SYNTHESIS, CHARACTERIZATION AND ANTI-FUNGAL ACTIVITY OF SOME NOVEL THIOSEMICARBAZIDES

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1 IJP 2014, 4(2), oopa et al. I: ITEATIAL JUAL F EEA I PAMAY AD EMITY Available online at esearch Article YTEI, AATEIZATI AD ATI-FUGAL ATIVITY F ME VEL TIEMIABAZIDE. oopa*, J. aravanan,. Mohan and ekha Parmesh Department of Pharmaceutical hemistry, PE ollege of Pharmacy, Bangalore-50, Karnataka, India. ABTAT 2-amino-3--(substituted carboxanilido)-4, 5-disubstituted thiophene J 1A 1L were synthesized using versatile Gewald reaction. First step was preparation of substituted cyanoacetanilides (omp. o J a, b & c) which were carried out by condensation of substituted anilines and cyano acetate which was then reacted with Methylenic ketone, ammonium acetate, glacial acetic acid, benzene, sulphur to produce 2-amino-3--(substituted carboxanilido)-4, 5-disubstituted thiophenes (J 1A 1L). Later the compound 2-amino-3--(substituted carboxanilido)-4,5-disubstituted thiophenes in DMF were treated with a & carbon di sulfide further the mixture was stirred with hydrazine hydrate to yield twelve substituted new thiosemicarbazides (J 2A-2L). The compounds were characterized I, 1 M spectral data and screened for anti-fungal activity. Keywords: Thiophenes, Thiosemicarbazides, pectral analysis, Antifungal activity. ITDUTI Most of the therapeutic agents are heterocyclic compounds; hence heterocyclic chemistry has been the most fruitful area for drug discovery. Among the heterocyclic compounds sulphur containing moieties have attracted maximum attention as they have several pharmacological activities as antimicrobial 1,2,5,7,8, antifungal 6,9,11, antibacterial 4, anticonvulsant 10, antitumor 13, antiinflammatory 14, antioxidant 12 activity and so on. imilarly thiosemicarbazide 6,7 derivatives also have been reported to possess various biological activities as antifungal 6,9,11. The therapeutic importance of these rings promoted us synthesize novel benzo (b)thiophenes by Gewald 3 reaction and their thiosemicarbazide derivatives. haracterize the compound by I and 1 M spectroscopic techniques and evaluate them for their antifungal activity. MATEIAL AD METD hemicals ubstituted anilines (o-anisidine, p-anisidine and o-toluene), ethyl cyanoacetate, glacial acetic acid, methylinic ketones (ethyl methyl ketone, cyclopentanone, cyclohexanone, cycloheptanone), benzene, ammonium acetate, sodium sulfate, sulphur, diethyl amine, ethanol, dimethyl formamide, sodium hydroxide, carbon di sulfide and ydrazine hydrate were obtained from local dealer. All other chemical used were of laboratory grade. Preparation of substituted cyanoacetanilides(omp. o J a, b & c) A mixture of substituted anilines (0.50 M) and ethyl cyano acetate (56.5 ml; 0.50 M) were taken in a conical flask, mixed well and heated on a heating mantle at º for 5-6 hrs. Then the reaction mixture was left at room temperature for overnight. The solid obtained was collected, washed with ethanol and dried. ecrystallization was done by acetone: water mixture 445

2 IJP 2014, 4(2), oopa et al. I: Preparation of 2-cyano-2-(methylidene)-substituted carboxanilides. A mixture of substituted cyano acetanilide (0.04 M), appropriate methylenic ketone (4.6 ml; 0.04 M), ammonium acetate (1 g) and glacial acetic acid (2 ml) in benzene (100 ml) was refluxed with an arrangement for continuous separation of water involving dean stark apparatus. After 8 hrs the reaction mixture was cooled, diluted with 10 ml benzene and washed with sodium carbonate solution (10% w/v in water) and water successively and dried over anhydrous sodium sulphate. The solvent was removed under vacuum. The intermediate crude product obtained was immediately processed for the next step. Preparation of 2- amino-3-- substituted carboxanilido - 4,5- disubstitutedthiophenes (omp. no. J 1A- 1L) To a mixture of 2-yano 2- (methylidene) - - substituted carboxanilides in alcohol (30 ml) was added sulphur (1.28 g; 0.04 M) with stirring maintaining the temperature between 40-45º during addition. Then to the reaction mixture, diethyl amine (4.0 ml) was added drop wise with stirring. The reaction mixture was stirred for 1 hr at 40-45º and chilled overnight. The solid obtained was filtered, washed with ethanol and recrystallized from isopropyl alcohol. Preparation of synthesis of 2-(3 thiosemicarbazide) -3-ubstituted carboxanilido-4,5-disubstituted thiophenes (J 2A- 2L) To a solution of 2-amino-3--(substituted carboxanilido)-4,5-disubstituted thiophenes (0.005M) in DMF (10 ml) was added a (0.01 M) & carbon di sulfide (0.75 ml). The mixture was stirred at for 1 hr. While stirring the reaction mixture was added hydrazine hydrate (0.01M) & stirring continued at 60 0 for 1hr more. n adding water a pale yellow solid separated out which is recrystallized from DMF: water. ATI-FUGAL TUDIE All the synthesized compounds were screened for their antifungal activity by agar diffusion method 15 at a conc of 50µg/ml against Aspergillus niger and candida albicans. After 24 hours of drug addition, Zone of inhibition was measured and recorded. Miconazole itrate at 50µg/ml was used as standard in the experiment. EULT From the antifungal activity results it was observed that all the compounds influenced the activity. Among the drugs tested for antifungal activities Table 5 showed that compounds J- 2A, J-2, J-2E, and J-2I exhibited potent activity by showing zone of inhibition ranging from 15mm-18mm. All these drugs showed potent activity against andida albicans and Aspergillus niger with high zone of inhibition. All other drugs showed moderate inhibititory properties against the test organism. Miconazole nitrate exhibited potent inhibitory properties against the entire test organism. DIUI From the I, 1 M, and Mass spectrum obtained, characterization of data has been done and given in table 2, 3 and 4. The formation of the new series of thiosemicarbazides were confirmed by the shift of I peaks between cm -1 as seen in the starting materials to cm -1 in the final compounds indicating the aryl cyclization due to the cyclic keto group and the presence of prominent peaks between cm -1 of - 2 in thiosemicarbazides compared to their starting compounds is sufficient to explain the formation of the new thiosemicarbazides. The M spectra of J-2D and J-2E, were indicated the formation of new compounds. The sharp singlet peaks at δ (ppm) = (J- 2D), δ = (J-2E), indicated the presence of -- group in the compounds. It will indicate the formation of new compounds. 446

3 IJP 2014, 4(2), oopa et al. I: EME ubstituted anilines cyano acetate ubstituted cyano acetanilide Methylinic ketone J a, b & c 3 / 3 4 Benzene, refluxed for 8hrs + DMF 2 a amino-3--substituted carboxanilido-4,5- disubstituted thiophenes (J 1A-1L) 1 hr stirring , ( 2 5 ) cyano-2(methylidine)--substituted carboxanilides Unstable compound 1 2 2, hr stirring (3' thiosemicarbazide)-3-substituted carboxamido-4,5-disubstituted thiophenes (J 2A-2L) Where: = p- 3, o- 3, o- 3 1, 2 = - 3, - ( 2 ) 3, - ( 2 ) 4, - ( 2 )

4 IJP 2014, 4(2), oopa et al. I: Table 1: Physical data of 2-yano-2-(methylidene) --substituted carboxanilides (J a, b & c), 2- amino-3--substituted carboxanilido-4,5-disubstituted thiophenes (J 1A 1L) and 2-(3 thiosemicarbazide) -3-ubstituted carboxanilido-4,5- disubstituted thiophenes (J 2A-2L) r. o. omp. o. tructure ecrystalization olvent M.W. (g) M.P. () % Yield TL olvent ystem f 1 J-a Me 2 acetone: water hloroform: acetate(8:2) 0.28 Me 2 J-b 2 acetone: water hloroform: acetate(8:2) J- 2 acetone: water hloroform: acetate J-1A Me Isopropyl alcohol hloroform: acetate J-1B Me 2 Isopropyl alcohol hloroform: acetate J-1 Me Isopropyl 2 alcohol hloroform: acetate J-1D Me 2 Isopropyl alcohol hloroform: acetate J-1E M e Isopropyl alcohol hloroform: acetate J-1F Me 2 Isopropyl alcohol hloroform: acetate

5 IJP 2014, 4(2), oopa et al. I: J-1G Me 2 Isopropyl alcohol hloroform: acetate J-1 Me 2 Isopropyl alcohol hloroform: cetate J- 2A Me DMF:Water (8:2) J-2B Me 2 DMF:Water J- 2 Me J-2D 16 J-2E 17 J-2F Me 2 Me 2 3 Me (9.5:0.5)

6 IJP 2014, 4(2), oopa et al. I: J-2G 19 J Me Me (8:2) J-2I J-2J J-2K 3 2 DMF:Water (9.2:0.8) J-2L 3 2 DMF:Water

7 IJP 2014, 4(2), oopa et al. I: omp. o. Table 2: (pectral data) of 2-yano-2-(methylidene) --substituted carboxanilides (J a, b and c) tructure λ max (nm) I (KBr) cm -1 1 M (DM) J-a Me (--); (Ar- ); (Ali-); (-); (=); (=); (bend); (-); (-). J-b Me (--); (Ar- ); (Ali-); 2250 (); 1697 (=); 1541 (bend); 1543(=); 1123(-); 824(-) (-) (--); (Ar- 3 ); (Ali-); 2263,24 J-c (); (=); (-bend); (-); 877(-); 723.4(-). Table 3: (pectral data) 2- amino-3--substituted carboxanilido-4,5-disubstituted thiophenes (J 1A 1L) General structure omp. o. tructure λ max (nm) I (KBr) cm -1 1 M (DM) J-1A Me & (- 2); (--); (Ar- ); (Ali-); (=); (=); (-). J-1B Me & (-2); 3294 (--); 3024 (Ar-); 2929 (Ali-); 1656 (=); 1521 (=); 1242 (-). J-1 Me & (- 2); 3270 (--); 3043 (Ar-); 2941 (Ali-); 1658 (=); 1522 (=); 1239 (-); 824(-). 451

8 IJP 2014, 4(2), oopa et al. I: J-1D J-1E J-1F Me Me Me & (- 2); 3267 (--); 3143 (Ar-); (Ali-); 1652 (=); 1527 (=); 1239 (-) & (- 2); (--); (Ar- ); (Ali-); (=); (=); (-) & (- 2); (--); (Ar- ); (Ali-); (=); (=); (-). 8.52(s, 1, -, g); 7.53 (d, 2, Ar-, h,k); 6.84 (d, 2, Ar-, i,j); 5.36 (s, 2, 2, f); 3.78 (s, 3, - 3, ); 2.81(t, 2, - 2-, e); 2.61 (t, 2, - 2-, a); 1.83 (s, 2, - 2-, d); 1.65 (t, 4,- 2,b,c). 8.44(d, 1, Ar-,h ); 8.29 (s, 1,-, d); 7.02(m, 2, Ar-, f,g); 6.89(d, 1, Ar-,e)5.95 (br, 2, 2, c); 3.89 (s, 3, - 3, ); 2.33(s, 3, - 3, b); 2.20 (s, 3, - 3, a). J-1G Me & (- 2); (--); (Ar- ); (Ali-); (=); (=); 1170 (-); 836 (-). J-1 Me (--); 3011 (Ar-); 2972 (Ali-); 1647 (=); 1540 (=); 1292 (-); 824 (-). J-1I & (- 2); (--); (Ar- ); (Ali-); (=); (=); 826 (-). J-1J & 3328(- 2); 3239 (- -); (Ar-); 2927 (Ali -); 1659 (=); 1540 (=); 823 (-). J-1K & (- 2); (-); (Ar- ); 2924 (Ali-); 1643 (=); 1540 (=); 827 (-). J-1L (--); 2927 (Ali-); 1669 (=); 1566 (=); 1220(-); 1086 (- amine); 832 (-). 452

9 IJP 2014, 4(2), oopa et al. I: Table 4: 2-(3 thiosemicarbazide) -3-ubstituted carboxamido-4,5- substituted thiophenes(j 2A-2L) General structure J 2A-2L omp. o. tructure λ max (nm) I (KBr) cm -1 1 M (Dl 3) J-2A Me & (- 2); 3288 (--); 3040 (Ar-); (Ali-); (=); 1518 (=); 1253 (-); 799.6(-). - J-2B Me & (- 2); 3294 (--); 3024 (Ar- ); 2929 (Ali-); 1656 (=); 1521 (=); 1242 (-). - J-2 Me & 3298(- 2); 3250(--); 3034 (Ar-); (Ali-); 1668(=); 1522 (=); 1239 (-); 809(-). J-2D Me & (- 2),3267 (--); 3143 (Ar-); 2941 (Ali-); 1652 (=); 1527 (=); 1239 (-0); 820 (-) (s, 1, --,g); 9.0(s, 1, -, f); 8.53 (s, 1, -, 5); 7.53 (d, 2, Ar-, 1,4); 6.84 (d, 2, Ar-, 2,3); 5.36 (s, 2, 2, h); 3.78 (s, 3, - 3, ); 2.81(t, 2, - 2-, e); 2.61 (t, 2, - 2-, a); 1.83 (s, 2, - 2-, d); 1.65 (t, 4,- 2,b,c). J-2E M e & (- 2) (--); (Ar-); (Ali-); (=); 1553 (=); 1259 (-0); 816 (- ) (s, 1, --,d); 9.10(s, 1, -, c); 8.44(d, 1, Ar-,4); 8.29 (s, 1,-,5); 7.02(m, 2, Ar-, 2,3); 6.89(d, 1, Ar-,1); 5.95 (br, 2, 2, e); 3.89 (s, 3, - 3, ); 2.33 (s, 3, - 3, b); 2.20 (s, 3, - 3, a). 453

10 IJP 2014, 4(2), oopa et al. I: J-2F Me & (- 2); 3326 (--); 3027(Ar- );2924(Ali-); 1650 (=); 1532 (=); (-); 814 (-). J-2G J-2 Me Me & 3245(- 2); 3239 (--); 3026 (Ar-); 1656 (=); 1539 (=); 1246 (-); 831 (-) & (- 2); 3254 (--); 3011 (Ar- ); 2972 (Ali-); 1647 (=); 1540 (=); 1292 (-); 822 (-). J-2I & (- 2); (-); (Ar-);2998(Ali- ); 1656(=); (=); (- ); (-). J-2J J-2K J-2L & (- 2); 3279 (--); 3098(Ar-); (Ali-); 1659 (=); 1540 (=); 1246 (-); 824 (-) & (- 2);3322(--); (Ar-); 2924 (Ali- ); 1643 (=); 1540 (=); 1220 (-); 815 (-) & (- 2); (--); (Ar- ); (Ali-); (=); (= ); 1220(-); 1086 (- amine); (-). 454

11 IJP 2014, 4(2), oopa et al. I: Table 5: Antifungal activity data of 2-(3 thiosemicarbazide) -3-ubstituted carboxamido-4,5- substituted thiophenes (J 2A-2L) J 2A-2L Zone of inhibition (mm) ompound ode 1& 2 Aspergillusniger andida albicans J 2A p- 3 ( 3) J 2B p- 3 ( 2) J 2 p- 3 ( 2) J 2D p- 3 ( 2) J 2E o- 3 ( 3) J 2F o- 3 ( 2) J 2G o- 3 ( 2) J 2 J 2I J 2J J 2K J 2L o- 3 o- 3 o- 3 o- 3 o- 3 ( 2) 5 ( 3) 2 ( 2) 3 ( 2) 4 ( 2) 5 Miconazole nitrate Dose concentration: 50 μg/0.1 ml A : o activity ontrol : DMF (Dimethyl formamide) LUI From the antifungal results, it was observed that the presence of methyl group on heterocyclic nucleus as in J-2A, 2, 2E and 2I exhibited considerable activity against Aspergillus niger and andida albicans employed on par with the standards used. AKWLEDGEMET The authors are thankful to Management, PE ollege of Pharmacy for providing necessary facilities. EFEEE 1. aravanan J et al. ynthesis of some 3- substituted amino-4,5- tetramethylene 455

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