Synthesis and Biological Evaluation of New Tetrahydrobenzothiophene Derivatives
Lianpao Wu
Student
Degree Thesis in Chemistry 30 ECTS
Master’s Level
Supervisors: Fredrik Almqvist, Dang The Hung
Examiner: Andreas Larsson
Department of Chemistry
Umeå university
Abstract
In a high throughput screening of 17,500 compounds, two hit compounds containing a
tetrahydrobenzothiophene core were identified as interesting biofilm inhibitors of
Escherichia Coli UTI89.Therefore, a small library of this type of compounds has been
synthesized and tested for their activity against the biofilm formation of this bacteria
to explore their structure activity-relationships (SARs) for further study.
The small library of novel class of tetrahydrobenzothiophene compounds included
amide, sulfonamide and sulfonylurea derivatives. Thirteen compounds have been
successfully synthesized from methyl 2-amino-6-phenyl-4, 5, 6, 7-tetrahydrobenzo[b]
thiophene-3-carboxylate, which was prepared from Gewald three-component reaction.
From biological data, compounds 4 and 8 were shown to have significant antibiofilm
activity, and preliminary information on the structure activity-relationships of this
class of compounds has been obtained for further investigation.
Keywords
Tetrahydrobenzothiophene, biofilm, Escherichia Coli, structure activity-relationships,
antibacterial, Gewald reaction
II
III
List of abbreviations Å ångström
Ac acetyl
Ar aryl
Boc tert-butoxycarbonyl
Boc2O di (tert-butyl) dicarbonate
DBU 1,8-diazabicyclo[5.4.0]undecene-7
DCC N, N'-Dicyclohexylcarbodiimide
DMF Dimethylformamide
DMSO Dimethyl sulfoxide
e.g. for example
equiv. equivalent(s)
et al. et alii (Latin for “and others”)
etc. et cetera (Latin for “and the rest”)
Et ethyl
h Hours
IR Infrared
LC-MS liquid chromatography-mass spectrometry
Me methyl
MeCN acetonitrile
mL millilitre
mM millmole
MWI Microwave irradiation
NMR Nuclear Magnetic Resonance
Ph phenyl
rt. Room temperature
SAR structure activity-relationship
TBTU O-(Benzotriazol-1-yl)-N, N, N′, N′-tetramethyluronium
tetrafluoroborate
t-Bu tert-butyl
TEA Triethylamine
TFA Trifloroacetic acid
THF Tetrahydrofuran
TLC Thin layer chromatography
UPEC uropathogenic Escherichia Coli
IV
V
Table of contents
1. Introduction ................................................................................................................ 1
1.1 Antibiotic resistance of bacteria and biofilm formation ...................................... 1
1.2. Identification of two hits against biofilm formation ........................................... 2
1.3. Application of Gewald reaction to the synthesis of tetrahydrobenzothiophene
scaffold ............................................................................................................... 2
1.4. Amide bond formation by TBTU ....................................................................... 3
1.5. Objectives ........................................................................................................... 4
2. Results and Discussion .............................................................................................. 5
2.1. Chemistry ............................................................................................................ 5
2.1.1. Modifications of the functional group at the C-2 position ........................... 5
2.2.2. Modifications of the functional group at the C-3 position ........................... 8
2.2. Biological evaluation ........................................................................................ 11
3. Conclusion and future perspectives ......................................................................... 13
5. Acknowledgement ................................................................................................... 14
6. Experimental Section ............................................................................................... 15
7. References ................................................................................................................ 26
VI
1
1. Introduction
1.1 Antibiotic resistance of bacteria and biofilm formation
The war between human and bacteria is still continuing. Nowadays, infectious
diseases are one of the biggest threats to human health.[1,2]
One key problem is the
bacterial evolution of resistance to antibacterial agents. For example when the first
antibiotic sulfonamide was developed in 1930s it saved a large number of people’s
lives. However, the bacteria resistant to sulfonamide appeared about ten years later.
The same thing happened to many antibiotic drugs (i.e. penicillin, streptomycin,
methicillin and linezolid) which were developed decades later.[3]
To win the war,
several approaches for antimicrobial therapy have been studied. It has been mentioned
that targeting bacterial virulence is one of the promising methods. Compared to
traditional antibiotic strategies, which inhibit bacterial growth or kill bacteria, this
approach can slow down the resistance processes, since it decreased the selective
pressure which promotes the growth of antibiotic resistant bacterial strains. In
addition, it has no or little influence on the normal human microbiota.[4-6]
Bacterial adhesion is one of the virulence properties of almost all microbes.[7,8]
Gram
negative bacteria like uropathogenic Escherichia Coli (UPEC) can produce pili and
curli, which help UPEC adhere to host cells and biofilm formation, related to the
recurrent infections and antibiotic resistance.[9-13]
Biofilms are thin layers of microorganisms and can be formed on biotic surfaces (such
as plants and animals) or on abiotic surfaces (such as minerals and surface of dead
organisms). They have their own communication and defense systems.[14,15]
The
biofilm formation is very complex, including five stages as shown in Figure 1.[16]
Many infectious diseases, such as endocarditis and urinary tract infection, are
persistent infection due to the biofilm formation.[5]
Therefore, the quest for novel
biofilm inhibitors is still rapidly growing.
Figure1. Five stages involved in the formation of biofilm. (A) Initial reversible attachment on
solid surface. (B) Other bacteria continued binding to form matrix. (C) Maturation phase:
cells become layered and effects of quorum sensing begin. (D) Clusters reach maximum
thickness. (E) Planktonic bacteria released from matrix dispersion.[16]
2
1.2. Identification of two hits against biofilm formation
Through a high throughput screening of 17,500 compounds, two hits (A and B)
containing a tetrahydrobenzothiophene core were found to have significant activity
against the formation of biofilm of Escherichia Coli UTI89 (Figure 2).
Figure 2. Chemical structures of two hits compounds.
1.3. Application of Gewald reaction to the synthesis of
tetrahydrobenzothiophene scaffold Since discovered in 1961, the Gewald reaction has become an useful method for the
synthesis of the 2-aminothiophene scaffold.[17]
This three-component reaction
involved condensation of a carbonyl compound, an activated nitrile and elemental
sulfur in the present of a base in alcohol or DMF (Scheme 1). Besides the classical
method, several modified approaches (such as solid-supported or microwave
accelerated Gewald reaction) have also been developed.[18,19]
Scheme 1. A general synthesis of 2-aminothiophene scaffold derived from Gewald reaction.
Although the full mechanism of Gewald reaction is not very clear [18]
the most
plausible mechanism is a base-promoted mechanism (Scheme 2).[19]
It involves four
main steps as shown below.
3
Scheme 2. The plausible mechanism of Gewald three-component reaction.
The first step is a Knoevenagel condensation between a carbonyl compound and an
activated nitrile to produce intermediate e. The second step is an addition of elemental
sulfur in the present of a base to give intermediate g. The third step is an intra-
molecular ring closure reaction. Nucleophilic attack by sulfur to the triple bond of the
cyano group give the cyclic imino product i, which in principle has equilibrium with
the tautomeric form 2-aminothiophene j. It was proved that the 2-aminothiophene
occurs exclusively in the amino form.[20-22]
1.4. Amide bond formation by TBTU
Amide functional group plays an important role in medicinal chemistry and biological
chemistry. Generally, it can be directly formed from amines and activated carboxylic
acids. There are myriad coupling reagents (such as DCC, TBTU, etc.) that can be used
to convert the carboxyl group to more reactive functional group such as anhydride,
acyl halide or active esters.[23,24]
TBTU (O-(Benzotriazol-1-yl)-N, N, N', N'-
tetramethyluronium tetrafluoroborate) is one of the less expensive coupling reagents,
which has been widely used in the amide bond formation.[25]
The mechanism of this reaction included several steps: addition of the carboxylate to
TBTU followed by decomposition of the intermediate and addition of the released
anion d to the carbonyl center to give an urea derivative and an activated ester g, and
finally addition of an amine to the activated ester g to form the amide h (Scheme
3).[24]
4
Scheme 3. The proposed mechanism of amide bond formation catalyzed by TBTU.
1.5. Objectives
Regardless of the functional groups at C-2 and C-3 positions, a preliminary study on
analogs of hits A and B showed that derivatives derived from hit A displayed higher
activity than those derived from hit B. Therefore, based on the structure of hit A,
several analogs with C-2 and C-3 modifications have been synthesized and
biologically evaluated to obtain information on the relationship of their structure and
activity against biofilm formation of Escherichia Coli UTI89 for further study.
Scheme 4. Synthesis of methyl 2-amino-6-phenyl-4, 5, 6, 7-tetrahydrobenzo[b]thiophene-3-
carboxylate via Gewald reaction. Reagents and conditions: (a) Et2NH, S8, MeOH, room
temperature, 23 h, 82%.
The core scaffold methyl 2-amino-6-phenyl-4, 5, 6, 7-tetrahydrobenzo[b]thiophene-3-
carboxylate 3 was synthesized via Gewald three-component reaction (Scheme 4).
From 3, thirteen analogs (A, 4-8, 10-11, 13, 15-18) have been synthesized (Figure 3).
5
Figure 3. Structures of target compounds.
2. Results and Discussion
2.1. Chemistry
2.1.1. Modifications of the functional group at the C-2 position
Five compounds 4, 5, 6, 7 and hit A were successfully synthesized from compound 3
and appropriate acid anhydrides (Scheme 5).[26]
Though the reactivity of the amine
group at C-2 position is quite weak due to its conjugation with the aromatic core
skeleton, the reactions still worked very well with a set of reactive anhydrides. As
shown in table 1, the yields of all compounds are higher than 65%.
6
Scheme 5. Modifications of hit A at the C-2 position. Reagents and conditions: (a) 1.0 equiv.
appropriate acid anhydrides, CH2Cl2, refluxed under N2; (b) NH3 , MeOH, 0 ºC → 110 ºC
(MWI), 1 h, 91%; (c) 2.5 equiv. methanesulfonyl chloride, 2.0 equiv. TEA, CH2Cl2, 0 ºC, 20
min, 96%; (d) 2.0 equiv. sodium methoxide, MeOH, 0 ºC→ 40 ºC overnight, 74%; (e) 1.0
equiv. benzenesulfonyl chloride, 2.0 equiv. TEA, CH2Cl2, 0 ºC→ 50 ºC, 2 days, 47%; (f) 1.0
equiv. 4-methyl benzenesulfonyl cyanide, 1.0 equiv. TEA, toluene, 110 ºC oil bath, 20 h, 75%.
Table 1. Variation of anhydrides in the synthesis of compounds 4-7 and hit A.
Entry Anhydride R2 Product Yield (%)
1
4 69%
2
5 98%
3
6 68%
4
7 65%
5
A 89%
7
Since the anhydride for synthesis of compound 8 is not available at the same reaction
condition as mentioned above, other synthetic pathway was adopted (Scheme 6).
Scheme 6. Synthetic route to compound 8. Reagents and conditions: (a) Ether, 0 ºC to room
temperature, overnight; (b) TEA, CH2Cl2, 0 ºC to room temperature, 5 min, 76%; (c) TFA,
CH2Cl2, 0 ºC to room temperature, 21 h, 60%.
Compound 8 was synthesized via a synthetic sequence shown in scheme 6. First, the
reaction between oxalyl chloride and tert-butanol was done to give intermediate 21.
Then treatment of compound 3 with the intermediate 21 provided compound 3c,
which was treated with CF3COOH to afford compound 8 in 60 % yield after
recrystalization with methanol.[27]
The anhydride for synthesis of compound 9 needs a very hard condition and it is not
stable at room temperature (decomposes to CO2 and ketene),[28,29]
so compound 9 was
attempted to be synthesized from propanedioyl dichloride and tert-butanol. However,
the synthesis of the tert-butyl 3-chloro-3-oxopropanoate intermediate was not
successful due to a possible reason that the propanedioyl dichloride is quite unstable
and very reactive. On the other hand, direct coupling of compound 3 and
propanedioyl dichloride in a base solution also failed. Hence, another synthetic
pathway which was reported by Ju-Yeon Lee et al[30]
was tried (Scheme 7). However,
no product was formed, probably due to the low reactivity of the amine group within
compound 3 compared to aniline in the reported method.
Scheme 7. Synthetic route to compound 9. Reagents and conditions: Meldrum’ acid, Xylene
refluxed overnight.
8
Scheme 5 also shows the pathway for synthesis of target compound 10. The methanol
solution of starting material 4 was saturated with ammonia gas, and this solution was
heated at 110 ºC by microwave for 1 h to give target compound 10 in excellent yield
91%.
To investigate the importance of a base on mesylation reaction of 3, several bases
were tried,[31,32]
but the dimesylated product was observed. Thus, several conditions
were tried (Table 2), but only small amount of the product from entries 3, 4 and 5
were observed.
Table 2. Screening different bases for synthesis of compound 11.
Entry Base Reaction conditions (a) Results
1 TEA CH2Cl2, rt, 30 min dimesylation
2 pyridine CH2Cl2, rt, overnight dimesylation
3 pyridine CH2Cl2, 0 ºC , 9 h mesylation and dimesylation
4 DBU CH2Cl2, -40 ºC →0 ºC, 30 min mesylation and dimesylation
5 TEA CH2Cl2, -40 ºC, 2 h→0 ºC, 0.5 h mesylation and dimesylation
Due to difficulty in purification and low yield of product, another reaction condition
was tried (Scheme 5). To completely convert the starting material to the disubstituted
product, 2.5 equivalent of methanesulfonyl chloride was used for the reaction. Then,
the dimesylated product was mono cleavaged with sodium methoxide and good yield
(74%) of compound 11 was obtained.[33]
The synthesis of compound 12 via treatment of 3 and benzenesulfonyl chloride in the
present of different bases (e.g. TEA, pyridine and sodium carbonate) was also tried.
However, only disubstituted product 3b was obtained (Scheme 5) by using TEA. On
the other hand, a lot of starting material was observed when pyridine or sodium
carbonate was employed in the reaction. This result shows that this reaction is a
thermodynamic control reaction, and the dimer form is the more stable product.
The diarylsulfonylurea 13 was obtained in good yield (75%) by overnight reflux of 4-
methyl benzenesulfonyl cyanide, compound 3 and TEA in toluene.[34]
2.2.2. Modifications of the functional group at the C-3 position
To synthesize compound 14, several reducing agents such as LiAlH4, NaBH4 and
LiBH4 were tried to find out the best one for direct reduction of compound 4[35-38]
(Scheme 8). However, no desired product was obtained and many by-products were
observed. The reduction was also tried through other synthetic pathways [36, 39, 40]
such
as reduction of 3, 3d, 3f and 3g by different conditions, but no desired products were
obtained (Scheme 8).
9
Scheme 8. Synthesis of compound 14. Reagents and conditions: (a) 1.0 equiv. succinic
anhydride, CH2Cl2, refluxed 28 h, 69%; (b) (i) 4.0 eq NaBH4, 4.0 eq MeOH, 0.1 eq
NaB(OAC)3H, THF, 0 ºC → rt, 19 h or (ii) 1.5 eq LiBH4, 1.5 eq MeOH, Et2O, refluxed, 21 h
or (iii) 10.0 eq LiBH4, 1.5 eq MeOH, THF, 65 ºC (MWI), 45 min or (iv) 2.0 eq LiAlH4 (10%
in THF), THF, 0 ºC → rt , 4 h or (v) 2.0 eq LiAlH4 (10% in THF), THF, 65 ºC (MWI), 45
min; (c) 10.0 equiv. 10.0 M NaOH (aq), THF: MeOH: H2O= 5:2:10 refluxed overnight; (d)
4.0 equiv. LiAlH4 in THF (2.0 M), THF, 0 ºC→ 65 ºC (MWI), 2 h; (e) 9.0 equiv. BH3 in THF
(1.0 M), THF, 0 ºC→ 65 ºC (MWI) 1 h; (f) 2.1 equiv. Boc2O, 0.1 equiv. DMAP, dioxane, 80
ºC, 3 h then 3.0 equiv. N2H4.H2O, 40 ºC for 1.5 h, 81%; (g) 2.0 equiv. LiAlH4 in THF (2.0 M),
THF, 0 ºC→ 65 ºC (MWI), 1 h; (h) 3.0 equiv. BH3 in THF (2.0 M), 29 h.
To the best of my knowledge, there is only one patent by Gillen, Kevin James and his
co-workers,[41]
describing synthesis of the primary alcohol compound whose system is
similar to compound 14. The synthetic route used in the patent was adopted with
some modifications (Scheme 9). In order to prepare the aldehyde compound 3l, the
amino group of the starting compound 3 was first protected by an acetyl group,[42]
then followed by hydrolysis 3j, decarboxylation 3k and formylation 3l. However,
when compound 3k was treated with Vilsmeier reagent, an intramolecular ring
closure reaction was observed to give by-product 3m, so the aldehyde 3l could not be
obtained.
10
Scheme 9. Another synthetic pathway for compound 14. Reagents and conditions: (a) 2.5
equiv. acetyl chloride, pyridine, CH2Cl2, 0 ºC → rt, 85%; (b) 10.0 equiv. 10.0 M NaOH (aq),
THF: MeOH: H2O= 5:2:10 refluxed 9 h, 93%; (c) 1.5 equiv. Cu, quinoline, MWI 200 ºC, 10
min, 54%; (d) 1.0 equiv. DMF, 1.0 equiv. POCl3, CH3CH2Cl2, rt → 85 ºC 3 h, then
CH3COONa (aq), 1.5 h, 19%.
The target amide compounds (15-18) were synthesized from compound 3 via a
synthetic sequence including N-Boc protection, hydrolysis, amide bond formation,[43]
N-Boc-deprotection[44]
and acylation (Scheme 10). To ensure complete carbamolyation
of compound 3, two more equivalent of di-tert-butyl dicarbonate are required since
one equivalent of di-tert-butyl dicarbonate resulted in a mixture of starting material,
mono-Boc and di-Boc products. Because compound 3n is sensitivity to nucleophiles,
one of the carbamate groups as well as any excess Boc2O could be converted to tert-
butyl hydrazinecarboxylate by adding excess hydrazine. Moreover, the by-product
tert-butyl hydrazinecarboxylate could be easily removed by silica column, and
compound 3f was achieved in very good yield (81% from 3).[43]
In the coupling reaction either aliphatic or aromatic amines were used in the present
of TEA and the amide coupling reagent TBTU. The yield of some products is quite
low. It is possibly due to the low reactivity of the acid at C-3 position, induced by the
amine group at C-2 position. The amine group is an excellent electron donating group
that decreases the electronpilicity of the carbonyl carbon at C-3 position. The reaction
rate was in the order of morpholine > isopropylamine > aniline > benzyl amine at the
same reaction condition, possibly due to different nucleophilicity.
11
Scheme 10. Synthetic pathway of the target compounds 15-18. Reagents and conditions: (a)
2.1 equiv. Boc2O, 0.1 equiv. DMAP, dioxane, 80 ºC, 3 h; (b) 3.0 equiv. N2H4.H2O, 40 ºC for
1.5 h, 81% (from 3); (c) 10.0 equiv. 10.0 M NaOH (aq), THF: MeOH: H2O= 5:2:10 refluxed
overnight, 85%; (d) 1.0 equiv. TBTU, 1.0 equiv. 2.0 equiv. TEA, ethyl acetate, R1NH2, stirred
at rt; (e) 1.0 ml TFA , 1.0 ml CH2Cl2 stirred at rt; (f) 1.0 equiv. succinic anhydride, CH2Cl2,
refluxed 3 h-7 h 40 min.
Due to stability of 23b-23d, only 23a was purified after treatment with TFA. Finally
these deprotected products were converted to 15-18 by treatment with an equimolar
amount of succinic anhydride (Table 3).
Table 3. Variation of amines in the synthesis of target compounds 15-18.
Entry R1NH2 Product Yield (%)
1
15 27%
2
16 75%
3
17 30%
4
18 65%
2.2. Biological evaluation All the synthesized compounds were evaluated for their activity against biofilm
formation of Escherichia Coli UTI89 using a pili-dependent biofilm assay. In the
biofilm assay, the percentage inhibitory activity of all synthesized compounds was
measured at concentrations of 200 M, 100 M and 50 M. Subsequently,
compounds with good inhibition rate (> 50% at 50 M) were further tested at lower
12
concentrations (25, 12.5, 6.3 and 3.1 M) to determine their ED50 values. In addition,
a growth inhibition assay was also performed to validate that active compounds are
really true inhibitors of biofilm formation and not acting as bactericidal agents. The
results of this evaluation are outlined in table 4.
Table 4. Activity against the growth and biofilm formation of Escherichia Coli UTI89.
Comp.
No.
R1 R2 ED50 of biofilm formation (µM)
growth (µM)
A OMe
25 >50
4 OMe
15 >50
5 OMe
25 >50
6 OMe
25 >50
7 OMe
25 >100
8 OMe
12.5 >100
10 OMe
25 >100
11 OMe
No activity >100
13 OMe
25 >100
15
No activity >100
16
25 >100
17
No activity >100
18
No activity >100
Considering the modification of the cyclic acid group at C-2 position of hit A by other
acid moieties, the data showed that this transformation results in compounds (4-8)
with retained or improved activity. Among them compounds 4 and 8 displayed higher
inhibitory effect on biofilm formation than the hit A, suggesting that an acyclic acid
group is more favorable for the biological activity than the cyclic acid counterpart.
Also, a retained activity in the sulfonylurea analog 13 suggested that modification of
13
the acid moiety at C-2 position by an acid bioisostere strategy may provide potent
biofilm inhibitors.
Regarding the modification of the methyl ester at C-3 position by other amide groups
(15-18) while keeping an acyclic acid moiety constant, the data revealed that only
compound 16 derived from an aryl amine is retained activity compared to the hit A,
suggesting that few amide groups at C-3 position would be tolerated if further
modification on this position is considered.
3. Conclusion and future perspectives
Based on the structure of hit A, several tetrahydrobenzothiophene derivatives (A, 4-8,
10-11, 13, 15-18) have been successfully synthesized to investigate their preliminary
structure-activity relationships. An attempt to transform the ester group into a primary
alcohol was not successful, although several strategies have been employed. A
possible reason is due to a low reactivity of the methyl ester group located in a
conjugate system.
All the synthesized compounds were tested for their antibiofilm activity by evaluation
of their inhibitory effects on biofilm formation of Escherichia Coli UTI89 using a
pili-dependent biofilm assay. As expected, the preliminary information on their SARs
has been observed as a guidance for further investigation. Compared to hit A, several
biofilm inhibitors with improved activity (e.g. 4 and 8) have been investigated by a
simple replacement of the cyclic acid group by an acyclic acid moiety at the C-2
position.
The biological data of the synthesized compounds in the present study suggests that
further modification should focus on other ester groups at C-3 position or acid
bioisostere groups at C-2 position.
14
5. Acknowledgement
Herein I would like to express my deepest gratitude and heartfelt thanks to all the
people who have guided, supported and helped me in various ways during the past
two years.
Firstly, I sincerely thank my supervisor, Professor Fredrik Almqvist, for giving me
this interesting project to work with, for the advices at the beginning of this project
and for the discussions during this work.
Secondly, I would also thank my second supervisor, Dr. Dang The Hung, for all the
helpful advices and discussions and helps in the lab, for your patient guidance during
my thesis writing.
I am also grateful to:
All group members including Christoffer, Magnus, Munawar, Syam, Krishna,
Karl and Lina in the Almqvist’s group, thank you all for being good friends and for
pleasant time. A special thanks to Syam and Magnus for working with me in the lab
during many Saturdays.
The Study Administrator: Barbro and my previous Study Counselor: Bertil. Thank
you for helping me with all sorts of matters when I met during my study.
I wish to thank all my good friends and teachers in China for everlasting care and
support. I also want to thank all my lecturers, classmates and friends in Umeå for
making my days here colorful and memorable. I will keep this memory in my heart
forever.
Last, I would like to thank my parents, who encourage me all the time in my life, and
give me endless love and bless. And I also thank my brother for his supporting and
encouraging me all the time!
15
6. Experimental Section
Unless otherwise stated all the reactions were carried out in oven-dried glassware. All
solvents and reagents were commercially (Sigma-Aldrich, Fluka and Lancaster).The
solvents were dried over 4 Å molecular sieves. Infrared spectra were collected on a
Perkin-Elmer FT-IR Spectrometer. Analytical thin layer chromatography (TLC) was
performed on Merck Silica gel 60 F254 plates. Flash column chromatography was
performed on Sigma-Aldrich silica gel.60. Microwave reactions were carried out in
Biotage Initiator using Smith Process Vial TM
sealed with Teflon septa and an
aluminum crimp top. 1H NMR was recorded at 293 K and 400 MHz and
13C NMR
was recorded at 100 MHz. The spectra were calibrated using the residual peak of
solvent as internal standard [CDCl3 (CHCl3 δH 7.26 ppm, CDCl3 δC 77.1 ppm),
DMSO-d6 (DMSO-d5 δH 2.49 ppm, DMSO-d6 δC 40.0 ppm), MeOH-d4 (CD2HOD δH
3.31 ppm, CD3OD δC 49.0 ppm)].
Methyl 2-amino-6-phenyl-4, 5, 6, 7-tetrahydrobenzo
[b]thiophene-3-carboxylate (3)
To a stirring mixture of phenylcylohexanone (2.0 g, 0.02 mol), methyl cyanoacetate
(1.23 mL, 0.014 mol) and powdered sulfur (0.45 g, 0.014 mol) in methanol (10 mL),
diethylamine (0.72 mL, 0.007 mol) was added dropwise. The mixture was kept
stirring at RT for 22 h. The precipitate product was collected and washed with MeOH.
The mother liquid was recrystalized, a white solid 3 (2.9 g, 82%) was obtained. IR
3445, 3321, 1665 cm-1
; 1H NMR (400 MHz, CDCl3) δ 7.42-7.31 (m, 2H, Ar-H)
7.28-7.21 (m, 3H, Ar-H), 5.98 (bs, 2H, NH2), 3.83 (s, 3H, COOCH3), 3.08-2.93 (m,
2H), 2.85-2.62 (m, 3H), 2.17-2.06 (m, 1H), 1.99-1.85 (m, 1H); 13
C NMR (100 MHz,
CDCl3) δ 166.4, 162.1, 146.0, 132.2, 128.4 (2C), 126.9 (2C), 126.3, 117.0, 105.4,
50.6, 40.8, 32.4, 30.0, 27.2; LC-MS Rf (min) = 5.80, LC-MS m/z (ES+) calculated for
C16H18NO2S [M+H] 288: found 288.
The general procedure for synthesis of compounds A, 4-7
To a solution of compound 3 (208.0 mg, 0.81 mol) in dry CH2Cl2 (10 mL) was added
an equimolar amount of the appropriate acid anhydrides. The mixture was kept at
refluxing for 2~28 h under an atmosphere of nitrogen. The mixture was concentrated
under reduced pressure, and the solid was recrystalized from methanol.
Methyl 2-[[(6-carboxy-3-cyclohexen-1-yl)carbonyl]amino]-6-phenyl-4, 5, 6, 7-
tetrahydrobenzo[b]thiophene-3-carboxylate (A)
The light yellow solid was recrystalized from methanol. A white solid A (81.8 mg,
89%) was obtained. IR 3252, 2913, 1691, 1657, 1558, 1243, 1206 cm-1
; 1H NMR
(400 MHz, DMSO-d6) δ 12.3 (bs, 1H, NH), 11.2 (s, 1H, COOH), 7.34-7.28 (m, 4H,
Ar-H), 7.24-7.18 (m, 1H, Ar-H), 5.69 (m, 2H, -CH2-CH=CH-CH2-), 3.84 (s, 3H,
COOCH3), 3.18-3.11 (m, 1H), 3.10-3.04 (m, 1H), 3.00-2.82 (m, 3H), 2.78-2.66 (m,
2H), 2.48-2.30 (m, 4H), 2.05-1.97 (m, 1H), 1.93-1.80 (m, 1H); 13
C NMR (100 MHz,
DMSO-d6) δ 174.7, 171.0, 166.2, 146.1, 130.5, 128.8 (3C), 127.3 (3C), 126.7, 126.2,
125.9, 124.9, 110.9, 52.2, 40.2, 39.5, 39.4, 31.8, 30.0, 26.6, 25.7; LC-MS Rf (min) =
5.87, LC-MS m/z (ES-) calculated for C24H24NO5S [M-H] 438: found 438.
16
Methyl 2-[(3-carboxypropanoyl)amino]-6-phenyl-4, 5, 6, 7-tetrahydrobenzo
[b]thiophene-3-carboxylate (4)
The light yellow solid was recrystalized from methanol. A white solid 4 (221.0 mg,
69%) was obtained. IR 3193, 3026, 1688, 1665, 1534 cm-1
; 1H NMR (400 MHz,
DMSO-d6) δ 12.25 (bs, 1H, COOH), 10.96 (s, 1H, NH), 7.33-7.18 (m, 5H, Ar-H),
3.82 (s, 3H, COOCH3), 3.00-2.81 (m, 3H), 2.77-2.65 (m, 4H), 2.60-2.53 (m, 2H),
2.04-1.95 (m, 1H), 1.91-1.79 (m, 1H); 13
C NMR (100 MHz, DMSO-d6) δ 173.4,
169.1, 165.2, 146.3, 145.6, 130.1, 128.3 (2C), 126.8 (2C), 126.2, 125.5, 110.7, 51.5,
39.7, 31.3, 30.7, 29.5, 28.6, 26.1; LC-MS Rf (min) = 5.15, LC-MS m/z (ES-)
calculated for C20H20NO5S [M-H] 386: found 386.
Methyl 2-[(4-carboxybutanoyl)amino]-6-phenyl-4, 5, 6, 7-tetrahydrobenzo
[b]thiophene-3-carboxylate (5)
The product was purified by silica gel column chromatography using ethyl DCM:
MeOH= 98:2 (0.1% CH3COOH) → 97:3 (0.1% CH3COOH) as eluent. The solvents
were removed under reduced pressure and the solid was recrystalized from methanol.
Compound 5 (82.6mg, 98%) was obtained as a white solid. IR 3252, 2953, 1713,
1660, 1651 cm-1
; 1H NMR (400 MHz, CDCl3) δ 11.28 (s, 1H, NH), 7.35-7.28 (m, 2H,
Ar-H), 7.25-7.24 (m, 1H, Ar-H), 7.24 -7.19 (m, 2H, Ar-H), 3.86 (s, 3H, COOCH3),
3.04-2.87 (m, 3H), 2.83-2.70 (m, 2H), 2.60 (t, J = 7.40 Hz, 2H), 2.50 (t, J = 7.40 Hz,
2H), 2.16-2.03 (m, 3H), 1.96-1.83 (m, 1H); 13
C NMR (100 MHz, CDCl3) δ 178.5,
169.2, 167.0, 147.8, 145.7, 130.4, 128.5 (2C), 126.9 (2C), 126.4, 126.3, 111.1, 51.5,
40.5, 35.4, 32.9, 32.1, 30.1, 26.6, 20.6; LC-MS Rf (min) = 5.67, LC-MS m/z (ES-)
calculated for C21H22NO5S [M-H] 400: found 400.
Methyl 2-[(2-carboxybenzoyl)amino]-6-phenyl-4, 5, 6, 7-tetrahydrobenzo
[b]thiophene-3-carboxylate (6)
The product was purified by silica gel column chromatography using DCM: MeOH=
97:3 as eluent. The solvents were removed under reduced pressure and the yellow oil
was obtained. The yellow oil was recrystalized from methanol. Compound 6 (61.9
mg, 68%) was obtained as a yellow solid. IR 3025, 2917, 1660, 1561 cm-1
; 1H
NMR (400 MHz, CDCl3) δ 11.71 (s, 1H, NH), 8.06 (d, J = 3.76 Hz, 1H, Ar-H), 7.70-
7.62 (m, 2H, Ar-H), 7.62-7.56 (m, 1H, Ar-H), 7.37-7.30 (m, 2H, Ar-H), 7.29-7.27 (m,
1H, Ar-H), 7.25-7.20 (m, 2H, Ar-H), 3.84 (s, 3H, COOCH3), 3.08 -2.93 (m, 3H),
2.87-2.74 (m, 2H), 2.18-2.10 (m, 1H), 2.00-1.87 (m, 1H); 13
C NMR (100 MHz,
CDCl3) δ 170.0, 167.0, 165.6, 147.7, 145.7, 136.0, 132.7, 131.5, 130.8, 129.4, 128.5
(2C), 127.8, 127.0, 126.9 (2C), 126.4, 125.7, 112.0, 51.6, 40.5, 32.1, 30.1, 26.6; LC-
MS Rf (min) = 5.80, LC-MS m/z (ES-) calculated for C24H20NO5S [M-H] 434: found
434.
Methyl 2-[[(1E)-3-carboxy-1-oxo-2-propen-1-yl]amino]-6-phenyl-4, 5, 6, 7-
tetrahydrobenzo[b]thiophene-3-carboxylate (7)
The yellow solid was recrystalized from methanol. Compound 7 (52.8 mg, 65%) was
obtained as a yellow solid. IR 3188, 2923, 2492, 1710, 1677, 1591 cm-1
; 1H NMR
(400 MHz, CDCl3) δ 14.5 (bs, 1H, NH), 12.1 (s, 1H, COOH), 7.37-7.30 (m, 2H, Ar-
H), 7.28-7.26 (m, 2H, Ar-H), 7.25-7.22 (m, 1H, Ar-H), 6.55-6.42 (d, J = 16.00 Hz, 2H,
17
-CH=CH-COOH), 3.93 (s, 3H, COOCH3), 3.10 -2.96 (m, 3H), 2.89-2.75 (m, 2H),
2.20-2.11 (m, 1H), 2.00-1.87 (m, 1H); 13
C NMR (100 MHz, CDCl3) δ 167.1, 163.8,
162.0, 145.2, 144.6, 131.8, 130.3, 129.6, 128.6 (2C), 126.8 (2C), 126.6, 114.5, 77.2,
52.1, 40.3, 32.2, 29.8, 26.5; LC-MS Rf (min) = 5.65, LC-MS m/z (ES-) calculated for
C20H18NO5S [M-H] 384: found 384.
Methyl 2-[(2-tert-butoxy-2-oxo-acetyl)amino]-6-phenyl-4, 5, 6, 7-
tetrahydrobenzo[b]thiophene-3-carboxylate (3c)
The mixture of butanol (148.2 mg, 2.00 mmol) and dry ether (1.0 mL) was cooled to 0
ºC and then the solution (cooled to 0 ºC) of oxalyl chloride (0.17 mL, 2.00 mmol) in
dry ether (4.0 mL) was added dropwise to the mixture. The resulting mixture was
stirred at 0 ºC for 20 min then stirred at RT overnight. The solvent was removed the
under reduced pressure, and colourless oil 21 was obtained. Then it was dissolved in
dry DCM (8.6 mL) to prepare 0.23M solution. Compound 3 (60.0 mg, 0.21 mmol)
and 58.0 µL TEA were mixed in dry DCM (5.0 mL), and then tert-butyl 2-chloro-2-
oxoacetate 21 (0.23 M, 1.0 mL) was added dropwise to the mixture at 0 ºC under N2.
The mixture was stirred at 0 ºC for 5 min and then washed with saturated NaHCO3
(10 mL) and extracted by DCM (3x20 mL).The organic phase was dried over Na2SO4
(s).The solvent was evaporated, and the residue was purified by silica gel column
chromatography using DCM: MeOH= 99:1 as eluent. The solvents were removed
under reduced pressure to give compound 3c (66.0mg, 76%) as yellow foam. IR
3241, 2915, 1754, 1729, 1693 cm-1
; 1H NMR (400 MHz, CDCl3) δ 12.4 (s, 1H,
NH), 7.39-7.28 (m, 2H, Ar-H), 7.28-7.26 (m, 1H, Ar-H), 7.25-7.14 (m, 2H, Ar-H),
3.91 (s, 3H, COOCH3), 3.15-2.91 (m, 3H), 2.90-2.72 (m, 2H), 2.21-2.07 (m, 1H),
2.00-1.84 (m, 1H), 1.62 (s, 9H, CO(CH3)3); 13
C NMR (100 MHz, CDCl3) δ 166.1,
158.2, 153.9, 145.6, 145.5, 131.5, 128.5 (2C), 128.1, 126.8 (2C), 126.4, 113.4, 85.3,
51.7, 40.5, 32.3, 29.9, 27.7 (3C), 26.6; LC-MS Rf (min) = 6.50, LC-MS m/z (ES-)
calculated for C22H24NO5S [M-H] 414: found 414.
Methyl 2-[(carboxycarbonyl)amino]-6-phenyl-4, 5, 6, 7-tetrahydrobenzo
[b]thiophene-3-carboxylate (8)
The solution of compound 3c (63.0 mg, 0.15 mmol) in DCM (6.0 mL) was cooled to
0 ºC, and trifluoroacetic acid (2.0 mL) was added dropwise to the mixture. The
mixture was stirred at RT for 21 h, and then the solvents were evaporated under
reduced pressure and the residue was coevaprorated with Et2O (10x 10 mL) to give a
yellow solid crude which was recrystalized from MeOH. Compound 8 (33 mg, 60%)
was obtained as a yellow solid. IR 3233, 2936, 1765, 1675 cm-1
; 1H NMR (400
MHz, CDCl3) δ 12.5 (s, 1H, NH), 7.35-7.29 (m, 2H, Ar-H), 7.27-7.20 (m, 3H, Ar-H),
3.93 (s, 3H, COOCH3), 3.11-2.93 (m, 3H), 2.87-2.74 (m, 2H), 2.19-2.11 (m, 1H),
1.98-1.87 (m, 1H); 13
C NMR (100 MHz, CDCl3) δ 165.9, 158.5, 153.8, 145.3, 144.1,
132.1, 129.3, 128.6 (2C), 126.8 (2C), 126.5, 115.0, 52.0, 40.4, 32.3, 29.8, 26.5; LC-
MS Rf (min) = 5.39, LC-MS m/z (ES-) calculated for C18H16NO5S [M-H] 358: found
358.
Methyl 2-[[(3-aminocarbonyl)propanoyl]amino]-6-phenyl-4, 5, 6, 7-
tetrahydrobenzo[b]thiophene-3-carboxylate (10)
18
A solution of compound 4 (60.0 mg, 0.21 mmol) in MeOH (5.0 mL) was bubbled
with NH3 (g) in a seal tube for 0.5 h until the solution became clear. The reaction
mixture was heated by microwave irradiation for 1 h at 110 ºC, and the solvent was
evaporated. The concentrated crude was washed with brine and extracted with ethyl
acetate (3x20 mL), dried over Na2SO4 (s), filtered and evaporated. The residue was
purified by silica gel column chromatography using MeOH: DCM= 5:95 (1% HAC)
as eluent. The solvents were evaporated under reduced pressure and compound 10
(54.8 mg, 91%) was obtained as a white solid. IR 3194, 3026, 2923, 1689, 1665,
1533 cm-1
; 1H NMR (400 MHz, CDCl3) δ 11.30 (s, 1H, NH), 7.35-7.29 (m, 2H, Ar-
H), 7.27-7.26 (m, 1H, Ar-H), 7.25-7.20 (m, 2H, Ar-H), 3.87 (s, 3H, COOCH3), 3.05-
2.88 (m, 3H), 2.71-2.85 (m, 6H), 2.17-2.08 (m, 1H), 1.96-1.85 (m, 1H); 13
C NMR
(100 MHz, CDCl3) δ 177.3, 168.3, 166.9, 147.7, 145.7, 130.5, 128.5 (2C), 126.9 (2C),
126.4, 126.3, 111.2, 51.5, 40.5, 32.1, 30.9, 30.0, 28.8, 26.6; LC-MS Rf (min) = 5.37,
LC-MS m/z (ES-) calculated for C20H21N2O4S [M-H] 385: found 385.
Methyl 2-[bis(methylsulfonyl)amino]-6-phenyl-4, 5, 6, 7-
tetrahydrobenzo[b]thiophene-3-carboxylate (3a)
Compound 3 (60.0 mg, 0.21 mmol) and TEA (58.0 µL, 0.42 mol) was mixed in dry
DCM (2.5 mL), and the mixture was cooled to 0 ºC then a solution of
methanesulfonyl chloride (16.0 µL in 1.5 mL DCM) was added dropwise thereto.
After 20 min the solution turned slight yellow, and HCl (1.0 mL, 0.5 M) was added.
The mixture was extracted by DCM (3x15 mL) and the solvents were removed under
reduced pressure to give compound 3a (73.6 mg, 96%) as white foam. IR 1717
cm-1
; 1H NMR (400 MHz, CDCl3) δ 7.37-7.31 (m, 2H, Ar-H), 7.27-7.26 (m, 1H, Ar-
H), 7.25-7.22 (m, 2H, Ar-H), 3.88 (s, 3H, COOCH3), 3.50 (s, 3H, -SO2CH3), 3.45 (s,
3H, -SO2CH3), 3.15 -2.98 (m, 3H), 2.92-2.80 (m, 2H), 2.21-2.12 (m, 1H), 1.99-1.87
(m, 1H); 13
C NMR (100 MHz, CDCl3) δ 162.5, 145.1, 138.2, 135.4, 134.0, 131.7,
128.7 (2C), 126.8 (2C), 126.7, 51.8, 42.9, 42.7, 40.3, 33.2, 29.5, 26.7; LC-MS Rf (min)
= 5.57, LC-MS m/z (ES+) calculated C18H19NO6S3 for [M-CH2] 429: found 429.
Methyl 2-[(methylsulfonyl)amino]-6-phenyl-4, 5, 6, 7-tetrahydrobenzo
[b]thiophene-3-carboxylate (11)
A mixture of compound 3a (30.1 mg, 0.06 mmol) and sodium methoxide (3.7 mg,
0.13 mmol) in MeOH (6.0 mL) was stirred at 0 ºC with for 3 h and continued to
stirred at 40 ºC overnight. The mixture was washed with HCl (10.0 mL, 1.0 M) and
extracted with DCM. The organic phase was dried over Na2SO4 (s), filtrated and
concentrated under reduced pressure. The residue was purified by silica gel column
chromatography using ethyl acetate: heptane= 12:88 as eluent. The solvents were
evaporated under reduced pressure to give compound 11 (18.4 mg, 74%) as a white
solid. IR 3135, 1660 cm-1
; 1H NMR (400 MHz, CDCl3) δ 10.19 (s, 1H, NH), 7.36-
7.29 (m, 2H, Ar-H), 7.27-7.25 (m, 1H, Ar-H), 7.25-7.21 (m, 2H, Ar-H), 3.87 (s, 3H,
COOCH3), 3.09 (s, 3H, -SO2CH3), 3.05 -2.86 (m, 3H), 2.82-2.70 (m, 2H), 2.18-2.08
(m, 1H), 1.97-1.84 (m, 1H); 13
C NMR (100 MHz, CDCl3) δ 166.3, 148.5, 145.4,
132.1, 128.6 (2C), 126.8 (2C), 126.5, 126.3, 113.0, 51.7, 40.5, 39.7, 33.3, 29.8, 26.8;
LC-MS Rf (min) = 5.67, LC-MS m/z (ES-) calculated for C17H18NO4S2 [M-H] 364:
found 364.
19
Methyl 2-[bis(phenylsulfonyl)amino]-6-phenyl-4, 5, 6, 7-
tetrahydrobenzo[b]thiophene-3-carboxylate (3b)
To a cooled solution (0 ºC) of compound 3 (60.0 mg, 0.21 mmol) and TEA (58.0 µL,
0.42 mol) in dry DCM (2.5 mL) was added dropwise a solution of benzenesulfonyl
chloride (16.0 µL in 1.5 mL DCM). The solution was stirred at this temperature for 20
min then at RT overnight under an atmosphere of nitrogen. Due to the presence of a
lot of starting material, the mixture was refluxed in 50 ºC oil bath for two days. After
cooling to RT, HCl (1.0 mL, 0.5 M) was added. The mixture was extracted with DCM
(3x15 mL), dried over Na2SO4 (s) and filtered, the solvents were evaporated under
reduced pressure. The residue was purified by silica gel column chromatography
using ethyl acetate: heptane= 1:4 as eluent. Compound 3b (28.1 mg, 47%) was
obtained as a yellow solid. IR 1717 cm-1
; 1H NMR (400 MHz, CDCl3) δ 8.05-7.98
(m, 4H, Ar-H), 7.71-7.66 (m, 2H, Ar-H), 7.60-7.54 (m, 4H, Ar-H), 7.38-7.33 (m, 2H,
Ar-H), 7.29-7.26 (m, 2H, Ar-H), 7.25-7.22 (m, 1H, Ar-H), 3.21 (s, 3H, COOCH3),
3.14-3.00 (m, 3H), 2.92-2.78 (m, 2H), 2.20-2.12 (m, 1H), 2.00-1.88 (m, 1H); 13
C
NMR (100 MHz, CDCl3) δ 161.8, 145.3, 139.5, 139.3, 138.6, 135.7, 134.6, 134.0,
134.0, 131.9, 129.1 (2C), 129.0 (2C), 128.9 (4C), 128.6 (2C), 126.8 (2C), 126.6, 50.9,
40.3, 33.2, 29.6, 26.4; LC-MS Rf (min) = 6.17, LC-MS m/z (ES+) calculated for
C28H26NO6S3 [M+H] 568: found 568.
Methyl 2-[[(4-methylphenyl)sulfonylamino)carbonyl]amino]-6-phenyl-4, 5, 6, 7-
tetrahydrobenzo[b]thiophene-3-carboxylate (13)
To a stirring solution of compound 3 (30.0 mg, 0.10 mmol) and TEA (29.0 µL, 0.10
mmol) in dry toluene (5.0 mL) was added 4-methylbenzenesulfonyl cyanide (16.0 µL,
0.10 mmol). The reaction mixture was refluxed for 20 h under an atmosphere of
nitrogen. The mixture was concentrated under reduced pressure, and the residue was
recrystalized from methanol to afford a white solid 13 (38.0 mg, 75%). IR 3273,
3231, 1668, 1535 cm-1
; 1H NMR (400 MHz, CDCl3) δ 11.64 (s, 1H, NH), 8.07 (s, 1H,
NH), 7.92 (s, 1H, Ar-H), 7.90 (s, 1H, Ar-H), 7.35-7.28 (m, 5H, Ar-H), 7.25-7.19 (m,
2H, Ar-H), 3.96 (s, 3H, COOCH3), 3.09-2.85 (m, 3H), 2.85-2.69 (m, 2H), 2.42 (s, 3H,
Ar-CH3), 2.18-2.09 (m, 1H), 1.95-1.84 (m, 1H); 13
C NMR (100 MHz, CDCl3) δ 165.9,
148.0, 146.9, 145.6, 145.2, 136.1, 131.3, 130.1 (2C), 128.5 (2C), 127.4 (2C), 126.8
(2C), 126.7, 126.4, 112.2, 51.8, 40.4, 32.0, 30.0, 26.6, 21.6; LC-MS Rf (min) = 6.09,
LC-MS m/z (ES+) calculated for C24H25N2O5S2 [M+H] 485: found 485.
General procedure for hydrolysis of the methyl esters
The compound to be hydrolyzed was dissolved in a mixture of V (THF: Methanol: water)
=5:2:10 and aqueous NaOH (10.0 M, 10.0 eq), and the mixture was refluxed
overnight. Solvent was removed under reduced pressure, and the resulting sodium
carboxylate was filtered off and dissolved in water. The free acid was precipitated by
addition of HCl (3.0 M), filtered off, washed with water and then concentrated from
methanol.
2-Amino-6-phenyl-4, 5, 6, 7-tetrahydrobenzo
[b]thiophene-3-carboxylic acid (3d)
20
By following the general procedure for hydrolysis of the methyl esters, compound 3
(219.7 mg, 0.76 mmol) was converted to compound 3d (189.9 mg, 91%). IR:
3456, 3341, 1628, cm-1
; 1H NMR (400 MHz, MeOH-d4) δ 7.33-7.25 (m, 4H, Ar-
H), 7.23-7.13 (m, 1H, Ar-H), 3.13-3.04 (m, 1H), 3.02-2.93 (m, 1H), 2.83-2.60 (m,
3H), 2.07-1.99 (m, 1H), 1.94-1.82 (m, 1H); 13
C NMR (100 MHz, MeOH-d4) δ 158.7,
146.6, 133.3, 128.0 (2C), 126.5 (2C), 125.7, 115.8, 48.0, 47.8, 41.2, 32.2, 30.3, 26.9;
LC-MS Rf (min) = 5.15, LC-MS m/z (ES-) calculated for C15H14NO2S [M-H] 272:
found 272.
Methyl 2-(tert-butoxycarbonylamino)-6-phenyl-4, 5, 6, 7-
tetrahydrobenzo[b]thiophene-3-carboxylate (3f)
Compound 3 (720.0 mg, 2.50 mmol), Boc2O (1145.0 mg, 5.25 mmol) and DMAP
(30.5 mg, 0.25 mmol) were mixed in dioxane (20 mL), and the mixture was stirred in
an oil bath (80 ºC) for 3h under an atmosphere of nitrogen.Then N2H4.H2O (0.36 ml,
7.50 mmol) was added, and the mixture was stirred at 40 ºC for 1.5 h. The solvents
were removed under reduced pressure and the residue was purified by silica gel
column chromatography with 0-10% ethyl acetate-Heptane to give a white solid 3f
(786.1 mg, 81%). IR 3242, 1715, 1658 cm-1
; 1H NMR (400 MHz, CDCl3) δ 10.28
(bs, 1H, NH), 7.33-7.28 (m, 2H, Ar-H), 7.27-7.25 (m, 1H, Ar-H), 7.24-7.18 (m, 2H,
Ar-H), 3.83 (s, 3H, COOCH3), 3.05-2.84 (m, 3H), 2.81-2.69 (m, 2H), 2.14-2.07 (m,
1H), 1.95-1.85 (m, 1H), 1.51 (s, 9H, CO(CH3)3); 13
C NMR (100 MHz, CDCl3) δ
166.7, 152.2, 150.3, 145.8, 130.7, 128.5 (2C), 126.9 (2C), 126.4, 124.6, 109.6, 81.9,
51.3, 40.6, 32.1, 30.0, 28.2 (3C), 26.7; LC-MS Rf (min) = 7.12, LC-MS m/z (ES+)
calculated for C21H26NO4S [M+H] 388: found 388.
2-(tert-Butoxycarbonylamino)-6-phenyl-4, 5, 6, 7-tetrahydrobenzo
[b]thiophene-3-carboxylic acid (3g)
By following the general procedure for hydrolysis of the methyl esters, compound 3f
(673.0 mg, 1.73 mmol) was converted to compound 3g (551.2 mg, 85%) as a light
brown solid. IR 3281, 2924, 1719, 1636 cm-1
; 1H NMR (400 MHz, DMSO-d6) δ
13.05 (bs, 1H, COOH), 10.4 (s, 1H, NH), 7.33-7.28 (m, 4H, Ar-H), 7.24-7.18 (m, 1H,
Ar-H), 2.99-2.90 (m, 2H), 2.88-2.80 (m, 1H), 2.74-2.63 (m, 2H), 2.02-1.94 (m, 1H),
1.90-1.79 (m, 1H), 1.48 (s, 9H, CO(CH3)3); 13
C NMR (100 MHz, DMSO-d6) δ 167.6,
151.7, 149.0, 146.2, 131.4, 128.8 (2C), 127.3 (2C), 126.7, 124.6, 110.8, 82.2, 40.2,
31.9, 30.0, 28.2(3C), 26.8; LC-MS Rf (min) = 6.05, LC-MS m/z (ES-) calculated for
C20H22NO4S [M-H] 372: found 372.
Methyl 2-acetylamino-6-phenyl-4, 5, 6, 7-tetrahydrobenzo
[b]thiophene-3-carboxylate (3i)
Acetyl chloride (94.0 µL, 1.33 mmol) was added to a solution of compound 3 (151.2
mg, 0.52 mmol) and pyridine (2.3 mL) in DCM (9.2 mL) at 0 ºC. The yellow mixture
was stirred at RT for 45 min and the solution became clear. The solvents were
removed under reduced pressure and the residue was taken up in ethyl acetate and
washed with HCl (1.0 M).The organic phase was dried over Na2SO4 (s), filtered and
the solvent was evaporated under reduced pressure. The residue was purified by silica
gel column chromatography using ethyl acetate: heptane= 1:4 as eluent. Compound 3i
(145.5 mg, 85%) was obtained as white foam. IR 3280, 1672, 1600 cm-1
; 1H NMR
21
(400 MHz, CDCl3) δ 11.23 (s, 1H, NH), 7.35-7.28 (m, 2H, Ar-H), 7.26 -7.20 (m, 3H,
Ar-H), 3.86 (s, 3H, COOCH3), 3.05-2.87 (m, 3H), 2.82-2.70 (m, 2H), 2.25 (m, 3H,
COCH3), 2.15-2.07 (m, 1H), 1.95-1.84 (m, 1H); 13
C NMR (100 MHz, CDCl3) δ 167.0
(2C), 148.1, 145.7, 130.3, 128.5 (2C), 126.9 (2C), 126.4, 126.1, 110.8, 51.4, 40.5,
32.1, 30.1, 26.6, 23.7; LC-MS Rf (min) = 5.99, LC-MS m/z (ES+) calculated for
C18H20NO3S [M+H] 330: found 330.
2-Acetylamino-6-phenyl-4, 5, 6, 7-tetrahydrobenzo
[b]thiophene-3-carboxylic acid (3j)
By following the general procedure for hydrolysis of the methyl esters, compound 3i
(145.5 mg, 0.44 mmol) was converted to compound 3j (129.7 mg, 93%) as a light
yellow solid. IR 3281, 2931, 1670, 1638 cm-1
; 1H NMR (400 MHz, DMSO-d6) δ
13.06 (bs, 1H, COOH), 11.16 (s, 1H, NH), 7.34-7.26 (m, 4H, Ar-H), 7.23-7.17 (m,
1H, Ar-H), 3.02-2.79 (m, 3H), 2.76-2.64 (m, 2H), 2.20 (s, 3H, COCH3), 2.03-1.93 (m,
1H), 1.92-1.78 (m, 1H); 13
C NMR (100 MHz, DMSO-d6) δ 167.4, 167.3, 146.9,
146.3, 130.9, 128.8 (2C), 127.3 (2C), 126.6, 125.5, 111.8, 40.3, 31.9, 30.1, 26.7, 23.8;
LC-MS Rf (min) = 5.31, LC-MS m/z (ES-) calculated for C17H16NO3S [M-H] 314:
found 314.
2-Acetylamino-6-phenyl-4, 5, 6, 7-tetrahydrobenzo
[b]thiophene (3k)
Compound 3j (102.1 mg, 0.32 mmol) and copper powder (30.7 mg, 0.48 mmol) were
mixed in quinoline (13.4 mL), and the mixture was heated in a Biotage Smith Creator
microwave at 200 ºC for 10 min. The mixture was diluted with H2O (10 mL),
acidified to pH=1 with HCl (5.0 M) and extracted with ethyl acetate (3x15 mL). The
organic phase was dried over Na2SO4 (s), filtered and evaporated under reduced
pressure. The residue was purified by silica gel column chromatography using ethyl
acetate: heptane= 1:4→ 3:7 as eluent. The solvents were evaporated under reduced
pressure and compound 3k (47.4 mg, 54%) was obtained as a white yellow solid. IR
3267, 1635 cm-1
; 1H NMR (400 MHz, CDCl3) δ 7.74 (s, 1H, Ar-H), 7.27-7.22 (m,
2H, Ar-H), 7.21-7.19 (m, 1H, Ar-H), 7.18 -7.12 (m, 1H, Ar-H), 6.30 (s, 1H, NH),
2.99-2.84 (m, 2H), 2.76-2.67 (m, 1H), 2.65-2.54 (m, 2H), 2.10 (s, 3H, COCH3), 2.04-
1.98 (m, 1H), 1.94-1.81 (m, 1H); 13
C NMR (100 MHz, CDCl3) δ 166.7, 146.0, 135.7,
131.6, 128.5 (2C), 127.9, 126.9 (2C), 126.3, 113.1, 41.2, 32.2, 30.2, 25.6, 23.3; LC-
MS Rf (min) = 5.20, LC-MS m/z (ES+) calculated for C16H18NOS [M+H] 272: found
272.
2-Chloro-7-phenyl-5, 6, 7, 8-tetrahydro[1]benzothieno[2, 3-b]pyridine (3m)
Phosphorus oxychloride (6.1 µL, 0.6 mmol) was added dropwise to a stirring solution
of DMF (5.1 µL, 0.6 mmol) in 1, 2-dichloroethane (0.5 mL) at 0 ºC, and a solution of
compound 3k (18.1 mg, 0.06 mmol) in dry 1, 2-dichloroethane (1.0 mL) was added
dropwise to the mixture. And the mixture was stirred at RT for 15 min then refluxed
at 85 ºC for 3.5 h. After cooling to RT, a solution of sodium acetate (209.5 mg, 2.55
mmol, in 2.0 mL H2O) was added to the mixture, and the solution was heated at 50 ºC
for 1.5 h. After cooling, the solution was diluted with DCM (10 mL) and extracted
with DCM (3x 10 mL).The organic phase was separated, washed with saturated
sodium bicarbonate solution, dried over Na2SO4 (s), filtered and evaporated under
22
reduced pressure. The residue was purified by silica gel column chromatography
using DCM: heptane= 1:1 as eluent. The solvents were evaporated under reduced
pressure and compound 3m (3.8 mg, 19%) was obtained as a white yellow solid. IR
1582, 1522, 1492, 1426, 1350, 1139, 1121 cm-1
; 1H NMR (400 MHz, CDCl3) δ
7.76 (d, J = 4.12 Hz, 1H, Ar-H), 7.37-7.32 (m, 2H, Ar-H), 7.31-7.23 (m, 4H, Ar-H),
3.22-3.11 (m, 2H), 3.05-2.95 (m, 1H), 2.94-2.75 (m, 2H), 2.30-2.22 (m, 1H), 2.13-
2.01 (m, 1H); 13
C NMR (100 MHz, CDCl3) δ 160.4, 146.8, 145.1, 137.6, 131.7,
130.0, 128.6 (2C), 126.8 (2C), 126.7, 126.6, 119.7, 40.8, 33.3, 29.4, 23.5; LC-MS Rf
(min) = 6.45, LC-MS m/z (ES+) calculated for C17H15ClNS [M+H] 300: found 300.
The general procedure for synthesis of compounds 22a-22d
Compound 3g (80.0 mg, 0.21 mmol), TBTU (69.6 mg, 0.21 mmol) and TEA (70.0
µL, 0.42 mmol) were mixed in ethyl acetate (8.0 mL) .After the mixture was stirred at
RT for 10 min, two equimolar amount of the appropriate amine was added dropwise.
The mixture was stirred for 3 h, 3.5 h, 2 h, and 4.5 h respectively (in the order from
22a to 22d). After dilution with 15 mL ethyl acetate and adding 20 mL H2O, the
organic layer was separated, extracted with ethyl acetate (3x30 mL), dried over
Na2SO4 (s) and evaporated.
N-isopropyl 2-(tert-butoxycarbonylamino)-6-phenyl-4, 5, 6, 7-tetrahydrobenzo
[b]thiophene-3-carboxamide (22 a)
The product was purified by silica gel column chromatography using ethyl acetate:
heptane= 1:10 as eluent. The solvents were evaporated under reduced pressure and
compound 22a (48.2 mg, 54%) was obtained as white solid. IR 3263, 3155, 1702,
1615, 1522 cm-1
; 1H NMR (400 MHz, CDCl3) δ 11.02 (s, 1H, NH), 7.37-7.29 (m, 2H,
Ar-H), 7.28-7.26 (m, 1H, Ar-H), 7.25-7.21 (m, 2H, Ar-H), 5.67 (d, J = 3.6 Hz, 1H, -
NHCH(CH3)2), 4.28-4.17 (m, 1H, -NHCH(CH3)2), 3.07-2.89 (m, 2 H), 2.86-2.71 (m,
3H), 2.24 -2.15 (m, 1H), 2.04-1.92 (m, 1H), 1.50 (s, 9H, CO(CH3)3), 1.24 (d, J= 8 Hz,
3H, -NHCH(CH3)2), 1.25 (d, J= 8 Hz, 3H, -NHCH(CH3)2); 13
C NMR (100 MHz,
CDCl3) δ 165.4, 152.5, 147.4, 145.3, 128.6 (2C), 126.8 (3C), 126.5, 125.6, 112.5,
81.3, 41.4, 40.2, 32.1, 30.1, 28.2 (3C), 26.9, 23.0, 22.9; LC-MS Rf (min) = 6.60, LC-
MS m/z (ES-) calculated for C23H29N2O3S [M-H] 413: found 413.
N-phenyl 2-(tert-butoxycarbonylamino)-6-phenyl-4, 5, 6, 7-tetrahydrobenzo
[b]thiophene-3-carboxamide (22b)
The product was purified by silica gel column chromatography using ethyl acetate:
heptane= 6:94 as eluent. The solvents were evaporated under reduced pressure and
compound 22b (41.6 mg, 43%) was obtained as a white solid. IR 3382, 2984,
1709, 1636, 1518 cm-1
; 1H NMR (400 MHz, CDCl3) δ 10.82 (s, 1H, NH), 7.58-7.47
(m, 3H, Ar-H), 7.41-7.30 (m, 4H, Ar-H), 7.30-7.25 (m, 2 H, Ar-H), 7.19-7.11 (m, 1H,
Ar-H), 3.13-3.03 (m, 1H), 3.03-2.88 (m, 3H), 2.87-2.78 (m, 1H), 2.44-2.17 (m, 1H),
2.16-1.89 (m, 1H), 1.50 (s, 9H, CO(CH3)3); 13
C NMR (100 MHz, CDCl3) δ 164.4,
152.5, 148.7, 145.1, 137.3, 129.1 (2C), 128.6 (2C), 126.9 (2C), 126.6, 126.5, 126.1,
124.7, 120.8 (2C), 112.6, 81.7, 40.2, 32.1, 30.1, 28.2 (3C), 27.0; LC-MS Rf (min) =
6.59, LC-MS m/z (ES-) calculated for C26H27N2O3S [M-H] 447: found 447.
23
[2-(tert-butoxycarbonylamino)-6-phenyl-4, 5, 6, 7-tetrahydrobenzo
[b]thien-3-yl]-4-morpholinylmethanone (22c)
The product was purified by silica gel column chromatography using ethyl acetate:
heptane = 1:4 → 1:1 as eluent. The solvents were evaporated under reduced pressure
and compound 22c (44.4 mg, 47%) was obtained as a white solid. IR 3282, 3198,
1714, 1605, 1534 cm-1
; 1H NMR (400 MHz, CDCl3) δ 8.05 (bs, 1H, NH), 7.34-7.29
(m, 2H, Ar-H), 7.28-7.26 (m, 1H, Ar-H), 7.24-7.19 (m, 2H, Ar-H), 3.75-3.41 (m, 8H,
-NHCH2CH2O-), 3.09-2.91 (m, 2H), 2.84-2.74 (m, 1H), 2.63-2.49 (m, 2H), 2.13-2.05
(m, 1H), 1.96-1.85 (m, 1H), 1.51 (s, 9H, CO(CH3)3); 13
C NMR (100 MHz, CDCl3) δ
166.7, 152.4, 145.5, 139.8, 129.0, 128.5 (2C), 127.4, 126.8 (2C), 126.4, 117.1, 81.7,
67.1 , 67.0, 45.9, 44.9, 40.8, 32.0, 29.8, 28.2 (3C), 24.9; LC-MS Rf (min) = 5.57, LC-
MS m/z (ES+) calculated for C24H31N2O4S [M+H] 443: found 443.
N-benzyl 2-(tert-butoxycarbonylamino)-6-phenyl-4, 5, 6, 7-tetrahydrobenzo
[b]thiophene-3-carboxamide (22d)
The product was purified by silica gel column chromatography using ethyl acetate:
heptane= 1:10 as eluent. The solvents were evaporated under reduced pressure and
compound 22d (64.4 mg, 65%) was obtained as colourless oil. IR 3029, 2972,
1708, 1618, 1561 cm-1
; 1H NMR (400 MHz, CDCl3) δ 11.06 (s, 1H, NH), 7.39-7.26
(m, 7H, Ar-H), 7.25-7.24 (m, 2H, Ar-H), 7.24-7.20 (m, 1H, Ar-H), 6.21-6.13 (m, 1H,
NH), 4.61 (d, J = 2.78 Hz, 2H, Ar-CH2NH-), 3.05-2.90 (m, 2H), 2.86-2.69 (m, 3H),
2.20-2.12 (m, 1H), 2.03-1.91 (m, 1H), 1.51 (s, 9H, CO(CH3)3); 13
C NMR (100 MHz,
CDCl3) δ 166.1, 152.5, 148.1, 145.2, 138.1, 128.8 (2C), 128.6 (2C), 127.6, 127.5
(2C), 126.8 (2C), 126.5, 125.7, 112.0, 81.5, 43.5, 40.1, 32.0, 30.0, 28.2 (4C), 26.9;
LC-MS Rf (min) = 6.65, LC-MS m/z (ES+) calculated for C27H31N2O3S [M+H] 463:
found 463.
2-Amino-N-(isopropyl)-6-phenyl-4, 5, 6, 7-tetrahydrobenzo
[b]thiophene-3-carboxamide (23a)
Compound 22a (28.0mg, 0.067mmol) was dissolved in dry DCM (1.0 mL), and TFA
(1.0 mL) was added dropwise. The mixture was stirred at RT for 20 min, diluted with
DCM (20 mL), quenched with saturated NaHCO3 solution, washed with brine (15
mL) and extracted with DCM (3x10 mL). The solvent was evaporated under reduced
pressure. The product was purified by silica gel column chromatography using ethyl
DCM: MeOH= 95:5 (0.5% TEA) as eluent. The solvents were evaporated, compound
23a (14.4 mg, 68%) was obtained as yellow oil. IR 3243, 3205, 2924, 1560 cm-1
; 1H NMR (400 MHz, CDCl3) δ 7.37-7.29 (m, 2H, Ar-H), 7.28-7.26 (m, 1H, Ar-H),
7.25-7.18 (m, 2H, Ar-H), 5.50 (d, J = 3.8 Hz, 1H, -NHCH (CH3)2), 4.27-4.16 (m, 1H,
-NHCH(CH3)2), 3.06-2.97 (m, 1H), 2.86 -2.66 (m, 4H), 2.20-2.11 (m, 1H), 2.01-1.89
(m, 1H), 1.21 (d, J= 8 Hz, 3H, -NHCH (CH3)2), 1.23 (d, J= 8 Hz, 3H, -NHCH
(CH3)2); 13
C NMR (100 MHz, CDCl3) δ 165.7, 158.7, 145.5, 128.9, 128.6 (2C),
128.5, 126.8 (2C), 126.4, 118.4, 108.9, 41.0, 40.4, 32.3, 30.1, 27.3, 23.1; LC-MS Rf
(min) = 5.39, LC-MS m/z (ES+) calculated for C18H23N2OS [M+H] 315: found 315.
The general procedure for synthesis of compounds 15-18
24
An equimolar amount of succinic anhydride was added to a stirring mixture of
compounds 23a, 23b, 23c, or 23d in dry CH2Cl2 (3.0 mL).The mixture was refluxed
under an atmosphere of nitrogen for 7.5 h, 5 h, 3 h and 1.5 h respectively. After
dilution with 20 mL DCM, the mixture was washed with brine (20 mL) and extracted
with DCM (3x20 mL). The organic layer was separated, dried over Na2SO4 (s) and
filtered. The solvent was removed under reduced pressure, and purified by silica gel
column chromatography.
N-Isopropyl 2-[(3-carboxypropanoyl)amino]-6-phenyl-4, 5, 6, 7-tetrahydrobenzo
[b]thiophene-3-carboxamide (15)
The crude was purified by silica gel column chromatography using ethyl DCM:
MeOH= 97.5:2.5 (0.5% CH3COOH) as eluent two times. Compound 23a (14.4 mg,
0.05 mmol) was converted to compound 15 (5.0 mg, 27%) as yellow oil. IR 3297,
3257, 1709, 1681, 1600 cm-1
; 1H NMR (400 MHz, CDCl3) δ 12.01 (s, 1H, NH), 7.30-
7.23 (m, 2H, Ar-H), 7.22-7.19 (m, 1H, Ar-H), 7.18-7.13 (m, 2H, Ar-H), 5.72 (d, J =
3.8 Hz, 1H, -NHCH (CH3)2), 4.20-4.09 (m, 1H, -NHCH(CH3)2), 3.01-2.85 (m, 2H),
2.80-2.65 (m, 7H), 2.18-2.09 (m, 1H), 1.98-1.86 (m, 1H), 1.20 (d, J= 8 Hz, 3H, -
NHCH (CH3)2), 1.18 (d, J= 8 Hz, 3H, -NHCH (CH3)2); 13
C NMR (100 MHz, CDCl3)
δ 176.2, 168.6, 165.4, 145.1, 128.9, 128.6 (2C), 127.4, 126.8 (2C), 126.6, 126.5,
113.9, 41.7, 40.1, 32.0, 31.0, 30.1, 28.9, 26.7, 22.9 (2C); LC-MS Rf (min) = 5.15, LC-
MS m/z (ES-) calculated for C22H25N2O4S [M-H] 413: found 413.
N-Phenyl 2-[(3-carboxypropanoyl)amino]-6-phenyl-4, 5, 6, 7-tetrahydrobenzo
[b]thiophene-3-carboxamide (16)
Compound 22b (41.6 mg, 0.05 mmol) was dissolved in dry DCM (1.0 mL) at RT, and
TFA (1.0 mL) was added dropwise. The mixture was stirred at RT for 20 min, and
diluted with DCM (20 mL), and the solvent was removed under reduced pressure.
Following the general procedure for synthesis of compounds 15-18, and the crude was
purified by silica gel column chromatography using ethyl DCM: MeOH= 99.8 :0.2
(0.1% CH3COOH) → 99 :1 (0.1% CH3COOH) as eluent two times. The solvents were
evaporated under reduced pressure and compound 16 (17.6 mg, 75%) was obtained as
a yellow solid. IR 1719, 1666, 1619 cm-1
; 1H NMR (400 MHz, DMSO-d6) δ 12.14
(bs, 1H, NH), 10.76 (bs, 1H, NH), 9.94 (s, 1H, COOH), 7.72 (s, 1H, Ar-H), 7.70 (s,
1H, Ar-H), 7.37-7.29 (m, 6H, Ar-H), 7.26-7.19 (m, 1H, Ar-H), 7.10-7.04 (t, J = 7.38
Hz, 1H, Ar-H), 3.02-2.70 (m, 5H), 2.68-2.60 (m, 2H), 2.57-2.50 (m, 2H), 2.05-1.97
(m, 1H), 1.93-1.81 (m, 1H); 13
C NMR (100 MHz, DMSO-d6) δ 174.1, 169.6, 163.5,
146.2, 139.6, 129.8, 129.0 (2C), 128.8 (2C), 127.3 (2C), 126.7, 126.3, 123.8, 120.4
(2C), 120.0, 32.1 (2C), 30.7, 30.1, 29.3 (2C), 25.4; LC-MS Rf (min) = 5.42, LC-MS
m/z (ES-) calculated for C25H23N2O4S [M-H] 447: found 447.
[2-[(3-carboxypropanoyl)amino]-6-phenyl-4, 5, 6, 7-tetrahydrobenzo
[b]thien-3-yl]-4-morpholinylmethanone (17)
Compound 22c (44.4 mg, 0.1 mmol) was dissolved in dry DCM (1.0 mL) at RT, and
TFA (1.0 mL) was added dropwise. The mixture was stirred at RT for 10 min, and
diluted with DCM (20 mL), and the solvent was removed under reduced pressure.
Following the general procedure for synthesis of compounds 15-18, and the crude was
purified by silica gel column chromatography using ethyl DCM: MeOH= 98 :2 (0.1%
25
CH3COOH) → 97 :3 (0.1% CH3COOH) as eluent two times. The solvents were
evaporated under reduced pressure and compound 17 (13.5 mg, 30%) was obtained as
colorless oil. IR 3242, 3173, 2921, 1678, 1578, 1542 cm-1
; 1H NMR (400 MHz,
CDCl3) δ 9.51 (s, 1H, COOH), 7.35-7.26 (m, 2H, Ar-H), 7.24-7.18 (m, 3H, Ar-H),
3.74 -3.42 (m, 8H, -NHCH2CH2O-), 3.06-2.91 (m, 2H), 2.82-2.74 (m, 4H), 2.68-2.58
(m, 1H), 2.57-2.46 (m, 2H), 2.15-2.05 (m, 1H), 1.99-1.85 (m, 1H); 13
C NMR (100
MHz, CDCl3) δ 176.8, 168.9, 167.4, 145.4, 136.6, 128.9, 128.5 (2C), 128.2, 126.8
(2C), 126.5, 117.1, 66.9 (2C) , 51.9, 45.0, 40.8, 31.9, 30.7, 29.7, 28.7, 24.8; LC-MS
Rf (min) = 4.64, LC-MS m/z (ES-) calculated for C23H25N2O5S [M-H] 441: found
441.
N-Benzyl 2-[(3-carboxypropanoyl)amino]-6-phenyl-4, 5, 6, 7-tetrahydrobenzo
[b]thiophene-3-carboxamide (18)
Compound 18 (64.4 mg, 0.09 mmol) was dissolved in dry DCM (1.0 mL) at RT, and
TFA (1.0 mL) was added dropwise. The mixture was stirred at RT for 10min, and
diluted with DCM (20 mL), and the solvent was removed under reduced pressure.
Following the general procedure for synthesis of compounds 15-18, the crude was
purified by silica gel column chromatography using ethyl DCM: MeOH= 99 :1 (0.1%
CH3COOH) → 97 :3 (0.1% CH3COOH) as eluent two times. The solvents were
evaporated under reduced pressure and compound 18 (24.7 mg, 55%) was obtained as
a white solid. IR 1725, 1681, 1593 cm-1
; 1H NMR (400 MHz, DMSO-d6) δ 12.21
(bs, 1H, NH), 11.24 (s, 1H, COOH), 8.09 (t, J = 5.88 Hz, 1H, Ar-CH2NH-), 7.37-7.28
(m, 8H, Ar-H), 7.28-7.17 (m, 2H, Ar-H), 4.56-4.39 (m, 2H, Ar-CH2NH-), 3.01-2.70
(m, 5H), 2.66-2.60 (m, 2H), 2.55-2.51 (m, 2H), 2.05-1.97 (m, 1H), 1.95-1.81 (m, 1H); 13
C NMR (100 MHz, DMSO-d6) δ 174.0, 169.3, 165.4, 146.1, 141.2, 139.9, 129.1,
128.8 (2C), 128.7 (2C), 127.6 (2C), 127.3 (2C), 127.1, 126.7, 126.3, 117.4, 43.0, 32.0,
31.0, 30.1, 29.2 (2C), 25.8; LC-MS Rf (min) = 5.34, LC-MS m/z (ES-) calculated for
C26H25N2O4S [M-H] 461: found 461.
26
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28
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