Anticancer And Antibacterial Studies Of New Asymmetric Triazoles

A key intermediate 4,5-diphenyl-4H-1,2,4-triazole-3-thiol(1) was synthesized from base catalyzed dehydrative cyclization of phenyl thiosemicarbazide. The 1,2,4-triazole-3-thiol (1) was converted to 3-thio acid hydrazide (3a,b) via 3-thio ethyl esters (2a,b) intermediates. Then 3-thiol acid hydrazide (3a, b) were converted to asymmetric bis-1,2,4-triazole (5a-l) via thiosemicarbazide intermediate (4a-l). The synthesized compounds were characterized by IR and NMR spectral studies.Synthesized compounds 5a-l were evaluated for in vitro antioxidant activity by DPPH radical scavenging assay method and evaluation of in vitro anticancer activity by MTT assay method against three cancer cell lines, HT29 (human adenocarcinoma), EAC (Ehrich Ascites carcinoma) and MDA-231 (Human breast cancer).

All the synthesized compounds were subjected to in vitro antibacterial activity against Bacillus subtilus (ATCC 6633), Staphylococcus aureus (ATCC-25923), Escherichia coli (ATCC-25922), and Pseudomonas aeruginosa (ATCC- 27853) and their zone of inhibition is determined.IntroductionCancer is the second leading cause of death worldwide, following heart diseases. World Health Organization has estimated 12 million deaths worldwide due to cancer in 2030. Although progress has been steadily made in cancer research to reduce mortality and improve survival, cancer still accounts for nearly 1 in every 4 deaths in the world (Jemal A et al 2009). Triazoles are known to have a large spectrum of potential anticancer, antimitotic and antifungal properties (yashwant et al 2010). Dimeric analogues of various heterocyclic compounds are drawing much attention in the recent past.

Many dimeric compounds designed as bis-DNA intercalators were evaluated as anti-cancer agents. Dimers of more Dimers of more lipophilic compounds have shown potent and broad spectrum activity against human solid tumor cell lines both in culture and as xenografts in nude mice. Some of the bis-intercalators were found to possess high selective toxicity against human colon carcinoma (Yong et al., 2005; Denny, 2003). Asymmetric Synthesis (Fucheng Qu et.al 1999), anticancer (Shivarama Holla et. al 2001, Spicer et al., 2000; Dabholkar and Ansari, 2008), (Al-Masoudi et al 2004), (De-Chang Zhang et al 2006), (Krzysztof Sztanke et al. 2008).

Many of the bis- 1,2,4-triazoles have also been reported to possess wide spectrum of biological activity (Holla et al., 2000; Holla et al., 2002; Ghorab et al., 2000; Al-Soud and Al-Masoudi, 2004). The triazole derivatives vorozole, letrozole, and anastrozole are non-steroidal drugs used for the treatment of breast cancer (Clemons et al., 2004).Based on the above literature, the authors have tried to design the bis-triazole derivatives by incorporating various modifications using groups such as substitution of other heterocyclic ring at 5th position through oxyalkyl or alkyl linkages (Demirbas et al 2004), Doubling the heterocyclic ring as well as replacement of chlorine by fluorine or by ethoxy, methoxy and by isopropyl groups.

On the other hand, the effectiveness of commercially available anti-microbials has become unreliable, due to the emergence of resistant microorganisms like methicillin resistant Staphylococcus aureus (MRSA), chloroquineresistant Plasmodium falciparam, multi-drug-resistant Mycobacterium tuberculosis, and vancomycin-resistant Enterococcus faecium (VRE) (Rostom et al., 2009). Hence such type of infections continue to be the driving force for the search and discovery of novel, more potent and selective non-traditional anti-microbial agents with the less likeliness of development of cross-resistance. Based on these reports, efforts are being made in the synthesis and biological activity of symmetric and asymmetric bis heterocycles, in particular 1,2,4- triazoles.

In view of these finding, we propose to synthesized asymmetric bis-1,2,4-triazole and evaluate for in vitro anticancer and antibacterial activity.Results and discussionChemistrySynthesis of the intermediate and target compounds was accomplished according to the steps depicted in Scheme 1. A key intermediate 4,5-diphenyl-4H-1,2,4-triazole-3-thiol (1) was synthesized from base catalyzed dehydrative cyclization of phenyl thiosemicarbazide. The compound 4, 5-diphenyl-4H-1,2,4-triazol-3-ylthio-ethylesters (2a,b) were synthesized from 4,5-diphenyl-4H-1, 2, 4-triazole-3-thiol (1) by treating it with ethyl chloroacetate/ ethyl chloroformate using dry acetone and K2CO3. Refluxing (2a, b) with excess of hydrazine hydrate in absolute ethanol afforded respective acid hydrazides (3a, b) in good yield.

The compound (4a-l) was prepared by treating the acidhydrazides (3a, b) with using six different substituted isothiocyanates namely phenyl, p-flurophenyl, p-chlorophenyl, p-tolyl, isopropyl and p-ethoxyphenyl isothiocyanate. The target compounds (5a-l) were synthesized by base catalyzed dehydrative cyclization of (4a-l). The structures of all the compounds were elucidated on the basis of elemental analysis, IR, 1H-NMR, 13C-NMR, and mass spectral data.SCHEME OF SYNTHESISThe IR spectra of compound 1 exhibits the absence of signals at 1660 cm−1 corresponds C=O, thus confirming the formation of cyclized compound.

Further, the1H-NMR spectrum shows the complex multiplet for aromatic protons appears between 7.02-7.89ppm. The broad singlet at 14.12 ppm is due to NH attached to C=S reveals the further confirmation the formation of compound. In the 13C-NMR spectrum of the same compound, the signals at 153.22 and 169.56 ppm were due to the C3 and C5 of the triazole moiety.

The chemical shifts at 130.74, 129.86, 128.74 and 127.15 were assigned to the aromatic carbon atoms of the phenyl ring. The mass spectrum shows molecular ion peak (M+) and base peak at 253 m/z.The IR spectrum of compounds 2a exhibits the presence of signals at 1728 cm−1 indicating the presence of a C=O, thus confirming the formation of ester compound. 1H-NMR spectrum of compound 2a the complex multiplet for aromatic protons appears between 7.33-7.56ppm.

The singlet at 4.08ppm integrates for 2H of CH2 group; where as the quadrate at 4.14ppm integrates for 2H of OCH2 group. The triplet at 1.20ppm accounts for the 3H of CH3 group. In the 13C-NMR spectrum of the same compound, the chemical shift was observed at 169.50 ppm, this was assigned to the carbonyl carbon of the ester compound.

The signals at 147.62 and 153.10 ppm were due to the C3 and C5 of the triazole moiety. The chemical shifts at 130.77, 129.91, 128.84 and 127.50ppm were assigned to the aromatic carbon atoms of the phenyl ring. The signals at 60.06 and 14.13 ppm were assigned to carbon atom of the -CH2 and -CH3 groups of the diethyl carbamoyl side chain. The carbon atoms of -SCH2 of the side chain resonated at 32.50 ppm.The IR spectrum of the 3a exhibits the absorption band for C=O group (amide) at 1660 cm−1 and for NH group at 3320 cm-1 indicates the formation of the compound 3a. The 1H-NMR Spectrum 3a shows the complex multiplet for aromatic protons appears between 7.32-7.55ppm.

The doublet at 4.28-4.31ppm integrates for 2H of NH2 group; whereas the singlet at 9.32ppm integrates for 1H of NH. The singlet at 3.89ppm accounts for the 2H of SCH2 group. The absence of signals for alkyl group and presence of peaks for NH, NH2 group confirms formation of compound 3a. The 13C-NMR spectrum of the compound 3a showed a signal at 170.34 ppm due to the -CONH functional group. The signal at 153.18 and 147.64 ppm was attributed to the C3 and C5 of the triazole moiety.

The chemical shifts at 130.94, 129.71, 128.80, and 127.18 were assigned to the aromatic carbon atoms of the phenyl ring. The carbon atom of -SCH2 of the side chain resonated at 40.92 ppm.The IR spectrum of the 4a exhibits signals at 1703 cm-1 which indicating the presence of a C=O. The 1H-NMR spectrum of the 4a shows the complex multiplet for aromatic protons appears between 7.02-7.55ppm. A singlet at 4.12ppm accounts for the 2H of SCH2 group and three singlet peaks of thiosemicarbazide appears between 10.21-11.20ppm indicates the formation compounds. This further confirmed the formation of compound 4a.

The 13C-NMR spectrum of the compound 4a showed a signal at 170.34 ppm due to the -CONH functional group, it also shows the signal at 181.53 due to C=S. The signal at 153.18 and 147.64 ppm was attributed to the C3 and C5 of the triazole moiety. The chemical shifts at 137.14, 130.73, 129.34, 128.67, and 127.53, 126.45 and 124.82 were assigned to the aromatic carbon atoms of the phenyl ring.

The carbon atom of -SCH2 of the side chain resonated at 41.52 ppm.The IR spectrum of the 5a exhibits the absence of signals at 1703 cm−1 indicating the lack of a C=O, thus confirming the formation of cyclized compound. The 1H-NMR spectrum of 4a exhibits the complex multiplet for aromatic protons appears between 7.22-7.55ppm. The broad singlet at 13.84ppm is due to NH attached to C=S.

The absence of NH singlet of the thiosemicarbazide and presence of NH attached to C=S peak, further confirms the formation of compound 5a. The 13C-NMR spectrum of the compound 5a showed a signal at 168.92 and 161.32 ppm which was attributed to the C3 and C5 of one of the triazole moiety and other triazole moiety shows signals at 153.76 and 148.73 ppm. The chemical shifts at 130.28, 129.61, 128.54, and 127.82ppm were assigned to the aromatic carbon atoms of the phenyl ring.

The carbon atom of -SCH2 of the side chain resonated at 27.92 ppm.Similar explanation for assigning the carbon holds good for the rest of the compounds. In conclusion, 1H-NMR and 13C-NMR spectral data were consistent with the proposed structures. The mass spectra of all the triazole derivatives were analyzed under ESI conditions.

Molecular ions were observed in the form of M+. Most of the compounds yield abundant molecular ions in the form of M+ peaks. Similarly, the elemental analyses of all the compounds have been performed and the data given under the physical data in Table 1.Table 1 Physical data of synthesized compoundscompoundRnMol. wtMol. formulayieldMP(0C)Elemental analysis calculated(found)CH1

-

-

253C14H11N3S75202-20466.38(66.21)4.38(4.29)2a

-

1339C18H17N3O2S64116-11863.70(63.62)5.05(4.98)2b

-

0312C16H14N3O2S65104-10661.52(61.63)4.52(4.40)3a

-

1325C16H15N5OS72184-18659.06(58.95)4.65(4.55)3b

-

0311C15H13N5OS65202-20457.86(57.72)4.21(4.15)4aPhenyl1460C23H20N6S2O83212-21459.98(59.83)4.38(4.23)4b4-fluorophenyl1478C23H19N6S2OF85240-24257.72(57.55)4.00(3.87)4c4-chlorophenyl1494C23H19N6S2OCl78232-23455.81(55.68)3.87(3.69)4d4-methoxyphenyl1490C24H22N6S2O282252-25458.76(58.65)4.52(4.41)4e4-ethoxyphenyl1504C25H24N6S2O275262-26457.91(57.85)4.32(4.21)4fisopropyl1426C20H22N6S2O72236-23859.38(59.25)4.76(4.78)4gPhenyl0446C22H18N6S2O78222-22459.19(59.30)3.98(3.92)4h4-fluorophenyl0464C22H17N6S2OF73230-23255.09(55.24)4.78(4.65)4i4-chlorophenyl0481C22H17N6S2OCl78238-24056.73(56.87)3.94(4.03)4j4-methoxyphenyl0476C23H20N6S2O270242-24457.47(57.65)4.76(4.65)4k4-ethoxyphenyl0490C24H22N6S2O268254-25655.09(55.24)4.56(4.68)4lisopropyl0412C19H20N6S2O70218-22058.74(58.83)4.92(4.78)5aPhenyl1442C23H18N6S278190-19462.42(62.30)4.10(4.02)5b4-fluorophenyl1460C23H17FN6S271220-22259.98(59.80)3.72(3.60)5c4-chlorophenyl1477C23H17ClN6S274226-22860.73(60.58)4.18(4.04)5d4-methoxyphenyl1472C24H20N6OS264232-23461.00(60.89)4.27(4.15)5e4-ethoxyphenyl1486C25H22N6OS270242-24461.71(61.55)4.56(4.40)5fisopropyl1408C20H20N6S267216-21858.80(58.45)4.93(4.8205gPhenyl0428C22H16N6S272252-25461.66(61.47)3.76(3.58)5h4-fluorophenyl0446C22H15FN6S274210-21259.18(59.04)3.39(3.25)5i4-chlorophenyl0463C22H15ClN6S270228-23060.43(60.29)3.84(3.70)5j4-methoxyphenyl0458C23H18N6OS268236-23860.24(60.12)3.96(3.78)5k4-ethoxyphenyl0472C24H20N6OS270226-22861.23(61.11)4.27(4.15)5lisopropyl0394C19H18N6S268218-22057.84(57.69)4.60(4.43)BIOLOGICAL ACTIVITYIn vitro antioxidant activityIn the present study, asymmetric bis-1,2,4-triazoles derivatives were evaluated for their free radical scavenging activity using the DPPH radical assay method. Reduction of DPPH radicals can be measured at 517nm (Roopan et al 2008, Yukesk et al 2006).

Different derivatives of bis-1,2,4-triazoles reduced DPPH radicals significantly. The activity of bis-1,2,4-triazoles derivatives was compared with ascorbic acid as standard. Compounds 5a, 5b, 5c, 5d, 5g, 5h, 5i and 5k shows % scavenging activity ranging from 53-85%. The tested compounds shown more than 50% of radical scavenging activity were selected for in vitro anticancer activity.Table 2 Antioxidant activity data of the synthesized compounds (5a-l)S.No.Compounds% Free Radical Scavenging Activityat 40 µg/ml15a8525b7235c6545d5355e3465f4275g6385h6095i64105j28115k69125l3913Ascorbic acid (std)94In vitro anticancer activityThe synthesized compounds 5a, 5b, 5c, 5d, 5g, 5h, 5i and 5k were screened for in vitro anticancer activity concentration against three human cancer cell lines, respectively HT29 (human adenocarcinoma), EAC (Ehrich Ascites carcinoma) and MDA-231 (Human breast cancer) by MTT assay method (Molinari et al., 2009, Manjula et al., 2009).

5-flurouracil was used as standard and DMSO was used as solvent control. The percentage inhibition and IC50 of all the tested compounds are given in table 3. Based on the IC50 value it has been observed that compounds shows good to poor anticancer activity at different concentration. Among the tested compound 5a, 5b and 5c shows good activity against EAC cancer cell line, where as compound 5a and 5b shows moderate activity against HT-29 and MDA-231 cancer cell line. The compound 5a with phenyl group at the 4th position of 1,2,4-triazole ring system shows IC50 value 203.36 µg/ml against the EAC (ehrich ascities carcinoma), which is lesser than the standard drug(IC50 454.48 µg/ml). At the 4th position of 1,2,4-triazole ring with 4-flurophenyl substitution 5b shows IC50 value 181.31 µg/ml, with p-methoxy phenyl 5c shows IC50 value 228.32 µg/ml.

It is observed that substitution on the phenyl ring increases the anticancer activity against EAC cancer cell line, where as the compound 5a with phenyl group at the 4th position of 1,2,4-triazole ring system shows IC50 value 55.49 µg/ml against the HT-29 (adenocarcinoma) and IC50 value 98.57 µg/ml against the MDA-231(breast cancer) which are more than the standard drug (IC50 value 10.04 µg/ml). At the 4th position of 1,2,4-triazole ring, with 4-flurophenyl substitution 5b shows IC50 value 46.66 µg/ml and 126 µg/ml against HT-29 and MDA-231 cancer cell lines.

It is observed that substitution on the phenyl ring shows moderate activity against HT-29 and MDA-231 cancer cell line.Table 3: Anticancer activity data of synthesized compoundsS.No.Cpd codeConc.µg/mlHT-29(adenocarcinoma)EAC(ehrich ascites carcinoma)MDA-231(breast cancer)% cytoto-xicityIC50µg/ml% cytoto-xicityIC50µg/ml% cytoto-xicity1Standard(5-flurouracil)20090.0010.0424.27454.4880.1710080.9816.3069.355067.8311.4143.262552.639.8030.131039.643.3819.8825a20081.2955.4946.54203.3670.3510073.6434.1760.745064.2927.6443.122532.1714.1328.731028.048.7419.1635b20082.7446.6650.39181.3160.3410071.1338.2753.425062.7429.1340.122542.3917.1227.311029.348.3417.6345c20054.23166.5642.13228.3237.8310040.1732.7430.795029.3424.7318.392517.1315.149.591010.327.134,5455d20034.73277.7021.72460.2315.0410028.6316.7815.245024.389.569.732511.674.325.41102.372.364.9365g20027.04281.3219.12604.6236.7310017.7110.2124.12509.238.6717.13259.845.5611.13108.323.89-0.3875h20021.04324.7917.12588.7523.7310017.7112.6520.12508.2310.6713.13257.848.5610.13106.325.893.3885i20016.04389.4314.12679.3920.7310012.7110.6517.12506.237.6710.13253.843.567.1310-0.321.894.3895k20012.04465.7612.73712.6516.261008.219.2911.18503.346.948.174251.782.545.2910-1.321.322.89In vitro antibacterial activityThe synthesized compounds were evaluated for their in vitro antibacterial activity against S.aureas, B.substilus, E.coli, P.aeruginosa by cup plate method (Rostom et al 2009., Hugo et al 1997). Ampicillin was used as standard and DMSO was used as solvent control.

The antibacterial activity data of the synthesized compounds are given in Table 4. The tested compounds show good to moderate antibacterial activity at 100 μg/ml concentration with the zone of inhibition in the range of 05-25 mm, the standard drug ampicillin has a zone of inhibition in the range of 28-32 mm for the four bacterial species. Among the tested compounds 5b, 5d show good antibacterial activity with zone of inhibition in the range of 21-26 mm at 100mg/ml concentration against the tested organisms. 5a, 5c, 5h and 5i show moderate antibacterial activity with zone of inhibition in the range of 16-23 mm at 100mg/ml concentration against the tested organisms. Rest of the derivatives shows weak antibacterial activity with the zone of inhibition in the range of 05-14 mm at 100mg/ml concentration against the tested organisms.Table 4: Antibacterial activity data of the synthesized compounds (5a-l)S.

No.Cpd. CodeZone of inhibition (in mm)S.aureasB.substilusE.coli15a16181725b25232235c20211745d22211955e12131665f08100975g13111485h21191995i201719105j050609115k121610125l17191513Ampicillin282726ConclusionA series 5-[(4,5-diphenyl-4H-1,2,4-triazol-3-ylthio)methyl]-4-substituted-4H-1,2,4-triazole-3-thiol (5a-f), 5-(4,5-diphenyl-4H-1,2,4-triazol-3-ylthio)-4-substituted-4H-1,2,4-triazole-3-thiol (5g-l) were synthesized as per the scheme I. The synthesized compounds were characterized by IR, 1H-NMR data and mass spectral data and consistent with the proposed structure. The in vitro anticancer activity of the synthesized compounds was determined by MTT assay method against the three human cancer cell line namely HT-29 (human adenocarcinoma), EAC (ehrich ascites carcinoma) and MDA-231 (human breast cancer) and the synthesized compounds were evaluated for their in vitro antibacterial activity against S.aureas, B.substilus, E.coli, P.aeruginosa by cup plate method.

The antimicrobial and cytotoxic results suggest that synthesized compounds 5a and 5b could be considered as possible dual antimicrobial-anticancer candidates that deserve further investigation and derivatization in order to explore the scope and limitation of their biological activities.ExperimentalMaterials and methodsThe melting points were determined in open glass capillaries and are uncorrected. IR spectra were recorded on Shimadzu FT-IR 8400-S spectrophotometer by KBr pellet technique. Elemental analyses were performed and found values are within 0.4% of theoretical values unless otherwise noted.

1H-NMR and 13C-NMR spectra were recorded on AMX-400 NMR spectrophotometer at 400 MHz using DMSO-d6 as the solvent and tetramethylsilane (TMS) as internal standard. The chemical shifts are expressed in δ ppm. The splitting patterns were designated as follows; s: singlet; d: doublet; q: quartet; m: multiplet. LCMS were recorded by using Shimadzu LCMS-2010A instrument by ESI.Synthesis of 4, 5-diphenyl-4H-1,2,4-triazole-3-thiol (1)Phenyl thiosemicarbazide (0.01 mole, 2.71 g) was added portion wise to 15 ml of 2N NaOH solution and resulting solution was refluxed for 6 h. After the completion of reaction, the reaction mixture was allowed to cool and filtered. The filtrate was acidified with 2N HCl.

The solid obtained by acidification was filtered, washed with water, dried and recrystallized from absolute ethanol. The physical data is presented in Table 1.IR (cm-1): 3103 (NH), 3031 (Ar CH) and 1546 (C=N); 1H-NMR δ: 14.12 (s, 1H, SH), 7.02-7.89 (m, 10H, Ar-CH); 13C-NMR δ: 153.22 (C3 of triazole), 169. 56 (C5 of triazole), 130.74 (C1 of phenyl ring), 129.86 (C2 and C6 of phenyl ring), 128.74 (C3 and C5 of phenyl ring), 127.15 (C4 of phenyl ring). MS m/z: 253.Synthesis of Ethyl 2-(4, 5-diphenyl-4H-1,2,4-triazol-3-ylthio)acetate (2a)A mixture of 1 (0.01 mole, 2.53 g), ethyl chloroacetate (0.01 mole,1.22 ml) and 1 g of anhydrous potassium carbonate in 50 ml dry acetone were refluxed for 8 h on water bath.

The reaction mixture was cooled and the solvent removed under reduced pressure. The residual mass was triturated with ice water to remove potassium carbonate and extracted with ether (3 - 50 ml) and the ether layer was washed with 10% sodium hydroxide solution (3 - 30 ml) followed by water (3 - 30 ml) and then dried over anhydrous sodium sulfate and evaporated to dryness to get crude solid. The solid obtained was filtered, dried and recrystallized from absolute ethanol.

The physical data is presented in Table 1.IR (cm-1): 1728 (C=O) and 3031 (Ar CH); 1H-NMR δ: 7.33-7.56 (m, 10H, Ar-H), 4.08 (s, 2H, SCH2), 4.14 (q, 2H, CH2), 1.20 (t, 3H, CH3); 13C-NMR δ: 169.50 (C=O), 147.62 (C3 of triazole), 153.10 (C5 of triazole), 130.77 (C1 of phenyl ring), 129.91 (C2 and C6 of phenyl ring), 128.84 (C3 and C5 of phenyl ring), 127.50 (C4 of phenyl ring), 60.06 (-CH2), 14.13 (-CH3), 32.50 (-SCH2) ; MS m/z: 339.Synthesis of O-ethyl S-4,5-diphenyl-4H-1,2,4-triazol-3-yl carbonothioate (2b)The experimental procedure was repeated as with 2b using 1 (0.01 mole, 2.53 g), ethyl chloroformate (0.01 mole, 1.22 ml) and anhydrous K2CO3 in 50 ml dry acetone. The physical data is presented in Table 1.IR (cm-1): 1763 (C=O) and 3064 (Ar CH); 1H-NMR δ: 7.39-7.63 (m, 10H, Ar-H), 4.32 (q, 2H, CH2), 1.32 (t, 3H, CH3); 13C-NMR δ: 168.54 (C=O), 147.34 (C3 of triazole), 154.56 (C5 of triazole), 130.89 (C1 of phenyl ring), 129.78 (C2 and C6 of phenyl ring), 128.23 (C3 and C5 of phenyl ring), 127.85 (C4 of phenyl ring), 61.85 (-CH2), 14.89 (-CH3); MS m/z: 312.Synthesis of 2-(4,5-diphenyl-4H-1,2,4-triazol-3-ylthio)acetohydrazide (3a)A mixture of 2a (0.1 mole, 3.39 g) and hydrazine hydrate 99% (0.1 mole, 5 ml) in 25 ml of ethanol was reflux for 6 h on water bath.

The excess of solvent was removed and reaction mixture was left overnight at room temperature and the solid separated was collected by filtration. The solid obtained was filtered, dried and recrystallized from absolute ethanol. The physical data is presented in Table 1.IR (cm-1): 1660 (C=O, amide), 3320 (NH); 1H-NMR δ: 9.32 (s, 1H, NH), 7.32-7.55 (m, 10H, Ar-H), 4.28-4.31(d, 2H, NH2), 3.89 (s, 2H, SCH2); 13C-NMR δ: 170.34 (-CONH), 153.18 (C3 of triazole), 147.64 (C5 of triazole), 130.94 (C1 of phenyl ring), 129.71 (C2 and C6 of phenyl ring), 128.80 (C3 and C5 of phenyl ring), 127.18 (C4 of phenyl ring), 40.92 (-SCH2); MS m/z: 325.Synthesis of 2-(4,5-diphenyl-4H-1,2,4-triazol-3-ylthio)hydrazide (3b)The experimental procedure was repeated as with 3b using 2b (0.1 mole, 3.39 g) and hydrazine hydrate (0.1mole, 5 ml,) in absolute ethanol (25 ml).

The physical data is presented in Table 1.IR (cm-1): 1697 (C=O, amide), 3101 (NH); 1H-NMR δ: 10.02. (s, 1H, NH), 7.34-7.82 (m, 10H, Ar-H), 4.16-4.45(d, 2H, NH2); 13C-NMR δ: 169.72 (-CONH), 153.178 (C3 of triazole), 147.28 (C5 of triazole), 131.64 (C1 of phenyl ring), 129.59 (C2 and C6 of phenyl ring), 128.67 (C3 and C5 of phenyl ring), 127.89 (C4 of phenyl ring), 40.26 (-SCH2); MS m/z: 311.Synthesis of the 1-[2-(4,5-diphenyl-4H-1,2,4-triazole-3-ylthio)acetyl]-4-phenyl thiosemicarbazide (4a).An equimolar quantity of 3a (0.01 mole, 3.25 g) and phenyl isothiocyanate were separately dissolved in minimum quantity of absolute ethanol. The solution of phenyl isothiocyanate was poured into the solution of hydrazide with continuous stirring. The reaction mixture was refluxed for 8 h. The white solid obtained on cooling the reaction mixture to room temperature was filtered, dried and recrystallized from dichloromethane.

The physical data is presented in Table 1.IR (cm-1): 1243 (C=S), 1703 (C=O); 1H-NMR δ: 11.20 (d, 1H, NH), 10.80 (d, 1H, NH), 10.21(s, 1H, NH), 7.02-7.55 (m, 15H, Ar-H), 4.12 (s, 2H, SCH2); 13C-NMR δ: 170.34 (-CONH), 181.53 (C=S), 153.18 (C3 of triazole), 147.64 (C5 of triazole), 137.14 (C1 of phenyl ring), 130.73 (C2 and C6 of phenyl ring), 129.34 (C3 and C5 of phenyl ring), 128.67 (C4 of phenyl ring), 41.52 (-SCH2); MS m/z: 460.Synthesis of 1-[2-(4,5-diphenyl-4H-1,2,4-triazole-3-ylthio)acetyl]-4-(4-flurophenyl) thiosemicarbazide (4b)The experimental procedure was repeated as with 3a (0.01 mole, 3.25 g) and 4-flurophenyl isothiocyanate in 25 ml absolute ethanol. The physical data is presented in Table 1.IR (cm-1): 1256 (C=S), 1675 (C=O); 1H-NMR δ: 11.12 (d, 1H, NH), 10.65 (d, 1H, NH), 10.13 (s, 1H, NH), 7.10-7.63 (m, 14H, Ar-H), 4.21 (s, 2H, SCH2); 13C-NMR δ: 170.68 (-CONH), 180.63 (C=S), 153.91 (C3 of triazole), 148.23 (C5 of triazole), 137.23 (C1 of phenyl ring), 130.78 (C2 and C6 of phenyl ring), 128.69 (C3 and C5 of phenyl ring), 128.52 (C4 of phenyl ring), 41.62 (-SCH2); MS m/z: 478.Synthesis of 1-[2-(4,5-diphenyl-4H-1,2,4-triazole-3-ylthio)acetyl]-4-(4-chlorophenyl) thiosemicarbazide (4c)The experimental procedure was repeated as with 3a (0.01 mole, 3.25 g) and 4-chlorophenyl isothiocyanate in 25 ml absolute ethanol.

The physical data is presented in Table 1.IR (cm-1): 1248 (C=S), 1692 (C=O); 1H-NMR δ: 11.63 (d, 1H, NH), 10.73 (d, 1H, NH), 10.76 (s, 1H, NH), 7.22-7.86 (m, 14H, Ar-H), 4.14 (s, 2H, SCH2); 13C-NMR δ: 170.67 (-CONH), 180.29 (C=S), 152.45 (C3 of triazole), 148.97 (C5 of triazole), 137.38 (C1 of phenyl ring), 131.25 (C2 and C6 of phenyl ring), 128.17 (C3 and C5 of phenyl ring), 127.64 (C4 of phenyl ring), 40.18 (-SCH2); MS m/z: 494.Synthesis of 1-[2-(4,5-diphenyl-4H-1,2,4-triazole-3-ylthio)acetyl]-4-(4-methoxyphenyl) thiosemicarbazide (4d)The experimental procedure was repeated as with 3a (0.01 mole, 3.25 g) and 4-methoxyphenyl isothiocyanate in 25 ml absolute ethanol. The physical data is presented in Table 1.IR (cm-1): 1246 (C=S), 1682 (C=O); 1H-NMR δ: 11.15 (d, 1H, NH), 10.45 (d, 1H, NH), 10.72 (s, 1H, NH), 7.19-7.76 (m, 14H, Ar-H), 4.10 (s, 2H, SCH2), 3.73 (s, 3H, OCH3); 13C-NMR δ: 169.57 (-CONH), 181.32 (C=S), 152.87 (C3 of triazole), 147.67 (C5 of triazole), 137.92 (C1 of phenyl ring), 130.75 (C2 and C6 of phenyl ring), 128.78 (C3 and C5 of phenyl ring), 127.17 (C4 of phenyl ring), 40.78 (-SCH2), 55.93 (CH3); MS m/z: 490.Synthesis of 1-[2-(4,5-diphenyl-4H-1,2,4-triazole-3-ylthio)acetyl]-4-(4-ethoxyphenyl) thiosemicarbazide (4e)The experimental procedure was repeated as with 3a (0.01 mole, 3.25 g) and 4-ethoxyphenyl isothiocyanate in 25 ml absolute ethanol.

The physical data is presented in Table 1.IR (cm-1): 1253 (C=S), 1690 (C=O); 1H-NMR δ: 11.43 (d, 1H, NH), 10.78 (d, 1H, NH), 10.67 (s, 1H, NH), 7.25-7.76 (m, 14H, Ar-H), 4.24 (s, 2H, SCH2), 3.98 (q, 2H, CH2), 1.33 (t, 3H, CH3); 13C-NMR δ: 170.27 (-CONH), 180.54 (C=S), 152.12 (C3 of triazole), 148.74 (C5 of triazole), 137.14 (C1 of phenyl ring), 129.29 (C2 and C6 of phenyl ring), 128.61 (C3 and C5 of phenyl ring), 127.56 (C4 of phenyl ring), 41.36 (-SCH2), 64.72 (OCH2), 14.8 (CH3); MS m/z: 504.Synthesis of 1-[2-(4,5-diphenyl-4H-1,2,4-triazole-3-ylthio)acetyl]-4-(isopropyl) thiosemicarbazide (4f)The experimental procedure was repeated as with 3a (0.01 mole, 3.25 g) and isopropyl isothiocyanate in 25 ml absolute ethanol. The physical data is presented in Table 1.IR (cm-1): 1250 (C=S), 1685 (C=O); 1H-NMR δ: 11.37 (d, 1H, NH), 10.21 (d, 1H, NH), 10.65 (s, 1H, NH), 7.12-7.63 (m, 10H, Ar-H), 4.18 (s, 2H, SCH2), 3.97 (m, 1H, CH), 1.36 (d, 6H, 2CH3); 13C-NMR δ: 169.94 (-CONH), 181.23 (C=S), 151.34 (C3 of triazole), 148.67 (C5 of triazole), 137.84 (C1 of phenyl ring), 129.78 (C2 and C6 of phenyl ring), 127.45 (C3 and C5 of phenyl ring), 126.32 (C4 of phenyl ring), 40.65 (-SCH2), 48.23 (CH), 23.54 (CH3); MS m/z: 426.Synthesis of 1-[2-(4,5-diphenyl-4H-1,2,4-triazole-3-ylthio)formyl]-4-phenyl thiosemicarbazide (4g)The experimental procedure was repeated as with 3b (0.01 mole, 3.25 g) and phenyl isothiocyanate in 25 ml absolute ethanol.

The physical data is presented in Table 1.IR (cm-1): 1247 (C=S), 1688 (C=O); 1H-NMR δ: 11.02 (d, 1H, NH), 10.45 (d, 1H, NH), 10.86 (s, 1H, NH), 7.24-7.74 (m, 15H, Ar-H); 13C-NMR δ: 169.78 (-CONH), 180.46 (C=S), 151.78 (C3 of triazole), 148.23 (C5 of triazole), 137.62 (C1 of phenyl ring), 128.28 (C2 and C6 of phenyl ring), 127.71 (C3 and C5 of phenyl ring), 126.34 (C4 of phenyl ring); MS m/z: 446.Synthesis of 1-[2-(4,5-diphenyl-4H-1,2,4-triazole-3-ylthio)formyl]-4-(4-fluorophenyl) thiosemicarbazide (4h)The experimental procedure was repeated as with 3b (0.01 mole, 3.25 g) and 4-fluorophenyl isothiocyanate in 25 ml absolute ethanol. The physical data is presented in Table 1.IR (cm-1): 1245 (C=S), 1683 (C=O); 1H-NMR δ: 11.16 (d, 1H, NH), 10.54 (d, 1H, NH), 10.87 (s, 1H, NH), 7.12-7.64 (m, 14H, Ar-H); 13C-NMR δ: 168.58 (-CONH), 181.49 (C=S), 150.23 (C3 of triazole), 148.67 (C5 of triazole), 137.78 (C1 of phenyl ring), 129.18 (C2 and C6 of phenyl ring), 128.12 (C3 and C5 of phenyl ring), 127.82 (C4 of phenyl ring); MS m/z: 464.Synthesis of 1-[2-(4,5-diphenyl-4H-1,2,4-triazole-3-ylthio)formyl]-4-(4-chlorophenyl) thiosemicarbazide (4i)The experimental procedure was repeated as with 3b (0.01 mole, 3.25 g) and 4-chlorophenyl isothiocyanate in 25 ml absolute ethanol.

The physical data is presented in Table 1.IR (cm-1): 1254 (C=S), 1702 (C=O); 1H-NMR δ: 11.23 (d, 1H, NH), 10.35 (d, 1H, NH), 10.97 (s, 1H, NH), 7.32-7.81 (m, 14H, Ar-H); 13C-NMR δ: 168.17 (-CONH), 180.15 (C=S), 151.75 (C3 of triazole), 148.67 (C5 of triazole), 137.43 (C1 of phenyl ring), 130.14 (C2 and C6 of phenyl ring), 129.23 (C3 and C5 of phenyl ring), 128.65 (C4 of phenyl ring); MS m/z: 481.Synthesis of 1-[2-(4,5-diphenyl-4H-1,2,4-triazole-3-ylthio)formyl]-4-(4-methoxyphenyl) thiosemicarbazide (4j)The experimental procedure was repeated as with 3b (0.01 mole, 3.25 g) and 4-methoxyphenyl isothiocyanate in 25 ml absolute ethanol. The physical data is presented in Table 1.IR (cm-1): 1252 (C=S), 1689 (C=O); 1H-NMR δ: 11.15 (d, 1H, NH), 10.43 (d, 1H, NH), 10.83 (s, 1H, NH), 7.23-7.78 (m, 14H, Ar-H), 3.79 (s, 3H, OCH3); 13C-NMR δ: 169.23 (-CONH), 181.34 (C=S), 152.45 (C3 of triazole), 148.74 (C5 of triazole), 137.12 (C1 of phenyl ring), 131.56 (C2 and C6 of phenyl ring), 129.92 (C3 and C5 of phenyl ring), 128.34 (C4 of phenyl ring), 54.27 (OCH3); MS m/z: 476.Synthesis of 1-[2-(4,5-diphenyl-4H-1,2,4-triazole-3-ylthio)formyl]-4-(4-ethoxyphenyl) thiosemicarbazide (4k)The experimental procedure was repeated as with 3b (0.01 mole, 3.25 g) and 4-ethoxyphenyl isothiocyanate in 25 ml absolute ethanol.

The physical data is presented in Table 1.IR (cm-1): 1243 (C=S), 1693 (C=O); 1H-NMR δ: 11.27 (d, 1H, NH), 10.56 (d, 1H, NH), 10.79 (s, 1H, NH), 7.28-7.78 (m, 14H, Ar-H), 3.79 (q, 2H, CH2), 1.12 (t, 3H, CH3); 13C-NMR δ: 169.83 (-CONH), 180.73 (C=S), 151.76 (C3 of triazole), 148.63 (C5 of triazole), 137.18 (C1 of phenyl ring), 132.38 (C2 and C6 of phenyl ring), 129.72 (C3 and C5 of phenyl ring), 128.67 (C4 of phenyl ring), 65.27 (OCH2), 15.34 (CH3); MS m/z: 490.Synthesis of 1-[2-(4,5-diphenyl-4H-1,2,4-triazole-3-ylthio)formyl]-4-isopropyl thiosemicarbazide (4l)The experimental procedure was repeated as with 3b (0.01 mole, 3.25 g) and isopropyl isothiocyanate in 25 ml absolute ethanol. The physical data is presented in Table 1.IR (cm-1): 1245 (C=S), 1690 (C=O); 1H-NMR δ: 11.32 (d, 1H, NH), 10.23 (d, 1H, NH), 10.84 (s, 1H, NH), 7.37-7.86 (m, 10H, Ar-H), 4.04 (m, 1H, CH), 1.43 (d, 6H, 2CH3); 13C-NMR δ: 169.74 (-CONH), 181.45 (C=S), 150.67 (C3 of triazole), 148.57 (C5 of triazole), 137.39 (C1 of phenyl ring), 131.63 (C2 and C6 of phenyl ring), 129.75 (C3 and C5 of phenyl ring), 128.73 (C4 of phenyl ring), 48.56 (CH), 23.73 (CH3); MS m/z: 412.Synthesis 5-[(4,5-diphenyl-4H-1,2,4-triazol-3-ylthio)methyl]-4-phenyl-4H- 1,2,4- triazole-3-thiol (5a).The compound 4a (0.01 mole, 4.60 g) was added portion wise to 15 ml of 2N NaOH solution and resulting solution was refluxed for 6 h. After the completion of reaction, the mixture was allowed to cool and filtered.

The filtrate was acidified with 2N HCl. The solid obtained by acidification was filtered, washed with water, dried and recrystallized from absolute ethanol. The physical data is presented in Table 1.IR (cm-1): 1496 (C=N); 1H-NMR δ: 13.84 (s, 1H, SH), 7.22-7.55 (m, 15H, Ar-H), 4.27 (s, 2H, SCH2); 13C-NMR δ: 168.92 (C3 of triazole), 161.32 (C5 of triazole), 130.28 (C1 of phenyl ring), 129.61 (C2 and C6 of phenyl ring), 128.54 (C3 and C5 of phenyl ring), 127.82 (C4 of phenyl ring), 27.92 (-SCH2); MS m/z: 442.Synthesis of 5-[(4,5-diphenyl-4H-1,2,4-triazol-3-ylthio)methyl]-4-(4-fluorophenyl)-4H-1,2,4-triazole-3-thiol (5b)The experimental procedure was repeated as with 4b (0.01 mole, 4.78 g) and 2N NaOH and 2N HCl.

The physical data is presented in Table 1.IR (cm-1): 1514 (C=N); 1H-NMR δ: 13.83 (s, 1H, SH), 7.25-7.55 (m, 14H, Ar-H), 4.16 (s, 2H, SCH2); 13C-NMR δ: 167.73 (C3 of triazole), 154.67 (C5 of triazole), 131.43 (C1 of phenyl ring), 130.34 (C2 and C6 of phenyl ring), 129.23 (C3 and C5 of phenyl ring), 128.78 (C4 of phenyl ring), 28.23 (-SCH2); MS m/z: 460.Synthesis of 5-[(4,5-diphenyl-4H-1,2,4-triazol-3-ylthio)methyl]-4-(4-chlorophenyl)-4H-1,2,4-triazole-3-thiol (5c)The experimental procedure was repeated as with 4c (0.01 mole, 4.94 g) and 2N NaOH and 2N HCl. The physical data is presented in Table 1.IR (cm-1): 1512 (C=N); 1H-NMR δ: 13.72 (s, 1H, SH), 7.21-7.54 (m, 14H, Ar-H), 4.25 (s, 2H, SCH2); 13C-NMR δ: 167.56 (C3 of triazole), 149.35 (C5 of triazole), 132.24 (C1 of phenyl ring), 130.45 (C2 and C6 of phenyl ring), 129.56 (C3 and C5 of phenyl ring), 128.86 (C4 of phenyl ring), 27.16 (-SCH2); MS m/z: 477.Synthesis of 5-[(4,5-diphenyl-4H-1,2,4-triazol-3-ylthio)methyl]-4-(4-methoxyphenyl)-4H-1,2,4-triazole-3-thiol (5d)The experimental procedure was repeated as with 4d (0.01 mole, 4.90 g) and 2N NaOH and 2N HCl.

The physical data is presented in Table 1.IR (cm-1): 1496 (C=N); 1H-NMR δ: 13.67 (s, 1H, SH), 7.31-7.65 (m, 14H, Ar-H), 4.30 (s, 2H, SCH2), 3.79 (s, 3H, OCH3); 13C-NMR δ: 167.56 (C3 of triazole), 149.32 (C5 of triazole), 131.47 (C1 of phenyl ring), 130.87 (C2 and C6 of phenyl ring), 129.27 (C3 and C5 of phenyl ring), 128.74 (C4 of phenyl ring), 27.91 (-SCH2), 55.92 (-0CH3); MS m/z: 472.Synthesis of 5-[(4,5-diphenyl-4H-1,2,4-triazol-3-ylthio)methyl]-4-(4-ethoxyphenyl)-4H-1,2,4-triazole-3-thiol (5e)The experimental procedure was repeated as with 4e (0.01 mole, 5.04 g) and 2N NaOH and 2N HCl. The physical data is presented in Table 1.IR (cm-1): 1493 (C=N); 1H-NMR δ: 13.73 (s, 1H, SH), 7.26-7.73 (m, 14H, Ar-H), 4.18 (s, 2H, SCH2), 3.98 (q, 2H, OCH2), 1.33 (t, 3H, CH3); 13C-NMR δ: 168.12 (C3 of triazole), 149.83 (C5 of triazole), 132.36 (C1 of phenyl ring), 131.34 (C2 and C6 of phenyl ring), 129.28 (C3 and C5 of phenyl ring), 128.47 (C4 of phenyl ring), 27.23 (-SCH2), 64.75 (-CH2), 14.82 (-CH3); MS m/z: 486.Synthesis of 5-[(4,5-diphenyl-4H-1,2,4-triazol-3-ylthio)methyl]-4-isopropyl-4H-1,2,4-triazole-3-thiol (5f)The experimental procedure was repeated as with 4f (0.01 mole, 4.26 g) and 2N NaOH and 2N HCl.

The physical data is presented in Table 1.IR (cm-1): 1489 (C=N); 1H-NMR δ: 13.53 (s, 1H, SH), 7.28-7.56 (m, 10H, Ar-H), 4.41 (s, 2H, SCH2), 4.01(m, 1H, CH), 1.45 (d, 6H, 2CH3); 13C-NMR δ: 168.67 (C3 of triazole), 149.38 (C5 of triazole), 132.63 (C1 of phenyl ring), 131.43 (C2 and C6 of phenyl ring), 129.82 (C3 and C5 of phenyl ring), 128.42 (C4 of phenyl ring), 27.41 (-SCH2); MS m/z: 408.Synthesis of 5-(4,5-diphenyl-4H-1,2,4-triazol-3-ylthio)-4-phenyl-4H-1,2,4-triazole-3-thiol (5g)The experimental procedure was repeated as with 4g (0.01 mole, 4.46 g) and 2N NaOH and 2N HCl. The physical data is presented in Table 1.IR (cm-1): 1545 (C=N); 1H-NMR δ: 13.72 (s, 1H, SH), 7.25-7.63 (m, 15H, Ar-H); 13C-NMR δ: 168.34 (C3 of triazole), 160.23 (C5 of triazole), 131.43 (C1 of phenyl ring), 129.16 (C2 and C6 of phenyl ring), 128.73 (C3 and C5 of phenyl ring), 127.28 (C4 of phenyl ring); MS m/z: 428.Synthesis of 5-(4,5-diphenyl-4H-1,2,4-triazol-3-ylthio)-4-(4-fluorophenyl)-4H-1,2,4-triazole-3-thiol (5h)The experimental procedure was repeated as with 4h (0.01 mole, 4.64 g) and 2N NaOH and 2N HCl.

The physical data is presented in Table 1.IR (cm-1): 1545 (C=N); 1H-NMR δ: 13.34 (s, 1H, SH), 7.21-7.56 (m, 14H, Ar-H); 13C-NMR δ: 167.37 (C3 of triazole), 154.76 (C5 of triazole), 131.34 (C1 of phenyl ring), 130.62 (C2 and C6 of phenyl ring), 129.32 (C3 and C5 of phenyl ring), 128.87 (C4 of phenyl ring); MS m/z: 446.Synthesis of 5-(4,5-diphenyl-4H-1,2,4-triazol-3-ylthio)-4-(4-chlorophenyl)-4H-1,2,4-triazole-3-thiol (5i)The experimental procedure was repeated as with 4i (0.01 mole, 4.81 g) and 2N NaOH and 2N HCl. The physical data is presented in Table 1.IR (cm-1): 1497 (C=N); 1H-NMR δ: 14.08 (s, 1H, SH), 7.17-7.49 (m, 14H, Ar-H); 13C-NMR δ: 167.56 (C3 of triazole), 149.35 (C5 of triazole), 132.24 (C1 of phenyl ring), 130.45 (C2 and C6 of phenyl ring), 129.56 (C3 and C5 of phenyl ring), 128.86 (C4 of phenyl ring); MS m/z: 463.Synthesis of 5-(4,5-diphenyl-4H-1,2,4-triazol-3-ylthio)-4-(4-methoxyphenyl)-4H-1,2,4-triazole-3-thiol (5j)The experimental procedure was repeated as with 4j (0.01 mole, 4.76 g) and 2N NaOH and 2N HCl.

The physical data is presented in Table 1.IR (cm-1): 1512 (C=N); 1H-NMR δ: 13.76 (s, 1H, SH), 7.02-7.55 (m, 14H, Ar-H), 3.82 (s, 3H, OCH3); 13C-NMR δ: 168.65 (C3 of triazole), 150.23 (C5 of triazole), 131.74 (C1 of phenyl ring), 130.78 (C2 and C6 of phenyl ring), 129.72 (C3 and C5 of phenyl ring), 128.67 (C4 of phenyl ring), 55.29 (-OCH3); MS m/z: 458.Synthesis of 5-(4,5-diphenyl-4H-1,2,4-triazol-3-ylthio)-4-(4-ethoxyphenyl)-4H-1,2,4-triazole-3-thiol (5k)The experimental procedure was repeated as with 4k (0.01 mole, 4.90 g) and 2N NaOH and 2N HCl. The physical data is presented in Table 1.IR (cm-1): 1498 (C=N); 1H-NMR δ: 14.12 (s, 1H, SH), 7.18-7.63 (m, 14H, Ar-H), 3.67 (q, 2H, OCH2), 1.54 (t, 3H, CH3); 13C-NMR δ: 168.34 (C3 of triazole), 150.33 (C5 of triazole), 132.63 (C1 of phenyl ring), 131.43 (C2 and C6 of phenyl ring), 129.67 (C3 and C5 of phenyl ring), 128.35 (C4 of phenyl ring), 64.36 (-CH2), 14.21 (-CH3); MS m/z: 472.Synthesis of 5-(4,5-diphenyl-4H-1,2,4-triazol-3-ylthio)-4-isopropyl-4H-1,2,4-triazole-3-thiol (5l)The experimental procedure was repeated as with 4l (0.01 mole, 4.12 g) and 2N NaOH and 2N HCl.

The physical data is presented in Table 1.IR (cm-1): 1509 (C=N); 1H-NMR δ: 13.72 (s, 1H, SH), 7.28-7.56 (m, 10H, Ar-H), 4.12(m, 1H, CH), 1.57 (d, 6H, 2CH3); 13C-NMR δ: 168.83 (C3 of triazole), 150.84 (C5 of triazole), 132.74 (C1 of phenyl ring), 131.83 (C2 and C6 of phenyl ring), 129.12 (C3 and C5 of phenyl ring), 128.78 (C4 of phenyl ring), 32.82 (CH), 24.45 (CH3); MS m/z: 394.Biological activityIn vitro antioxidant activityIn vitro antioxidant activity was carried out by DPPH radical scavenging assay method (Roopan et al 2008, Yukesk et al 2006). A stock solution of Ascorbic acid was prepared by dissolving 10mg in 10ml of Methanol from this a serial dilution of concentration 20, 40, 60, 80 and 100 µg/ml were prepared. Similarly A stock solution of Synthesized compounds were prepared by dissolving 10mg in 10ml of Methanol from this a serial dilutions of concentration 20, 40, 60, 80 and 100 µg/ml were prepared.

The assay was carried out using UV- spectrophotometer at 517 nm. To 1ml of various concentrations of synthesised compounds, 1ml DPPH solution (40µg/ml) was added into the test tube. The solution was incubated at 37 0C for 30 min and the absorbance of each solution was measured at 517 nm against the corresponding test and standard blanks. Experiment was performed in triplicate.The percentage free radical scavenging activity can be calculated by the formula given below.Control - Test% Scavenging = -------------------- X 100ControlIn vitro cytotoxicity activityIn vitro cytotoxicity activities of the synthesized compounds against three human cancer cell lines were HT-29(adenocarcinoma), EAC (ehrich ascites carcinoma) and MDA-231(Breast cancer).

The cell lines were procured from National Centre for Cell Sciences, Pune, India, were cultured in DMEM medium supplemented with 10% FBS, 1% L-glutamine, and 50 µg/ml gentamicin sulfate in a CO2 incubator in a humidified atmosphere of 5%CO2 and 95% air. The in vitro cytotoxicity was determined using a standard MTT assay (Molinari et al., 2009, Manjula et al., 2009, Purohit et al 2010). Briefly, the exponentially growing cells were plated in 96-well plates (104 cells/well in 100 µl of medium) and incubated for 24 h for attachment.

The test compounds were prepared prior to the study by dissolving in 0.1% DMSO and diluted with medium. The cells were then exposed to different concentration of test compounds (200, 100, 50, 25 and 10 µg/ml) in a volume of 100 µl/well. The cells in the growth control wells received only the same volume of medium containing 0.1%DMSO.

After 72 h of exposure, the medium was removed and the cell cultures were incubated with 100 µl of MTT reagent (0.1%) for 4 h at 370C. The pink colored formazan was dissolved in 100 µl of DMSO and absorbance of each well was read in an ELISA micro plate reader at 570 nm. The experiment was performed in triplicate and the percentage cytotoxicity was calculated using the following formula.The drug concentration that causes 50% cell growth inhibition after 72 h of continuous exposure to the test compounds (IC50) was determined by plotting the graph of concentration of the drug against the percent cytotoxicity and performing the regression analysis. The IC50 values of the test compounds are shown in Table 3.In vitro antibacterial activitySynthesized compounds were evaluated for in vitro antibacterial activity at 100mg/ml concentration by cup plate method (Rostom et al 2009., Hugo et al 1997).

The test organisms used for antibacterial activity were Staphylococcus aureus, Bacillus substilus (grams positive) and Pseudomonas aeruginosa, Escherichia coli (grams negative).Test compounds and reference standards were dissolved in DMSO and sterile distilled water, respectively, for the preparation of stock and working solutions. Sterile nutrient agar plates were prepared by pouring the sterile agar into Petri dishes in aseptic conditions. Overnight cultures of bacteria organisms were adjusted to 106 c.f.u. ml-1 according to the Mac-Farland turbidity standards. Each standardized test organism culture (0.5ml) was spread on to the agar plates.

Required numbers of cavities were made by using a sterile borer of diameter 6 mm on the agar plates. The solution containing 100μg/ml concentrations of the test compounds and reference standards were placed on the respective cavities. Cavity containing DMSO was used as solvent control.

The plates were maintained at 4°C for 1 h to allow the diffusion of the solution into the medium and incubated at 37°C for 24h. After the incubation period, the zone of inhibition was measured in mm.Acknowledgments Authors are thankful to The Principal, JSS College of Pharmacy, Mysore, India for providing necessary facilities. Authors also thankful to The Director, NMR research centre, Indian Institute of Science, Bangalore for spectral data. Thanks are due to Mr.

V.M. Chandrashekhar, Asst. Professor, Department of Pharmacology, HSK college of Pharmacy, Bagalkot, India for carrying out anti-cancer activity.