Note. Antibacterial and antitubercular activities of some diphenyl hydrazones and semicarbazones

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1 Indian Journal of Chemistry Vol. 49B, ctober 00, pp ote Antibacterial and antitubercular activities of some diphenyl hydrazones and semicarbazones A S aja*, A K Agarwal, Mahajan, S Pandeya & S Ananthan Pharmaceutical Chemistry esearch Laboratory, Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi 005, India asraja.phe@itbhu.ac.in Tuberculosis Antimicrobial Acquisition and Coordinating Facility, Southern esearch Institute, Birmingham, Alabama, USA eceived March 008; accepted (revised) 9 January 00 Condensation of phenoxy or 4-bromophenoxy acetic acid hydrazide and substituted aryl semicarbazides with appropriate carbonyl compounds yield corresponding hydrazones and semicarbazones, respectively. The chemical structure of the synthesized compounds has been confirmed by UV, I and H M spectral data. All the compounds have been investigated for their antibacterial activity against ten pathogenic strains and antitubercular activity against Mycobacterium tuberculosis H7 v. Compound 4f, Acetic acid, phenoxy-,[-(4-chlorophenyl) ethylidene] hydrazide has been found to exhibit 80% inhibition in the preliminary antitubercular screening (MIC >6.5 μg/ml). Keywords: Hydrazones, semicarbazones, synthesis, antibacterial, antitubercular Tuberculosis (TB) ranks among the most important burdens on human health, not only due to the large number of cases (~9 million/year worldwide) but also because about one quarter of sufferers dies most of them young adults. Drug resistance and multidrugresistant tuberculosis is perceived as a growing hazard to human health worldwide. The perceived threat of drug-resistant TB is enormous. The biggest menace is multidrug-resistant TB caused by strains resistant to at least IH and rifampicin, the two principal first line drugs used in combination chemotherapy. Therefore, the development of potent new antitubercular drugs without cross resistance with known antimycobacterial properties is urgently needed. Semicarbazones with HCH =C< pharmacophore have been reported to possess a wide range of biological properties such as antimicrobial,4, antiviral 5 anti-hiv 6 and anticonvulsant 7 actions. Several thiosemicarbazides 8, hydrazides 9 and hydrazones 0, and its cyclic derivatives were identified as potent antitubercular agents. In view of these facts and in continuation of our research work -5, we prepared a new series of hydrazones and semicarbazones bearing -CH CH=C and -CH=CH functionalities, respectively. ecently, we reported the anticonvulsant activity of some of these diphenyl hydrazones and 4- nitrophenyl semicarbazones 6. The diphenyl hydrazones were prepared in a study of structural modification of semicarbazones and found to possess less or no anticonvulsant activity than semicarbazones. In extension of our earlier work, the diphenyl hydrazones and semicarbazones were further evaluated for their antimicrobial properties and the results are presented here. The diphenyl hydrazones (4a-r) were synthesized from phenoxy or 4-bromophenoxy acetic acid as depicted in Scheme I. The semicarbazones were obtained from substituted anilines as shown in Scheme II. The synthesized compounds were investigated for their antimicrobial activity against Staphylococcus aureus, Aeromonas hydrophilia, Bacillus subtilis, Vibrio cholerae, Enterococcus faecalis, Shigella flexeneri, Enterobacter, Plesiomonas shigelloids, Klebsiella pneumoniae, Escherchia coli and Mycobacterium tuberculosis H7 v. esults and Discussion Synthesis of phenoxy or 4-bromophenoxy acetic acid hydrazones (Scheme I) was carried out by following the method reported by us earlier 6. The 4- substituted phenyl semicarbazones were obtained by following the Scheme II. A solution of 4-substituted aniline and sodium cyanate was stirred together to precipitate substituted phenyl urea. Hydrazinolysis of the urea with hydrazine hydrate gave good yield (78%) of semicarbazides, which were further on treatment with appropriate carbonyl compound yielded the semicarbazones (8a-d). The spectral analysis of all the compounds was done by I and H M and the spectral data were consistent with the assigned structures. The MIC data of the compounds, which have shown activity against the tested microbes, are

2 TES 85 C H C Me CH H a: H b: Br Conc. H S 4 C H C H H Glacial CH CH 4a-4r 4a:,, ; 4b:,,, =H 4c:,, ; = CH 4d:,, ; = CH 4e:,, ; 4f:, ; = CH ; 4g:, ; = CH ; 4h:,, ; = (CH) 4i:, ; = CH ; 4j:, ; = CH ; = Ph 4k: = Br;, ; 4l: = Br;,, 4m: = Br;,, 4n: = Br; = CH ;, 4o: = Br; ;, 4p: = Br; = CH ; ; = Ph 4q: = Br; = CH ; ; = 4r: = Br; = CH ;, Scheme I H H ac 5 Glacial CH CH 6 H H 8a-8d 4 H Glacial CH CH 4 H H 7 8a: ; = CH ;, 4 ; = CH 8b: = F;,, 4 ; = CH 8c: = F; ;,, 4 = CH 8d: = ; ;, 4, = CH Scheme II

3 86 IDIA J. CHEM., SEC B, CTBE 00 Table I MIC (μg/ml) data of the compounds tested against bacterial strain(s) Micro organisms Compounds 4a 4b 4c 4d 4f 4h 4i 4n 4r 8b TM SM Staphylococcus aureus Aeromonas hydrophilia Bacillus subtilis Vibrio cholerae Enterococcus faecalis Shigella flexeneri Enterobacter Plesiomonas shigelloids Klebsiella pneumoniae Escherchia coli *The ( ) sign indicates that the compounds were active at MIC of more than 5000 μg/ml. Compounds 4e, 4g, 4j, 4k, 4l, 4m, 4o, 4p, 8a, 8c and 8d have shown no activity in the initial screening against all strains tested. reported in Table I. It has been observed that most of the diphenyl hydrazones have exhibited mild to moderate antibacterial activity towards the selected bacterial strains and the results were comparable to standard drugs used. Compounds 4a, 4c, 4f, 4n and 8b have exhibited broad spectrum activity by showing activity against both Gram-negative and Grampositive bacteria at MIC ranging from.5 to 5000 μg/ml. In general, it was observed that the hydrazones (4a-r) exhibited better antibacterial profile than that of semicarbazones (8a-d). The in vitro antitubercular screening was conducted at 6.5 μg/ml against Mycobacterium tuberculosis H7 v (ATCC 794) in BACTEC B medium using a broth micro dilution method, the Microplate Alamar Blue Assay (MABA). The MIC and percentage inhibition data of the compounds, which have shown activity against the pathogenic bacterium, are presented in Table II. The compounds 4f, 4c and 4d exhibited substantial antitubercular activity in the initial screening, particularly 4f Acetic acid, phenoxy-[-(4-chlorophenyl) ethylidene] hydrazide showed 80% inhibition (MIC >6.5 μg/ml) and emerged as the most active compound in the present series. Experimental Section The melting points were determined in open capillary tubes using Thomas Hoover melting point apparatus and are uncorrected. The purity of the Table II Percentage inhibition of compounds against Mycobacterium tuberculosis H7 v at MIC (6.5μg/mL) Compd* % Inhibition 4a 6 4b 4 4c 6 4d 44 4e 8 4f 80 4g 9 4h 5 4i 4j 7 4k 9 4l 6 4n 0 4p 9 4q 5 4s 5 8a 7 8b ifampin 95 * Compounds 4m, 4o, 4r, 8c and 8d showed no inhibition at a dose of >6.5 μg/ml. The standard drug rifampin was tested at 0.5μg/mL. compounds was checked by TLC using chloroform and methanol (9:). UV spectra were scanned on JASC model 7800 UV/VIS spectrophotometer.

4 TES 87 Infra-red spectra were determined on JASC FT/I- 500 Infrared spectrophotometer by KBr disc method. H M spectral studies were done on Jeol FX90Q FT spectrophotometer using DMS-d 6 and CDCl as solvents and TMS as internal reference. Synthesis The synthesis of diphenyl hydrazones (4a-r) was accomplished by the condensation of phenoxy or 4- bromophenoxy acetic acid hydrazide with appropriate carbonyl compound (Scheme I). The physicochemical characterization data of diphenyl hydrazones were reported by us earlier 6. The semicarbazones (8a-d) were obtained from appropriate 4-substituted aniline by the reaction with sodium cyanate and followed by treatment with hydrazine hydrate to yield corresponding phenyl urea and phenyl semicarbazide respectively. The treatment of later with appropriate aryl aldehyde yielded the corresponding semicarbazones (Scheme II). The representative synthetic procedures along with the chemical data of intermediates and final products are described below. Synthesis of substituted phenylurea The substituted aniline (0. mole) was dissolved in glacial acetic acid (0 ml) and diluted with water (00 ml). To this, an equimolar quantity (0. mole) of sodium cyanate in warm water (50 ml) was added with constant stirring. The reaction-mixture was allowed to stand for hr and the solid precipitate formed was filtered off and dried after recrystallization from boiling water. 4-Chloro--methylphenylurea 6a: Yield 78%, m.p. 8 C; 4-itrophenylurea 6b: Yield 78%, m.p. 40 C; 4-Fluorophenylurea 6c: Yield 74%, m.p. 67 C; I (KBr): 40 ( -H), 755 (-C=), 80 cm - (C 6 H 5 -F); H M (CDCl ): δ 6 (s, H, C 6 H 4 - H ), (m, 4H, p-f-c 6 H 4 ), 0.09 (s, H, - CH-, D exchangeable). Synthesis of substituted phenyl semicarbazides 7,8 To a solution of substituted phenylurea (0.0 mole) in ethanol (0 ml), an equimolar quantity of hydrazine hydrate was added. The reaction-mixture was made alkaline by adding sodium hydroxide. The reaction-mixture was refluxed for - hr and the precipitate obtained after cooling was filtered under suction and recrystallized from ethanol. 4-Chloro-(-methyl)-phenylsemicarbazide 7a: Yield 78%, m.p. 90 C; (KBr): 70 ( -H), 85 (CH -), 68 (-C=), (C-), 05 (C 6 H 5 -Cl), 845 cm - (C 6 H 5 -H); H M (CDCl ): δ.4 (s, H, - -CH CH C), (m, 4H, p-cl-c 6 H 4 ), 8.8 (s, H, -CH-, D exchangeable). 4-itrophenylsemicarbazide 7b: Yield 78%, m.p C; 4-Fluorophenylsemicarbazide 7c: Yield 78 %, m.p. 6 C; I (KBr): 40 ( -H), 755 (-C=), 70 cm - (C 6 H 5 -F); H M (CDCl ): δ 6 (s, H, C 6 H 4 -H, D exchangeable), (m, 4H, p-f-c 6 H 4 ), 0.09 (s, H, -CH-, D exchangeable). Synthesis of semicarbazones 8a-d A mixture of substituted phenyl semicarbazide (0.0 mole) and appropriate carbonyl compound (0.0 mole) in ethanol (0 ml) was refluxed together in the presence of few drops of glacial acetic acid. After hr, the precipitate obtained was filtered off and recrystallized from ethanol. -methyl-4-chlorophenyl-,(4'-methoxyphenylmethylene) semicarbazide 8a: Yield 55%, m.p. 60 C, I (KBr): 70 ( o -H, amide), 85 (CH - ), 68 (-C=), (C-), 05 (C 6 H 5 -Cl), 845 cm - (C 6 H 5 -H); H M (CDCl ): δ.4 (s, H, - CH ),.9 (s, H, -CH ), (s, 4H, -CH - C 6 H 4 ), (m, 4H, p-cl-c 6 H 4 ), 9.59 (s, H, - CH-, D exchangeable), Anal. Calcd for C 6 H 6 Cl : C, 50.0; H, 4.9;,.00. Found: C, 50.6; H, 4.;, 0.90%. 4-Fluorophenyl-,(4'-methoxyphenylmethylene) semicarbazide 8b: Yield 74%, m.p. 78 C, I (KBr): 40 ( o -H, C 6 H 5 -H), 0 ( o -H, amide), 75 (-C=), 570 (C=), 45 (C 6 H 5 -F), 80 cm - (C 6 H 5 - H); H M (CDCl ): δ.95 (s, H, -CH ), 6.0 (s, H, C 6 H 5 -H), (s, 4H, p-ch --C 6 H 4 ), (m, 4H, p-f-c 6 H 4 ), 0.09 (s, H, -CH-, D exchangeable), Anal. Calcd for C 6 H 6 Cl : C, 55.5; H, 4.0;,.9. Found: C, 55.7; H, 4.0;,.8%. 4-Fluorophenyl-,(',4',5'-trimethoxyphenylmethylene) semicarbazide 8c: Yield 8%, m.p. 40 C, I (KBr): 480 ( o -H, C 6 H 5 -H), 70 ( o - H, amide), 78 (-C=), 60 (C=), 76 (C 6 H 5 -F), 840 cm - (C 6 H 5 -H); H M (CDCl ): δ.95 (s, H, - CH ), 6.0 (s, H, C 6 H 5 -H), (s, 4H, p- (CH ) -C 6 H 4 ), (m, 4H, p-f-c 6 H 4 ), 0.09 (s, H, -CH-, D exchangeable). Anal. Calcd for C 6 H 6 Cl : C, 58.7; H, 5.8;,.09. Found: C, 58.66; H, 5.4;,.4%. 4-itrophenyl-,(',4',5'-trimethoxyphenylmethylene) semicarbazide 8d: Yield 90%, m.p. 0 C, I (KBr): 400 ( o -H, C 6 H 5 -H), 70 ( o -H,

5 88 IDIA J. CHEM., SEC B, CTBE 00 amide), 70 (-C=), 60 (C=), 55 (C 6 H 5 -), 855 cm - (C 6 H 5 -H); H M (CDCl, ): δ.95 (s, H, - CH ), 6.0 (s, H, C 6 H 5 -H), (s, 4H, p- (CH ) -C 6 H 4 ), (m, 4H, p- -C 6 H 4 ), 0.09 (s, H, -CH-, D exchangeable), Anal. Calcd for C 6 H 6 Cl : C, 54.50; H, 4.80;, Found: C, 54.55; H, 4.7;, 4.88%. Antibacterial screening The in vitro antibacterial activity against ten pathogenic strains of bacteria was performed by agar double dilution technique 9. The microbial strains were procured from the Department of Microbiology, Institute of Medical Sciences, BHU, Varanasi. The stock solution of standard and test compounds was prepared in dimethyl sulfoxide (DMS) and subsequent dilutions were made with the same solvent. Mueller Hint Agar Media (Hi Media) was used to subculture various strains of microbes. ormal saline was used to prepare the inoculum of the bacteria to be used for the antibacterial study. Under aseptic conditions, the diluted test solutions with different concentration (5000 to 78. μg/ml) were added to the vials (previously sterilized) containing 00 discs and the disc were placed on the numbered plates. The plates were then incubated at 7 C for 4 hr. The MIC, the lowest concentration of the test drug that completely inhibited the growth of bacteria, determined and is presented in Table I. Sulphamethoxazole (SM) and trimethoprim (TM) were used as standard drugs. Antitubercular screening The in vitro antitubercular screening was carried out at Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF), Southern esearch Institute, Birmingham, Alabama. The primary screening was conducted at 6.5 μg/ml against Mycobacterium tuberculosis H7 v (ATCC 794) in BACTEC B medium using a broth micro dilution assay, the Microplate Alamar Blue Assay (MABA) 0. Compounds exhibiting fluorescence were tested in the BACTEC 460 radiometric system. The MIC and the percentage inhibition data of the compounds are reported in Table II. eferences Dye C, Williams B G, Espinal M A & aviglions M C, Science, 95, 00, 04. Tomioka H, Kekkaku, 77, 00, 57. Pandeya S, Souwmyalakshmi V & ath G, Indian Drugs, 8(), 00, Desai M J & Desai K K, Asian J Chem, (), 999,07. 5 Asis S E, Bruno A M, Conti G M & Gaozza C H, Farmaco, 5(6), 996, Mishra V, Pandeya S, De Clercq E, Pannecouque C & Witvrouw M, Pharm Acta Helv, 7(4), 998, 5. 7 Dimmock J & Baker G D, Epilepsia, 5(), 994b, Wahab A, Arzneimittelforschung, 9(), 979, Baneryu A, Dubnau E, Quemard A, Balasubramaniam V, Um K S, Wilson T, Collins, De Lisle G & Jacobs W Jr, Science, 6, 994, 7. 0 Dilanyen E & Arsenyam F G, Khim Farm Zh, 0(6), 996, 6. Buu-Hoi g Ph, Xuang g D, am g D, Binon F & oyer, J Chem Soc, 95, 58. za H, Joshi D & Parekh H, Indian J Chem, 7B, 998, 8. Pandeya S, aja A S & Stables J P, J Pharm Pharmaceut Sci, 5(), 00, 4. 4 Pandeya S, aja A S & ath G, Indian Drugs, 4(), 00, 4. 5 Pandeya S, aja A S & ath G, Indian J Chem, 45B, 006, Pandeya S, Agarwal A K, Singh A & Stables J P, Acta Pharmaceutica, 5 (), 00, 5. 7 Pandeya S, Manjula H & Stables J P, Pharmazie, 56(), 00,. 8 Pandeya S, Mishra V, Ponnilavarasan I & Stables J P, Pol J Pharmacol, 5, 000, 8. 9 Barry A, Antibiotics in Laboratory Medicine, edited by V Lorian, P.. (The Williams and Wilkins Co, Baltimore), Collins L A & Franzblau S G, Antimicrob Agents Chemother, 4, 997, 004.