Corresponding Author:
S. S. Panda
Department of Pharmaceutical Chemistry, Manipal College of Pharmaceutical Sciences, MAHE Manipal-576 104, India
E-mail: [email protected]
Date of Submission 14 July 2006
Date of Revision 30 January 2008
Date of Acceptance 25 March 2008
Indian J. Pharm Sci. 2008, 70 (2): 208-215  

Abstract

A number of chalcones were synthesized by reacting indole-3-aldehyde, prepared by Vilsemeir Haack reaction with 4-substituted acetophenone in NaOH solution in ethanol. These chalcones were immediately reacted with urea, thiourea and guanidine hydrochloride in presence of concentrated hydrochloric acid as reagent to obtain the corresponding hydroxy, thio and amino pyrimidines. The synthesized heterocyclics were characterized on the basis of physical, chemical tests and spectroscopic data and were tested for the acute antiinflammatory activity, antioxidant, antibacterial activity using carragenan-induced rat paw oedema method, DPPH (diphenylpicrylhydrazyl) radical scavenging method and cup plate method using Muller-Hinton agar media respectively. Evaluation of the compounds revealed remarkable antiinflammatory activity reflected by their ability to reduce the carragenan-induced inflammation in rats, appreciable antioxidant activity and also antibacterial activity was observed.

Keywords

Indole, pyrimidine, antiinfl ammatory, antioxidant, antibacterial

Due to interesting activity of various substituted pyrimidines as biological agents [1-3], considerable attention has focused on this class. The pharmaceutical importance of these compounds lies in the fact that they can be effectively used as analgesics, antiinflammatory, anticonvulsant, insecticidal, herbicidal, antitubercular, anticancer and antidiabetic agents. The ability of indole to exhibit antiinflammatory, antimicrobial, antifungal activities [4-6] prompted the selection of indole as starting compound.

In the light of these interesting biological activities, it appeared of interest to synthesize some new hydroxy/ thio/amino pyrimidine derivatives. Indole-3-aldehyde (2) prepared by Vilsemeir Haack reaction from indole (1) was reacted with 4-substituted acetophenones (a-j) in ethanolic NaOH to obtain chalcones (3a-j), which were condensed with urea, thiourea and guanidine hydrochloride in presence of cyclising agent, concentrated hydrochloric acid to obtain hydroxy pyrimidines (4a-j) thio pyrimidines (5a-j) amino pyrimidines (6a-j) respectively. The synthetic sequence leading to the formation of targeted compounds is depicted in scheme 1.

Figure

Scheme 1: Synthesis of bioactive molecules

The constitution of the products has been supported by element analyses, chemical tests, IR, NMR and Mass spectral data. All the compounds were evaluated for antiinflammatory activity using the carrageenaninduced rat paw edema model, antioxidant activity using DPPH and antibacterial activity against four different strains of Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Escherichia coli at different concentration of 5 µg/ml, 10 µg/ml, 50 µg/ml and 100 µg/ml.

Materials and Methods

All the melting points were determined using open capillary tubes in scientific melting point apparatus and are uncorrected. UV values were obtained by using Shimadzu UV/Vis Spectrophotometer, UV- 1601PC, at Manipal College of Pharmaceutical Sciences, Manipal. IR spectra of the synthesized compounds were scanned using a Shimazdu-Fourier Transform Infrared Spectrophotometer-8300 and KBr press at Manipal College of Pharmaceutical Sciences. PMR spectra of the synthesized compounds were recorded on 300 MHz spectrophotometer at IISC Bangalore. Mass spectra of the synthesized compounds were recorded on Shimazdu-GC-MS QP 5050A at Manipal College of Pharmaceutical Sciences. Progress, purity of the reaction and the intermediates were analyzed using precoated TLC plate and using UV chamber. Male Wistaar rats (150-200 g) were procured and the antiinflammatory activity was carried out on approval of Institutional Animal Ethics Committee, KMC, Manipal.

Indole-3-aldehyde (2)

In a 100 ml round bottom flask, Vielsmeier Haack complex was prepared using phosphorus oxychloride (1 ml) and N,N-dimethylformamide (3.15 ml) and was stirred using magnetic stirrer at 10-15º. Indole (0.01 mol) was dissolved in minimum quantity of N,N-dimethylformamide and added with stirring at 10-15º in Vielsmeier Haack complex. The reaction mixture was allowed to concentrate at 45º and kept for 30 minutes and then poured into ice cold water (100 ml), a clear red coloured solution was obtained, 10% sodium hydroxide (20 ml) was added to get the precipitate. This was boiled for 1 min and filtered, on cooling crystals were formed. Then these crystals were separated by filtration. Recrystallization was done using aqueous DMF. Melting point of obtained product was found to be 192-194º, Rf 0.52 (petroleum ether:ethyl acetate, 80:20), yield 76%.The formation of the aldehyde was confirmed by the Tollen?s test and 2,4-dinitrophenylhydrazine test. The compound was characterized by comparing the malting point and PMR with that reported in literature [7].

1-(4-substituted phenyl)-3-indolyl-2-propen-1-one(3a-j)

Equimolar quantities (0.01 mol) of indole-3-aldehyde and substituted acetophenones were taken in 100 ml conical flask and dissolved in 20 ml of ethanol to this (0.03 mol) of NaOH in minimum quantity of water was added. The mixture was stirred on a magnetic stirrer and the reaction was monitored with TLC. Reaction mixture was diluted with water and acidified with concentrated hydrochloric acid. The precipitated chalcone was filtered and recrystallized from absolute ethanol. The purity of chalcones was tested with thin layer chromatography using solvent system: petroleum ether (60-80º):ethyl acetate [70:30]

4-(Indol-3-yl)-6-(4-substituted phenyl)-2-substituted pyrimidines

Chalcone (0.01 mol) and urea, thiourea or guanidine hydrochloride (0.01 mol) were dissolved in absolute alcohol (20 ml). Few drops of concentrated HCl were added and the reaction mixture was refluxed and the reaction was monitored by TLC. After completion of reaction, it was poured into 250 ml of ice cold water and kept for some time. The crude solid was filtered and subjected to column chromatography. Elution with solvent system ethyl acetate/petroleum ether (60- 80º)] gave pure compound as white solid. The entire synthetic route has been shown in scheme 1.

Characteristics of 4a

IR (KBr) cm-1: 3396 (OH), 3341 (NH), 3048 (Ar-H), 1598 (C=N); PMR (DMSO-d6, d in ppm): 11.2 (s, 1H, OH, D2O exchangeable), 10.9 (s, 1H, NH, D2O exchangeable), 8.3-7.7(m, 5H, Ar-H), 7.6 (s, 1H, pyrimidine proton), 7.3 (s, 1H, indolyl proton), 7.2- 6.9(m, 4H, Ar-H),; m/z: 287 [M]+, 185, 171, 116, 102, 77, 54.

Characteristics of 4b

IR (KBr) cm-1: 3426 (OH), 3401 (NH2), 3381 (NH), 3143 (Ar-H), 1608 (C=N); PMR (DMSO-d6, d in ppm): 11.3 (s, 1H, OH, D2O exchangeable), 10.8 (s, 1H, NH, D2O exchangeable), 8.4-7.7(m, 4H, Ar-H), 7.5 (s, 1H, pyrimidine proton), 7.3 (s, 1H, indolyl proton), 7.1-6.7(m, 4H, Ar-H), 5.2(s, 2H, NH2, D2O exchangeable ; m/z: 302 [M]+• 185, 116, 77, 54.

Characteristics of 4c

IR (KBr) cm-1: 3395 (OH), 3341 (NH), 3108 (Ar-H), 1592 (C=N), 530 (C-Br); PMR (DMSO-d6, d in ppm): 11.2 (s, 1H, OH, D2O exchangeable), 10.7 (s, 1H, NH, D2O exchangeable), 8.3-7.8(m, 4H, Ar-H), 7.6 (s, 1H, pyrimidine proton), 7.3 (s, 1H, indolyl proton), 7.2-6.9(m, 4H, Ar-H),; m/z: 368 [M+2]+•?, 366 [M]+• 185, 11, 116, 77.

Characteristics of 4d

IR (KBr) cm-1: 3396 (OH), 3346 (NH), 3047 (Ar-H), 1590 (C=N), 743 (C-Cl); PMR (DMSO-d6, d in ppm): 11.1 (s, 1H, OH, D2O exchangeable), 10.9 (s, 1H, NH, D2O exchangeable), 8.4-7.7(m, 4H, Ar-H), 7.5 (s, 1H, pyrimidine proton), 7.3 (s, 1H, indolyl proton), 7.2-6.7(m, 4H, Ar-H); m/z: 324 [M+2]+•?, 322 [M]+• 85, 171, 137, 116, 77.

Characteristics of 4e

IR (KBr) cm-1: 3426 (OH), 3381 (NH), 3015 (Ar-H), 1611 (C=N); PMR (DMSO-d6, d in ppm): 11.3 (s, 1H, OH, D2O exchangeable), 10.8 (s, 1H, NH, D2O exchangeable), 9.5 (s, 1H, OH, D2O exchangeable), 9.3 (s, 1H, OH, D2O exchangeable), 8.3-7.8(m, 4H, Ar-H), 7.6 (s, 1H, pyrimidine proton), 7.3 (s, 1H, indolyl proton), 7.1-6.8(m, 3H, Ar-H); m/z: 319 [M]+• 185, 171, 134, 116, 77, 26

Characteristics of 4f

IR (KBr) cm-1: 3394 (OH), 3346 (NH), 3058 (Ar-H), 1599 (C=N); PMR (DMSO-d6, d in ppm): 11.2 (s, 1H, OH, D2O exchangeable), 10.7 (s, 1H, NH, D2O exchangeable), 8.3-7.7(m, 4H, Ar-H), 7.5 (s, 1H, pyrimidine proton), 7.3 (s, 1H, indolyl proton), 7.1- 6.9(m, 4H, Ar-H),; m/z: 305 [M]+• 171, 120, 116, 77, 54, 26.

Characteristics of 4g

IR (KBr) cm-1: 3376 (OH), 3337 (NH), 3067 (Ar-H), 1613 (C=N); PMR (DMSO-d6, d in ppm): 11.3 (s, 1H, OH, D2O exchangeable), 10.9 (s, 1H, NH, D2O exchangeable), 8.5-7.7(m, 4H, Ar-H), 7.6 (s, 1H, pyrimidine proton), 7.3 (s, 1H, indolyl proton), 7.2-7.0 (m, 4H, Ar-H), 2.2 (s, 3H, CH3); m/z: 301 [M]+• 245, 185, 116, 77, 26

Characteristics of 4h

IR (KBr) cm-1: 3396 (OH), 3221 (NH), 3053 (Ar-H), 1571 (C=N); PMR (DMSO-d6, d in ppm): 11.2 (s, 1H, OH, D2O exchangeable), 10.9 (s, 1H, NH, D2O exchangeable), 8.1-7.7 (m, 4H, Ar-H), 7.6 (s, 1H, pyrimidine proton), 7.3 (s, 1H, indolyl proton), 7.2-6.9 (m, 4H, Ar-H), 3.8 (s, 3H, OCH3); m/z: 317 [M]+• 279, 185, 171, 132, 116, 54, 26

Characteristics of 4i

IR (KBr) cm-1: 3396 (O-H), 3341 (NH), 3048 (Ar-H), 1598 (C=N); PMR (DMSO-d6, d in ppm): 11.2 (s, 1H, OH, D2O exchangeable), 10.9 (s, 1H, NH, D2O exchangeable), 9.8 (s, 1H, OH, D2O exchangeable), 8.3-7.7 (m, 4H, Ar-H), 7.5 (s, 1H, pyrimidine proton), 7.3 (s, 1H, indolyl proton), 7.1-6.7 (m, 4H, Ar-H),; m/z: 303 [M]+• 185, 171, 118, 116, 77, 26.

Characteristics of 4j

IR (KBr) cm-1: 3399 (O-H), 3338 (NH), 3088 (Ar-H), 1606 (C=N), 1501 (NO2); PMR (DMSO-d6, d in ppm): 11.3 (s, 1H, OH, D2O exchangeable), 10.6 (s, 1H, NH, D2O exchangeable), 8.3-7.6(m, 4H, Ar-H), 7.4 (s, 1H, pyrimidine proton), 7.3(s, 1H, indolyl proton), 7.2-6.8(m, 4H, Ar-H),; m/z: 332 [M]+• 242, 185, 171, 147, 116, 77, 26.

Characteristics of 5a

IR (KBr) cm-1: 3238 (NH), 3088 (Ar-H), 1609 (C=N), 822 (SH); PMR (DMSO-d6, d in ppm): 12.8 (s, 1H, SH, D2O exchangeable), 10.8 (s, 1H, NH, D2O exchangeable), 8.4 (s, 1H, pyrimidine proton), 8.3-7.6(m, 5H, Ar-H), 7.3(s, 1H, indolyl proton), 7.1-6.8(m, 4H, Ar-H); m/z: 303 [M]+• 201, 102, 116, 78, 54.

Characteristics of 5b

IR (KBr) cm-1: 3424 (NH2), 3235 (NH), 3075 (Ar-H), 1611 (C=N), 824 (SH); PMR (DMSO-d6, d in ppm): 12.9 (s, 1H, SH, D2O exchangeable), 10.7 (s, 1H, NH, D2O exchangeable), 8.4 (s, 1H, pyrimidine proton), 8.3-7.4(m, 4H, Ar-H), 7.3(s, 1H, indolyl proton), 7.0-6.6(m, 4H, Ar-H), 5.31 (s, 2H, NH2, D2O exchangeable); m/z: 318 [M]+• 201, 187, 117, 77, 36.

Characteristics of 5c

IR (KBr) cm-1: 3201 (NH), 3067 (Ar-H), 1568 (C=N), 822 (SH), 528 (C-Br); PMR (DMSO-d6, d in ppm): 12.8 (s, 1H, SH, D2O exchangeable), 10.7 (s, 1H, NH, D2O exchangeable), 7.9 (s, 1H, pyrimidine proton), 8.1-7.5(m, 4H, Ar-H), 7.4(s, 1H, indolyl proton), 7.2- 6.9(m, 4H, Ar-H); m/z: 384 [M+2]+•?, 382 [M]+• 201, 187, 181, 116,77.

Characteristics of 5d

IR (KBr) cm-1: 3237 (NH), 3089 (Ar-H), 1610 (C=N), 824 (SH), 752 (C-Cl); PMR (DMSO-d6, d in ppm): 12.7 (s, 1H, SH, D2O exchangeable), 10.8 (s, 1H, NH, D2O exchangeable), 8.4 (s, 1H, pyrimidine proton), 8.2-7.6(m, 4H, Ar-H), 7.3(s, 1H, indolyl proton), 7.2-6.5(m, 4H, Ar-H); m/z: 340 [M+2]+•?, 338 [M]+• 201, 137, 116, 77.

Characteristics of 5e

IR (KBr) cm-1: 3230 (NH), 3088 (Ar-H), 1609 (C=N), 825 (SH); PMR (DMSO-d6, d in ppm): 12.7 (s, 1H, SH, D2O exchangeable), 10.7 (s, 1H, NH, D2O exchangeable), 9.7 (s, 1H, OH, D2O exchangeable), 9.5 (s, 1H, OH, D2O exchangeable), 8.5 (s, 1H, pyrimidine proton), 8.3-7.7(m, 4H, Ar-H), 7.3(s, 1H, indolyl proton), 7.1-6.7(m, 4H, Ar-H); m/z: 335 [M]+• 187, 134, 116, 77, 54.

Characteristics of 5f

IR (KBr) cm-1: 3237 (NH), 3076 (Ar-H), 1608 (C=N), 825 (SH); PMR (DMSO-d6, d in ppm): 12.9 (s, 1H, SH, D2O exchangeable), 10.8 (s, 1H, NH, D2O exchangeable), 8.4 (s, 1H, pyrimidine proton), 8.2-7.6(m, 4H, Ar-H), 7.2(s, 1H, indolyl proton), 7.2-6.9(m, 4H, Ar-H); m/z: 321 [M]+• 201, 120, 116, 77, 34.

Characteristics of 5g

IR (KBr) cm-1: 3240 (NH), 3090 (Ar-H), 1611 (C=N), 822 (SH); PMR (DMSO-d6, d in ppm): 12.8 (s, 1H, SH, D2O exchangeable), 10.8 (s, 1H, NH, D2O exchangeable), 8.5 (s, 1H, pyrimidine proton), 8.3-7.6(m, 4H, Ar-H), 7.3(s, 1H, indolyl proton), 7.0- 6.7(m, 4H, Ar-H), 2.3 (s, 3H, CH3); m/z: 317 [M]+• 201, 187, 166, 77, 54.

Characteristics of 5h

IR (KBr) cm-1: 3246 (NH), 3089 (Ar-H), 1608 (C=N), 821 (SH); PMR (DMSO-d6, d in ppm): 12.9 (s, 1H, SH, D2O exchangeable), 10.9 (s, 1H, NH, D2O exchangeable), 8.4 (s, 1H, pyrimidine proton), 8.1-7.6(m, 4H, Ar-H), 7.3(s, 1H, indolyl proton), 7.2- 6.6(m, 4H, Ar-H), 3.9 (s, 3H, OCH3); m/z: 333 [M]+• 201, 187, 132, 116, 77, 54.

Characteristics of 5i

IR (KBr) cm-1: 3238 (NH), 3088 (Ar-H), 1614 (C=N), 825 (SH); PMR (DMSO-d6, d in ppm): 12.8 (s, 1H, SH, D2O exchangeable), 10.8 (s, 1H, NH, D2O exchangeable), 9.6 (s, 1H, OH, D2O exchangeable), 8.3 (s, 1H, pyrimidine proton), 8.3-7.4(m, 4H, Ar-H), 7.3(s, 1H, indolyl proton), 7.2-6.8(m, 4H, Ar-H); m/z: 319 [M]+• 201, 187, 156, 118, 116, 77.

Characteristics of 5j

IR (KBr) cm-1: 3231 (NH), 3096 (Ar-H), 1618 (C=N), 1535 (NO2), 824 (SH); PMR (DMSO-d6, d in ppm): 12.7 (s, 1H, SH, D2O exchangeable), 10.7 (s, 1H, NH, D2O exchangeable), 8.4 (s, 1H, pyrimidine proton), 8.0-7.5(m, 4H, Ar-H), 7.3(s, 1H, indolyl proton), 7.1-6.6(m, 4H, Ar-H); m/z: 348 [M]+• 201, 147, 116, 77.

Characteristics of 6a

IR (KBr) cm-1: 3412 (NH2), 3201 (NH), 3096 (Ar-H), 1610 (C=N); PMR (DMSO-d6, d in ppm): 10.7 (s, 1H, NH, D2O exchangeable), , 8.4-7.9 (m, 5H, Ar-H), 7.8 (s, 1H, pyrimidine proton), 7.6-7.5 (m, 4H, Ar-H), 7.3(s, 1H, indolyl proton), 5.9 (s, 2H, NH2, D2O

Characteristics of 6b

IR (KBr) cm-1: 3422 (NH2), 3201 (NH), 3093 (Ar-H), 1610 (C=N); PMR (DMSO-d6, d in ppm): 10.9 (s, 1H, NH, D2O exchangeable), , 8.5-7.9 (m, 5H, Ar-H), 7.8 (s, 1H, pyrimidine proton), 7.6-7.5 (m, 4H, Ar-H), 7.3(s, 1H, indolyl proton), 5.9 (s, 2H, NH2, D2O exchangeable); m/z: 301 [M]+• 184, 146, 116, 77, 34.

Characteristics of 6c

IR (KBr) cm-1: 3404 (NH2), 3214 (NH), 3096 (Ar-H), 1614 (C=N), 531 (C-Br); PMR (DMSO-d6, d in ppm): 10.8 (s, 1H, NH, D2O exchangeable), , 8.4-7.9 (m, 5H, Ar-H), 7.8 (s, 1H, pyrimidine proton), 7.7-7.4 (m, 4H, Ar-H), 7.3(s, 1H, indolyl proton), 6.1 (s, 2H, NH2, D2O exchangeable); m/z: 367 [M+2]+•?, 365 [M]+• 184, 170, 116, 77.

Characteristics of 6d

IR (KBr) cm-1: 3411 (NH2), 3215 (NH), 3089 (Ar-H), 1609 (C=N), 728 (C-Cl); PMR (DMSO-d6, d in ppm): 10.8 (s, 1H, NH, D2O exchangeable), , 8.4-7.9 (m, 5H, Ar-H), 7.8 (s, 1H, pyrimidine proton), 7.6-7.5 (m, 4H, Ar-H), 7.3(s, 1H, indolyl proton), 6.3 (s, 2H, NH2, D2O exchangeable); m/z: 323 [M+2]+•?, 321 [M]+• 184, 137, 116, 77.

Characteristics of 6e

IR (KBr) cm-1: 3412 (NH2), 3203 (NH), 3099 (Ar-H), 1610 (C=N); PMR (DMSO-d6, d in ppm): 10.7 (s, 1H, NH, D2O exchangeable), , 8.5-8.0 (m, 4H, Ar-H), 7.8 (s, 1H, pyrimidine proton), 7.6-7.5 (m, 3H, Ar-H), 7.2(s, 1H, indolyl proton), 6.5 (s, 2H, NH2, D2O exchangeable); m/z: 318 [M]+• 170, 134, 116, 77, 54.

Characteristics of 6f

IR (KBr) cm-1: 3412 (NH2), 3200 (NH), 3095 (Ar-H), 1611 (C=N); PMR (DMSO-d6, d in ppm): 10.9 (s, 1H, NH, D2O exchangeable), , 8.3-7.9 (m, 4H, Ar-H), 7.7 (s, 1H, pyrimidine proton), 7.6-7.5 (m, 4H, Ar-H), 7.3(s, 1H, indolyl proton), 6.0 (s, 2H, NH2, D2O exchangeable); m/z: 304 [M]+• 256, 184, 120, 116, 77.

Characteristics of 6g

IR (KBr) cm-1: 3416 (NH2), 3201 (NH), 3100 (Ar-H), 1610 (C=N); PMR (DMSO-d6, d in ppm): 10.7 (s, 1H, NH, D2O exchangeable), , 8.4-7.9 (m, 4H, Ar-H), 7.8 (s, 1H, pyrimidine proton), 7.6-7.4 (m, 4H, Ar-H), 7.3(s, 1H, indolyl proton), 6.9 (s, 2H, NH2, D2O exchangeable); m/z: 300 [M]+• 256, 184, 170, 116, 77, 34.

Characteristics of 6h

IR (KBr) cm-1: 3408 (NH2), 3220 (NH), 3055 (Ar-H), 1571 (C=N); PMR (DMSO-d6, d in ppm): 10.8 (s, 1H, NH, D2O exchangeable), , 8.2-7.8 (m, 4H, Ar-H), 7.7 (s, 1H, pyrimidine proton), 7.3-7.0 (m, 4H, Ar-H), 7.4 (s, 1H, indolyl proton), 6.8 (s, 2H, NH2, D2O exchangeable); m/z: 316 [M]+• 184, 132, 116, 77, 54.

Characteristics of 6i

IR (KBr) cm-1: 3422 (NH2), 3201 (NH), 3095 (Ar-H), 1615 (C=N); PMR (DMSO-d6, d in ppm): 10.7 (s, 1H, NH, D2O exchangeable), , 8.5-7.9 (m, 4H, Ar-H), 7.8 (s, 1H, pyrimidine proton), 7.7-7.5 (m, 4H, Ar-H), 7.2(s, 1H, indolyl proton), 6.1 (s, 2H, NH2, D2O exchangeable); m/z: 302 [M]+• 184, 170, 118, 116, 77, 54.

Characteristics of 6j

IR (KBr) cm-1: 3410 (NH2), 3216 (NH), 3091 (Ar-H), 1617 (C=N), 1533 (NO2); PMR (DMSO-d6, d in ppm): 11.0 (s, 1H, NH, D2O exchangeable), , 8.4-7.9 (m, 4H, Ar-H), 7.8 (s, 1H, pyrimidine proton), 7.7-7.4 (m, 4H, Ar-H), 7.3(s, 1H, indolyl proton), 5.9 (s, 2H, NH2, D2O exchangeable); m/z: 331 [M]+• 184, 170, 147, 116, 77, 54, 35.

Antiinfl ammatory activity

Carrageenan-induced rat paw oedema method was employed for evaluating the antiinflammatory activity of 4f, 4j, 5b, 5h, 6c and 6d at 200mg/kg body weight in Wistar rats (weighing 150-200 g). The rat paw oedema was produced by the method of Winter et al [8]. The percentage inhibition of inflammation was calculated by applying Newbould formula [9]. The results were found to be statistically significant against control at P< 0.05 by applying Scheffe?s Post Hoc method.

Antioxidant activity

All of the thirty compounds synthesized were analyzed for the antioxidant activity using DPPH radical scavenging method.

Antimicrobial activity

The compounds 4a-j, 5a-j and 6a-j were screened for their antibacterial activity against pathogenic organisms Staphylococcus aureus, Bacillus subtilis Pseudomonas aeruginosa and Escherichia coli at concentration of 5 µg/ml, 10 µg/ml, 50 µg/ml and 100 µg/ml using ciprofloxacin as standard. DMSO was used as solvent control, Muller-Hinton agar medium was used as culture medium. The method employed was cup-plate method [10,11]. The zones of inhibition formed were measured in mm.

Results and Discussion

All the physical characteristics of the synthesized copound are presented in Table 1. The purified products were screened for antiinflammatory, antioxidant and antibacterial activity. Six compounds were screened for antiinflammatory activity using carrageenan-induced rat paw oedema method. Out of them 4f, 5band 6c have shown better antiinflammatory activity than the standard drug, ibuprofen (Table 2). All products were screened for antioxidant activity using DPPH radical scavenging method. Compounds 4b, 4f, 4h, 5b, 5h, 6e and 6h have exhibited good antioxidant activity which is comparable with that of standard drug, ascorbic acid (Table 3). All products were screened for antibacterial activity towards different strains of Staphylococcus aureus, Bacillus subtilis Pseudomonas aeruginosa and Escherichia coli at different concentration of 5 µg/ml, 10 µg/ml, 50 µg/ml and 100 µg/ml compared to standard drug, ciprofloxacin. The compounds 4b, 4c, 4d, 4f, 4h 4j, 5b, 5c, 5d, 5f, 5h, 5j, 6d, 6f, 6h and 6j showed very good zone of inhibition at the concentration of 5-10 µg/ml against different bacteria (Table 4). Based on the structure activity relationships, it can be concluded that the presence of halogen group at 4 positions i.e. 4c, 4d, 4f, 5c, 5d, 5f, 6d and 6f showed good activity towards gram negative bacteria. Presence of nitro group and methoxy group at 4 position i.e. 4h, 4j, 5h, 5j, 6h, and 6j displayed good activity towards Gram positive bacteria.

Comp. R λmax Yield M.P Rf.
code     (%)a (û) Valueb
4a -H 278 77 146-148 0.58
4b -NH2 (p) 261.9 56 198-199 0.60
4c -Br (p) 252.6 48 201-203 0.62
4d -Cl (p) 255.6 64 223-225 0.63
4e -OH (o,p) 279.8 54 226-228 0.64
4f -F (p) 276.4 46 215-217 0.60
4g -CH  (p) 275.4 35 210-212 0.65
4h 3 -OCH3 (p) 277.8 69 286-288 0.59
4i -OH (p) 280.1 53 185-186 0.56
4j -NO2 (p) 281.4 70 256-258 0.61
5a -H 276.8 72 270-272 0.65
5b -NH2 (p) 262.3 65 223-225 0.58
5c -Br (p) 252.8 65 216-218 0.59
5d -Cl (p) 255.6 62 239-240 0.61
5e -OH (o,p) 278.5 76 205-207 0.65
5f -F (p) 275.6 53 310-312 0.59
5g -CH 3 (p) 276.6 29 218-220 0.63
5h -OCH 3 (p) 276.4 68 290-292 0.57
5I -OH (p) 277.8 46 245-247 0.64
5j -NO2 (p) 280.8 76 269-271 0.61
6a -H 277.8 45 248249 0.66
6b -NH  (p) 264.8 63 225-227 0.65
6c 2 -Br (p) 255.0 65 256-258 0.63
6d -Cl (p) 250.8 75 241-242 0.59
6e -OH (o,p) 279.8 50 295-297 0.58
6f -F (p) 276.2 48 247-249 0.66
6g -CH3 (p) 277.1 25 290-292 0.62
6h -OCH3  (p) 277.4 62 223-225 0.58
6i -OH (p) 276.3 49 201-202 0.56
6j -NO2 (p) 279.4 62 270-272 0.57

Table 1: Physical Data of Synthesized Compounds

Group Dose in µg Meanedema±SE % Reduction in edema volume
Control - 0.406±0.047 -
Ibuprofen 200 0.170±0.019a 68.08
4f 200 0.153±0.021ab 69.5
4j 200 0.268±0.022a 32.9
5b 200 0.105±0.019ab 73.7
5h 200 0.188±0.014a 52.6
6c 200 0.126±0.014ab 70.7
6d 200 0.175±0.026a 36.6

Table 2: Results of Antiinflammatory Studies of Compounds

                                            
Compd. % inhibition at various concentrations in µg/ml
1000 500 250 100 50 25 10
4a 62.0 54.3 46.7 39.6 27.6 19.0 13.2
4b 73.1 64.5 52.2 41.3 32.3 26.7 10.2
4c 56.8 49.3 38.8 29.4 20.1 14.6 08.5
4d 49.4 40.2 32.1 25.7 19.7 12.4 06.3
4e 52.5 46.8 37.3 28.9 21.2 16.7 10.7
4f 68.9 57.8 43.2 37.6 29.7 15.1 09.2
4g 50.1 44.8 38.9 33.4 24.3 17.2 09.3
4h 79.6 67.5 60.9 53.5 34.6 24.3 17.6
4i 63.2 56.7 49.8 40.7 29.5 18.3 11.3
4j 50.6 44.8 39.9 30.3 22.8 17.5 10.3
5a 64.3 57.2 47.8 36.5 29.5 19.3 13.2
5b 80.6 67.5 61.9 53.5 34.6 26.3 17.8
5c 55.6 44.5 37.3 25.7 18.6 14.2 10.6
5d 48.9 37.8 29.5 20.7 13.6 10.8 05.5
5e 43.2 33.5 27.4 21.1 17.3 11.4 06.4
5f 58.3 45.9 36.8 28.9 20.7 16.8 11.1
5g 62.3 56.7 45.3 36.7 24.8 16.3 11.3
5h 73.7 66.8 54.5 43.3 30.2 27.8 19.7
5I 54.1 46.3 39.4 27.9 19.8 13.7 08.4
5j 51.5 46.8 35.3 27.9 20.2 17.7 11.7
6a 39.8 33.7 26.8 20.1 14.3 09.8 05.3
6b 54.3 42.5 36.7 25.2 16.6 12.2 09.6
6c 62.8 55.4 42.6 37.7 29.8 21.3 12.1
6d 51.7 46.9 36.8 29.9 21.1 16.9 10.2
6e 79.8 47.9 38.7 28.9 22.3 16.8 11.7
6f 50.7 41.3 32.8 23.2 18.7 11.4 07.3
6g 53.5 46.6 34.3 26.9 21.2 16.8 11.7
6h 78.9 46.8 39.8 27.7 19.6 11.8 07.5
6i 41.2 34.5 27.6 20.5 15.3 10.2 05.1
6j 55.9 43.7 36.3 28.5 21.1 14.3 06.3

Table 3: Results of Antioxidant Studies of Compounds

Compd.   S.aureus       B. subtilis       E.coli

P

  50*   100*   50*   100*   50*   100*   50*
4a 12   16   -   -   14   16   11
4b 22   26   -   09   14   17   17
4c 14   20   -   -   12   14   14
4d 13   19   -   -   -   10   13
4e -   14   -   -   -   -   10
4f 14   18   -   -   14   16   14
4g -   10   -   -   10   15   12
4h 14   21   -   -   12   14   13
4i 12   16   -   10   -   -   10
4j 20   22   13   24   13   16   16
5a 10   13   -   -   10   11   10
5b 16   19   12   16   11   13   13
5c 13   16   -   10   13   18   13
5d 10   17   -   -   11   16   10
5e -   -   -   08   -   -   -
5f 11   19   -   -   14   17   11
5g 12   21   -   -   10   11   -
5h 17   25   10   12   15   16   11
5I 13   19   11   13   13   15   -
5j 10   16   12   19   12   13   15
6a -   10   -   -   11   14   10
6b 18   24   14   18   10   12   -
6c 17   23   -   -   13   17   13
6d 12   19   -   -   10   12   12
6e 11   16   -   10   10   13   10
6f 16   22   09   13   15   16   17
6g 19   14   -   -   -   10   10
6h 23   29   12   16   13   15   16
6i 18   21   -   -   -   11   11
6j 27   31   11   23   12   16   17
Ciprofloxacin 29   33   18   21   17   20   18

Table 4: Results of Antibacterial Studies of Compounds Zone of Growth Inhibition

Acknowledgements

The authors are thankful to IISC, Bangalore for carrying out PMR and elemental analysis. We are also grateful to the Principal, Manipal College of Pharmaceutical Sciences, Manipal, for providing all the facilities.

References