd. (%) for C18H16O5: C, 69.22; H, 5.16; Found: C,
69.12; H, 5.11.
97
98
Chapter-4
BIOLOIGICAL ACTIVITIES
Isocoumarins, dihydroisocoumarins and related compounds were tested for the
following activities.
4.1. Antioxidant Studies
4.2. Anti-inflammatory Studies
4.3. Herbicide studies
4.4. Fungicide studies
4.5. Insecticide Studies
4.6. Antifungal studies
4.7. Antibacterial studies
4.8. Brine shrimp lethality (Artemia salina) studies
4.9. Cytotoxicity and Antiviral Activities against different Cell Culture
a. Vero cell culture
b. HeLa cell Culture
c. E6SM cell Culture
4.10. Anti-HIV Studies
4.11. Anti-HBV studies
4.12. Anti-cancer studies
4.13. Antimetastatic studies
4.1 Antioxidant Studies
4.1.1 DPPH radical scavenging assay
Radical scavenging activity of compounds against stable DPPH (2,2-
diphenyl-2-picrylhydrazyl hydrate, Sigma–Aldrich Chemie, Steinheim, Germany)
was determined spectrophotometrically. When DPPH reacts with an antioxidant
compound, which can donate hydrogen, it is reduced. The changes in colour
99
(from deep-violet to light-yellow) were measured at 515 nm on a UV–Vis light
spectrophotometer (Spectronic Genesys 8, Rochester, USA). Radical
scavenging activity of compounds was measured by slightly modified method of
Brand-Williams as described below. Compound solutions were prepared by
dissolving an appropriate amount of each compound in ethanol. The solution of
DPPH in ethanol was prepared just before UV measurements. 1.5 mL of this
solution was mixed with 0.5 mL of compound solutions in 1 dm path length
disposable microcuvettes (final mass ratio of compound with DPPH was
approximately 3:1). The samples (at 1, 10, 100, 500 and 1000 mM) were kept in
the dark for 30 min at room temperature and then the decrease in absorption was
measured. For the control absorption of blank sample containing the same
amount of ethanol and DPPH solution was measured. The experiment was
carried out in triplicate. Radical scavenging activity was calculated by the
following formula:
AB-AA
AB
% inhibition =
Where
AB is the absorption of blank sample (t = 0 min);
AA is the absorption of tested extract solution (t = 15 min).
4.1.2 Procedure
The DPPH (2,2-diphenyl-2-picrylhydrazyl hydrate, Sigma-Aldrich Chemie,
Steinheim, Germany) radical scavenging effect was evaluated according to basic
method. Ethanol solution (0.5 mL) of various sample concentration (1, 10, 100,
500 and 1000 µM) was added to 1.5 mL DPPH ethanol solution (66.66 µM) 199.
After mixing gently and leaving for 30 min at room temperature in ambar vials,
the absorbance was measured at 515 nm using a spectrophotometer200-201.
Values IC50 of inhibition by all compounds was determined by comparison with a
control group. The antioxidant activity of each sample was expressed in terms of
100
IC50 (µM) required to inhibit DPPH radical formation by 50% and calculated from
the log-dose inhibition curve.
Table 4.1: Antioxidant activities of isocoumarins 146(a–g), keto-acids 147(a–g), hydroxy-acids 148(a–g) and 3,4-dihydroisocoumarins 149(a–g)
Compound 1µM 10µM 100µM 500µM 1000µM IC50 144e 7.281 22.056 52.248 49.471 51.586 93.3 144f 11.563 25.910 51.820 50.106 51.797 93.3 144g 6.747 30.120 44.578 49.683 51.586 563.4 146a 13.735 8.675 22.169 29.175 30.655 > 1000 146b -4.819 14.458 13.494 15.011 15.011 > 1000 146c -0.917 0.917 12.156 38.991 44.954 > 1000 146d 9.639 16.386 19.759 23.890 42.918 > 1000 146e 16.488 13.919 13.919 11.628 14.588 > 1000 146f 9.639 16.386 19.759 23.890 42.918 > 1000 146g 4.096 12.771 44.096 50.529 52.431 462.6 147a 6.210 26.124 50.749 50.106 53.277 97 147b 16.060 13.919 44.754 54.123 55.603 322.4 147c 4.795 27.837 44.754 50.529 52.008 463.3 147d 5.060 18.795 44.096 46.101 51.374 862 147e 6.638 13.490 50.964 50.529 53.700 97.5 147f 16.060 21.842 52.034 50.951 52.008 94.5 147g 6.747 30.120 44.578 49.683 51.586 563.4 148a 16.060 21.842 52.034 50.951 52.008 94.5 148b 6.638 13.490 50.964 50.529 53.700 97.5 148c 4.096 12.771 44.096 50.529 52.431 462.6 148d 4.795 27.837 44.754 50.529 52.008 433.3 148e 11.349 20.557 50.107 50.529 52.008 99.5 148f 3.614 8.434 45.301 53.066 53.066 337.2 148g 5.060 18.795 44.096 46.101 51.374 862 149a -4.587 5.275 38.991 45.872 45.872 > 1000 149b -0.917 0.917 12.156 38.991 44.954 > 1000 149c -3.211 5.734 8.716 34.633 49.312 > 1000 149d 34.699 17.349 34.940 12.474 15.645 > 1000 149e 7.066 10.278 7.281 0.000 0.211 > 1000 149f 10.361 2.410 -2.892 13.108 15.645 > 1000 149g 34.699 17.349 34.940 12.474 15.645 > 1000
Quercetin - - - - - 29.4
101
4.1.3 Discussion
The radical scavenging effects of synthetic compounds against stable free
radical DPPH (2,2-diphenyl-2-picrylhydrazyl hydrate) were measured
spectrophotometrically. Values for their IC50 are shown in Table 4.1. Compounds
144a, 144b (starting acids), 147a, 147e, 147f (keto-acids), 148a, 148b and 148e (hydroxyl-acids) were found to be good radical scavengers with IC50 below 100,
i.e 93.3, 93.3, 97.0, 97.5, 94.5, 94.5, 93.3 and 99.5 µM, respectively. Compound
144a, 144b, 148b was the most effective DPPH scavenger with an IC50 value of
93.3. Isocoumarins 146(a–g) and 3,4-dihydroisocumarins 149(a–g) were
considerably inactive with IC50 values over 1000 µM. R
O
OOH
R
OH
OOH
DPPH. DPPH.
DPPHHDPPHH
R
O.
OOH
R
OH
OOH
.
H. DelocalizationRate determining step
R
OH
OOH
.
DPPH.
DPPH.
R
OH
OOH
Donation of a second hydrogen atom
DimerizationDPPH
Complexation
R
OH
OOH
R
OH
OHO
4DPPH.R
OH
OOH
R
OH
OHO
Scheme 4.1: Proposed mechanism for keto-acid/ hydroxy-acid and DPPH
reaction
102
The contribution of the halogens in the aromatic ring when they guard a
disposition ortho or meta the scavenging of the free radical was enhance, when
electronic disposition was modified, also the antioxidant activity is modified. Part
of a certain structure and particularly halogens position in the molecule determine
antioxidant properties; these proprieties depend on the ability to donate hydrogen
or electron to a DPPH free radical 202-203.
4.2 Anti-inflammatory Studies
Inflammation is a complex phenomenon involving interrelationships
between humoral or cellular reactions and a number of inflammatory mediators. It
is a usual symptom covering different pathologies, and there are still many
questions to be answered in order to understand the inflammatory process as
well as a need for better-tolerated and more efficient non-steroidal anti-
inflammatory drugs. In the pathways of the inflammatory process, the implication
of free radicals is particularly important. It has also been reported that anti-
inflammatory drugs may be effective in the prevention of free radical-mediated
damage 204.
4.2.1. Experimental animals
Adult male Wistar CD-1 mice with a body weight ranging from 20 to 25 g
were used. All animals had free access to food and water and were kept on a
12/12 h light –dark cycle.
4.2.2. TPA-induced mouse ear edema
Mouse ear edema was evaluated following the protocol previously
described205, using groups of three male CD-1 mice. Edema was induced by
103
topical application of 2.5 mg per ear of TPA dissolved in EtOH. Solutions of the
compounds (1 mg/ear) and the standard drug indomethacin (1 mg/ear) as
reference, dissolved in different solvents according to their solubility,
respectively, were applied to both sides of the right ear (10 mL each side)
simultaneously with TPA. The ear swelling was measured before TPA application
and 4 h after, and the edema was expressed as the increase in thickness.
4.2.3 Discussion
The isocoumarins 146(a–g), keto-acids 147(a–g), hydroxy-acids 148(a–g) and 3,4-dihydroisocoumarins 149(a–g) were evaluated by measuring their anti-
inflammatory activity against TPA-induced inflammation in mice, and the
inhibitory activities were compared with that of indomethacin, a commercially
available anti-inflammatory drug. At a dose of 1.0 mg/ear, the compounds 144g,
146b, 147d, 147e, 148a and 148e showed good activity while the remaining
compounds showed lower or nearly equal activity compared to that of
indomethacin (91.35% at a dose of 1.0 mg/ear). For anti-inflammatory evaluation,
these compounds did not achieve a structural similar analysis as for the
antioxidant activity, because, in case of anti-inflammatory activity we have other
factors that intervene in the biological response (solubility, absorption,
distribution, etc.). Due to the lack of exact data related to the solubility,
absorption and distribution of each compound tested, it is difficult to clearly show
the structure–activity relationship concerning the anti-inflammatory activity.
However, we can qualitatively evaluate the activity by taking account of these
factors.
This Part of the chapter has been published: Ghulam Qadeer, Nasim H.
Rama, M. L. Garduno-Ramırez. Synthesis and anti-inflammatory activity of
fluorinated isocoumarins and 3,4-dihydroiscoumarins. J. Fluorine Chem.,
2007, 128, 641-6.
104
Table 4.2: Anti-inflammatory activity of isocoumarins 146(a–g), keto-acids 147(a–g), hydroxy-acids 148(a–g) and 3,4-dihydroisocoumarins 149(a–g)
Compound % of inhibition of
inflammation (1 mg/ear)
Compound
% of inhibition of inflammation
(1 mg/ear)
144e 70 147f 63.4 144f 21.5 147g 81.8 144g 105.2 148a 93.1 146a -12.5* 148b 81.8 146b 103.7 148c -64.9* 146c 25.3 148d 35.1 146d 64.8 148e 93.0 146e 64.8 148f 81.8 146f 30.8 148g -64.8 146g 82.1 149a 88.2 147a 31.3 149b 86.1 147b 85.4 149c -14.8* 147c 81.0 149d -54.7 147d 92.2 149e -54.7* 147e 92.1 149f 29.3
INDOMETACIN 91.4 149g -212.5 * Values with negative sign represent pro-inflammatory activity
The in vivo anti-inflammatory activity of these compounds indicated these
compounds as a possible candidate for the development of new drugs to treat
symptoms associated with inflammatory diseases, such as osteoarthritis and
arteriosclerosis. Further studies on the assessment of the COX-1/COX-2
selectivity index and inhibitory potency are in progress.
4.3 Herbicide Studies
Weeds compete with crops for sunshine, water, nutrients, and physical
space and are thus capable of greatly influencing the growth of crops and
undermining both crop quality and yield. Also, many weeds are the harbor or nest
of pathogens, viruses, and pests, which may result in the occurrence and spread
105
of plant diseases and insect pests in crops. Herbicides, as the main weed control
tool, play a very important role in modern agriculture. Since the discovery of the
herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) by Zimerman and Hitchcock,
the agrochemical industry has successfully developed a wide array of herbicides
with various chemical structures and modes of action206. However, an inevitable
problem associated with the use of herbicides is the occurrence of herbicide-
resistant weeds9. For example, the widespread use of herbicides, such as
chlorsulfuron, atrazine, diclofop-methyl, and paraquat, has caused herbicide
resistance in many weeds. Therefore, it is necessary to develop efficient
herbicides with novel structures and modes of action to overcome the resistance
of weeds.
4.3.1 Procedure Compound with certain concentration was dissolved in acetone or DMF
and diluted with water, and the solutions were added to certain amount of soil as
soil treatment in order that the concentration of test compound was 750 kg/ha,
after this, the weed seeds were geminated, each pot contains 20-30 seeds and
each experiment triplicated. The plant was cultured in the green house at 25-
27°C. After 4 weeks of growth the fresh weight was measured and the inhibition
percentage was calculated according to the corresponding control. For foliate
spray, after the growth of weed for 2 weeks, 750 kg/ha of solution was sprayed,
and the weeds were cultured for another 2 weeks, again fresh weight was
measured and the inhibition percentage was calculated according to the
corresponding control.
106
Table 4.3a: Herbicide activity (% inhibition) of the Difluorophenylisocoumarins
146(a–d), keto-acids 147(a–d), hydroxy-acids 148(a–d) and 3,4-
dihydroisocoumarins 149(a–d)
AR BC EC DS Compound
Density g / hect ST FS ST FS ST FS ST FS
146a 750 - - - 2.9 2.43 - - -
146b 750 - - 21.3 6.6 - 4.2 - -
146c 750 - - 14.1 24.0 18.9 21.7 24.5 9.8
146d 750 - - 10.6 51.9 6.1 2.3 - -
147a 750 - - - 2.9 2.4 - - -
147b 750 - - - 22.6 - - - -
147c 750 1.7 20.0 9.2 - 10.81 8.49 4.9 1.9
147d 750 - - 39.3 - 8.2 9.95 - -
148a 750 - - - - - - - -
148b 750 - - - - - - - -
148c 750 - 8.0 19.02 12.7 9.4 6.6 1.64 15.6
148d 750 - 8.0 22.7 16.5 5.4 22.6 18.0 7.8
149a 750 13.0 - 1.12 16.0 3.3 - - -
149b 750 - - - - - 16.6 - -
149c 750 - - - - - - - -
149d 750 13.0 - - - - - - -
Clorsulfuron 750 100 100 100 100 100 100 100 100
ST = Soil treatment FS = Foliar spray
Key: (-); No activity
The weeds used for this experiment were Brassica campestris (BC), Echinochloa crusgalli (EC), Amaranthus retroflexus L (AR) and Digitaria.
sanguinalis(L.)Scop (DS).
107
Table 4.3b: Herbicide activity (% inhibition) of the Dichlorobenzoic acid 144(e-g), dichlorophenylisocoumarins 146(e–g), keto-acids 147(e–g), hydroxy-acids 148(e-
g) and 3,4-dihydroisocoumarins 149(e–g)
AR BC EC DS Compound
Density g / hect ST FS ST FS ST FS ST FS
144e 750 - - 19.1 12.6 7.9 17.7 - -
144f 750 - 20.3 75.2 21.1 26.4 - - -
144g 750 - - 29.2 35.5 14.4 17.6 3.3 -
146e 750 - - - 26.55 - 20.6 - -
146f 750 15.9 15.1 94.9 47.9 - - - -
146f 375 10.1 - 73.0 30.4 - - - -
146f 188 - - 33.7 19.7 - - - -
146f 94 - - 1.69 1.74 - - - -
146g 750 - - 1.5 8.8 5.7 10.3 - -
147e 750 - - 23.2 2.9 18.4 17.9 - -
147f 750 - - 88.5 88.2 20.9 10.3 - -
147g 750 - - 67.9 33.3 19.6 0.4 - -
148e 750 13.0 - 94.3 35.5 23.7 - 11.1 22.
148f 750 1.4 - 79.7 33.1 19.0 14.1 15.1 -
148g 750 - - 78.2 35.2 - 10.7 - -
149e 750 - - - 19.4 5.6 - 1.3 -
149f 750 - - - 52.8 - - - 21.1
149g 750 - - 19.8 - 7.7 14.5 - -
Clorsulfuron 750 100 100 100 100 100 100 100 100
ST = Soil treatment FS = Foliar spray
Key: (-); No activity
108
4.3.2 Discussion
From the biological assay results in Table 4.3a and 4.3b, which
summarize the herbicide activity of the target compounds, some showed a
excellent herbicide activity in the pre-emergence treatment (soil treatment) than
post-emergence (foliar treatment). Compounds 144f (2,3-dichlorobenzoic acid),
146f [3-(2′,3′-dichlorophenyl)isocoumarin)], 147(f-g) (dichloro-keto-acids) and
148(e-g) (dichloro-hydroxy-acids) exhibited considerable inhibitory activity
against Brassica campestris as compared to Amaranthus retroflexus L,
Echinochloa crus-galli and Digitaria sanguinalis(L.)Scop in pre-emergence (soil
treatment). Even the inhibitory activity of compounds 146f and 148e reaches
95% at 750 g /hectare. The inhibitory activity of compound 146f has also been
measured at a concentration of 375, 188 and 94 g/hectare. It has been found that
with a decrease in concentration per hectare, the activity decreases. The other
synthesized compounds also show little herbicidal activity. Although we have no
information about the mode of action of these compounds, further research on
the modification of their structure and the mode of action is in progress.
A general overview of herbicide activity for difluoro- and dichlorophenyl-
isocoumarins and related compounds show significant herbicide activity as
compared to difluoroisocoumarin series.
4.6 Fungicide Studies
Over the last two decades, there has been a dramatic increase in the rate
of superficial and invasive fungal infections207-211. Modern agriculture relies on
effective control of fungal diseases to increase crop yield and quality and
consequently increase crop value212. No single fungicide can be used for all
disease situations and the widespread use of fungicides can select for fungicide
resistant pathogens. Therefore, there is need for safer and more cost-effective
109
fungicides, which are easier to use and provide better performance against
resistant pathogens213.
4.4.1 Procedure
Using fungi growth inhibition method for fungicide activity determination as
described by Fan 214. Compound with 500 µg mL-1 of concentration was dissolved
in water by 0.1mL of assistance of DMF and then 500 µg mL-1 of compound in
agar plate was prepared, the fungi was inoculated and cultured in the culture
tank at 24°C-26°C, the diameter of fungi spread was measured two days later,
growth inhibition was calculated by corresponding control.
Table 4.4a: Fungicidal activity (% inhibition) of the Difluorophenylisocoumarins
146(a–d), keto-acids 147(a–d), hydroxy-acids 148(a–d) and 3,4-
dihydroisocoumarins 149(a–d)
Compound Conc. (µg ml-1) FO CA AS GZ PP PA
146a 50 10.9 4.6 18.2 5.8 55.0 - 146b 50 - 4.6 31.8 34.8 40.0 - 146c 50 - - 18.8 16.0 34.4 - 146d 50 - 20.0 45.5 14.7 38.5 35.3 147a 50 - - - - - - 147b 50 - 4.6 63.6 8.7 52.5 - 147c 50 - - 31.3 4 42.6 - 147d 50 - - 9.1 - 34.6 11.8 148a 50 - - - - - - 148b 50 - - - - - - 148c 50 12.0 20.0 18.8 20 42.6 - 148d 50 12.0 13.3 12.5 24 39.3 - 149a 50 22.3 40.9 27.3 31.9 32.5 - 149b 50 2.3 9.1 22.7 11.6 18.8 - 149c 50 - - - - - - 149d 50 5.1 - 50.0 8.7 50.0 -
Amistar 50 100 100 100 100 100 100 Key: (-); No activity
110
The isocoumarins, dihydroisocoumarins and related compounds were
tested for their fungicidal bioassay. The results are reported as linear growth
inhibition (LGI) against some plant pathogens, e.g
FO = Fusarium oxysporum CA = Cercospora arachidicola
AS = Alternaria solani GZ = Gibberella zeae
PP = Physalospora piricola PA = Phoma asparagi
Table 4.4b: Fungicidal activity (% inhibition) of the Dichlorobenzoic acid 144(e-g), dichlorophenylisocoumarins 146(e–g), keto-acids 147(e–g), hydroxy-acids 148(e-
g) and 3,4-dihydroisocoumarins 149(e–g)
Key: (-); No activity
4.4.2 Discussion
From the biological assay results in Table 4.4a, which summarize the
fungicidal activity of the difluorophenylisocoumarins 146(a–d), keto-acids 147(a–d), hydroxy-acids 148(a–d) and 3,4-dihydroisocoumarins 149(a–d). The
Compound Concentration(µg mL-1) FO CA AS GZ PP PA
144e 50 8.0 13.6 100 11.6 57.5 - 144e 20 - - 20.0 - 24.1 - 144f 50 - 4.6 100 11.6 41.3 - 144f 20 - - - - 16.7 - 144g 50 - - 54.5 11.6 42.5 - 146e 50 2.3 4.6 36.4 23.2 47.5 - 146f 50 2.2 - - 5.8 48.8 - 146g 50 - 10.0 27.3 - 19.2 - 147e 50 - 20.0 24.2 26.5 38.5 23.5 147f 50 - 4.6 22.7 5.8 25.0 - 147g 50 - 10.0 18.2 - 26.9 - 148e 50 5.1 - 9.1 14.5 43.8 - 148f 50 - 10.0 9.1 - 21.2 - 148g 50 8.0 - 50.0 23.2 48.8 - 149e 50 8.0 - 22.7 5.8 43.8 - 149f 50 19.4 18.2 31.8 40.6 41.3 - 149g 50 - 60.0 21.2 11.8 53.9 35.5
Amistar 50 100 100 100 100 100 100
111
compounds 146a, 147b and 149d exhibited considerable inhibitory activity
against Alternaria solani and Physalospora piricola. The other synthesized
compounds also show fungicidal activity, but that activity is not significant.
Table 4.4b summarizes the fungicidal activity of the dichlorobenzoic acid
144(e-g), dichlorophenylisocoumarins 146(e–g), keto-acids 147(e–g), hydroxy-
acids 148(e-g) and 3,4-dihydroisocoumarins 149(e–g). The compounds 144(e-g),
148f and 149g exhibited considerable inhibitory activity against Alternaria solani,
Gibberella zeae and Physalospora piricola Even the inhibitory activity of
compounds 144e and 144f reaches 100% at 50µg/ml. The other synthesized
compounds also show fungicidal activity, but that activity is not significant.
4.5 Insecticidal Studies 4.5.1 Procedure
Weighing 10mg sample into a 50mL of glass beaker, then 20mL of
acetone was added, the maize leaf was dipped in the sample solution for 5
seconds, and the leaf was put in a petri dish with 10 cm of diameter to evaporate
all solvents. Nine pieces of maize leaves were cut short and put into the petri
dish of 10 cm diameter containing 10 Mythimna separata with 4 instars, the death
rate of insect was detected 24h and 96h later experiment. For Culex pipiens
pallens larva experiment, the 4 instars of insect was dipped into the water
solution for 24 hours, the death rate of insect was detected.
This Part of the chapter has been published: Ghulam Qadeer, Nasim H. Rama,
Zhi-Jin Fan, Bin Liu and Xiu-Feng Liu. Synthesis, herbicidal, fungicidal and
insecticidal activities of dichlorophenylisocoumarins and 3,4-dihydroiscoumarins.
J. Braz. Chem. Soc. 2007, 18(6), 1176-82
112
Table 4.5: Insecticidal bioactivity of the Difluorophenyisocoumarins 146(a-d) and
difluorophenyl-3,4-dihydroisocoumarins 149(a-d)
Death rate of Mythimna
separata % Death rate of mosquito
larva % Compd.
Concentration24h and
96h Concentration 24h
Status of Mythimna
separata survive
146a 500µg/mL 0 5µg/mL 0 Survive regularly
146b 500µg/mL 0 5µg/mL 0 Survive regularly
146c 500µg/mL 0 5µg/mL 0 Survive regularly
146d 500µg/mL 0 5µg/mL 0 Survive regularly
149a 500µg/mL 0 5µg/mL 0 Survive regularly
149b 500µg/mL 0 5µg/mL 0 Survive regularly
149c 500µg/mL 0 5µg/mL 0 Survive regularly
149d 500µg/mL 0 5µg/mL 0 Survive regularly
control Acetone 0 Acetone 0 Survive regularly
4.5.2 Discussion
All the synthesized compounds were tested for insecticide activity but
none of the synthesized compounds showed any insecticide effects on the test
insects.
4.6 Antifungal Studies Isocoumarins, dihydroisocoumarins and related compounds were tested by
agar tube dilution method215 for their in vitro antifungal bioassay. Results were
reported as linear growth inhibition (LGI) against some human pathogens, e.g
TL = Trichphyton longifusus CG = Candida glabrata
113
CA = Candida albicans MC = Microsporum canis
FS = Fusarium solani AF = Aspergillus flavus
Linear Growth Inhibition results of dichlorophenyl- and difluorophenyl-
isocoumarins and their 3,4-dihydroderivatives are given in table 4.6.
Correlation between Structure and Antifungal Activities Biological study of the natural products with medicinally useful
properties and some of their derivatives indicates possible relationship of the
chemical structure and over-all biological behavior of these compounds. In the
following section, results of biological activities of various types of synthesized
compounds e.g. isocoumarins, keto-acids, hydroxy-acids and
dihydroisocoumarins have been discussed in detail.
4.6.1 Isocoumarin 146(a-g) Linear growth inhibition data of isocoumarins 146(a-g) against different
pathogens in antifungal bioassay is depicted in table 4.6. The isocoumarin
(146d) showed significant activity against Trichphyton longifusus (70%). These
Isocoumarins 146(a-g) are found to be active against Trichphyton longifusus, and
Microsporum canis, show a little activity against Fusarium solani but are
completely inactive against Candida albicans, Aspergillus flavus and Candida
glabrata.
4.6.2 Keto- acids 147(a-g) Table 4.6 illustrates comparison of antifungal activities of keto-acids
147(a-g). Keto-acids 147c and 147f showed excellent acitivites [73% (147c) and
75 % (147f)] as compared to standard Miconazole (70%) against Trichphyton
longifusus. keto acids 147d, 147f and 147g also show significant acitivity [75% (147d), 85% (147f), 70% (147g)] agianst Microsporum canis. In general, activity
of the keto-acids is greater as compared to isocoumarin. Dichloroketo-acid
114
(147f) is more active than difluoro-keto-acid (147c). These keto-acids are also
completely inactive against Candida albicans, Aspergillus flavus and Candida
glabrata.
TABLE 4.6: Antifungal activity of isocoumarins 146(a–g), keto-acids 147(a–g), hydroxy-acids 148(a–g) and 3,4-dihydroisocoumarins 149(a–g) as linear growth
inhibition (%) at 200 µg/ml of madia SDA
Name of Fungi Compound TL CG CA MC FS AF 146a 45 - - - - - 146b 34 - - 55 35 - 146c - - - 50 - - 146d 70 - - 65 - - 146e 65 - - 60 - - 146f 50 - - - 35 - 146g - - - 55 - - 147a 55 - - 65 - - 147b - - - - - - 147c 73 - - 40 - - 147d 65 - - 75 - - 147e - - - 30 - - 147f 75 - - 85 - - 147g 65 - - 70 15 - 148a 65 - - 66 - - 148b 56 - - 75 - - 148c 78 - - 54 - - 148d 43 - - - - - 148e 34 - - 73 - - 148f 26 - - 35 - - 148g 70 - - 80 - - 149a 67 - - 53 - 20 149b 23 - - 20 - 149c 54 - - 46 - - 149d 65 - - 80 - - 149e 35 - - 30 - 25 149f 55 - - 20 20 - 149g 50 - - 40 - 20
Std. Drugs (Miconazole)
Inhibition (% )
70 110.8 110.8 98.4 73.25 Amphotericin
B 20
Key (-) = NO Activity
115
4.6.3 Hydroxy-acids 148(a-g)
Table 4.6, exhibits results of antifungal activities of hydroxy-acids 148(a-g). Hydroxy-acids 148c and 148g showed excellent activities [78% (148c) and 80
% (148g)] as compared to standard Miconazole (70%) against Trichphyton
longifusus. Hydroxy acids 148b, 148e and 148g also show significant acitivity
[75% (148b), 73% (148e), 80% (148g)] agianst Microsporum canis. In general,
dichlorohydroxy-acids are found to be more active than difluorohydroxy-acids.
These hydroxy-acids are also completely inactive against Candida albicans,
Aspergillus flavus and Candida glabrata. As a whole, these hydroxy-acids are
found less active as compared to keto-acid.
4.6.4 3,4-Dihydroisocoumarins 149(a-g)
Table 4.6 shows comparison of activities of dihydroisocoumarins 149(a-g). Dihydroisocoumarins are less active among the tested isocoumarin, keto-
acids and hydroxyl-acids against Trichphyton longifusus and Microsporum canis.
But dihydroisocoumarins 149a, 149e and 149g showed excellent activity [20% (149a), 25% (149e), 40% (149g)] against Aspergillus flavus while standard drug
inhibition (Amphotericin B) is 20%, whereas isocoumarin, keto-acids and
hydroxy-acids are completely inactive against Aspergillus flavus. These
dihydroisocoumarins show similar behavior against Candida albicans and
Candida glabrata as that of isocomarin, keto-acids and hydroxy-acids.
4.6.5 General Comparison of Antifungal Activities
A general overview upon activities show that some tested compounds
(147c, 147f, 148c, 148g, 149a, 149e, 149g) have shown some higher activity
against some pathogens as compared to standard drugs, however when
comparing activity of these compounds with each other, it can be concluded that
activity of keto-acids is relatively higher than isocoumarin, hydroxy-acids and
dihydroisocoumarins against Trichphyton longifusus and Microsporum canis.
116
Thus order of antifungal activity of these compounds is
Keto-acids > hydroxy-acids > isocoumarin> dihydroisocoumarins
4.8 Antibacterial Studies
isocoumarins 146(a–g), keto-acids 147(a–g), hydroxy-acids 148(a–g) and 3,4-dihydroisocoumarins 149(a–g) were tested for their anti-bacterial activity
against various bacteria by adopting agar well diffusion method (carron et al).
Bacteria cultures used were
i. Escherichia coli (EC)
ii. Bacillhas subtilis (BS)
iii. Shigella flexenari (SF)
iv. Staphylococcus aureus (SA)
v. Pseudomonas aeruginosa (PA)
vi. Slamonella typhi (ST)
Imipenem was used as standard drug. 24 Hours old culture containing
approximately 104-106 colony forming unit (CFU) was spread on the surface of
muller Hinton agar (MHA) plates. Wells were created in the medium with the help
of a sterile metallic borer. Test samples of different concentrations were added in
their respective wells. Experimental plates were incubated at 37oC for 24 hours
and zones of inhibition were measured and compared with standard drug.
Results are shown in table 4.7.
4.7.1 Discussion
A general overview upon antibacterial activity show that some tested
compounds (146e, 146f, 147e, 147f, 148c, 148d, 148e, 148f, 148g, 149a, 149b,149e, 149g) have shown significant activity against some pathogens but
somewhat lower activity as compared to standard drugs, however when
117
comparing activity of these compounds with each other, it can be concluded that
all type of compounds i.e. isocoumarin, keto-acides, hydroxy-acids and 3,4-
dihydroisocoumarins show a little activity.
TABLE 4.7: In vitro antibacterial activity of isocoumarins 146(a–g), keto-acids 147(a–g), hydroxy-acids 148(a–g) and 3,4-dihydroisocoumarins 149(a–g) as
linear growth inhibition (%) at 200 µg/ml of madia SDA
Key: (-) = NO Activity
Name of Bacterias Compound EC BS SF SA PA ST 146a 15 12 - 12 - - 146b 14 11 - 15 15 - 146c - - 14 10 - 15 146d - - - - - - 146e 10 21 12 20 - - 146f 24 17 - - 15 - 146g - - 12 10 - - 147a 12 - - - - - 147b - - - - - - 147c 20 - - 10 - - 147d 15 10 17 - 16 12 147e 11 - - 23 - - 147f - - - 9 - 20 147g - - 12 14 - 17 148a 10 - - 16 18 - 148b 07 12 - 15 - - 148c - - 21 14 - - 148d 13 - 12 - 23 - 148e 21 24 - 13 - - 148f 03 - 12 30 - 12 148g 08 - - 20 - - 149a - 22 20 13 11 - 149b 23 - 17 - 20 11 149c 14 12 - 16 - - 149d - - 11 20 - 13 149e 15 - 25 13 - - 149f 15 15 - 20 12 - 149g - 14 13 12 13 11
Std. Drug ( Imipenem) Inhibition
(% ) 30 33 27 33 24 25
118
4.8 Brine shrimp (Artemia salina) lethality studies
The procedure for the brine shrimp bioassay generally followed the
method developed by Solis et al. (1993) with some modifications. Brine shrimp
eggs (Artemia salina) obtained locally were hatched in artificial sea water using a
petri dish (34 g sea salts/litre deionized water) by incubation under a 60 W lamp,
providing direct light and warmth (24–26°C). Mycotoxin solutions were prepared
in acetonitrile–water (1:1) and microlitre volumes (amounts calculated on the
basis of a final well volume of 200 µl) were transferred to 96-well plates and air-
dried over night. After complete evaporation of the solvent the toxins were
redissolved in 100-µl-sea water. The actual concentrations of the well solutions
were determined by HPLC–MS analyses (quantification based on isotope dilution
with deuterated FB1 as internal standard; Hartl et al., 1999) and considered with
the data evaluation. Each plate included negative controls consisting of 100-µl
acetonitrile–water (1:1) and 200-µl sea water. Diacetoxyscirpenol (DAS) served
as a positive control (40 µl of a 10 µg/ml solution per well). For each toxin three
assays were performed, with six different dose levels and eight replicates per
dose (for NCM-FB1 only one assay was done due to limited amounts of the
compound).
After an incubation time of 24 hr, the hatched nauplii were separated from
the shells and remaining cysts using a Pasteur pipette and transferred to fresh
sea water. This was facilitated by attracting the shrimps to one side of the petri
dish with a light source. 100 µl of this solution containing 10–20 organisms were
pipetted into each well, resulting in a final well volume of 200 µl. To prevent
moisture loss the plates were covered with Parafilm (American National Can,
Greenwich, USA) and then incubated for 48 hr under direct light at 24–26°C and
shaken at 140 rpm. After 2 days of incubation the plates were examined under a
binocular microscope (18-fold magnification) and the numbers of dead (i.e. non-
motile) nauplii in each well were counted. The total numbers of shrimps per well
were determined onto addition of 100-µl acetonitrile.
119
A general overview upon Brine shrimp (Artemia salina) lethality activity
show that only compound 146c (isocoumarin) show positive lethality and all
remaining compound including keto-acids and hydroxy-acids have no
cytotoxicity.
Table 4.8: Brine shrimp (Artemia salina) lethality bioassay of Difluorophenyl
isocoumarins 146(a-d) and 3,4-dihydroisocoumarins 149(a-d)
Compound Dose (µg/ml)
No of Shrimps
No. of survivors
LD50 (µg/ml)
LD50 (µg/ml) Remarks
100 30 28 10 30 30 146a 1 30 30
-
7.46
No Cytotoxicity
100 30 28 10 30 30 146b 1 30 30
-
7.46 No Cytotoxicity
100 30 1 10 30 19 146c 1 30 29
13.12 7.46 Postive
Lethality
100 30 28 10 30 30 146d 1 30 30
--
7.46 No Cytotoxicity
100 30 28 10 30 30 149a 1 30 30
-
7.46 No Cytotoxicity
100 30 28 10 30 30 149b 1 30 30
-
7.46 No Cytotoxicity
100 30 28 10 30 30 149c
1 30 30
-
7.46 No Cytotoxicity
100 30 29 10 30 30 149d 1 30 30
-
7.46 No Cytotoxicity
Standard drug: Etoposide Key: (-) - No activity
120
4.9 Antiviral Studies
Antiviral activity against VZV, CMV, HIV-1, HIV-2, vaccinia virus, vesicular
stomatitis virus, Coxsackie virus B4, respiratory syncytial virus, parainfluenza-3
virus, reovirus-1, Sindbis virus and Punta Toro virus was determined essentially
as described previously216-218.
4.9.1 Viruses and cells
The origin of the viruses was as follows: herpes simplex virus type
1 (strain KOS), herpes simplex virus type 2 (strain G) (see reference 8); vaccinia
virus, vesicular stomatitis virus, coxsackievirus type B-4, Sindbis virus, measles
virus, and poliovirus type 1 (see reference 10); reovirus type 1 (ATCC VR-230),
Semliki forest virus (ATCC VR-67), and parainfluenza virus type 3 (ATCC VR-93)
(American Type Culture Collection, Rockville, Md.). The virus stocks were grown
in primary rabbit kidney cells (herpes simplex types 1 and 2, vaccinia virus, and
vesicular stomatitis virus), Vero cells (measles virus, reovirus, coxsackievirus,
and Semliki forest virus), HeLa cells (polio virus), chicken embryo cells (Sindbis
virus), or human embryonic lung cells (parainfluenza virus). The Vero and HeLa
cell lines used in this study were regularly examined for mycoplasma
contamination and found to be mycoplasma free.
4.9.2 Inhibition of virus-induced cytopathogenicity in vitro Confluent cell cultures in microtiter trays were inoculated dilution that
proved infective for 50% of the cell cultures. After 1 h of virus adsorption to the
cells, residual virus was removed and replaced by cell culture medium (Eagle
minimal essential medium) containing 3% fetal calf serum and various
concentrations of the test compounds. Viral cytopathogenicity was recorded as
soon as it reached completion in the untreated virus-infected cell cultures, i.e., at
1 to 2 days for vesicular stomatitis; at 2 days for Semliki forest, coxsackie, and
polio; at 2 to 3 days for vaccinia, herpes simplex types 1 and 2, and Sindbis; and
121
at 6 to 7 days for reo, parainfluenza, and measles viruses. The antiviral activity of
the compounds is expressed as the concentration required inhibiting viral
cytopathogenicity by 50%.
4.9.3 Cytotoxicity Cytotoxicity measurements were based on two parameters:
(i) Alteration of normal cell morphology and
(ii) Inhibition of host cell macromolecule (DNA, RNA,
and protein) synthesis.
To evaluate cell morphology, confluent cell cultures which had not been
infected but were treated with various concentrations of the test compounds were
incubated in parallel with the virus-infected cell cultures and examined
microscopically at the same time as viral cytopathogenicity was recorded for the
virus-infected cell cultures. A disruption of the cell monolayer, e.g., rounding up
or detachment of the cells, was considered as evidence for cytotoxicity. To
measure inhibition of host cell macromolecule synthesis, the cells were seeded in
Linbro microtiter tray wells (at 300,000 to 400,000 cells per well) in Eagle minimal
essential medium containing 10% fetal calf serum, various concentrations of the
test compounds, and 2.5 µCi of [methyl-3H]thymidine, [5-3H]uridine, or [4,5-
3H]leucine per ml and allowed to proliferate for 16 h at 37°C. The cells were then
treated with 5% ice-cold trichloroacetic acid, washed with 95% ethanol (five
times), air dried and counted for radioactivity in 7.5 ml of Lipoluma scintillation
fluid.
4.9.4 Antiviral activity in vivo
In vivo assays were carried out with both vesicular stomatitis and
coxsackievirus type B-4 in new born (2-day-old) NMRI mice and with vesicular
stomatitis virus in young weaned (25-day-old) NMRI mice. The NMRI mice were
obtained from the Animal Production Center (Proefdierencentrum) of the
Katholieke Universiteit Leuven. Newborn (2-day-old) mice were inoculated
subcutaneously with vesicular stomatitis virus at 4 PFU/0. 1ml per mouse (PFU,
122
as determined in mouse L-929 cell cultures) or coxsackievirus type B-4 at 5
CCID50 per 0.1 ml per mouse (CCID50, as determined in Vero cells). The mice
then received either a single intra-peritoneal injection of the compound (in 0.1 ml
physiological saline) at 1 h post infection or repeated intra-peritoneal injections of
the compound at 1 h and 1 and 2 days post infection. Young (25-day-old) mice,
weighing 11 to 13 g, were inoculated intranasally with vesicular stomatitis virus at
40 PFU/0.02 ml per mouse and then received either a single intra-peritoneal
injection of the compound (in 0.5 ml of physiological saline) at 1 h post infection
or repeated intra-peritoneal injections of the compound at 1 h and 1, 2, 3, and 4
days post infection.
Table 4.9a: Cytotoxicity and antiviral activities of Dichlorobenzoic acids 144(e-g), dichlorophenylisocoumarins 146(e-g), keto-acids 147(e-g), hydroxy-acid 148(e-
g) and 3,4-dihydroisocoumarins 149(e-g) in HeLa cell Culture
Minimum inhibitory concentrationb (µg/ml)
Compound
Minimum cytotoxic
concentrationa (µg/ml)
Vesicular stomatitis
virus Coxsackie virus B4
Respiratory syncytial
virus 144e 400 >80 (400) >80 (400) >80 144f >400 >400 400 >400 144g ≥400 >400 >400 >400 146e 400 >80 >80 80 146f 80 >16 >16 >16 146g 80 >16 >16 >16 147e ≥3.2 3.2 >3.2 >3.2 147f >400 400 >400 80 147g 400 >80 >80 >80 148e >400 240 >400 >400 148f >400 240 >400 >400 148g >400 >400 >400 >400 149e 400 >80 >80 >80 149f 80 >16 >16 >16 149g 80 >16 >16 >16
Brivudin >400 >400 >400 >400 (S)-DHPA >400 >400 >400 >400 Ribavirin >400 16 240 9.6
123
Table 4.9b: Cytotoxicity and antiviral activities of Dichlorobenzoic acids 144(e-g), dichlorophenylisocoumarins 146(e-g), keto-acids 147(e-g), hydroxy-acid 148(e-
g) and 3,4-dihydroisocoumarins 149(e-g) in E6SM cell Culture
Minimum inhibitory concentrationb (µg/ml)
Compound Minimum cytotoxic
concentrationa (µg/ml)
Herpes simplex virus-1 (KOS)
Herpes simplex virus-2
(G)
Vaccinia virus
Vesicular stomatitis
virus
Herpes simplex virus-1
TK- KOS ACVr
144e >400 240 240 >400 >400 240 144f >400 400 >400 >400 >400 240 144g >400 400 >400 >400 >400 240 146e ≥80 >80 >80 >80 >80 >80 146f 80 >16 >16 >16 >16 >16 146g ≥1.6 >1.6 >1.6 >1.6 >1.6 >1.6 147e 3.2 >0.64 >0.64 >0.64 >0.64 >0.64 147f ≥400 >400 240 240 >400 >400 147g ≥400 >400 >400 >400 >400 >400 148e 80 >16 >16 >16 >16 >16 148f 80 >16 >16 >16 >16 >16 148g >400 >400 >400 >400 >400 >400 149e 400 >80 >80 >80 >80 >80 149f 400 >80 >80 >80 >80 >80 149g 400 >80 >80 >80 >80 >80
Brivudin >400 0.128 80 3.2 >400 240 Ribavirin >400 240 240 9.6 >400 240 Acyclovir >400 0.0768 0.128 >400 >400 9.6
Ganciclovir >100 0.0192 0.032 >100 >100 0.48
4.9.4 Discussion
The dichlorophenylisocoumarins 146(e-g) and their 3,4-dihydroderivatives
149(e-g) were examined for their antiviral potential in a number of virus assay
systems, each adapted to its optimal cell substrate. In primary rabbit kidney cells
(Table 4.9a), the antiviral assays were performed with herpes simplex virus type
1 and type 2 as well as vaccinia virus and vesicular stomatitis virus as the
challenge viruses. No specific antiviral effects (i.e minimal antivirally effective
124
concentration ≥ 5-fold lower than minimal cytotoxic concentration) were noted for
any of the compounds against any of the viruses evaluated.
Table 4.9c: Cytotoxicity and antiviral activities of Dichlorobenzoic acids 144(e-g), dichlorophenylisocoumarins 146(e-g), keto-acids 147(e-g), hydroxy-acid 148(e-
g) and 3,4-dihydroisocoumarins 149(e-g) in Vero cell culture
Minimum inhibitory concentrationb (µg/ml)
Compound Minimum cytotoxic
concentrationa (µg/ml)
Para-influenza-
3 virus
Reovirus-1
Sindbis virus
Coxsackievirus B4
Punta Torovirus
144e 400 >80 >80 >80 >80 >80 144f 400 >80 >80 >80 >80 >80 144g 400 >80 >80 >80 >80 >80 146e 80 >16 >16 >16 >16 >16 146f 16 >3.2 >3.2 >3.2 >3.2 >3.2 146g 16 >3.2 >3.2 >3.2 >3.2 >3.2 147e 400 >80 >80 (400) >80 >80 >80 147f ≥400 >400 400 >400 >400 >400 147g 400 >80 >80 >80 >80 (240) >80 148e 400 >80 >80 >80 >80 >80 148f 3.2 >0.64 >0.64 >0.64 >0.64 >0.64 148g 400 >80 >80 >80 >80 >80 149e 3.2 >0.64 >0.64 >0.64 >0.64 >0.64 149f 400 >80 >80 >80 >80 >80 149g 400 >80 >80 >80 >80 >80 144e 80 >16 >16 >16 >16 >16
Brivudin >400 >400 >400 >400 >400 >400 (S)-DHPA >400 >400 400 >400 >400 >400 Ribavirin >400 80 48 >400 >400 16
a Required to cause a microscopically detectable alteration of normal cell morphology b Required to reduce virus induced cytopathogenicity by 50%
4.10 Anti-HIV studies219-225
Anti-HIV activity and cytotoxicity measurement in MT-4 cells were based on
viability of cells222 that had been infected or not infected with HIV and then
127
exposed to various concentrations of the test compounds. After the MT-4 cell
were allowed to proliferate for 5 days, the number of viable cell was quantified by
a tetrazolium-based colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) method in 96-well microtrays. While the cytopathic
effects of HIV-1 (IIIB) and HIV-2 (ROD) in MT-4 cells was measured by the MTT
procedure24.
4.10.1 Discussion The dichlorophenylisocoumarins 146(e-g) and their 3,4-dihydro-
isocoumarins 149(e-g) were examined for their anti-HIV activity and only some
trace of anti-HIV activity with the dichlorophenyl-3,4-dihyroisocoumarin 149(e-g) was detected. These compounds could be considered as a lead for further
synthesis. One of the first issues to address is to improve the solubility of the
compounds in aqueous medium.
4.11 Anti-HBV Studies
Hepatitis B virus (HBV)-infected hepatitis is one of the most common
infectious diseases in the world. More than 400 million people worldwide are
chronically infected by the hepatitis B virus226. HBV infection causes liver
diseases such as cirrhosis and may be eventually hepatocellular carcinoma.
Current clinical therapies for HBV infections with interferon-a, lamivudine, and
ribavirin have limited efficacy in a significant proportion of patients and often
result in severe side effects227-229. Thus it is urgently needed to develop more
effective and reliable therapeutics for the treatment of HBV infection.
4.11.1 Testing principle: 2.2.15 cells for hepatitis B virus carrier, the
samples inhibition of hepatitis B virus DNA replication and produce HBsAg,
HBeAg capacity.
128
4.11.2 Testing Materials and Methods Cell lines: 2.2.15 cells; by keeping the room.
Sample: A sample before dissolved in DMSO dubbed appropriate concentration
measured using culture medium for three times diluted, a total of eight dilutions.
Positive control: lamivudine (3TC), Glaxo Wellcome Company.
Major reagents: hepatitis B virus antigen and e antigen s RIA detection kit,
Beijing North Biotechnology Institute; α 32PdCTP, Demopoulos Biological
Engineering Limited.
Test methods : 2.2.15 cell types of 96-well plate, 36 hours after dilution by the
above were added to samples and a positive control, cells were located Kong,
plus 96 hours after the drugs were replaced after dilution containing different
concentrations of the culture fluid samples, Dosing eight days after admission,
respectively Set cell supernatants and 2.2.15 cells, using RIA cell supernatant
detection of HBsAg, HBeAg secretion, dot blot method to detect cell replication of
HBV DNA levels were calculated IC50 and SI.
Table 4.11: Anti-HBV activity of Dichlorophenylisocoumarins 146(e-g), keto-
acids 147(e-g), hydroxy-acid 148(e-g) and 3,4-dihydroisocoumarins 149(e-g)
HBsAg HBeAg DNA
Replication Compounds TC50
µg/ml IC50 µg/ml SI IC50
µg/ml SI IC50 µg/ml SI
146e 8.903 - - - - - - 146f ≥500 - - - - 17.48 3.18 146g ≥500 - - - - - - 147e 38.52 - - - - - - 147f 55.56 - - - - - - 147g 55.56 - - - - 13.57 3.17 148e 240.37 - - - - - - 148f 166.67 - - - - - - 148g 166.67 - - - - 15.23 2.89 149e 96.23 - - - - - - 149f ≥500 - - - - 23.34 5.23 149g ≥500 - - - - - -
lamivudine 1552.12 - - - - 86.59 17.92
129
4.11.3 Discussion
Dichlorophenylisocoumarins 146(e-g), keto-acids 147(e-g), hydroxy-acid 148(e-g) and 3,4-dihydroisocoumarins 149(e-g) were evaluated for their
cytotoxicities and anti-HBV activities, namely the ability to inhibit the replication of
HBV DNA and the production of HBsAg and HBeAg in HBV-infected 2.2.15 cells.
The results are summarized in Table 4.11. As shown in Table 4.11, none of the
evaluated compounds exhibited inhibitory effects on HBV.
4.12 Anti-Cancer Studies against breast cell (MCF-7) 4.12.1 Cell culture
MCF-7/AZ is a variant of the human mammary carcinoma cell family MCF-
7230. The cells are maintained on tissue culture plastic substrate (Nunc) in a
mixture of Dulbecco’s modified eagle’s medium (DMEM) and HAMF12 (50/50)
(Invtrogen, Carlsbad, CA) supplemented with 250 IU/ml penicillin, 100 µg/ml
streptomycin (Invitrogen) and 10% fetal bovine serum (FBS ) (Invitrogen), at
37°C in a humidified atmosphere containing 10% CO2.
4.12.2 Assay for cell viability
Cell viability was tested in accordance with Romijin et al.231. Briefly,
mitochondrial dehydrogenase activities were measured by an MTT-reagent
(Sigma, St. Louis, MO). Cells were seeded in microtiter plates at an initial density
of 1.5 × 104 cells in 200 µl culture medium and treated with increasing
concentrations of each compound. In each experiment, eight well were used to
determine the mean O.D. referring to cell viability.
4.12.3 Discussion
From the results given in table 4.12 for toxicity against the breast cancer
cells (MCF-7). Compounds 146a, 146c, 147a, 147b and 149d showed activity at
100 - 400 micromolar concentrations.
130
Table 4.12: Anti-cancer activity against breast cell of Dichlorophenyl-
isocoumarins 146(e-g), keto-acids 147(e-g), hydroxy-acid 148(e-g) and 3,4-
dihydroisocoumarins 149(e-g)
Concentration (microM) Compd. 0 2.5 25 50 100 400 % Viability 100 108 118 112 60 --- 146a
% std 6 2 7 6 --- % Viability 100 120 120 104 90 89 146b
% std 4 5 7 9 2 % Viability 100 97 96 95 94 51 146c % std 7 6 5 6 13 % Viability 100 75 80 82 70 0 146d % std 7 6 11 7 --- % Viability 100 104 90 87 77 48 147a % std 6 2 8 4 8 % Viability 100 92 82 80 80 68 147b % std 5 8 8 4 4 % Viability 100 98 95 91 90 82 147c % std 7 5 7 6 8 % Viability 100 103 98 92 91 89 147d % std 4 8 9 4 8 % Viability 100 93 93 88 80 79 148a % std 7 8 9 6 2 % Viability 100 91 90 89 86 48 148b % std 4 8 4 3 5 % Viability 100 89 86 86 75 71 148c % std 4 9 7 9 8 % Viability 100 100 91 90 85 83 148d % std 8 3 5 7 8 % Viability 100 96 93 82 83 77 149a % std 5 6 7 5 9 % Viability 100 93 93 90 86 85 149b % std 8 4 7 7 7 % Viability 100 75 80 82 83 80 149c % std 7 6 8 5 9 % Viability 100 92 85 80 79 68 149d % std 5 8 8 4 4
131
4.13 Anti-metastatic Studies 4.13.1 Procedure
This was performed as described previously232. Briefly, six-well plates
were filled with 1.25ml neutralized type I collagen (0.09%)(Upstate
Biotechnology, Lake Placid, NY) and incubated for 1 h at 37°C to allow
gelfication. Non-invasive into collagen type I 233 and served as control for
invasiveness as compared to untreated MCF-7/Az cells. Single cell
suspensions were prepared with trypsin/ EDTA, mixed with the different
compound solutions, seed on top of collagen type I gel and cultured at 37°C
for 24 h. Numbers of cells penetrating into the gel or remaining at the surface
were counted in 12 field of 0.157mm234, using an inverted microscope
controlled by a computer programme. The invasion index expresses the
percentage of invading cells over the total number of cells.
4.13.2 Discussion
From the results given in Table 4.13, for inhibition of invasion of these
cells into collagen, which is an indication of anti-metastatic activity. 3-(3′,4′-
Difluorophenyl)isocoumarin 146d showed excellent activity. It is worth to look
further into possible anti-metastatic properties of this compound.
132
Table 4.13: Anti-metastatic activity of Dichlorophenylisocoumarins 146(a-d), keto-
acids 147(a-d), hydroxy-acid 148(a-d) and 3,4-dihydroisocoumarins 149(a-d)
Compounds
Collagen type I invasion
Treatment Corresponding OD80
Cells MCF-7/AZ 146a 17 3 146b 16 2 146c 16 2 146d 2 1 147a 15 2 147b 17 2 147c 16 1 147d 16 2 148a 18 3 148b 17 2 148c 18 2 148d 18 3 149a 18 2 149b 16 1 149c 16 2 149d 16 2
+Control 19 1
146
Chapter-5 INTRODUCTION
Heterocyclic chemistry has now become a separate field of chemistry with
long history, present society and future prospects. The earliest compounds
known to mankind were of heterocyclic origin. Life, like ours, is totally
dependent on the heterocyclic compounds, it takes birth with purine /
pyrimidine bases, nourishes on carbohydrates and in case of disease, is
cured from medicines, many of which are heterocyclic in nature. Today, the
heterocyclic chemistry delivers reagents and synthetic methods of its own
traditional activity in synthesis of drugs, pesticides and detergents as well as
into the related fields such as biochemistry, polymers and material sciences.
5.1 1,2,4-Triazole
The presence of three nitrogen hetero-atoms in five-membered ring
systems defines an interesting class of compounds, the triazole. This may be
of two types, the 1,2,3-triazoles (1) and the 1,2,4-triazoles (2).
NN
NH
NHN
N(2)
NN
NH
NHN
N(1)
The name triazole was first given to the carbon nitrogen ring system
C2N3H3 by Bladin who described its derivatives in early 1885, although the
structures reported slightly incorrect1-2. An alternative name, pyrrodiazole was
given by Andreocci in 1889 regarding it as a member of a class of compounds
analogous to pyrrole.
A little interest emerged in this field from about 1925 to 1946. The
successors of Andreocci carried out most intensive investigations of the
chemistry of 1,2,4-triazoles. The chemical industry got renewed attention in
the synthesis of both simple and fused triazole systems after the discovery
that certain triazoles capable of inhibiting fog formation in photographic
147
emulsions and some others being useful herbicides and convulsants3. All
triazoles are of synthetic origin and there is no triazole ring system detected
as yet in nature.
5.2 Chemistry of 1,2,4-triazoles
1,2,4-Triazole systems possess some important features.
5.2.1 Aromaticity and stability
The stability of 1,2,4-triazole nucleus is an inherent property of its
aromatic nature. An aromatic sextet is formed by contribution of one π
electron from each atom joined by double bonds and the remaining two
electrons from a nitrogen atom. Such a system is stabilized by resonance and
though the triazole nucleus may be represented by tautomeric forms, each
tautomer is capable of extended resonance and its structure is more correctly
represented as a hybrid to which the following canonical forms contribute3.
NHN
N
NHN
N
NHN
N
NHN
N
NHN
N
NHN
N
It is also necessary to consider the tautomeric form where the imino
hydrogen atom is at the 4-position. The canonical forms that contribute to this
resonance hybrid are given below3.
NN
NH
NN
NH
NN
NH
NN
NH
⋅⋅ --
This representation makes the assumption that the triazole nucleus
actually consists of two hybrid structures, each representing an individual
tautomeric form. In modern theories such a view is incorrect. A more suitable
148
expression is to regard 1,2,4-triazoles as a true aromatic system, stabilized by
resonance and represented below3.
NN
N
HN
N
N
HN
N
N
H
NN
N
H
It is not intended to represent the charges on a nitrogen atom and on
the hydrogen atom as separate, complete charges but merely as a slight,
overall negative charge on the ring, balanced by a corresponding positive
charge on the hydrogen atom.
5.2.2 Amphoteric nature 1,2,4-Triazoles are amphoteric in nature, forming salts with acids as well as bases. 5.2.3 Tautomerism in triazoles
Tautomerism is possible in both the structural isomers of triazoles.
a. Tautomerism in 1,2,3-triazoles
1,2,3-Triazoles have two tautomeric forms, 1H-1,2,3-triazole (3) and
2H-1,2,3-triazole (4).
N3
N2N1
N3
N2N1
(3) (4)
H H
b. Tautomerism in 1,2,4-triazoles
1,2,4-Triazoles exhibit two tautomeric forms namely [4H]-1,2,4-
triazoles (5) and [1H]-1,2,4-triazoles (6).
N
N
NH
N
HN
N
(5) (6) The higher stability for tautomer (6) is indicated by temperature
coalescene studies, x-rays studies, basicity measurements, dipole moment
studies, NMR-spectra and theoretical methods.
149
c. Tautomerism in substituted-1,2,4-triazoles
Among the substituted 1,2,4-triazoles, 3-mercapto-1,2,4-triazoles exist
in two tautomeric forms, because the labile hydrogen may be attached either
to the nitrogen or the sulfur atom. It exhibits thione-thiol tautomeric forms
shown below. This compound exists predominantly in thione (7) form4-5.
N NH
N SRH
N N
N SHRH
1 2 21
44
(7) (8)
Chloro-1,2,4-triazoles exist as 3-chloro-1H-1,2,4-triazole (9a), 3-chloro-
4H-1,2,4-triazole (9b) and 5-chloro-1H-1,2,4-triazole (9c). These tautomers
have the stability order; 9a > 9c > 9b according to physical and theoretical
calculations6.
In case of bromo-1,2,4-triazoles, the possible tautomeric forms are, 3-
bromo-1H-1,2,4-triazole (10a), 3-bromo-4H-1,2,4-triazole (10b) and 5-bromo-
1H-1,2,4-triazole (10c). According to physical and theoretical calculations, the
tautomer (10a) and (10c) are of similar energy and the most stable tautomer
is (10c). These calculations agree with the results of Flammang et al 7.
N NH
NX
N N
NXH
N
NX
HN(a) (b) (c)
9) X= Cl10) X = Br
d. Spectroscopic evidence of tautomerism
Generally, the mixtures of tautomers are formed by the compounds
having free NH group. In the dominant isomer, the position of NH proton in
NMR spectra is generally unknown but sometimes spectroscopic comparison
with alkylated compounds is beneficial. It has been shown that alkylation and
150
acylation of 1,2,4-triazole leads to 1-substituted compounds and in the
absence of other information, the tautomeric mixtures are represented by 1-H
form in both 1,2,3-triazoles and 1,2,4-triazoles8.
5.3 Spectroscopy of 1,2,4-triazole
Ultraviolet, infrared and nuclear magnetic resonance spectroscopic studies are very informative about the structure of 1,2,4-triazoles and their derivatives.
5.3.1 Ultraviolet spectroscopy The unsubstituted 1,2,4-triazole (11) shows a very weak absorption at
205 nm in the ultraviolet absorption spectrum. Bathochromic shift occurs in N-
acetyl-1,2,4-triazole (12), with the absorption band being located at 221.5
nm9. A similar shift in the absorption maximum of 3,5-dimethyl-1,2,4-triazole
(13) appears on conversion into N-acetyl-3,5-dimethyl-1,2,4-triazole (14)10.
N
N
N N
NH
N N
N
N
(12) (13) (14)
N
NH
N
(11)O O
Cyclopentadiene has an absorption maximum at 238.5 nm and by
replacing carbon-carbon unsaturation with carbon-nitrogen unsaturation, a
known hypsochromic shift occurs, therefore, the lower value obtained for
1,2,4-triazoles is understandable11.
A large hyperchromic effect occurs on the acetylation of triazole and its
derivatives which may be compared qualitatively to the similar effect observed
in passing from benzene to acetophenone2.
In case of 5-substituted-3-mercapto-1,2,4-triazoles, the thione-thiol
tautomeric forms can also be differentiated by UV spectroscopy. The
ultraviolet spectra of an ethanolic solution of 5-aryl-3-mercapto-1,2,4-triazoles
usually show two absorption maxima at 252-256 nm and 288-298 nm. The
151
absorption at 288-298 nm is due to the presence of the chromophoric C=S
group4-5.
5.3.2 Infrared spectroscopy
The infrared spectroscopy is also very useful in characterization of
triazole ring. The absorptions in the region of 1570-1550 cm-1 due to N=N and
in the region of 1640-1560 cm-1 due to C=N functions12 are the diagnostic
features. 4-Amino-1,2,4-triazoles show the characteristic strong N–H
stretching of a primary amine at 3400-3200 cm-1.
In 5-substituted-3-mercapto-1,2,4-triazoles, the thione-thiol tautomeric
forms can also be differentiated in the IR spectra by the presence of C=S
absorption band at about 1325-1300 cm-1 for thione and by characteristic SH
absorption band at about 2600-2550 cm-1 for thiol forms13-14.
The N–H stretching vibrations at 3165 cm-1 and 3450 cm-1 have also
been found supportive of thione-thiol equilibrium. 4-Amino-1,2,4-triazoles
have been characterized by the appearance of N–H bands in the regions of
3200-3100 cm-1. For NH2 group, the absorption bands appear at about 3400-
3300 cm-1.
5.3.3 NMR and mass spectrometry 13C NMR is a powerful tool to characterize 1,2,4-triazol-3-ones. In the
spectrum of 1,2,4-triazol-3-ones two values for chemical shifts are obtained,
one at about 164-173 ppm for imine (C=N) and the other at 150-160 ppm for
carbonyl (C=O) carbon15-16.
In EIMS of 1,2,4-triazoles, a strong molecular ion peak is always
observed and the cleavage of bonds between N1–N2 and N4–C5 has been
observed usually. The triazole ring also undergoes N1–N2 and C3–N4
cleavage17.
152
5.4 Applications and biological activities
1,2,4-Triazole and its derivatives are an important class of compounds
which possess diverse agricultural, industrial and biological activities18-19,
including anti-microbial 20-21, sedative22-23, anticonvulsant22-23, anticancer 24-25,
anti-inflammatory26, diuretic27-28, antibacterial 29-31, hypoglycemic 32-33,
antitubercular34-35 and antifungal36-37. In recent years, the synthesis of these
heterocyclic compounds has received considerable attention38-43. This wide
range of applications has been covered by more than sixty papers in the
literature, many in the form of patents.
Some important 1,2,4-triazoles along with their applications are as
follows.
NN
NC2H5
NN
N SHCH2PhN
NN
NH
NH
NH
O
Anticonvulsant44 Dopamine-β-hydroxylase Antihypertensive inhibitor 45 activity46
NN
N N N
HN
NH
NN
N
SH
CH3
N
H
C6H5
Insecticide47 Anti-inflammatory48 Antimicrobial49
N N
NHS
NO2 Antiameobic50
5.4.1 Agricultural applications
In the plant protection technology, the research has been promoted to
discover more efficient pesticides to tackle new challenging problems. In order
153
to selectively control the growth of weeds, a whole range of azole herbicides
has been developed52 exhibiting high levels of activity, application flexibility,
crop tolerance and low levels of toxicity to mammals. Triazoles play an
important role among this classs of heterocycles. A series of 1,2,4-triazole
derivatives have been patented and extensively employed51. One example of
a herbicidal and pesticidal 1,2,4-triazole is given below.
NN
N NH2
O
O
N N
H
N(MeO)(S)PO
(16) (17) Herbicidal52 Pesticidal53-54
5.4.2 Pharmacological applications Over the last few decades, the biological and pharmaceutical properties
of 1,2,4-triazoles have created considerable interest in their synthesis and
chacterization 55-60. 1,2,4-Triazole and its derivatives possess widely differing
activities e.g., bacteriostate 61, bactericide 62, antifungal 63-64, sedative 65, anti-
carcinogen66, tuberculostatic67, anti-inflammatory68,diuretic69, antiviral70,
muscle relaxant71 and antihuman immunodeficiency virus (HIV)72. The
pathogenic fungi cause life threatening infections that have become
increasingly common during the past two decades. Fungal infections are
common in individuals with immuno-compromised hosts, such as patients
undergoing anticancer chemotherapy or organ transplants and patients with
AIDS. Three major fungal infections in immuno-compromised individuals are
candidosis, aspergillosis and cryptococcosis73-74.Whereas the most
widespread human superficial and cutaneous fungal infections are
dermatomycoses such as, toenails and tinea pedis. The common antifungal
agents currently used in clinic are azoles (such as fluconazole, ketoconazole,
and itraconazole), polyenes (such as amphotericin B) nystatin75,
echinocandins (such as caspofungin and micafungin) 76 and allylamines (such
154
as naftifine and terbinafine) 77. In antifungal chemotherapy, azoles having
fungistatic and broad-spectrum activities are used widely against most yeasts
and filamentous fungi. Flouconazole is preferred as first line antifungal
chemotherapy with relatively low toxicity but is not effective against anvasive
aspergillosis and has suffered severe drug resistance78-79. An improvement of
fluconazole is itraconazole, having a broader antifungal spectrum and better
tolerance but its variable oral absorption and low bioavailability has hampered
its use. The second generation of azoles such as voriconazole80,
posaconazole81 and ravuconazole82, have been developed with improved
profiles. They are noted for their broad antifungal spectrum, low toxicity, and
improved pharmacodynamic profiles.
Glycosylated triazole derivatives like 1-β-D-ribofuranosyl-[1H]-1,2,4-
triazole-3-carboxamide (Virazol)83 belong to the highly potent drugs against
DNA- and RNA-viruses84. Moreover, this compound shows antitumor activity 85 just as the anomeric 1-(2,3,4,6-tetra-O-acetyl-D-glucopyranosyl)-5-nitro-
[1H]-1,2,4-triazole86. The therapeutic use of non-steroidal anti-inflammatory
drugs (NSAIDs) which are used in treatment of a number of arthritic diseases
such as rheumatoid arthritis and osteoarthritis is limited because of their side
effects, such as, gastrointestinal haemorrhage and ulceration87. So, new
drugs having potent anti-inflammatory activity with minimum side effects have
been developed.
5.4.3 Industrial applications
a. Chemical Industry Some selected triazoles have been used as light emitting diodes
(Electroluminescent devices) 88-89. Some triazole systems have extensive use
in the separation of silver from other metal cations in liquid membrane
systems90.
In addition, these compounds are used as synthetic dyes and bleaching
agents91. Moreover, the inks having smooth writing properties also contain
triazole derivatives e.g, 3-amino-5-mercapto-1,2,4-triazole92. These
155
compounds have also been reported as inhibitors of corrosion of copper,
brass, aluminium and steel in marine environment93 and inhibit fog formation
in photographic emulsions94, plant growth inhibitors95 and herbicides96.
b. Textile industry The triazole derivatives have many applications in textile industry e.g,
sodium salt of a sulphonated triazole derivative possesses good detergent
action and N-benzylated aminotriazoles (18) have useful properties in
inhibiting the acid fading of dyestuff97.
N N
NR
RNH
(18)
c. Cotton industry In the cotton industry, 3-amino-1,2,4-triazole under its trade name
Amizol, has been used as a commercial defoliant for a number of years98.
5.5 Synthetic approaches towards 1,2,4-trizoles The early methods of preparation of 1,2,4-triazoles were simple and low
yields were obtained but they made the nucleus available for study within a
year of the original discovery by Bladin. These have now been replaced by
later modifications99 and by more efficient methods100-102.
5.5.1 From semicarbazides
A method of practical importance involves synthesis of 1-aryltriazoles
from 1-arylsemicarbazides. This is illustrated by the formation of 3-hydroxy-1-
phenyl-1,2,4-1H-triazole (19) from 1-phenylsemicarbazide and boiling
anhydrous formic acid. By heating (19) to over 200˚C with phosphorus
pentasulfide103, 1-phenyl-1,2,4-1H-triazole (20) is obtained in 80% yield.
156
(19) (20)
HNHN
O
NH2
NH2
HNO
O NNH
N
O
NN
NHO O
HP2S5
200°C
5.5.2 From triazine
The reaction of s-triazine with a substituted hydrazine salt104 gives
substituted 1,2,4-triazoles. For example, from phenyl hydrazine
hydrochloride 1-phenyl-1,2,4-1H-triazole is obtained in 83% yield. The
reaction proceeds by the formation of a substituted formamidrazone as a
result of ring cleavage of s-triazine, which reacts immediately with another
molecule of triazine to yield the substituted triazole.
N
N N 3NH2NHR.HCl HCNNHR
NH2.HCl
3
NN N
N
N
NR
NH4Cl3
R=Phenyl
5.5.3 From thiosemicarbazides
Usually, 1,2,4-triazoles are formed by the cyclization of a preformed
nucleus of the following types:
NCN
CN
CNN
NType A Type B
C
The former type of ring closure is the most efficient method of
synthesis of C-mono-substituted triazoles. In this way, a triazole containing a
mercapto or hydroxyl group is obtained which then may be removed by
oxidation. Thus, the cyclization of 1-acetylthiosemicarbazide with sodium
methoxide in methanol results in the formation of 5-mercapto-3-methyl-1,2,4-
triazole (21) which readily losses the mercapto group on oxidation with nitric
acid105 to form 3-methyl-1,2,4-triazole (22) 106-107.
157
NHH2N
NH
NN
NH
NN
NH
(21) (22)
ORO
S
HS
It is interesting that 4-benzoyl-1-carbamoyl-3-thiosemicarbazide (23) under various reaction conditions forms different products: with 20% sodium
hydroxide it yields 5-mercapto-3-phenyl-1,2,4-triazole (24) that is normal
cyclization product of 4-acylthiosemicarbazides with alkali; with concenterated
sulfuric acid at room temperature it forms 3-benzamido-5-hydroxy-1,2,4-
triazole (25) by the elimination of hydrogen sulfide; and a hot acetic
anhydride-acetic acid mixture results in the formation of 3-benzamido-5-
methyl-1,2,4-triazole (26)108.
NN
NH
NaOH H2SO4
(CH3CO)2O
CH3COOH
NN
NH
HO(23)(24)
(25)
HS H2N NH
HN
HN
O
S ONH
O
NN
NH
(26)
NHO
Dimova et al.109 synthesized a series of 4-substituted 5-aryl-1,2,4-
triazoles by cyclization of the corresponding substituted thiosemicarbazides.
Nair et al.110 reported an extensive review on the synthesis of 1,2,4-
triazoles and thiazoles from thiosemicarbazide and its derivatives.
5.5.4 From benzalsemicarbazones with ferric chloride
Oxidation of benzalsemicarbazone with ferric chloride solution readily
gives 3-aroyl-5-hydroxy-1,2,4-triazole (27) which is also obtained by the
158
oxidation of a mixture of benzaldehyde and azodicarbamide (28). The
hydroxyl group is removed on fusion of hydroxyl triazole with phosphorus
pentasulphide forming 3 (or 5)-substituted triazole (29)111.
RHC NH2
HN
N NCRN
NH
FeCl3
125-130°C1 hr
NCRN
NH
P2S5
N N
H2N NH2
130°C1hr
O
HO
FeCl3
OO
O
(27) (28)
(29)
5.5.5 From carboxylic acid hydrazides
The condensation of carboxylic acid hydrazides with carbon disulphide
in ethanolic potassium hydroxide yields potassium 3-aroyldithiocarbazates
(30) that is directly converted to 4-amino-4H-1,2,4-triazole-3-thione (31) with
an excess of hydrazine112. The methylation of (30) with methyl iodide provided
the S-alkylated derivatives (32) that also cyclize to (31) with hydrazine.
RHN
NH2
O
CS2, KOH
C2H5OHR N
H
HN SK
S
O
R NH
HN S
S
O
CH3I
NHN
N SNH2
R
NH2NH2
NH2NH2
(30)
(31) (32)R= C6H11, C6H5, 4-FC6H4
5.5.6 From 1,3,4-oxadiazol-5-thione
1,3,4-Oxadiazol-5-thiones are converted into 4-amino-1,2,4-triazol-5-
thiones with hydrazine hydrate 113-116. Thus, Reid and Heindel114 indicated that
the 5-aryl-2-1,3,4-oxadiazol-5-thione (33) recycled to form 4-amino-1,2,4-
triazole-3-thione (34) with hydrazine hydrate.
159
NHN
OAr S
NH2NH2NHN
NAr SNH2
(33) (34)
5.5.7 From thiosemicarbazides and carbonyl compounds
The cyclocondensation of 2,4-disubstituted thiosemicarbazides with
carbonyl functions forms triazoles. Reaction of 4-(2-methylallyl)-2-
phenylthiosemicarbazide (35) with ketones in the presence of catalytic
amount of sulfuric acid afforded 3,3-dimethyl-4-(2-methylallyl)-1-phenyl-1,2,4-
triazolidin-5-thione (36) 117.
NRNH2
NHR1
SNHN
N R3SR1
R2COR3
(36)
H2SO4
RR2
(35)
5.5.8 From thiocarbohydrazides and carbohydrazides
The condensation of thiocarbohydrazides (37) with aliphatic and
aromatic carboxylic acids is the choiest method for the preparation of 3-
alkyl/aryl-4-amino-∆2-5-mercapto-1,2,4-triazoline118-121.
The reaction is improved by using carboxylic acids at their melting
points, resulting in the preparation of 3-alkyl / aryl-4-amino-5-mercapto-1,2,4-
triazole (38) 122.
NHNH2
NHNH2
SNN
N RHSNH2
RCOOH
Melting temperature
(38)(37)
The carbohydrazide (39) on treatment with acid forms 4-amino-3-
methyl-∆2-1,2,4-triazolin-5-one (40).
160
NHNH2
NHNH2
ONHN
NONH2
MeCOOH
(40)(39)
5.5.9 From thiosemicarbazides with benzoyl chloride
Thiosemicarbazide with benzoyl chloride in boiling pyridine or alkali
undergo benzoylation and cyclization resulting in the formation of 4-benzoyl-3-
phenyl-∆2-5-mercapto-1,2,4-triazoline (41)123.
NHNH2
NH2
SNN
N PhHSPhCOCl
(41)
pH>7
O Ph
5.5.10 From phenylthiosemicarbazide with ethylphenyl-
imidate hydrochloride
The reactions of 4-phenylthiosemicarbazide with ethylphenylimidate
hydrochloride illustrated the formation of 3,4-diphenyl-∆2-1,2,4-triazoline-5-
thione (42)124.
NHNH2
NHPhS
NHN
N PhSPh
EtOC(Ph)=NH.HClpH>7
(42)
5.5.11 From condensation of a nitrile and a hydrazide 3,5-Disubstituted 1,2,4-triazoles (43) are synthesized from the
condensation of a nitrile and a hydrazide in a convenient and efficient one
step base catalysed synthesis described by Kap-Sun Yeung and co-workers
from Bristol-Myers Squibb 125. Under the reaction conditions, a diverse range
of functionality and heterocycles are tolerated.
161
R1 CN H2NNH R2
O K2CO3, C4H9OH
150°C, 1-14hN
N
NHR1
R2R1,R2 = aryl, hetroaryl 12 compounds
(34-83%)(43)
5.5.12 From isothiocyanates Isothiocyanates on condensation with acylhydrazide affords hydrazine
carbothioamides which are cyclized to 3-mercapto-1,2,4-triazoles (44) under
basic conditions126.
(44)
R1N
CS
+R2 N
H
NH2
O
R2 NH
HN
HN
R1
O
SN N
NR2
R1
SHC2H5OH NaOH
The hydrazide derivatives on refluxing with phenylisothiocyanate in
absolute ethanol yield the corresponding phenylthiosemicarbazides (45), which may be cyclized127 in the presence of NaOH.
N N
R1R
NaOHN N
R R1
(45)
HN NH2
O N N
R1R
HN N
HO
NH
SNCS
N NH
N S
5.5.13 From aromatic nitriles Aromatic nitriles (46) on reaction with hydrazine dihydrochloride in the
presence of hydrazine hydrate under microwave irradiation give 3, 5-
disubstituted-4-amino-(4H)-1,2,4-triazoles (47) 128-129.
(46) (47)
N N
NAr
ArNH2
Ar-CN + N2H4.2HClNH2-NH2.H2O
Ethylene glycol
162
5.5.14 Solid phase synthesis of triazoles
There are, so far, only a few published studies about the solid-phase
synthesis of substituted 1,2,4-triazoles. Katritzky reported the synthesis of tri-
substituted 1,2,4-triazoles on a solid support based on the condensation
reaction between an acyl hydrazide and substituted amidines130. The yields
were 37-90% and the purities depended on the substituents of the triazole
core.
This procedure enables the alkylation of the 1-position, giving the
trisubstituted 1,2,4-triazoles, but suffers from the nontraceless nature of the
reaction sequence. Hence, the synthesized 1,2,4-triazoles contain the 4-
hydroxyphenyl linker of the starting Wang resin. 3,4,5-Trisubstituted 1,2,4-
triazoles were also prepared on solid supports131.
A traceless synthesis of 3,5-disubstituted 1,2,4-triazoles has been
developed on polymeric supports132, using immobilized mesoionic 1,3-
oxazolium 5-oxides (münchnones) as key intermediates in the 1,3-dipolar
cycloaddition reaction, as shown below.
O
O
O OHN
O
OH
O
R
O
N
O
OH
O
R
R1 O
O O
N NN
R1
R
HN NN
R1
R
163
5.5.15 Synthesis of 1,2,4-triazoles under microwave irradiation
Microwave irradiation has become a widely used method to synthesize
many useful organic chemicals rapidly, with good yields and high
selectivity133-147. A great many relevant works suggest only a thermal nature of
the microwave action, which means that microwaves are considered to be a
method to heat chemical reagents rapidly and without any overheating. Some
other works describe specific non-thermal effects, and these effects are likely
to exist. Sometimes the effects are thought to be only specific forms of heat
effects, but not always.
Kappe et al.148-149 have used this method extensively for the synthesis
of their organic molecules; meanwhile Molteni and Ellis150 reviewed the work
carried out since 1994 in the field of microwave-assisted synthesis of
heterocyclic compounds and reactions in which a heteroatom is directly
participating in the bond forming process that gives rise to a heterocyclic core.
A novel one-step synthesis of thiazolo-[3,2-b]-1,2,4-triazoles (50) was
reported from the reaction of chalcones (48) with bis(1,2,4-triazolyl)sulfoxide
(49)151 . Symmetrical 3,5-substituted 4-amino-1,2,4-triazoles (53) are quickly
prepared from aromatic aldehydes (51) via nitriles (52) by two-step reactions
without any separation under microwave irradiation for each several
minutes152.
R1 R2
O+
N
NN
S NO N
N
Toluene, 90°C-TrH-H2O
NN
N
S
R2
R1
O
(48) (49) (50)
Ar
O
NO
MWNH2OH.HCl
MWNH2NH2.2HClNH2NH2.H2O
HO(CH2)2OH N N
NHHNArAr
N N
NAr
ArNH2
Ar
N
(51) (52) (53)
164
Condensation of acid hydrazide (54) with S-methylisothioamide
hydroiodide (55) and ammonium acetate on the surface of silica gel under
microwave irradiation afforded 1,2,4-triazoles (56)153.
R1
O
NH
NH2+
R2
S
NH2+ I-
NH2OAc,SiO2
(C2H5)3N, MW N N
HN
R2
R1
(54) (55) (56)
An efficient microwave-assisted one-pot and three-component
synthesis of substituted 1,2,4-triazoles (57) has been achieved utilizing
substituted primary amines154 .
O
NH
NH2 + NO
O+ RNH2
MW
N N
NR
(57)
Kidwai et al.155 have synthesized new antifungal azoles including 1,2,4-
triazole derivatives from substituted hydrazide (58) using various solid
supports under microwave irradiation.
NH
NHRO
NH O
NH4SCN
Basic/ NeutralAlumina, MW N
H O
NH
NNR
S
(58)
A simple and fast synthesis of 6-aryl-3-substituted 5H-[1,2,4]-triazolo-
[4,3-b][1,2,4]-triazoles (60) in high yields has been developed by microwave
assisted heterocyclization of N-(3-methylthio-5-substituted 4H-1,2,4-triazol-4-
yl)benzene- carboximidates (59)156.
165
HN
NH
NNN
R
S
NN
N
N
NH
R
(59) (60) 5.6 1,3,4-Thiadiazoles
1,3,4-Thiadiazoles (61) are five membered heterocyclic compounds
having two nitrogen and one sulfur atom in a symmetrical structure. The first
representative of this group was discovered by Emil Fischer in 1882. Since
then 1,3,4-thiadiazoles have received a great deal of attention, mainly
because of their diversity of pharmacological properties157.
NN
SR R'
(61) Tautomerism
The substituted 1,3,4-thiadiazoles also exibit tautomerism e.g,
mercapto-substituted thiadiazoles show thiol and thione tautomeric forms. The
tautomerism influences the reactivity of the thiadiazoles, which has been
demonstrated for polymerization processes and substitution reactions at
different moieties158-159.
For 2-mercapto-5-methyl-1,3,4-thiadiazole (McMT), spectral data
indicate that the thione tautomer exists predominantly in DMSO solution as
well as in the solid state.
NHN
S S
NN
S SH
2,5-Dimercapto-1,3,4-thiadiazole, is present as thione-thiol tautomer
(62) in solid state while in solution a solvent dependent equilibrium is believed
to exists between the thione-thiol and thione-thione (63) forms, the former
166
being the prevailing species in polar solvents shown by vibrational
spectroscopy and X-ray structural analysis160.
NHHN
S SS
NHN
S SHS
NN
S SHHS
(63) (62)
Some important applications of 1,3,4-thiadiazoles are :
NN
S SHHS
NN
S NH2
NN
S SN N
Corrosion inhibitor161 Antitumor and Ulcer Inhibitor163
Neoplasma Inhibitor162
NN
SN Cl
NN
S NHRNN
O
NN
S NH
Insecticidal164 Antibacterial165 H2-antagonist166
5.7 Applications
1,3,4-Thiadiazole derivatives are important in industry, medicine and
agriculture.
a. Biological applications
Thiadiazole ring displays a broad spectrum of biocidal activities
possibly by virtue of NCS toxophoric moiety. Its derivative 2-amino-1,3,4-
thiadiazole is a cyclic analogue of thiosemicarbazone, which often displays
diverse physiological activites. 2,5-Disubstituted-1,3,4-thiadiazole derivatives
have been found to possess biocidal activities including antifungal167-168
167
antibacterial167-169, anti-inflammatory170, antituberculosis171, anticonvulsant172,
radioprotective173, and anticancer 174.
1,3,4-Thiadiazole derivatives possess interesting biological activity
probably conferred to them by the strong aromaticity of this ring system175,
which leads to great in vivo stability and generally, a lack of toxicity for higher
vertebrates, including humans. When diverse functional groups that interact
with biological receptors are attached to this ring, compounds possessing
oustanding properties are obtained. Except for some antibacterial
sulfonamides (albucid and globucid), no longer used clinically, but which
possessed historical importance 175. The most interesting examples are
constituted by 5-amino-l,3,4-thiadiazole derivatives such as the thiol (64a), a
compound used as radioprotective agent 176, as well as an investigational
antitumor 177 and gastroprotective 178 drug; acetazolamide (64b), which was
the first non-mercurial diuretic drug179-182 used clinically thereafter as
antiglaucoma 183, antiepileptic184 and antiulcer drug 185 together with a large
series of its congeners derived from 5-amino-1,3,4-thiadiazol-2-sulfonamide
(64c) 182.
NN
S ZRHN
64a. R = H, Z= SH64b. R = Ac, Z= SO2NH264c. R = H, Z= SO2NH2
These compounds have also been investigated as complexing
agents180-183 in order to obtain substances with a diversified biological activity,
conferred to them among others by the presence of the metal ions, except for
that of the thiadiazole ligand. Thus, some metal complexes of ligands of type
64a-c have been recently reported as in vitro inhibitors of the zinc enzyme
carbonic anhydrase181-183 whereas in vivo studies showed good antiepileptic
action for some Cu(II) and Zn(II) complexes of the sulfonamide type
ligands182. Finally, some 2,5-disubstituted-l,3,4-thiadiazoles as well as their
Cu(II) complexes were reported to act as fungitoxic agents 183
168
b) Industrial applications
1,3,4-Thiadiazole, 2,5-bis(tert-nonyldithio) is used to formulate finished
greases and lubricating oils including industrial, gear, automatic transmission
and some types of automotive crankcase, heavy duty diesel and medium
speed diesel oils. In these applications, it is used as an ashless copper
corrosion inhibitor and extreme pressure (EP) agent 183.
1,3,4-Thiadiazole, 2,5-bis(tert-nonyldithio) is also used as a sulfur
deactivator, corrosion inhibitor and antioxidant in gasoline, heating oil and
Liquefied Petroleum Gas 184.
c. Chemical industry
i) Synthesizing other chemicals
1,3,4-Thiadiazoles with amines or alkalis readily produce salts of strong
bases. For example, 2,5-dimercapto-1,3,4-thiadiazole (DMcT), also known as
bismuthiol is used to synthesize salts and polymers. On treatment with
ammonia or pyridine it generates monoammonium and monopyridine salts,
while with hydrazine or hydrazine hydrate gives both mono- and dihydrazine
salts185. Its heavy metal salts may be prepared in a polar solvent such as
ethanol. The products are polymeric complexes186. Polymers may be
produced by treating DMcT with a sulfur chloride such as S2Cl2 in strongly
alkaline solution at temperatures up to 100 °C187. Oxidative polymerization of
2,5-Dimercapto-1,3,4-thiadiazole gives poly(1,3,4-thiadiazole-2,5-diyldithio)
(65).
NN
S SS
n(65)
ii) Flame and scorch retardants
Fire retardant compositions for wildland fire suppression are based on
salts of thiosulfuric acid and contain substituted 1,3,4-thiadizoles presumably
as a stabilizer or the corrosion inhibitor188. Chemonic industries, Inc., USA,
169
patented colored liquid fire retardant compositions for aerial application to
vegetation. The compositions were apparently based on ammonium
polyphosphate and contained DMcT189. Substituted 1,3,4-thiadizoles or its
sodium or other metal salts were used as a viscosity stabilizer for the
galactomannan gum thickener for a fire retardant composition based on
ammonium phosphate and/or ammonium sulfate190.
iii) Adhesion improver
Substituted 1,3,4-thidizoles salts were used in compositions to
improve heat-resistant adhesion between steel cord and rubber in tires191.
Use of 1,3,4-thidiazole derivtives as the cross linking agent improved
compositions for bonding and sealing chlorinated polyethylene roofing
membranes used for flat roofs 192.
iv) Analytical reagent
Substituted 1,3,4-thidizole is a chelating agent that is used to
determine metals in industrial, environmental, and biological samples (e.g.),
lead193, copper194, aluminum, arsenic, nickel, and selenium 195, and cadmium
and zinc196. Maxwell and Smyth reported satisfactory use in the determination
of cadmium, lead, and zinc in river waters by anodic stripping voltammetry197.
v) Purification and waste treatment
1,3,4-Thidizoles salts and bentonite or zeolite sorbents are used for
heavy metal ion removal from industrial and municipal wastewaters198. A
potential wastewater treatment process is reported for cadmium removal by
complexation and polymerization with derivatives of 1,3,4-thiadizoles199. The
complexing property of DMcT is used to remove trace metal contamination
(cadmium, cobalt, iron (III), lead, nickel, and zinc) from commercial ethanol for
use as engine fuel 200. It has also been employed as a trapping agent for
sulfonic acids from penicillin 201.
170
vi) Biocides
The biocidal compositions of certain thiol compounds such as 1,3,4-
thiadiazole derivtives in which antimony, arsenic, or bismuth is complexed
with it are prepared229. The biocidal compositions were said to be useful as a
disinfectant, a preservative, bactericidal, bacteriostatic, antibiofilm, antifungal,
and antiviral agents 202.
The thiadiazole fungicides are active against leaf blight of rice and
canker of oranges203.
vii) Photography
DMcT is a general photographic chemical204. DMcT and its disodium
salt were used to form silver salts that stabilized photographic layers205. It (up
to 1%) was used in the composition of a silver halide nuclear emulsion234. The
salts of substituted 1,3,4-thiadizoles are also used in silver-based
photographic materials for metal reliefs, printed circuits, and printing plates 206. Moreover, 1,3,4-thiadizole derivatives are mentioned as a component of
the novel ascorbic acid developer liquid for silver halide film processing in a
more recent Fuji Photo Film Co. patent 207.
5.8 Synthetic approaches towards 1,3,4-thiadiazoles
Several methods for the synthesis of 1,3,4-thiadiazoles are reported in
the literature. Some of them are given below.
5.8.1 From thiosemicarbazide
i) A standard method for the preparation of 1,3,4-thiadiazoles (66) is
dehydrative cyclization of acylthiosemicarbazide
R1 NH
HN R2
O
S
H2SO4 NN
SR1 R2
(66)
171
Different acidic reagents have been used for dehydration e.g. sulfuric
acid 208, phosphoric acid, acetic anhydride 209-210 and phosphorus halides211.
ii) The condensation of thiosemicarbazide with benzoic acid (67) in
phosphorus oxychloride gives 1,3,4-thiadiazole in 94% yield212.
OH
OH2N N
HNH2
S
NN
S NH2ArPOCl31h, 70°C(67)
iii) 5-Amino-[1,3,4]-thiadiazole derivatives can be prepared from the
reaction of p-anisaldehyde (68) with thiosemicarbazide to give an
intermediate, followed by cyclization in the presence of ferric chloride in
aqueous solution 213
H2N NH
NH2
S
NN
S NH2H3CO
C2H5OH
FeCl3H2O
H3CO
CHON
HN NH2
H3COS
(68)
+
iv) The dehydration of thiosemicarbazides with acetyl chloride followed
by hydrolysis of the acetamide gives amino-1,3,4-thiadiazoles.
NH
HN NH2
O
S
NN
S NH2
C10H21O C10H21O
CH3COClHCl
5.8.2 From diacylhydrazide
2,5-Disubstituted-1,3,4-thiadiazole has been prepared by the reaction
of diacylhydrazide (69) with phosphorus pentasulphide 214.
172
R1HN N
HR2
O
O
P2S5NN
SR1 R2
+
(69) 5.8.3 From dithiocarbazinic acid derivatives 215
a. Dithiocarbazinic acid derivatives (70) on reaction with carbon
disulphide yield 2, 5- dimercapto-1,3,4-thiadiazole (71).
RHN
NH
SH
S
CS2NN
S SHS
R
+
(70) (71)
b. 2-Mercapto-1,3,4-thiadiazoles can also be obtained when
dithiocarbazinic acids react with aliphatic aldehydes.
RHN
NH
SH
S NN
S CH3HS
RCH3CHO+
5.8.4 From fluorous Lawesson’s reagent
1,3,4-Thiadiazoles are synthesized from N’-acylbenzohydrazide (72) by using Fluorous Lawesson’s reagent (73) in THF at 55°C within 6hrs 216.
NH
HN CH3
O
O
NN
S CH3LR
(72)
P
P
S
S
O(CH2)4(CF2)6F
F(F2C)6(H2C)4O
Fluorous Lawesson's reagent (LR)(73)
173
5.9 Isatin derivatives-Indolinones 5.9.1 Isatin
Isatin (1H-indole-2,3-dione) was first obtained by Erdman and Laurent
in 1841 as a product from the oxidation of indigo by nitric and chromic acids.
NH
O
O
(74)
The synthetic versatility of isatin has led to the extensive use of this
compound in organic synthesis. Three reviews have been published regarding
the chemistry of this compound: the first by Sumpter, in 1954217, a second by
Popp in 1975218, and the third on the utility of isatin as a precursor for the
synthesis of other heterocyclic compounds219. The synthetic versatility of
isatin has stemmed from the interest in the biological and pharmacological
properties of its derivatives. These properties are more fully detailed in the
supplementary material.
In nature, isatin is found in plants of the genus Isatis220, in Calanthe
discolor LINDL 221 and in Couroupita guianensis Aubl 222, and has also been
found as a component of the secretion from the parotid gland of Bufo frogs223,
and in humans as it is a metabolic derivative of adrenaline224-226. Substituted
isatins are also found in plants, for example the melosatin alkaloids
(methoxyphenylpentyl isatins) obtained from the Caribbean tumorigenic plant
Melochia tomentosa227-229 as well as from fungi: 6-(3’-methylbuten-2’-yl)isatin
was isolated from Streptomyces albus230 and 5-(3’-methylbuten-2’-yl)isatin
from Chaetomium globosum231. Isatin has also been found to be a component
of coal tar232.
Isatin has been known for about 150 years and has been recently
found, like oxindole and endogenous poly-functional heterocyclic compounds,
to exhibit biological activity in mammals233. Isatin also is a synthetically
174
versatile substrate that can be used to prepare a large variety of heterocyclic
compounds, such as indoles and quinolines, and as a raw material for drug
synthesis 234. Isatin is further known to be a color reagent for the amino acid
proline, forming a blue derivative235. This property has been exploited for the
determination of this amino acid in pollens236 and other vegetable materials237
using paper chromatography or for the detection of polymer-bound
compounds possessing proline residues 238. Some isatin derivatives exhibit
antiplasmodial activity239. Schiff bases and Mannich bases of isatin are known
to possess a wide range of pharmacological properties including antibacterial,
240-242 anticonvulsants, 243-244 anti-HIV245-248, antifungal 249-252 and antiviral
activity253. Bis-Schiff bases are characterized by their capacity to completely
co-ordinate a metal ion, forming chelate rings 254. The Schiff bases of isatin
have also been used as ligands for complexation of metals such as copper
II255. These complexes catalyzed the oxidation of carbohydrates. Bis-Schiff
bases can act as inhibitors of human α-thrombin256. Recently it has been
reported that a bis-imine of isatin has antimicrobial properties 257 and affects
cell viability258.
5.9.2 Indolinones
Oxindoles (2-indolinones) are a class of heterocyclic compounds found
in many natural products259-260 and in a number of marketed drugs261-264. Of
particular interest are 3-substituted 3-hydroxyoxindoles. This substructure is
encountered in a large variety of natural alkaloids with a wide spectrum of
biological activities. Especially 3-alkyl-substituted 3-hydroxyoxindoles occur
frequently in nature, e.g., convolutamydines(75)265, 267, donaxaridine(76) 266-
267, maremycins(77) 268 , dioxibrassinine (78) 269, celogentin K (79) 270 and 3’-
hydroxyglucoisatisins (80) 271.
175
NH
OBr
BrOH
R
75. Convolutamydine A (R = COCH3)75. Convolutamydine E (R = CH2OH)
N
NH
HN
R
OO
S
HH
77. Maremycin A (R = OH)77. Maremycin B (R = OH)
NH
HOHN
S
S
O
78. Dioxibrassinine
NH
HO
NH
O
76. Danxaridine
NH
HO
O
O
O
NOSO3SO
OH
HO HO
OH
-
80. 3-hydroxyglucoisatisin
NH
O
79. Celogentin K
HO
NH
NHOHN
NH
O
O
OHNO
HNNH2
NHO
HNNH
OCO2H
NHN
5.9.3 Synthetic approaches towards indolinones
The products of partial reduction of isatin, dioxindole and oxindole,
have been widely used in organic synthesis, especially in the development of
new drugs. Some natural products also belong to these classes of
compounds, for instance dioxibrassinin272.
Oxindoles can be prepared by the reduction of either dioxindoles or
isatins. The reductions have been performed by using red phosphorous and
iodic acid273, by use of H2S in a pyridine/co-solvent mixture274. It can also be
prepared by reduction of the isatin-3-ethylene thioketal with Raney nickel275 or
by the Wolf-Kishner reaction276-279. Lower molecular weight alcohols such as
EtOH or iPrOH are used as solvents which lead to high yields of the desired
product280. It has however been found that isatin can be reduced to the
corresponding oxindoles in high yields (76-92%) by the use of hydrazine
hydrate as the solvent in the absence of any additional base281-282. A
chromatographic method for the quality control of oxindoles, frequently used
as raw materials for pharmaceutical products, using normal phase HPLC has
been developed283.
176
Indigo, isoindigo and indirubin are natural pigments bearing the
oxindole motif and have considerable economical importance. As a
consequence, synthetic methodologies have been developed for the obtention
of these pigments and analogues. Indigo and monothioindigo can be obtained
from the reaction of isatin with P4S10 284. Isoindigos have been prepared by
an acid catalyzed reaction of isatin and oxindole derivatives285-286 and from the
reaction of N-methylisatoic anhydride or N-methylisatin with sodium
phosphonates287-288.
Isoindigos and thioisoindigos have also been prepared from the
reaction of isatin with Lawesson’s reagent289. Indirubins, which are described
as effective antileukemic agents, can be prepared from isatin and indican, a
compound extracted in high yield from Baphicacanthus cusia290, or from isatin
and N-methyl-O-acetylindoxyl 291 and from isatin and 3-hydroxyindole292
Pyrrolo-indigo compounds can be prepared by the condensation of
isatin with pyrrolin-4-ones293; and thionapthene indigo dyes (Thioindigo
Scarlet) are obtained from hydroxythionapthenes and isatin in acidic media294.
In a reverse sense, isatin has been identified as one of the products of the
oxidation of indigo by nitric acid and light. This process may be involved in the
fading of indigo in museum collection objects295 and denim jeans296-297. The
same conversion can be realized by ozonolysis298, acidic bromate299 or by a
chemiluminescent autoxidation of indigo300. N-Methylisatin is also obtained in
the photooxidation of N-methylindole-3-acetic acid301.
Isoindigo, obtained from isatin (74) and oxindole (81), is converted
diastereoselectively into diazacrisenodiones (82) by reduction with Zn/AcOH,
and subsequent acid-catalyzed rearrangement285 as given below.
177
NH
O
O
(74)
NH
O+CH3COOH, HCl
Heat(100%)
NH
O
HN
O
Zn, HCl
NH
O
HN
OHCl (4mol/ L)
(85 %)
HN
NH
O
OH
H
(81)
(82) An efficient and versatile method for stereoselective synthesis of (E)-
3,3-(diarylmethylene)indolinones (83) by a palladium-catalyzed tandem Heck-
carbocyclization/Suzuki-coupling sequence is presented here as a synthetic
approach towards indolinones302.
NH
O
IR
Pd(PPh3)4, CuTCArB(OH)2, rt
RNH
O
Ar
(83)
A new synthesis of 3-[(4-amido)pyrrol-2-yl]-2-indolinones has been
developed, where the amide side chain was installed prior to pyrrole
formation. This strategy precludes the need to use any coupling reagents to
install the amide side chain. This process includes a zinc-free alternative to
the Knorr pyrrole synthesis303.
5.10 Plan of work
Observed through the literature review, 1,2,4-triazoles and 1,3,4-
thiadiazoles are important from medicinal, industrial and agricultural point of
view. The presence of a mercapto group at 3-position of 1,2,4-triazoles has
been found to show an increase in the antimicrobial activities. Moreover, we
178
know that a chiral drug interacts with a chiral receptor site; the two
enantiomers of the drug interact differently and may lead to different effects.
In view of all these facts, it was planed to utilize carboxylic acid hydrazides in
the synthesis of substituted 1,2,4-triazoles-3-thione and substituted 1,3,4-
thiadiazoles.
In order to achieve the objectives of the present work, following
synthetic plan was devised (Scheme 5.1).
R OH
O CH3OH
R O
O H2NNH2
H2OR N
H
ONH2
NC S
R'
R
O
NH
HN
S
HN
R'
NaOH
Conc H2SO4
NN
N
NN
S
R SH
NHR
R'
R'
Acid Ester HydrazideIsothiocyanat
Thiosemicarbazide1,2,4-Triazole
1,3,4-Thiadiazole
NH
O
OR''
NH
O
NR''
HN
R
O
5-Haloisatin
Isatin derivatives(Indolinones)
Scheme 5.1
The plan which is illustrated by scheme was based on the following known
principles:
1. Carboxylic acids undergo esterification in acidic medium in the
presence of an alcohol.
2. Esters of carboxylic acids are converted to their respective hydrazides
with hydrazine monohydrate.
179
3. Isothiocyanates are formed from amines involving salts of
dithiocarbamate as an intermediate.
4. Condensation of carboxylic acid hydrazides with isothiocyanates yields
thiosemicarbazides.
5. Intramolecular dehydrative cyclization of substituted
thiosemicarbazides in basic medium affords the corresponding
substituted 3-mercapto-1,2,4-triazoles.
6. Cyclization of substituted thiosemicarbazides in acidic medium gives
the corresponding substituted 1,3,4-thiadiazoles.
The synthesized hydrazides are also used for the synthesis of oxindols
(2-indolinones), by condensation with halo-isatins in the presence of ethanol
solvent.
The characterization of the synthesized compounds was carried out by
spectroscopic techniques, like UV, IR, 1H NMR, 13C NMR and Mass
spectrometry. The synthesized indolinones, 4-substituted-1,2,4-triazol-3-ones
and thiadiazoles were screened for their antifungal, antibacterial, herbicidal,
insecticidal, fungicidal, plant growth regulatory activity and antiviral activities
180
Chapter- 6 RESULTS AND DISCUSSION
This chapter deals with the results and discussions of the synthetic aspects of
the target compounds. Firstly the synthesis of carboxylic acid hydrazides was
carried out by the condensation of carboxylic esters of corresponding acids with
hydrazine hydrate (80%). The carboxylic acid hydrazides were then reacted with
2-methoxyphenylisothiocyanate, 4-methoxyphenylisothiocyanate and cyclohexyl-
isothiocyanate to obtain corresponding 1,4-disubstituted thiosemicarbazides.
Each of the isothiocyanate was prepared by the reaction of corresponding amine
with carbon disulphide in the presence of ammonium hydroxide and lead nitrate.
These thiosemicarbazides were subjected to dehydrative cyclization in basic
medium and acidic medium to furnish corresponding 1,2,4-triazoles and 1,3,4-
thiadiazoles.
The synthesized hydrazides were also used for the synthesis of oxindols
(2-indolinones), by condensation with halo-isatins in the presence of ethanol
solvent.
6.1 Synthesis of methyl / ethyl esters 85(a-r)
The methyl esters were synthesized by the reaction of corresponding
substituted benzoic acids with methanol/ethanol in the presence of a catalytic
amount of sulfuric acid 304 (Scheme 6.1). The esters were characterized on the
basis of their physical constants and IR spectral data (Table 6.1).
CH3OH / H+
ORC2H5OH / H+
Reflux 5-6 hrs84(a-r) 85(a-r)
X C-OHO
R
X C-OMeO
R
[Yield = 77- 92%]
181
a. X = 0 R = 2,5-F2
b. X = 0 R = 3,5-F2
c. X = 0 R = 2,6-F2
d. X = 0 R = 3,4-(OCH3)2
e. X = 0 R = 3,5-(OCH3)2
f. X = 0 R = 2,4-(OCH3)2
g. X = 0 R = 2,6-(OCH3)2
h. X = CH2 R = 4-Cl
i. X = CH2 R = 4-F
j. X = (CH2)2 R = 3,4,5-(OCH3)3
k. X = (CH2)2 R = 4-OCH3
l. X = OCH2 R = 4-Br
m. X = O(CH2)2 R = 2,4-Cl2
n. X = O(CH2)3 R = 2,4-Cl2
o. X = OCH2 R = 2,4-Cl2
p. X = CH2= CH2 R = 4-OCH3
q. X = CH2 R = 3-OCH3
r. X = SCH2 R = 2,4-Cl2
Scheme 6.1: Synthesis of esters 85 (a-r)
Esters 85(a-r) can be characterized by the disappearance of broad peak
of acid in the range of 3200- 2400 cm-1. The IR spectral data of compounds 85(a-r) is tabulated in table 6.1.
182
Table 6.1: Physical constants and IR spectral data of methyl esters 85(a-r)
νmax (cm-1) Compd M.P (°C) Yield
(%) Rf * Ar–H Sp3 C–H C=O C=C C-O
85a Oil 92 0.72 3060 2942 1726 1585 1290 85b Oil 85 0.50 3058 2940 1727 1582 1309 85c Oil 87 0.71 3061 2943 1728 1590 1313 85d 58-62 79 0.81 3089 2901 1740 1610 1304 85e 41-43 77 0.79 3063 2896 1735 1625 1315 85f 116-121 80 0.74 3085 2890 1738 1596 1325 85g 87-90 81 0.74 3030 2989 1725 1645 1296 85h Oil 78 0.79 3056 2910 1711 1569 1285 85i Oil 83 0.59 3101 3001 1729 1578 1387 85j Oil 89 0.68 3048 3010 1725 1596 1269 85k Oil 79 0.78 3084 2986 1751 1586 1316 85l Oil 89 0.75 3104 2916 1742 1610 1296
85m Oil 82 0.83 3089 2986 1726 1566 1310 85n Oil 83 0.77 3125 2896 1722 1577 1296 85o Oil 82 0.76 3110 2975 1745 1587 1283 85p Oil 78 0.75 3056 2974 1720 1650 1300 85q 125-126 89 0.69 3178 2986 1731 1612 1285 85r Oil 87 0.63 3018 2946 1735 1625 1310 * Petroleum ether: ethylacetate (4:1)
The synthesis of two of the esters 85g and 85q, is also confirmed by x-ray
crystallography, as given below.
6.1.1 Crystal structure of Ethyl 2-(3′-methoxyphenyl)acetate 85q
Ethyl 2-(3′-methoxyphenyl)acetate (85q) (Fig. 6.1) shows no unusual
features. The diherdral angle between the planar ester group and benzene ring is
74.81 (2).
As can be seen from the packing diagram (Fig. 6.2), the Ethyl 2-(3′-
methoxyphenyl)acetate (85q) is stacked along a axis and elongated along the b
axis. Dipole-dipole and Van der waals interactions are effective in the molecular
packing.
183
Fig. 6.1: Crystal structure of Ethyl 2-(3′-methoxyphenyl)acetate 85q
Fig. 6.2: Packing diagram for Ethyl 2-(3′-methoxyphenyl)acetate
Crystal data C11H14O3 Dx = 1.223 Mg m−3 Mr = 195.23 Melting point: 398(1) K Monoclinic, P21/n Mo Kα radiation λ = 0.71073 Å a = 14.868 Å Cell parameters from 1609 reflections b = 4.851 Å θ = 2.9–23.6º
184
c = 15.428 Å µ = 0.09 mm−1 β = 107.70º T = 293 (2) K V = 1060.1 Å3 Block, colourless Z = 4 0.40 × 0.30 × 0.30 mm F000 = 420
Geometric parameters (°A,°)
Selected bond lengths: O1—C2 1.596 (6) C6—C10 1.401 (12) O2—C4 1.460 (9) C1—C2 1.369 (9)
C1—C8 1.414 (9) O3—C10 1.344 (10) O3—C12 1.409 (11) C4—C11 1.480 (12)
Selected bond angles:
C10—C7—H7 119.7 C2—C1—C8 133.9 (6) C8—C7—H7 119.7
C7—C10—C6 119.7 (7) O2—C4—C11 106.4 (6)
Selected torsional bond angles: C9—C5—H5 119.2 H12A—C12—H12B 109.5 C5—C6—C10 118.7 (7) C10—C7—C8 120.6 (7) C4—O2—C2—C1 176.1 (5) C7—C8—C9—C5 1.2 (12) C4—O2—C2—O1 −5.2 (9)
C6—C5—C9—C8 −1.6 (14) C12—O3—C10—C7 170.4 (8) C9—C5—C6—C10 2.2 (13) C10—C7—C8—C1 178.0 (7) C2—C1—C8—C9 175.6 (7) C5—C6—C10—C7 −2.5 (11) C2—C1—C8—C7 −4.0 (12)
This Part of the chapter has been published: Ghulam Qadeer, Nasim Hasan
Rama, Murat Tas and Okan Zafer Yesilel; Crystal structure of Ethyl-2-(3-
methoxyphenyl)acetate; Acta Cryst.; 2007, E63 ,o4678.
185
6.1.2 Crystal structure of Methyl 2,6-dimethoxybenzoate (85g)
A perspective view of methyl 2,6-dimethoxybenzoate (85g) is shown in
Fig. 6.3. Bond lengths and angles can be regarded as normal. The ester group
is perpendicular by 91.88 (19) out of the plane of the central aromatic ring.
Fig. 6.3: Crystal structure of Methyl 2,6-dimethoxybenzoate (85g)
Fig. 6.4: Packing diagram for Methyl 2,6-dimethoxy benzoate
186
π-π Stacking interaction can be observed in the crystal packing (Fig. 6.4).
Carboxylate atom C7 is located above the centre of the aromatic ring of a
neighboring parallel molecule (at x −1, y, z) at a distance of 3.585 A °.
Crystal data C10H12O4 Dx = 1.317 Mg m−3 Mr = 196.20 Melting point: 450(1) K Orthorhombic, Pbca Mo Kα radiation λ = 0.71073 Å a = 7.2306 (9) Å Cell parameters from 1609 reflections b = 14.1058 (17) Å θ = 2.9–23.6º c = 19.403 (2) Å µ = 0.10 mm−1 V = 1978.9 (4) Å3 T = 100 (2) K Z = 5 Block, colourless F000 = 832 0.40 × 0.30 × 0.30 mm
Geometric parameters (°A, °)
Selected Bond lengths: O1—C7 1.3335 (16) C4—C5 1.379 (2) O1—C8 1.4548 (17) O4—C6 1.3680 (17)
O4—C10 1.4397 (17) C1—C6 1.3951 (19) C1—C2 1.398 (2) C1—C7 1.498 (2)
Selected Bond angles: C7—O1—C8 116.06 (12) O2—C7—O1 123.88 (14) C2—O3—C9 117.32 (12) O2—C7—C1 124.86 (13)
H8B—C8—H8C 109.5 C4—C3—C2 118.84 (16) C5—C4—C3 122.38 (16) C4—C5—C6 118.39 (16)
Selected torsional bond angles: C9—O3—C2—C3 −1.7 (2) C4—C5—C6—O4 179.56 (13) C6—C1—C2—O3 −179.47 (12) C2—C1—C6—O4 179.59 (13)
C7—C1—C2—C3 178.67 (13) C7—C1—C6—C5 −177.57 (13) O3—C2—C3—C4 179.34 (13) C8—O1—C7—O2 −2.0 (2)
This Part of the chapter has been published: Ghulam Qadeer, Nasim Hasan
Rama, Mohammad Azad Malik and Madeleine Helliwell; Crystal structure of
Methyl 2,6-dimethoxybenzoate; Acta Cryst. 2007, E63, o3027.
187
6.2 Synthesis of hydrazides 86(a-r)
Methyl / Ethyl esters 85(a-r) of substituted benzoic acids were converted
to the corresponding hydrazides 86(a-r) by refluxing with hydrazine hydrate
(80%) in methanol305 (Scheme 3.2). The synthesized hydrazides 86(a-r) were
recrystallized from aqueous ethanol and characterized by their physical
constants, IR (Table 6.2), 1H NMR, 13C NMR (Table 6.3), Mass spectrometry
and X-ray crystallography.
[Yield = 72 - 88%]85(a-r) 86(a-r)X C-OMe
OR
X C-NHO
R
NH2
NH2-NH2.H2OCH3OHReflux4-7hrs
Scheme 6.2: Synthesis of substituted hydrazides 86 (a-r)
a. X = 0 R = 2,5-F2
b. X = 0 R = 3,5-F2
c. X = 0 R = 2,6-F2
d. X = 0 R = 3,4-(OCH3)2
e. X = 0 R = 3,5-(OCH3)2
f. X = 0 R = 2,4-(OCH3)2
g. X = 0 R = 2,6-(OCH3)2
h. X = CH2 R = 4-Cl
i. X = CH2 R = 4-F
j. X = (CH2)2 R = 3,4,5(OCH3)3
k. X = (CH2)2 R = 4-OCH3
l. X = OCH2 R = 4-Br
m. X = O(CH2)2 R = 2,4-Cl2
n. X = O(CH2)3 R = 2,4-Cl2
o. X = OCH2 R = 2,4-Cl2
p. X = CH2= CH2 R = 4-OCH3
q. X = CH2 R = 3-OCH3
r. X = SCH2 R = 2,4-Cl2
The IR spectra of hydrazides 86(a-r) exhibited a characteristic absorption
band for primary NH2, in the region 3547-3309 cm-1, while absorption band for
the secondary NH was observed in the range 3476-3198 cm-1. A strong
absorption in the region 1662-1618 cm-1 was assigned to the carbonyl group of
amide linkage. The IR spectral data of hydrazides 86(a-r) is given in table 6.2.
188
Table 6.2: Physical constants and IR spectral data of hydrazides 86(a-r)
υmax (cm-1) S.No m.p (°C) Yield (%) Rf* NH2 NH C=O C=C C–O
86a 141-142 78 0.80 3312 3312 b 1626 1597 1249 86b 158-159 74 0.87 3326 3286 1628 1595 1262 86c 244-246 88 0.75 3316 3316 b 1632 1579 1260 86d 118 82 0.81 3335 3296 1661 1596 1255 86e 165-166 73 0.78 3410 3325 1656 1578 1268 86f 101-102 72 0.80 3350 3210 1668 1560 1256 86g 160-161 79 0.87 3361 3256 1659 1589 1254 86h 102-104 79 0.75 3396 3241 1654 1596 1256 86i 158-160 81 0.81 3385 3274 1674 1574 1253 86j 121-122 78 0.78 3345 3196 1648 1585 1241 86k 125-126 78 0.80 3341 3341 1659 1574 1248 86l 110-112 74 0.87 3252 3252b 1658 1572 1249
86m 164-165 82 0.75 3345 3210 1654 1574 1254 86n 154-155 78 0.81 3385 3202 1652 1547 1247 86o 63-65 74 0.78 3374 3216 1663 1569 1257 86p 151-152 88 0.80 3341 3189 1639 1547 1251 86q 204-206 82 0.87 3298 3195 1658 1601 1239 86r 60-62 72 0.75 3347 3347b 1645 1540 1281
*petroleum ether: ethylacetate (4:1) b broad band
The formation of hydrazide 86(a-r) from the respective methyl/ethyl esters
was confirmed in the 1H NMR spectra by the appearance of NH2 proton signal in
the region of 2.00-2.50 ppm and NH proton signal in the region of 8.50- 9.98ppm
and by the disappearance of –OCH3 proton signals in the region of 3.77-
3.90ppm. In all these compounds 86(a-r), the integration curve indicated three
protons in the aromatic region confirming the presence of phenyl ring. In all these
compounds having two fluorine atoms as a substituent, splitting due to coupling
between fluorine and the hydrogens was also observed. The structures are futher
confirmed by 13C NMR (Table 6.3), mass spectrometry and X-ray
crystallography. The detail of spectral data of more hydrazides is given in chapter
7 (Experimental).
189
Table 6.3: 1H NMR and 13C NMR spectral data of 3,5-Difluorophenylhydrazides
O
HN
NH2
12 3
4
56
F
F
δ (ppm) and multiplicity Carbon
1H NMR 13C NMR
C=O ---- 164.3 OCH3 ---- 56.3 C-1 ---- 137.18 C-2 7.39-7.55(m) 107.02 C-3 ---- 163.58 C-4 7.39-7.55(m) 110.74 C-5 ---- 161.02 C-6 7.39-7.55(m) 110.57 NH 9.98 (s) ---- NH2 4.60(s) ----
6.2.1 Crystal structure of 3,5-difluorobenzohydrazide (86b)
The molecular structure of 3,5-difluorobenzohydrazide (86b) is shown in
Fig. 6.5. Bond distances and angles are within expected ranges. The hydrazidic
group C7/O1/N1/N2 is planar (maximum displacement being 0.007 (3) Å for C7)
and nearly coplanar with the benzene ring [the dihedral angle being 9.27 (10) °].
The crystal packing is stabilized by N—H···O and N—H···N intermolecular
hydrogen interactions (Fig. 6.6).
190
Fig. 6.5: Crystal structure of 3,5-Difluorobenzohydrazide (86b)
Fig. 6.6: Packing diagram showing the hydrogen bonding.
Crystal data C7H6F2N2O F000 = 352 Mr = 172.14 Dx = 1.558 Mg m−3 Orthorhombic, P212121 Melting point: 374(1) K Hall symbol: P 2ac 2ab Mo Kα radiation λ = 0.71073 Å a = 3.8635 (4) Å µ = 0.14 mm−1 b = 6.4199 (6) Å T = 293 (2) K c = 29.589 (3) Å Block, pale yellow V = 733.89 (13) Å3 0.42 × 0.30 × 0.26 mm Z = 4
191
Geometric parameters (°A, °)
Selected bond lengths: C1—C2 1.380 (3) C5—C6 1.385 (2) C6—C7 1.500 (2) C7—N1 1.337 (2)
N1—H1 0.88 (2) C4—F2 1.355 (2) C4—C5 1.382 (2) N2—H3 0.85 (2)
Selected bond angles: C2—C1—C6 118.14 (16) C5—C6—C1 120.34 (14) C5—C6—C7 116.48 (14) F1—C2—C3 118.51 (17)
N2—N1—H1 114.8 (12) F2—C4—C5 117.72 (19) N1—N2—H2 107.8 (14) C3—C4—C5 123.62 (17)
Selected torsional bond angles: C6—C1—C2—F1 178.82 (17) C4—C5—C6—C7 −177.92 (18) F1—C2—C3—C4 −178.66 (18) C1—C6—C7—O1 −178.34 (16)
C2—C3—C4—C5 −0.1 (3) C5—C6—C7—N1 178.82 (15) F2—C4—C5—C6 178.36 (19) C6—C7—N1—N2 −173.47 (14)
Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A N1—H1···N2i 0.88 (2) 2.14 (2) 2.9733 (19) 158.4 (16) N2—H2···O1ii 0.93 (2) 2.05 (2) 2.9737 (19) 175 (2) N2—H3···O1 0.85 (2) 2.38 (2) 2.7240 (17) 104.6 (17) N2—H3···O1iii 0.85 (2) 2.42 (2) 3.024 (2) 128.8 (17)
Symmetry codes: (i) x−1/2, −y+3/2, −z; (ii) x−1/2, −y+5/2, −z
(iii) x+1/2, −y+5/2, −z
This Part of the chapter has been published: Ghulam Qadeer, Nasim Hasan
Rama, Muhammad Zareef and Wai-Yeung Wong; Crystal structure of 3,5-
Difluorobenzohydrazide , Acta Cryst, 2007, E63, o1176–o1177.
192
6.2.2 Crystal structure of 2,6-dimethoxybenzohydrazide (86g)
Fig. 6.7: Crystal structure of 2,6-Dimethoxybenzohydrazide (86g)
The molecular structure of 2,6-dimethoxybenzohydrazide (86g) is shown
in Fig 6.7. Bond distances and angles are within expected ranges. The
hydrazidic group C9/O3/N1/N2 is planar (maximum displacement being 0.007 (3)
Å for C8) and perpendicular with the benzene ring [the dihedral angle being
88.27 (10) °]. Crystal data C9H12N2O3 Dx = 1.257 Mg m−3 Mr = 196.21 Melting point: 244(2) K Orthorhombic, Pbca Mo Kα radiation λ = 0.71073 Å a = 7.2598 (5) Å Cell parameters from 1520 reflections b = 14.2558 (11) Å θ = 2.7–24.9º This Part of the chapter has been published: Ghulam Qadeer, Nasim Hasan
Rama and Wen Tong Chen; Crystal structure of 2,6-Dimethoxybenzohydrazide
Acta Cryst. 2007, E63, o2892.
193
c = 20.0412 (11) Å µ = 0.10 mm−1 V = 2074.1 (2) Å3 T = 293 (2) K Z = 8 Block, colorless F000 = 832 0.16 × 0.14 × 0.06 mm
Geometric parameters (°A, °)
Selected bond lengths: O1—C2 1.366 (3) C2—C3 1.393 (4) C7—H7B 0.9600 N2—H2A 0.8600 C7—H7C 0.9600
C8—H8A 0.9600 C1—C2 1.393 (3) C8—H8B 0.9600 C1—C9 1.506 (3) C8—H8C 0.9600
Selected bond angles: C2—O1—C7 118.4 (3) C4—C5—H5A 120.7 C9—N2—N1 118.5 (2) H7B—C7—H7C 109.5 O1—C2—C3 125.5 (3) O2—C8—H8B 109.5 C3—C2—C1 119.5 (3)
H8A—C8—H8B 109.5 C2—C3—C4 118.5 (3) O2—C8—H8C 109.5 C2—C3—H3A 120.7 C3—C4—H4A 118.6 N2—C9—C1 112.7 (2) C4—C5—C6 118.6 (3)
6.2.3 Crystal structure of 3,4-dimethoxybenzohydrazide (86d)
Fig. 6.8: Crystal structure of 3,4-Dimethoxybenzohydrazide (86d)
194
The molecular structure of (86d) is shown in Fig. 6.8. Bond distances and
angles are within expected ranges. The dihedral angle between the planar
hydrazidic group (C7/O1/N1/N2) and the benzene ring (C1—C6) is 63.27 (3) °. The crystal structure is stabilized by N–H···O hydrogen bonding (Fig. 6.9).
Fig. 6.9: Packing diagram showing the hydrogen bonding
Crystal data C9H12N2O3 Dx = 1.396 Mg m−3
Dm = 1.375 Mg m−3 Mr = 196.21 Melting point: 391(1) K Monoclinic, P2(1)/c Mo Kα radiation λ = 0.71073 Å a = 13.610 (3) Å Cell parameters from 927 reflections b = 8.9130 (19) Å θ = 2.8–25.1º c = 7.9780 (17) Å µ = 0.11 mm−1 β = 105.266 (4) º T = 100 (2) K V = 933.6 (3) Å3 Block, white Z = 4 0.25 × 0.20 × 0.20 mm F000 = 416
195
Geometric parameters (°A, °)
Selected bond lengths: C1—C6 1.385 (3) C7—N1 1.334 (2) C1—C2 1.402 (2) C8—O2 1.434 (2)
C8—H8B 0.9800 C2—H2 0.9500 C3—C4 1.413 (3) C9—H9A 0.9800
Selected bond angles: C6—C1—C2 119.75 (18) O2—C8—H8A 109.5 C6—C1—C7 122.13 (18) O2—C8—H8B 109.5 C4—C5—C6 120.17 (18)
C7—N1—N2 121.93 (17) C1—C6—C5 120.27 (19) N1—N2—H2A 105.5 (14) C1—C6—H6 119.9 N1—N2—H2B 105.3 (14)
Selected torsional bond angles: C6—C1—C2—C3 1.8 (3) C4—C5—C6—C1 −0.9 (3) C7—C1—C2—C3 177.49 (17) C6—C1—C7—O1 147.43 (18) C1—C2—C3—O2 178.76 (16) C2—C1—C7—O1 −28.1 (3)
O2—C3—C4—C5 179.36 (16) C1—C7—N1—N2 175.03 (17) C2—C3—C4—C5 −1.0 (3) C2—C3—O2—C8 10.0 (3) O3—C4—C5—C6 −179.15 (18) C4—C3—O2—C8 −170.31 (15)
Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A
N1—H1···O1i 0.88 2.10 2.894 (2) 150
N2—H2A···O1ii 0.90 (2) 2.17 (2) 2.944 (2) 144.0 (18)
Symmetry codes: (i) −x, y+1/2, −z−1/2; (ii) x, −y+3/2, z−1/2.
This Part of the chapter has been published: Ghulam Qadeer, Nasim Hasan
Rama, Mohammad Azad Malik and James Raftery; Crystal structure of 3,4-
Dimethoxybenzohydrazide; Acta Cryst. 2007, E63, o3026.
196
6.2.4 Crystal structure of 2-(2′,4′-dichlorophenylsulfanyl)acto-
hydrazide (86r)
Fig. 6.10: Crystal structure of 2-(2′,4′-Dichlorophenylsulfanyl)actohydrazide (86r)
The molecular structure of (86r) is shown in Fig. 6.10. Bond distances and
angles are within expected ranges. The dihedral angle between the planar
hydrazidic group (C8/O1/N1/N2) and benzene ring (C1—C6) is 91.07 (3) °. Two
centrosymmetrically related N1—H1A···O1 (N1···O1, 3.078 A, H1A···O1, 2.666 A,
N1—H1A···O1, 110.8) hydrogen bonds form a dimer (Fig. 6.11).
Fig. 6.11: Packing diagram showing the hydrogen bonding
197
Crystal data
2(C8H8Cl2N2O1S1) Z = 2 Mr = 251.13 F000 = 240 Triclinic, P1 Dx = 1.657 Mg m−3 Hall symbol: -P 1 Melting point: 333(2) K a = 7.350 (5) Å Mo Kα radiation λ = 0.71073 Å b = 8.133 (6) Å Cell parameters from 1520 reflections c = 8.545 (6) Å θ = 2.7–24.9º α = 94.802 (10) º µ = 0.82 mm−1 β = 90.140 (9) º T = 293 (2) K γ = 98.492 (10) º Block, colourless V = 503.4 (6) Å3 0.15 × 0.14 × 0.14 mm
Geometric parameters (°A, °)
Selected bond lengths: Cl1—C6 1.749 (3) C1—C2 1.376 (4) Cl2—C4 1.729 (3) C1—H1C 0.9300
N1—H1B 0.8600 C5—H5A 0.9300 N2—C8 1.334 (4) C7—C8 1.502 (4)
Selected bond angles: C3—S1—C7 117.5 (2) C5—C4—Cl2 119.6 (2) N2—N1—H1A 120.0 S1—C7—C8 110.3 (2) C2—C1—H1C 120.3 S1—C7—H7A 109.6 C1—C2—C3 121.7 (3)
C8—C7—H7A 109.6 C1—C2—H2B 119.2 S1—C7—H7B 109.6 C3—C2—H2B 119.2 C8—C7—H7B 109.6 S1—C3—C2 126.5 (3) H7A—C7—H7B 108.1
This Part of the chapter has been published: Ghulam Qadeer, Nasim Hasan
Rama and Wen-Tong Chen Crystal structure of 2-(2,4-Dichlorophenylsulfanyl)-
acetohydrazide ; Acta Cryst. 2007, E63, o2932.
198
6.2.5 Crystal structure of (E)-3-(4′-methoxyphenyl)acrylo-
hydrazide (86p)
Fig. 6.12: Crystal structure of (E)-3-(4′-Methoxyphenyl)acrylohydrazide (86p)
The molecular structure of (E)-3-(4′-methoxyphenyl)acrylohydrazide (86p)
is shown in Fig. 6.12. Bond distances and angles are within expected ranges.
The dihedral angle between the planar hydrazidic group (C10/O2/N1/N2) and the
benzene ring (C1—C6) is 73.93 (3) °.
Crystal data
C10H12N2O2 Dx = 1.213 Mg m−3 Dm = 1.213 Mg m−3 Dm measured by not measured
Mr = 192.20 Melting point: 477(2) K Monoclinic, P2 (1)/c Mo Kα radiation λ = 0.71069 Å a = 18.661 (5) Å Cell parameters from 691 reflections b = 4.842 (5) Å θ = 3.5–23.6º c = 12.041 (5) Å µ = 0.09 mm−1 β = 106.774 (5) º T = 293 (2) K V = 1041.7 (12) Å3 Block, colorless Z = 4 0.30 × 0.20 × 0.10 mm F000 = 400
199
Geometric parameters (°A, °)
Selected bond lengths: O2—C10 1.233 (2) C4—C3 1.377 (3) N1—C10 1.3319 (19) C4—C8 1.512 (2)
N1—N2 1.4132 (17) C8—H8 0.9700 O1—C7 1.424 (3) C7—H7A 0.9600
Selected bond angles: C10—N1—N2 123.06 (12) C4—C5—H5 118.9 C10—N1—H1N 117.4 (12) O2—C10—N1 121.96 (14) C1—C6—H6 120.0 C6—C1—O1 124.95 (17)
C4—C8—H8 108.9 C6—C1—C2 119.31 (16) O1—C7—H7A 109.5 O1—C1—C2 115.74 (16) O1—C7—H7B 109.5 C5—C4—C3 116.80 (17)
Selected torsional bond angles: N2—N1—C10—O2 −1.4 (2) C2—C1—C6—C5 −0.5 (3) O2—C10—C9—C8 40.0 (2)
C1—C2—C3—C4 −0.4 (3) C3—C4—C8—C9 82.5 (3) O1—C1—C2—C3 −178.76 (18)
This Part of the chapter has been published: Ghulam Qadeer, Nasim Hasan
Rama, Zhong-Min Su; Crystal structure of (E)-3-(4′-Methoxyphenyl)acrylo-
hydrazide; Acta Cryst. 2007, E63, o2989.
200
6.2.6 Crystal structure of 3-(3′,4′,5′-trimethoxyphenyl)propane-
hydrazide (86j)
Fig. 6.13: Crystal structure of 3-(3′,4′,5′-Trimethoxyphenyl)propanohydrazide
(86j)
The molecular structure of 3-(3′,4′,5′-trimethoxyphenyl)propanohydrazide
(86j) is shown in Fig. 6.13. Bond distances and angles are within expected
ranges. The dihedral angle between the planar hydrazidic group (C9/O1/N1/N2)
and the benzene ring (C1—C6) is 61.23 (2) °.
X-ray analysis reveals that the asymmetric unit contains two independent
molecules. N—H···O and C—H···O hydrogen bonds link the molecules into
201
layers. Molecules in adjacent layers are linked via N—H···O hydrogen bonds
(Fig. 6.14).
Fig. 6.14: Packing diagram showing the H-bonding Crystal data C12H18N2O4 Dx = 1.364 Mg m−3
Dm = 1.323 Mg m−3 Dm measured by not measured
Mr = 254.28 Melting point: 398(1) K Monoclinic, P2 (1)/n Mo Kα radiation λ = 0.71073 Å a = 9.7770 (14) Å Cell parameters from 2527 reflections b = 20.189 (3) Å θ = 2.4–24.6º c = 12.7300 (19) Å µ = 0.10 mm−1 β = 99.618 (3) º T = 100 (2) K V = 2477.4 (6) Å3 Plate, white Z = 8 0.20 × 0.20 × 0.05 mm F000 = 1088
Geometric parameters (°A, °)
Selected bond lengths: C1—C6 1.387 (2) C14—H14 0.9500
C1—C2 1.389 (2) C16—O7 1.3814 (19)
202
C4—O3 1.3844 (18) C18—H18 0.9500
C4—C5 1.392 (2) C19—C20 1.529 (2)
Selected bond angles: C6—C1—C2 119.66 (16) O7—C16—C15 120.21 (16) C6—C1—C7 123.41 (16) O7—C16—C17 120.41 (16) C20—C19—H19A 108.3 O4—C5—C4 115.25 (15)
O1—C9—N1 122.40 (16) O7—C23—H23A 109.5 O1—C9—C8 122.22 (16) O7—C23—H23B 109.5 N1—C9—C8 115.37 (16)
Selected torsional bond angles: C6—C1—C2—C3 −1.8 (3) C7—C1—C2—C3 174.91 (16) C1—C2—C3—O2 −178.38 (16) C1—C2—C3—C4 0.0 (3) O2—C3—C4—O3 −2.5 (2) C2—C3—C4—O3 179.08 (14) O3—C4—C5—C6 −179.06 (15) C3—C4—C5—C6 −1.9 (2)
O5—C21—N3—N4 −1.5 (3) C16—C15—O6—C22 169.91 (15) C13—C14—C15—C16 0.8 (3) C14—C15—O6—C22 −9.7 (2) O6—C15—C16—O7 −3.3 (2) C17—C16—O7—C23 −85.0 (2
Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A N4—H4B···O6i 0.924 (18) 2.491 (19) 3.401 (2) 168.2 (15) N2—H2B···O5 1.016 (19) 2.110 (19) 3.045 (2) 152.1 (14) N2—H2A···O3ii 0.883 (17) 2.618 (17) 3.453 (2) 158.2 (15) N2—H2A···O2ii 0.883 (17) 2.377 (17) 3.067 (2) 135.2 (14) N4—H4A···O1 0.997 (19) 2.131 (19) 3.084 (2) 159.5 (15) N3—H3···O1iii 0.88 2.02 2.8914 (18) 171 N1—H1···O5iv 0.88 2.01 2.8719 (18) 166 Symmetry codes: (i) −x+3/2, y−1/2, −z+1/2; (ii) −x+3/2, y−1/2, −z+3/2; (iii) −x+2,
−y, −z+1; (iv) −x+1, −y, −z+1.
This Part of the chapter has been published: Ghulam Qadeer, Nasim Hasan
Rama, Mohammad Azad Malik and James Raftery; Crystal structure of 3-
(3′,4′,5′-Trimethoxyphenyl)propanohydrazide; Acta Cryst., 2007, E63 , o3025.
203
6.2.7 Crystal structure of 3-(4′-methoxyphenyl)propanohydrazide
(86k)
Fig. 6.15: Crystal structure of 3-(4′-Methoxyphenyl)propanohydrazide (86k)
The molecular structure of 3-(4′-methoxyphenyl)propanohydrazide (86k)is
shown in Fig. 6.15. Bond distances and angles are within expected ranges. The
dihedral angle between the planar hydrazidic group (C9/O1/N1/N2) and the
benzene ring (C1—C6) is 81.27 (3) °. The crystal structure is stabilized by N–
H···O and N–H···N hydrogen bonding (Fig. 6.16).
Fig. 6.16: Packing diagram showing the H-bonding
204
Crystal data C10H14N2O2 F000 = 416 Mr = 194.23 Dx = 1.276 Mg m−3
Dm = 1.253 Mg m−3 Dm measured by not measured
Monoclinic, P21/c Melting point: 383(2) K Hall symbol: -P 2ybc Mo Kα radiation λ = 0.71073 Å a = 18.519 (9) Å Cell parameters from 691 reflections b = 4.816 (2) Å θ = 3.4–23.6º c = 11.884 (6) Å µ = 0.09 mm−1 β = 107.521 (7) º T = 100 (2) K V = 1010.7 (8) Å3 Block, colorless Z = 4 0.30 × 0.20 × 0.10 mm
Geometric parameters (°A, °) Selected bond lengths: C1—C2 1.390 (5) C7—H7B 0.9900 C1—C6 1.393 (4) C8—C9 1.498 (5)
C1—C7 1.502 (5) C10—H10B 0.9800 C5—C6 1.381 (4) C10—H10C 0.9800
Selected bond angles: C2—C1—C6 116.4 (3) H7A—C7—H7B 107.8 C2—C1—C7 121.9 (3) O2—C4—C5 115.2 (3) O2—C10—H10B 109.5
C5—C6—C1 122.3 (3) C5—C6—H6 118.8 C9—N1—N2 122.9 (3) C1—C6—H6 118.8 C4—O2—C10 116.5 (3)
Selected torsional bond angles: C6—C1—C2—C3 1.2 (5) C2—C1—C7—C8 −98.0 (4) C7—C1—C2—C3 −178.8 (3) C6—C1—C7—C8 82.1 (4)
C3—C4—O2—C10 −4.6 (5) C2—C1—C6—C5 −1.0 (5) C5—C4—O2—C10 174.4 (3) C7—C1—C6—C5 179.0 (3)
This Part of the chapter has been published: Ghulam Qadeer, Nasim Hasan
Rama, Mohommad Azad Malik and James Raftery; Crystal structure of 3-(4′-
Methoxyphenyl)propanohydrazide; Acta Cryst. 2007, E63 , o3061.
205
Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A N1—H1···O1i 0.88 2.04 2.883 (3) 159 N2—H2A···N2ii 0.89 (3) 2.35 (4) 3.174 (4) 154 (3) N2—H2B···O1iii 0.88 (3) 2.28 (4) 3.112 (4) 158 (3)
Symmetry codes: (i) x, y+1, z; (ii) −x+1, y−1/2, −z+3/2; (iii) −x+1, −y+1, −z+1.
6.3 Synthesis of isothiocyanate 87(a-c)306
Three substituted phenyl isothiocyanates namely 2-methoxyphenyl-
isothiocyanate (87a), 4-methoxyphenylisothiocyanate (87b) and cyclohexyl-
isothiocyanate (87c) were prepared by the reaction of corresponding amine with
ammonical carbon disulphide followed by the reaction with lead nitrate. The
isothiocyanates were finally recovered by steam distillation. The general
synthetic scheme is given below.
[Yield = 47 - 70%]
+ CS2 MeOHNH4OHRHN
S
S-NH4+
R NC
S
Stirring12hr
Pb(NO3)2Overnight stirring
+ +
Isothiocyanate
RNH2
87a) R = 2-MeO(C6H4)-87b) R = 4-MeO(C6H4)-87c) R = -C6H11
Amine
87(a-c)
Scheme 6.3: Synthesis of Isothiocyanate 87 (a-c)
All the three isothiocyanates 87(a-c) were obtained in about 50% yield. All
isolated isothiocyanates were purified by column chromatography on silica gel
column in acetone: hexane (7:3). Each isolated isothiocyanate showed a single
spot on TLC which indicated its purity. All the three isothiocyanates showed
206
characteristic IR absorption band in the region of 2106-2035 cm-1. Thus presence
of N=C=S group in the IR spectrum was indicated by a broad intense peak at
2106 cm-1.
Table 6.4: IR data of Cyclohexyl- and substituted methoxyphenylisothiocyanate
87(a-c)
The formation of isothiocyanates 87(a-c) from the amine were confirmed by 1H NMR spectra by the absence of any proton signal for N-H of the amine. In
case of substituted methoxyphenylisothiocyanate, a single peak for methoxy
protons appeared at 3.79 ppm. The 1H NMR spectral data of isothiocyanate
87(a-c) are given in table 6.5. The 13C NMR spectra of isothiocyanates show
absorption at 155-165ppm due to carbon of isothiocyanate group. In case of
substituted methoxyisothiocyanates a peak in range of 45-55ppm is observed.
The 13C NMR data of isothiocyanates 103(a-e) is also given in table 6.5.
The structure is also confirmed by mass spectrometry. These Isothiocyanates
87(a-c) give M+2 and M+4 peaks due to presence of sulphur atom. The mass
spectral data from isotopic peaks confirmed the formation of Isothiocyanate. In
case of isothiocyanates molecular ion peak is base peak. The molecular ion peak
for the methoxyphenylisothiocyanate is 165 (100%) and 141(100%) for
cyclohexyl- isothiocyanate.
υmax (cm-1) Code M.P.
(°C) Yield Rf sp3 C-H stretching
sp2 C-H stretching N=C=S C=C
87a oil 49% 0.79 2967 & 2940 3069 2035
broad 1591, 1495 &
1457
87b oil 47% 0.81 2957,
2907 & 2836
3053 2106 broad
1603, 1503 & 1460
87c oil 50% 0.82 2941 3063 2065 broad ----
207
Table 6.5: 1H NMR data of Cyclohexyl- and substituted methoxyphenyl -
isothiocyanate 87 (a-c)
1H NMR
δ (ppm)
13C NMR
δ (ppm) Code Substitutent
-OCH3
protons
Aromatic
protons
Aliphatic
carbon
Aromatic
carbon
Isothio-
cyanate
carbon
87a 2-methoxyphenyl 3.29 6.89-7.28 51.5 116.50-135.54 162.51
87b 4-methoxyphenyl 3.24 6.81- 7.28 55.58 114.33-133.80 158.57
87C Cyclohexyl ---- 1.32-1.88 ---- 23.23-33.20 129.58
6.4 Synthesis of thiosemicarbazides 88(a-w)
The thiosemicarbazides 88(a-w) were synthesized by the reaction of the
corresponding carboxylic acid hydrazides and isothiocyanate in methanol307-308
(Scheme 6.3). Eighteen different hydrazides 86(a-r) were selected to be
condensed with three substituted isothiocyanate to bring diversity in biological
activity of the final products. The thiosemicarbazides are intermediate for the
synthesis of heterocycles (1,2,4-triazoles and 1,3,4-thiadiazoles).
[Yield = 78 - 85%]86(a-r) 87(a-c) 88(a-w)
X C-NHO
R
NH2 +R′
NC S
O
NH
HN
S
HN
R′Reflux
6 hrsX
R
Scheme 6.4: Synthesis of substituted Thiosemicarbazides 88 (a-w)
88a. X = 0 R = 3,5-F2 R′ = -C6H11 88b. X = O(CH2)2 R = 2,4-Cl2 R′ = -C6H11 88c. X = 0 R = 3,4-(OCH3)2 R′ = -C6H11 88d. X = OCH2 R = 2,4-Cl2 R′ = -C6H11
208
88e. X = (CH2)2 R = 3,4,5-(OCH3)3 R′ = -C6H11 88f. X = (CH2)2 R = 4-OCH3 R′ = -C6H11 88g. X = OCH2 R = 4-Br R′ = 2-MeO(C6H4)- 88h. X = 0 R = 2,5-F2 R′ = 2-MeO(C6H4)- 88i. X = OCH2 R = 2,4-Cl2 R′ = 2-MeO(C6H4)- 88j. X = 0 R = 3,5-F2 R′ = 2-MeO(C6H4)- 88k. X = 0 R = 3,4-(OCH3)2 R′ = 2-MeO(C6H4)- 88l. X = 0 R = 2,6-(OCH3)2 R′ = 2-MeO(C6H4)- 88m. X = 0 R = 3,5-(OCH3)2 R′ = 2-MeO(C6H4)- 88n. X = (CH2)2 R = 3,4,5(OCH3)3 R′ = 2-MeO(C6H4)- 88o. X = (CH2)2 R = 4-OCH3 R′ = 2-MeO(C6H4)- 88p. X = O(CH2)2 R = 2,4-Cl2 R′ = 4-MeO(C6H4)- 88q. X = O(CH2)3 R = 2,4-Cl2 R′ = 4-MeO(C6H4)- 88r. X = (CH2)2 R = 4-OCH3 R′ = 4-MeO(C6H4)- 88s. X = OCH2 R = 4-Br R′ = 4-MeO(C6H4)- 88t. X = OCH2 R = 2,4-Cl2 R′ = 4-MeO(C6H4)- 88u. X = 0 R = 2,5-F2 R′ = 4-MeO(C6H4)- 88v. X = (CH2)2 R = 3,4,5-(OCH3)3 R′ = 4-MeO(C6H4)- 88w. X = 0 R = 3,4-(OCH3)2 R′ = 4-MeO(C6H4)-
The synthesized compounds were recrystallized from a mixture of ethyl
acetate and petroleum ether. The physical data of compounds 88(a-w) is
tabulated in table 6.6. The conversion to thiosemicarbazides 88(a-w) was
indicated in the IR spectra by the appearance of a carbonyl absorption in the
region of 1692-1665 cm-1 and an absorption in the region 1246-1224 cm-1 for C=S
group. The characteristic absorption bands for three secondary N–H groups were
observed in the region of 3388-3141 cm-1. The IR spectral data of the
synthesized thiosemicarbazides 88(a-w) is presented in table 6.6.
209
Table 6.6: Physical constants and IR spectral data of Thiosemicarbazides 88(a-w)
υmax (cm-1) Compd mp (°C) *Rf Yield (%) N-H C=O C=S C=C
88a 131-133 0.37 85 3250-3145 1653 1265 1600, 1568
88b 149-150 0.34 83 3271-3149 1659 1251 1612, 1585
88c 185-186 0.38 80 3351-3115 1666 1239 1613, 1561
88d 189-190 0.35 79 3290-3009 1654 1255 1607, 1560
88e 174-176 0.37 84 3400-3145 1658 1244 1598, 1547
88f 133-135 0.34 85 3411-3205 1673 1242 1625, 1588
88g 165-167 0.34 80 3385-3216 1685 1255 1615, 1566
88h 153-154 0.37 78 3285-3210 1670 1245 1605, 1549
88i 156-157 0.40 85 3385-3216 1671 1248 1586, 1539
88j 149-150 0.39 83 3315-3116 1645 1265 1587, 1542
88k 173-175 0.41 80 3410-3016 1660 1251 1587, 1565
88l 180-182 0.35 79 3400-3260 1663 1261 1625, 1541
88m 165-167 0.37 84 3305-3011 1667 1250 1603, 1561
88n 174-175 0.40 85 3445-3211 1656 1255 1621, 1545
88o 189-191 0.34 80 3383-3116 1668 1235 1601, 1520
88p 186-187 0.37 78 3250-3100 1658 1247 1632, 1568
88q 155-156 0.39 85 3311-3019 1670 1257 1604, 1555
88r 165-167 0.35 83 3432-3210 1675 1252 1605, 1546
88s 141-143 0.42 80 3326-3100 1663 1254 1599, 1525
88t 134-135 0.35 79 3142-3001 1675 1248 1605, 1542
88u 119-120 0.37 84 3254-3006 1663 1245 1605, 1509
88v 164-165 0.34 85 3330-3016 1645 1236 1625, 1555
88w 148-149 0.41 80 3400-3180 1668 1262 1596, 1524
*Petroleum ether: Acetone; (6:4)
Formation of these thiosemicarbazides 88(a-w) from the reaction of
isothiocyanate 87(a-c) and acid hydrazides 86(a-r) was confirmed by 1H NMR
210
spectral data. Signals for NH proton of amide type linkage appeared in the range
of 10-11ppm and signals for two NH proton of thiourea type linkage appeared in
the range of 9-10 ppm. The 1H NMR data of a representativie
thiosemicarbazides 88m is given in the table 6.7. The formation of these
thiosemicarbazide were further confirmed from 13C NMR by the appearance of
new peaks in aromatic region and C=O peak in the range of 160-170ppm and
C=S peak in the range of 155-165ppm. The 13C NMR data of the
thiosemicarbazide 88m is given in the table 6.7
Table 6.7: 1H NMR and 13C NMR spectral data of Thiosemicarbazide 88m
HN
NH
S
NH
O
O
1 2
34
5
61′2′
3′4′5′
6′
OO
AB
1H NMR 13C NMR
Proton δ(ppm),intensity,multiplicity and coupling constants (J) Carbon δ(ppm)
NH 10.60 (s) C=S 181.5 NH 9.81 (s) C=O 166.3 NH 9.22 (s) ---- ---- H-1 ---- C-1 126.5 H-2 ---- C-2 152.6 H-3 7.19 (4H, m) C-3 111.9 H-4 7.19 (4H, m) C-4 126.1 H-5 7.19 (4H, m) C-5 120.3 H-6 7.19 (4H, m) C-6 128.2 H-1′ ---- C-1′ 134.8 H-2′ 6.93 (2H, t, J = 1.8 Hz) C-2′ 106.2 H-3′ ---- C-3′ 160.8 H-4′ 6.72 (1H, t, J = 2.1 Hz) C-4′ 104.2 H-5′ ---- C-5′ 160.8 H-6′ 6.93 (2H, t, J = 1.8 Hz) C-6′ 106.2
-OCH3 (B) 3.80 (6H, s) -OCH3 (B) 56.2 -OCH3 (A) 3.75(3H, s) -OCH3 (A) 55.9
211
In mass fragmentation of 1-(3,5-dimthoxybenzoyl)-4-(2-methoxyphenyl)-
thiosemicarbazides 88m (Sheme 6.4), the molecular ion peak appeared at m/z
361 which confirmed the formation of 1-(3,5-dimthoxybenzoyl)-4-(2-
methoxyphenyl)thiosemicarbazides 88m and base peak appears at m/z 167.
.
HN
NH
S
NHO
O
O O
(m/z = 361, 12.4%)
S
NHO
(m/z = 167, 100 %)
OCN
(m/z = 133, 62.1 %)
O
(m/z = 108, 38.9 %)
HN
NH
SO
O O
(m/z = 240, 15.3 %)
O
O O
(m/z = 166, 35.3 %)
O O
(m/z = 138, 07.6 %)
HN
N NO
O
O O
HNNH2
O
O O
NHO
O O
(m/z = 196, 28.7%)
-SH2
-
-NCS-SNH2
O-
-NHNCS
-CO
HN
NH2O
O O
-NH2
SNH
O-
(m/z = 327, 4.4%)
(m/z = 180, 32.4%)
+
+
+
+
+ .+ .
+ .+ .
+ .
..
.
+
(88m)
Scheme 6.5: Mass fragmentation patern of thiosemicarbazide 88m
The elemental analysis of the 1-(3,5-dimthoxybenzoyl)-4-(2-
methoxyphenyl)thiosemicarbazides 88m, showed the best agreement of found
elemental percentage composition with calculated values as shown in table 6.8.
Table 6.8: Elemental analysis data of thiosemicarbazide 88m
Compd. % C H N S
Calculated 56.50 5.30 11.63 8.87 88m
Found 56.12 5.78 11.31 8.45
212
The structure of 1-(3,5-dimthoxybenzoyl)-4-(2-methoxyphenyl)thiosemi-
carbazides 88m is finally confirmed by XRD analysis as explained below.
6.4.1 Crystal structure of 1-(3,5-Dimethoxybenzoyl)-4-(2-methoxyphenyl)thiosemicarbazide (88m)
Fig. 6.17: Crystal structure of 1-(3,5-Dimethoxybenzoyl)-4-(2-
methoxyphenyl)thiosemicarbazide (88m)
In the molecule of 1-(3,5-Dimethoxybenzoyl)-4-(2-methoxyphenyl)-
thiosemicarbazide (Fig. 6.17), the bond lengths and angles are in normal ranges.
The dihedral angle between the planar rings is 63.4 (2). The crystal structure of
(I) is stabilized by intramolecular N—-H···N and intermolecular N—H··· S
hydrogen bonds. Intermolecular N—H···S hydrogen bonds link the independent
molecules into dimers (Fig. 6.18). Dipole–dipole and van der Waals interactions
are also effective in the molecular packing in the crystal structure.
213
Fig. 6.18: Packing diagram show the dipole–dipole and van der Waals
interactions are also in the crystal structure.
Crystal data C17H19N3O4S Dx = 1.371 Mg m−3 Mr = 361.41 Melting point: 435(2) K Monoclinic, P2 (1)/c Mo Kα radiation λ = 0.71073 Å a = 15.371 (6) Å Cell parameters from 1520 reflections b = 14.775 (6) Å θ = 2.7–24.9º c = 7.904 (3) Å µ = 0.21 mm−1
β = 102.835 (6) º T = 293 (2) K V = 1750.3 (12) Å3 Block, colorless Z = 4 0.46 × 0.26 × 0.20 mm F000 = 760
This Part of the chapter has been published: Ghulam Qadeer, Muhammad
Hanif, Nasim Hasan Rama and Wen-Tong Chen; Crystal structure of 1-(3′,5′-
Dimethoxybenzoyl)-4-(2′-methoxyphenyl)thiosemicarbazide; Acta Cryst. 2007,
E63, o3051.
214
Geometric parameters (°A, °)
Selected bond lengths: S1—C13 1.6769 (17) C4—C5 1.383 (3) O1—C5 1.369 (2) N1—C6 1.415 (2)
C17—H17B 0.9600 C3—C4 1.381 (3) C17—H17C 0.9600 C3—H3A 0.9300
Selected bond angles: C5—O1—C15 117.66 (17) C8—C9—C10 119.24 (15) C8—O2—C16 118.15 (15) C8—C9—H9A 120.4 C10—O3—C17 117.66 (14) C10—C9—H9A 120.4 C13—N1—C6 130.71 (15)
O3—C10—C11 124.11 (16) C13—N1—H1B 114.6 C1—C6—C5 119.90 (17) H17A—C17—H17C 109.5 C9—C8—C7 120.89 (16) H17B—C17—H17C 109.5
6.4.2 Crystal structure of 1-[2-(2,4-Dichlorophenoxy)acetyl]-4-cyclohexylthiosemicarbazide 88d
Fig. 6.19: Crystal structure of 1-[2-(2,4-Dichlorophenoxy) acetyl]-4-
cyclohexylthiosemicarbazide 88d
215
In 1-[2-(2,4-dichlorophenoxy)acetyl]-4-cyclohexylthiosemicarbazide (Fig. 6.19), the bond lengths and angles are in normal ranges. The thiosemicabazide
group is approximately planar and forms a dihedral angles of 88.03 (5)° with the
benzene ring. The crystal structure is stabilized by intermolecular N—-H···O and
N—H··· S hydrogen bonding. Dipole–dipole and van der Waals interactions are
also effective in the molecular packing in the crystal structure.
Crystal data
C15H19Cl2N3O2S F000 = 784 Mr = 376.29 Dx = 1.476 Mg m−3
Dm = 1.429 Mg m−3 Dm measured by not measured
Monoclinic, P21/c Melting point: 462(1) K Hall symbol: -P 2ybc Mo Kα radiation λ = 0.71073 Å a = 15.4180 (16) Å Cell parameters from 2164 reflections b = 12.1530 (13) Å θ = 2.1–27.5º c = 9.285 (1) Å µ = 0.52 mm−1 β = 103.299 (2) º T = 100 (2) K V = 1693.1 (3) Å3 Block, colorless Z = 4 0.25 × 0.20 × 0.15 mm
Geometric parameters (°A, °)
Selected bond lengths:
C1—O1 1.367 (3) C10—C11 1.511 (4) C1—C2 1.387 (4) C7—H7A 0.9900
C8—O2 1.222 (3) C8—N1 1.350 (3) N1—N2 1.382 (3) C10—N3 1.475
This Part of the chapter has been published: Ghulam Qadeer, Nasim Hasan
Rama, Mohammad Azad Malik, Javeed Akhtar and James Raftery; Crystal
structure of 1-[2-(2,4-Dichloro-phenoxy)acetyl]-4-cyclohexylthiosemicarbazide,
Acta Cryst. 2007, E63, o3503.
216
Selected bond angles: O1—C1—C2 125.0 (2) C12—C11—H11A 109.6 N1—C8—C7 115.0 (2)
C14—C15—H15B 109.6 N2—N1—H1 119.3 N3—C10—C15 110.4 (2)
Selected torsional bond angles: O1—C1—C2—C3 179.1 (2) C11—C12—C13—C14 55.5 (3) C6—C1—C2—C3 −0.5 (4) N2—C9—N3—C10 −176.2 O1—C1—C6—Cl2 1.4 (3)
S1—C9—N3—C10 4.4 (4) C2—C1—C6—Cl2 −178.9 C11—C10—N3—C9 87.9 (3) O1—C7—C8—O2 −173.6 C10—C11—C12—C13 −57.3 (3)
Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A N2—H2A···O2i 0.88 2.06 2.926 (3) 166 N3—H3A···S1ii 0.88 2.60 3.430 (2) 157
Symmetry codes: (i) x, −y+1/2, z+1/2; (ii) x, −y+1/2, z−1/2
6.4.3 Crystal structure of 1-[3-(4-methoxyphenyl) propanoyl]-4-(2-methoxyphenyl)thiosemicarbazide (88o)
Fig. 6.20: Crystal structure of 1-[3-(4-Methoxyphenyl)propanoyl]-4-(2-
methoxyphenyl)thiosemicarbazide 880
217
Crystal data C18H21N3O3S Z = 4 Mr = 359.44 F000 = 760 Triclinic, P1 Dx = 1.322 Mg m−3 a = 9.0495 (16) Å Mo Kα radiation λ = 0.71073 Å b = 13.397 (2) Å Cell parameters from 3145 reflections c = 15.364 (3) Å θ = 2.3–26.3º α = 78.810 (3) º µ = 0.20 mm−1 β = 87.164 (3) º T = 100 (2) K γ = 81.269 (3) º Block, colourless V = 1805.7 (6) Å3 0.50 × 0.30 × 0.30 mm 6.4.4 Crystal structure of 1-(3,5-Difluorobenzoyl)-4-cyclohexyl-
thiosemicarbazide (88a)
Fig. 6.21: Crystal structure of 1-(3,5-Difluorobenzoyl)-4-
cyclohexylthiosemicarbazide 88a
Crystal data C16H23F2N3O2S2 Z = 6 Mr = 391.49 F000 = 1236 Hexagonal, P6 (1) Dx = 1.351 Mg m−3 a = 13.0740 (13) Å Mo Kα radiation λ = 0.71073 Å b = 13.0740 (13) Å Cell parameters from 1479 reflections
218
c = 19.507 (4) Å θ = 3.3–19.3º α = 90º µ = 0.31 mm−1 β = 90º T = 100 (2) K γ = 120º Block, white V = 2887.6 (7) Å3 0.20 × 0.20 × 0.05 mm
Geometric parameters (°A, °)
Selected bond lengths: C11—H11A 0.9900 C2—H2 0.9500 C12—H12A 0.9900 C3—F1 1.361 (4) C12—H12B 0.9900
C3—C4 1.379 (5) C13—C14 1.552 (5) C4—C5 1.362 (5) C13—H13A 0.9900
Selected bond angles: C2—C1—C6 119.6 (4) C13—C12—C11 110.3 (4) C2—C1—C7 124.1 (4) C12—C13—H13A 109.5 N3—C9—C14 110.1 (3) S2—C16—H16A 109.5 N3—C9—C10 112.2 (3)
S2—C16—H16B 109.5 C14—C9—C10 110.6 (3) H16A—C16—H16B 109.5 C11—C10—C9 110.3 (3) H11A—C11—H11B 108.0 C15—S2—C16 98.2 (3)
Selected torsional bond angles: C6—C1—C2—C3 0.0 (6) C14—C9—C10—C11 57.6 (4) C7—C1—C2—C3 175.6 (4) F2—C5—C6—C1 −179.3 N3—C8—N2—N1 −4.3 (6) C2—C1—C6—C5 −0.4 (6)
S1—C8—N2—N1 179.4 (3) C7—C1—C6—C5 −176.3 C14—C9—N3—C8 −151.6 C6—C1—C7—N1 −170.9 C10—C9—N3—C8 84.8 (5) N3—C9—C10—C11 −179.1
Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A N1—H1···O2 0.88 1.91 2.769 (4) 165 N1—H1···S2 0.88 3.01 3.738 (3) 141 N2—H2A···O2i 0.88 2.15 2.882 (4) 140 N3—H3···O1ii 0.88 1.97 2.823 (4) 163
Symmetry codes: (i) x−y, x−1, z+1/6; (ii) y+1, −x+y+1, z−1/6
219
6.5 Synthesis of Substituted 1,2,4-Triazol-3- thiones 89(a-t)
The respective 1,2,4-triazol-3-thiones 89(a-t) were synthesized by
refluxing the thiosemicarbazide in 4N aqueous sodium hydroxide solution307-308.
The products were purified by recrystallization from an appropriate solvent.
[Yield = 42 - 75%]
O
NH
HN
S
HN
R′
88(a-w)
X
RNaOH
NN
N SH
R′89(a-t)
X
R
Scheme 6.6: Synthesis of substituted Triazoles 89 (a-t)
89a. X = OCH2 R = 2,4-Cl2 R′ = -C6H11 89b. X = (CH2)2 R = 3,4,5-(OCH3)3 R′ = -C6H11 89c. X = 0 R = 3,5-F2 R′ = -C6H11 89d. X = 0 R = 3,5-(OCH3)2 R′ = -C6H11 89e. X = (CH2)2 R = 3,4,5-(OCH3)3 R′ = 2-MeO(C6H4)- 89f. X = 0 R = 3,5-(OCH3)2 R′ = 2-MeO(C6H4)- 89g. X = 0 R = 2,5-F2 R′ = 2-MeO(C6H4)- 89h. X = OCH2 R = 2,4-Cl2 R′ = 2-MeO(C6H4)- 89i. X = 0 R = 3,5-F2 R′ = 2-MeO(C6H4)- 89j. X = OCH2 R = 4-Br R′ = 2-MeO(C6H4)- 89k. X = (CH2)2 R = 4-OCH3 R′ = 2-MeO(C6H4)- 89l. X = O(CH2)2 R = 2,4-Cl2 R′ = 2-MeO(C6H4)- 89m. X = (CH2)2 R = 3,4,5-(OCH3)3 R′ = 4-MeO(C6H4)- 89n. X = 0 R = 3,5-(OCH3)2 R′ = 4-MeO(C6H4)- 89o. X = 0 R = 2,5-F2 R′ = 4-MeO(C6H4)- 89p. X = OCH2 R = 2,4-Cl2 R′ = 4-MeO(C6H4)- 89q. X = 0 R = 3,5-F2 R′ = 4-MeO(C6H4)- 89r. X = OCH2 R = 4-Br R′ = 4-MeO(C6H4)- 89s. X = (CH2)2 R = 4-OCH3 R′ = 4-MeO(C6H4)- 89t. X = O(CH2)2 R = 2,4-Cl2 R′ = 4-MeO(C6H4)-
220
Mechanism
R NH
HN N
R'
O
S
OH-
H -H2O
R NH
HN N
R'
O
S-
NHHN
N SR
-O
R'H
OH
NHN
N SRHO
R'
H
-H2O
NHN
N SR
R' Scheme 6.7: Mechanism of dehydrative cyclization of Thiosemicarbazide in
basic medium
The physical and IR spectral data of compounds 88(a-t) is tabulated in table 6.9. The cyclization of thiosemicarbazides to 1,2,4--triazol-3-thiones was
indicated in the IR spectrum by the appearance of a single absorption for N–H
and disappearance of carbonyl absorption. In addition, a band appeared in the
region 1531-1481 cm-1 attributed to C=N group. The absorption bands in the
region of 1601-1411cm-1 were assigned to C=C absorptions of the aromatic ring.
Formation of the triazoles 89(a-t) was confirmed by 1H NMR. In 1H NMR
signals for N-H proton of triazoles appeared at the range of 13.0-14.5 ppm.
These protons gave broad signal and are highly deshielded. Disappearance of
other signals of N-H proton confirmed the formation of the triazoles. 1H NMR data
of the triazoles 89f is given in table 6.10. The formation of the triazoles 89(a-t) was also confirmed by disappearance of peak due to C=O in the range of 155-
165 ppm in 13C NMR spectrum. A new peak due to C=N appeared in the range of
158-168ppm. The 13C NMR spectral data of the triazole 89f is also given in the
table 6.10.
221
Table 6.9: Physical and IR spectral data of 1,2,4-Triazoles 89(a-t)
νmax (cm-1) Compd. mp (°C) Yield *Rf
N-H Sp3 C-H
C=N C=C C=S
89a 178-180 58% 0.36 3409 3038-2873 1509 1591-1484 1271
89b 234-235 61% 0.31 3401 3032-2863 1501 1601-1471 1276
89c 221-223 67% 0.25 3411 3018-2853 1516 1589-1481 1255
89d 120-122 51% 0.36 3431 3090-2893 1521 1571-1454 1266
89e 218-219 54% 0.31 3403 3038-2873 1511 1601-1455 1256
89f 197-198 49% 0.35 3391 3108-2907 1501 1581-1474 1241
89g 133-134 75% 0.34 3361 3056-2940 1531 1584-1484 1244
89h 201-202 58% 0.40 3419 3081-2813 1498 1586-1456 1286
89i 169-170 61% 0.38 3307 3015-2903 1507 1588-1414 1247
89j 154-155 67% 0.36 3333 3101-2804 1513 1594-1384 1271
89k 204-205 51% 0.31 3291 3104-2903 1521 1571-1464 1265
89l 165-166 54% 0.35 3341 3054-2813 1491 1601-1484 1279
89m 169-170 56% 0.34 3249 3038-2933 1481 1551-1481 1257
89n 153-154 75% 0.39 3289 3031-2873 1497 1571-1464 1275
89o 173-175 58% 0.36 3381 3001-2874 1502 1601-1414 1245
89p 222-223 42% 0.31 3341 3018-2813 1514 1587-1411 1255
89q 229-230 66% 0.35 3349 3008-2803 1481 1571-1454 1248
89r 156-158 75% 0.34 3425 3108-2913 1521 1551-1435 1247
89s 102-103 58% 0.29 3401 3010-2870 1519 1601-1489 1276
89t 208-209 42% 0.28 3388 3038-2873 1505 1581-1444 1245
*Petroleum ether: acetone; (6:4)
222
Table 6.10: 1H NMR and 13C NMR data of the triazole 89f
NNH
N
SO
O
1
23
4
5 6
1
23
4
56
O ′
′′
′
′ ′
A
B
Carbon 1H NMR 13C NMR
NH 14.07 (1H, s) ----
C=S ---- 169.41
C=N ---- 151.00
1 ---- 121.42
2 ---- 155.07
3 7.39(1H, dd, J = 7.8, 1.5Hz) 113.37
4 7.09(1H, ddd, J = 7.5, 1.2Hz) 123.63
5 7.49(1H, ddd, J = 9.0,1.5Hz) 121.42
6 7.17(1H, dd, J = 7.8, 0.6Hz) 128.00
1′ ---- 121.42
2′ 6.45(1H, d, J = 2.1Hz) 105.58
3′ ---- 160.66
4′ 6.52 (1H, dd, J = 2.4Hz) 102.76
5′ ---- 160.66
6′ 6.45(1H, d, J = 2.1Hz), 105.58
OCH3 (B) 3.78 (6H, s) 56.65
OCH3 (A) 3.63 (3H, s) 55.31
223
In mass spectrum of 5-(3,5-Dimethoxyphenyl)-4-(2-methoxyphenyl)-4H-
1,2,4-triazole-3-thione 89f, the molecular ion peak appeared at m/z 343 which
confirmed the formation of 5-(3,5-Dimethoxyphenyl)-4-(2-methoxyphenyl)-4H-
1,2,4-triazole-3-thione 89f and base peak appeared at m/z 310 (Scheme 6.8)
NHN
NS O
O
O
(m/z = 343, 60.0 %)
NN
NHS O
O
O
NN
N O
O
O-SH
(m/z = 310, 100 %)
N
N O
O
O
NC
SH-
(m/z = 284, 06.7 %)
N
N O
O
(m/z = 177, 42.2 %)N
NO
O
O
(m/z = 178, 03.2 %)N
O
O
HN
NCSO
-H
(m/z = 163, 06.5 %)N
O
O
NO
O
N
N SHO
NC
SH-
NHO-H
N
N SO
(m/z = 148, 22.5 %)
(m/z = 180, 02.5 %)
(m/z = 122, 06.0 %)
(m/z = 179, 02.0 %)
O
+ +
+
++
.
.
.
.
.
+ . + .
+ .
+ .
+ . .
Scheme 6.8: Mass fragmentation pattern of Triazole 89f
224
Finally the structure of 5-(3,5-Dimethoxyphenyl)-4-(2-methoxyphenyl)-4H-
1,2,4-triazole-3-thione 89f was confirmed by XRD analysis, details are given
below.
6.5.1 Crystal structure of 4-(2-methoxyphenyl)-5-(3,5-dimethoxy-
phenyl)-2H-1,2,4-triazole-3(4H)-thione (89f)
Fig. 6.22: Crystal structure of 4-(2-Methoxyphenyl)-5-(3,5-dimethoxyphenyl)-2H-
1,2,4-triazole-3(4H)-thione 89f
225
The C1=S1 bond length [1.6782 (14) A °] compares with 1.6773 (19) A ° in
4-(4-chlorophenyl)-3-(furan-2-yl)-1H-1,2,4- triazole-5(4H)-thione309 and 1.668 (5)
A °in 4-amino-3-(1,2,3,4,5-pentahydroxypentyl)-1H-1,2,4-triazole-5(4H)-thione310.
In the triazole ring, the N2 ═C1 bond [1.3385 (17) A °] shows double-bond
character. In the crystal structure, all bond lengths and angles are comparable
with those observed in related structures309-310. The triazole ring is planar within
0.002 A °. the triazole group is almost idealy planar. 2-methoxyphenyl ring is
almost perpendicular and 3,5-dimethoxyphenyl ring is planar to the triazole ring.
It form inversion related dimers via N—H···S hydrogen bonds. The structure is
further stabilized by intermolecular-stacking interactions down the b axis. N2—
H2···S1 hydrogen bonds link molecules of title compound into infinite chains
extending along the b axis of the unit cell.
Crystal data C17H17N3O3S Z = 2 Mr = 343.40 F000 = 360 Triclinic, P1 Dx = 1.425 Mg m−3
Dm = 1.411 Mg m−3 Dm measured by not measured
Hall symbol: -P1 Melting point: 470(1) K a = 8.8950 (8) Å Mo Kα radiation λ = 0.71073 Å b = 9.3510 (8) Å Cell parameters from 3874 reflections c = 10.5510 (9) Å θ = 2.3–28.3º α = 94.3650 (10) º µ = 0.22 mm−1 β = 102.4520 (10) º T = 100 (2) K γ = 108.9240 (10) º Block, white V = 800.44 (12) Å3 0.35 × 0.30 × 0.25 mm
Geometric parameters (°A, °)
Selected bond lengths: C1—N2 1.3385 (17) C10—C11 1.3862 (19) C1—N1 1.3785 (16) C10—C15 1.4084 (18) C1—S1 1.6782 (14)
C16—O2 1.4337 (16) C5—H5 0.9500 N2—N3 1.3674 (15) C9—H9B 0.9800 N2—H2 0.8800
226
Selected bond angles: N2—C1—N1 103.64 (11) C12—C11—H11 120.6 N2—C1—S1 128.55 (10) C12—C13—C14 118.86 (13) N1—C2—C10 128.40 (11)
C12—C13—H13 120.6 C8—C3—C4 121.02 (12) C14—C13—H13 120.6 C10—C11—H11 120.6 C14—O3—C17 116.86 (11)
Selected torsional bond angles: C8—C3—C4—O1 −179.51 (12) N1—C3—C4—O1 −2.65 (17) C4—C3—N1—C2 72.67 (17)
N1—C2—N3—N2 −0.04 (14) C15—C14—O3—C17 1.57 (19) C13—C14—C15—C10 0.75 (19)
Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A N2—H2···S1i 0.88 2.45 3.2624 (12) 154
Symmetry codes: (i) −x+1, −y+1, −z+1.
This Part of the chapter has been published: Ghulam Qadeer, Nasim Hasan
Rama, Javeed Akhtar, Mohammad Azad Malik, and James Raftery; Crystal
structure of 3-(3,5-dimethoxyphenyl)-4-(2-methoxyphenyl)-2H-1,2,4-triazole-
3(4H)-thione; Acta Cryst. 2007, E63, o3629.
227
6.5.2 Crystal Structure of 5-(3,4,5-trimethoxyphenylethyl)-4-(2-
methoxyphenyl)-2H-1,2,4-triazole-3(4H)-thione (89e)
Fig. 6.23: Crystal Structure of 5-(3,4,5-Trimethoxyphenylethyl)-4-(2-
methoxyphenyl)-2H-1,2,4-triazole-3(4H)-thione 89e
228
Crystal data C20H23N3O4S Z = 2 Mr = 401.47 F000 = 424 Triclinic, P1 Dx = 1.294 Mg m−3 Hall symbol: -P1 Melting point: 491(1) K a = 8.6368 (6) Å Mo Kα radiation λ = 0.71073 Å b = 10.5422 (7) Å Cell parameters from 5348 reflections c = 11.6944 (8) Å θ = 2.4–26.4º α = 91.7330 (10)º µ = 0.19 mm−1 β = 92.9550 (10)º T = 100 (2) K γ = 104.0750 (10)º Rectangular, colourless V = 1030.44 (12) Å3 0.55 × 0.35 × 0.30 mm
Geometric parameters (°A, °) Selected bond lengths: S1—C2 1.6775 (14) C7—C8 1.393 (2) O1—C7 1.3650 (18) C14—C19 1.3792 (19) N3—C1 1.3789 (18)
C1—C3 1.4892 (18) C16—C17 1.388 (2) C3—C4 1.5266 (19) C17—C18 1.384 (2) C4—C5 1.5122 (18)
Selected bond angles: C7—O1—C11 116.44 (13) C5—C10—H10 120.3 C8—O2—C12 113.01 (11) C9—C10—H10 120.3 C5—C6—C7 120.17 (13) C9—C8—C7 119.96 (12)
O4—C20—H20C 109.5 O3—C9—C10 124.48 (13) H20A—C20—H20C 109.5 C8—C9—C10 120.34 (13) H20B—C20—H20C 109.5 C5—C10—C9 119.45 (13)
Selected torsional bond angles: C1—N1—N2—C2 1.85 (15) C6—C7—C8—C9 −0.4 (2) N2—N1—C1—N3 −0.12 (15) C13—O3—C9—C8 −172.13 (13) N2—N1—C1—C3 −178.15 (13)
C2—N3—C14—C19 72.32 (18) C14—N3—C2—S1 3.5 (2) C1—N3—C14—C19 −107.68 (15) N1—C1—C3—C4 0.6 (2) C2—N3—C14—C15 −107.91 (15)
229
Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A N2—H2N···O2i 0.878 (17) 1.890 (18) 2.7558 (15) 168.4 (15)
Symmetry codes: (i) x, y, z+1
6.5.3 Crystal Structure of 3-(4-Methoxyphenethyl)-4-(2-methoxy- phenyl)-1H-1,2,4-triazole-5(4H)-thione (89k)
Fig. 6.24: Crystal Structure of 3-(4-Methoxyphenethyl)-4-(2-methoxyphenyl)-1H-
1,2,4-triazole-5(4H)-thione 89k
Crystal data C18H19N3O2S Z = 4 Mr = 341.42 F000 = 720 Monoclinic, P21/n Dx = 1.279 Mg m−3 Hall symbol: -P 2yn Mo Kα radiation λ = 0.71073 Å a = 8.3664 (4) Å µ = 0.20 mm−1 b = 19.2172 (10) Å T = 293 (2) K
230
c = 11.2800 (6) Å Block, colorless β = 102.1330 (10)º 0.38 × 0.30 × 0.24 mm V = 1773.07 (16) Å3
Geometric parameters (°A, °)
Selected bond lengths: C1—O1 1.431 (2) C10—H10A 0.9700 C1—H1A 0.9600 C10—H10B 0.9700 C1—H1B 0.9600
C11—H11B 0.9700 C2—C3 1.386 (2) C12—C17 1.365 (2) C2—C7 1.389 (2) C12—C13 1.382 (2)
Selected bond angles: O1—C1—H1A 109.5 C10—C11—H11A 109.5 O1—C1—H1B 109.5 C12—C11—H11B 109.5
H1A—C1—H1B 109.5 C14—C13—H13A 119.1 C12—C11—H11A 109.5
Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A N2—H1···S1i 0.823 (18) 2.468 (18) 3.2864 (14) 173.3 (19) C6—H6A···O2ii 0.93 2.57 3.278 (2) 133
Symmetry codes: (i) −x, −y+2, −z+2; (ii) −x+3/2, y+1/2, −z+3/2.
This Part of the chapter has been published: Ghulam Qadeer, Muhammad
Hanif, Nasim Hasan Rama and Wai-Yeung Wong; Crystal structure of 3-(4-
Methoxyphenethyl)-4-(2-methoxyphenyl)-1H-1,2,4-triazole-5(4H)thione; Acta
Cryst. 2007, E63, o3502.
231
6.5.4 Crystal Structure of 3-(4-bromophenoxymethyl)-4-(4-methoxyphenyl)-1H-1,2,4-triazole-5(4H)-thione (89r)
Fig. 6.25: Crystal Structure of 3-(4-Bromophenoxymethyl)-4-(4-methoxyphenyl)-
1H-1,2,4-triazole-5(4H)-thione 89r
Crystal data C16H14BrN3O2S Z = 2 Mr = 392.27 F000 = 396 Triclinic, P1 Dx = 1.659 Mg m−3 a = 7.4590 (6) Å Mo Kα radiation λ = 0.71073 Å
232
b = 7.7920 (7) Å Cell parameters from 3998 reflections c = 15.2870 (13) Å θ = 2.7–28.2º α = 97.3230 (10)º µ = 2.76 mm−1 β = 96.3740 (10)º T = 100 (2) K γ = 114.9470 (10)º Block, white V = 785.49 (12) Å3 0.20 × 0.20 × 0.15 mm
Geometric parameters (°A, °)
Selected bond lengths: Br1—C1 1.906 (2) C9—S1 1.677 (2) C1—C6 1.379 (3) C10—C11 1.378 (3)
N1—N2 1.374 (2) C9—N2 1.340 (3) N2—H2A 0.8800 C9—N3 1.379 (3)
Selected bond angles: C6—C1—C2 121.5 (2) C15—C10—N3 119.38 (18) C6—C1—Br1 118.95 (17) C10—C11—C12 119.71 (19)
C4—O1—C7 118.82 (16) C11—C10—C15 121.17 (19) C13—O2—C16 117.65 (17) C11—C10—N3 119.44 (18)
Selected torsional bond angles: C6—C1—C2—C3 −1.1 (3) N3—C8—N1—N2 −0.2 (2) Br1—C1—C2—C3 177.71 (17)
C7—C8—N1—N2 177.55 (19) C1—C2—C3—C4 0.2 (3) N3—C9—N2—N1 −0.3 (2)
Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A N2—H2A···S1i 0.88 2.37 3.2446 (18) 174
Symmetry codes: (i) −x, −y+2, −z+1
233
6.5.5 Crystal Structure of 3-(2,4-dichlorophenoxymethyl)-4-(4-methoxyphenyl)-1H-1,2,4-triazole-5(4H)-thione (89p)
Fig. 6.26: Crystal Structure of 3-(2,4-Dichlorophenoxymethyl)-4-(4-
methoxyphenyl)-1H-1,2,4-triazole-5(4H)-thione 89p
Crystal data C16H13Cl2N3SO2 Z = 2 Mr = 382.64 F000 = 394 Triclinic, P1 Dx = 1.588 Mg m−3 a = 7.6529 (12) Å Mo Kα radiation λ = 0.71073 Å b = 7.6658 (12) Å Cell parameters from 1128 reflections c = 15.520 (3) Å θ = 2.9–27.4º α = 94.180 (3)º µ = 0.58 mm−1 β = 100.835 (3)º T = 100 (2) K γ = 113.856 (3)º Block, white V = 806.5 (2) Å3 0.23 × 0.12 × 0.10 mm
234
Geometric parameters (°A, °)
Selected bond lengths C1—O2 1.375 (4) C9—S1 1.674 (3) C1—C6 1.389 (4) C10—C11 1.371 (4) C1—C2 1.396 (4) C10—C15 1.385 (4)
C10—N3 1.453 (4) C2—Cl1 1.731 (3) C11—C12 1.387 (4) C3—C4 1.387 (4) C11—H11 0.9500 C3—H3 0.9500
Selected bond angles C3—C2—Cl1 119.2 (3) C11—C12—C13 120.1 (3) C1—C2—Cl1 119.7 (2) C11—C12—H12 119.9 C2—C3—C4 118.3 (3) C13—C12—H12 119.9 C2—C3—H3 120.9
O1—C13—C14 115.3 (3) C4—C3—H3 120.9 O1—C13—C12 124.7 (3) C5—C4—C3 121.7 (3) C14—C13—C12 120.0 (3) C5—C4—Cl2 120.0 (3)
Selected torsional bond angles Cl1—C2—C3—C4 179.3 (2) C8—N1—N2—C9 0.4 (3) C2—C3—C4—C5 −1.9 (5) N2—C9—N3—C8 0.1 (3) C2—C3—C4—Cl2 177.6 (2) S1—C9—N3—C8 179.7 (2)
C3—C4—C5—C6 2.1 (5) S1—C9—N3—C10 0.6 (5) C4—C5—C6—C1 −0.5 (5) N1—C8—N3—C9 0.2 (4) O2—C1—C6—C5 179.2 (3) C2—C1—C6—C5 −1.3 (5)
Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A N2—H2···Cl3i 0.88 2.39 3.269 (3) 174
Symmetry codes: (i) −x+1, −y+1, −z
235
6.6 Synthesis of thiadiazoles
Thiadiazoles 90(a-j) were synthesized by treating thiosemicarbazides with
concentrated H2SO4 at low temperature. Stirring continued over night and the
mixture was poured on crushed ice yielded thiadiazoles311. These thiadiazoles
were purified by recrystallization with suitable solvent. Physical data of these
thiadiazoles is given in table 6.11.
[Yield = 49 - 61%]
O
NH
HN
S
HN
R′
88(a-w)
X
RConc. H2SO4
NN
S NH
90(a-j)
X
RR′
Scheme 6.9: Synthesis of substituted Thidiazoles 90 (a-j)
90a. X = OCH2 R = 2,4-Cl2 R′ = -C6H11 90b. X = 0 R = 3,5-F2 R′ = -C6H11 90c. X = 0 R = 3,5-(OCH3)2 R′ = 2-OMe (C6H4)- 90d. X = 0 R = 3,4-(OCH3)2 R′ = 2-OMe (C6H4)- 90e. X = (CH2)2 R = 3,4,5-(OCH3)3 R′ = 2-OMe (C6H4)- 90f. X = O(CH2)2 R = 2,4-Cl2 R′ = 2-OMe (C6H4)- 90g. X = O(CH2)3 R = 2,4-Cl2 R′ = 2-OMe (C6H4)- 90h. X = OCH2 R = 2,4-Cl2 R′ = 2-OMe (C6H4)- 90i. X = O(CH2)3 R = 2,4-Cl2 R′ = 4-OMe (C6H4)- 90j. X = OCH2 R = 2,4-Cl2 R′ = 4-OMe (C6H4)-
Mechanism
R NH
O: HN
HN
SR'
H+
R NH
OHN
HN
SR'
+H
S
NHNNH
R'RHO
S
NHNNH
R'R
H2O
H+
+
-H2OS
NNNH
R'R
H
+-H+
S
NNNH
R'R
Scheme 6.10: Mechanism of dehydrative cyclization of the Thiosemicarbazides
in acidic medium
236
Thiadiazoles 90(a-j) were characterized by IR spectra by disappearance
of peaks of thiourea type linkage of thiosemicarbazides. A peak due to N-H
stretch appeared in the range of 3300-3400 -1cm. A carbonyl peak of amide type
linkage also disappeared in the range of 1600-1680 -1cm. C=N stretch appeared
in the range of 1500-1510-1cm. The physical and IR data of thiadiazoles 90(a-j) is given in the table 6.11
Table 6.11: Physical and IR spectral data of Thiadiazoles 90(a-j)
νmax (cm-1) Compd
mp
(°C) Rf
Yield(%)
NH stretch
C-H stretch C=N
stretch C=C
90a 134-135 0.34 49 3301 3038-2873 1501 1601-1384
90b 198-199 0.38 51 3309 3021-2881 1511 1611-1385
90c 147-148 0.36 48 3291 3008-2821 1515 1591-1383
90d 189-191 0.39 58 3241 3018-2913 1521 1601-1414
90e 201-202 0.37 61 3315 3010-2893 1491 1615-1484
90f 178-179 0.35 59 3311 3039-2803 1508 1595-1384
90g 197-198 0.37 55 3206 3008-2870 1523 1594-1481
90h 188-189 0.35 57 3191 3031-2913 1503 1600-1484
90i 145-146 0.37 55 3299 3038-2945 1508 1594-1381
90j 111-112 0.37 54 3401 3058-2870 1531 1597-1394
1H NMR spectra confirmed the cyclization of thiosemicarbazide to
thiadiazole. In 1H NMR signal for N-H proton of thiadiazoles appeared at the
range of 13.0-14.5 ppm. These protons gave broad signals and were highly
deshielded. Disappearance of other signals of N-H proton confirmed the
formation of thiadiazoles. 1H NMR data of a representative thiadiazoles 90c is
given in table 6.12. The formation of thiadiazoles 90(a-j) was also confirmed by 13C NMR data. In 13C NMR, thiadiazoles were characterized by the
237
disappearance of C=O peak of amide in the range of 165ppm. The 13C NMR data
of thiadiazole 90c is also given in the table 6.12.
Table 6.12: 1H and 13C NMR data of 2-Methoxyphenylamino-5-(3′,5′-
dimethoxyphenyl)-1,3,4-thiadiazole 90c
S
NN
O
O
HN
O1 2
34
56
123
4
5 6
′′′
′
′ ′
AB
Carbons 1H NMR 13C NMR
NH 8.97(1H, s) ----
C=N ---- 178.45
C=N ---- 155.27
1 ---- 134.09
2 ---- 150.84
3 7.29(1H, dd, J = 7.8, 2.0Hz) 116.58
4 7.05(1H, ddd, J = 8.1, 1.8Hz) 121.37
5 7.35(1H, ddd, J = 8.4,1.5Hz) 123.54
6 7.17 (1H, dd, J = 7.5, 1.6Hz) 116.58
1′ ---- 135.23
2′ 6.39(1H, d, J = 2.1Hz) 105.71
3′ ---- 165.54
4′ 6.59(1H, dd, J = 4.5, 2.4Hz) 99.71
5′ ---- 165.54
6′ 6.39(1H, d, J = 2.1Hz) 105.71
OCH3 (B) 3.77(6H, s) 56.54
OCH3 (A) 3.73(3H, s) 55.78
238
In mass spectrum of 2-methoxyphenylamino-5-(3′,5′-dimethoxyphenyl)-
1,3,4-thiadiazole 90c, the molecular ion peak appeared at m/z 347 which
confirmed the formation of 2-methoxyphenylamino-5-(3′,5′-dimethoxyphenyl)-
1,3,4-thiadiazole 90c and base peak appeared at m/z 111 (Scheme 6.11).
N N
SNH
O
O Cl
(m/z = 347,10.0%)
N N
SNH2
O
O
(m/z = 237, 3.5%)
N N
SO
O
(m/z = 222, 2.5%)
-NH2
NS
NH2
NHO
O
(m/z = 74, 23.4%)
Cl
(m/z = 111,100%)
N
SO
O
(m/z = 195, 2%)
-N
SO
O
(m/z = 181, 31.2%)
N
SNH
Cl
(m/z = 184,10%)N
O
O
(m/z = 163, 36.3%)
O
O
(m/z = 137, 4.0%)
NO
O
Cl
CN
NC
O
O
S
NH(m/z = 195, 10.3%)
Cl
CNNH
(m/z = 152, 20.2%)
NCSO
O
-NCl
CHN
(m/z = 138, 21.5%)
+
++
++
-CN
.
..
.+ .
+ .
+ .+ .
+ .
+ .
+ .
+ .
Scheme 6.11: Mass fragmentation pattern of 2-Methoxyphenylamino-5-(3′,5′-
dimethoxyphenyl)-1,3,4-thiadiazole 90c
239
6.7 Synthesis of indolinones
Indolinones 91(a-o) and 92(a-o) were synthesized by treating substituted
hydrazides with haloisatin in ethanol. These indolinones were purified by
recrystallization with suitable solvent. Physical data of these indolinones is given
in table 6.13.
[Yield = 79 - 91%]
X
R
NH
ONH2
NH
O
OR''
NH
O
NR''
HN
X
O
86(a-o) 91(a-ο)92(a-ο)
+
R″ = Cl, Br
C2H5OH
R
91a. X = 0 R = 3,5-F2 R″ = Cl 91b. X = 0 R = 2,5-F2 R″ = Cl 91c. X = 0 R = 2,6-F2 R″ = Cl 91d. X = 0 R = 3,4-(OCH3)2 R″ = Cl 91e. X = 0 R = 3,5-(OCH3)2 R″ = Cl 91f. X = 0 R = 2,4-(OCH3)2 R″ = Cl 91g. X = 0 R = 2,6-(OCH3)2 R″ = Cl 91h. X = CH2 R = 4-Cl R″ = Cl 91i. X = CH2 R = 4-F R″ = Cl 91j. X = (CH2)2 R = 3,4,5(OCH3)3 R″ = Cl 91k. X = (CH2)2 R = 4-OCH3 R″ = Cl 91l. X = OCH2 R = 4-Br R″ = Cl 91m. X = O(CH2)2 R = 2,4-Cl2 R″ = Cl 91n. X = O(CH2)3 R = 2,4-Cl2 R″ = Cl 91o. X = OCH2 R = 2,4-Cl2 R″ = Cl 92a. X = 0 R = 2,5-F2 R″ = Br 92b. X = 0 R = 3,5-F2 R″ = Br 92c. X = 0 R = 2,6-F2 R″ = Br 92d. X = 0 R = 3,4-(OCH3)2 R″ = Br 92e. X = 0 R = 3,5-(OCH3)2 R″ = Br 92f. X = 0 R = 2,4-(OCH3)2 R″ = Br 92g. X = 0 R = 2,6-(OCH3)2 R″ = Br 92h. X = CH2 R = 4-Cl R″ = Br 92i. X = CH2 R = 4-F R″ = Br
240
92j. X = (CH2)2 R = 3,4,5(OCH3)3 R″ = Br 92k. X = (CH2)2 R = 4-OCH3 R″ = Br 92l. X = OCH2 R = 4-Br R″ = Br 92m. X = O(CH2)2 R = 2,4-Cl2 R″ = Br 92n. X = O(CH2)3 R = 2,4-Cl2 R″ = Br 92o. X = OCH2 R = 2,4-Cl2 R″ = Br
All the synthesized compounds were characterized by their physical
constants, IR (Table 6.13), 1H NMR, 13C NMR (Table 6.14), mass spectrometry
and X-ray crystallography. The IR spectra of indolinones exhibitied a strong
absorption in the region 1662-1618 cm-1 was assigned to the carbonyl group of
amide linkage, while absorption band for the secondary NH was observed in the
range 3476-3198 cm-1. The physical and IR spectral data of few indolinones are
given in table 6.13.
Table 6.13: Physical constants and IR spectral data of indolinones 91(a-i)
υmax (cm-1) S.No m.p (°C)
Yield (%)
NH strech
C-H strech
C=O C=N C=C
91a 178-179 89 3281 3084 1710 1677 1623, 1460 91b 165-166 81 3412 3115 1719 1670 1622, 1308 91c 212-213 84 3419 3118 1713 1678 1627, 1278 91d 145-146 70 3252 2936 1711 1674 1594, 1463 91e 112-113 85 3249 2845 1709 1670 1598, 1461 91f 201-202 80 3345 3138 1710 1675 1594, 1468 91g 155-156 85 3255 3132 1712 1670 1592, 1463 91h 139-140 89 3216 2853 1727 1691 1596, 1470 91i 188-189 76 3262 3149 1720 1678 1602, 1449
The formation of indolinones 91(a-o) and 92(a-o) from the respective
hydrazide and haloisatin was confirmed in the 1H NMR spectra by the
appearance of two NH proton signal in the region of 10.0- 12.0ppm. The
structures were futher confirmed by 13C NMR (Table 6.14). In 13C NMR, a signal
at δ 130-140ppm for C=N confirmed the formation of indolinones. The 1H NMR
241
and 13C NMR of a representative indolinone 91a is given in table 6.14. The
detail of spectral data of other indolinones is given in chapter 7 (Experimental). Table 6.14: 1H NMR and 13C NMR spectral data of (Z)-N'-(5-Chloro-2-oxoindolin-
3-ylidene)-3,5-difluorobenzohydrazide 91a
O
NHN
F
F
NH
O
Cl
12
345
67
4a
7a
1
2
34
56
′
′
′′
′′
δ (ppm) and multiplicity Carbon
1H NMR 13C NMR
C=O ---- 171.3 2 ---- 164.2 3 ---- 136.7 4 8.11(1H, s) 127.1 4a ---- 116.8 5 ---- 127.4 6 7.68-7.43(4H, m) 132.8 7 6.93(1H, d, J = 8.7Hz) 126.0 7a ---- 143.3 1′ ---- 141.7
2′ 7.68-7.43(4H, m) 108.0 3′ ---- 164.7 4′ 7.68-7.43(4H, m) 112.2
5′ ---- 164.7 6′ 7.68-7.43(4H, m) 108.0
NH 10.98(1H, s) ----
242
NH 11.78(1H, s) ----
In mass spectrum of (Z)-N'-(5-chloro-2-oxoindolin-3-ylidene)-3,5-difluoro-
benzohydrazide 91a, the molecular ion peak appeared at m/z 307 which
confirmed the formation of (Z)-N'-(5-chloro-2-oxoindolin-3-ylidene)-3,5-
difluorobenzohydrazide 91a and base peak appeared at m/z 194 (Scheme 6.12).
HN
O
NCl
HN
F
FO
-CO
NCl
HN
F
FO
NH
NCl
NHHN
O
NHN
HNCl
-CO
NHCl
NH
Cl
NH
Cl
(m/z = 335, 56.3%, Cl35)
+
(m/z = 307, 10.3%,Cl35)
+
++
+
+ +
(m/z = 166, 52.3%,Cl35)(m/z = 194, 100%,Cl35)
(m/z = 139, 15.3%,Cl35)(m/z = 152, 0.3%,Cl35)
(m/z = 75, 6.3%)(m/z = 115, 3.3%,Cl37)
(m/z = 309, 4.3%,Cl37)
(m/z = 337, 16.3%,Cl37)
(m/z = 168, 12.3%,Cl37)(m/z = 196, 33.2%,Cl37)
(m/z = 154, 0.3%,Cl37)
(m/z = 113, 55.3%,Cl35)
F
FO
F
FO
F
FO
(m/z = 141, 90.3%)
HN
O
NHN
Cl
(m/z = 131, 90.3%,Cl37)
+. +.
.
.
.
243
Scheme 6.12: Mass fragmentation pattern of (Z)-N'-(5-Chloro-2-oxoindolin-3-
ylidene)-3,5-difluorobenzohydrazide (91a) Finally the structure of 91a was confirmed by XRD analysis. The
detail is given below.
6.7.1 Crystal Structure of (Z)-N'-(5-chloro-2-oxoindolin-3-ylidene) -3,5-difluorobenzohydrazide (90a)
Fig. 6.27: Crystal Structure of (Z)-N'-(5-Chloro-2-oxoindolin-3-ylidene)-3,5-
difluorobenzohydrazide (90a)
Crystal data C15H8ClF2N3O2 V = 1365.4 (2) Å3 Mr = 335.69 Z = 4 Monoclinic, P21/c Mo Kα a = 3.7932 (4) Å µ = 0.32 mm−1 b = 30.394 (3) Å T = 293 (2) K c = 11.8600 (11) Å 0.30 × 0.25 × 0.10 mm β = 93.034 (2)º
Geometric parameters (°A, °)
244
Selected bond lengths C1—F1 1.356 (2) C9—O2 1.229 (2) C1—C2 1.359 (3) C9—N3 1.349 (3) C1—C6 1.377 (3) C10—C15 1.366 (3) C2—C3 1.392 (3)
C10—C11 1.400 (3) C2—H2A 0.9300 C10—N3 1.413 (3) C3—C4 1.389 (3) C11—C12 1.385 (3) C3—C7 1.491 (3) C12—C13 1.379 (3)
Selected bond angles C3—C2—H2A 120.5 C12—C11—C8 132.36 (19) C4—C3—C2 119.6 (2) C10—C11—C8 106.79 (17) C4—C3—C7 123.44 (19)
C12—C13—Cl1 119.54 (16) F2—C5—C6 117.9 (2) C14—C13—Cl1 118.77 (17) F2—C5—C4 118.23 (19) C15—C14—C13 120.8 (2)
Selected torsional bond angles C2—C1—C6—C5 −0.3 (4) C11—C10—C15—C14 −0.9 (3) N3—C10—C15—C14 178.6 (2)
C2—C3—C7—O1 9.1 (3) C11—C8—N2—N1 179.2 (2) C9—C8—N2—N1 −2.9 (3)
Hydrogen-bond geometry (Å, °) D—H···A D—H H···A D···A D—H···A N1—H1···O2 0.81 (2) 2.05 (2) 2.747 (3) 144 (2) N3—H2···O1i 0.86 (2) 2.17 (3) 2.890 (2) 142 (2) C15—H15A···O1i 0.93 2.44 3.130 (3) 131
Symmetry codes: (i) x+1, −y+1/2, z+1/2.
245
Chapter-7 EXPERIMENTAL
7.1 Substrates and reagents Substituted carboxylic acids were products of Aldrich. Chloroform and
methanol were supplied by Lab Scan. Diethyl ether and dimethyl sulfoxide were
products of Riedel de Haёn. Magnesium sulphate was obtained from Fluka and
ethanol and ethyl acetate were obtained from commercial sources.
7.2 Purification of solvents All the solvents were used after necessary purification and drying
according to the standard procedures. The dried solvents were stored over
molecular sieves (4oA). A brief account of purification procedures employed is as
follow.
i. Methanol Calcium oxide (250g), freshly activated in a muffle furnace at 300-400 oC,
was introduced into a round bottom flask containing one litre of methanol. It was
refluxed for 6 hours and distilled at 68 oC.
ii. Absolute ethanol Calcium oxide (250g), freshly activated in a muffle furnace at 300-400 oC,
was introduced into a round bottom flask containing one litre of ethanol. It was
refluxed for 6 hours and then distilled at 77-78 oC.
iii. Acetone
Anhydrous calcium chloride (200g) was introduced into a round bottom
flask containing one litre of acetone and it was left for 4~5 hours. Pure acetone
was distilled at 56 oC.
246
iv. Ethylacetate
In one litre of ethylacetate, 50ml of acetic anhydride and 10~15 drops of
conc.H2SO4 were added and refluxed for 5 hours. It was fractionated and treated
with 25g of potassium carbonate. It was filtered and distilled over 40g of calcium
hydride at 77 oC.
v. Diethyl ether
Diethyl ether was first distilled over anhydrous calcium chloride. The
distillate was refluxed on sodium wire, using benzophenone as an indicator.
When the colour of ether turned dark green, the mixture was distilled and stored
over molecular sieves (4 oA).
vi. Dimethyl sulphoxide
Calcium hydride (200g) was added to dimethyl sulfoxide (1000 ml) and
allowed to stand overnight. The solvent was filtered, fractionally distilled over
calcium hydride and stored on molecular sieves (4 oA).
7.3 Instrumentation The Rf-values were determined using pre-coated silica gel aluminium
packed plates Kiesal 60 F254 from Merck (Germany). Melting points of the
compounds were measured in open capillaries using Gallenkamp melting point
apparatus (MP-D) and are uncorrected. The IR spectra were recorded on FTS
3000 MX, Bio-Rad Merlin (Excalibur Model) spectrophotometer. The 1H-NMR
spectra were run on a Bruker 300 MHz NMR spectrometer in DMSO solution
using TMS as an internal standard. MS were recorded on Agilent technologies
6890N (GC) and an inert mass selective detector 5973 mass spectrometer.
247
7.4 General procedure for the synthesis of esters
Substituted benzoic acids (0.2 moles) were dissolved in methanol/ethanol
(50 mL) in a round bottom flask equipped with a reflux condenser and a calcium
chloride guard tube. Concentrated sulfuric acid (2 mmoles) was added and the
reaction mixture subjected to reflux for 8-10 hours and monitored by thin layer
chromatography. After completion of the reaction, the excess alcohol was
removed under reduced pressure and resulting oil was poured into water. The
oily layer was separated and the aqueous portion extracted with diethyl ether (3 ×
50 mL). The combined organic layers were washed with dilute solution of sodium
carbonate (100 mL) to remove unreacted acid. The organic layer was dried over
anhydrous sodium sulphate. The solvent was removed on a rotary evaporator
after filtration.
Methyl 2,5-difluorobenzoate (85a): Yield: 92%; Oil; Rf: 0.77 (Petroleum ether :
ethylacetate, 4:1); IR (NaCl Cell, νmax, cm-1): 3060 (sp2 CH Stretching), 2942 (sp3
CH Stretching), 1726 (C=O), 1585 (C=C), 1290 (C-O).
Methyl 3,5-difluorobenzoate (85b): Yield: 85%; Oil; Rf: 0.88 (Petroleum ether :
ethylacetate, 4:1)); IR (NaCl Cell, νmax, cm-1): 3058 (sp2 CH Stretching), 2940
(sp3 CH Stretching), 1727 (C=O), 1582 (C=C), 1309 (C-O). Methyl 2,6-difluorobenzoate (85c): Yield: 87%; Oil; Rf: 0.74 (Petroleum ether :
ethylacetate, 4:1); IR (NaCl Cell, νmax, cm-1): 3061 (sp2 CH Stretching), 2943 (sp3
CH Stretching), 1728 (C=O), 1590 (C=C), 1313 (C-O). Methyl 3,4-dimethoxybenzoate (85d): Yield: 79%; m.p: 60-61 °C (lit. 59-62°C);
Rf: 0.57 (Petroleum ether : ethylacetate, 4:1); IR (KBr, νmax, cm-1): 3089 (sp2 CH
Stretching), 2901 (sp3 CH Stretching), 1740 (C=O), 1610 (C=C), 1304 (C-O).
248
Methyl 3,5-dimethoxybenzoate (85e): Yield: 77%; m.p: 40-41°C (Lit. 41-43 °C);
Rf: 0.67 (Petroleum ether : ethylacetate, 4:1); IR (KBr, νmax, cm-1): 3063 (sp2 CH
Stretching), 2889 (sp3 CH Stretching), 1735 (C=O), 1625 (C=C), 1315 (C-O). Methyl 2,4-dimethoxybenzoate (85f): Yield: 80%; Oil; Rf: 0.76 (Petroleum ether
: ethylacetate, 4:1) (solvent for recrystallization: ethanol); IR IR (NaCl Cell, νmax,
cm-1): 3085 (sp2 CH Stretching), 2890 (sp3 CH Stretching), 1738 (C=O), 1596
(C=C), 1325 (C-O). Methyl 2,6-dimethoxybenzoate (85g): Yield: 81%; m.p: 87-88oC (Lit. 87-90oC)
[267]; Rf: 0.72 (Petroleum ether : ethylacetate, 4:1); IR (KBr, νmax, cm-1): 3030
(sp2 CH Stretching), 2989 (sp3 CH Stretching), 1725 (C=O), 1645 (C=C), 1296
(C-O).
Ethyl 2-(3-methoxyphenyl)acetate (85h): Yield: 78%; m.p: 125-126oC; Rf: 0.70
(Petroleum ether : ethylacetate, 4:1); IR (KBr, νmax, cm-1): 3056 (sp2 CH
Stretching), 2910 (sp3 CH Stretching), 1711 (C=O), 1569 (C=C), 1285 (C-O).
Methyl 2-(4-chlorophenyl)acetate (85i): Yield: 83%; Oil ; Rf: 0.71 (Petroleum
ether: ethylacetate, 4:1); IR (NaCl Cell, νmax, cm-1): 3101 (sp2 CH Stretching),
3001 (sp3 CH Stretching), 1729 (C=O), 1578 (C=C), 1387 (C-O). Methyl 2-(4-flourophenyl)acetate (85j): Yield: 89%; Oil; Rf: 0.71 (Petroleum
ether : ethylacetate, 4:1); IR (NaCl Cell, νmax, cm-1): 3048 (sp2 CH Stretching),
3010 (sp3 CH Stretching), 1725 (C=O), 1596 (C=C), 1269 (C-O). Methyl 3-(3,4,5-trimethoxyphenyl)propionate (85k): Yield: 79%; m.p: 88-90 oC;
Rf: 0.71 (Petroleum ether: ethylacetate, 4:1); IR (KBr, νmax, cm-1): 3084 (sp2 CH
Stretching), 2986 (sp3 CH Stretching), 1751 (C=O), 1586 (C=C), 1316 (C-O).
249
Methyl 3-(4-methoxyphenyl)propionate (85l): Yield: 89%; m.p: 36-37 oC (Lit.
37-41oC); Rf: 0.71 (Petroleum ether : ethylacetate, 4:1); IR (KBr, νmax, cm-1): 3104
(sp2 CH Stretching), 2916 (sp3 CH Stretching), 1742 (C=O), 1610 (C=C), 1296
(C-O). Methyl 2-(4-bromophenoxy)acetate (85m): Yield: 82%; Oil ; Rf: 0.71
(Petroleum ether : ethylacetate, 4:1); IR (NaCl Cell, νmax, cm-1): 3089 (sp2 CH
Stretching), 2986 (sp3 CH Stretching), 1726 (C=O), 1566 (C=C), 1310 (C-O). Methyl 2-(2,4-dichlorophenoxy)propionate (85n): Yield: 83%; Oil; Rf: 0.71
(Petroleum ether: ethylacetate, 4:1); IR (NaCl Cell, νmax, cm-1): 3125 (sp2 CH
Stretching), 2896 (sp3 CH Stretching), 1722 (C=O), 1577 (C=C), 1296 (C-O).
Methyl 4-(2,4-dichlorophenoxy)butyrate (85o): Yield: 82%; Oil; Rf: 0.71
(Petroleum ether : ethylacetate, 4:1); IR (NaCl Cell, νmax, cm-1): 3110 (sp2 CH
Stretching), 2975 (sp3 CH Stretching), 1745 (C=O), 1587 (C=C), 1283 (C-O). Methyl 2-(2,4-dichlorophenoxy)acetate (85p): Yield: 78%; m.p: Rf: 0.71
(Petroleum ether : ethylacetate, 4:1); IR (NaCl Cell, νmax, cm-1): 3056 (sp2 CH
Stretching), 2974 (sp3 CH Stretching), 1720 (C=O), 1550 (C=C), 1300 (C-O).
Methyl 3-(4-methoxyphenyl)acrylate (85q): Yield: 89%; m.p: 86-87°C (lit. 88-
89°C); Rf: 0.71 (Petroleum ether : ethylacetate, 4:1); IR (NaCl Cell, νmax, cm-1):
3178 (sp2 CH Stretching), 2986 (sp3 CH Stretching), 1731 (C=O), 1612 (C=C),
1286 (C-O). Methyl 2-(2,4-dichlorophenylthio)acetate (85r): Yield: 87%; Oil; Rf: 0.71
(Petroleum ether : ethylacetate, 4:1); IR (NaCl Cell, νmax, cm-1): 3018 (sp2 CH
Stretching), 2946 (sp3 CH Stretching), 1735 (C=O), 1525 (C=C), 1310 (C-O).
250
7.5 General procedure for the synthesis of hydrazides
The respective ester (0.02 moles) was dissolved in methanol (100 mL) in
a round bottom flask fitted with a reflux condenser and a calcium chloride drying
tube. Hydrazine hydrate (80%, 0.04 moles) was added slowly and the reaction
was monitored by thin layer chromatography. After completion of the reaction,
the reaction mixture was concentrated under reduced pressure. The resulting
crude solid was filtered, washed with water and recrystallized from aqueous
ethanol.
2,5-Difluorobenzohydrazide (86a): Yield: 78%; m.p: 141-142oC; Rf: 0.80
(petroleum ether : acetone, 8:2); IR (KBr, νmax, cm-1): 3312 (NH stretching), 1626
(C=O), 1597 (C=C); 1H NMR (DMSO-d6, δ ppm ): 9.64 (1H, s, NH), 7.30-7.41
(3H, m, Ar-H), 4.58 (2H, s, NH2); 13C NMR (DMSO-d6, δ ppm ): 162.5(C=O),
[158.5, 155.3, 124.9, 119.1, 118.3, 116.5] (Ar-C); GCMS (DMF, m/z, %): 172 (22,
M+), 152 (3), 141 (100), 113 (52), 93 (4), 74 (2), 63 (20), 50 (5), 31 (6); Anal. Cald
for C7H6F2N2O : C, 48.84; H, 3.51; N, 16.27. Found: C, 48.71; H, 3.55; N, 16.22.
3,5-Difluorobenzohydrazide (86b): Yield: 74%; m.p: 158-159 oC; Rf: 0.87
(petroleum ether: acetone, 8:2); IR (KBr, νmax, cm-1): 3286 (NH stretching), 1628
(C=O), 1595 (C=C); 1H NMR (DMSO-d6, δ ppm ): 9.98 (1H, s, NH), 7.39-7.55
(3H, m, Ar-H), 4.60 (2H, s, NH2); 13C NMR (DMSO- d6, δ ppm ): 164.3 (C=O),
[163.5, 161.0, 137.1, 110.7), 110.5, 107.0] (Ar-C); GCMS (DMF, m/z, %): 172
(22, M+), 141 (100), 113 (71), 93 (4), 74 (2), 63 (29), 50 (5), 31 (6). Anal. Cald for
C7H6F2N2O : C, 48.84; H, 3.51; N, 16.27. Found: C, 48.81; H, 3.53; N, 16.23.
2,6-Difluorobenzohydrazide (86c): Yield: 88%; m.p: 244-246 oC; Rf: 0.75
(petroleum ether : acetone, 8:2); IR (KBr, νmax, cm-1): 3316 (NH stretching), 1632
(C=O), 1579 (C=C); 1H NMR (DMSO-d 6, δ ppm ): 9.73 (1H, s, NH), 7.35-7.55
251
(3H, m, Ar-H), 4.50 (2H, s, NH2); 13C NMR (DMSO-d6, δ ppm ): 164.0 (C=O),
[161.5, 161.0, 136.1, 112.7, 111.5, 109.0](Ar-C). GCMS (DMF, m/z, %):172
(11.02, M+), 141 (100), 113 (45.34), 93 (14.33), 74 (02.32), 63 (29.90), 50
(05.10), 31 (06.52). Anal. Cald for C7H6F2N2O : C, 48.84; H, 3.51; N, 16.27.
Found: C, 48.84; H, 3.43; N, 16.20.
3,4-Dimethoxybenzohydrazide (86d): Yield: 82%; m.p: 118 oC; Rf: 0.81
(petroleum ether : acetone, 8:2); IR (KBr, νmax, cm-1): 3296 (NH stretching), 1661
(C=O), 1596 (C=C), 1255 (C-O). 1H NMR (DMSO-d 6, δ ppm ): 9.67 (1H, s, NH),
7.48 (1H, d, J = 2.1Hz, H-6 ), 7.45 (1H, d, J = 3Hz, H-2), 7.05 (1H, d, J = 8.4Hz,
H-5), 4.50 (2H, s, NH2) 3.79 (6H, s, 2 × OCH3); 13C NMR (DMSO-d 6, δ ppm ):
166.17 (C=O), [151.5, 148.6, 125.9, 120.5, 110.9, 105.2](Ar-C), 55.8 (OCH3).
GCMS (DMF, m/z, %): 196 (13, M+), 165 (100), 137 (22), 122 (13), 107 (8), 92
(5), 79 (12), 63 (4), 51 (7), 31 (2). Anal. Cald for C9H12N2O3 : C, 55.09; H, 6.16; N,
14.28; Found: C, 55.12; H, 6.13; N, 14.21.
3,5-Dimethoxybenzohydrazide (86e): Yield: 73%; m.p: 165-166oC; Rf: 0.78
(petroleum ether : acetone, 8:2); IR (KBr, νmax, cm-1): 3410, 3325 (NH stretching),
1656 (C=O), 1578 (C=C), 1268 (C-O). 1H NMR (DMSO-d6, δ ppm ): 9.76 (1H, s,
NH), 6.99 (1H, d, J = 2.1 Hz, H-4), 6.62 (2H, dd, J = 3.0 Hz, H-2,6), 4.49 (2H, s,
NH2), 3.78 (6H, s, 2 × OCH3); 13C NMR (DMSO-d6, δ ppm ): 165.8 (C=O), [160.7,
160.5, 135.7, 105.2, 105.0, 103.4](Ar-C), 55.7 (OCH3). GCMS (DMF, m/z, %):196
(11, M+), 165 (100), 137 (32), 122 (23), 107 (18), 92 (35), 79 (12), 63 (14), 51
(27), 31 (5). Anal. Cald for C9H12N2O3 : C, 55.09; H, 6.16; N, 14.28; Found: C,
55.11; H, 6.23; N, 14.24.
2,4-Dimethoxybenzohydrazide (86f): Yield: 72%; m.p: 101-102oC; Rf: 0.80
(petroleum ether : acetone, 8:2); IR (KBr, νmax, cm-1): 3350, 3210 (NH
stretching), 1668 (C=O), 1560 (C=C), 1256 (C-O). 1H NMR (DMSO-d6, δ ppm ):
9.69 (1H, s, NH), 7.77 (1H, d, J = 2.1 Hz, H-6 ), 6.67 (1H, d, J = 3.0 Hz, H-5),
6.41 (1H, d, J = 8.4 Hz, H-5), 4.72 (2H, s, NH2) 3.87 (6H, s, 2 × OCH3); 13C NMR
252
(DMSO-d6, δ ppm ): 164.8 (C=O), 163.2, [159.2, 132.4, 113.3, 106.2, 98.9] (Ar-
C), 56.0 (OCH3). GCMS (DMF, m/z, %):196 (15, M+), 165 (100), 151 (39), 138
(19), 119 (43), 105 (10), 91 (15), 77 (18), 65 (4), 55 (4), 32 (3). Anal. Cald for
C9H12N2O3 : C, 55.09; H, 6.16; N, 14.28; Found: C, 55.05; H, 6.11; N, 14.19.
2,6-Dimethoxybenzohydrazide (86g): Yield: 79%; m.p: 160-161oC; Rf: 0.87
(petroleum ether : acetone, 8:2); IR (KBr, νmax, cm-1): 3361, 3256 (NH
stretching), 1659 (C=O), 1589(C=C), 1254 (C-O). 1H NMR (DMSO- d 6, δ ppm ):
9.75(1H, s, NH), 6.95(1H, t, J = 7.1 Hz, H-4 ), 6.60(1H, d, J = 8.0 Hz, H-3,5),
4.51(2H, s, NH2), 3.75(6H, s, 2 × OCH3); 13C NMR (DMSO-d6, δ ppm ):
166.1(C=O), [160.9, 160.7, 135.7, 107.1, 105.5, 103.4](Ar-C), 55.8(OCH3).
GCMS (DMF, m/z, %): 196 (24, M+), 165 (100), 137 (14), 122 (23), 107 (8), 92
(5), 77 (22), 63 (4), 51 (7), 31 (2). Anal. Cald for C9H12N2O3 : C, 55.09; H, 6.16; N,
14.28; Found: C, 55.12; H, 6.13; N, 14.21.
2-(3-Methoxyphenyl)acetohydrazide (86h): Yield: 79%; m.p: 102-104oC; Rf:
0.75 (petroleum ether : acetone, 8:2); IR (KBr, νmax, cm-1): 3396, 3241 (NH
stretching), 1654 (C=O), 1596 (C=C), 1256 (C-O); 1H NMR (DMSO- d 6, δ ppm ):
8.97 (1H, s, NH), 6.99 (1H, dd, J = 8.4, 2.1 Hz, H = 5’), 6.75-6.35 (3H, m, H = 2’,
4’,6’), 4.34 (2H, s, NH2), 3.84 (3H, s, OCH3), 3.29 (2H, s, CH2); 13C NMR (DMSO-
d 6, δ ppm ): 169.6 (C=O), [158.7, 135.5, 131.2, 121.5, 111.2](Ar-C), 56.3(OCH3),
40.7(CH2). GCMS (DMF, m/z, %): 180 (22, M+), 149 (25), 121 (100), 107 (12), 89
(44), 73 (6), 63 (20), 51 (5), 32 (64). Anal. Cald for C9H12N2O2 : C, 59.99; H, 6.71;
N, 15.55; Found: C, 59.92; H, 6.75; N, 15.51.
2-(4-Chlorophenyl)acetohydrazide (86i); Yield: 81%; m.p: 158-160oC; Rf: 0.81
(petroleum ether : acetone, 8:2); IR (KBr, νmax, cm-1): 3385, 3274 (NH stretching),
1674 (C=O), 1574 (C=C); 1H NMR (DMSO- d 6, δ ppm ): 9.23 (1H, s, NH), 7.35
(2H, d, J = 8.4 Hz, H = 2’, 6’), 7.26 (2H, d, J = 8.4 Hz, H = 3’, 5’), 4.22 (2H, s,
NH2), 3.34 (2H, s, CH2); 13C NMR (DMSO- d 6, δ ppm ): 169.6 (C=O), [135.7,
253
131.5, 131.2, 128.5](Ar-C), 40.7(CH2). GCMS (DMF, m/z, %): 184 (22, M+), 152
(35), 125 (100), 99 (12), 89 (44), 73 (6), 63 (20), 51 (5), 32 (64). Anal. Cald for
C8H9ClN2O : C, 52.04; H, 4.91; N, 15.17; Found: C, 52.09; H, 4.95; N, 15.11.
2-(4-Flourophenyl)acetohydrazide (86j): Yield: 78%; m.p: 121-122oC; Rf: 0.78
(petroleum ether : acetone, 8:2); IR (KBr, νmax, cm-1): 3345, 3196 (NH
stretching), 1648 (C=O), 1585 (C=C); 1H NMR (DMSO-d6, δ ppm ): 9.25 (1H, s,
NH), 7.27 (2H, m, H = 2’, 6’), 7.10 (2H, m, H = 3’, 5’), 4.22 (2H, s, NH2), 3.39 (2H,
s, CH2); 13C NMR (DMSO-d 6, δ ppm ): 169.9 (C=O), [163.0, 132.8, 131.2, 131.1,
115.4, 115.2](Ar-C), 40.6(CH2); GCMS (DMF, m/z, %):168 (28, M+), 136 (37),
109 (100), 89 (2), 83 (24), 63 (10), 57 (15), 32 (34). Anal. Cald for C8H9FN2O : C,
57.14; H, 5.39; N, 16.66; Found: C, 57.04; H, 5.34; N, 16.61.
3-(3,4,5-Trimethoxyphenyl)propionatohydrazide (86k): Yield: 78%; m.p: 125-
126 oC; Rf: 0.80 (petroleum ether : acetone, 8:2); IR (KBr, νmax, cm-1): 3341 (NH
stretching), 1659 (C=O), 1574 (C=C), 1248 (C-O). 1H NMR (DMSO-d 6, δ ppm ):
8.99 (1H, s, NH), 6.49 (2H, s, Ar-H), 4.20 (2H, s, NH2), 3.74 (9H, s, 3 × OCH3),
2.32 (2H, t, CH2), 2.75 (2H, t, CH2); 13C NMR (DMSO-d 6, δ ppm ): 171.3(C=O),
[153.14, 137.4, 136.0, 105.9](Ar-C), 56.16-(OCH3), 31.8 & 35.1(2 × CH2). GCMS
(DMF, m/z, %): 254 (68, M+), 223 (17), 207 (7), 181 (100), 165 (4), 148 (14), 136
(5), 121 (4), 105 (3), 91 (3), 77 ( 7), 65 (4). Anal. Cald for C12H8N2O4 : C, 56.68;
H, 7.13; N, 11.02; Found: C, 56.97; H, 7.11; N, 11.07.
3-(4-Methoxyphenyl)propionatohydrazide (86l): Yield: 74%; m.p: 110-112 oC;
Rf: 0.87 (petroleum ether : acetone, 8:2); IR (KBr, νmax, cm-1): 3352 (NH
stretching), 1658 (C=O), 1572 (C=C), 1249 (C-O). 1H NMR (DMSO-d 6, δ ppm ):
8.98 (1H, s, NH), 7.09 (2H, d, J = 8.4 Hz, H-2’.6’), 6.86 (2H, dd, J = 1.8, 8.4 Hz,
H-3’.5’), 4.16 (2H, s, NH2), 3.39 (3H, s, OCH3), 2.50 (2H, t, CH2), 2.27 (2H, t,
CH2); 13C NMR (DMSO-d6, δ ppm ): 171.3 (C=O), [157.9,133.5, 129.5, 114.1](Ar-
C), 55.4 (OCH3), 30.6 & 35.8 (2 × CH2). GCMS (DMF, m/z, %): 194 (25), 162
254
(14), 134 (10), 121 (100), 105 (03), 91(13), 77 ( 9), 65 (4). Anal. Cald for
C10H14N2O2 : C, 61.84; H, 7.27; N, 14.42; Found: C, 61.97'; H, 7.21; N, 14.37.
3-(4-Bromophenoxy)acetatohydrazide (86m): Yield: 82%; m.p: 164-165 oC; Rf:
0.75 (petroleum ether : acetone, 8:2); IR (KBr, νmax, cm-1): 3345, 3210 (NH
stretching) 1654 (C=O), 1574(C=C), 1254 (C-O). 1H NMR (DMSO-d 6, δ ppm ):
9.39 (1H, s, NH), 7.43 (2H, d, J = 8.4 Hz, H-2’.6’), 6.92 (2H, d, J = 8.4 Hz, H-
3’.5’), 4.48 (2H, s, NH2) , 3.42 (2H, s, CH2); 13C NMR (DMSO-d 6, δ ppm ):
166.91 (C=O), [157.5, 132. 3, 117.2, 113.0](Ar-C), 66.7 (CH2). GCMS (DMF, m/z,
%): 246 [30, M+ (Br81)], 244 [(30, M+ (Br79)], , 185 & 187 (23), 172 & 174 (100),
155 & 157 (55), 143 & 145 (5), 117 & 119 (2), 73 & 75 (26), 63 & 65 (14), 45 (80).
Anal. Cald for C10H11BrN2O2 : C, 39.21; H, 3.70; N, 11.43; Found: C, 39.37'; H,
3.54 ; N, 11.37.
2-(2,4-Dichlorophenoxy)propionatohydrazide (86n): Yield: 78%; m.p: 154-155 oC; Rf: 0.81 (petroleum ether: acetone, 8:2); IR (KBr, νmax, cm-1): 3385, 3202 (NH
stretching) 1652 (C=O), 1547 (C=C), 1247 (C-O). 1H NMR (DMSO- d 6, δ ppm ):
9.35 (1H, s, NH), 7.57 (1H, d, J = 2.7 Hz, H-3’), 7.34 (1H, dd, J = 11.4, 2.4 Hz, H-
5’), 6.98 (1H, d, J = 9.0 Hz, H-6’), 4.75 (1H, q, CH), 4.32 (2H, s, NH2), 1.47 (3H,
d, CH3); 13C NMR (DMSO- d 6, δ ppm ): 169.6 (C=O), [152.4, 129.9, 128.4,
125.5, 123.5, 116.4](Ar-C), 74.30 (CH), 19.06 (CH3). GCMS (DMF, m/z, %): 250
(3, M+ (Cl37)), 248 (5, M+ (Cl35)), 191 (23, Cl37), 189 (40, Cl35), 164 (20 Cl37), 162
(32, Cl35), 154 (25), 145 (24), 133 (7), 125 (22), 109 (26), 98 (4), 87 (100), 75
(11), 59 (68). Anal. Cald for C9H10Cl2N2O2 : C, 43.40; H, 04.05; N, 11.25; Found:
C, 43.47; H, 04.02; N, 11.20.
2-(2,4-Dichlorophenoxy)butyratohydrazide (86o): Yield: 74%; m.p: 63-65 oC;
Rf: 0.78 (petroleum ether : acetone, 8:2); IR (KBr, νmax, cm-1): 3374, 3216 (NH
stretching), 1663 (C=O), 1569 (C=C), 1257 (C-O). 1H NMR (DMSO-d 6, δ ppm ):
9.03 (1H, s, NH), 7.53 (1H, d, J = 2.1 Hz, H-3’), 7.33 (1H, dd, J = 2.4, 11.1 Hz, H-
5’), 7.13 (1H, d, J = 8.7 Hz, H-6’), 4.17 (2H, t, CH2), 4.03 (2H, s, NH2), 2.21 (2H,
255
t, J = 7.5 Hz, CH2), 1.94 (2H, m, CH2). ); 13C NMR (DMSO-d 6, δ ppm ): 171.5
(C=O), [153.4, 129.6, 128.5, 124.7, 122.8, 115.4](Ar-C), [68.81, 30.06, 25.06]
(CH2). GCMS (DMF, m/z, %): 264 (2, M+, Cl37 ), 262 (2, M+
, Cl35), 233 (3, Cl37 ),
231 (2, Cl35 ), 177 (5, Cl37), 175 (2, Cl35), 164 (20, Cl37), 162 (32, Cl35), 147 (3,
Cl37), 145 (24, Cl35), 135 (3, Cl37), 133 (7, Cl35), 111 (4, Cl37), 109 (6, Cl35), 103
(4, Cl37), 101 (100, Cl35), 69 (11), 59 (68). Anal. Cald for C10H12Cl2N2O2 : C,
45.65; H, 4.60; N, 10.65; Found: C, 45.55; H, 4.63; N, 10.62.
2-(2,4-Dichlorophenoxy)acetohydrazide (86p): Yield: 88%; m.p: 151-152 oC;
Rf: 0.80 (petroleum ether : acetone, 8:2); IR (KBr, νmax, cm-1): 3341, 3189 (NH
stretching), 1639 (C=O), 1547 (C=C), 1251 (C-O). 1H NMR (DMSO-d 6, δ ppm ):
9.26 (1H, s, NH), 7.53 (1H, d, J = 2.1 Hz, H-3’), 7.33 (1H, dd, J = 2.4, 11.1 Hz, H-
5’), 7.07 (1H, d, J = 8.7 Hz, H-6’), 4.61 (2H, s, CH2), 4.37 (2H, s, NH2); 13C NMR
(DMSO-d 6, δ ppm ): 166.4 (C=O), [153.0,129.7, 128.4, 125.4, 122.9, 115.6](Ar-
C), 67.5 (CH2); GCMS (DMF, m/z, %): 236 (12, M+, Cl37 ), 234 (32, M+
, Cl35), 201
(8, Cl37 ), 199 (22, Cl35 ), 177 (27, Cl35), 175 (35, Cl37), 164 (60, Cl37), 162 (92,
Cl35), 147 (33, Cl37), 145 (44, Cl35), 135 (8, Cl37), 133 (17, Cl35), 111 (44, Cl37),
109 (36, Cl35), 75 (34, Cl37), 73 (100, Cl35), 63 (21), 45 (88), 31 (29). Anal. Cald
for C8H8Cl2N2O2 : C, 40.88; H, 03.43; N, 11.92. Found: C, 40.73; H, 03.14; N,
11.82.
3-(4-Methoxyphenyl)acrylohydrazide (86q): Yield: 82%; m.p: 204-206 oC ; Rf:
0.87 (petroleum ether: acetone, 8:2); IR (KBr, νmax, cm-1): 3298, 3195 (NH
stretching), 1658 (C=O), 1601 (C=C), 1239 (C-O). 1H NMR (DMSO-d 6, δ ppm ):
8.98 (1H, s, NH), 7.09 (2H, d, J = 8.4 Hz, H-2’.6’), 6.86 (2H, d, J = 8.4, Hz, H-
3’.5’), 4.16 (2H, s, NH2), 3.39 (3H, s, OCH3), 2.50 (2H, d, J = 8.4, CH), 2.27 (2H,
d, J = 8.1, CH); 13C NMR (DMSO-d6, δ ppm ): 171.3 (C=O), 157.9, 143.9, 133.5,
129.5, 117.5, 114.1, 55.4 (OCH3); GCMS (DMF, m/z, %): 192 (21), 161 (10), 133
(21), 120 (100), 107 (1), 91 (13), 77 (9), 65(4). Anal. Cald for C10H12N2O2 : C,
62.49; H, 06.29; N, 14.57; Found: C, 62.97; H, 06.21; N, 14.37.
256
2-(2,4-Dichlorophenylthio)acetohydrazide (86r): Yield: 72%; m.p: 60-62 oC; Rf:
0.75 (petroleum ether : acetone, 8:2); IR (KBr, νmax, cm-1): 3347 (NH stretching)
1645 (C=O), 1540 (C=C), 1281 (C-O). 1H NMR (DMSO-d 6, δ ppm ): 9.26 (1H, s,
NH), 7.53 (1H, d, J = 2.1 Hz, H-3’), 7.33 (1H, dd, J = 11.1, 2.4, Hz, H-5’), 7.07
(1H, d, J = 8.7 Hz, H-6’), 4.51 (2H, s, CH2), 4.37 (2H, s, NH2); 13C NMR (DMSO-
d 6, δ ppm ): 166.4 (C=O), [153.0, 129.7, 128.4, 125.4, 122.9, 115.6] (Ar-C),
43.5 (CH2); GCMS (DMF, m/z, %): 252 (10, M+, Cl37 ) 250 (12, M+
, Cl35), 217 (6,
Cl37 ), 215 (12, Cl35 ), 193 (17, Cl35), 191 (31, Cl37), 180 (20, Cl37), 178 (52, Cl35),
163 (53, Cl37), 161 (54, Cl35), 151 (8, Cl37), 149 (12, Cl35), 127 (44.36, Cl37), 125
(36.58, Cl35), 91 (34.32, Cl37), 89 (100, Cl35), 79 (21.24), 61 (88.36), 47 (29).
Anal. Cald for C8H8Cl2N2OS: C, 38.26; H, 03.21; N, 11.15; S, 12.77; Found: C,
38.06; H, 03.29; N, 11.11; S, 12.25.
7.6 General procedure for the synthesis of isothiocyanate
The substituted amine (0.25 moles) was dissolved in methanol (18 mL) in
a round bottom flask fitted with a reflux condenser. The whole assembly was
placed in an ice bath. Carbon disulfide (0.38 moles) and ammonia solution (1.0
mole) was added slowly. The temperature of the reaction mixture was maintained
at 15°C (not allowed to rise above 30°C) and stirred for 12h. After stirring, the
contents of the flask were transferred to a beaker. A solution of lead nitrate was
prepared in water prepared and added to the above mixture and stirred
overnight. The precipitates of lead sulphide formed along with isothiocyanate.
The isothiocyanate isolated by steam distillation. The isothiocyanate extracted by
using ethylacetate which was evaporated on rotary to get isothiocyanate. 2-Methoxyphenylisothiocyante (87a): Yield: 45%; Oily liquid; Rf: 0.82 (n-
hexane : ethyl acetate 8:2); IR (NaCl Cell, νmax, cm-1): 3069 (sp2 CH stretch),
2967, 2940 (sp3 CH), 2035b (N=C=S), 1591, 1495, 1457 (C=C); 1H NMR (CDCl3,
257
δ ppm ): 7.31 (1H, d, J = 8.2Hz, Ar-H), 7.21 (1H, t, J = 7.9 Hz, Ar-H), 6.9 (1H, d, J
= 8.1 Hz, Ar-H ), 6.7 (1H, t, J = 8.3 Hz, Ar-H), 3.84 (3H, s, OCH3); 13C NMR (CDCl3, δ ppm ): 158.0 (C=S), [138.2, 130.5, 128.8, 123.1, 117.8, 113.3] (Ar-C),
54.5(OCH3); GC-MS(CHCl3, m/z.(%)): 165 (100), 150 (45), 132 (51), 122 (90), 78
(12), 63 (15), 51 (11), 45 (6), 39 (10); Anal. Cald for C8H7NOS: C, 58.16; H,
4.27; N, 8.48; S, 19.41; Found: C, 58.15; H, 4.20; N, 8.47; S, 19.49.
4-Methoxyphenylisothiocyante (87b): Yield: 44%; Oil; Rf: 0.81 (n-hexane :
ethyl acetate 8:2); IR (NaCl Cell, νmax, cm-1): 3053 (sp2 CH), 2957, 2907, 2836
(sp3 CH), 2106, b (N=C=S), 1603, 1503, 1460 (C=C); 1H NMR (CDCl3, δ ppm):
7.18 (2H, dt, J = 9.0, 3.3 Hz, Ar-H ), 6.86 (2H, dt, J = 9.0, 3.3 Hz, Ar-H), 3.80
(3H, s, OCH3); 13C NMR (CDCl3, δ ppm): 158.5(C=S), [133.8, 126.9,126.5, 123.5,
115.1, 114.8, 114.3] ( Ar-C), 55.5(OCH3); GC-MS (CHCl3, m/z, (%): 165 (100),
150(35), 132(59), 122(90), 82 (25 ), 78(25), 63(15), 51(11), 45(6), 39(10). Anal.
Cald for C8H7NOS: C, 58.16; H, 4.27; N, 8.48; S, 19.41; Found: C, 58.15; H,
4.20; N, 8.47; S, 19.49.
Cyclohexylisothiocyante (87c): Yield: 55%; Yellow oil; Rf: 0.68 (Petroleum
ether : ethyl acetate, 4:1); IR (KBr, νmax, cm-1): 2084, 2943-2970, 1454-1601. 1H
NMR (CDCl3, δ ppm ): δ 3.70-3.62(1H, m), 1.88-1.32(10H, m); 13C NMR (CDCl3):
δ 129.5(C=S), [55.3, 33.2, 24.9, 23.2](Cyclohexyl-H). Anal. Cald for C7H11NS: C,
59.53; H, 7.85; N, 9.92; S, 22.70; Found: C, 59.33; H, 7.84; N, 9.95; S, 22.77.
7.7 General procedure for the synthesis of Thiosemicarbazides
The substituted carboxylic acid hydrazide (6.8 mmoles) was dissolved in
methanol (30ml) and the substituted isothiocyanate (6.6 mmoles), separately
dissolved in methanol (10ml), was added dropwise with continuous stirring. The
reaction mixture was refluxed for 10-12 h. and monitored by TLC. After
consumption of the starting materials, the mixture was cooled to room
258
temperature. Then, methanol was evaporated on rotary leaving behind crude
thiosemicarbazide as an oil that get solidified later on by cooling and
recrystallized from a mixture of ethylacetate and petroleum ether to yield
thiosemicarbazides 88(a-w).
1-(3,5-Difluorobenzoyl)-4-cyclohexylthiosemicarbazide (88a): Yield: 85%;
m.p: 131-133; Rf: 0.37 (Petroleum ether : acetone; 6:4); IR (KBr, νmax, cm-1):
3250-3145 (NH Stretching), 1653 (C=O), 1600, 1568 (C=C), 1265 (C=S); 1HNMR
(DMSO-d6, δ ppm ): δ 10.45 (1H, s, NH), 9.27 (1H, s, NH), 8.94 (1H, s, NH),
7.64-7.48 (3H, m, H-2,4,6), 4.13 (1H, m, H-1′), 1.78-1.04 (10H, m, cyclohexyl-H); 13C NMR (DMSO- d 6, δ ppm ): 164.0 (C=O), [162.7, 160.9, 136.4, 111.6, 108.0,
107.5] (Ar-C), [53.6, 36.2, 32.3, 31.2, 25.6, 25.4] (cyclohexyl-C); GCMS (DMF,
m/z, %): 313 (6, M+), 171 (100), 141 (13), 107 (8), 92 (5), 79 (12), 63 (4), 51 (7),
39 (2); Anal. Cald for C14H17F2N3OS : C, 53.66; H, 5.47; N, 13.41; S, 10.23;
Found: C, 53.61; H, 5.35; N, 13.44; S, 10.29.
1-[2-(2,4-Dichlorophenoxy)propanoyl]-4-cyclohexylthiosemicarbazide (88b):
Yield: 83%; m.p: 149-150°C; Rf: 0. 34 (Petroleum ether : acetone; 6:4); IR (KBr,
νmax, cm-1): 3271-3149 (NH Stretching), 1659 (C=O), 1585 (C=C), 1251 (C=S); 1H
NMR (DMSO-d6, δ ppm ): 9.91 (1H, s, NH), 9.57 (1H, s, NH), 9.28 (1H, s, NH),
7.65-7.05 (3H, m, H-3′,5′,6′), 4.77 (1H, q, CH), 3.31 (1H, m, H-1), 1.79-1.07
(13H, m, cyclohexyl H); 13C NMR (DMSO-d6, δ ppm ): 167.0 (C=O), [153.9,
129.8, 128.3, 125.4, 122.8, 115.7] (Ar-C), 70.88 (CH), [53.3, 32.3, 25.6, 25.3]
(Cyclohexyl-C), 20.13 (CH3); GCMS (DMF, m/z, %): 390 (3, M+), 247 (100), 217
(18), 172 (43), 144 (24), 141 (13), 111 (59), 83 (18), 60 (11), 79 (12), 63 (4), 51
(7), 39 (2); Anal. Cald for C16H21Cl2N3O2S : C, 49.23; H, 5.42; N, 10.77; S, 8. 21;
Found: C, 49.27; H, 5.45; N, 10.73; S, 8. 28.
1-(3,4-Dimethoxybenzoyl)-4-cyclohexylthiosemicarbazide (88c): Yield: 80%;
m.p: 185-186; Rf: 0.38 (Petroleum ether : acetone; 6:4); IR (KBr, νmax, cm-1):
259
3351-3115 (NH Stretching), 1666 (C=O), 1561 (C=C), 1239 (C=S); 1H NMR
(DMSO-d 6, δ ppm ): 10.05 (1H, s, NH), 9.37 (1H, s, NH), 8.84 (1H, s, NH), 7.54-
7.41 (3H, m, H-2,5,6), 4.10 (1H, s, H-1′), 1.71-1.09 (10H, m, cyclohexyl-H), 3.71
(6H, s, OCH3); 13C NMR (DMSO-d 6, δ ppm ): 166.0 (C=O), [158.7, 155.9, 136.4,
121.6, 118.0, 111.5] (Ar-C), 55.8 (OCH3), [53.6, 36.2, 31.3, 31.2, 25.6, 25.4]
(Cyclohexyl-C); GCMS (DMF, m/z, %): 337 (3, M+), 247 (100), 217 (18), 172
(43), 144 (24), 141 (13), 111 (59), 83 (18), 60 (11), 79 (12), 63 (4), 51 (7), 39 (2); Anal. Cald for C16H23N3O3S : C, 56.95; H, 6.87; N, 12.45; S, 9.50; Found: C,
56.93; H, 6.81; N, 12.49; S, 9.55.
1-[2-(2,4-Dichlorophenoxy)acetyl]-4-cyclohexylthiosemicarbazide (88d):
Yield: 79%; m.p: 189-190°C; Rf: 0.35 (Petroleum ether : acetone; 6:4); IR (KBr,
νmax, cm-1): 3290-3009 (NH Stretching), 1654 (C=O), 1560 (C=C), 1255 (C=S); 1H
NMR (DMSO-d 6, δ ppm ): 9.95 (1H, s, NH), 9.47 (1H, s, NH), 9.18 (1H, s, NH),
7.62-7.08 (3H, m, H-3′,5′,6′), 4.74 (2H, s, CH2), 3.07 (1H, s, H-1), 1.79-1.07
(10H, m, cyclohexyl-H); 13C NMR (DMSO-d6, δ ppm ): 167.0 (C=O), [152.9,
129.8, 128.3, 125.4, 122.8, 115.7] (Ar-C), 66.88 (CH2), [53.3, 32.3, 25.6, 25.3]
(Cyclohexyl-C). GCMS (DMF, m/z, %): 376 (21, M+), 232 (100), 230 (14), 202
(2), 172 (11), 144 (42), 141 (22), 111 (13), 77 (8), 63 (4), 51 (7); Anal. Cald for
C15H19Cl2N3O2S : C, 47.88; H, 5.09; N, 11.17; S, 8.52; Found: C, 47.83; H, 5.01;
N, 11.14; S, 8.62.
1-[3-(3,4,5-Trimethoxyphenyl)propanoyl]-4-cyclohexylthiosemicarbazide (88e): Yield: 84%; m.p: 174-176; Rf: 0.37 (Petroleum ether : acetone; 6:4); IR
(KBr, νmax, cm-1): 3400-3145 (NH Stretching), 1658 (C=O), 1598 (C=C), 1244
(C=S); 1H NMR (DMSO-d6, δ ppm ): 10.17 (1H, s, NH), 9.64 (1H, s, NH), 9.04
(1H, s, NH), 7.91 (1H, d, J = 2.1 Hz, H-6′), 7.76 (1H, d, J = 2.1 Hz, H-2′), 4.10
(1H, s, H-1), 3.73 (9H, s, 3 × OCH3), 2.81 (2H, t, J = 7.5 Hz, CH2), 2.51 (2H, t, J
= 8.1 Hz, CH2), 1.71-1.09 (10H, m, cyclohexyl-H); 13C NMR (DMSO-d 6, δ ppm ):
180.5 (C=S), 176.2 (C=O), [152.6, 136.2, 132.1, 105.9, 105.8] (Ar-C), 56.15
260
(OCH3), [53.6, 36.2, 35.4, 31.3, 31.3, 31.2, 25.6, 25.4] (Cyclohexyl & alphitic-C);
GCMS (DMF, m/z, %): 395 (3, M+), 253 (100), 238 (22), 195 (11), 157 (21), 141
(13), 107 (9), 92 (5), 79 (12), 63 (4), 51 (7); Anal. Cald for C19H29N3O4S : C,
57.70; H, 7.39; N, 10.92; S, 8.11; Found: C, 57.77; H, 7.32; N, 10.95; S, 8.17.
1-[3-(4-Methoxyphenyl)propanoyl]-4-cyclohexylthiosemicarbazide (88f): Yield: 85%; m.p: 133-135; Rf: 0.33 (Petroleum ether : acetone; 6:4); IR (KBr,
νmax, cm-1): 3411-3205 (NH Stretching), 1673 (C=O), 1588 (C=C), 1242 (C=S);
1H NMR (DMSO-d6, δ ppm ): 10.87 (1H, s, NH), 9.77 (1H, s, NH), 9.53 (1H, s,
NH), 7.29 (2H, d, J = 8.4 Hz, H-2′,6′), 7.17 (2H, d, J = 8.4 Hz, H-3′,5′), 3.74 (3H,
s, OCH3), 2.65 (2H, t, CH2), 2.51 (1H, m, CH2), 2.33 (1H, m, H-1), 1.79-1.19
(10H, m, cyclohexyl-H); 13C NMR (DMSO-d 6, δ ppm ): 169.7 (C=O), [156.9,
133.4, 132.3, 129.6, 114.2] (Ar-C), 55.61 (OCH3), [53.6, 36.2, 35.7, 31.3, 31.2),
29.9, 25.6, 25.4] (Cyclohexyl & aliphetic-C); GCMS (DMF, m/z, %): 335 (7, M+),
301 (3), 237 (21), 193 (100), 178 (12), 163 (15), 141(52), 135 (8), 112 (10), 99
(5), 83 (9); Anal. Cald for C17H25N3O2S : C, 60.87; H, 7.51; N, 12.53; S, 9.56;
Found: C, 60.75; H, 7.57; N, 12.45; S, 9.52.
1-[2-(4-Bromophenoxy)acetyl]-4-(2-methoxyphenyl)thiosemicarbazide (88g): Yield: 80%; m.p: 141-142oC; Rf: 0.34 (petroleum ether : acetone, 6:4); IR
(KBr, νmax, cm-1): 3385-3216 (NH Stretching), 1685 (C=O), 1605 (C=C), 1255
(C=S); 1H NMR (DMSO-d 6, δ ppm ): 9.95 (1H, s, NH), 9.69 (1H, s, NH), 9.32
(1H, s, NH), 7.81-6.65 (8H, m, Ar-H), 4.51 (2H, s, H-1), 3.79 (3H, s, OCH3); 13C
NMR (DMSO-d 6, δ ppm ): 167.1 (C=O), [158.4, 157.6, 133.7, 128.5, 128.3,
123.4, 118.8, 111.8] (Ar-C), 68.55 (CH2), 56.6 (OCH3); GCMS (DMF, m/z, %): 410(19, M+), 377 (2), 287 (25), 244 (100), 228 (15), 213 (45), 185 (11), 165
(100), 133 (2), 123 (13), 107 (8); Anal. Cald for C16H16BrN3O3S : C, 46.84; H,
3.93; N, 10.24; S, 7.82; Found: C, 46.89; H, 3.97; N, 10.19; S, 7.89.
1-(2,5-Difluorobenzoyl)-4-(2-methoxyphenyl)thiosemicarbazide (88h): Yield:
78%; m.p: 153-154oC; Rf: 0.37 (petroleum ether : acetone, 6:4); IR (KBr, νmax,
261
cm-1): 3285-3210 (NH Stretching), 1670 (C=O), 1605 (C=C), 1245 (C=S); 1H
NMR (DMSO-d 6, δ ppm ): 10.62 (1H, s, NH), 9.92 (1H, s, NH), 9.19 (1H, s, NH),
7.96-7.52 (3H, m, H-3′,4′,6′), 7.18 (1H, dd, J = 7.5 Hz, H-5), 7.06 (1H, d, J = 7.8
Hz, H-3), 6.94 (1H, dd, J = 7.8 Hz, H-4), 6.73 (1H, d, J = 7.8 Hz, H-6), 3.79 (3H,
s, OCH3); 13C NMR(DMSO-d 6, δ ppm ): 165.3 (C=O), [160.8, 153.6, 129.8,
126.2, 123.5, 120.2, 115.9] (Ar-C), 55.1 (OCH3); GCMS (DMF, m/z, %): 337 (11,
M+), 303 (11), 215 (2), 172 (5), 165 (100), 156 (5), 141(54), 133 (14), 123 (19),
113 (8), 107 (21); Anal. Cald for C15H13F2N3O2S : C, 53.41; H, 3.88; N, 12.46; S,
9.91; Found: C, 53.49; H, 3.79; N, 12.44; S, 9.98.
1-[2-(2,4-Dichlorophenoxy)acetyl]-4-(2-methoxyphenyl)thiosemicarbazide (88i): Yield: 85%; m.p: 156-157oC; Rf: 0.40(petroleum ether : acetone, 6:4); IR
(KBr, νmax, cm-1): 3385-3216 (NH Stretching), 1671 (C=O), 1586 (C=C), 1248
(C=S); 1H NMR (DMSO-d6, δ ppm ): 10.05 (1H, s, NH), 9.61 (1H, s, NH), 9.02
(1H, s, NH), 7.88-6.89 (7H, m, Ar-H), 4.11 (2H, s, CH2), 3.76 (3H, s, OCH3); 13C
NMR (DMSO-d 6, δ ppm ): 169.1(C=O), [153.4, 151.6, 129.7, 128.5, 128.5,
126.4, 124.8, 122.8, 120.2, 115.5, 111.8] (Ar-C), 68.75 (CH2), 56.1 (OCH3); GCMS (DMF, m/z, %): 400 (3, M+), 365 (20), 277 (28), 234 (56), 218 (4), 203
(23), 175 (19), 165 (100), 133 (2), 123 (12), 107 (8); Anal. Cald for
C16H15Cl2N3O3S : C, 48.01; H, 3.78; N, 10.50; S, 8.01; Found: C, 48.11; H, 3.74;
N, 10.51; S, 8.07.
1-(3,5-Difluorobenzoyl)-4-(2-methoxyphenyl)thiosemicarbazide (88j): Yield:
83%; m.p: 149-150oC; Rf: 0.39 (petroleum ether : acetone, 6:4); IR (KBr, νmax,
cm-1): 3315-3116 (NH Stretching), 1645 (C=O), 1587 (C=C), 1265 (C=S); 1H
NMR (DMSO-d 6, δ ppm ): 10.52 (1H, s, NH), 9.72 (1H, s, NH), 9.39 (1H, s, NH),
7.83-7.22 (7H, m, Ar-H), 3.84 (3H, s, OCH3); 13C NMR (DMSO-d6, δ ppm ):
165.3 (C=O), [160.5, 155.3, 129.8, 127.2, 123.5, 120.2, 115.9, 111.9] (Ar-C),
56.1 (OCH3); GCMS (DMF, m/z, %): 337 (29, M+), 303 (2), 215 (12), 172 (15),
165 (100), 156 (9), 141 (50), 133 (15), 123 (21), 113 (51), 107 (25); Anal. Cald
262
for C15H13F2N3O2S : C, 53.41; H, 3.88; N, 12.46; S, 9.91; Found: C, 53.47; H,
3.79; N, 12.42; S, 9.93.
1-(3,4-Dimethoxybenzoyl)-4-(2-methoxyphenyl)thiosemicarbazide (88k): Yield: 80%; m.p: 173-175oC; Rf: 0.41 (petroleum ether : acetone, 6:4); IR (KBr,
νmax, cm-1): 3410-3016 (NH Stretching), 1660 (C=O), 1587 (C=C), 1251(C=S); 1H
NMR (DMSO-d 6, δ ppm ): 10.25 (1H, s, NH), 9.89 (1H, s, NH), 9.42 (1H, s, NH),
7.79 (1H, dd, J = 8.1, 2.1 Hz, H-6′), 7.21-7.05 (4H, m, H-3,4,5,6), 6.95 (1H, d, J
= 2.4 Hz, H-2′), 6.72 (1H, d, J = 8.1Hz, H-5′), 3.88 (6H, s, OCH3), 3.71 (3H, s,
OCH3); 13C NMR (DMSO-d 6, δ ppm ): 166.5 (C=O), [160.3, 153.6, 133.8, 128.3,
126.5, 120.2, 111.9, 106.1, 104.2] (Ar-C), 56.1-55.9 (OCH3); GCMS (DMF, m/z,
%): 361 (24, M+), 327 (25), 239 (20), 196 (19), 180 (26), 165 (100), 165 (21), 137
(12), 123 (13), 107 (8); Anal. Cald for C17H19N3O4S : C, 56.50; H, 5.30; N, 11.63;
S, 8.87; Found: C, 56.60; H, 5.33; N, 11.50; S, 8.85.
1-(2,6-Dimethoxybenzoyl)-4-(2-methoxyphenyl)thiosemicarbazide(88l): Yield: 79%; m.p: 180-182oC; Rf: 0.35 (petroleum ether : acetone, 6:4); IR (KBr,
νmax, cm-1): 3400-3260 (NH Stretching), 1663 (C=O), 1625, 1541 (C=C), 1261
(C=S); 1H NMR (DMSO-d6, δ ppm ): 10.65 (1H, s, NH), 9.71 (1H, s, NH), 9.35
(1H, s, NH), 7.79 (1H, dd, J = 8.4Hz, H-4′), 7.63 (1H, dd, J = 8.1Hz, H-5), 7.01-
6.89 (4H, m, H-3,4,3′,5′), 6.62 (1H, d, J = 8.1Hz, H-6), 3.95 (6H, s, OCH3), 3.70
(3H, s, OCH3); 13C NMR (DMSO-d 6, δ ppm ): 164.5 (C=O), [158.4, 158.0, 135.5,
128.2, 126.3, 121.2, 111.9, 106.1](Ar-C), 56.22-55.74(OCH3); GCMS (DMF, m/z,
%): 361 (6, M+), 327 (20), 239 (33), 196 (92), 180 (26), 165 (100), 137 (12), 123
(13), 107 (8); Anal. Cald for C17H19N3O4S : C, 56.50; H, 5.30; N, 11.63; S, 8.87;
Found: C, 56.54; H, 5.29; N, 11.62; S, 8.78.
1-(3,5-Dimethoxybenzoyl)-4-(2-methoxyphenyl)thiosemicarbazide(88m): Yield: 84%; m.p: 165-167oC; Rf: 0.37 (petroleum ether : acetone, 6:4); IR (KBr,
νmax, cm-1): 3305-3011 (NH Stretching), 1667 (C=O), 1561 (C=C), 1250 (C=S); 1H
263
NMR (DMSO-d 6, δ ppm ): 10.60 (1H, s, NH), 9.81 (1H, s, NH), 9.22 (1H, s, NH),
7.90 (1H, d, J = 2.4Hz, H-2′,6′), 7.19-7.02 (4H, m, H-3,4,5,6), 6.72 (1H, dd, J =
2.1 Hz, H-4′), 3.80 (6H, s, OCH3), 3.75 (3H, s, OCH3); 13C NMR (DMSO-d 6, δ
ppm ): 166.3 (C=O), [160.8, 152.6, 134.8, 128.2, 126.5, 120.2, 111.9, 106.1,
104.2] (Ar-C), 56.1-5.9 (OCH3); GCMS (DMF, m/z, %): 361(12, M+), 327 (41),
239 (02), 196 (10), 180 (26), 165 (100), 165 (21), 137 (12), 123 (13), 107 (8); Anal. Cald for C17H19N3O4S : C, 56.50; H, 5.30; N, 11.63; S, 8.87; Found: C,
56.59; H, 5.34; N, 11.53; S, 8.81.
1-[3-(3,4,5-Trimethoxyphenyl)propanoyl]-4-(2-methoxyphenyl)thiosemi- carbazide (88n): Yield: 85%; m.p: 174-175oC; Rf: 0.40 (petroleum ether :
acetone, 6:4); IR (KBr, νmax, cm-1): 3445-3211 (NH Stretching), 1656 (C=O), 1525
(C=C), 1255 (C=S); 1H NMR (DMSO-d 6, δ ppm ): 10.07 (1H, s, NH), 9.74 (1H, s,
NH), 8.97 (1H, s, NH), 7.91 (1H, s, H-6′), 7.16-6.97 (4H, m, H-3,4,5,6), 6.55 (1H,
s, H-2′), 3.79 (9H, s, OCH3), 3.62 (3H, s, OCH3), 2.85 (2H, t, J = 7.5 Hz, CH2),
2.54 (2H, t, J = 8.1 Hz, CH2); 13C NMR (DMSO-d6, δ ppm ): 180.9 (C=S),
176.5(C=O), [153.2, 152.6, 137.2, 136.1, 128.1, 127.3, 126.5, 120.2, 111.8,
105.9, 105.8] (Ar-C), 60.3-56.1 (OCH3), 35.4 (CH2), 31.3 (CH2); GCMS (DMF,
m/z, %): 419 (9, M+), 385 (2), 254 (10), 238 (15), 223 (45), 195 (11), 165 (100),
133 (12), 123 (13), 107 (5); Anal. Cald for C20H25N3O5S : C, 57.26; H, 6.01; N,
10.02; S, 7.64; Found: C, 57.21; H, 6.11; N, 10.12; S, 7.69.
1-[3-(4-Methoxyphenyl)propanoyl]-4-(2-methoxyphenyl)thiosemicarbazide (88o): Yield: 80%; m.p: 189-191oC; Rf: 0.34 (petroleum ether : acetone, 6:4); IR
(KBr, νmax, cm-1): 3383-3116 (NH Stretching), 1668 (C=O), 1520 (C=C), 1235
(C=S); 1H NMR (DMSO-d 6, δ ppm ): 9.99 (1H, s, NH), 9.67 (1H, s, NH), 9.53
(1H, s, NH), 7.43 (2H, d, J = 8.4 Hz, H-2′,6′), 7.29 (2H, d, J = 8.4 Hz, H-3′,5′),
6.84-6.33 (4H, m, H-3,4,5,6), 3.83 (3H, s, OCH3), 3.73 (3H, s, OCH3), 2.87(2H, t,
J = 7.1 Hz, CH2), 2.56 (2H, t, J = 7.5 Hz, CH2); 13C NMR (DMSO-d 6, δ ppm ):
264
169.3 (C=O), [157.8, 157.4, 132.7, 129.6, 128.5, 126.3, 123.3,114.2] (Ar-C),
55.6-55.4 (OCH3), 35.7 (CH2), 29.9 (CH2); GCMS (DMF, m/z, %): 359 (4, M+),
325 (22), 237 (13), 194 (17), 178 (25), 167 (100), 163 (45), 135 (23), 123 (9), 107
(18); Anal. Cald for C18H21N3O3S : C, 60.15; H, 5.89; N, 11.69; S, 8.92; Found: C,
60.19; H, 5.75; N, 11.65; S, 8.91.
1-[2-(2,4-Dichlorophenoxy)propanoyl]-4-(2-methoxyphenyl)thiosemi-
carbazide (88p): Yield: 78%; m.p: 186-187°C; Rf: 0.37 (Petroleum ether :
acetone; 6:4); IR (KBr, νmax, cm-1): 3250-3100 (NH Stretching), 1658 (C=O),
1568 (C=C), 1247 (C=S); 1H-NMR (DMSO-d 6, δ ppm ): 9.98 (1H, s, NH), 9.55
(1H, s, NH), 9.38 (1H, s, NH), 7.63-7.15 (3H, m, H-3′,5′,6′), 4.79 (1H, q, CH),
6.80-6.39 (2H, m, H-3,4,5,6), 3.80 (3H, s, OCH3), 2.03 (3H, d, CH3); 13C NMR
(DMSO-d 6, δ ppm ): 167.4 (C=O), 157.2, 153.5, 129.8, 128.3), 125.4, 124.3,
122.8, 115.7](Ar-C), 74.25 (CH), 19.13 (CH3); GCMS (DMF, m/z, %): 414 (3, M+),
380 (21), 291(25), 248 (54), 232 (20), 216 (19), 189 (21), 167 (100), 133 (22),
123 (3), 107 (3); Anal. Cald for C17H17Cl2N3O3S : C, 49.28; H, 4.14; N, 10.14; S,
7. 74; Found: C, 49.18; H, 4.10; N, 10.17; S, 7. 70.
1-[4-(2,4-Dichlorophenoxy)butanoyl]-4-(2-methoxyphenyl)thiosemi- carbazide (88q): Yield: 85%; m.p: 155-156oC; Rf: 0.39(petroleum ether:
acetone, 6:4); IR (KBr, νmax, cm-1): 3311-3019 (NH Stretching), 1670 (C=O),
1605 (C=C); 1H NMR (DMSO-d 6, δ ppm ): 10.05 (1H, s, NH), 9.61(1H, s, NH),
9.02 (1H, s, NH), 7.88-6.89 (7H, m, Ar-H), 4.11 (2H, t, J = 6.3 Hz, CH2), 3.76
(3H, s, OCH3), 2.39 (2H, t, J = 7.2 Hz, CH2), 2.02 (2H, t, J = 7.2 Hz, CH2); 13C
NMR (DMSO-d 6, δ ppm ): 169.1 (C=O), [153.4, 151.6, 129.7, 128.5, 128.5,
126.4, 124.8, 122.8, 120.2, 115.5, 111.8] (Ar-C), 68.7 (CH2), 56.1 (OCH3), 30.0
(CH2), 24.7 (CH2); GCMS (DMF, m/z, %): 428(7, M+), 394 (11), 322 (21), 262
(15), 246 (33), 231(15), 203 (31), 167(100), 133 (2), 123 (13), 107 (8); 63 (5), 51
(7), 31 (2); Anal. Cald for C18H19Cl2N3O3S : C, 50.47; H, 4.47; N, 9.81; S, 07.49;
Found: C, 50.47; H, 4.45; N, 9.88; S, 7.55.
265
1-[3-(4-Methoxyphenyl)propanoyl]-4-(4-methoxyphenyl)thiosemicarbazide (88r): Yield: 83%; m.p: 165-167oC; Rf: 0.35 (petroleum ether : acetone, 6:4); IR
(KBr, νmax, cm-1): 3432-3210 (NH Stretching), 1675 (C=O), 1605 (C=C), 1252
(C=S); 1H NMR (DMSO-d 6, δ ppm ): 9.87 (1H, s, NH), 9.47 (1H, s, NH), 9.33
(1H, s, NH), 7.23 (2H, d, J = 8.4Hz, H-2′,6′), 7.14 (2H, d, J = 8.4 Hz, H-3′,5′),
6.89 (2H, d, J = 8.4Hz, H-3,5), 6.83 (2H, d, J = 8.4 Hz, H-2,6), 3.74 (3H, s,
OCH3), 3.69 (3H, s, OCH3), 2.79 (2H, t, J = 7.5 Hz, CH2), 2.46 (2H, t, J = 7.5 Hz,
CH2); 13C NMR (DMSO-d 6, δ ppm ): 171.7 (C=O), [157.9, 157.2, 133.4, 132.3,
129.6, 114.2] (Ar-C), 55.6-55.4 (OCH3), 35.7 (CH2), 29.9 (CH2); GCMS (DMF,
m/z, %): 359 (19, M+), 325 (20), 237 (8), 194 (17), 178 (21), 167 (100), 163 (23),
135 (29), 123 (19), 107 (1); Anal. Cald for C18H21N3O3S : C, 60.15; H, 5.89; N,
11.69; S, 8.92; Found: C, 60.11; H, 5.79; N, 11.65; S, 8.99.
1-[2-(4-Bromophenoxy)acetyl]-4-(4-methoxyphenyl)thiosemicarbazide (88s): Yield: 80%; m.p: 141-143oC; Rf: 0.42 (petroleum ether : acetone, 6:4); IR (KBr,
νmax, cm-1): 3326-3100 (NH Stretching), 1663 (C=O), 1525 (C=C), 1254 (C=S);
1H-NMR (DMSO-d 6, δ ppm ): 10.24 (1H, s, NH), 9.56 (2H, s, NH), 7.47 (2H, d, J
= 8.7 Hz, H-3′,5′), 7.24 (2H, d, J = 8.7Hz, H-2′,6′), 6.98 (2H, d, J = 8.4Hz, H-3,5),
6.90 (2H, d, J = 8.7 Hz, H-2,6), 4.61 (2H, s, CH2), 3.76 (3H, s, OCH3); 13C NMR
(DMSO-d 6, δ ppm ): 171.7 (C=O), [157.5, 157.3, 132.5, 129.6, 128.9, 117.4,
113.7] (Ar-C), 66.4 (CH2), 55.6 (OCH3); GCMS (DMF, m/z, %): 410 (21, M+), 377
(14), 287 (20), 244 (13), 228 (19), 213 (45), 185 (11), 165 (100), 133 (12), 123
(13), 107 (15); Anal. Cald for C16H16BrN3O3S : C, 46.84; H, 3.93; N, 10.24; S,
7.82; Found: C, 46.89; H, 3.95; N, 10.27; S, 7.88.
1-[2-(2,4-Dichlorophenoxy)acetyl]-4-(4-methoxyphenyl)thiosemicarbazide (88t): Yield: 79%; m.p: 134-135oC; Rf: 0.35 (petroleum ether : acetone, 6:4); IR
(KBr, νmax, cm-1): 3142-3001 (NH Stretching), 1675 (C=O), 1605 (C=C), 1248
(C=S); 1H NMR (DMSO-d 6, δ ppm ): 10.20 (1H, s, NH), 9. 60 (2H, s, NH), 7.60-
6.89 (7H, m, Ar-H), 4.78 (2H, s, CH2), 3.74 (3H, s, OCH3); 13C NMR (DMSO-d 6,
266
δ ppm ): 169.4 (C=O), [160.2, 157.3, 132.2, 129.8, 129.7, 128.4, 128.3, 116.1,
114.8] (Ar-C), 67.4 (CH2), 55.9 (OCH3); GCMS (DMF, m/z, %): 400 (11, M+), 365
(27), 277 (13), 234 (43), 218 (4), 203 (29), 175 (19), 165 (100), 133 (2), 123 (12),
107 (8); Anal. Cald for C16H15Cl2N3O3S : C, 48.01; H, 3.78; N, 10.50; S, 8.01;
Found: C, 48.07; H, 3.71; N, 10.40; S, 8.11.
1-(2,5-Difluorobenzoyl)-4-(4-methoxyphenyl)thiosemicarbazide (88u): Yield:
84%; m.p: 119-120oC; Rf: 0.37 (petroleum ether : acetone, 6:4); IR (KBr, νmax,
cm-1): 3254-3006 (NH Stretching), 1663 (C=O), 1509 (C=C), 1245 (C=S); 1H
NMR (DMSO-d 6, δ ppm ): 10.66 (1H, s, NH), 9.95 (1H, s, NH), 9.23 (1H, s, NH),
7.52-7.38 (3H, m, H-3′,4′,6′), 7.16 (2H, d, J = 7.5 Hz, H-3,5), 7.06 (2H, d, J = 7.8
Hz, H-2,6), 3.81 (3H, s, OCH3); 13C NMR (DMSO-d 6, δ ppm ): 166.3 (C=O),
[160.7, 153.6, 129.8, 126.2, 123.5, 120.2, 115.9] (Ar-C), 55.6 (OCH3); GCMS
(DMF, m/z, %): 337 (4, M+), 303 (19), 215 (22), 172 (15), 165 (100), 156 (5), 141
(54), 133 (14), 123 (19), 113 (8), 107 (21); Anal. Cald for C15H13F2N3O2S : C,
53.41; H, 3.88; N, 12.46; S, 9.91; Found: C, 53.46; H, 3.77; N, 12.42; S, 9.96.
1-[3-(3,4,5-Trimethoxyphenyl)propanoyl]-4-(4-methoxyphenyl)thiosemi- carbazide (88v): Yield: 85%; m.p: 164-165oC; Rf: 0.39 (petroleum ether :
acetone, 6:4); IR (KBr, νmax, cm-1): 3330-3016 (NH Stretching), 1645 (C=O),
1555 (C=C), 1236 (C=S); 1H NMR (DMSO-d 6, δ ppm ): 11.03 (1H, s, NH), 10.45
(1H, s, NH), 9.55 (1H, s, NH), 7.40 (1H, dd, J = 7.8 Hz, H-2, 6), 7.23 (1H, d, J =
8.1Hz, H-3,5), 7.15 (2H, s, H-2’,6’), 2.41 (2H, t, J = 6.9 Hz, CH2 ), 2.31 (2H, t, J =
6.9 Hz, CH2); 13C NMR (DMSO-d 6, δ ppm ): 187.8 (C=S), 167.5 (C=O), [161.0,
151.1, 150.7, 138.1, 136.2, 133.8, 130.1, 118.8, 110.2, 109.7, 105.1] (Ar-C),
55.8-56.0 (OCH3), 41.4 (CH2), 39.1 (CH2); GCMS (DMF, m/z, %): 419(17, M+),
385 (12), 254 (25), 238 (10), 223 (45), 195 (11), 165 (100), 133 (12), 123 (13),
107 (5); Anal. Cald for C20H25N3O5S : C, 57.26; H, 6.01; N, 10.02; S, 7.64;
Found: C, 57.19; H, 6.11; N, 10.22; S, 7.69.
267
1-(3,4-Dimethoxybenzoyl)-4-(4-methoxyphenyl)thiosemicarbazide (88w): Yield: 80%; m.p: 148-149oC; Rf: 0.40 (petroleum ether : acetone, 6:4); IR (KBr,
νmax, cm-1): 3400-3180 (NH Stretching), 1668 (C=O), 1596 (C=C), 1262 (C=S);
1H NMR (DMSO-d 6, δ ppm ): 10.37 (1H, s, NH), 9.68 (1H, s, NH), 9.57 (1H, s,
NH), 7.59 (1H, dd, J = 8.4, 1.8 Hz, H-6 ), 7.54 (1H, d, J = 1.8 Hz, H-2), 7.31 (2H,
d, J = 9.0 Hz, H-3’, 5’), 7.05 (1H, d, J = 8.4 Hz, H-5), 6.92 (2H, d, J = 9.0 Hz, H-
2’,6’), 3.88 (6H, s, OCH3), 3.82 (3H, s, OCH3); 13C NMR (DMSO-d6, δ ppm ):
166.0 (C=O), [157.1, 152.1, 127.9, 121.8, 113.6] (Ar-C), 55.8-56.0 (OCH3);
GCMS (DMF, m/z, %): 361(12, M+), 327 (32), 239 (27), 196 (19), 180 (26), 165
(100), 165 (21), 137 (12), 123 (13), 107 (8); Anal. Cald for C17H19N3O4S : C,
56.50; H, 5.30; N, 11.63; S, 8.87; Found: C, 56.57; H, 5.33 N, 11.69; S, 8.77.
7.8 General procedure for the synthesis of 1,2,4 Triazole-3-thiones
The respective thiosemicarbazide (1.4 mmol) was refluxed in 25mL of 4N
aqueous sodium hydroxide solution. The reaction was monitored by TLC. After
completion of reaction, the reaction mixture was cooled to room temperature and
filtered. The filtrate was neutralized with 6N hydrochloroic acid to precipitate the
triazole which was filtered and recrystalized from aqueous ethanol.
5-[1-(2,4-Dichlorophenoxy)methyl]-4-(cyclohexyl)-4H-1,2,4-triazole-3-thione (89a): Yield: 58%; m.p.: 178-180°C; Rf : 0.36 (Petroleum ether : acetone; 6:4); IR
(KBr, νmax, cm-1): 3409 (NH stretching), 3038-2873 (CH stretching), 1509 (C=N),
1591-1484 (C=C), 1271 (C=S); 1H NMR (DMSO-d 6, δ ppm ): 12.03 (1H, s, NH),
7.59 (1H, d, J = 2.1Hz, H-3′), 7.12 (1H, d, J = 7.8 Hz, H-5′), 6.82 (1H, d, J = 8.1
Hz, H-6′), 4.69 (2H, s, CH2), 3.23 (1H, s, H-1), 1.75-1.13 (10H, m, cyclohexyl H);
13C NMR (DMSO-d 6, δ ppm): 169.5 (C=S), 161.2 (C=N), [153.4, 152.3, 134.1,
130.8, 129.9] (Ar-C), 77.8 (CH2), [53.1, 32.4, 30.6, 25.2] (Cycylohexyl-C); GCMS
(DMF, m/z, %): 358 (10, M+), 357 (2), 325 (17), 298 (21), 216 (14), 215 (2), 201
268
(100), 175 (13), 141(8); Anal. Cald for C15H17Cl2N3OS : C, 50.28; H, 4.78; N,
11.73; S, 8.95; Found: C, 50.26; H, 4.70; N, 11.74; S, 8.90.
5-(3,4,5-Trimethoxyphenylethyl)-4-(cyclohexyl)-4H-1,2,4-triazole-3-thione (89b): Yield: 61%; m.p.: 234-235°C; Rf : 0.31 (Petroleum ether : acetone; 6:4); IR
(KBr, νmax, cm-1): 3101 (NH stretching), 3038-2873 (CH stretching), 1501(C=N),
1601-1384 (C=C), 1276 (C=S); 1H NMR (DMSO-d 6, δ ppm ): 13.04 (1H, s, NH),
6.33 (2H, s, H-2′,6′), 4.01 (1H, s, H-1), 1.71-1.07 (10H, m, cyclohexyl-H), 2.72-
2.54 (4H, m, 2 × CH2); 13C NMR (DMSO-d 6, δ ppm ): 169.5 (C=S), 152.4 (C=N),
[153.8, 135.1, 132.8, 105.9] (Ar-C), 60.3-56.1 (OCH3), [53.1, 32.4, 30.6, 27.6,
25.2](Cycylohexyl-C & CH2); GCMS (DMF, m/z, %): 377 (3, M+), 376 (10), 344
(23), 236 (12), 221 (100), 195 (3), 141(5), 83 (54); Anal. Cald for C19H27N3O3S :
C, 60.45; H, 7.21; N, 11.13; S, 0.49; Found: C, 60.48; H, 7.24; N, 11.17; S, 8.55.
5-(3,5-Difluorophenyl)-4-(cyclohexyl)-4H-1,2,4-triazole-3-thione (89c): Yield:
67%; m.p.: 221-223°C; Rf : 0.25 (Petroleum ether : acetone; 6:4); IR (KBr, νmax,
cm-1): 3411 (NH stretching), 3018-2853 (CH stretching), 1516 (C=N), 1589-1481
(C=C), 1255 (C=S); 1H NMR (DMSO-d 6, δ ppm ): 12.95 (1H, s, NH), 7.59 (2H,
ddd, J = 12.5, 2.4 Hz, H-2′,6′), 7.05 (1H, m, H-4′), 4.06 (1H, s, H-1), 1.70-1.03
(10H, m, cyclohexyl H); 13C NMR (DMSO-d 6, δ ppm ): 175.0 (C=S), 154.7
(C=N), [168.2, 132.8, 107.5, 106.7] (Ar-C), [56.3, 32.3, 29.6, 24.3] (Cycylohexyl-
C); GCMS (DMF, m/z, %): 295 (9, M+), 262 (17), 236 (12), 154 (13), 141 (100),
139 (12), 113 (50); Anal. Cald for C14H15F2N3S : C, 56.93; H, 5.12; N, 14.23;
S,10.86; Found: C, 56.85; H, 5.17; N, 14.28; S, 10.81.
5-(3,5-Dimethoxyphenyl)-4-(cyclohexyl)-4H-1,2,4-triazole-3-thione (89d): Yield: 51%; m.p.: 120-122°C; Rf : 0.36 (Petroleum ether : acetone; 6:4); IR (KBr,
νmax, cm-1): 3331 (NH stretching), 3090-2893 (CH stretching), 1521(C=N), 1571-
1454 (C=C), 1266 (C=S); 1H NMR (DMSO-d6, δ ppm ): 13.65 (1H, s, NH), 6.45
(1H, dd, J = 2.4 Hz, H-4′), 6.37 (2H, d, J = 2.1 Hz, H-2′,6′), 3.95 (1H, s, H-1),
269
3.73 (6H, s, OCH3), 1.66-1.13 (10H, m, cyclohexyl H); 13C NMR (DMSO-d 6, δ
ppm ): 173.2 (C=S), 152.0 (C=N), [162.6, 131.8, 104.5, 102.7](Ar-C), 56.2-55.4
(OCH3), [57.9, 33.3, 27.6, 25.3] ( Cycylohexyl-C); GCMS (DMF, m/z, %): 319 (29,
M+), 286 (21), 260 (7), 176 (5), 164 (21), 141 (100), 137 (14); Anal. Cald for
C16H21N3O2S: C, 60.16; H, 6.63; N, 13.16; S, 10.04; Found: C, 60.12; H, 6.69; N,
13.14; S, 10.17.
5-(3,4,5-Trimethoxyphenylethyl)-4-(2-methoxyphenyl)-4H-1,2,4-triazole-3-thione (89e): Yield: 47%; m.p.: 218-219°C; Rf : 0.31 (Petroleum ether : acetone;
6:4); IR (KBr, νmax, cm-1): 3301 (NH stretching), 3038-2873 (CH stretching), 1611
(C=N), 1601-1455 (C=C), 1256 (C=S); 1H NMR (DMSO-d6, δ ppm ): 13.67 (1H,
s, NH), 7.53 (1H, ddd, J = 7.8, 1.8 Hz, H-5), 7.26 (1H, dd, J = 7.8, 0.6 Hz, H-3),
7.18 (1H, dd, J = 7.8, 1.8 Hz, H-6), 7.09 (1H, ddd, J = 7.5, 0.9 Hz, H-4), 6.29 (2H,
s, H-2′,6′), 3.76-3.59 (12H, s, OCH3), 2.72-2.54 (4H, m, 2 × CH2); 13C NMR
(DMSO-d 6, δ ppm ): 168.3 (C=S), 152.4 (C=N), [154.9, 153.1, 136.2, 131.8,
130.6, 122.2, 121.2, 113.3, 105.9] (Ar-C), 60.3-56.1 (OCH3), 32.4 (CH2), 27.6
(CH2); GCMS (DMF, m/z, %): 401 (33, M+), 386 (10), 368 (22), 287 (9), 206 (3),
181 (100), 197 (6), 148 (13), 137 (7), 120 (4), 105(3), 92 (3),77 (11), 65 (5), 51
(4), 39 (2); Anal. Cald for C20H23N3O4S : C, 59.83; H, 5.77; N, 10.47; S, 7.99;
Found: C, 59.89; H, 5.71; N, 10.43; S, 8.05.
5-(3,5-Dimethoxyphenyl)-4-(2-methoxyphenyl)-4H-1,2,4-triazole-3-thione (89f): Yield: 49%; m.p.: 197-198°C; Rf : 0.35 (Petroleum ether : acetone; 6:4); IR
(KBr, νmax, cm-1): 3291 (NH stretching), 3108-2973 (CH stretching), 1501 (C=N),
1581-1474 (C=C), 1241 (C=S); 1H NMR (DMSO-d 6, δ ppm ): 14.07 (1H, s, NH),
7.49 (1H, ddd, J = 9.0,1.5 Hz, H-5), 7.39 (1H, dd, J = 7.8, 1.5 Hz, H-3), 7.17(1H,
dd, J = 7.8, 0.6 Hz, H-6), 7.09 (1H, ddd, J = 7.5, 1.2 Hz, H-4), 6.52 (1H, dd, J =
4.5, 2.4 Hz, H-4′), 6.45 (2H, d, J = 2.1 Hz, H-2′,6′), 3.58 (9H, s, OCH3); 13C NMR
(DMSO-d6, δ ppm ): 169.4 (C=S), 151.0 (C=N), [160.6, 155.0, 130.8, 128.0,
123.6, 121.4, 113.3, 105.5, 102.7] (Ar-C), 56.3-55.6 (OCH3); GCMS (DMF, m/z,
%): 343 (45, M+), 310 (100), 280 (2), 254 (3), 163 (8), 149 (15), 137(3), 120 (11),
270
105(7), 92 (6),77 (17), 65 (5), 51(4), 39 (2); Anal. Cald for C17H17N3O3S : C,
59.46; H, 4.99; N, 12.24; S, 9.34; Found: C, 59.49; H, 4.91; N, 12.28; S, 9.36.
5-(2,5-Difluorophenyl)-4-(2-methoxyphenyl)-4H-1,2,4-triazole-3-thione (89g): Yield: 75%; m.p.: 133-134°C; Rf : 0.34 (Petroleum ether : acetone; 6:4); IR (KBr,
νmax, cm-1): 3361 (NH stretching), 3056-2943 (CH stretching), 1531 (C=N), 1584-
1484 (C=C), 1244 (C=S); 1H NMR (DMSO-d6, δ ppm ): 13.46 (1H, s, NH), 7.61
(1H, dd, J = 12.5,2.1 Hz, H-2′), 7.49 (1H, ddd, J = 9.0,1.5 Hz, H-5), 7.39 (1H, dd,
J = 7.8, 1.5 Hz, H-3), 7.21 (2H, m, H-4′,5′), 7.17 (1H, dd, J = 7.8, 0.6 Hz, H-6),
7.09 (1H, ddd, J = 7.5, 1.2 Hz, H-4), 3.81 (3H, s, OCH3); 13C NMR (DMSO-d6, δ
ppm ): 174.0 (C=S), 153.6 (C=N), [162.2, 159.7, 130.8, 128.3, 122.6, 121.4,
118.2 113.5, 111.5] (Ar-C), 56.3 (OCH3); GCMS (DMF, m/z, %): 319 (9, M+), 286
(15), 260 (51), 180 (22), 166 (41), 153 (32), 139 (100), 113 (8), 107 (5); Anal.
Cald for C15H11F2N3OS : C, 56.42; H, 3.47; N, 13.16; S, 10.04; Found: C, 56.48;
H, 3.45; N, 13.10; S, 10.14.
5-[1-(2,4-Dichlorophenoxy)methyl]-4-(2-methoxyphenyl)-4H-1,2,4-triazole-3-thione (89h): Yield: 58%; m.p.: 201-202°C; Rf : 0.40 (Petroleum ether : acetone;
6:4); IR (KBr, νmax, cm-1): 3419 (NH stretching), 3081-2813 (CH stretching), 1498
(C=N), 1586-1456 (C=C), 1286 (C=S); 1H NMR (DMSO-d6, δ ppm ): 13.03 (1H,
s, NH), 7.66 (1H, s, H-3′), 7.49 (1H, d, J = 7.8 Hz, H-5′), 7.21 (1H, d, J = 8.1Hz,
H-6′), 7.19 (1H, ddd, J = 9.0,1.5 Hz, H-5), 7.13 (1H, dd, J = 7.8, 1.5 Hz, H-3),
7.07 (1H, dd, J = 7.8, 0.6 Hz, H-6), 6.89 (1H, ddd, J = 7.5, 1.2 Hz, H-4), 4.69
(2H, s, CH2), 3.85 (3H, s, OCH3); 13C NMR (DMSO-d6, δ ppm ): 165.0 (C=S),
152.3 (C=N), [160.4, 155.5, 143.1, 131.8, 130.9, 128.6, 124.6, 123.4, 122.4,
118.8, 115.5] (Ar-C), 79.2 (CH2), 56.8 (OCH3); GCMS (DMF, m/z, %): 382 (33,
M+), 348 (10), 221 (22), 216 (25), 203 (100), 175 (13), 166 (8), 107 (15); Anal.
Cald for C16H13Cl2N3O2S : C, 50.27; H, 3.43; N, 10.99; S, 8.39; Found: C, 50.24;
H, 3.49; N, 10.98; S, 8.49.
271
5-(3,5-Difluorophenyl)-4-(2-methoxyphenyl)-4H-1,2,4-triazole-3-thione (89i): Yield: 61%; m.p.: 169-170°C; Rf : 0.38 (Petroleum ether : acetone; 6:4); IR (KBr,
νmax, cm-1): 3300 (NH stretching), 3015-2903 (CH stretching), 1507 (C=N), 1588-
1414 (C=C), 1244 (C=S); 1H NMR (DMSO-d6, δ ppm ): 14.06 (1H, s, NH), 7.52
(2H, dd, J = 15.5, 2.8 Hz, H-2′,6′), 7.43 (1H, m, H-4′), 7.39 (1H, ddd, J = 8.4,1.5
Hz, H-5), 7.27 (1H, dd, J = 7.5, 1.8 Hz, H-3), 7.13 (1H, dd, J = 7.8, 1.5 Hz, H-6),
7.09 (1H, ddd, J = 7.5, 1.2 Hz, H-4), 3.88 (3H, s, OCH3); 13C NMR (DMSO-d6, δ
ppm ): 172.8 (C=S), 155.6 (C=N), [164.2, 159.0,133.0, 128.8, 123.2, 121.4,
113.5, 108.5, 106.3] (Ar-C), 55.1 (OCH3); GCMS (DMF, m/z, %): 319 (18, M+),
286 (9), 260 (42), 180 (20), 166 (68), 153 (21), 139 (100), 113 (13), 107 (9); Anal. Cald for C15H11F2N3OS : C, 56.42; H, 3.47; N, 13.16; S, 10.04; Found: C,
56.48; H, 3.42; N, 13.14; S, 10.13.
5-[1-(4-Bromophenoxy)methyl]-4-(2-methoxyphenyl)-4H-1,2,4-triazole-3-thione (89j): Yield:67%; m.p.: 154-155°C; Rf : 0.36 (Petroleum ether : acetone;
6:4); IR (KBr, νmax, cm-1): 3333 (NH stretching), 3101-2804 (CH stretching), 1513
(C=N), 1594-1384 (C=C), 1271 (C=S); 1H NMR (DMSO-d6, δ ppm ): 2.74 (1H, s,
NH), 7.71 (1H, dd, J = 8.4,1.5 Hz, H-3), 7.55 (2H, m, H-4,5), 7.19 (1H, d, J = 7.5
Hz, H-6), 6.90 (2H, d, J = 8.1 Hz, H-2′,6′), 6.73 (2H, d, J = 7.8 Hz, H-3′,5′), 4.71
(2H, s, CH2), 3.85 (3H, s, OCH3); 13C NMR (DMSO-d6, δ ppm ): 170.5 (C=S),
154.0 (C=N), [163.1, 158.9, 134.1, 133.9, 129.6, 127.6, 123.5, 118.3, 115.2] (Ar-
C), 81.2 (CH2), 56.0 (OCH3); GCMS (DMF, m/z, %): 392 (11, M+), 358 (10), 231
(12), 226 (23), 213 (100), 185 (19), 166 (8), 107 (15); Anal. Cald for
C16H14BrN3O2S : C, 48.99; H, 3.60; N, 10.71; S, 8.17; Found: C, 48.90; H, 3.69;
N, 10.73; S, 8.12.
5-[2-(4-Methoxyphenyl)ethyl]-4-(2-methoxyphenyl)-4H-1,2,4-triazole-3-thione (89k): Yield: 51%; m.p.: 204-205°C; Rf : 0.31 (Petroleum ether: acetone; 6:4); IR
(KBr, νmax, cm-1): 3291 (NH stretching), 3104-2903 (CH stretching), 1521 (C=N),
1571-1464 (C=C), 1265 (C=S); 1H NMR (DMSO-d6, δ ppm ): 13.42 (1H, s, NH),
7.52 (1H, dd, J = 8.4,1.5 Hz, H-4), 7.25 (2H, m, H-4,5), 7.10 (1H,d, J = 7.5 Hz, H-
272
6), 6.95 (2H, d, J = 8.1 Hz, H-2′,6′), 6.78 (2H, d, J = 7.8 Hz, H-3′,5′), 3.75 (3H, s,
OCH3), 3.68 (3H, s, OCH3), 2.71 (2H, t, J = 7.5 Hz, CH2), 2.53 (2H, t, J = 7.5 Hz,
CH2); 13C NMR (DMSO-d6, δ ppm ): 168.3 (C=S), 152.4 (C=N), [158.1, 154.9,
132.3, 131.8, 130.6, 129.6, 122.2, 121.9, 114.2, 113.2, 56.3-55.4 (OCH3), 30.8
(CH2), 27.6 (CH2); GCMS (DMF, m/z, %): 341(3, M+), 324 (17), 236 (5), 227
(43),188 (4), 134 (3), 121 (100), 105 (3), 92 (13),77 (21), 65 (7), 51(6), 39 (2);
Anal. Cald for C18H19N3O2S : C, 63.32; H, 5.61; N, 12.31; S, 9.39; Found: C,
63.36; H, 5.62; N, 12.39; S, 9.31.
5-[1-(2,4-Dichlorophenoxy)ethyl]-4-(2-methoxyphenyl)-4H-1,2,4-triazole-3-thione (89l): Yield: 54%; m.p.: 165-166°C; Rf : 0.35 (Petroleum ether : acetone;
6:4); IR (KBr, νmax, cm-1): 3341 (NH stretching), 3054-2813 (CH stretching), 1491
(C=N), 1601-1484 (C=C), 1279 (C=S); 1H NMR (DMSO-d 6, δ ppm ): 12.93 (1H,
s, NH), 7.62 (1H, s, H-3′), 7.59 (1H, d, J = 7.5 Hz, H-5′), 7.48 (1H, d, J = 8.4 Hz,
H-6′), 7.31 (1H, ddd, J = 9.0,1.5 Hz, H-5), 7.23 (1H, dd, J = 7.5, 1.5 Hz, H-3),
7.17 (1H, dd, J = 7.8, 0.6 Hz, H-6), 6.99 (1H, ddd, J = 7.5, 1.2 Hz, H-4), 4.41
(1H, s, CH2), 3.70 (3H, s, OCH3), 1.70 (3H, s, CH2); 13C NMR (DMSO-d 6, δ ppm
): 168.7 (C=S), 156.6 (C=N), [159.4, 151.5, 132.8, 129.9, 129.6, 129.6, 126.6,
124.4, 123.4, 116.8, 114.5] (Ar-C), 72.2 (CH2), 55.5 (OCH3), 15.2 (CH2); GCMS
(DMF, m/z, %): 396 (7, M+), 362 (21), 230 (9), 215 (16), 216 (100), 189 (32), 180
(13), 166 (3), 107 (15); Anal. Cald for C17H15Cl2N3O2S : C, 51.52; H, 3.82; N,
10.60; S, 8.09; Found: C, 51.58; H, 3.85; N, 10.67; S, 8.13.
5-(3,4,5-Trimethoxyphenylethyl)-4-(4-methoxyphenyl)-4H-1,2,4-triazole-3-thione (89m): Yield: 56%; m.p.: 169-170°C; Rf : 0.34 (Petroleum ether : acetone;
6:4); IR (KBr, νmax, cm-1): 3241 (NH stretching), 3038-2933 (CH stretching), 1481
(C=N), 1551-1481 (C=C), 1257 (C=S); 1H NMR (DMSO-d6, δ ppm ): 13.21(1H, s,
NH), 7.32 (1H, d, J = 7.8 Hz, H-3,5), 7.08 (1H, d, J = 7.5 Hz, H-2,6), 6.79 (1H, d,
J = 1.8 Hz, H-2′,6′), 3.84-3.61 (12H, s, OCH3), 2.73-2.50 (4H, m, CH2); 13C NMR
(DMSO-d 6, δ ppm ): 169.1 (C=S), 153.2 (C=N), [155.9, 151.1, 138.2, 134.8,
273
128.6, 126.2, 115.3, 105.9] (Ar-C), 58.3-56.1 (OCH3), 32.4 (CH2), 30.6 (CH2); GCMS (DMF, m/z, %): 401 (25, M+), 386 (19), 368 (20), 287 (19), 206 (23), 181
(100), 197 (66), 148 (33), 137 (37), 120 (14), 105 (13), 92 (13), 77 (18), 65 (5),
51(4), 39 (2); Anal. Cald for C20H23N3O4S : C, 59.83; H, 5.77; N, 10.47; S, 7.99;
Found: C, 59.80; H, 5.76; N, 10.35; S, 8.15.
5-(3,5-Dimethoxyphenyl)-4-(4-methoxyphenyl)-4H-1,2,4-triazole-3-thione (89n): Yield: 75%; m.p.: 153-154°C; Rf : 0.39 (Petroleum ether : acetone; 6:4);
IR (KBr, νmax, cm-1): 3189 (NH stretching), 3031-2873 (CH stretching), 1497
(C=N), 1571-1464 (C=C), 1275 (C=S); 1H NMR (DMSO-d6, δ ppm ): 13.37 (1H,
s, NH), 7.53 (1H, d, J = 9.0 Hz, H-3,5), 7.24 (1H, d, J = 7.5 Hz, H-2,6), 6.72 (1H,
dd, J = 4.5, 2.4 Hz, H-4′), 6.51 (2H, d, J = 2.1 Hz, H-2′,6′), 3.71-3.61 (9H, s,
OCH3); 13C NMR (DMSO-d6, δ ppm ): 168.7 (C=S), 151.4 (C=N), [163.4, 157.2,
132.8, 128.0, 127.6, 117.3, 108.5, 104.7] (Ar-C), 56.3-55.0 (OCH3); GCMS
(DMF, m/z, %): 343 (39, M+), 310 (100), 280 (12), 254 (13), 163 (18), 149 (25),
137 (30), 120 (10), 105(12), 92 (16),77 (11), 65 (15), 51(6), 39 (8); Anal. Cald for
C17H17N3O3S : C, 59.46; H, 4.99; N, 12.24; S, 9.34. Found: C, 59.46; H, 4.94; N,
12.25; S, 9.30.
5-(2,5-Difluorophenyl)-4-(4-methoxyphenyl)-4H-1,2,4-triazole-3-thione (89o): Yield: 58%; m.p.: 173-175°C; Rf : 0.36 (Petroleum ether : acetone; 6:4); IR (KBr,
νmax, cm-1): 3181 (NH stretching), 3001-2874 (CH stretching), 1502 (C=N), 1601-
1414 (C=C), 1245 (C=S); 1H NMR (DMSO-d6, δ ppm ): 2.09 (1H, s, NH), 7.54
(1H, dd, J = 13.0, 2.4 Hz, H-2′), 7.42 (1H, ddd, J = 11.3,1.0 Hz, H-3,5), 7.29 (2H,
m, H-4′,5′), 7.07 (1H, dd, J = 7.8, 0.6 Hz, H-2,6), 3.88 (3H, s, OCH3); 13C NMR
(DMSO-d6, δ ppm ): 175.1 (C=S), 154.38 (C=N), [160.5, 157.7, 129.3, 127.6,
121.4, 118.2, 116.5, 114.5, 113.5] (Ar-C), 56.7 (OCH3); GCMS (DMF, m/z, %): 319 (17, M+), 286 (20), 260 (35), 180 (29), 166 (13), 153 (32), 139 (100), 113
(18), 107 (15); Anal. Cald for C15H11F2N3OS : C, 56.42; H, 3.47; N, 13.16; S,
10.04; Found: C, 56.45; H, 3.49; N, 13.15; S, 10.16.
274
5-[1-(2,4-Dichlorophenoxy)methyl]-4-(4-methoxyphenyl)-4H-1,2,4-triazole-3-thione (89p): Yield: 42%; m.p.: 222-223°C; Rf : 0.31 (Petroleum ether : acetone;
6:4); IR (KBr, νmax, cm-1): 3341 (NH stretching), 3018-2813 (CH stretching), 1514
(C=N), 1587-1411 (C=C), 1255 (C=S); 1H NMR (DMSO-d6, δ ppm ): 12.73 (1H,
s, NH), 7.51 (1H, s, H-3′), 7.44 (1H, d, J = 7.8 Hz, H-5′), 7.29 (1H, d, J = 8.1 Hz,
H-6′), 7.14 (1H, d, J = 9.0, 1.5 Hz, H-3,5), 7.10 (1H, d, J = 7.8, 1.5 Hz, H-2,6),
4.55 (2H, s, CH2), 3.74 (3H, s, OCH3); 13C NMR (DMSO-d 6, δ ppm ): 167.0
(C=S), 153.3 (C=N), [156.4, 154.5, 132.4, 131.8, 130.9, 128.6, 126.6, 123.4,
118.8, 115.5] (Ar-C), 77.2 (CH2), 56.8 (OCH3); GCMS (DMF, m/z, %): 382 (29,
M+), 348 (19), 221 (32), 216 (20), 203 (100), 175 (33), 166 (81), 107 (19); Anal.
Cald for C16H13Cl2N3O2S : C, 50.27; H, 3.43; N, 10.99; S, 8.39; Found: C, 50.24;
H, 3.41; N, 10.95; S, 8.35.
5-(3,5-Difluorophenyl)-4-(4-methoxyphenyl)-4H-1,2,4-triazole-3-thione (89q): Yield: 66%; m.p.: 229-230°C; Rf : 0.35 (Petroleum ether : acetone; 6:4); IR (KBr,
νmax, cm-1): 3349 (NH stretching), 3008-2803 (CH stretching), 1481 (C=N), 1571-
1454 (C=C), 1248 (C=S); 1H NMR (DMSO-d6, δ ppm ): 13.36 (1H, s, NH), 7.61
(2H, dd, J = 15.5, 2.8 Hz, H-2′,6′), 7.47 (1H, m, H-4′), 7.31 (1H, ddd, J = 8.4,1.5
Hz, H-3,5), 7.07 (1H, dd, J = 7.8, 1.5 Hz, H-2,6), 3.81 (3H, s, OCH3); 13C NMR
(DMSO-d6, δ ppm ): 170.6 (C=S), 154.6 (C=N), [166.2, 157.0, 133.7, 128.8,
125.4, 115.5, 107.5, 106.3] (Ar-C), 55.9 (OCH3); GCMS (DMF, m/z, %): 319 (20,
M+), 286 (26), 260 (21), 180 (59), 166 (19), 153 (38), 139 (100), 113 (10), 107
(5); Anal. Cald for C15H11F2N3OS : C, 56.42; H, 3.47; N, 13.16; S, 10.04; Found:
C, 56.42; H, 3.46; N, 13.19; S, 10.03.
5-[1-(4-Bromophenoxy)methyl]-4-(4-methoxyphenyl)-4H-1,2,4-triazole-3-thione (89r): Yield: 75%; m.p.: 156-158°C; Rf : 0.34 (Petroleum ether : acetone;
6:4); IR (KBr, νmax, cm-1): 3425 (NH stretching), 3108-2913 (CH stretching), 1521
(C=N), 1551-1435 (C=C), 1247 (C=S); 1H NMR (DMSO-d 6, δ ppm ): 2.51 (1H, s,
NH), 7.68 (2H, d, J = 8.4 Hz, H-3,5), 7.53 (2H, d, J = 7.8 Hz, H-3′,5′), 6.23 (2H,
d, J = 8.4 Hz, H-2′,6′), 6.81 (2H, d, J = 7.8 Hz, H-2,6), 4.44 (2H, s, CH2), 3.74
275
(3H, s, OCH3); 13C NMR (DMSO-d6, δ ppm ): 169.3 (C=S), 153.2 (C=N), [160.1,
157.4, 135.1, 128.4, 127.6, 117.3, 115.2] (Ar-C), 78.2 (CH2), 56.43 (OCH3); GCMS (DMF, m/z, %): 392 (21, M+), 358 (61), 231 (32), 226 (12), 213 (100), 185
(12), 166 (28), 107 (9); Anal. Cald for C16H14BrN3O2S : C, 48.99; H, 3.60; N,
10.71; S, 8.17; Found: C, 48.97; H, 3.65; N, 10.63; S, 8.17.
5-[1-(4-Methoxyphenyl)ethyl]-4-(4-methoxyphenyl)-4H-1,2,4-triazole-3-thione (89s): Yield: 58%; m.p.: 102-103°C; Rf : 0.29 (Petroleum ether : acetone; 6:4); IR
(KBr, νmax, cm-1): 3201(NH stretching), 3010-2870 (CH stretching), 1519 (C=N),
1601-1489 (C=C), 1276 (C=S); 1H NMR (DMSO-d6, δ ppm ): 13.15 (1H, s, NH),
7.59 (1H, d, J = 8.4 Hz, H-2′,6′), 7.35 (2H, d, J = 7.8 Hz, H-3′,5′), 6.95 (2H, d, J =
8.1 Hz, H-3,5), 6.73 (2H, d, J = 7.8 Hz, H-2,6), 3.70-3.62 (6H, s, OCH3), 3.02
(2H, t, J = 7.5 Hz, CH2), 2.14 (2H, t, J = 7.5 Hz, CH2); 13C NMR (DMSO-d6, δ
ppm ): 167.8 (C=S), 155.4 (C=N), [157.6, 135.3, 130.1,128.6, 127.2, 117. 9, 117.
2, 115.2] (Ar-C), 56.8-55.3 (OCH3), 35.8 (CH2), 20.6 (CH2); GCMS (DMF, m/z,
%): 341(17, M+), 324 (20), 236 (15), 227 (32),188 (24), 134 (32), 121 (100), 105
(23), 92 (23),77 (29), 65 (71), 51 (46), 39 (12); Anal. Cald for C18H19N3O2S : C,
63.32; H, 5.61; N, 12.31; S, 9.39; Found: C, 63.39; H, 5.62; N, 12.35; S, 9.36.
5-[1-(2,4-Dichlorophenoxy)ethyl]-4-(4-methoxyphenyl)-4H-1,2,4-triazole-3-thione (89t): Yield: 42%; m.p.: 208-209°C; Rf : 0.28 (Petroleum ether : acetone;
6:4); IR (KBr, νmax, cm-1): 3201 (NH stretching), 3038-2873 (CH stretching), 1505
(C=N), 1581-1444 (C=C), 1245 (C=S); 1H NMR (DMSO-d 6, δ ppm ): 13.13 (1H,
s, NH), 7.58 (1H, s, H-3′), 7.49 (1H, d, J = 7.5 Hz, H-5′), 7.37 (1H, d, J = 8.4 Hz,
H-6′), 7.03 (1H, d, J = 7.8 Hz, H-3,5), 6.87 (1H, d, J = 7.5 Hz, H-2,6), 4.52 (2H,
s, CH2), 3.81 (3H, s, OCH3), 1.79 (2H, s, CH2); 13C NMR (DMSO-d6, δ ppm ):
169.5 (C=S), 154.6 (C=N), [158.4, 152.5, 132.5, 128.9, 128.6, 127.9, 126.6,
125.4, 124.4, 118.8, 115.5] (Ar-C), 72.2 (CH2), 55.5 (OCH3), 15.2 (CH2); GCMS
(DMF, m/z, %): 396 (14, M+), 362 (19), 230 (29), 215 (36), 216 (100), 189 (32),
180 (13), 166 (23), 107 (3);Anal. Cald for C17H15Cl2N3O2S : C, 51.52; H, 3.82; N,
10.60; S, 8.09. Found: C, 51.54; H, 3.80; N, 10.67; S, 8.11.
276
7.9 General procedure for the synthesis of 1,3,4- Thiadiazoles
Each thiosemicarbazide (0.1 mmol) was added in portion to conc. sulphuric
acid (15 mL, 98%) at 0°C. The reaction mixture was heated at 120°C in an oil
bath for 10 minutes. The mixture was then stirred for 1 h at room temperature.
The reaction mixture was then poured into crushed ice and followed by dropwise
addition of aqueous ammonia (33%) till the appearance of a solid. The crude
1,3,4-thiadiazole was filtered, washed with water and recrystallized from acetic
acid-water (1:10).
Cyclohexylamino-5-(3,4,5-trimethoxyphenethyl)-1,3,4-thiadiazole (90a): Yield: 49%; m.p.: 134-135°C; Rf : 0.34 (Petroleum ether : acetone; 6:4); IR (KBr,
νmax, cm-1): 3101 (NH stretching), 3038-2873 (CH stretching), 1501 (C=N), 1601-
1384 (C=C), 1276 (C=S); 1H NMR (DMSO-d 6, δ ppm ): 9.04 (1H, s, NH), 6.41
(2H, d, J = 2.4 Hz, H-2′,6′), 3.57 (1H, s, H-1), 1.87-1.17 (10H, m, cyclohexyl H),
2.92-2.74 (4H, m, 2 × CH2); 13C NMR (DMSO-d 6, δ ppm ): [173.5, 170.2] (C=N),
[153.5, 141.4, 134.8, 105.9] (Ar-C), 60.1-56.6 (OCH3), [55.2,34.2, 32.4, 29.6,
27.6, 22.2] (cyclohexyl-C & CH2 ); GCMS (DMF, m/z, %): 377 (19, M+), 295
(100), 279 (22), 221 (13), 156 (8), 98 (25), 58 (7); Anal. Cald for C19H27N3O3S :
C, 60.45; H, 7.21; N, 11.13; S, 8.49; Found: C, 60.42; H, 7.27; N, 11.13; S, 8.50.
Cyclohexylamino-5-(3,5-dimethoxyphenyl)-1,3,4-thiadiazole (90b): Yield:
51%; m.p.: 198-199°C; Rf : 0.38 (Petroleum ether : acetone; 6:4); IR (KBr, νmax,
cm-1): 3309 (NH stretching) , 3021-2881 (CH stretching), 1511 (C=N), 1611-
1385 (C=C); 1H NMR (DMSO-d6, δ ppm ): 10.05 (1H, s, NH), 6.39 (1H, dd, J =
3.2, 2.1 Hz, H-4′), 6.27 (2H, d, J = 2.1 Hz, H-2′,6′), 3.17 (1H, s, H-1), 3.88 (6H, s,
OCH3), 1.83-1.13 (10H, m, cyclohexyl H); 13C NMR (DMSO-d6, δ ppm ): [176.2,
167.2] (C=N), [163.3, 140.8, 107.5, 99.7] (Ar-C), 55.4 (OCH3), [54.9, 35.3, 27.1,
277
25.3] (cyclohexyl-C); GCMS (DMF, m/z, %): 319 (7, M+), 237 (100), 221 (13),
177 (21), 163 (23), 141 (8), 98 (25), 83 (6), 74 (79), 58 (7); Anal. Cald for
C16H21N3O2S : C, 60.16; H, 6.63; N, 13.16; S, 10.04; Found: C, 60.14; H, 6.63; N,
13.15; S, 10.15.
2-Methoxyphenylamino-5-(3,4,5-trimethoxyphenethyl)-1,3,4-thiadiazole (90c): Yield: 48%; m.p.: 147-148°C; Rf : 0.36 (Petroleum ether : acetone; 6:4); IR
(KBr, νmax, cm-1): 3291 (NH stretching), 3008-2821 (CH stretching), 1515 (C=N),
1591-1383 (C=C); 1H NMR (DMSO-d 6, δ ppm ): 9.87 (1H, s, NH), 7.57 (1H, ddd,
J = 7.8, 2.4Hz, H-5), 7.36 (1H, dd, J = 7.5, 1.8 Hz, H-3), 7.15 (1H, dd, J = 7.8, 1.8
Hz, H-6), 7.03 (1H, ddd, J = 7.5, 0.9 Hz, H-4), 6.59 (2H, s, H-2′,6′), 3.88-3.87
(12H, s, OCH3), 2.79-2.57 (4H, m, 2 × CH2); 13C NMR (DMSO-d6, δ ppm ):
[170.3, 156.5] (C=N), [153.1, 150.9, 138.2, 135.8, 132.2, 123.6, 120.2, 116.3,
107.9] (Ar-C), 59.3-56.5 (OCH3), 36.4 (CH2), 33.6 (CH2); GCMS (DMF, m/z, %):
401(33, M+), 295 (100), 279 (22), 235 (13), 221 (13), 165 (25), 156 (8), 107 (25),
58 (7); Anal. Cald for C20H23N3O4S : C, 59.83; H, 5.77; N, 10.47; S, 7.99; Found:
C, 59.85; H, 5.75; N, 10.47; S, 7.95.
2-Methoxyphenylamino-5-(3,5-dimethoxyphenyl)-1,3,4-thiadiazole (90d): Yield: 58%; m.p.: 189-191°C; Rf : 0.39(Petroleum ether: acetone; 6:4); IR (KBr,
νmax, cm-1): 3241 (NH stretching), 3018-2913 (CH stretching), 1521 (C=N), 1601-
1414 (C=C); 1H NMR (DMSO-d6, δ ppm ): 8.97 (1H, s, NH), 7.35 (1H, ddd, J =
8.4,1.5 Hz, H-5), 7.29 (1H, dd, J = 7.8, 2.0 Hz, H-3), 7.17 (1H, dd, J = 7.5, 1.6
Hz, H-6), 7.05 (1H, ddd, J = 8.1, 1.8 Hz, H-4), 6.59 (1H, dd, J = 4.5, 2.4 Hz, H-4′),
6.39 (2H, d, J = 2.1Hz, H-2′,6′), 3.77 (9H, s, OCH3); 13C NMR (DMSO-d6, δ ppm
): [178.4, 155.27] (C=N), [165.5, 150.8,135.2, 134.0, 123.5, 121.3, 116.5, 105.7,
99.7] (Ar-C), 56.5-55.7 (OCH3); GCMS (DMF, m/z, %): 343 (11, M+), 237 (100),
221 (13), 177 (21), 165 (13), 141 (8), 107 (45), 83 (6), 74 (79); Anal. Cald for
C17H17N3O3S : C, 59.46; H, 4.99; N, 12.24; S, 9.34; Found: C, 59.44; H, 4.94; N,
12.25; S, 9.34.
278
2-Methoxyphenylamino-5-[1-(2,4-Dichlorophenoxy)methyl]-1,3,4-thiadiazole (90e): Yield: 61%; m.p.: 201-202°C; Rf : 0.37 (Petroleum ether : acetone; 6:4); IR
(KBr, νmax, cm-1): 3315 (NH stretching), 3010-2893 (CH stretching), 1491 (C=N),
1615-1484 (C=C); 1H NMR (DMSO-d 6, δ ppm ): 9.05 (1H, s, NH), 7.62 (1H, s,
H-3′), 7.48 (1H, d, J = 7.5 Hz, H-5′), 7.25 (1H, d, J = 8.1 Hz, H-6′), 7.17 (1H, ddd,
J = 9.0, 1.5 Hz, H-5), 7.12 (1H, dd, J = 7.8, 1.5 Hz, H-3), 7.06 (1H, dd, J = 7.8,
1.6 Hz, H-6), 6.84 (1H, ddd, J = 7.5, 1.2 Hz, H-4), 4.69 (2H, s, CH2), 3.81 (3H, s,
OCH3); 13C NMR (DMSO-d6, δ ppm ): [164.3, 152.3] (C=N), [153.5, 149.4, 133.7,
131.8, 129.9, 125.6, 122.6, 120.4, 118.4, 118.2, 115.8] (Ar-C), 65.2 (CH2),
56.2(OCH3); GCMS (DMF, m/z, %): 282 (6, M+), 275 (100), 259 (16), 201 (24),
136 (38), 107 (12), 78 (25), 58 (4); Anal. Cald for C16H13Cl2N3O2S : C, 50.27; H,
3.43; N, 10.99; S, 8.39; Found: C, 50.24; H, 3.48; N, 10.95; S, 8.48.
2-Methoxyphenylamino-5-(3,5-Difluorophenyl)-1,3,4-thiadiazole (90f): Yield:
59%; m.p.: 178-179°C; Rf : 0.35(Petroleum ether : acetone; 6:4); IR (KBr, νmax,
cm-1): 3311 (NH stretching), 3939-2803 (CH stretching), 1508 (C=N), 1595-1384
(C=C); 1H NMR (DMSO-d6, δ ppm ): 8.98 (1H, s, NH), 7.57 (2H, dd, J = 15.5,
2.1 Hz, H-2′,6′), 7.49 (1H, m, H-4′), 7.38 (1H, ddd, J = 8.4, 1.5 Hz, H-5), 7.26
(1H, dd, J = 7.5, 1.8 Hz, H-3), 7.18 (1H, dd, J = 7.8, 1.5 Hz, H-6), 7.05 (1H, ddd,
J = 7.5, 1.2 Hz, H-4), 3.85 (3H, s, OCH3); 13C NMR (DMSO-d6, δ ppm ): [177.8,
154.6] (C=N), [164.4, 149.3, 138.0, 134.8, 123.2, 120.4, 118.5, 115.5, 112.5,
105.3] (Ar-C), 56.1 (OCH3); GCMS (DMF, m/z, %): 319 (22, M+), 213 (100), 197
(13), 221 (13), 171 (24), 165 (21), 153 (23), 107 (45), 74 (14); Anal. Cald for
C15H11F2N3OS : C, 56.42; H, 3.47; N, 13.16; S, 10.04; Found: C, 56.45; H, 3.46;
N, 13.10; S, 10.11.
4-Methoxyphenylamino-5-(3,4,5-trimethoxyphenethyl)-1,3,4-thiadiazole (90g): Yield: 55%; m.p.: 197-198°C; Rf : 0.37 (Petroleum ether : acetone; 6:4); IR
(KBr, νmax, cm-1): 3206 (NH stretching), 3008-2870 (CH stretching), 1523 (C=N),
1594-1481 (C=C); 1H NMR (DMSO-d6, δ ppm ): 9.12 (1H, s, NH), 7.30 (1H, d, J
279
= 7.8 Hz, H-3,5), 7.11 (1H, d, J = 7.5 Hz, H-2,6), 6.70 (1H, d, J = 1.8 Hz, H-2′,6′),
3.89-3.67 (12H, s, OCH3), 2.78-2.59 (4H, m, CH2); 13C NMR (DMSO-d6, δ ppm ):
[169.1, 153.9] (C=N), [151.7, 151.1, 137.2, 136.4, 133.8, 119.6, 116.3, 106.0]
(Ar-C), 57.0-56.6 (OCH3), 36.4 (CH2), 33.6 (CH2); GCMS (DMF, m/z, %): 401
(12, M+), 295 (100), 279 (13), 235 (6), 221 (13), 165 (35), 156 (18), 107 (25), 58
(7); Anal. Cald for C20H23N3O4S : C, 59.83; H, 5.77; N, 10.47; S, 7.99; Found: C,
59.87; H, 5.73; N, 10.38; S, 8.14.
4-Methoxyphenylamino-5-(3,5-dimethoxyphenyl)-1,3,4-thiadiazole (90h): Yield: 57%; m.p.: 188-189°C; Rf : 0.35 (Petroleum ether : acetone; 6:4); IR (KBr,
νmax, cm-1): 3191 (NH stretching), 3031-2913 (CH stretching), 1503 (C=N), 1600-
1484 (C=C); 1H NMR (DMSO-d6, δ ppm ): 9.05 (1H, s, NH), 7.53 (1H, d, J = 9.0
Hz, H-3,5), 7.24 (1H, d, J = 7.5Hz, H-2,6), 6.72 (1H, dd, J = 4.5, 2.4 Hz, H-4′),
6.51 (2H, d, J = 2.1 Hz, H-2′,6′), 3.71-3.61 (9H, s, OCH3); 13C NMR (DMSO-d6, δ
ppm ): [178.4, 153.4] (C=N), [164.2, 151.2, 136.8, 136.2, 118.0, 117.7, 105.5,
101.7] (Ar-C), 56.0-55.4 (OCH3); GCMS (DMF, m/z, %): 343 (25, M+), 237
(100), 221 (9), 177 (18), 165 (21), 141 (18), 107 (40), 83 (26), 74 (33); Anal. Cald
for C17H17N3O3S : C, 59.46; H, 4.99; N, 12.24; S, 9.34; Found: C, 59.49; H, 4.94;
N, 12.29; S, 9.32.
4-Methoxyphenylamino-5-[1-(2,4-dichlorophenoxy)methyl]-1,3,4-thiadiazole (90i): Yield: 55%; m.p.: 145-146°C; Rf : 0.37 (Petroleum ether : acetone; 6:4); IR
(KBr, νmax, cm-1): 3299 (NH stretching), 3038-2945 (CH stretching), 1508 (C=N),
1594-1381 (C=C); 1H NMR (DMSO-d 6, δ ppm ): 8.73 (1H, s, NH), 7.51 (1H, s,
H-3′), 7.48 (1H, d, J = 7.8 Hz, H-5′), 7.31 (1H, d, J = 8.1 Hz, H-6′), 7.17 (1H, d, J
= 9.0, 1.5 Hz, H-3,5), 7.12 (1H, d, J = 7.8, 1.5 Hz, H-2,6), 4.51 (2H, s, CH2),
3.84(3H, s, OCH3); 13C NMR (DMSO-d6, δ ppm ): [177.2, 153.3](C=N), [156.5,
152.4, 136.4, 131.8, 129.9, 125.6, 118.6, 118.3, 115.3](Ar-C), 66.2(CH2),
56.8(OCH3); GCMS (DMF, m/z, %): 282 (16, M+), 275(100), 259(10), 201(21),
280
136(45), 107(21), 78(19), 58(14); Anal. Cald for C16H13Cl2N3O2S : C, 50.27; H,
3.43; N, 10.99; S, 8.39; Found: C, 50.24; H, 3.44; N, 10.97; S, 8.36.
4-Methoxyphenylamino-5-(3,5-difluorophenyl)-1,3,4-thiadiazole(90j): Yield:
54%; m.p.: 111-112°C; Rf : 0.37 (Petroleum ether : acetone; 6:4); IR (KBr, νmax,
cm-1): 3401(NH stretching), 3058-2870 (CH stretching), 1531(C=N), 1597-
1394(C=C); 1H NMR (DMSO-d6, δ ppm ): 9.04(1H, s, NH), 7.64(2H, ddd, J =
15.5,2.1 Hz, H-2′,6′), 7.51(1H, m, H-4′), 7.28(1H, d, J = 7.8 Hz, H-3,5), 7.07(1H,
d, J = 7.5 Hz, H-2,6), 3.76(3H, s, OCH3); 13C NMR (DMSO-d6, δ ppm ): [178.6,
154.6](C=N), [166.2, 152.0, 137.7, 137.0, 121.2, 116.5, 116.5, 105.3](Ar-C),
56.6(OCH3); GCMS (DMF, m/z, %): 319 (19, M+), 213(100), 197(28), 221(34),
171(24), 165(39), 153(12), 107(33), 74(29); Anal. Cald for C15H11F2N3OS : C,
56.42; H, 3.47; N, 13.16; S, 10.04; Found: C, 56.45; H, 3.42; N, 13.14; S, 10.14.
7.10 General procedure for the synthesis of Indolinones
A mixture of haloisatin (0.01 moles) was refluxed with an equimolar
amount of the appropriate hydrazide (0.01 moles) in ethanol (10 mL) for 5 hours
and left to cool. The precipitated solid was filtered, washed with water, dried and
crystallized from the appropriate solvent.
(E)-N′-(5-Chloro-2-oxoindolin-3-ylidene)-3,5-difluorobenzohydrazide (91a):
Yield: 89%; m.p: 178-179 oC; IR (KBr, νmax, cm-1): 3281 (NH stretching), 3084
(sp2 CH stretching), 1710 (C=O), 1677 (C=N), 1623, 1460 (C=C); 1H NMR
(DMSO-d6, δ ppm ): 11.78(1H, s, NH), 10.98 (1H, s, NH), 8.11 (1H, s, H-4),
7.68-7.43 (4H, m, H-6,2′,4′,6′), 6.93 (1H, d, J = 8.7Hz, H-7); 13C NMR (DMSO-d
6, δ ppm ): 171.3, 164.7, 164.2, 143.3, 141.7, 136.7, 132.8, 127.4, 127.1, 126.0,
116.8, 112.9, 108.0; EIMS (m/z, %): 335 (48, M+), 307 (14), 290 (2), 287 (1), 250
(2), 244 (2), 216 (1), 194 (100), 166 (53), 141 (91), 113 (56), 102 (10), 75 (7), 63
(6), 51 (2); Anal. Cald for C15H8ClF2N3O2: C, 53.67; H, 2.40; N, 12.52; Found: C,
53.62; H, 2.45; N, 12.58.
281
(E)-N′-(5-Chloro-2-oxoindolin-3-ylidene)-2,5-difluorobenzohydrazide (91b):
Yield: 88%; m.p: 165-166 oC; IR (KBr, νmax, cm-1): 3412-3237 (NH stretching),
3115 (sp2 CH stretching), 1719 (C=O), 1622 (C=N), 1308 (C-O); 1H NMR
(DMSO-d6, δ ppm ): 11.44(1H, s, NH), 10.96(1H, s, NH), 8.13(1H, s, H-4), 7.72-
7.41(3H, m, H-3′,4′,6′), 7.08-6.79(2H, m, H-6,7); 13C NMR (DMSO-d6, δ ppm ):
170.5, 162.2, 160.7, 159.1, 145.3, 133.7, 132.8, 131.4, 129.1, 127.5, 124.0,
121.9, 120.8, 118.5, 116.9; EIMS (m/z, %): 335 (24, M+), 307 (24), 291 (1), 290
(1), 250 (1), 232 (2), 224 (1), 194 (55), 166 (37), 141 (100), 113 (44), 102 (12),
75 (7), 63 (7), 51 (1); Anal. Cald for C15H8ClF2N3O2: C, 53.67; H, 2.40; N, 12.52;
Found: C, 53.66; H, 2.43; N, 12.53.
(E)-N′-(5-Chloro-2-oxoindolin-3-ylidene)-2,6-difluorobenzohydrazide (91c):
Yield: 84%; m.p: 212-213oC; IR (KBr, νmax, cm-1): 3419-3232 (NH stretching),
3118 (sp2 CH stretching), 1713 (C=O), 1627 (C=N), 1278 (C-O); 1H NMR
(DMSO-d 6, δ ppm ): 11.45 (1H, s, NH), 10.71 (1H, s, NH), 8.00 (1H, s, H-4),
7.54-7.31 (3H, m, H-3′,4′,6′), 6.76 (2H, d, J = 14.4 Hz, H-6,7); 13C NMR (DMSO-
d6, δ ppm) : 169.8, 163.7, 160.2, 144.0, 136.9, 133.4, 131.9, 131.2, 130.1, 124.5,
120.2, 115.5, 110.5; EIMS (m/z, %): 335 (18, M+), 307 (9), 281 (7), 207 (13), 194
(72), 166 (48), 141 (100), 113 (94), 102 (17), 75 (22), 63 (37), 44 (24), 32 (17);
Anal. Cald for C15H8ClF2N3O2: C, 53.67; H, 2.40; N, 12.52; Found: C, 53.68; H,
2.48; N, 12.55.
(E)-N′-(5-Chloro-2-oxoindolin-3-ylidene)-3,4-dimethoxybenzohydrazide
(91d): Yield: 83%; m.p: 145-146oC; IR (KBr, νmax, cm-1): 3252 (NH stretching),
3101 (sp2 CH stretching), 1711 (C=O), 1652 (C=N), 1594-1463 (C=C), 1350 (C-
O); 1H NMR (DMSO-d6, δ ppm ): 11.49 (1H, s, NH), 10.24 (1H, s, NH), 7.60 (1H,
d, J = 2.0 Hz, H-4), 7.50-7.42 (3H, m, H-6,2′,6′), 7.19 (1H, d, J = 8.4 Hz, H-7),
6.99 (1H, d, J = 8.4 Hz, H-5′), 3.87-3.85 (6H, s, OCH3); 13C NMR (DMSO-d 6, δ
ppm ): 169.3, 163.4, 154.2, 149.9, 145.2, 133.5, 131.3, 130.8, 129.1, 128.0,
282
124.0, 121.3, 120.5, 116.5, 113.2, 56.42 (OCH3); EIMS (m/z, %):359 (3, M+), 330
(3), 279 (21), 261 (1), 194 (2), 182 (3), 167 (40), 149 (100), 113 (9), 104 (7), 71
(9), 57 (10), 55 (4); Anal. Cald for C17H14ClN3O4: C, 56.75; H, 3.92; N, 11.68;
Found: C, 56.70; H, 3.98; N, 11.63.
(E)-N′-(5-Chloro-2-oxoindolin-3-ylidene)-3,5-dimethoxybenzohydrazide
(91e): Yield: 85%; m.p: 112-113oC; IR (KBr, νmax, cm-1): 3249 (NH stretching),
3185 (sp2 CH stretching), 1670 (C=O), 1681 (C=N), 1598-1461 (C=C), 1307 (C-
O); 1H NMR (DMSO-d6, δ ppm ): 12.42 (1H, s, NH), 11.33 (1H, s, NH), 7.52 (1H,
d, J = 2.4 Hz, H-4), 7.39 (2H, dd, J = 2.0 Hz, H-2′,6′), 7.32 (1H, dd, J = 6.8, 2.4
Hz, H-6), 7.16 (1H, d, J = 8.8Hz, H-7), 6.92 (1H, dd, J = 2.0 Hz, H-4′), 3.35 (6H,
s, OCH3); 13C NMR (DMSO-d 6, δ ppm ): 170.4, 165.2, 162.2, 145.2, 137.0,
135.5, 133.3, 131.8, 124.1, 120.5, 119.5, 105.2, 56.7 (OCH3); EIMS (m/z, %):
359 (15, M+), 331 (14), 288 (24), 264 (2), 194 (5), 193 (3), 165 (100), 137 (25),
122 (35), 107 (9), 102 (8), 77 (7), 63 (5), 51 (2); Anal. Cald for C17H14ClN3O4: C,
56.75; H, 3.92; N, 11.68; Found: C, 56.76; H, 3.94; N, 11.67.
(E)--N′-(5-Chloro-2-oxoindolin-3-ylidene)-2,4-dimethoxybenzohydrazide
(91f): Yield: 80%; m.p: 201-202oC; IR (KBr, νmax, cm-1): 3345 (NH stretching),
3138 (sp2 CH stretching), 1715 (C=O), 1652 (C=N), 1594-1461 (C=C), 1350 (C-
O); 1H NMR (DMSO-d6, δ ppm ): 10.96 (1H, s, NH), 8.30 (1H, s, NH), 7.61 (1H,
d, J = 5.2 Hz, H-4), 7.44 (2H, dd, J = 14.2, 2.4 Hz, H-6,5′), 7.35 (2H, d, J = 13.2
Hz, H-7,6′), 6.94 (1H, d, J = 2.4 Hz, H-3′), 3.36 (6H, s, OCH3); 13C NMR (DMSO-
d6, δ ppm ): 169.0, 164.2, 161.6, 145.4, 134.5, 133.3, 132.0, 129.1, 124.1, 120.3,
118.5, 111.2, 107.2, 101.3, 56.4 (OCH3); EIMS (m/z, %): 359 (5, M+), 331 (8),
309 (2), 281 (3), 264 (3), 194 (4), 165 (100), 138 (8), 122 (15), 111 (9), 71 (18),
57 (21), 43 (10); Anal. Cald for C17H14ClN3O4: C, 56.75; H, 3.92; N, 11.68;
Found: C, 56.64; H, 3.95; N, 11.68.
283
(E)-N′-(5-Chloro-2-oxoindolin-3-ylidene)-2,6-dimethoxybenzohydrazide
(91g): Yield: 85%; m.p: 155-156oC; IR (KBr, νmax, cm-1): 3255 (NH stretching),
3132 (sp2 CH stretching) 1712 (C=O),1615 (C=N), 1592-1463 (C=C), 1342 (C-
O); 1H NMR (DMSO-d6, δ ppm ): 11.48 (1H, s, NH), 7.95 (1H, s, NH), 7.59 (1H,
d, J = 3.6 Hz, H-4), 7.44 (1H, dd, J = 16.8, 4.0 Hz, H-6), 7.09-6.81 (1H, m, H-
7,3′,4′,5′), 3.43 (6H, s, OCH3); 13C NMR (DMSO-d 6, δ ppm ): 169.4, 163.2,
160.2, 145.6, 135.2, 133.3, 132.8, 124.1, 121.0, 119.5, 108.5, 106.2, 56.0
(OCH3); EIMS (m/z, %): 359 (18, M+), 331 (17), 300 (2), 288 (1), 194 (5), 165
(100), 137 (24), 122 (29), 107 (6), 77 (4), 63 (4), 51 (2); Anal. Cald for
C17H14ClN3O4: C, 56.75; H, 3.92; N, 11.68; Found: C, 56.74; H, 3.95; N, 11.73.
(E)-N′-(5-Chloro-2-oxoindolin-3-ylidene)-2-(4-chlorophenyl)acetohydrazide
(91h): Yield: 89%; m.p: 139-140oC; IR (KBr, νmax, cm-1): 3216 (NH stretching),
3153 (sp2 CH stretching), 1727 (C=O),1685 (C=N), 1596-1470 (C=C), 1311 (C-
O); 1H NMR (DMSO-d6, δ ppm ): 10.93 (1H, s, NH), 8.29 (1H, s, NH), 7.94 (1H,
s, H-4), 7.56 (1H, d, J = 8.1 Hz, H-7), 7.21 (1H, d, J = 9.9 Hz, H-6), 6.89 (2H, d, J
= 12.3 Hz, H-2′,6′), 6.62 (2H, d, J = 13.5 Hz, H-3′,5′), 4.04 (2H, s, CH2); 13C NMR
(DMSO-d6, δ ppm ): 171.1, 166.4, 145.3, 134.5, 131.3, 130.8, 130.2, 129.1,
124.0, 120.5, 46.4 (CH2). EIMS (m/z, %): 347 (16, M+), 319 (5), 304 (2), 279 (3),
211 (3), 194 (43), 181 (205), 166 (26), 153 (56), 125 (100), 111 (13), 89 (20), 73
(22), 63 (11), 57 (10); Anal. Cald for C16H11Cl2N3O2: C, 55.19; H, 3.18; N, 12.07;
Found: C, 55.25; H, 3.18; N, 12.13.
(E)-N′-(5-Chloro-2-oxoindolin-3-ylidene)-2-(4-Flourophenyl)acetohydrazide
(91i) : Yield: 79%; m.p: 188-189 oC; IR (KBr, νmax, cm-1): 3262 (NH stretching),
3146 (sp2 CH stretching), 1720 (C=O), 1687 (C=N), 1602-1507 (C=C), 1313
(C=O); 1H NMR (DMSO-d6, δ ppm ): 11.35 (1H, s, NH), 10.93 (1H, s, NH), 7.66
(1H, s, H-4), 7.37 (2H, d, J = 8.0 Hz, H-6,7), 7.12 (2H, d, J = 8.4 Hz, H-2′,6′),
6.92 (2H, d, J = 8.4Hz, H-3′,5′), 4.02 (2H, s, CH2); 13C NMR (DMSO-d6, δ ppm ):
172.6, 165.3, 162.2, 146.4, 133.2, 132.0, 131.8, 129.9, 124.0, 120.1, 116.5,
284
115.2, 43.4 (CH2); EIMS (m/z, %): 331 (15, M+), 303 (6), 279 (2), 222 (1), 194
(46), 181 (2), 166 (24), 153 (9), 138 (21), 125 (12), 109 (100), 102 (11), 83 (12),
75 (7), 63 (4), 44 (1); Anal. Cald for C16H11ClFN3O2: C, 57.93; H, 3.34; N, 12.67;
Found: C, 57.98; H, 3.37; N, 12.62.
(E)-N′-(5-Chloro-2-oxoindolin-3-ylidene)-3-(3,4,5-trimethoxyphenyl)-
propionatehydrazide (91j): Yield: 86%; m.p: 150-151 oC; IR (KBr, νmax, cm-1):
3223 (NH stretching), 3107 (sp2 CH stretching), 1739 (C=O), 1614 (C=N), 1463
(C=C), 1342 (C-O); 1H NMR (DMSO-d6, δ ppm ): 10.42 (1H, s, NH), 8.45 (1H, s,
NH), 7.55 (1H, s, H-4), 7.39 (1H, dd, J = 8.4,2.0 Hz, H-6), 6.93 (1H, d, J = 8.0
Hz, H-7), 6.58 (1H, s, H-2′,6′), 3.75-3.35 (12H, s, OCH3), 2.88 (2H, s, CH2), 2.72
(2H, s, CH2); 13C NMR (DMSO-d6, δ ppm ): 169.1, 168.4, 152.5, 145.8, 137.2,
134.5, 133.3, 132.0, 130.5, 129.8, 124.2, 120.5, 106.5, 40.2 (CH2), 36.4 (CH2);
EIMS (m/z, %): 417 (32, M+), 400 (4), 372 (8), 358 (3), 341 (2), 313 (2), 236 (12),
223 (7), 194 (18), 181 (100), 166 (16), 138 (12), 102 (7), 77 (6), 65 (3), 51 (2);
Anal. Cald for C20H20ClN3O5: C, 57.49; H, 4.82; N, 10.06; Found: C, 57.46; H,
4.89; N, 10.00.
(E)-N′-(5-Chloro-2-oxoindolin-3-ylidene)-3-(4-methoxyphenyl)propionate-
hydrazide (91k): Yield: 83%; m.p: 198-199 oC; IR (KBr, νmax, cm-1): 3256 (NH
stretching), 3074 (sp2 CH stretching), 1746 (C=O), 1680 (C=N), 1645-1486
(C=C), 1391 (C-O); 1H NMR (DMSO-d 6, δ ppm ): 10.89 (1H, s, NH), 8.27 (1H, s,
NH), 7.94 (1H, s, H-4), 7.40 (1H, dd, J = 8.4, 1.6 Hz, H-6), 7.18 (1H, dd, J = 6.8,
2.0 Hz, H-2′,6′), 6.88 (1H, d, J = 8.4 Hz, H-7), 6.84 (1H, dd, J = 6.4, 1.6 Hz, H-
3′,5′), 3.35 (3H, s, OCH3), 2.88 (2H, s, CH2), 2.72 (2H, s, CH2); 13C NMR (DMSO-
d6, δ ppm ): 169.4, 168.2, 158.5, 144.8, 133.5, 133.4, 132.4, 130.5, 129.8, 128.5,
124.2, 120.5, 115.3, 39.2 (CH2), 36.0 (CH2); EIMS (m/z, %): 357 (18, M+), 340
(7), 312 (17), 298 (4), 277 (5), 194 (8), 176 (12), 166 (11), 121 (100), 91 (13), 77
(9), 65 (3), 51 (1); Anal. Cald for C18H16ClN3O3: C, 60.42; H, 4.51; N, 11.74;
Found: C, 60.46; H, 4.41; N, 11.75.
285
(E)-N′-(5-Chloro-2-oxoindolin-3-ylidene)-3-(4-bromophenoxy)acetato-
hydrazide (91l): Yield: 85%; m.p: 205-206oC; IR (KBr, νmax, cm-1): 3286(NH
stretching), 3074 (sp2 CH stretching), 1746 (C=O), 1645 (C=N), 1486 (C=C),
1391 (C-O); 1H NMR (DMSO-d 6, δ ppm ): 11.61 (1H, s, NH), 10.95 (1H, s, NH),
7.94 (1H, s, H-4), 7.46 (3H, d, J = 9.6 Hz, H-7, 3′,5′), 6.92 (3H, dd, J = 8.7 Hz, H-
6,2′,6′), 5.16 (2H, s, CH2); 13C NMR (DMSO-d6, δ ppm ): 164.6, 162.7, 157.6,
143.0, 132.5, 126.3, 126.2, 117.2, 116.6, 65.9 (CH2); EIMS (m/z, %): 409 (48,
M+,
Br81), 407 (38, M+,
Br79), 340 (7), 312 (17), 298 (4), 277 (5), 194 (8), 176 (12),
166 (11), 121 (100), 91 (13), 77 (9), 65 (3), 51 (1); Anal. Cald for
C16H11BrClN3O3: C, 47.03; H, 2.71; N, 10.28; Found: C, 47.07; H, 2.79; N, 10.23.
(E)-N′-(5-Chloro-2-oxoindolin-3-ylidene)-2-(2,4-dichlorophenoxy)aceto-
hydrazide(91m): Yield: 79%; m.p: 191-192oC; IR (KBr, νmax, cm-1): 3184 (NH
stretching), 3052 (sp2 CH stretching), 1726 (C=O), 1625 (C=N), 1489 (C=C),
1371 (C-O); 1H NMR (DMSO-d 6, δ ppm ): 11.48 (1H, s, NH), 7.94 (1H, s, NH),
7.58 (1H, s, H-4), 7.43 (2H, d, J = 8.4, 2.4 Hz, H-6,5′), 6.98 (1H, d, J = 8.0 Hz, H-
3′), 6.81 (2H, d, J = 8.0 Hz, H-7,6′), 3.82 (2H, s, CH2); 13C NMR (DMSO-d 6, δ
ppm ): 172.6, 168.7, 153.6, 145.0, 133.2, 132.3, 131.2, 129.2, 124.6, 120.2,
118.5, 67.9 (CH2); EIMS (m/z, %): 397 (02, M+), 362 (1), 264 (100), 231 (6), 219
(2), 194 (14), 166 (16), 162 (10), 138 (13), 111 (7), 102 (7), 69 (13), 41 (3). Anal.
Cald for C16H10Cl3N3O3: C, 48.21; H, 2.53; N, 10.54; Found: C, 48.25; H, 2.59; N,
10.50.
(E)-N′-(5-Chloro-2-oxoindolin-3-ylidene)-2-(2,4-dichlorophenoxy)propionato-
hydrazide (91n): Yield: 82%; m.p: 116-117 oC; IR (KBr, νmax, cm-1): 3226 (NH
stretching), 3096 (sp2 CH stretching), 1735 (C=O), 1615 (C=N), 1473 (C=C),
1206 (C-O); 1H NMR (DMSO-d 6, δ ppm ): 11.34 (1H, s, NH), 10.90 (1H, s, NH),
7.95 (1H, s, H-4), 7.79 (1H, s, H-4,3′), 7.60-7.36 (2H, m, H-6,5′), 6.78 (1H, d, J =
12.3 Hz, H-7,6′), 5.38 (1H, s, CH), 1.59 (2H, s, CH3); 13C NMR (DMSO-d6, δ ppm
286
): 173.6, 169.7, 151.6, 144.7, 133.4, 132.3, 131.2, 129.2, 125.6, 123.5, 119.9,
117.8, 75.9 (CH) 15.5 (CH3); EIMS (m/z, %): 409 (4, M+), 394 (5), 366 (8), 352
(8), 323 (9), 295 (12), 253 (16), 239 (17), 197 (20), 183 (21), 155 (23), 113 (33),
99 (38), 85 (100), 71 (97), 57 (78), 43 (18); Anal. Cald for C17H12Cl3N3O3: C,
49.48; H, 2.93; N, 10.18; Found: C, 49.46; H, 2.99; N, 10.15.
(E)-N′-(5-Chloro-2-oxoindolin-3-ylidene)-2-(2,4-dichlorophenoxy)butyrato-
hydrazide (91o): Yield: 89%; m.p: 133-134 oC; IR (KBr, νmax, cm-1): 3451 (NH
stretching), 3091 (sp2 CH stretching), 1740 (C=O), 1680 (C=N), 1448, 1429
(C=C), 1384 (C-O); 1H NMR (DMSO-d 6, δ ppm ): 10.97 (1H, s, NH), 8.31 (1H, s,
NH), 7.63-6.90 (6H, m, Ar-H), 5.29 (1H, s, CH2), 3.57 (1H, s, CH2), 2.51 (1H, s,
CH2); 13C NMR (DMSO-d 6, δ ppm ): 168.6, 166.7, 151.6, 146.1, 133.5, 132.3,
131.2, 130.2, 128.6, 124.2, 120.6, 117.8, 69.9 (CH2), 35.5 (CH2), 25.9 (CH2); EIMS (m/z, %): 427 (2, M+), 397 (1), 362 (1), 264 (100), 231 (4), 194 (14), 166
(15), 162 (9), 138 (3), 111 (7), 102 (7), 69 (13), 41 (4); Anal. Cald for
C18H14Cl3N3O3: C, 50.67; H, 3.31; N, 9.85; Found: C, 50.61; H, 3.32; N, 9.80.
(E)-N′-(5-Bromo-2-oxoindolin-3-ylidene)-3,5-difluorobenzohydrazide (92a):
Yield: 91%; m.p: 158-159 oC; IR (KBr, νmax, cm-1): 3228 (NH stretching), 3042
(sp2 CH stretching), 2996-2834, 1708 (C=O), 1595 (C=N), 1351 (C-O); 1H NMR
(DMSO-d 6, δ ppm ): 11.53 (1H, s, NH), 10.49 (1H, s, NH), 7.76 (1H, s, H-4),
7.62-7.45 (4H, m, H-6,2′,4′,6′), 6.97 (1H, d, J = 8.7 Hz, H-7); 13C NMR (DMSO-
d6, δ ppm ): 169.3, 165.7, 163.2, 145.3, 138.7, 134.7, 132.8, 125.4, 121.8, 119.8,
104.9; EIMS (m/z, %): 381 (25, M+, Br81), 379 (25, M+
, Br79), 353 (10, Br81), 351
(10, Br79), 301 (1), 240 (61, Br81), 238 (61, Br79), 212 (34, Br81), 210 (35, Br79),
184 (12, Br81), 182 (12, Br79), 141 (100), 113 (69), 103 (22), 63 (10); Anal. Cald
for C15H8BrF2N3O2: C, 47.39; H, 2.12; N, 11.05; Found: C, 47.38; H, 2.15; N,
11.02.
287
(E)-N′-(5-Bromo-2-oxoindolin-3-ylidene)-2,5-difluorobenzohydrazide (92b):
Yield: 88%; m.p: 139-140oC; IR (KBr, νmax, cm-1): 3231 (NH stretching), 3052 (sp2
CH stretching), 2983-2856, 1705 (C=O), 1625 (C=N), 1590-1359 (C=C), 1316
(C-O); 1H NMR (DMSO-d 6, δ ppm ): 11.23 (1H, s, NH), 10.79 (1H, s, NH), 7.81
(1H, s, H-4), 7.72-7.54 (3H, m, H-3′,4′,6′), 7.16-6.72 (2H, m, H-6,7); 13C NMR
(DMSO-d 6, δ ppm ): 169.2, 164.2, 159.2, 158.1, 144.3, 132.7, 130.8, 130.4,
127.5, 124.0, 121.9, 120.8, 119.3, 118.5, 116.9; EIMS (m/z, %): 381 (21, M+, Br81), 379 (21, M+, Br79), 353 (17, Br81), 351 (17, Br79), 301 (4), 273 (5), 240 (43,
Br81), 238 (43, Br79), 212 (22 Br81), 210 (22, Br79), 184 (5, Br81), 182 (5, Br79), 141
(100), 113 (39), 103 (12), 63 (6); Anal. Cald for C15H8BrF2N3O2: C, 47.39; H,
2.12; N, 11.05; Found: C, 47.32; H, 2.11; N, 11.03.
(E)-N′-(5-Bromo-2-oxoindolin-3-ylidene)-2,6-difluorobenzohydrazide (92c):
Yield:85%; m.p: 184-185oC; IR (KBr, νmax, cm-1): 3227 (NH stretching), 3025 (sp2
CH stretching), 2987-2850, 1715 (C=O), 1651 (C=N), 1597-1349 (C=C), 1326
(C-O); 1H NMR (DMSO-d 6, δ ppm ): 11.21 (1H, s, NH), 10.41 (1H, s, NH), 7.74
(1H, s, H-4), 7.54-7.31 (3H, m, H-3′,4′,6′), 6.71 (2H, d, J = 14.4 Hz, H-6,7); 13C
NMR (DMSO-d 6, δ ppm ): 168.3, 164.2, 161.2, 145.2, 136.2, 135.7, 133.2,
132.2, 120.2, 115.5, 111.5; EIMS (m/z, %): 381 (4, M+, Br81), 379 (4, M+, Br79),
353 (54, Br81), 351 (54, Br79), 301 (34), 273 (4), 240 (23, Br81), 238 (23, Br79),
212 (12, Br81), 210 (12, Br79), 184 (4, Br81), 182 (4, Br79), 141 (100), 113 (46), 103
(2), 63 (2); Anal. Cald for C15H8BrF2N3O2: C, 47.39; H, 2.12; N, 11.05; Found: C,
47.37; H, 2.19; N, 11.09.
(E)-N′-(5-Bromo-2-oxoindolin-3-ylidene)-3,4-dimethoxybenzohydrazide
(92d): Yield: 87%; m.p: 121-122 oC; IR (KBr, νmax, cm-1): 3223 (NH stretching),
3054 (sp2 CH stretching), 2992-2836, 1742 (C=O), 1678 (C=N), 1536 (C=C),
1308 (C-O); 1H NMR (DMSO-d 6, δ ppm ): 11.33 (1H, s, NH), 10.49 (1H, s, NH),
7.57 (1H, d, J = 2.4 Hz, H-4), 7.45-7.39 (3H, m, H-6,2′,6′), 7.11 (1H, d, J = 8.4
Hz, H-7), 6.93 (1H, d, J = 8.4 Hz, H-5′), 3.89-3.82 (6H, s, OCH3); 13C NMR
288
(DMSO-d6, δ ppm ): 168.3, 164.4, 153.2, 151.2, 145.2, 134.1, 133.5, 131.3,
127.9, 123.4, 120.3, 119.5, 117.2, 113.4, 56.5 (OCH3); EIMS (m/z, %): 405 (22,
M+, Br81), 403 (22, M+, Br79), 379 (8, Br81), 377 (8, Br79), 351 (5), 284 (2), 240 (19,
Br81), 238 (19, Br79), 178 (3), 165 (100), 137 (7), 122 (19), 77 (15); Anal. Cald for
C17H14BrN3O4: C, 50.51; H, 3.49; N, 10.40; Found: C, 50.55; H, 3.48; N, 10.43.
(E)-N′-(5-Bromo-2-oxoindolin-3-ylidene)-3,5-dimethoxybenzohydrazide
(92e): Yield: 80%; m.p: 204-205oC; IR (KBr, νmax, cm-1): 3228 (NH stretching),
3065 (sp2 CH stretching), 2999-2835, 1747 (C=O), 1678 (C=N), 1535 (C=C),
1306 (C-O); 1H NMR (DMSO-d 6, δ ppm ): 11.56 (1H, s, NH), 10.02 (1H, s, NH),
7.70 (1H, s, H-4), 7.56 (1H, dd, J = 8.4, 2.0 Hz, H-6), 6.98 (2H, d, J = 2.4 Hz, H-
2′,6′), 6.93 (1H, d, J = 8.0 Hz, H-7), 6.82 (1H, dd, J = 2.0 Hz, H-4′), 3.83 (6H, s,
OCH3); 13C NMR (DMSO-d 6, δ ppm ): 168.6, 162.6, 160.7, 141.4, 138.8, 135.2,
133.9, 123.1, 121.2, 119.3, 105.1, 104.3, 55.6 (OCH3). EIMS (m/z, %): 405 (15,
M+, Br81), 403 (15, M+, Br79), 379 (11, Br81), 377 (11, Br79), 351 (2), 284 (1), 240
(9, Br81), 238 (9, Br79), 178 (23), 165 (100), 137 (17), 122 (16), 77 (5). Anal. Cald
for C17H14BrN3O4: C, 50.51; H, 3.49; N, 10.40; Found: C, 50.55; H, 3.43; N,
10.41.
(E)-N′-(5-Bromo-2-oxoindolin-3-ylidene)-2,4-dimethoxybenzohydrazide
(92f): Yield: 86%; m.p: 167-168 oC; IR (KBr, νmax, cm-1): 3486-3211 (NH
stretching), 3075 (sp2 CH stretching), 2839, 1717 (C=O), 1652 (C=N), 1492
(C=C), 1238 (C-O); 1H NMR (DMSO-d 6, δ ppm ): 11.54 (1H, s, NH), 10.24 (1H,
s, NH), 7.72 (1H, s, H-4), 7.53 (1H, dd, J = 8.4, 2.0 Hz, H-6), 7.24 (1H, d, J = 8.4
Hz, H-6′), 6.99 (1H, d, J = 8.0 Hz, H-7), 6.70 (1H, dd, J = 7.8, 2.0 Hz, H-5′), 6.67
(1H, d, J = 2.1 Hz, H-3′), 3.88 (6H, s, OCH3); 13C NMR (DMSO-d6, δ ppm ):
168.2, 163.5, 161.7, 141.4, 134.2, 133.3, 128.1, 124.2, 121.2, 111.8, 106.3,
101.1, 55.3 (OCH3); EIMS (m/z, %): 405 (5, M+, Br81), 403 (5, M+, Br79), 377 (6,
Br81), 375 (6, Br79), 325 (2), 297 (3), 240 (1), 165 (100), 122 (7), 107 (6), 77 (3);
Anal. Cald for C17H14BrN3O4: C, 50.51; H, 3.49; N, 10.40; Found: C, 50.55; H,
3.45; N, 10.49.
289
(E)-N′-(5-Bromo-2-oxoindolin-3-ylidene)-2,6-dimethoxybenzohydrazide
(92g): Yield: 88%; m.p: 128-129 oC; IR (KBr, νmax, cm-1): 3416-3311 (NH
stretching), 3072 (sp2 CH stretching), 2839, 1717 (C=O), 1606 (C=N), 1592
(C=C), 1308 (C-O); 1H NMR (DMSO-d 6, δ ppm ): 11.78 (1H, s, NH), 10.49 (1H,
s, NH), 7.85 (1H, s, H-4), 7.62 (1H, dd, J = 8.0, 2.1 Hz, H-6), 7.41 (1H, d, J = 8.4
Hz, H-6′), 7.05 (1H, d, J = 8.0 Hz, H-7), 6.79 (1H, dd, J = 7.8, 2.0 Hz, H-5′), 6.71
(1H, d, J = 2.1 Hz, H-3′), 3.80 (6H, s, OCH3); 13C NMR (DMSO-d 6, δ ppm ):
169.6, 164.5, 161.7, 145.4, 134.2, 133.3, 124.7, 121.2, 119.8, 106.3, 56.4
(OCH3); EIMS (m/z, %): 405 (5, M+, Br81), 403 (6, M+, Br79), 377 (4, Br81), 375 (4,
Br79), 325 (1), 297 (2), 240 (1), 165 (100), 122 (9), 107 (4), 77 (2), 57 (2); Anal.
Cald for C17H14BrN3O4: C, 50.51; H, 3.49; N, 10.40. Found: C, 50.50; H, 3.44; N,
10.47.
(E)-N′-(5-Bromo-2-oxoindolin-3-ylidene)-2-(4-chlorophenyl)acetohydrazide
(92h): Yield: 85%; m.p: 137-138oC; IR (KBr, νmax, cm-1): 3577-3110 (NH
stretching), 3011 (sp2 CH stretching), 1636 (C=O), 1611 (C=N), 1561 (C=C),
1254 (C-O); 1H NMR (DMSO-d 6, δ ppm ): 11.81 (1H, s, NH), 10.29 (1H, s, NH),
7.99 (1H, s, H-4), 7.69 (1H, d, J = 8.1 Hz, H-7), 7.45 (1H, d, J = 9.9 Hz, H-6),
7.09 (2H, d, J = 12.3 Hz, H-2′,6′), 7.01 (2H, d, J = 13.5 Hz, H-3′,5′), 4.14 (2H, s,
CH2); 13C NMR (DMSO-d 6, δ ppm ): 171.4, 164.3, 145.2, 134.0, 133.9, 133.5,
131.3, 130.8, 124.1, 121.0, 119.0, 56.4 (OCH3), 49.2 (CH2); EIMS (m/z, %): 347
(16, M+), 319 (5), 304 (2), 279 (3), 211 (3), 194 (43), 181 (20), 166 (26), 153 (56),
125 (100), 111 (13), 89 (20), 73 (22), 63 (11), 57 (10); Anal. Cald for
C16H11BrClN3O2: C, 48.94; H, 2.82; N, 10.70; Found: C, 48.92; H, 2.84; N, 10.75.
(E)-N′-(5-Bromo-2-oxoindolin-3-ylidene)-2-(4-Flourophenyl)aceto-
hydrazide(92i): Yield: 81%; m.p: 157-158 oC; IR (KBr, νmax, cm-1): 3547-3410
(NH stretching), 1636 (C=O), 1600 (C=N), 1561 (C=C), 1231 (C-O); 1H NMR
(DMSO-d 6, δ ppm ): 11.19 (1H, s, NH), 10.81 (1H, s, NH), 7.79 (1H, s, H-4),
290
7.54 (2H, d, J = 8.0 Hz, H-6,7), 7.09 (2H, d, J = 8.4 Hz, H-2′,6′), 6.98 (2H, d, J =
8.4 Hz, H-3′,5′), 4.14 (2H, s, CH2); 13C NMR (DMSO-d 6, δ ppm ): 168.7, 165.5,
160.2, 145.5, 134.5, 133.8, 133.2, 130.1, 130.0, 124.1, 120.3, 119.5, 115.2, 56.7
(OCH3), 48.6 (CH2); EIMS (m/z, %): 375 (11, M+), 347 (8), 323(3), 266(2), 238
(49), 225(11), 210(24), 197(11), 182 (21), 169 (12), 153(100), 146(11), 127 (12),
119 (10), 107(5), 88 (1). Anal. Cald for C16H11BrFN3O2: C, 51.08; H, 2.95; N,
11.17; Found: C, 51.12; H, 2.94; N, 11.12.
(E)-N′-(5-Bromo-2-oxoindolin-3-ylidene)-3-(3,4,5-trimethoxyphenyl)-
propionatehydrazide (92j): Yield: 84%; m.p: 101-102 oC; IR (KBr, νmax, cm-1):
3272 (NH stretching), 2940, 2833, 1733 (C=O), 1654 (C=N), 1594-1460 (C=C),
1241 (C-O); 1H NMR (DMSO-d 6, δ ppm ): 10.90 (1H, s, NH), 8.37 (1H, s, NH),
7.65 (1H, s, H-4), 7.51 (1H, d, J = 7.6 Hz, H-7), 6.88 (1H, d, J = 10.4 Hz, H-6),
6.56 (1H, s, H-2′,6′), 3.74-3.36 (12H, s, OCH3), 2.87 (2H, s, CH2), 2.50 (2H, s,
CH2); 13C NMR (DMSO-d 6, δ ppm ): 169.3, 168.4, 152.6, 142.3, 137.5, 134.6,
134.0, 133.8, 123.7, 122.9, 119.5, 105.5, 55.7 (OCH3), 36.4 (CH2), 30.4 (CH2);
EIMS (m/z, %): 463 (33, M+, Br81), 461 (33., M+, Br79), 418 (7, Br81), 416 (7, Br79),
383 (5), 337 (2), 284 (1), 236 (13), 181 (100, Br81), 179 (11, Br79), 151 (4, Br81),
149 (4., Br79), 103 (4), 77 (3), 65 (2.); Anal. Cald for C20H20BrN3O5: C, 51.96; H,
4.36; N, 9.09; Found: C, 51.99; H, 4.39; N, 9.12.
(E)-N′-(5-Bromo-2-oxoindolin-3-ylidene)-3-(4-methoxyphenyl)propionate-
hydrazide (92k): Yield: 80%; m.p: 130-131 oC; IR (KBr, νmax, cm-1): 3201 (NH
stretching), 3092, 2836, 1734 (C=O), 1648 (C=N), 1597-1511 (C=C), 1375 (C-O);
1H NMR (DMSO-d 6, δ ppm ): 10.81 (1H, s, NH), 8.42 (1H, s, NH), 7.65 (1H, s,
H-4), 7.49 (1H, d, J = 8.4 Hz, H-6,7), 7.23 (1H, d, J = 6.8 Hz, H-2′,6′), 6.91 (1H,
d, J = 6.4 Hz, H-3′,5′), 3.38 (3H, s, OCH3), 2.92 (2H, s, CH2), 2.45 (2H, s, CH2); 13C NMR (DMSO-d 6, δ ppm ): 173.8, 167.0, 152.1, 143.9, 142.3, 138.8, 137.6,
126.3, 123.2, 122.9, 121.8, 116.5, 56.5 (OCH3), 35.7 (CH2), 30.7 (CH2); EIMS
(m/z, %): 403 (15, M+, Br81), 401 (15, M+, Br79), 358 (12, Br81), 356 (12, Br79),
291
277 (5), 241 (6, Br81), 239 (6, Br79), 214 (5, Br81), 212 (5, Br79), 176 (20), 121
(100), 91 (8) 77 (8), 57 (3); Anal. Cald for C18H16BrN3O3: C, 53.75; H, 4.01; N,
10.45; Found: C, 53.79; H, 4.11; N, 10.42.
(E)-N′-(5-Bromo-2-oxoindolin-3-ylidene)-3-(4-bromophenoxy)aceto-
hydrazide (92l): Yield: 81%; m.p: 155-156 oC; IR (KBr, νmax, cm-1): 3281 (NH
stretching), 3120, 2925, 1745-1713 (C=O), 1645 (C=N), 1585 (C=C), 1287 (C-O);
1H NMR (DMSO-d 6, δ ppm ): 10.96 (1H, s, NH), 8.33 (1H, s, NH), 7.94 (1H, s,
H-4), 7.55 (3H, d, J = 8.4 Hz, H-7), 7.46 (3H, dd, J = 8.8 Hz, H-6), 7.03-6.83 (4H,
m, H-2′,3′,5′,6′), 5.15 (2H, s, CH2); 13C NMR (DMSO-d 6, δ ppm ): 163.9, 162.1,
157.0, 142.7, 134.7, 132.0, 128.2, 116.7, 114.2, 64.7 (CH2); EIMS (m/z, %): 453
(15, M+, Br81), 451 (10, M+, Br79), 375 (3, Br81), 373 (3, Br79), 356 (2), 282 (3,
Br81), 280 (4, Br79), 240 (15, Br81), 242 (15, Br79), 212 (8, Br81), 210 (8, Br79), 157
(8, Br81), 155 (8, Br79), 73 (100), 44 (24); Anal. Cald for C16H11Br2N3O3: C,
42.41; H, 2.45; N, 9.27; Found: C, 42.47; H, 2.40; N, 9.22.
(E)-N′-(5-Bromo-2-oxoindolin-3-ylidene)-2-(2,4-dichlorophenoxy)aceto-
hydrazide (92m): Yield: 88%; m.p: 171-172 oC; IR (KBr, νmax, cm-1): 3189 (NH
stretching), 2948, 1749 (C=O), 1654 (C=N), 1596 (C=C), 1298 (C-O); 1H NMR
(DMSO-d 6, δ ppm ): 10.88 (1H, s, NH), 8.37 (1H, s, NH), 7.59 (1H, d, J = 1.8 Hz,
H-4), 7.52 (1H, dd, J = 5.8, 2.2 Hz, H-6), 7.34 (1H, dd, J = 6.2, 1.8 Hz, H-5′),
7.19 (1H, d, J = 2.2 Hz, H-3′), 6.88 (1H, d, J = 8.4 Hz, H-7), 6.83 (1H, d, J = 8.4
Hz, H-6′), 3.34 (2H, s, CH2); 13C NMR (DMSO-d 6, δ ppm ): 167.6, 163.7, 152.8,
142.5, 135.9, 134.5, 129.1, 127.9, 124.2, 122.2, 116.7, 114.8, 68.2 (CH2); EIMS
(m/z, %): 445 (15, M+, Br81), 443 (33, M+, Br79), 408 (53, Br81), 406 (39, Br79),
328 (37), 282 (9, Br81), 280 (12, Br79), 240 (95, Br81), 238 (100, Br79), 212 (53,
Br81), 210 (58, Br79), 162 (42, Br81), 160 (88, Br79), 145 (73), 73 (77). Anal. Cald
for C16H10BrCl2N3O3: C, 43.37; H, 2.27; N, 9.48; Found: C, 43.47; H, 2.20; N,
9.42.
292
(E)-N′-(5-Bromo-2-oxoindolin-3-ylidene)-2-(2,4-dichlorophenoxy)-
propionatohydrazide (92n): Yield: 83%; m.p: 196-198 oC; IR (KBr, νmax, cm-1):
3557-3419 (NH stretching), 1636 (C=O), 1601 (C=N), 1561 (C=C), 1238 (C-O);
1H NMR (DMSO-d 6, δ ppm ): 11.35 (1H, s, NH), 10.97 (1H, s, NH), 7.62-6.84
(6H, m, Ar-H), 5.47 (1H, s, CH), 1.61 (2H, d, J = 6.6 Hz, CH3); 13C NMR (DMSO-
d6, δ ppm ): 164.6, 162.7, 157.6, 143.0, 132.5, 126.3, 126.2, 117.2, 116.6, 65.9
(CH2); EIMS (m/z, %): 457 (51, M+, Br81), 445 (33, M+, Br79), 422 (68, Br81), 420
(52, Br79), 342 (7), 296 (41, Br81), 294 (53, Br79), 268 (97, Br81), 266 (100, Br79),
240 (89, Br81), 242 (89, Br79), 191 (58, Br81), 189 (95, Br79), 162 (29), 145 (27),
103 (27), 75 (14), 63 (9); Anal. Cald for C17H12BrCl2N3O3: C, 44.67; H, 2.65; N,
9.19; Found: C, 44.66; H, 2.61; N, 9.15.
(E)-N′-(5-Bromo-2-oxoindolin-3-ylidene)-2-(2,4-dichlorophenoxy)butyrato-
hydrazide (92o): Yield: 86%; m.p: 207-208oC; IR (KBr, νmax, cm-1): 3547-3410
(NH stretching), 1636 (C=O), 1608 (C=N), 1561 (C=C), 1248 (C-O); 1H NMR
(DMSO-d 6, δ ppm ): 11.39 (1H, s, NH), 10.98 (1H, s, NH), 7.60-6.85 (6H, m, Ar-
H), 5.32 (1H, s, CH2), 2.88 (1H, s, CH2), 2.72 (1H, s, CH2); 13C NMR (DMSO-d 6,
δ ppm ): 164.6, 162.7, 157.6, 143.0, 132.5, 126.3, 126.2, 117.2, 116.6, 65.9
(CH2); EIMS (m/z, %): 473 (3, M+, Br81), 471 (3, M+, Br79), 445 (1, Br81), 443 (4,
Br79), 388 (1, Br81), 386 (2, Br79), 310 (98, Br81), 308 (100, Br79), 232 (9, Br81),
230 (33, Br79), 212 (8, Br81), 210 (10, Br79), 162 (12), 103 (7), 69 (13), 63 (3);
Anal. Cald for C18H14BrCl2N3O3: C, 45.89; H, 3.00; N, 8.92; Found: C, 45.82; H,
3.02; N, 8.95.
293
Chapter 8
BIOLOGICAL ACTIVITIES A rapid advance in the development of new techniques for determining the
biological activity of synthetic and natural compounds has triggered a
renaissance in the drug development. Primary bioassay screening plays a very
important role in the drug development programme. These screenings acts as a
tool to conduct activity directed isolations of bioactive compounds for curing
humans and animals. Primary screening provides first indication for bioactivities
and thus helps in the selection of lead compounds for secondary screening for
detailed pharmacological evaluations.
Indolinones, Triazoles and thiadiazoles were tested for the following
activities.
8.1. Herbicide studies
8.2. Fungicide studies
8.3. Insecticide studies
8.4. Plant growth regulating studies
8.5. Cytotoxicity and Antiviral activities against different cell culture
a. Vero cell culture
b. HeLa cell Culture
c. HEL cell Culture
d. Anti-feline corona virus in CRFK cell cultures
e. Anti-feline herpes virus in CRFK cell cultures
f. Anti-influenza virus activity in MDCK cell cultures
8.6. Antifungal studies
8.7. Antibacterial studies
The detail of each bioassay screening is as follows.
294
8.1 Herbicide studies 8.1.1 Plant Material
Two broadleaf plants and two grasses were used to test the herbicidal
activity of compounds including rape (Brassica napus) (RA), amaranth pigweed
(Amaranthus retroflexus) (AR), barnyard grass (Echinochloa crusgalli (L.)
Beauv.) (BG) and Crab grass (Digitaria adscendens) (CG).
Seeds of amaranth pigweed, barnyard grass and crab grass were
reproduced outdoors and stored at 4°C. Seeds of rape were bought from Institute
of Crop, Tianjin Agroculture Science Academy.
8.1.2 Culture method
Seeds were planted in 7.5-cm-diameter disposable paper cup (250ml)
containing artificial mixed soil. Before plant emergence, the cups were covered
with plastic film to keep moist. Plants were grown in the green house. Fresh
weight of upground plants were measured 21 days after treatment.
8.1.3 Treatment
Dosage (activity ingredient) for each compound is 1500 grams per
hectare. Purified compounds were dissolved in 100µl N.N-dimethylformamide
with the addition of a little Tween 20, and then were sprayed using a laboratory
belt sprayer delivering at 750L/ha-spray-volume. The same amount of water was
sprayed as control.
a) Pre-emergency treatment: Compounds were sprayed immediately after
seeds planting. Two replicates each treatment.
b) Post-emergency treatment: Compounds were sprayed after the first true
leave expanding.
295
Table 8.1a: Herbicide activity (% inhibition) of the synthesized
Chloroisatin derivatives 91(a-o)
BG RA AR
CG
Compd.
Density g / hect
ST FS ST FS ST FS ST FS 91a 1500 - - - - - - - - 91b 1500 - - - - - - - - 91c 1500 - - 2.4 22.3 22.5 13.9 - - 91d 1500 - - - - - - - - 91e 1500 - - - - - - - - 91f 1500 - - - - - - - - 91g 1500 - - 2.9 - - 16.5 - - 91h 1500 - - - - - - - - 91i 1500 - - 2.4 - - - - - 91j 1500 - - 36.0 30.4 22.5 8.9 - - 91k 1500 - - 7.8 34.9 - - - - 91l 1500 - - - - - 38.2 - -
91m 1500 - - 5.1 - - - - - 91n 1500 - - - - - - - - 91o 1500 - - 5.1 - - - - -
Table 8.1b: Herbicide activity (% inhibition) of the synthesized
Bromoisatin derivatives 92(a-o)
BG RA AR
CG
Compd.
Density g / hect
ST FS ST FS ST FS ST FS 92a 1500 - - - - - - - - 92b 1500 - - - - - - - - 92c 1500 - - - - - 13.9 - - 92d 1500 - - 20.0 - - - - - 92e 1500 - - - - - - - - 92f 1500 - - - - - 34.9 - 44.3 92g 1500 - - 33.3 - - - - - 92h 1500 - - - - - - - - 92i 1500 - - - - - 21.5 - - 92j 1500 - - 29.3 - - - - - 92k 1500 - - - - - - - - 92l 1500 - - - - - 24.5 - 45.2
92m 1500 - - - - - - - - 92n 1500 - - 59.1 - - - - - 92o 1500 - - - - - - - -
Key: (-) - No activity
296
8.1.4 Analysis
The inhibition percent of upground fresh weight is used to describe the
control efficiency of compounds. Activity level:
A: ≥80%, B: 60~79%, C: 40~59%, D: ≤39%.
8.1.5 Discussion
From the biological assay results in Table 8.1a & 8.1b, which summarize
the herbicide activity of the synthesized compounds. From the results, it is infer
that a few compounds show herbicidal activity but not so significant.
8.2 Fungicide Studies 8.2.3 Disc method in vitro (DIV)
Disc method also called Agar dilution method. Including five kinds of fungi:
Fusarium wilt on cucumber (Fusarium oxysporum f. cucumerinum) (CF),
Speckle on peanut (Cercospora rachidicola) (PS), Tomato early blight
(Alternaria solani) (TB), Wheat scab (Gibberella zeae) (WS) and Apple
rootspot (Physalospora piricola) (AR).
8.2.2 Test concentration 50µg/ml. Detect mycelium expanding diameter 48h after treatment.
8.2.3 Against cucumber grey mould----Micro method in
vitro (MIV)
Spore suspension of cucumber grey mould (Botrytis cinerea) (CM)
contacted with compound solution to detect the inhibition activity of
compound to spore germination and growth of mycelium. Detect the result
by eyeballing 3 days after treatment.
297
8.2.4 Against wheat powdery mildew----In vivo (IV)
Summer spores of wheat powdery mildew (Erysiphe graminis f. sp.
Tritici) (WP) were sprayed on wheat plant (3-leaf) and 24h later
compound suspension was sprayed as well. Disease index was
detected by eyeballing 7days after treatment.
8.2.5 Activity level
A: ≥80% B: 60~79%
C: 40~59% D: ≤39%
Table 8.2a: Fungicidal activity (% inhibition) of the synthesized
Chloroisatin derivatives 91(a-o)
DIV MIV IV Compd. Conc. (µg/ml) WS TB PS AR CF CM WP
91a 50 8.3 6.2 - 3.3 7.1 - - 91b 50 5.6 6.2 16.0 6.7 - - - 91c 50 - 3.1 - - 14.2 - - 91d 50 2.5 - - - - - - 91e 50 13.5 2.3 - 3.5 39.2 - - 91f 50 33.2 - - - - - - 91g 50 - 9.3 12.9 5.1 10.8 - - 91h 50 12.1 - 13.5 0 7.1 - - 91i 50 5.6 12.4 - 54.9 - - - 91j 50 15.4 - 4.0 5.1 - - - 91k 50 - 9.3 - 16.5 - - - 91l 50 - 18.6 - - - - -
91m 50 8.3 - 16.0 - - - - 91n 50 22.3 - 16.0 - - - - 91o 50 - - - 5.1 7.1 - -
Key: (-) - No activity
298
Table 8.2b: Fungicidal activity (% inhibition) of the synthesized
Bromoisatin derivatives 92(a-o)
DIV MIV IV Compd. Conc. (µg/ml) WS TB PS AR CF CM WP
92a 50 8.3 6.2 - 3.3 7.1 - - 92b 50 5.6 6.2 16.0 6.7 - - - 92c 50 - 3.1 - - 14.2 - - 92d 50 2.5 - - - - - - 92e 50 13.5 2.3 - 3.5 39.2 - - 92f 50 33.2 - - - - - - 92g 50 - 9.3 12.9 5.1 10.8 - - 92h 50 12.1 - 13.5 - 7.1 - - 92i 50 5.6 12.4 - 54.9 - - - 92j 50 15.4 - 4.0 5.1 - - - 92k 50 - 9.3 - 16.5 - - - 92l 50 - 18.6 - - - - -
92m 50 8.3 - 16.0 - - - - 92n 50 22.3 - 16.0 - - - - 92o 50 - - - 5.1 7.1 - -
Key: (-) - No activity
8.2.6 Discussion
From the biological assay results in Table 8.2a and 8.2b, which
summarize the Fungicidal activity of the synthesized compounds. From the
results, it is inferred that a few compounds show fungicidal activity but not so
significant.
8.3 Insecticide Studies 8.3.1 Activity against Armyworm
Armyworm (Mythimna separata Walker)(AW),normal population feeding
indoor.
299
Leaf- dipped-method: Dip maize leaf blade into compound solution (200 µg/ml,
dissolved in acetone), feeding 4th worm. Mainly observing stomach poison
activity, contact poison activity and feeding condition as well. Motility of worm
was detected 24h after treatment.
8.3.2 Activity against Aphid
Aphid (Aphis laburni kaltenbch)(AL), normal population feeding indoor on
bean plant.
Dipping-method: Dip plant with nymphae into compound emulsion (200µg/ml,
containing acetone or other solvent and Sorpol-560 or other emulsifier) for 2 to 3
seconds, swung off the liquid drop and then insert into the foam base covered
with glass mantle. Mainly observing stomach poison activity, contact poison
activity and feeding condition as well. Motility of worm was detected 24h after
treatment.
8.3.3 Activity level
A: ≥80% B: 60~79%
C: 40~59% D: ≤39%
8.3.4 Discussion
Insecticidal activity of the synthesized compounds is summarized in
Table 8.3. From the results, it is inferred that only compound 91b show very little
insecticidal activity (10%) and remaining chloro- and bromoisatin derivatives
show no insecticidal activity.
300
Table 8.3: Insecticidal activity of the synthesized
Chloroisatin derivatives 91(a-o)
8.4 Plant growth regulating activity (PGR)
Modification of plant growth and development through the use of plant
growth regulators is becoming an increasingly important aspect of modern
agricultural practice. The availability of synthetic regulators that mimic the effect
of plant hormones has greatly facilitated this practice. Synthetic analogues of the
naturally occurring auxins, cytokinins and ethylene have been particularly useful
in this regard. However, most of the known plant growth regulators have
comparatively low physiological activity. The increase of the doses applied is
undesirable because of ecological reasons. All this imposes the search of new
high physiologically active substances.
Compd. Conc. (µg/ml) AW AL
91a 200 0 0 91b 200 10.0 0 91c 200 0 0 91d 200 0 0 91e 200 0 0 91f 200 0 0 91g 200 0 0 91h 200 0 0 91i 200 0 0 91j 200 0 0 91k 200 0 0 91l 200 0 0
91m 200 0 0 91n 200 0 0 91o 200 0 0
301
8.4.1 Cucumber cotyledon rhizogenesis method (CCR) Filter paper method: To detect auxin-like activity of compound.
Concentration: 10µg.ml-1.
Cucumber cultivar: JINYAN 4.
Cotyledons were prepared by cultured on agar for 3 days (26±1°C) in dark
after seed immersion. 0.3ml 100µg.ml-1 solution dissolved in DMF was dropped
on paper disc with diameter of 6cm, air dried and put into Petri dishes (6cm). 3ml
distilled water was added into the dishes and then 10 cotyledons was put into as
well. Counting the number of adventitious roots arised and calculate the
increasing percent of rhizogenesis 5days after treatment (26±1°C).
8.4.2 Activity level A: ≥80% B: 60~79%
C: 40~59% D: ≤39%
Table 8.4: Plant growth regulating activity of the synthesized
Chloroisatin derivatives 91(a-o) and Bromoisatin derivatives 92(a-o)
Chloro-isatin derivatives Bromo-isatin derivatives
Compd. Cucumber cotyledon rhizogenesis method
(CCR) Compd.
Cucumber cotyledon rhizogenesis method
(CCR) 91a 23.3 92a 16.6 91b 46.6 92b 13.3 91c 70.0 92c 63.3 91d 0 92d 6.6 91e 20.0 92e 63.3 91f -30.0 92f -10.0 91g 12.5 92g -10.0 91h 61.7 92h 23.3 91i 54.1 92i 79.2 91j 75.1 92j 24.2 91k 73.0 92k -20.0 91l 16.6 92l 0
91m 13.3 92m 51.9 91n 40.0 92n 0 91o -20.0 92o -100.0
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8.4.3 Discussion
Plant growth regulatory activity of the synthesized compounds has been
summarized in Table 8.4. From the results, it is inferred that compounds 91c, 91j, 91k and 92i show significant activity while the remaining compounds also
show activity but not so significant.
8.5 Antiviral Studies (For experimental detail, sec section 4.9 of this thesis)
Table 8.5a: Cytotoxicity and Antiviral activities of Chloroisatin derivatives
91(a-o) in HeLa cell Culture
EC50b (µg/ml)
Compound Minimum cytotoxic
concentrationa (µg/ml)
Vesicular stomatitis
virus Coxsackie virus B4
Respiratory syncytial
virus 91a 100 >20 >20 >20 91b ≥20 >20 >20 >20 91c 20 >4 >4 >4 91d 100 >20 >20 >20 91e 100 >20 >20 >20 91f 20 >4 >4 >4 91g 20 >4 >4 >4 91h 20 >4 >4 >4 91i 4 >0.8 >0.8 >0.8 91j ≥20 >20 >20 >20 91k 100 >20 >20 >20 91l 20 >4 >4 >4
91m 20 >4 >4 >4 91n 20 >4 >4 >4 91o ≥20 >20 >20 >20
D.S.-5000 >100 2.4 12 0.5 (S)-DHPA (µM) >250 30 >250 150 Ribavirin (µM) >250 10 250 10
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Table 8.5b: Cytotoxicity and Antiviral activities of Bromoisatin derivatives
92(a-o) in HeLa cell Culture
EC50b (µg/ml)
Compound Minimum cytotoxic
concentrationa (µg/ml)
Vesicular stomatitis
virus Coxsackie virus B4
Respiratory syncytial
virus 92a 20 >4 >4 >4 92b 100 >20 >20 >20 92c 0.8 >0.16 >0.16 >0.16 92d 100 >20 >20 >20 92e 20 >4 >4 >4 92f ≥20 20 >20 >20 92g 20 >4 >4 >4 92h 100 >20 >20 >20 92i >100 >100 >100 >100 92j 20 >4 >4 >4 92k 20 >4 >4 >4 92l 20 >4 >4 >4
92m 100 >20 >20 >20 92n ≥20 >20 >20 >20 92o 20 >4 >4 >4
D.S.-5000 >100 2.4 12 0.5 (S)-DHPA (µM) >250 30 >250 150 Ribavirin (µM) >250 10 250 10
304
Table 8.5c: Cytotoxicity and Antiviral activities of Chloroisatin derivatives
91(a-o) and Bromoisatin derivatives 92(a-o) in Vero cell cultures
EC50
b (µg/ml)
Compound Minimum cytotoxic
concentrationa (µg/ml)
Para-influenza-
3 virus
Reovirus-1
Sindbis virus
Coxsackievirus
B4
Punta Toro virus
91a >100 >100 >100 >100 >100 >100 91b 100 >20 >20 >20 >20 >20 91c 20 >4 >4 >4 >4 >4 91d 100 >20 >20 >20 >20 >20 91e 100 >20 >20 >20 >20 >20 91f ≥100 >100 >100 >100 60 >100 91g ≥0.8 >0.8 >0.8 >0.8 >0.8 >0.8 91h 20 >4 >4 >4 >4 >4 91i 20 >4 >4 >4 >4 >4 91j 20 >4 >4 >4 >4 >4 91k 100 >20 >20 >20 >20 >20 91l ≥20 >20 >20 >20 >20 >20
91m 20 >4 >4 >4 >4 >4 91n ≥20 >20 >20 >20 >20 >20 91o 100 >20 >20 >20 >20 >20 92a 100 >20 >20 >20 >20 >20 92b 20 >4 >4 >4 >4 >4 92c ≥100 >100 >100 >100 >100 60 92d 20 >4 >4 >4 >4 >4 92e 20 >4 >4 >4 >4 >4 92f >100 >100 >100 >100 >100 >100 92g ≥20 >20 >20 >20 >20 >20 92h 100 >20 >20 >20 >20 >20 92i ≥100 >100 >100 >100 >100 >100 92j 20 >4 >4 >4 >4 >4 92k 20 >4 >4 >4 >4 >4 92l ≥4 >4 >4 >4 >4 >4
92m >100 >100 >100 >100 >100 >100 92n 100 >20 >20 >20 >20 >20 92o 100 >20 >20 >20 >20 >20
D.S-5000 >100 >100 >100 20 12 >100 (S)-DHPA (µM) >250 >250 >250 >250 >250 >250 Ribavirin (µM) >250 150 250 250 >250 150
305
Table 8.5d: Cytotoxicity and Antiviral activities of Chloroisatin derivatives 91(a-o) and Bromoisatin derivatives 92(a-o) in HEL cell Culture
EC50b (µg/ml)
Compound Minimum cytotoxic
concentrationa (µg/ml)
Herpes simplex virus-1 (KOS)
Herpes simplex virus-2
(G)
Vaccinia virus
Vesicular stomatitis
virus
Herpes simplex virus-1
TK- KOS ACVr
91a >100 >100 >100 >100 >100 >100 91b 100 >20 >20 >20 >20 >20 91c 20 >4 >4 >4 >4 >4 91d 100 >20 >20 >20 >20 >20 91e 100 >20 >20 20 >20 >20 91f 100 >20 >20 >20 >20 >20 91g 0.8 >0.16 >0.16 >0.16 >0.16 >0.16 91h 20 >4 >4 >4 >4 >4 91i 20 >4 >4 >4 >4 >4 91j 100 >20 >20 >20 >20 >20 91k 100 >20 >20 >20 >20 >20 91l ≥4 >4 >4 >4 >4 >4
91m 0.8 >0.16 >0.16 >0.16 >0.16 >0.16 91n 100 >20 >20 >20 >20 >20 91o 100 >20 >20 >20 >20 >20 92a 100 >20 >20 >20 >20 >20 92b 100 >20 >20 >20 >20 >20 92c 100 >20 >20 >20 >20 >20 92d 100 >20 >20 20 >20 >20 92e 20 >4 >4 >4 >4 >4 92f 100 >20 >20 >20 >20 >20 92g 20 >4 >4 >4 >4 >4 92h 20 >4 >4 4 >4 >4 92i ≥20 >20 >20 >20 >20 >20 92j 4 >0.8 >0.8 >0.8 >0.8 >0.8 92k ≥0.8 >0.8 >0.8 >0.8 >0.8 >0.8 92l ≥0.8 >0.8 >0.8 >0.8 >0.8 >0.8
92m 100 >20 >20 >20 >20 >20 92n 100 >20 >20 >20 >20 >20 92o 100 >20 >20 >20 >20 >20
Brivudin (µM) >250 0.08 10 6 >250 50 Ribavirin (µM) >250 10 150 250 >250 150 Acyclovir (µM) >250 0.4 0.4 250 >250 50
Ganciclovir (µM) >100 0.03 0.03 100 >100 2 aRequired to cause a microscopically detectable alteration of normal cell morphology. bRequired to reduce virus-induced cytopathogenicity by 50 %.
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Table 8.5e: Cytotoxicity and anti-Feline Corona Virus (FIPV) and anti-Feline
Herpes Virus activity of chloroisatin derivatives 91(a-o) and Bromoisatin
derivatives 92(a-o) in CRFK cell cultures EC50
b (µg/ml)
Compounds CC50a (µg/ml) Feline Corona Virus
(FIPV) Feline Herpes Virus
91a >100 46.7 >100 91b >100 >100 >100 91c 12.9 >4 >4 91d 31.9 >20 >20 91e >100 >100 >100 91f >100 >100 >100 91g >100 >100 >100 91h 5.7 >4 >4 91i 17.6 >4 >4 91j 24.4 >20 >20 91k >100 >100 >100 91l >100 >100 >100
91m >100 >100 >100 91n >100 >100 >100 91o >100 >100 >100 92a >100 >100 81.2 92b >100 >100 >100 92c 42.5 >20 >20 92d 22.9 >20 >20 92e 11.8 >4 >4 92f >100 >100 >100 92g 96.7 >20 >20 92h 69.0 >20 >20 92i >100 >100 >100 92j 2.6 >0.8 >0.8 92k >100 >100 >100 92l >100 >100 >100
92m 5.7 >4 >4 92n >100 >100 >100 92o >100 >100 >100 HHA >100 3.2 6.4 UDA >100 6.6 5.2
Ganciclovir (µM) >100 >100 2.6 a 50% Cytotoxic concentration, as determined by measuring the cell viability with the colorimetric formazan-based MTS assay. b 50% Effective concentration, or concentration producing 50% inhibition of virus-induced cytopathic effect, as determined by measuring the cell viability with the colorimetric formazan-based MTS assay. CRFK cells: Crandell-Rees Feline Kidney cell
307
8.5f Anti-influenza virus activity and cytotoxicity in MDCK cell cultures 8.5.1 Procedure
The test compounds were evaluated for their antiviral activity against three
influenza virus subtypes [A/Puerto Rico/8/34(H1N1); A/Hong Kong/7/87 (H3N2)
and B/Hong Kong/5/72]. Antiviral activity was estimated from the inhibitory effect
on virus-induced cytopathic effect, as determined by microscopical examination
and/or the formazan-based MTS cell viability test. Cytotoxicity of the test
compounds was expressed as the compound concentration causing minimal
changes in cell morphology (MCC), or the concentration causing 50% cytotoxicity
(CC50), as determined by the MTS assay.
8.5.2 Discussion
Compounds 91(a-o) and 92(a-o) were evaluated for their anti-influenza
virus activity, and their activities were compared with those of oseltamivir
carboxylate (the active form of Tamiflu®), ribavirin, amantadine and rimantadin
(Table 8.5f). None of the compounds 91(a-o) and 92(a-o) were able to inhibit the
cytopathic effects of influenza A or B at subtoxic concentrations. On the contrary,
the reference compounds oseltamivir carboxylate and ribavirin were active
against influenza virus; their EC50 values are clearly lower than their MCC values
(concentrations causing minimal toxicity). For amantadine and rimantadine, the
best activity was seen with the H3N2 strain. These compounds are known to be
inactive against influenza B. Also, the H1N1 A/PR/8/34 strain that is used in our
tests is less sensitive to amantadine and rimantadine.
308
Table 8.5f: Cytotoxicity and Anti-influenza acticity of chloroisatin derivatives 91(a-o)
Cytotoxicity Antiviral EC50
c Influenza A
H1N1 subtype
Influenza A H3N2
subtype Influenza B
Compd. Conc. unit
Minimum cytotoxic
conc.a CC50
bvisualCOE score
MTSvisual CPE
score MTS
visualCPE
score MTS
91a µg/ml 100 >100 NA NA NA NA NA NA 91b µg/ml 20 >100 NA NA NA NA NA NA 91c µg/ml 20 30.3 NA NA NA NA NA NA 91d µg/ml 20 11.3 NA NA NA NA NA NA 91e µg/ml 4 2.0 NA NA NA NA NA NA 91f µg/ml ≥100 >100 NA NA NA NA NA NA 91g µg/ml ≥4 21.5 NA NA NA NA NA NA 91h µg/ml ≥20 42.9 NA NA NA NA NA NA 91i µg/ml 100 >100 NA NA NA NA NA NA 91j µg/ml 20 9.0 NA NA NA NA NA NA 91k µg/ml 20 100 NA NA NA NA NA NA 91l µg/ml 4 1.9 NA NA NA NA NA NA
91m µg/ml 100 >100 NA NA NA NA NA NA 91n µg/ml 100 >100 NA NA NA NA NA NA 91o µg/ml 4 2 NA NA NA NA NA NA
Oseltamivir carboxylate µM >100 >100 0.07 0.08 107 1.5 4 2.0
Ribavirin µM 100 94.6 9 12.4 9 8.2 9 5.5 Amantadin µM >100 >100 45 83.3 4 3.1 NA NA Rimantadin µM >100 >100 45 39.3 0.8 0.1 NA NA
309
Table 8.6g: Cytotoxicity and Anti-influenza acticity of Bromoisatin derivatives 92(a-o)
Cytotoxicity Antiviral EC50c
Influenza A H1N1
subtype
Influenza A H3N2
subtype Influenza B
Compd. Conc. unit
Minimum cytotoxic
conc.a CC50
bvisualCOE score
MTSvisual CPE
score MTS
visualCPE
score MTS
92a µg/ml 100 >100 NA NA NA NA NA NA 92b µg/ml 20 >100 NA NA NA NA NA NA 92c µg/ml 20 30.3 NA NA NA NA NA NA 92d µg/ml 20 11.3 NA NA NA NA NA NA 92e µg/ml 4 2.0 NA NA NA NA NA NA 92f µg/ml ≥100 >100 NA NA NA NA NA NA 92g µg/ml ≥4 21.5 NA NA NA NA NA NA 92h µg/ml ≥20 42.9 NA NA NA NA NA NA 92i µg/ml 100 >100 NA NA NA NA NA NA 92j µg/ml 20 9.0 NA NA NA NA NA NA 92k µg/ml 20 100 NA NA NA NA NA NA 92l µg/ml 4 1.9 NA NA NA NA NA NA
92m µg/ml 100 >100 NA NA NA NA NA NA 92n µg/ml 100 >100 NA NA NA NA NA NA 92o µg/ml 4 2 NA NA NA NA NA NA
Oseltamivir carboxylate µM >100 >100 0.07 0.08 107 1.5 4 2.0
Ribavirin µM 100 94.6 9 12.4 9 8.2 9 5.5 Amantadin µM >100 >100 45 83.3 4 3.1 NA NA Rimantadin µM >100 >100 45 39.3 0.8 0.1 NA NA
b50% Cytotoxic concentration, as determined by measuring the cell viability with the colorimetric formazan-based MTS assay. aMinimum compound concentration that causes a microscopically detectable alteration of normal cell morphology. c50% Effective concentration, or concentration producing 50% inhibition of virus-induced cytopathic effect, as determined by visual scoring of the CPE, or by measuring the cell viability with the colorimetric formazan-based MTS assay. MDCK cells: Madin Darby canine kidney cells. NA: not active at the highest concentration tested, or at subtoxic concentration
310
8.5.3 Conclusion
The reference compounds oseltamivir carboxylate (the active form of
Tamiflu®) and ribavirin were active against influenza virus; their EC50 values are
clearly lower than their MCC values (concentrations causing minimal toxicity).
For amantadin and rimantadin, the best activity was seen with the H3N2 strain
(middle column). These compounds are known to be inactive against influenza B
(right column). Also, the H1N1 A/PR/8/34 strain that is used in our tests is known
to be less sensitive to amantadine and rimantadine (left column).
Among the range of 30 compounds [chloroisatin 91(a-o) and bromoisatin
92(a-o)] tested, none was able to inhibit the cytopathic effects of influenza A or B
virus at subtoxic concentrations or the highest concentration tested (100 µg/ml).
8.6 Antifungal Studies During last two decades, the life threatening infections caused by
pathogenic fungi and bacteria become increasingly common, especially in
individuals immunocompromised patients with AIDS. Clinically, candidosis,
aspergillosis, and cryptococosis are major fungal infections in these patients.
Fungi also produced toxin in foods and cause poisoning with out being physically
present. Another problem caused by fungi is allergy due to their spores.
However, the current antifungal therapy suffers from drug related toxicity; sever
drug resistance, non optimal pharmacokinetics and serious drug interaction.
Therefore, there is an emergent need to develop novel antifungal drugs with
higher efficiency, broad spectrum and low toxicity. Therefore antifungal and
antibacterial activities of synthesized triazoles 89(a-t) and thiadiazoles 90(a-j) were carried out.
311
8.6.1 Antifungal activities of synthesized triazole 89(a-t) and thiadiazoles 90(a-j)
The agar tube dilution method was used for testifying the antifungal
activities of synthesized triazoles and thiadiazoles. The antifungal assay was
done against four different fungal strains, which are:
Aspergillus flavus
Mucor species
Aspergillus niger
Aspergillus fumigatus
These funguses were maintained on sabouraud dextrose agar (SDA)
medium at 4ºC.
8.6.2 Media for fungus Sabouraud dextrose agar (SDA) was used to grow fungus for inoculums
preparations. Its composition was:
Peptone complex = 10gm/L
Glucose = 40 gm/L
Agar =15 gm/L
8.6.3 Preparation of media for fungus Sabouraud dextrose agar (SDA) was prepared by dissolving 6.5 gm
/100mL in distilled water and PH was adjusted at 5.6. Contents were dissolved
and dispensed as 4 mL volume into screw capped tubes and were autoclaved at
121 ºC for 21 minutes.
312
8.6.4 Loading of samples Tubes were allowed to cool to 50ºC and non solidified SDA was loaded
66.6 µL of triazoles 89(a-t) and thiadizoles 90(a-j) with pipette from stocked
solution. This would give the final concentration of 200 µg/ml of the pure
compound in the media. Tubes were then allowed to solidify at room temperature
in slanting position. Tubes were prepared in triplicate for each fungus species. 8.6.5 Inoculation of fungus, incubation and measurement
of growth inhibition The tubes containing solidified media and tested triazoles 89(a-t) and
thiadizoles 90(a-j) were inoculated with 4mm diameter of inoculums, taken from
seven days old culture of fungus. Other media supplemented with DMSO and
terbinafine were used as negative and positive control respectively. All
experiments were done in three replicates. The tubes were incubated at 28ºC for
seven days. Growth in the media was determined by measuring linear growth
(cm) and growth inhibition was calculated with reference to negative control.
(cm) control negativein growth Linear m)in test/(cgrowth linear - (cm) control negativein growth Linear )Inhibition (%age Formula =
8.6.6 Activity level
Below 40% inhibition = low activity
40-60% inhibition = moderate activity
60-70% inhibition = good activity
70% inhibition = significant activity
313
Table 8.6a: Antifungal Assay of Triazoles 89(a-t)
% inhibition Compd. Aspergillus
flavus Aspergillus fumigatus
Aspergillus niger
Mucor. sp
89a 36.58 32.86 74.89 45.45 89b 49.08 45.10 45.41 11.03 89c 16.61 10.56 19.58 17.20 89d 48.48 20.87 29.16 7.90 89e 56.71 12.5 40.20 1.08 89f 39.48 17.78 43.33 5.73 89g 25.76 47.03 37.50 17.64 89h 9.60 15.97 12.91 13.41 89i 4.72 30.79 20.62 40.25 89j 18.59 20.10 8.0 18.72 89k 34.91 41.10 52.50 21.86 89l 46.79 29.63 66.56 10.28
89m 57.47 42.91 34.47 4.65 89n 40.20 39.48 1.08 49.08 89o 43.33 25.76 5.73 16.61 89p 37.50 9.60 17.64 48.48 89q 12.91 4.72 13.41 56.71 89r 20.62 34.91 40.25 39.48 89s 29.63 46.79 17.64 25.76 89t 42.91 57.31 13.41 20.10
Turbenafine 100 100 100 100
Table 8.6: Antifungal Assay of Thiadizoles 90(a-j)
% inhibition Compd. Aspergillus
flavus Aspergillus fumigatus
Aspergillus niger
Mucor. sp
89a 22.05 52.24 49.47 51.58 89b 25.91 51.82 50.10 51.79 89c 30.12 44.57 49.68 51.58 89d 8.67 22.16 29.17 30.65 89e 14.45 13.49 15.01 15.01 89f 10.91 12.15 38.99 44.95 89g 16.38 19.75 23.890 42.91 89h 13.91 13.91 11.62 14.58 89i 16.38 19.75 23.89 42.91 89j 12.77 44.09 50.52 52.43
Turbenafine 100 100 100 100
314
8.6.6 Interpretation of results
Synthesized triazoles 89(a-t) and thiadizoles 90(a-j) were screened for
their antifungal activities. In vitro evaluation of antifungal activity was carried out
by the agar tube dilution method. All triazoles and thiadizoles exhibited moderate
activity against four fungal strains compared to reference chemotherapeutic i.e.
terbinafine at tested concentration. Among these synthesized compounds, 89a
and 89l were most active against Aspergillus niger. All the other remaining
compounds also showed good to moderate activity against these four fungal
strains.
8.7 Antibacterial assay
Synthesized triazoles 89(a-t) and thiadizoles 90(a-j) were screened at 1
mg/mL in DMSO (initial concentration of compounds). Nutrient broth medium for
bacterial growth was prepared by dissolving 0.8g/100mL in distilled water & pH
was adjusted to 7.2 and then medium was autoclaved. To performed
antibacterial assay, Nutrient agar medium was prepared by dissolving 2g/100mL
in distilled water and PH was maintained at 7.2, and then medium was
autoclaved. To compare the turbidity of bacterial culture, McFarland 0.5 barium
sulphate was used.
Following bacterial strains were used:
Staphylococcus aurens (Gram positive)
Bacillus Subtilis (Gram positive)
Salmonella setubal (Gram negative)
Enterobacter aerogenes (Gram negative)
315
8.7.1.1 Method
Nutrient agar medium was prepared by above mention method. It was
sealed with 1mL of prepared oculum/100mL of prepared nutrient agar medium
and was shaked. Petri plates (14cm) were made by pouring 75mL of sealed
nutrient agar media and allow it to solidify. Eleven wells per plate were made with
sterile cork borer (8mm). Using micropipette, 100µL of test solutions were poured
in respective wells. To all samples two solutions for positive control
(Roxithromycin 1mg/mL, Cefixime-USP 1mg/mL and one negative control DMSO
was applied to each plate. These plates were incubated at 37ºC. After 24 hour
incubation diameter of the clear zone was measured.
8.7.2 Results These synthesized triazoles 89(a-t) and thiadizoles 90(a-j) show no
activity against above mentioned bacteria.
133
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