Synthesis, Spectral Characterization and Biological ...

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Synthesis, Spectral Characterization and Biological Studies of mercapto triazole Schiff bases S.A. Deodware a, S H Gaikwad b* , U. B. Chanshetti a, D. J. Sathe c a

Dept. of Chemistry, Arts, Science and Commerce College , Naldurg, Osmanabad (M.S.) India. b Dept. of Chemistry, Shri Shivaji Mahavidyalaya, Barshi, Solapur (M.S.) India. c Dept. of Chemistry, KIT’s College of Engineering, Kolhapur, (M.S.) India.

_____________________________________________________________________________________________ ABSTRACT Synthesis of a series of 4-(2’/3’/4’-nitrobenzelideneimino) -3-methyl/ethyl-5-mercapto-1,2,4-triazole by the condensation of 4-amino-5-mercapto-3-methyl/ethyl-1,2,4-triazole with 2/3/4 nitrobenzaldehyde. The synthesized compounds were characterized by CHN analysis, IR, 1H-NMR, UV-Visible and mass spectral studies. These compounds were tested for antimicrobial activities against Staphylococcus aureus, Pseudomonas aeruginosa, Aspergillus niger and Candida albicans. All the Schiff bases were also screened for their anticancer activity. Key words: 1,2,4-triazole; Schiff base; Spectral studies; antimicrobial and anticancer activity. _____________________________________________________________________________________________ INTRODUCTION Chemistry and pharmacological activity of such substituted triazole compounds prompt us to synthesize a series of new potentially active groups bearing to the 1,2,4-triazole nucleus. These observations paved new avenues to synthesize some 5-substituted 1,2,4-triazole-3-thiol derivatives containing Schiff bases with the view to investigate their strength as better chemotherapeutic agent. The chemistry of N-bridged heterocycles derived from 1,2,4-triazole has received considerable attention in recent years due to its use in different areas of biological activities and as industrial intermediates. The 1,2,4-triazole derivatives are known to exhibit antimicrobial [1-5], antitubercular [6], anticancer [7,8], anticonvulsant[9], anti-inflamatory and analgesic properties[10]. The arrangement of three basic nitrogen atoms in triazole ring induces the antiviral activities in the compounds containing triazole ring[11]. The 1,2,4-triazole nucleus has been incorporated into a wide variety of therapeutically interesting drug candidates H1/H2 histamin e- receptor blockers, cholinesterase active agents, CNS stimulants, antianxiety agents, sedatives(12) and compounds exhibiting activity such as antimycotic activity such as Fluconazole, Itraconazole and Voriconazole [13,14]. There are also some known drugs containing 1,2,4-triazole moiety, eg: Triazolam[15], Alprazalam [16], Etizolam[17], Furacylin[18], Ribavirin[19], Hexaconazole[20], Triadimefon[21], Mycobutanil[22], Rizatriptan[23], Propiconazole[24], Fluotrimazole[25]. A series of 1,2,4-triazole derivatives has been extensively employed in agriculture as herbicides[26] while certain 1,2,4-triazoles also find applications in the preparations of photographic plates, polymers and as analytical agents [27].

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Der Pharma Chemica, 2015, 7 (7):365-372 _____________________________________________________________________________

In this paper, we report that synthesis of a series of 4-(2’/3’/4’-nitrobenzelideneimino) -3-methyl/ethyl-5-mercapto1,2,4-triazole by condensation reactions of 4-amino-5-mercapto-3-methyl/ethyl-1,2,4-triazole and 2/3/4 nitrobenzaldehyde were used as basic precursors and ethanol as a solvent. The synthesized compounds were characterized by CHN analysis, IR, 1H-NMR, UV-Vis, Mass spectral studies, and their biological (antimicrobial and anticancer) activities. MATERIALS AND METHODS All the reagents/chemicals used were of analytical grade. Solvents were dried and distilled before use according to standard procedures [28,30].The purity of the compounds was confirmed by thin layer chromatography using merk silica gel 60 F254 coated alumina plates and visualized by exposure to iodine vapors. IR spectra were recorded on Thermo Fisher scientific model Nicolet IS10. 1H NMR spectra were recorded in DMSO on Broker Avalce-400MHz using TMS as internal standard. Mass spectra were recorded on Micro mass Q-T micro (TOF MS ES+). Electronic spectra were recorded on Shimadzu UV-3600 using DMSO. 2.2 Synthesis of Schiff bases 3-substituted-4-amino-5-mercapto-1, 2, 4-triazoles were synthesized in accordance with the literature- reported method [28,29,30]. Schiff bases were prepared by refluxing a mixture of appropriate mercaptotriazoles (0.01mol) with 2/3/4 nitrobenzaldehyde (0.01mol) in ethanol for four hours and cooled. After cooling, the colored solid products were filtered and washed with cold ethanol and vacuum dried over CaCl2. The purity of the products was checked by TLC. Names of synthesized Schiff bases and their representations are as follows 1) 4-(2’-nitro benzelideneimino)-3-methyl-5-mercapto-1,2,4-triazole ( HL-1). 2) 4-(3’-nitro benzelideneimino)-3-methyl-5-mercapto-1,2,4-triazole (HL-2). 3) 4-(4’-nitro benzelideneimino)-3-methyl-5-mercapto-1,2,4-triazole (HL-3). 4) 4-(2’-nitro benzelideneimino)-3-ethyl-5-mercapto-1,2,4-triazole (HL-4). 5) 4-(3’-nitro benzelideneimino)-3-ethyl-5-mercapto-1,2,4-triazole (HL-5). 6) 4-(4’-nitro benzelideneimino)-3-ethyl-5-mercapto-1,2,4-triazole (HL-6). SchemeN

N

N H

N

CHO N

N

+ R

N

SH

N H2

3-substituted-4-amino 5-mercapto-1,2,4-triazole

R

N

HC

N

NO2

Nitro benzaldehyde

SH

R

HC

N O2

Nitro 4-(2’-nitro benzelideneimino) -3 substituted-5-mercapto-1,2,4 Triazole

N N

N O2

tautomeric form

Where R = -CH3 or -C2H5 RESULTS AND DISCUSSION 3.1 Elemental and physical constant data of Schiff bases Theoretical and experimental values of elemental analysis of compounds are in good agreement with each other confirming the stoichiometry of synthesized Schiff bases. Elemental and physical constant data of Schiff bases is listed in table 1.

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3.2 1H NMR Spectroscopic studies The 1H-NMR spectra of the ligands show –SH proton signal at δ 13.27-13.7 ppm A signal at δ 10.62-11.07 ppm is observed due to azomethine proton. The aromatic protons in the ligands appear as multiplate at δ 7.27-8.29 ppm. An additional signal at 2.22-2.39 ppm is due to –CH3 group of the ligands in HL-1 to HL-3 and isomeric =C-CH3 signals for these compounds are observed at 2.6-2.63 ppm. For the ligands HL-4 to HL-6 quartet was observed at δ 2.61-2.78 ppm due to –CH2 – of -CH2- CH3 and triplet is observed at δ 1.25-1.38 ppm on account of –CH3 of CH2-CH3. NMR data of Schiff bases are as in table 2. 3.3 IR Spectroscopic studies The IR spectra provided valuable information about the possible co-ordination sites. The IR spectrum of the ligands showed a number of important absorption bands. The ligands do exhibit tautomerism (39) and one can expect both signals due to νS-H and νC=s at 2753- 2797, 1114-1176cm-1 respectively. An absorption band at ~1590 cm-1 is the presence of C=N in the molecule. The absorption bands corresponding to the nitro group are seen at 1523-1592 cm-1 and 1315-1387 cm-1 respectively. The –NH band observed at 3049-3096 cm-1 clearly give an evidence of establishment of thione ↔ thiol tautomerism. IR spectral data in cm-1 of Schiff bases HL-1 to HL-6 are as shown in table 3. 3.4 Electronic spectral analysis Electronic spectra of all ligands are characterized by two bands in the UV-visible region. The bands occurring in the range 245-260 nm are due to low or medium energy π→π* transition within aromatic moieties. These transitions may originate due to perturbed local excitation of the phenyl group [39]. Another intense band in the lower energy region of the spectra between 322-378 nm is attributed to the n→π* transition of azomethine group. Electronic spectral data of compounds are as shown in table 4. 3.5 Mass Spectroscopic studies The mass spectra of all the synthesized compounds were recorded. All the spectra exhibited parent peak due to molecular ion (M+). The proposed molecular formula of these compounds was confirmed by comparing their molecular formula weights with the m/z values. The molecular ion peaks obtained are as follows m/z 264(HL-1 to HL-3), while 278(HL-4 to HL-6). These data correspond with the proposed molecular formula for these compounds. In addition to the peaks due to the molecular ions, the spectra exhibited peaks assignable to various fragments arising from the thermal cleavage of the compounds have been observed. Table 1: Elemental and physical constant data of Schiff bases.

Sr. No 1 2 3

Name of Schiff bases

HL-1 HL-2 HL-3

4

HL-4

5

HL-5

6

HL-6

Mol. Formula C10H9N5S02 C10H9N5S02 C10H9N5S02 C11H11N5S02 C11H11N5S02 C11H11N5S02

M.P. (oC)

Mol. Wt.

Color

263

346

Pale yellow

263

235

colorless

263

230

Dark yellow

312

318

Pale yellow

252

318

colorless

318

Dark yellow

283

Elemental analysis C% H% N% Found Found Found (calc.) (calc.) (calc.) 45.20 3.52 26.48 (45.63) (3.42) (26.61) 45.78 3.45 26.70 (45.63) (3.42) (26.61) 45.52 3.55 26.65 (45.63) (3.42) (26.61) 47.52 3.78 25.38 (47.65) (3.97) (25.27) 47.71 3.81 25.21 (47.65) (3.97) (25.27) 47.55 3.94 25.22 (47.65) (3.97) (25.27)

Table2: NMR data of Schiff bases Sr. no. 1 2 3 4 5 6

Schiff bases HL-1 HL-2 HL-3 HL-4 HL-5 HL-6

NMR signals in(δ ppm) 2.28(S,3H,CH3), 2.61(S,3H,isomeric=C-CH3), 7.27-7.99 (m,4H,Ar-H),11.07(S,1H,N=CH), 13.27(S,1H,SH) 2.22(S,3H,CH3), 2.63(S,3H,isomeric=C-CH3), 7.38-7.92 (m,4H,Ar-H),10.62(S,1H,N=CH), 13.54(S,1H,SH) 2.39(S,3H,CH3), 2.6(S,3H,isomeric=C-CH3), 8.04-8.29 (m,4H,Ar-H),10.75(S,1H,N=CH), 13.64(S,1H,SH) 2.61(q,2H, -CH2-CH3), 1.25(t,3H,-CH2-CH3), 7.32-8.02 (m,4H,Ar-H),10.87(S,1H,N=CH), 13.7(S,1H,SH) 2.78(q,2H,-CH2-CH3), 1.38(t,3H,-CH2-CH3), 7.52-8.12 (m,4H,Ar-H),10.68(S,1H,N=CH), 13.34(S,1H,SH) 2.7(q,2H,-CH2-CH3), 1.28(t,3H,-CH2-CH3), 7.75-8.28 (m,4H,Ar-H),10.79(S,1H,N=CH), 13.61(S,1H,SH).

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Der Pharma Chemica, 2015, 7 (7):365-372 _____________________________________________________________________________ Table 3 : IR spectral data of the Schiff bases (cm-1) Sr. no.

Name of Schiff bases

1 2 3 4 5 6

HL-1 HL-2 HL-3 HL-4 HL-5 HL-6

νC=N

νSH

1590 1589 1579 1580 1593 1602

νC=S

2760 2753 2770 2777 2779 2797

1114 1176 1170 1158 1152 1161

νN-H 3067 3072 3096 3090 3049 3057

Symm 1337 1387 1342 1358 1335 1315

νNO2 asymm 1523 1525 1592 1535 1525 1519

Table 4: UV-visible λmax values in nm of Schiff bases Sr. No. 1 2 3 4 5 6

Schiff base HL-1 HL-2 HL-3 HL-4 HL-5 HL-6

π→π* 284 245 257 252 250 260

n→π* 338 352 335 322 378 330

Table 5: Antimicrobial activity of Schiff bases Sr. No.

Schiff base .

1

HL-1

2

HL-2

3

HL-3

4

HL-4

5

HL-5

6

HL-6

Conc. (mg/ml) 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0 0.2 0.4 0.6 0.8 1.0

Staphylococcus aureus +++ +++ +++ +++ +++ ++ +++ +++ +++ ++ ++ ++ +++ +++ +++ +++ ++ ++ +++ +++ +++ ++ + + +++

Pseudomonas aeruginosa

Aspergillus niger -

Candida albicans

+++ +++ +++ +++ +++ ++ ++ ++ + ++ ++ +++ +++ + + + + + + ++ ++ 0.2 +++ +++ 0.4 +++ +++ 0.6 ++ ++ 0.8 ++ ++ 1.0 ++ +++ Less than 5mm – Inactive (-), 6-10mm - weakly active(+), 11-14mm - moderately active(++), 15mm and above - highly active(+++).

Table : 6

++ ++ ++ ++ +++ +++ +++ +++ +++ +++ +++ +++ + ++ ++ + + + ++

Anticancer studies of compounds on MCF-7 using Sulforhodamine B assay Schiff base HL-1 HL-2 HL-3 HL-4 HL-5 HL-6

LC50 >100 >80 >100 >80 >80 >80

TGI >100 >80 >100 >80 >80 >80

GI50 64.1 55.4 55.7 64.6 46.9 41.5

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(a)

(b) Fig.1:

1

H NMR spectra of HL-3 (a) and HL-6 (b)

Fig.2: IR spectra of HL-2 Schiff base.

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Fig.3: UV spectra of HL-1 Schiff base

Fig.4: Mass spectra of HL-2 Schiff base.

4. Antibacterial activity studies The antibacterial and antifungal activity of the Schiff bases were tested on Staphylococcus aureus, Pseudomonas aeruginosa, Aspergillus niger and Candida albicans. The method used for antibacterial activity was Agar wellDiffusion method [31] and for antifungal activity was Agar-Ditch method [32, 33]. The stock solution 1mg/ml was prepared and was used to prepare concentrations of 0.8, 0.6, 0.4, 0.2 mg/ml. The bacteria and fungi were inoculated on the surface of nutrient agar and Sabouraud’s agar, respectively. The various concentrations of the compounds were inoculated in the wells prepared on the agar plates. The plates were incubated at room temperature for 24 hours. In order to clarify the effect of DMSO on the biological screening, separate studies were carried out with DMSO and showed no activity against any bacteria and fungi. The results are as summarized in the table5. All the compounds are inactive against Aspergillus niger and weak to moderately active against Candida albicans while highly active against staphylococcus aureus and Pseudomonas aeruginosa. The results were compared with standard antibiotics like Gentamycine and Streptomycin.

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5. Anticancer activity studies All the newly synthesized compounds were screened for their anticancer activity. The anticancer activity was measured in vitro for the newly synthesized compounds using the Sulforhodamine B stain (SRB) assay method [3436] The cell line used in the present investigation is MCF-7 (Breast). In the current protocol each cell line is inoculated on a pre-incubated microtitler plate. The test agents are added at a single concentration and the culture is incubated for 48h. The endpoint determinations are made with Sulforhodamine B, a protein binding dye. Results for each test agents are reported as the percent growth of the cells tested. The compounds which reduce the growth of any one of the cell lines to 32% or less (negative numbers indicate cell kill) are passed on for evolution over a 5-log dose range. In the present screening program the compounds HL-1 to HL-6 possessed growth percentage to less than 32% are regarded as active compounds [37-38]. HL-1 and HL-3 were active against breast cancer MCF-7 cell line at the concentration10-4 M while HL-2, HL-4, HL5 were active at the concentration 8x10-6 M and HL-6 was active at 4x10-6M and 8x10-6M. The results of anticancer activity of all synthesized compounds are shown in table 6. CONCLUSION The results show that Schiff bases have a tautomeric structure (Scheme), which means that they exist in the solid state in forms. The structure of the synthesized Schiff bases was confirmed by IR, 1HNMR, mass spectral data and CHN analysis. The HL-1 to HL-6 compounds are highly active against Staphylococcus aureus and Pseudomonas aeruginosa while they are inactive against Aspergillusniger and weakly to moderately active against Candida albicans. The core outcome of our work is that every single compound is highly active against breast cancer cell line MCF-7. Acknowledgement The authors are thankful to ACTREC, Tata memorial centre, Kharghar, New Mumbai, India for providing anticancer activity. REFERENCES [1] B.S. Holla, R. Gonslves, S. Shenoy , IT Frarmaco, 1998, 53, 574-578. [2] B. S.Holla, B Veerendra, M. K. Shivnanda, N .S. Kumari, Ind. J.Chem, 2003, 42B. 2010-2014. [3] M. Ashok, B. S. Holla, J. Pharmacol, Toxicol, 2007, 2(3), 256-263. [4] D. J. Prasad, B.Poojari, B.S.Holla, N. S. kumari, Eur. J. Med. Chem.,2009, 44, 551-557. [5] G. T. Zitouni, Z. A. Kaplanckli, M. T. Yildiz P Chevallet, D. Kaya, Eur. J. Med. Chem, 2005, 40, 607-613. [6] K. Walczak, A Gondela, J. Suwinski, Eur J. Med. Chem., 2004, 39, 849-853. [7] B. S. Holla, K. N. Poojary, B. S. Rao, M. K. Shivananda, Eur J. Med. Chem.,2002, 37, 511-517. [8] B. S. Holla, B. Veerendra, M. K. Shivananda, B. Poojary, Eur, J. Med. Chem., 2003, 38, 759-767. [9] M. Amir, K. Shikha, Eur J. Med chem.,2004, 39, 535-545. [10] Almasirad, S. A. Tabatabai, M. Faizi, A. Kebriaeezadeh, N Mehrabi, A Dalvandi, A. Shafiee, Bioorg. Med. Chem. Lett, 2004, 14, 6057-6059. [11] K. Masuda, T. Toga, Chem Abstr. ,1976, 84, 12173. [12] E. Schreier, Helv. Chim acta , 1976, 10;59(2) 585-606. [13] The Merck index, 12th ed. Whitehouse Station, NJ: Merck & Co, Inc., 1996:761 [14] J. Haber , Cas Lek; Cek , 2001, 140, 596-602. [15] Brucato, A. copoola, S. Gianguzza, P.M Provenzano, Bull. Soc Ital. Sper., 1978, 54, 1051. [16] D.L. Coffen, R.I. Fryer, U.S Patent,3 849 434, 1974; chem. Abstr. ,1975, 82, 730044 V. [17] M. Shiroki, T. Tahara, K. Araki, Jap. Patent, 75100096 ,1975; Chem. Abstr. 1976, 84, 59588k. [18] F, D Povelista, A.G Gural, Antibiotiki Moscow 1973, 18, 71; Chem. Abstr. 78, 1973, 93044. [19] R.W. Sidwell, L.B Allen, J.H. Hoffman , J.T. Witkowsti, L.N Simon , Proc Soc . Exp. Biol. Med., 1975, 148, 854-858. [20] M.C. Shepherd, Proceedings of Birtish Crop protection conference Brigntion 1986, 1, 19. [21] H. Lye, Modern Selective Fungicides, Longman Scientific Technical, Harlow, 1987. pp. 13–30. [22] P. Efthimiadis, Proceedings of British crop Protection Conference Brighton, 1988,3, 887. [23] C. Hart, Drugs for migraine, In: Modern Drug Discovery,Ame. Chem. Soc., 1999. [24] G. V. Reet., J. Heeres , L. Wals, Ger. Patent 1976, 2,551 560; Chem. Abstr. ,1976, 85, 94368.

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