CHAPTER-2

Synthesis, Characterization and Biological Studies of Novel Schiff

base ligands of 3-amino-2-methyl-4(3H)-Quinazolinone

INTRODUCTION

Schiff bases, named for Hugo Schiff [1], are formed when any primary amine reacts with

an aldehyde or a ketone under specific conditions. Structurally, a Schiff base (also known as

imine or azomethine) is a nitrogen analogue of an aldehyde or ketone in which the carbonyl

group (C=O) has been replaced by an imine or azomethine group. Schiff bases are important

class of compounds in medicinal and pharmaceutical field. They show biological applications

including antibacterial [2-4], antifungal [5, 6] and antitumor activity [7, 8]. The imine group

present in such compounds has been shown to be critical to their biological activities[9, 10].

Schiff bases play an important role in inorganic chemistry as they easily form stable

complexes with most transition metal ions. Schiff base ligands are considered ‘‘privileged

ligands” because of their preparation. Schiff base ligands are able to coordinate many different

metals [11-14] and to stabilize them in various oxidation states.

Schiff bases have been used extensively as ligands in the field of coordination chemistry,

some of the reasons are that the intramolecular hydrogen bonds between the (O) and the (N)

atoms which play an important role in the formation of metal complexes and that Schiff base

compounds show photochromism and thermochromism in the solid state by proton transfer from

the hydroxyl (O) to the imine (N) atoms [15]. A large number of Schiff bases and their

complexes have been investigated for their interesting and important properties, such as their

ability to reversibly bind oxygen catalytic activity in the hydrogenation of olefins, photochromic

properties and complexing ability towards some toxic metals,

Schiff bases are considered as a very important class of organic compounds which have

wide applications in many biological aspects. Due to their multiple implications, the transition

metal complexes with Schiff bases, as ligands, are of paramount scientific interest [16]. Schiff

bases with donors (N, O, S, etc.) have structural similarities with natural biological systems and

due to the presence of imine group, are utilized in elucidating the mechanism of transformation

and rasemination reaction in biological systems [17-19]. Schiff base complexes have been used

as drugs. Moreover, it is well known that some drug activities, when administered as metal

complexes, are being increased [20], and several Schiff base complexes have also been shown to

inhibit tumor growth [21]. The effect of the presence of various substituents in the phenyl rings

of aromatic Schiff bases on their antimicrobial activity has been reported [22].

A vast number of quinazolines have been synthesized for biological screening, and a

variety of activities were observed. Several derivatives are being used clinically. A few of the

many references for each activity are provided, such as antidepressants [23], hypotensive activity

[24], analgesic [25], antithrombic [26], anticoagulant [27], antifibrillatory [28], arteriosclerosis

[29] and anti-inflammatory activity [30].

LITERATURE REVIEW

Over the past decade, the synthesis of privileged classes of heterocyclic molecules has

become one of the prime areas of research in synthetic organic chemistry [31]. These privileged

structures have gained much attention, owing to their potential role as ligands, which are capable

of binding multiple biological targets [32]. Among the nitrogen-containing privileged class of

molecules, substituted quinazolinones and quinazolines are considered as important therapeutic

scaffolds [33-34].

Heterocyclic moieties can be found in a large number of compounds which display

biological activity. The biological activity of the compounds is mainly dependent on their

molecular structures (Elzahany et al. 2008) [35].

Schiff bases are important class of compounds due to their flexibility, structural

similarities with natural biological substances and also due to presence of imine (-N=CH-) which

imports in elucidating the mechanism of transformation and rasemination reaction in biological

system (Rajavel et al. 2008) [36]. These novel compounds could also act as valuable ligands

whose biological activity has been shown to increase on complexation (Mohamed et al. 2006)

[37].

Quinolinones, an important class of heterocyclic compounds are part of the quinoline

alkaloid family and are also known for their diverse biological activity [38]. Quinolinone

heterocycles have emerged as potential therapeutic agents and are the basis of many medicinal

drugs used in the treatment of heart failure, cancer and inflammatory diseases [39]. They are also

used as antibacterial, antifungal, anti-inflammatory, antitubercular, anticancer, antineoplastic,

anti-ischemic, antiallergic, antihypertensive and anti-ulcerative agents [40-44]. Recently, a novel

series of quinolinones have shown potent inhibitory activity against human immunodeficiency

virus type-1 (HIV-1) virus and also exhibited promising activity against several non-nucleoside

reverse transcriptase inhibitors (NNRTIs) [45].

It has been more than a century since the initial studies on 4(3H)-quinazolinones [46],

and they are well known as biologically active compounds [47]. 4(3H)-Quinazolinone has been

identified as an important class of heterocyclic compounds in medicinal chemistry, having

anticonvulsant [48], antihypertensive [49], antidiabetic [50], and anti-tumor activity [51].

Antimicrobial and antihistaminic activities have also been documented [52]. A number of

syntheses of these types of compounds have previously been reported.

Recently, Mayer and co-workers [53] have reported a solid-phase synthesis approach to

2-alkyl substituted analogs. Villalgordo and co-workers [54] have described a solid-phase

synthesis based on an aza Wittig-mediated annulations strategy; however, a mixture of two

isomers was formed. More recently, a paper by Gopalsamy and Yang [55] have presented a

related solid-phase synthesis of 2-amino-4(3H)-quinazolinones. Robert Lieby [56] has given the

synthesis of 3-amino-4(3H)-quinazolinones from N-(2-carbomethophenyl) imidate esters. The

analytical and physical data confirmed the structure of synthesized 4(3H)-quinazolinones. David

Connolly and co-workers [57] have been reviewed the synthesis of quinazolinones and

quinazolines by using various different synthetic methods.

The literature survey reveals that no preparative methods have been reported on 3-amino-

2-methyl-4(3H)-quinazolinone derivatives and also their characterization. Therefore, in the

present investigation, the author has made an effort to synthesize Schiff’s base ligands of the

cited derivatives and also characterized by employing elemental analysis, IR, 1H-NMR and mass

spectral studies.

EXPERIMENTAL

Materials and methods

All the chemicals and solvents were of AnalaR grade. 3-amino-2-methyl-4(3H)-

quinazolinone was procured from Sigma-Aldrich, Bangalore and used as received. The

spectroscopic grade solvents were used as supplied by commercial sources without any further

purification. Thin layer chromatography was performed using Silica Gel G (Merck Index) coated

on glass plates and the spots were visualized by exposure to iodine. All melting points (m.p.)

were determined with a Büchi 530 melting point apparatus in open capillaries and are

uncorrected. Elemental analysis was performed on a Carlo-Erba 1160 elemental analyzer.

Infrared spectra were recorded in the range 4000-200 cm-1 on a JASCO FTIR-8400

spectrophotometer using Nujol mulls between polyethylene sheets. 1HNMR spectra were

obtained on a Varian AC 400 spectrometer. EI-MS were determined on Varian 1200L model

mass spectrometer.

PROCEDURES

Synthesis of Schiff base ligands

3-(2-hydroxybenzylideneamino)-2-methylquinazolin-4(3H)-one (L1)

A volume of 25 mL methanolic solution of 3-amino-2-methyl-4(3H)-quinazolione (1.75

g, 10 mmol) was slowly added to a 15 mL of 2-hydroxybenzaldehyde (1.31 g, 10 mmol in

methanol). The reaction mixture was stirred for 30 min and then refluxed for 4h. The completion

of reaction was monitored by TLC. The solvent was removed by distillation. The solid product

obtained was recrystallized from ethanol to yield L1.

Yield : 71%; mp: 167 °C; FT-IR (nujol, ν/cm-1): 3067, 2974, 2367 (C-H), 1695 (C=O),

1655 (C=N), 3434 (O-H); 1H-NMR (400 MHz, DMSO-d6) δ: 2.50 (s, CH3, 3H, C8), 8.71 (s, CH,

1H, -N=CH-), 10.03 (s, OH, 1H), 7.83-7.34 (m, Ar-H, 8H, Aromatic protons); Mass(m/z): 280

[M++1, 79%]; Elemental analysis(%) for C16H13N3O2 : Expt.(calcd), C 68.43 (68.5), H 4.58

(4.61), N 14.9 (15.02).

2-methyl-3-(pyridine-2-ylmethyleneamino)quinazolin-4(3H)-one (L2)

In a round bottom (RB) flask, a mixture of 3-amino-2-methyl-4(3H)-quinazolione (1.75

g, 10 mmol in 25 mL methanol) and 2-pyridinecarboxaldehyde (1.08 g, 10 mmol in 15 mL

methanol) was heated under reflux for 4h with initial stirring of 30 min. The product obtained

was concentrated under vacuum, filtered off and recrystallized from ethanol to give L2.

Yield : 63%; mp: 175 °C; FT-IR (nujol, ν/cm-1): 3088, 2924, 2161 (C-H), 1682 (C=O),

1612 (C=N); 1H-NMR (400 MHz, DMSO-d6) δ: 2.72 (s, CH3, 3H, C8), 9.22 (s, CH, 1H, -

N=CH-), 8.76 (d, CH, 1H, N-CH=CH), 7.87-7.43 (m, Ar-H, 8H, Aromatic protons); Mass(m/z):

265 [M+, 83%]; Elemental analysis(%) for C15H12N4O2: Expt.(calcd), C 68.18 (68.27), H 4.28

(4.31), N 21.22 (21.32).

3-(2-hydroxy-3-methoxybenylideneamino)-2-methylquinolin-4(3H)-one (L3)

An equimolar quantities of methanolic solution of both 3-amino-2-methyl-4(3H)-

quinazolione (1.75 g, 25 mL of 10 mmol) and 4-hydroxy-3-methoxybenzaldehyde (1.59 g,15 mL

of 10 mmol ) was stirred for 30 min and refluxed for 4h. The solvent was removed under vacuum

and the solid product obtained was filtered, dried and crystallized from ethanol.

Yield : 61%; mp: 217 °C; FT-IR (nujol, ν/cm-1): 3063, 2987, 2912 (C-H), 1666 (C=O),

1589 (C=N), 3414 (O-H); 1H-NMR(400 MHz, DMSO-d6) δ: 2.76 (s, CH3, 3H, C8), 9.11 (s, CH,

1H, -N=CH-), 3.35 (s, CH3, 3H, meta to methoxy), 10.59 (s, OH, 1H), 7.78-7.04 (m, Ar-H, 7H,

Aromatic protons); Mass(m/z): 310 [M+, 61%]; Elemental analysis(%) for C17H15N3O3 :

Expt.(calcd.), C 66.01 (66.12), H 4.93 (5.03), N 13.58 (13.79).

3-((5-ethylthiophene-2-yl)methyleneamino)-2-methylquinazolin-4(3H)-one (L4)

A mixture of 3-amino-2-methyl-4(3H)-quinazolione (1.75 g, 10 mmol in 25 mL

methanol) and 5-ethyl-2-thiophenecarboxaldehyde (1.43 g, 10 mmol in 15 methanol) was

gradually stirred for 30 min and refluxed for 4h. Evaporation of solvent under reduced pressure

gave the solid product, which was filtered and crystallized from ethanol.

Yield : 79%; mp: 183 °C; FT-IR (nujol, ν/cm-1): 2358, 2144 (C-H), 1655 (C=O), 1595 (C=N); 1H-NMR (400 MHz, DMSO-d6) δ: 2.77 (s, CH3, 3H, C8), 8.25 (s, CH, 1H, -N=CH-), 7.76-7.26

(m, Ar-H, 7H, Aromatic protons); Mass(m/z): 310[M+, 61%]; Elemental analysis(%) for

C16H15N3OS: Expt.(calcd.), C 64.62 (65.13), H 5.08 (5.14), N 14.13 (15.61).

BIOLOGY

Antimicrobial activity

Even though pharmacological industries have produced a number of new antibiotics in

the last three decades, resistance to these drugs by microorganisms has increased. In general,

bacteria have the genetic ability to transmit and acquire resistance to drugs, which are utilized as

therapeutic agents [58]. From 1980 to 1990, Montelli and Levy [59] have documented a high

incidence of resistant microorganisms in clinical microbiology in Brazil. This fact has also been

verified in other clinics around all over world. To contribute in the field of bioinorganic

chemistry, consequently, the compounds synthesized have been evaluated for their antibacterial

and antifungal actions.

Schiff bases are reported to show a variety of biological activities like antibacterial

(Daisley and Shah, 1984) [60] and antifungal (Piscopo et al. 1987) [61]. Quinazolinones are

reported to have antibacterial (Guersoy and Illhan, 1995) [62], antifungal (Mishra and Gupta,

1982) [63] activities.

The antimicrobial activity of a drug or a test compound is evaluated by a wide range of

techniques. In any technique, the principle is the preparation of a concentration gradient of the

drug/ test compound in a nutrient medium and the observation of growth of the organism taking

place when the medium is seeded with test organism and incubated. There are two important

methods employed for antimicrobial sensitivity testing of a compound, namely i) Disc diffusion

technique and ii) Minimum inhibitory concentration technique. Both these techniques give

reproducible results of reasonable degree of accuracy and also they involve very simple

operation. These two techniques have been followed in the present investigation.

The in vitro antimicrobial screening effects of the synthesized ligands (L1-L4) were

evaluated against four bacteria namely Bacillus Subtilis, Escherichia coli, Staphylococcus aureus

and Ralstonia solanacearum and three fungi namely Aspergillus niger, Aspergillus flavus and

Alternaria solani by disc diffusion method using nutrient agar medium for antibacterial studies

and potato dextrose agar medium for antifungal studies [64-66].

Determination of MIC

Minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial

compound that will inhibit the visible growth of microorganisms after overnight incubation. This

evaluation was done according to Muroi and Kubo, 1996 [67]. Aliquots of 100 μL of resistant

bacteria cultures (106 cells/ mL) grown in 10 mL of nutrient broth for 6 h were inoculated in

nutrient broth supplemented with the respective antibiotics (50 μg/mL) with different

concentrations of test samples. The concentration for test samples ranged from 100-500 μg/mL.

Chloramphenicol (standard antibacterial drug) was used at the sub-inhibitory concentration

(50 μg/mL). After 48 h, the optical density of each sample was documented and compared to

those of MIC to verify any synergistic effect among the tested compounds.

Disc diffusion method

The bacteria and fungi were sub-cultured in the agar and potato dextrose agar medium

and were incubated for 24 h for bacteria and 48 h for fungi at 37 °C. Standard antibacterial drug

(Chloramphenicol) and antifungal drug (Fluconazole) were used for comparison. The discs

having a diameter of 4 mm were soaked in the test solutions and were placed on an appropriate

medium previously seeded with organisms in petri plates and stored in an incubator at the above

mentioned period of time. Antimicrobial activity of all the synthesized ligands was evaluated by

measuring the zone of growth inhibition against the test organisms with zone reader (Hi

antibiotic zone scale). A solvent dimethyl formamide (DMF) was used as a negative control.

Chloramphenicol (standard antibiotic) and Fluconazole (standard antifungal drug) were used as

positive control. The stock solution (1 mg/ mL) of the test compounds was prepared in DMF.

Each test was performed in triplicate in individual experiments and the average is reported.

Radical scavenging activity

Nitrogen heterocyclic compounds have been used widely in the pharmaceutical industry

because of their perfect biological activities [68-70]. Quinazolinones are excellent reservoir of

bioactive substances. A number of biological activities are associated with quinazolinones

especially antioxidant activity [71-73]. Reactive oxygen species (ROS) such as superoxide

anions, hydrogen peroxide, hydroxyl and nitric oxide radicals, play an important role in oxidative

stress related to the pathogenesis of various important diseases [74]. Antioxidants act as a major

defense against radical mediated toxicity by protecting the damages caused by free radicals.

Antioxidant agents are effective in the prevention and treatment of complex diseases, like

atherosclerosis, stroke, diabetes, Alzheimer’s disease and cancer [75]. Flavonoids and phenolic

compounds are widely distributed in plants which have been reported to exert multiple biological

effects including antioxidant, free radical scavenging abilities, anti-inflammatory and

anticarcinogenic [76]. This has attracted a great deal of research interest in natural antioxidants.

A number of synthetic compounds such as quinazolines [77], triazoles [78] and pyrazole [79]

have also been extremely exploited for antioxidant activity.

Radical scavenging activity of the Schiff bases (L1-L4) were investigated

spectrophotometrically using 1,1-diphenyl-2-picrylhydrazyl (DPPH) [80]. The DPPH radical has

been widely used to evaluate the free radical scavenging capacity of different antioxidants [81,

82]. With this method, it is possible to determine the antiradical power of an antioxidant activity

by measurement of the decrease in the absorbance of DPPH at 518 nm. Resulting from a color

change from purple to yellow the absorbance decreased when the DPPH is scavenged by an

antioxidant, through donation of hydrogen to form a stable DPPH molecule. In the radical form

this molecule had an absorbance at 518 nm, which disappeared after acceptance of an electron or

hydrogen radical from an antioxidant compound to become a stable diamagnetic spin paired

molecule [83]. The odd electron in the DPPH free radical gives a strong absorption maximum at

518 nm and is purple in color. The color turns from purple to yellow as the molar absorptivity

(optical density) of the DPPH radical at 518 nm reduces when the odd electron of DPPH radical

becomes paired with hydrogen radical from a free radical scavenging antioxidant to form the

reduced DPPH-H. The resulting decolorization is stoichiometric with respect to the number of

electrons captured. Various concentrations of the experimental compounds were added to

solution of DPPH in methanol (125 μM, 2 mL) and in each case the final volume was made up to

4 mL with water. The solution was shaken and incubated at 37 ºC for 30 min in dark. The

decrease in absorbance of DPPH was measured at 518 nm. Percentage inhibition was calculated

by comparing the absorbance values of control and test samples.

Anthelmintic activity

Helminth infections are among the most widespread infections in humans, distressing a

huge population of the world. Parasitic diseases cause ruthless morbidity affecting principally

population in endemic areas [84]. Hence, there is an increasing demand towards anthelmintics.

Drug resistance is a major problem in the chemotherapy of many parasitic diseases due to

helminths both in humans and in farm animals [85]. Resistance to anthelmintics involves several

groups of mechanisms: changes of the sites for binding of drugs [86], detoxifying processes

including increased activity of several more or less specific enzymes [87] and increased drug

efflux by membrane transporters. While the first two mechanisms mainly explain the

development of resistance against single groups of anthelmintic molecules, the third mechanism

has been the aim of increasing investigations during the last decade given its importance in

multiple resistances to several groups of anthelmintics simultaneously.

The anthelmintic assay was carried out as per the method of Ajaiyeoba et al [88]. The

assay was performed in vitro using adult earthworm (Pheretima posthuma) owing to its

anatomical and physiological resemblance with the intestinal roundworm parasites of human

beings for preliminary evaluation of anthelmintic activity [90-91]. Test samples was prepared at

the concentration, 5 mg/ mL in DMF and six worms i.e., Pheretima posthuma, of approximately

equal size (3-5 cm in length and 0.1-0.2 cm in width were used for all the experimental protocol)

were placed in each nine cm petri dish containing 25 mL of above test solution. Piperazine

citrate (5 mg/ mL) was used as reference standard and DMF as control [92-94]. All the test

solution and standard drug solution were prepared freshly before starting the experiments.

Observations were made for the time taken for paralysis was noted when no movement of any

sort could be observed except when the worms were shaken vigorously. Time for death of worms

were recorded after ascertaining that worms neither moved when shaken vigorously nor when

dipped in warm water(50 °C).

RESULTS AND DISCUSSION

The Schiff base ligands (L1-L4) were synthesized by the condensation of 3-amino-2-

methyl-4(3H)quinazolione with different aromatic aldehydes in 1:1 molar proportion in

methanol. The Schiff bases were soluble in methanol, ethanol, DMSO, acetone and insoluble in

water. The compounds were purified by repeated recrystalization from ethanol and then dried.

The results of elemental analysis (C, H, N) with molecular formulae and the melting points of the

prepared ligands are presented in the synthesis section and Table 1. The results obtained are in

good agreement with those calculated for the suggested formulae and the melting points are

sharp, indicating the purity of the prepared Schiff bases. The structures of the Schiff bases are

confirmed by physical and spectral data and are given below (Figure 1).

Figure 1: Structure of Schiff base ligands.

Table 1: Analytical and physical data of quinazolin-4(3H)-one Schiff base ligands.

Compound

Ligand

Melting

Point

(°C)

Yield

(%)

Elemental found (calcd)

Molecular

Formula

(Mol. Wt)

C (%)

H (%)

N (%)

S(%)

L1 C16H13N3O2

(279)

167-169

71

68.43

(68.5)

4.58

(4.61)

14.9

(15.02)

-

L2 C15H12N4O2

(264)

175-177

63

68.18

(68.27)

4.28

(4.31)

21.22

(21.32)

-

L3 C17H15N3O3

(309)

215-217

61

66.01

(66.12)

4.93

(5.03)

13.58

(13.79)

-

L4 C16H15N3OS

(297) 183-185 79

64.62

(65.13)

5.08

(5.14)

14.13

(15.61)

10.77

(10.91)

IR spectra

The IR spectra of the ligands (L1-L4) under investigation were recorded by nujol method

on JASCO FT-IR spectrophotometer in the frequency range of 4000-400 cm−1. The important

diagnostic bands in the IR spectra were assigned and the bands positions are compiled in the

synthesis part.

The IR spectra of L1-L4 are shown in Figures 2a-d and the important stretching

frequencies of various functional groups are given in Table 2. The IR spectra of all the Schiff

bases showed a broad peak in the range of 3088-2144 cm-1 for CH stretching. A highly intense

peak in the range of 1593-1603cm-1 was observed in all the ligands which can be attributed to

azomethine (C=N) stretching. A broad peak of less intensity in the region 1656-1684cm-1 was

found in all the spectra of ligands and can be assigned for C=O stretching. A comparison of FT-

IR spectrum of 3-amino-2-methyl-4(3H)-quinazoloinone with the spectra of ligands (L1-L4)

showed the formation of Schiff bases (C=N) stretching.

Table 2: The important diagnostic IR absorption bands (in cm-1) of quinazolin-4(3H)-one

Schiff base ligands.

Ligands ν (O-H) ν (C-H) ν (C=N) ν (C=O)

L1 3434 3067 & 2974 1603 1663

L2 - 3088 & 2924 1597 1684

L3 3414 3063 & 2987 1586 1650

L4 - 3299 & 3188 1574 1656

Figure 2a: IR spectrum of 3-(2-hydroxybenzylideneamino)-2-methylquinazolin-4(3H)-one (L1).

0

110

50

100

4000 600100020003000

%T

Wavenumber [cm-1]

Figure 2b: IR spectrum of 2-methyl-3-(pyridine-2-ylmethyleneamino)quinazolin-4(3H)-one (L2).

Figure 2c: IR spectrum of 3-(2-hydroxy-3-methoxybenylideneamino)-2-methylquinolin-4(3H)-one (L3).

0

110

50

100

4000 600100020003000

%T

Wavenumber [cm-1]

0

110

50

100

4000 600100020003000

%T

Wavenumber [cm-1]

Figure 2d: IR spectrum of 3-((5-ethylthiophene-2-yl)methyleneamino)-2-methylquinazolin-4(3H)-one (L4).

1H-NMR spectra

A review of the literature revealed that NMR spectroscopy has been proven to be useful

in establishing the nature and structure of many Schiff bases, as well as their metal complexes in

solution. The NMR spectra of Schiff bases were recorded in d6-dimethylsulfoxide (DMSO)

solution, using tetramethylsilane (TMS) as an internal standard on VARIAN-400 NMR

spectrometer. Chemical shifts were reported as δ-values in parts per million (ppm) relative to

Si(CH3)4 as relative reference (δ = 0 ppm) and to the solvent as internal reference.

The chemical shift data of all the ligands (L1-L4) are presented in Table 3. 1H-NMR

spectra of L1-L4 are shown in Figures 3a-d.

Table 3: 1H-NMR spectral data of Schiff base ligands.

Compounds δ (OH) δ (CH=N) δ (Ar-H) δ (CH3)

C16H13N3O2 10.03 8.71 7.83-7.34 2.50

C15H12N4O2 - 8.76 7.87-7.43 2.72

C17H15N3O3 10.59 9.11 7.78-7.04 2.76

C16H15N3OS - 8.25 7.76-7.26 2.77

-10

110

0

50

100

4000 600100020003000

%T

Wavenumber [cm-1]

Figure 3a: 1H-NMR spectrum of 3-(2-hydroxybenzylideneamino)-2-methylquinazolin-

4(3H)-one (L1).

Figure 3b: 1H-NMR spectrum of 2-methyl-3-(pyridine-2-ylmethyleneamino)quinazolin-4(3H)-

one (L2).

Figure 3c: 1H-NMR spectrum of 3-(2-hydroxy-3-methoxybenylideneamino)-2-

methylquinolin-4(3H)-one (L3).

Figure 3d: 1H-NMR spectrum of 3-((5-ethylthiophene-2-yl)methyleneamino)-2-

methylquinazolin-4(3H)-one (L4).

Mass spectra

The mass spectrum was recorded on electron ionization (EI) mode on VARIAN-1200 L

model spectrometer. The mass spectra of ligands L1-L4 are compiled in synthetic part and the

spectra of each ligand are given in Figures 4a-d.

Figure 4a: Mass spectrum of 3-(2-hydroxybenzylideneamino)-2-methylquinazolin-4(3H)-one

(L1).

Figure 4b: Mass spectrum of 2-methyl-3-(pyridine-2-ylmethyleneamino)quinazolin-4(3H)-one

(L2).

Figure 4c: Mass spectrum of 3-(2-hydroxy-3-methoxybenylideneamino)-2-methylquinolin-

4(3H)-one (L3).

Figure 4d: Mass spectrum of 3-((5-ethylthiophene-2-yl)methyleneamino)-2-

methylquinazolin-4(3H)-one (L4).

Antimicrobial results

In the present study, the antimicrobial activity of the Schiff base ligands (L1-L4) were

evaluated against two Gram-positive (E.coli and R.solanacearum), two Gram-negative bacteria

(B.subtilis and S.aureus) and three fungi (A. niger, A.flavus and A. solani). Minimum inhibitory

concentration (MIC) of these ligands against bacteria and fungi was determined by the method

given by Nomiya et al. [95]. Standard antibiotics namely Chloramphenicol and standard

antifungal drug Fluconazole were used for comparison with antibacterial and antifungal activities

shown by compounds (Table 4). All the ligands possessed good antibacterial activity against

Gram positive bacteria (E.coli and R.solanacearum) and antifungal activity against A.

niger and A. solani. However, the ligands exerted moderate to poor activity against B.subtilis,

S.aureus and A. flavus. Keeping in view, the rising problems of antimicrobial resistance, these

chemical compounds may be used for formulating as novel chemotherapeutic agents.

Table 4: Antimicrobial activity of Schiff base ligands L1-L4.

Compounds

Zone of inhibition (in mm)*

Antibacterial activity Antifungal activity

B.subtilis E.coli S.aureus R.solanacearum A.niger A. flavus A.solani

L1 11 14 12 08 07 05 08

L2 09 13 11 14 09 08 03

L3 21 19 22 23 21 13 19

L4 17 15 19 20 11 09 14

Chloramphenicol 29 26 25 32 - - -

Fluconazole - - - - 27 23 25

*average of three replicates

Antioxidant results

The free radical scavenging activity of Schiff bases was tested by their ability to bleach

the stable radical DPPH. The activity was monitored by following the absorption at 518 nm in a

visible spectrophotometer. In the presence of any free radical scavenger, this odd electron pair up

and causes the diminishing of absorption band which is proportional to the number of electrons

taken up. The activity was studied at different concentrations (0.1–0.4 mmol) for each

compound. The variation in activity is represented in graphs (Figure 5). The graph was plotted

with percentage antioxidant activity (AA %) on the y-axis and concentration on the x-axis. The

scavenging ability of the synthesized compounds was compared with ascorbic acid as a standard.

Compounds L1 and L3 having a better scavenging ability. This may be due to the presence of

hydroxyl group. Rest of the compounds showed moderate antioxidant activity (Fig 2). Radical

scavenging activity was expressed as a percentage and was calculated using the following

formula:

where Asample is the absorbance of the test sample and Acontrol is the absorbance of the control.

Figure 5: Scavenging activity of Schiff base ligands.

Anthelmintic activity

From the observations made, all the Schiff base ligands produced a significant

anthelmintic activity and the results are shown in Table 5. The anthelmintic activity of the

prepared ligand was compared with standard drug (piperazine citrate). The dimethyl formamide

was used as a control. The extent of activity shown by the ligands was found to be poor than that

of the standard drug, Piperazine citrate which justifies its activity. It could be concluded that the

Schiff bases (L1-L4) is having poor anthelmintic activity.

Table 5: Results of Anthelmentic Activity of Schiff base ligands (L1-L4).

Compound

Concentration

in mg/ mL

Time taken for paralysis

(in min)

Time taken for death (in

min)

Dimethyl formamide

(control)

- No effect till ten hours No effect till ten hours

Piperazine citrate (standard) 5 10 10

L1 5 25 29

L2 5 27 32

L3 5 21 24

L4 5 19 26

CONCLUSION

The work has approached towards the synthetic and biological approach of these

wonder molecules, 4(3H)-quinazolinone derivatives. The preparation procedure follow in this

work for the synthesis of title compounds offers reduction in the reaction time, operation

simplicity, cleaner reaction and easy work-up. All these Schiff base ligands are insoluble in

water but soluble in organic solvents, DMF, DMSO, CH3Cl and THF. Elemental analyses

confirms the chemical composition of the synthesized compounds while FT-IR and 1H-NMR

spectroscopy confirms the functional groups, particularly -HC=N, C=O and O-H groups, of the

compounds. All spectroscopic analysis confirmed the proposed structures for these compounds.

Antibacterial data have shown that the synthesized compounds have a significant biological

activity against the tested microorganisms. The antioxidant activity of 3-(2-

hydroxybenzylideneamino)-2-methylquinazolin-4(3H)-one (L1) and 3-(2-hydroxy-3-

methoxybenylideneamino)-2-methylquinolin-4(3H)-one (L3) showed better activity than 2-

methyl-3-(pyridine-2-ylmethyleneamino)quinazolin-4(3H)-one (L2) and 3-((5-ethylthiophene-2-

yl)methyleneamino)-2-methylquinazolin-4(3H)-one (L4) due to the presence of hydroxyl group

in the compounds L1 and L3. The anthelmintic activity of all the ligands showed poor to

moderate activity against Indian earth worm Pheretima posthuma.

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