Synthesis, Characterization and Bioevaluation of New Tetrahydroquinazolines

새로운 Tetrahydroquinazoline의 합성, 구조 결정 및 생물학적 평가

Abstract

The synthesis, characterization and bioevaluation of five new tetrahydroquinazolines was reported. Initially cyclohexanones were prepared and they were used as synthons to get the target molecules. These were condensed with substituted benzaldehydes and the resulting chalcones were treated with guanidine hydrochloride . All the molecules were non-toxic to human cells and showed significant antibacterial activity.

새로운 다섯개의 tetrahydroquinazolines 의 합성과 구조 결정 및 생물학적 평가는 보고 되었다. 최초에 준비된 cyclohexanones는 목표분자를 얻기 위한 synthon으로 사용되어졌다. 이들은 치환된 벤즈알데히드와 결합되어졌고, 얻어진 chalcone은 구아니딘 염산염으로 처리했다. 모든 분자들은 인간세포에 무독하며 상당한 항균성을 보여줬다.

INTRODUCTION

A number of plants belonging to the families Acanthaceae, Cruciferae, Malvoceae and Rutaceae are known to contain quinazoline alkaloids.1 Quinazoline derivatives2 possess a broad spectrum of biological activities such as antidiabetic,3 anti-convulsant,4 analgesic,5 antibacterial,6 protein tyrosine kinase inhibitors,7 EGFR inhibitors,8 PDGFR phosphorylation inhibitors,9 CNS depressants10 and antitumor activity.11 Furthermore, the heterocyclic core constitutes more than 40 alkaloids12 isolated from natural products and some show interesting biological profiles such as antimalarial13 and diuretic14 properties. Keeping this in view it was proposed to synthesize new tetrahydroquinazoline derivatives and study the influence of substituents on biological activities. A simple and convenient synthesis of five new quinazolines 11-15 have been reported (Schemes 1 and 2).

2 - Butanone 1 was condensed with benzaldehyde 2 in presence of dry HCl gas , yielded pure compound 3 which was again condensed with benzaldehyde in presence of 20% aq. NaOH at 5 ℃. The compound 4, thus formed was crystallized from methanol and its structure was confirmed by physical and spectral data.15 Cycloaddition of compound 4 to ethyl cyanoacetate in presence of sodium ethoxide resulted compound 5 as white coloured crystals. The I.R. spectra exhibited bands at 1705-1720 (cyclic C=O), 1725-1755 (C=O ester), 2235 and 2260 cm-1 (CN). The 1HNMR spectra can be rationalized by presuming that the two aryl groups at 2 and 6 positions are in cis-1,3-di-equatorial arrangement in the preferred rigid chair conformation of cyclohexanone ring, although a number of dynamic forms do exist.15 Thus, the 1HNMR spectral data is assigned as, δ 0.60-0.75 (t, 3H, CH3 CH2-O),0.9 (d, 3H, CH3), 2.72-2.83 (dd,1H, He-eq), 3.18-3.46 (m, 2H, Hc+He-ax), 3.65-3.84 (m, 4H, Hb+Hf+OCH2), 7.2-7.4 (m, 10H, aromatic protons).The formation of compound 5 is further proved by its 13C NMR data viz., δ11.994 (1C, CH3), 13.527 (1C, OCH2CH3), 43.956 (1C, Cc), 45.718 (1C, Cf), 49.401(1C, Ce), 56.280 (1C, Ca), 60.238 (1C, Cb), 62.633 (1C, OCH2), 116.780 (1C, CN), 128.212, 128.667, 128.815, 128.967, 135.861, 136.624 (12C, Ar-C’s), 166.634 (1C, COO), 206.939 (1C, Cd).

Scheme 1

Scheme 2

Compound 5 on condensation with substituted benzaldehydes, yielded chalcones 6-10 which were crystallized from methanol as yellow needles. The thin layer chromatography of these chalcones showed characteristic colour spots with methanol – sulphuric acid (9 : 1) as spraying reagent. They also exhibited the characteristic colour test with antimony trichloride.16 Further the formation of chalcones is conformed by their spectral data. The synthesis of tetrahydroquinazolines 11-15 was accomplished by condensing chalcones 6-10 with guanidine hydrochloride in alkaline medium (Scheme 2).

Compound 11 analyzed for C33H32N4O4, was well supported by its spectral data. The I.R. spectrum showed the absorption at 1685 and 1597 cm-1 characteristic17 of the C=N and C=C stretch of the pyrimidine system and a sharp peak at 3492 cm-1 indicating the NH stretchings of the amino group. The characteristic out of plane CH bending18 was observed at 1002 and 805 cm-1.The formation of compound 11 is further confirmed by its FAB mass spectrum. Mass spectrum showed the characteristic [M]+ ion at m/z 548(5%). The other fragment ions of m/z 307(16%), 240(21%), 209(18%), 195(58%), 165(16%), 154(100%, base peak), 135(80%), 120(18%), 107(36%), 91(48%), 69(38%), 55(44%) were observed. The fragmentation pattern was presented in the Chart-1. These fragmentations were characteristic for tetrahydroquinazolines.19-24 The 1H and 13C NMR data is in accordance with the structure. The 13C NMR data was explained as 16.622 (1C, CH3), 37.499 (1C, Ce), 37.763 (1C, Ch), 44.015(1C, Cg), 50.319 (1C, Cf), 53.510 (1C, OCH3), 67.476 (1C, OCH2), 116.704 (1C, CN), 156.238-129.520 (22C, Ar-C’s), 167.369 (1C, C=O). Thus, the formation of compound 11 was characterized.

Chart 1

 

RESULTS AND DISCUSSION

Tetrahydroquinazolines 11-15 were tested for cytotoxic activity at NFMC, Bharathidasan University, Tiruchirapally, India. Studies were performed by using Dye exclusion method. All the compounds 11-15 are non-toxic to PBMC and two different cancer cell lines (Jurkat & Raji) from 5 μg to 800 μg level (Table 1).

Table 1.*Anticancer Activity Testing was performed by Dye exclution method. Bioactivity assay was performed in a 96 Well tissue culture plate (Greiner, Germany). A constant number of cancerous (Jurkat, Raji & PBMC) cell suspension and desired concentrations of the compounds and media were added into each well. The plate was incubated at 37 ℃ in CO2 incubator with 5% CO2. The number of live cells were counted after every 12 hrs by using inverted phase contrast microscope (Nikon TM) and the Newbauer’s counting chamber by dye exclution method.

The minimum inhibitory concentrations (MIC) of quinazoline derivatives 11-15 were obtained against five representative Gram-positive organisms and four Gram-negative organisms. It has been observed that all the derivatives exhibited interesting biological activity however, with a degree of variation. The potencies of these molecules varied somewhat, depending upon the nature of their substituent(s) on the phenyl moiety in the 4-position. The methoxy analogue 11 displayed good zone of inhibition for B. subtilis and moderately active on B.pumilus and E.faecalis whereas it is inactive against S.faecalis and M.luteus (Table 2, entry 1) at MIC 20 μg/ml and it showed good activity, almost equal to that of Benzyl Penicillin against B. subtilis at 200 μg/ml concentration (Table 2, entry 4). Surprisingly and in contrast to compound 11, the N,N-dimethyl amino derivative 12 is inactive against B. subtilis, B. pumilus and E.faecalis whereas it is active against S.faecalis and M.luteus (Table 2, entry 5) at MIC 20 μg/ml and it is equally active as Benzyl Penicillin against S.faecalis and M.luteus at 200 μg/ml dose (Table 2, entry 8). Substitution of both methoxy and hydroxyl groups on phenyl ring (14) increases the activity of the molecule and this is evidenced by the fact that it inhibited all Gram-positive organisms except B. subtilis at MIC 20 μg/ml (Table 2, entry 13) and at 200 μg/ml concentration (Table 2, entry 16) it exhibits inhibitory activity, similar to Benzyl Penicillin against all Gram-positive organisms except B. subtilis . All these interpretations were better visualized in Figs. 1 and 2.

Table 2.*Negative Control DMSO, no activity, #Solutions of different concentrations of compounds 11-15 were prepared in DMSO and tested against Gram-positive bacteria. The antibacterial activity of the test compounds 11-15 was compared with Benzyl Penicillin. All the compounds are non-toxic to human cells at the above dose.

The level of activity of tetrahydroquinazolines 11-15 towards Gram-negative organisms is slightly less than to that observed with Gram-positive organisms (Figs 1-4). Compound 12, which contain N,Ndimethyl amino substituent, displayed good antibacterial activity against E.Coli, P.vulgaris and K. pneumoniae at all concentrations (Table 3, entry 5-8). The methoxy analogue 11 exhibits inhibitory zones against P.marginalis and E.Coli but it is inactive against P.vulgaris and K.pneumoniae at MIC 20 μg/ml (Table 3, entry 1). Quinazoline 14 was inactive against all Gram-negative organisms at MIC 20 μg/ml (Table 3, entry 13) and less potent than other molecules at remaining concentrations (Table 3, entry 14-16). Moreover, this finding is also in sharp contrast with the findings of Gram-positive organisms for 14 (Table 2, entry 13-16). The Chloro substituted analogue 15 displayed MIC 50 μg/ml against all Gram-negative organisms except K.pneumoniae (Table 3, entry 17) and it was slightly more potent than other molecules at 100 μg/ml and 200 μg/ml (Table 3, entry 19, 20), concentrations against P.marginalis.

Fig. 1.Effect of quinazolines 11-15 against B. subtilis, B. pumilus and E. faecalis at 20 μg-200 μg concentration. Benzyl Penicillin is used as standard reference to compare the activity. Compounds 11-15 were non-toxic to normal human PBMC and Jurkat and Raji (cancer cell lines) from 5 μg-800μg level.

Fig. 2Effect of quinazolines 11-15 against S. faecalis and M. luteus at 20 μg-200 μg concentration. Benzyl Penicillin is used as standard reference to compare the activity. Compounds 11-15 were non-toxic to normal human PBMC and Jurkat and Raji (cancer cell lines) from 5 μg-800 μg level.

Fig. 3.Effect of quinazolines11-15 against P. marginalis and E. coli at 20 μg-200 μg concentration. Benzyl Penicillin is used as standard reference to compare the activity. Compounds 11-15 were non-toxic to normal human PBMC and Jurkat and Raji (cancer cell lines) from 5 μg-800 μg level.

Fig. 4.Effect of quinazolines 11-15 against P. vulgaris and K. pneumoniae at 20 μg-200 μg concentration. Benzyl Penicillin is used as standard reference to compare the activity. Compounds 11-15 were non-toxic to normal human PBMC and Jurkat and Raji (cancer cell lines) from 5 μg-800 μg level.

Table 3.*Negative Control DMSO, no activity, # Solutions of different concentrations of compounds 11-15 were prepared in DMSO and tested against Gram-negative bacteria. The antibacterial activity of the test compounds 11-15 was compared with Benzyl Penicillin. All the compounds are non-toxic to human cells at the above dose.

 

CONCLUSIONS

We have herein reported the activity of new tetrahydroquinazoline derivatives 11-15 to inhibit the bacterial activity of the Gram-positive organisms and Gram-negative organisms along with their cytotoxic profile. We have synthesized and tested a set of variously substituted quinazolines and the best results were obtained with compound 14 in case of Gram-positive bacteria and compound 12 is more potent in case of Gram-negative bacteria. All compounds 11-15 are non-toxic from 5 μg to 800 μg level and showed moderate to good antibacterial activity. The difference in potency of the compounds, was most probably the result of substituent differences on the phenyl moiety. This is in good agreement with previous findings that a quinazoline ring with appropriate substituents are the dominant factors for multiple biological activities. So we conclude that, the newly synthesized tetrahydro quinazolines are non-toxic antibacterial agents. Actually our aim is to synthesize anticancer agents and the studies are in progress. The present compounds possess non-toxic nature with antibacterial properties.

 

EXPERIMENTAL

Melting points of the compounds were recorded on an electro-thermal apparatus and were uncorrected. IR-spectra in KBr were recorded in NICO-LET AVATAR-320-FT-IR spectrophotometer. Elemental analysis was carried out on CHNS OEA 1108 elemental analyzer. 1H NMR spectra were recorded on BRUKER AMX-400 spectrometer operating at 400 MHz. 13C NMR spectra were recorded on BRUKER AMX-400 spectrometer at operating frequency 100 MHz. Mass spectra were recorded on either FINNIGAN MAT 1020B or MICRO MASS VG 70-70H spectrometer operating at 70 ev using direct inlet system. The GCMS spectra were recorded on SHIMADZU-QP-5050A instrument. The purity of the compounds were checked on TLC and HPLC (SHIMADZU-LC 6A) using Shimpack CLC-Sil column and Shimpack CLC-ODS column using UV detector. Starting materials and solvents were purchased from Acros, Merck or Aldrich.

Synthesis of 3-methyl-4-phenylbut-3-en-2-one (3)

In a 100 ml two - necked round - bottomed flask benzaldehyde (0.10 mmol) and 2-butanone (0.02 mmol) were taken. The contents were cooled to 0-5 ℃ and then dry HCl gas was passed until it was saturated and turns to red colour. The reaction mixture was stirred for 8 hrs and the layer formed at the bottom was separated and rejected. The crude product was dilute with benzene, washed with NaHSO3 solution followed by water. The organic layer was separated, dried with anhydrous Na2SO4 and evaporated. The residue when distilled under reduced pressure yielded pure compound, which was solidified on keeping in a refrigerator for 2-3 days. B.p. 100-103 ℃/5 mm (lit17, b.p., 127-130 ℃/12 mm, m.p. 38 ℃), yield 55%. HPLC purity : 100%, GCMS purity: 96.96%; 1HNMR (CDCl3/TMS): δ2.0 (s, 3H, Hc), 2.4 (s, 3H, Ha), 7.2-7.4 (m, 5H, Ar-H’s), 7.5 (s, 1H, Hd). G.C.M.S (%):160 [M+] (65%), 159 [M-H]+ (85), 145(25), 115(100%, base peak), 91(40), 63(13), 51(23), 43(80).

Synthesis of 2-methyl-1,5-diphenylpenta-1,4-dien-3-one (4)

A solution of 20% aq. NaOH (50 ml) was added drop wise to an ice-cold solution of compound 3 (0.10 mmol) and benzaldehyde (0.12 mmol) in ethanol stirred for 1-2 hrs. Then the reaction mixture was brought to room temperature and the stirring was continued for 5-7 hrs. The contents were poured into cold water and acidified with acetic acid and extracted with ether. The ethereal layer was washed with NaHCO3 solution followed by water and dried with anhydrous Na2SO4. Then the solvent was evaporated and the residue was distilled under reduced pressure, which was solidified on keeping at room temperature for 12 hrs.B.p. 192-198 ℃/5 mm (lit15. 180-182 ℃/0.45 mm), yield 68%. HPLC purity : 99%, GCMS purity : 96.12%; 1HNMR (CDCl3/TMS): δ2.2 (s, 3H, Hb), 7.3-7.6 (m, 10H, Ar-H’s), 7.6-7.8 (dd, 2H, Hd+He), 7.85 (s, 1H, Ha). GCMS(%): 248 [M]+ (60%), 247 [M-H]+(35), 205 (18%), 144(10), 131(30), 116(100%, basepeak), 103(40), 77(35), 51(20).

Synthesis of ethyl 1-cyano-3-methyl-4-oxo-2,6-diphenylcyclohexanecarboxylate (5)

Compound 4 (0.05 mmol), ethyl cyanoacetate (0.05 mmol) and absolute ethanol (50 ml) were taken in a 100 ml round bottomed flask. To this 15 ml of 10% NaOEt solution was added and refluxed for 5 hrs. The contents were concentrated and poured in ice cold water, acidified with acetic acid and extracted with ether. The ethereal layer was washed with NaHCO3 solution followed by water and dried over anhydrous MgSO4 and the solvent was stripped off by distillation. The crude product was chromatographed over silicagel with n-hexane and ethyl acelate (95:5) as eluent. Further, the product was recrystallised from methanol, which yielded white coloured crystals. M.p.: 128 ℃, yield 68%, molecular formula: C23H23NO3. Elemental analysis: Found (%): C, 76.32; H, 6.35; N 3.82, Required (%): C, 76.43; H, 6.41; N, 3.88. 1H NMR (CDCl3/TMS): δ0.60-0.75 (t, 3H, CH3CH2O), 0.9 (d, 3H, CH3), 2.72-2.83 (dd, 1H, He-eq), 3.18-3.46 (m, 2H, Hc+ He-ax), 3.65-3.84 (m, 4H, Hb+Hf+OCH2), 7.2-7.4 (m, 10H, Ar-H’s).13C NMR : 11.994 (1C, CH3), 13.527 (1C, OCH2CH3), 43.956 (1C, Cc), 45.718 (1C, Cf), 49.401 (1C, Ce), 56.280 (1C, Ca), 60.238 (1C, Cb), 62.633 (1C, OCH2), 116.780 (1C, CN), 128.212, 128.667, 128.815, 128.967, 135.861, 136.624 (12C, Ar-C’s) 166.634 (1C, COO), 206.939 (1C, Cd).I.R (νmax): 1708, 1728, 2247, 960, 785 cm-1 .

Synthesis of Chalcones-General Procedure

A mixture of compound 5 (0.01 mmol), substituted benzaldehydes (0.01 mmol) in ethanol (25 ml) and aqueous potassium hydroxide (15 g in 15 ml of water) was stirred for 2 hrs and kept at room temperature for 24 hours. The reaction mixture was neutralized with acetic acid and diluted with water. The solid thus obtained was filtered, washed with water and crystallized from appropriate solvent and characterized by using spectral data and elemental analysis. Compound 5 was condensed with veratraldehyde, 4-(dimethylamino) benzaldehyde, 4-hydroxy benzaldehyde, vanillin, 4-chlorobenzaldehyde to furnish the respective chalcones 6-10.

Synthesis of Ethyl 2-amino-6-cyano-4-(3,4-dimethoxyphenyl)-8-methyl-5, 7-diphenyl- 5,6,7,8-tetrahydroquinazoline-6-carboxylate (11)

Compound 6 (1 mmol) and guanidine hydrochloride (1 mmol) were refluxed together in 10% ethanolic potassium hydroxide (12 ml) for 5 hrs, progress of the reaction was monitored by TLC. The excess of the solvent was removed in vaccum, extracted with chloroform and washed with water. The combined organic layers were dried over anhydrous Na2SO4, filtered and distilled under reduced pressure to give a gummy product, which was chromatographed over silicagel column and recrystallised from chloroform / methanol as yellow needles, m.p.: 238 ℃, yield 61%, molecular formula : C33H32N4O4. Elemental Analysis : Found (%) : C, 71.96; H, 5.69; N, 10.14%, Required (%) : C, 72.26; H, 5.84; N, 10.22%. 1HNMR (d6-acetone/TMS): δ0.76-0.88 (m,6H,CH3+OCH2CH3), 2.41-2.56 (m,1H, Hh), 3.05(br.s, 2H, NH2), 3.6(d,1H, Hg), 3.8 (s, 6H, 2 x OCH3), 3.9-4.0 (m, 2H, -OCH2CH3), 4.2 (s, 1H, He), 7.25-7.43 (m, 13H, Ar-H’s).I.R. (νmax): 3492, 2972, 2646, 2253, 1715, 1597, 1506, 1271, 1228, 1131, 1002, 921, 765, 701, 601, 515 cm-1.

Synthesis of Ethyl 2-amino-6-cyano-8- methyl-4-[4-(dimethylamino) phenyl]-5,7-diphenyl-5,6,7,8-tetrahydroquinazoline-6-carboxylate (12)

M.p.: 238 ℃, yield 59%, molecular formula : C33H33N5O2 .Elemental Analysis : Found (%) : C, 74.33; H, 6.18; N, 13.08%, Required (%) : C, 74.57; H, 6.22; N, 13.18%. 1HNMR (d6-acetone/TMS): δ 0.74-0.86 (m, 6H, CH3+OCH2 CH3), 2.40-2.58 (m, 1H, Hh), 3.0 (br. s, 2H, NH2), 3.15 (s, 6H, NMe2), 3.6 (d, 1H, Hg), 3.8-4.0 (m, 2H, -OCH2CH3), 4.0 (s,1H, He), 7.2-7.5 (m, 14H, Ar-H’s).I.R. (νmax): 3384, 3165, 2965, 2915, 1694, 1626, 1438, 1359, 1190, 1106, 1049, 968, 881 cm-1.

Synthesis of Ethyl 2–amino-6-cyano-4-(4-hydroxy phenyl)-8-methyl-5,7-diphenyl-5,6,7,8-tetrahydroquinazoline-6-carboxylate (13)

M.p.: 198 ℃, yield 63%, molecular formula : C31H28N4O3 . Elemental Analysis : Found (%) : C, 72.75; H, 5.51; N, 11.02%, Required (%) : C, 73.81; H, 5.56; N, 11.12%. 1HNMR (d6-acetone/TMS): δ0.56 (t, 3H, CH3CH2O-), 0.7 (d, 3H, CH3), 2.31-2.59 (m, 1H, Hh), 2.9 (s, 2H, NH2), 3.38 (d, 1H, Hg), 3.65- 3.85 (m, 2H, -OCH2CH3), 4.1 (s, 1H, He), 7.08-7.8 (m, 14H, Ar-H’s), 9.87 (s, 1H, -OH). I.R. (νmax): 3441, 3356, 3206, 1680, 1608, 1494, 1290, 1174, 1068, 970, 885, 784 cm-1.

Synthesis of Ethyl 2-amino-6-cyano-4-(4-hydroxy-3-methoxy phenyl)-8-methyl-5,7-diphenyl-5,6,7,8-tetrahydroquinazoline-6-carboxylate(14)

M.p: 256℃, yield 57%, molecular formula : C32H31N4O4. Elemental Analysis :Found (%) : C, 71.46; H, 5.72; N, 10.36% , Required (%) : C, 71.77; H, 5.79; N, 10.48%. 1HNMR (d6-acetone/TMS): δ0.74-0.86 (m, 6H, CH3+OCH2CH3), 2.40-2.56 (m, 1H, Hg), 3.0 (br.s, 2H, NH2), 3.6 (d, 1H, Hg), 3.81 (s, 3H, -OCH3), 3.85-4.0 (m, 2H, -OCH2CH3), 4.05 (s, 1H, He), 7.41-7.6 (m, 13H, Ar-H’s), 9.6 (s, 1H, -OH). I.R. (νmax): 3448, 2968, 2670, 2284, 1724, 1579, 1498, 1290, 1246, 1180, 1056, 980, 764, 710, 615, 540 cm-1.

Synthesis of Ethyl 2-amino-4-(4-chlorophenyl)-6-cyano-8-methyl-5,7-diphenyl-5,6,7,8-tetrahydroquinazoline-6-carboxylate (15)

M.p.: 208 ℃, yield 64%, molecular formula : C31H27N4O2Cl. Elemental Analysis: Found (%) : C, 71.16; H, 5.09; N, 10.46; Cl, 6.65 %, Required (%) : C, 71.27; H, 5.18; N, 10.73, Cl, 6.71%. 1HNMR (d6-acetone/TMS): δ0.68 (t, 3H, CH3CH2O-), 0.8 (d, 3H, CH3), 2.34-2.62 (m, 1H, Hh), 2.85 (s, 2H, NH2), 3.36 (d, 1H, Hg), 3.68-3.82 (m, 2H, -OCH2CH3), 4.0 (s, 1H, He), 7.28-7.46 (m,14H, Ar- H’s). I.R. (νmax): 3368, 3340, 1684, 1605, 1524, 1349, 1268, 1026, 956, 849, 715 cm-1.

Cytotoxic studies - Dye exclution method

The experiments were carried out in the laboratory of Prof. T. Tirunala Sundari, Dept. of Microbiology, Bharathidasan University, Tiruchirapally, India. Anticancer Activity Testing was performed by Dye exclution method. Bioactivity assay was performed in a 96 Well tissue culture plate (Greiner, Germany). Various concentrations of the compound ranging from 5 μg to 800 μg were made. A constant number of cancerous (Jurkat, Raji & PBMC) cell suspension and a constant volume of the complete media were added into each well. Desired concentrations of the compounds were added into each well and the volume was made constant, using complete media. Negative (solvent used for dissolving the compound) and positive (known immunomodulator) controls were also maintained. The plate was incubated at 37 ℃ in CO2 incubator with 5% CO2. The number of live cells were counted after every 12 hours by using inverted phase contrast microscope (Nikon TM) and the Newbauer’s counting chamber by dye exclution method.

Antimicrobial activity studies-Cup Plate Method

Sterilized molten nutrient agar medium was inoculated with 50 μL of the test organism aseptically. The temperature of the molten medium should be below 45 ℃. The inoculated medium was poured in the assay – plate and kept on a horizontal surface to avoid non-uniform solidification of the medium. Cups of 8 mm diameter were made at equidistance with bore-puncher. Solutions of different concentrations of compounds 11-15 were prepared in DMSO. 50 μL of test solutions were introduced into the cups using micropipette. These plates were covered and kept at 5-10 ℃ for 3 hours for better diffusion of the solution into the medium. They were then incubated at 37 ℃ for 18 hrs in an incubator and the diameters of inhibition zones were measured in mms (Tables 2 & 3). DMSO alone was kept as control, which did not have any inhibition zone. The antibacterial activity of the test compounds 11-15 was compared with Benzyl Penicillin.