Indian Journal of Chemistry Vol. 52B, August 2013, pp 1176-1181
Note Synthesis and antimicrobial activities of some new 1,2,4-triazole derivatives Shobha Desai*a, Umesh Laddib, Rajani Bennurc & Shankar Bennurc a
Department of Chemistry, SS Arts College and T P Science Institute, Sankeshwar 591 313, India
b
Department of Chemistry, Channabasaveshwar Institute of Technology, NH 206 (BH Road), Gubbi 572 216, India c
Department of Chemistry, Karnatak University, Dharwad 580 003, India E-mail:
[email protected]
Received 6 September 2011; accepted (revised) 13 May 2013 The triazole nucleus is one of the most important and well known heterocycle which is a common and integral feature of a variety of natural products and medicinal agents. The wide spectrum of antimicrobial, antiviral and potent pharmacological activities of triazole and their derivatives has established them as medicinally significant scaffolds. In view of these reports and in continuation of the search for triazole derivatives with better antimicrobial activities, herein is reported the synthesis and antimicrobial activities of some new triazole derivatives with biologically active sulphonamide moiety at the 3rd position. The structures of the newly synthesised compounds have been established on the basis of their spectrochemical data and elemental analysis. All the compounds have been screened for antimicrobial activities against Escherichia coli, Bacillus cirroflagellosus, Aspergillus niger and Colletotrichum capsici by cup plate method using Cotrimoxazole and Diflucan as standards.
Keywords: 1,2,4-Triazoles, antimicrobial activity, sulphonamide, pharmacophore, biologically active
It is well known that, azoles are an emerging class of drugs, wherein either an imidazole or a triazole group is joined to an asymmetric carbon atom as their functional pharmacophore (e.g., Ketaconazole, Fluconazole, Itraconazole, Ravuconazole, Voriconazole and Posaconazole). Encouraged by these systematic serendipitous reports, established pharmacological activities such as antimicrobial1-3,6, antiinflammatory4,5, analgesic6, anticancer7 activities and in continuation of the search for triazole derivatives with better antimicrobial activities8-10, herein is reported the synthesis and antimicrobial activities of some new 1,2,4-triazole derivatives. All the compounds were screened for their antimicrobial activities against Escherichia coli, Bacillus
cirroflagellosus, Aspergillus niger and Colletotrichum capsici by cup plate method using Cotrimoxazole (Trimethoprim 500 mg and Sulphamethoxazole 800 mg) and Diflucan as standards. The synthetic route for the same is depicted as Scheme I. In the present investigation, 3-β-[(N-benzenesulphonyl/tosyl)-4-(un)substituted anilino] ethyl-4anilino-5-mercapto-1,2,4-triazoles 3a-f, were obtained by refluxing potassium salts of β-[(N-benzenesulphonyl/tosyl)-4-(un)substituted anilino]propionyl dithiocarbazinates11 with phenyl hydrazine. Condensation of triazoles 2a-f (Ref 11) with aliphatic/aromatic acids in phosphoryl chloride yielded 3-β-[(N-benzenesulphonyl/tosyl)-4-(un)substituted anilino]ethyl-6-H/CH3/C6H5/p-NO2C6H4s-triazolo(3,4-b)(1,3,4)thiadiazoles 4a-d′. Condensation of two moles of triazoles 2a-f, with one mole of oxalic acid in phosphoryl chloride yielded 6,6′-bis-{β[(N-benzenesulphonyl/tosyl)-4-(un)substituted anilino]ethyl-s-triazolo(3,4-b)(1,3,4) thiadiazoles} 5a-f. The reaction of triazoles 2a-f, with hydrazine hydrate, yielded 3-β-[(N-benzenesulphonyl/tosyl)-4-(un)substituted anilino]ethyl-4-amino-5-hydrazino-1,2,4triazoles 6a-f. 3-β-[(N-Benzenesulphonyl/tosyl)-4(un)substituted anilino]ethyl-s-triazolo(3,4-b)(1,3,4) thiadiazole-6-(5H)-thiones 7a-f, were obtained by refluxing triazoles 2a-f, with alcoholic potassium hydroxide and carbon disulphide. Cyclocondensation of triazoles 2a-f, with benzoin yielded 5(H)-3-β-[(Nbenzenesulphonyl/tosyl)-4-(un)substituted anilino]ethyl-6,7-diphenyl-s-triazolo(3,4-b)(1,3,4)thiadiazines 8a-f. 3-β-[(N-Benzenesulphonyl/tosyl)-4-(un)substituted anilino]ethyl-6-phenyl-s-triazolo(3,4-b)(1,3,4)thiadiazines 9a-f, were obtained by cyclocondensation of triazoles 2a-f, with phenacyl bromide. Synthetic route to the title compounds is depicted in Scheme I. The structures of the newly synthesised compounds were confirmed by elemental and spectral (IR, 1H NMR and mass) analysis. Antimicrobial Activity After establishing the physicochemical properties, all the compounds were screened for antimicrobial activities against Gram negative bacterium Escherichia coli, Gram positive bacterium Bacillus cirroflagellosus and fungi Aspergillus niger and
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Scheme I
Colletotrichum capsici by cup plate method12. Antimicrobial activity was calculated as relative inhibition with reference to the standard. DMF was used as solvent control. Cotrimoxazole (Trimethoprim 500 mg and Sulphamethoxazole 800 mg) and Diflucan (Flucanazole) were used as standards for antibacterial and antifungal respectively. Antimicrobial activity is expressed as relative percent inhibition RI, in comparison with the standard. Majority of the triazole derivatives showed moderate to minimum antibacterial activity against both Escherichia coli, and Bacillus cirroflagellosus. However, the same compounds showed interesting
antifungal activity against Aspergillus niger and Colletotrichum capsici, with more pronounced activity against the Colletotrichum capsici strain. These results substantiate the fact that triazoles are better antifungal agents. Results and Discussion From the antimicrobial activity data of all the triazole derivatives, the following structure activity relationship can be generalised. 3-β-[(N-Benzenesulphonyl/tosyl)-4-(un)substitutedanilino]ethyl-4-anilino-5-mercapto-1,2,4- triazoles 3a-f, have shown greater antifungal activity against C.
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capscici (141.41-261.62%). The same compounds, however have shown minimum to moderate antimicrobial activity in the range of 28.20 to 70.00% RI, against E. coli and A. niger. Same triazoles, however, exhibited either minimum activity (1.2823.38%, RI) or were devoid of any activity against B. cirroflagellosus. Condensation of 3-β-[(N-benzenesulphonyl/tosyl)4-(un)substituted anilino]ethyl-4-amino-5-mercapto(4H)-1,2,4-triazoles11 2a-f, with acids has decreased the antimicrobial activity. Majority of the compounds have shown better antifungal activity against C. capscici (4i, 4w, 4c′ have RI of 126.93). In the same series, only compound 4j has exhibited maximum antibacterial activity (RI of 93.07) against E. coli, Only four compounds 4o, 4b′ (78.76%) and 4h, 4j (RI of 72.2%) have exhibited moderate degree of antifungal activity against A. niger. All the compounds have shown minimum antibacterial activity against. B. cirroflagellosus (RI in the range of 1.28-48.98). Conversion of 3-β-[(N-benzenesulphonyl/tosyl)-4(un)substituted anilino]ethyl-4-amino-5-mercapto(4H)-1,2,4-triazoles 2a-f, to 6,6′-bis-{β-[(N-benzenesulphonyl/tosyl)-4-(un)- substituted anilino]ethyl-striazolo(3,4-b)(1,3,4)thiadiazoles} 5a-f, has lowered the antimicrobial activity. Only compound 5f has shown an increased antifungal activity of RI 188.89% against C. capscici. The same compound has shown moderate degree of antimicrobial activity with RIs of 78.76% and 72.36% against A. niger and B. cirroflagellosus respectively. Condensation of 3-β-[(N-benzenesulphonyl/tosyl)4-(un)substituted anilino]ethyl-4-amino-5-mercapto(4H)-1,2,4-triazoles 2a-f, with hydrazine hydrate to 3β-[(N-benzene-sulphonyl/tosyl)-4-(un)substituted anilino]ethyl-4-amino-5-hydrazino-1,2,4-triazoles 6a-f, has decreased the antimicrobial activity against all the four strains, except 6e (RI : 79.90, 77.55, 72.20 and 156.66 against E. coli, B. cirroflagellosus, A. niger and C. capscici respectively). However, all the compounds have exhibited better antifungal activity, even greater than the standard fluconazole against C. capscici (100-302%). Cyclocondensation of 3-β-[(N-benzenesulphonyl/tosyl)-4-(un)substituted anilino]ethyl-4-amino-5mercapto-(4H)-1,2,4-triazoles 2a-f, to 3-β-[(Nbenzenesulphonyl/tosyl)-4-(un)-substituted anilino]ethyl-s-triazolo(3,4-b)(1,3,4)thiadiazole-6-(5H)-thiones 7a-f, has lowered the activity. Only compounds 7a and 7b have shown better antifungal activity of RI,
206% and 75.76% against C. capscici. All the compounds exhibited minimum antimicrobial activity against E. coli, B. cirroflagellosus and A. niger. 5(H)-3-β-[(N-Benzenesulphonyl/tosyl)-4(un)substituted anilino]ethyl-6,7-diphenyl-s-triazolo (3,4b)(1,3,4)thiadiazines 8a-f, have exhibited better antifungal activity against C. capscici, (RI: 87.54302%). The same compounds however exhibited minimum to moderate antimicrobial activity against E. coli, B. cirroflagellosus and A. niger. Cyclocondensation of 3-β-[(N-benzenesulphonyl/tosyl)-4-(un)substituted anilino]ethyl-4-amino-5mercapto-(4H)-1,2,4-triazoles 2a-f, with phenacyl bromide to 3-β-[(N-benzenesulphonyl/tosyl)-4(un)substituted anilino]ethyl-6-phenyl-s-triazolo(3,4b)(1,3,4)-thiadiazines 9a-f has also reduced the antimicrobial activity, except 9e (RI, 223.91% and 79.1% against C. capscici and E. coli respectively). To summarise, all the compounds have exhibited minimum antibacterial activity against B. cirroflagellosus and minimum to moderate antimicrobial activity against E. coli and A. niger. However, the same compounds have exhibited significant antifungal activity against C. capscici. Experimental Section Melting points were determined in open capillaries and are uncorrected. IR spectra in KBr, were recorded on a Perkin-Elmer Spectrophotometer and 1H NMR spectra on a Varian 300 MHz NMR spectrometer using TMS as an internal standard (chemical shift in δ, ppm). Mass spectra were recorded on Finnigan Mat 8230 spectrometer. The starting materials, potassium salt of β-[(Nbenzenesulphonyl/tosyl)-4-(un)substituted anilino]propionyl dithiocarbazinates 1a-f, and 3-β-[(Nbenzenesulphonyl/tosyl)-4-(un)substituted anilino]ethyl-4-amino-5-mercapto-(4H)-1,2,4-triazoles 2a-f, were prepared as per the literature methods11. Structures of the compounds were established on the basis of elemental and spectral analysis. Physicochemical properties of all the newly synthesised compounds are enumerated in Table I and spectral details (IR, 1H NMR and mass spectra) are followed by the procedures. General procedure for the preparation of 3-β-[(Nbenzenesulphonyl/tosyl)-4-(un)-substituted anilino]ethyl-4-anilino-5-mercapto-1,2,4-triazoles 3a-f Potassium salt of β-[(N-benzenesulphonyl/tosyl)-4(un)substituted anilino]propionyldithio-carbazinate(s)
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Table I — Physicochemical characterization of compounds 3a-f – 9a-f* Compd
R1
R2
R3
m.p. (°C)
Yield (%)
Compd
R1
R2
R3
m.p. (°C)
Yield (%)
4b′ 4c′ 4d′
Cl Cl Cl H H CH3 CH3 Cl Cl H H CH3 CH3 Cl Cl H H CH3 CH3 Cl Cl H H CH3 CH3 Cl Cl H H CH3 CH3 Cl Cl
CH3 CH3 CH3 H CH3 H CH3 H CH3 H CH3 H CH3 H CH3 H CH3 H CH3 H CH3 H CH3 H CH3 H CH3 H CH3 H CH3 H CH3
C6H5 CH2C6H5 p-NO2C6H4 — — — — — — — — — — — — — — — — — — — — — — — — — — — — — —
130-32 95-96 216-18 200-02 182-84 188-89 172-74 128-29 116-17 170-72 204-05 152-54 140-42 110-11 120-21 158-60 196-98 198-99 202-04 108-10 190-92 70-71 68-70 75-76 92-93 85-86 90-91 139-40 124-25 198-99 >300 100-01 97-98
34 37 36 60 65 60 64 60 62 70 75 60 64 50 52 68 69 60 64 63 62 40 39 40 54 49 52 35 49 48 50 49 50
H H — 170-71 30 3a H CH3 — 90-91 35 3b CH3 H — 80-82 34 3c CH3 CH3 — 145-46 32 3d Cl H — >300 31 3e Cl CH3 — 130-32 28 3f H H H 95-96 30 4a H H CH3 105-06 40 4b H H C6H5 111-12 45 4c H H CH2C6H5 96-97 40 4d H H p-NO2C6H4 179-80 50 4e H CH3 H 165-67 40 4f H CH3 CH3 97-98 38 4g H CH3 C6H5 196-97 39 4h H CH3 CH2C6H5 160-61 37 4i H CH3 p-NO2C6H4 155-56 36 4j CH3 H H 170-72 34 4k CH3 H CH3 150-51 35 4l CH3 H C6H5 >290 34 4m CH3 H CH2C6H5 >280 32 4n CH3 H p-NO2C6H4 170-71 37 4o CH3 CH3 H 160-62 36 4p CH CH CH 165-66 35 4q 3 3 3 CH3 CH3 C6H5 190-92 37 4r CH3 CH3 CH2C6H5 220-21 34 4s CH3 CH3 p-NO2C6H4 180-81 37 4t Cl H H 195-96 37 4u Cl H CH3 198-99 35 4v Cl H C6H5 215-17 34 4w Cl H CH2C6H5 178-79 36 4x Cl H p-NO2C6H4 175-76 38 4y Cl CH3 H 180-81 34 4z 4a′ Cl CH3 CH3 138-39 35 *All the compounds gave satisfactory C, H and N analysis
1a-f (0.01 mol) was dissolved in water (10 mL). To this clear solution was added phenyl hydrazine (0.01 mol) and the resulting mixture was refluxed at 15560°C in an oil bath for 5-6 hr (till the evolution of H2S gas ceased). The clear solution was treated with decolourising charcoal, filtered, cooled in ice and then carefully acidified with acetic acid. The precipitated solid was filtered, washed with water, dried and purified by recrystallization from ethanol. Yield: 28-
5a 5b 5c 5d 5e 5f 6a 6b 6c 6d 6e 6f 7a 7b 7c 7d 7e 7f 8a 8b 8c 8d 8e 8f 9a 9b 9c 9d 9e 9f
35%; 3c: 1H NMR (DMSO-d6): δ 2.30 (s, 3H, CH3), 2.40 (s, 1H, NH), 2.90 (t, 2H, CH2CH2), 3.89 (t, 2H, CH2CH2), 6.87-7.63 (m, 14H, ArH); 3d: IR (KBr): 3100 (NH), 2970 (CH2), 1640 (>C=CC=NC-N), 1230, 1150 (>C=S), 1380 cm-1 (SO2); MS: m/z (%) 463 (25), 450 (35), 428 (25), 412 (29), 390 (63), 274 (60), 235 (65), 155 (40), 106 (27), 91 (60), 79 (42), 63 (15); 3e: IR (KBr): 3300 (NH), 2920 (CH2), 1660 (>C=CC=NC-N), 1230, 1160 (>C=S), 1380 cm-1 (SO2); 1 H NMR: (DMSO-d6): δ 2.50 (s, 1H NH), 2.94 (t, 2H, CH2CH2), 3.99 (t, 2H, CH2CH2), 6.90-7.65 (m, 14H, ArH). General procedure for the preparation of 3-β-[(Nbenzenesulphonyl/tosyl)-4-(un)substituted anilino]ethyl-6-H/CH3/C6H5/p-NO2C6H4-s-triazolo(3,4-b)(1,3,4)thiadiazoles, 4a-d′ A mixture of 3-β-[(N-benzenesulphonyl/tosyl)-4(un)substituted anilino]ethyl-4-amino-5-mercapto-(4H)1,2,4-triazoles 2a-f, (0.005 mol), formic acid 100% (0.5 mL) and POCl3 (8 mL) were heated together at 100°C in an oil bath for 1 hr. The reaction mixture was cooled, poured onto crushed ice and the pH of the solution was brought to 7 by the addition of liquor ammonia. The precipitated solid was filtered, washed thoroughly with cold water, dried and purified by recrystallization from absolute alcohol. 4f: IR (KBr): 2930 (CH2), 1630 (>C=CC=NC-N), 1340 cm-1 (SO2); 4h: 1H NMR (DMSO-d6): δ 2.40 (s, 3H, CH3), 2.00 (t, 2H, CH2CH2), 3.90 (t, 2H, CH2CH2), 6.8 - 8.20 (m, 14H, ArH); 4m: IR (KBr): 2920 (CH2), 1600 (>C=CC=NC-N), 1340 cm-1 (SO2); 4o: IR (KBr): 2920 (CH2), 1680 (>C=CC=NC-N), 1390 cm-1 (SO2); MS: m/z (%) 496 (23), 454 (60), 390 (63), 346 (19), 307 (19), 274 (17), 261 (23), 239 (58), 193 (25), 154 (92), 136 (83), 107 (40), 91 (71),77 (48), 65 (21); 4v: 1H NMR (DMSO-d6): δ 2.5 (s, 3H, CH3), 2.95 (t, 2H, CH2CH2), 3.89 (t, 2H, CH2CH2), 6.65-7.95 (m, 9H, ArH). General procedure for the preparation of 6,6′-bis{β-[(N-benzenesulphonyl/tosyl)-4-(un)substituted anilino]ethyl-s-triazolo(3,4-b)(1,3,4)thiadiazoles}, 5a-f The title compounds 5a-f, were synthesised by reacting a mixture of triazole, 2a-f, (0.02 mol), oxalic acid (0.01 mol) and POCl3 (8 mL) following the above procedure. 5f: IR (KBr): 2920 (CH2), 1625 (>C=CC=NC-N), 1380 cm-1 (SO2); 1H NMR (DMSO-d6): δ 2.45 (s, 3H, CH3), 2.60 (t, 2H, CH2CH2), 3.40 (t, 2H, CH2CH2), 7.1-7.80 (m, 8H, ArH); MS: m/z (%) 563 (17), 406 (10), 360 (38), 139 (16), 127 (40), 91(25). General procedure for the preparation of 3-β-[(Nbenzenesulphonyl/tosyl)-4-(un) substituted anilino]ethyl-4-amino-5-hydrazino-1,2,4-triazoles, 6a-f Triazole, 2a-f, (0.01 mol) and hydrazine hydrate 99% (0.01 mol) in absolute ethanol (50 mL) were
refluxed on a water bath, till the evolution of H2S ceased. The solid obtained was filtered, washed with cold water, dried and purified by recrystallization from ethanol to obtain the crystals of 6a-f. 6c: IR (KBr): 3520, 3220 (NH2), 3010 (NH), 2880 (CH2), 1630 (>C=CC=NC-N), 1390 cm-1 (SO2); 1H NMR (DMSO-d6): δ 2.33 (s, 3H, CH3), 4.66 (s, 2H, NH2), 11.31 (br,s, 1H, NH), 2.30 (t, 2H, CH2CH2), 3.98 (t, 2H, CH2CH2), 6.88-7.60 (m, 9H, ArH); 6a: MS: m/z (%) 344 (10), 307 (20), 289 (15), 246 (20), 234 (50), 136 (45), 120 (15), 106 (25), 93 (20), 79 (30), 65 (15), 51 (10). General procedure for the preparation of 3-β-[(Nbenzenesulphonyl/tosyl)-4-(un)-substituted anilino]ethyl-s-triazolo(3,4-b)(1,3,4)thiadiazole-6-(5H)thiones, 7a-f A mixture of triazole, 2a-f (0.01 mol), CS2 (0.015 mol) and a solution of KOH (0.015 mol) in absolute ethanol (50 mL) was refluxed until the evolution of H2S ceases (8 hr). The reaction mixture was concentrated, cooled and poured onto crushed ice and carefully acidified with acetic acid. The resultant precipitate was filtered, washed with cold water, dried and purified by recrystallization from ethanol. 7b: IR (KBr): 2915 (CH2), 1620 (>C=CC=NC-N), 1380 (SO2), 3210 (NH), 1190, 1100 cm1 (>C=S); MS: m/z (%) 375 (19), 260 (25), 154 (33), 136 (44), 106 (48), 91 (48), 77 (46), 39 (27); 7c: IR (KBr): 2900 (CH2), 1610 (>C=CC=NC-N), 1380 (SO2), 3210 (NH), 1190,1100 cm1 (>C=S); 1H NMR (DMSO-d6): δ 2.34 (s, 3H, CH3), 4.70 (s, 1H, -NH-C=S), 3.27 (t, 2H, CH2CH2), 4.05 (t, 2H, CH2CH2), 6.82-7.69 (m, 9H, ArH). General procedure for the preparation of 5(H)-3β-[(N-benzenesulphonyl/tosyl)-4-(un)-substituted anilino]ethyl-6,7-diphenyl-s-triazolo(3,4-b)(1,3,4)thiadiazines, 8a-f A mixture of triazole, 2a-f, (0.01 mol) and benzoin (0.01 mol), in absolute ethanol (30 mL) was heated to get a clear solution. To this hot solution, 2N KOH (5 mL) was added and the reaction mixture was refluxed for 5 hr and cooled poured on crushed ice. The light yellow precipitate that separated, was filtered, dried and purified by recrystallization from aqueous ethanol (80%). 8b: IR (KBr): 2920 (CH2), 1670 (>C=CC=NC-N), 1340 cm-1 (SO2); 8e: 1H NMR (DMSO-d6): δ 2.60 (t, 2H, CH2CH2), 3.89 (t, 2H, CH2CH2), 6.30 (s, 2H, -S-CH2-), 6.90-8.02 (m, 14H, ArH); MS: m/z (%) 494 (33), 439 (35), 362 (37),
NOTES
306 (29), 192 (52), 157 (56), 117(83), 79 (87), 39 (90). General procedure for the preparation of 3-β-[(Nbenzenesulphonyl/tosyl)-4-(un)-substituted anilino]ethyl-6-phenyl-s-triazolo(3,4-b)(1,3,4)thiadiazines, 9a-f A suspension of triazoles 2a-f, (0.01 mol) and phenacyl bromide (0.013 mol) in absolute ethanol (25 mL) was heated under reflux for 8 hr. Thereafter, aqueous Na2CO3 was added and the reaction mass cooled. The precipitated solid was filtered, dried and purified by recrystallization from ethanol. 9b: IR (KBr): 3320 (NH), 3070 (CH2), 1665 (>C=CC=NC-N), 1320 cm-1 (SO2); MS: m/z (%) 550 (15), 507 (20), 421 (60), 364 (15), 313 (16), 289 (42), 249 (25), 211 (65), 154 (42), 89 (25), 79 (52), 63 (12), 51 (12), 39(12). Acknowledgement Authors thank the Heads of RSIC-CDRI, Lucknow, TIFR-Mumbai and RSIC-IIT, Mumbai for spectral assistance. Authors SRD and UVL are thankful to UGC and CSIR-New Delhi for financial assistance in the form of FIP and SRF respectively. The authors
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thank the Chairman, Department of Chemistry, Karnatak University, Dharwad for providing necessary facilities. References 1 Mathew V, Keshavayya J, Vaidya V P & Giles D, Eur J Med Chem, 42(6), 2007, 823. 2 Parmar K, Prajapati S, Patel R, Joshi S & Patel R, Int J Chem Tech Res, 3(2), 2011, 761. 3 Bonde C G, Peepliwal A & Gaikwad N J, Arch Pharm (Weinheim), 343(4), 2010, 228. 4 El Shehry M F, Abu-Hashem A A & El-Telbani E M, Eur J Med Chem, 45(5), 2010, 1906. 5 Mathew V, Keshavayya J & Vaidya V P, Eur J Med Chem, 41(9), 2006, 1048. 6 Ravindra K C, Vagdevi H M & Vaidya V P, Indian J Chem, 47B, 2008, 1271. 7 Sunil D, Isloor A M & Shetty P, Der Pharma Chemica, 1(2), 2009, 19. 8 Hosur M C, Talawar M B, Laddi U V, Bennur R S & Bennur S C, Indian J Chem, 34B, 1995, 707. 9 Talawar M B, Laddi U V, Somannavar Y S, Bennur R S & Bennur S C, Indian J Heterocycl Chem, 5, 1996, 215. 10 Laddi U V, Desai S R, Bennur R S & Bennur S C, Indian J Chem, 40B, 2001, 828. 11 Desai S R, Laddi U V, Bennur R S, Patil P A & Bennur S C, Indian J Pharm Sci, 73(1), 2011, 115. 12 Seely H W & Van Demark P J, Microbes in Action (Freeman W H and Co, San Francisco, USA), 1972.
