g a r a m a n i j O h r l 1,2,4,-Triazoller / 1,2,4-Triazoles

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1 e r l n i j ri h Ar c r þ t ý s r Re m i g in l Synthesis, Chrcteriztion nd Antimicrobil Evlution of Substituted 1,2,4-Trizole Thiones Contining Pyrzole Moiety Değiştirilmiş 1,2,4-Trizol Tiyonlrını İçeren Pirzol Prçlrının Sentezi, Krkterize Edilmesi ve Antimikrobiyl Açıdn Değerlendirilmesi 1,2,4,-Trizoller / 1,2,4-Trizoles Desbttin Venkt ryn Ro 1, Aluru Rghvendr Guru Prsd 2, Ydti rsimh Spoorthy 1 Dhrmpuri Rghunth Ro 3, Lkshmn Ro Krishn Ro Rvindrnth 1 1 Deprtment of Chemistry, Sri Krishndevry University, Anntpur, A.P. 2 Fculty of Science nd Technology, ICFAI Foundtion for Higher Eduction, Hyderbd, A.P. 3 Extension nd Trining Division, tionl Institute of utrition, Indin Council for Medicl Reserch, Hyderbd, A.P. Indi Özet Amç:4-(3-((değiştirlmiş)-1H-pyrzol-3-yl-methylsulphonyl)-5-((3,5,6- trichloro-pyridin-2-yl-oxymeth- yl)-[1,2,4]trizol 4 yl-methyl)-morpholine (8-g) olrk dlndırıln yeni mddeyi sentezlemek ve ntimikrobiyl ktivitelerini değerlendirmek. Gereç ve Yöntem: Yeni sentezlenen ürünlerin kimysl ypılrı IR, 1H MR, kitle spektrometrisi ve elementl nliz verileri ile ydınltılmıştır. Stphylococcus ureus CCS 2079, Bcillus Cereus, CCS 2106, Escherichi coli CCS 2065, Pseudomns eruginos CCS 2200, Aspergillus niger CCS 1196 ve Cndid lbicns CCS 2106 yönelik nti mikrobiyl ktiviteleri disk difüzyon yöntemi ile belirlenmiş ve minimum inhibitör konsntrsyonlrı broth dilüsyon metodu ile gösterilmiştir. Bulgulr: Ürünlerin elementl nlizi göstermiştir ki deneyler yoluyl bulunn değerler teorik olrk hesplnn değerlere çok ykındır. Her vkd kritik fonksiyone gruplrın IR ve 1H MR kitle spektrometresel düzenlenmeleri belirlenmiştir. Kitle spektrl frgmntsyon ntimikrobiyl ktivite çısındn trnn son ürünün oluşumunu kesinleştirmiştir. Her bir ürünün ntibkteriyel ve ntifungl ktiviteleri değerlendirilmiş ve sunulmuştur. Ürün serilerinin her bir üyesinin test edilen mikroplr krşı ktif olduğu bulunmuş, özellikle 8C türevinin dh iyi ntimikrobiyl ktivite gösterdiği izlenmiştir. Trtışm: Ürünler yeniden 1,2,4-trizollerin temel dvrnışını isbt eder şekilde belirgin ntifungl ktivite göstermişlerdir. Anhtr Kelimeler 1,2,4,-Trizoller; Krkterizsyon; Antimikrobiyl Aktivite; Minimum İnhibitör Konsntrsyon Abstrct Aim: To synthesize series of novel compounds nmely, 4-(3-((substituted)- 1H-pyrzol-3-yl-methylsulphonyl)-5-((3,5,6-trichloro-pyridin-2-yl-oxymethyl)-[1,2,4]trizol 4 yl-methyl)-morpholine (8-g) nd evlute their ntimicrobil ctivity. Mteril nd Method: The chemicl structures of newly synthesized compounds were elucidted by IR, 1 H MR, mss spectrl nd elementl nlysis dt. Their ntimicrobil ctivities ginst Stphylococus ureus CCS 2079, Bcillus Cereus, CCS 2106, Escherichi coli CCS 2065, Pseudomns eruginos CCS 2200, Aspergillus niger CCS 1196 nd Cndid lbicns CCS 2106 were investigted by employing disk diffusion method nd minimum inhibitory concentrtion ws found out by broth dilution method. Results: Elementl nlysis of the compounds indicted tht the vlues found by the experiments were very close to theoreticlly clculted vlues. IR nd 1 H MR spectrl ssignments of criticl functionl groups were indicted in ech cse. Mss spectrl frgmenttion confirmed the formtion of the finl product to be screened for ntimicrobil ctivity. Antibcteril nd ntifungl ctivity of ech of the compounds were evluted nd presented. Ech member of the series of compounds ws found to be ctive ginst tested microbes in prticulr, the derivtive 8C ws found to exhibit better ntimicrobil ctivity. Discussion: The compounds demonstrted significnt ntifungl ctivity once gin evidencing the bsic trit of 1,2,4-trizoles. Keywords 1,2,4,-Trizoles; Chrcteriztion; Antimicrobil Action; Minimum Inhibitory Concentrtion DI: /JCAM.2323 Received: Accepted: Printed: J in Anl Med 2015;6(5): Corresponding Author: Aluru Rghvendr Guru Prsd, Fculty of Science nd Technology, ICFAI Foundtion for Higher Eduction, Hyderbd, A.P. Indi. E-Mil: 590 Journl of inicl nd Anlyticl Medicine

2 1,2,4,-Trizoller / 1,2,4-Trizoles Introduction In spite of the vilbility of lrge number of chemotherpeutic gents, there is n ever incresing demnd for the new clss of ntibiotics due to the combintion of incresing number of infectious diseses nd the incresing number of multi drug resistnt microbes. Though extensive reserch leding to the invention of new drugs is tking plce, 1,2,4-trizoles nucleus hs received substntil ttention owing to their diverse phrmcologicl importnce [1,2]. The verstile biologicl ctivities of trizoles re due to their bility to bind with vriety of enzymes nd receptors in living system vi diverse non covlent interctions [3]. In ddition, trizole compounds re effective ntifungl gents. Mny such hevily mrketed modern-dy ntifungl drugs contining trizole nucleus nmely, fluconzole [4], voriconzole [5], itrconzole [6], rvuconzole [7] etc., re reported in the literture. The bsic nitrogen of zole ring would be tightly bound to the heme iron of the fungl cytochrome P450 preventing substrte nd oxygen binding, thus they ct s cytochrome P450 14α-demethylse inhibitors [8]. Trizole nucleus hs been phrmcologiclly significnt scffold in mny drug ctegories such s ntimycobil [9], ntiinflmtory [10,11], ntioxidnt [12], ntivirl [13], ntituberculr [13], nti HIV [13], ntitumor [14], ntimicrobil [11,15], nlgesic [11], nticovsculnt [16] gents, etc. These dys, the fusion of medicinlly importnt heterocyclic rings to prospective phrmceuticl cndidtes is mjor strtegy to chieve greter ctivity nd wider medicinl spectrum. Keeping this in view, the uthors herewith report the synthesis nd phrmcologicl evlution of 1,2,4-trizole thiones fused with pyrzole, n nother medicinlly importnt heterocyclic ring system hving brod phrmcologicl spectrum [17-23]. A number of compounds contining diverse phrmcologicl importnce is reported from these lbortories [24,25] Mteril nd Method All Chemicls nd regents were procured from Rnbxy Lbortories Ltd, Chemicl Division, Indi. The stndrd bcteril nd fungl strins were procured from tionl Centre for Cell Sciences, Pune, Indi. UV-Visible spectrophotometer mnufctured by Shimdzu Corportion, Jpn ws used for trnsprency mesurements. Infrred spectr of compounds were recorded on Perkin-Elmer FT-IR Spectrometer. 1 H MR spectr were recorded on JEL (300MHz) Spectrometer using TMS s n internl stndrd. Mss spectr were recorded on Mss Spectrometer JEL sx-102. Synthetic Procedures Synthesis of Methyl-2-(3,5,6-trichloro-pyridin-2-yloxy)-cette (2) Thionyl chloride (4.35 ml, 58.6 mmol, 3.0 eq) ws dded to solution of 2-(3,5,6-trichloropyridin-2-yl-oxy) cetic cid (1) (5.0 g, 19.5 mmol, 1 eq.) in methnol (50 ml) t 0 C nd ws heted to 80 C for 2 hours. The rection mixture ws cooled to room temperture nd concentrted under reduced pressure to remove methnol, diluted with cold wter nd filtered. The solid so obtined ws dried under vcuum to get Methyl-2-(3,5,6- trichloro-pyridin-2-yloxy) cette (2 Synthesis of 2-(3,5,6-trichloro-pyridin-2-yloxy)-cetohydrzide(3 Hydrzine hydrte (2.08 g, 41.6 mmol, 2.5 eq.) ws dded to solution of Methyl-2(3,5,6-trichloro-pyridin-2-yloxy) cette (2) (4.5 g, 16.6 mmol, 1 eq.) in ethnol (50 ml) nd ws refluxed for 16 hours. The rection mixture ws cooled to 0 C, filtered nd the solid so obtined ws wshed with ethnol to get pure 2-(3,5,6-trichloro-pyridin-2-yl-oxy) ceto hydrzide (3 Synthesis of 1-(2-(3,5,6-trichloro-pyridin-2-yloxy)-cetyl)-thio semicrbzide (4 Potssium thiocynte (1.08 g, mmol, 3.0 eq.) ws dded to solution of 3 (1.0 g, 3.70 mmol, 1 eq.) in cetic cid (15 ml) nd ws heted to 80 C for 3 hours. The rection mixture ws cooled to room temperture, diluted with wter, filtered nd the solid so obtined ws wshed with cold wter to get 1-(2-(3,5,6-trichloro-pyridin-2-yloxy)cetyl)thiosemicrbzide (4 The smple ws recrystllized from mixture of dimethyl formmide nd wter (1:1 Synthesis of 5-((3,5,6-trichloro-pyridin-2-yloxy)-methyl)-2H- 1,2,4-trizole-3(4H)-thione (5) KHC 3 (4.5 g, 45.7 mmol, 2 eq) dissolved in 5 ml wter ws dded to stirred solution of 1-(2-(3,5,6-trichloro-pyridin-2- yloxy)cetyl) thiosemi crbzide (4) (7.5 g, 22.8 mmol, 1 eq.) in mixture of DMF : H 2 (96:24 ml) nd ws stirred for 16 hours. The rection mixture ws filtered, the filtrte ws cidified with 2 H to ph 5 nd extrcted with ethyl cette. The orgnic lyer ws wshed with wter, brine solution, dried over nhydrous sodium sulphte nd the solvent ws removed under reduced pressure to get crude 5-((3,5,6-trichloro-pyridin-2-yloxy) methyl-2h-1,2,4-trizole-3(4h)-thione (5 Synthesis of 2-((5-(1,5-diethyl)-1H-pyrzol-3-yl)-methylthio)- 4H-[1,2,4]trizol-3-yl)-methoxy)-3,5,6-trichloro-pyridine (6) 5-((3,5,6-trichloro-pyridin-2-yloxy)methyl-2H-1,2,4-trizole-3(4H)-thione (5) (200.0 mg, 0.64 mmol, 1 eq.) nd 3-(chloromethyl)1,5-diethyl-1H-pyrzole (94.0 mg, 0.70 mmol, 1.1 eq.) were dded to solution of HC 3 (108.0 mg, 1.29 mmol, 2.0 eq.) in ethnol (10 ml) nd stirred for 48 hours t room temperture. The rection mixture ws concentrted under reduced pressure to remove excess ethnol, diluted with wter nd extrcted with ethyl cette. The orgnic lyer ws wshed with wter, brine solution, dried over nhydrous sodium sulphte nd the solvent ws removed under reduced pressure to get crude product, 2-((5-((1,5-diethyl-1H-pyrzol-3-yl) methylthio)-4h-1,2,4-trizol-3-yl)methoxy)-3,5,6-trichloro pyridine (6 Similr procedure ws followed to for the synthesis of 6b-6g. Synthesis of 4 (3 ((1,5-diethyl) 1H pyrzol 3 yl-methylthio 5 ((3,5,6 trichloro-pyridine 2 yl-oxy)-methyl)-[1,2,4]trizol 4 yl)-methyl)-morpholine (7) A mixture of 6 (0.1 mol), morpholine (0.15 mol) nd wter (20 ml) were stirred to obtin cler solution. HCH (0.05 mol) nd DMF were dded to bove mixture in ice cold condition, stirred for 2 hours in n ice bth nd left overnight t room temperture. White solid so obtined ws isolted nd recrystllized from ethnol to give 7. The rection procedure leding to the formtion of 7 ws extended for the synthesis of 7b 7g. Synthesis of 4-(3-(1,5-diethyl-1H-pyrzol-3-yl-methylsulphonyl)- 5-((3,5,6-trichloro-pyridin-2-yl-oxymethyl)-[1,2,4]trizol 4 ylmethyl)-morpholine (8) Journl of inicl nd Anlyticl Medicine 591

3 1,2,4,-Trizoller / 1,2,4-Trizoles A solution of 0.95 g of (0.02 mol) of 7 in glcil cetic cid (50 ml) ws tken in 250 ml of round bottom flsk fitted with reflux condenser. The solution ws heted to boil, 8 ml of 36% H 2 2 ws dded nd refluxed for 2 hours. The rection mixture ws cooled, filtered nd the solid so obtined ws recrystllized from 95% ethnol to give 8. The rection procedure leding to HCH DMF (1) (6) (7) H C C S 2, MeH 2 Me 2 H HH HH o C EtH (2) (3) KSC 80 o C AcH H S R S HC H H 3 2 DME : H 2 EtH H H KHC 3 H S R (5) (4) S R 30% H 2 2 AcH the formtion of 8 ws extended for the synthesis of 8b 8g. The rection sequence is outlined in Scheme I. Results nd Discussion Substituted 1,2,4-trizole thiones were chrcterized by elementl nlysis, IR nd, 1 H MR nd mss spectrl dt. The detils re given in the following lines. Chrcteriztion of Methyl-2-(3,5,6-trichloro-pyridin-2-yloxy)- cette (2) Moleculr formul, Yield, Element culted% (Found%): C 8 H 6 -, 86%, C (35.12); H 2.24 (2.51); (39.53); (5.01), IR ν mx in cm 1 (Group): 2980 (liphtic - ); 1610 (>C=); 1645 (ester >C=), 1 HMR (300 MHz, DMS-d 6 ) δ ppm: 8.81 (s, 1H, Ar-H); 5.18 (s, 2H, C- ); 3.7 (s, 3H, C-CH 3 Chrcteriztion of 2-(3,5,6-trichloro-pyridin-2-yloxy)-cetohydrzide (3) Moleculr formul, Yield, Element culted% (Found%): C 7 H , 66%, C (31.61); H 2.24 (2.54); (39.11); (15.45), IR ν mx in cm 1 (Group): 3225 (>H); 2980 (liphtic - - ); 1654 (>C= of CH), 1HMR (300 MHz, DMS-d 6 ) δ ppm: 9.2 (s, 1H, H); 8.81 (s, 1H, Ar-H); 5.18 (s, 2H, C- ); 3.1 (s, 2H, H 2 Chrcteriztion of 1-(2-(3,5,6-trichloro-pyridin-2-yloxy)- cetyl)-thio semicrbzide (4) Moleculr formul, Yield, Element culted% (Found%): (8) 2 S R Scheme 1. Synthesis of 4-(3-((substituted)-1H-pyrzol-3-yl-methylsulphonyl)-5- ((3,5,6-trichloro-pyridin-2-yl-oxymethyl)-[1,2,4]trizol 4 yl-methyl)-morpholine (8 Tble 1. The detils of substituents in vrious 1,2,4-trizoles synthesized compound R 8 3-(chloromethyl)-1,5-dimethyl-1H-pyrzole 8b 3-(chloromethyl)-1,5-diethyl-1H-pyrzole 8c 5-tert-butyl-3-(chloromethyl)-1-methyl-1H-pyrzole 8d 3-(chloromethyl)-1-methyl-1H-pyrzole 8e 3-(chloromethyl)-1-ethyl-1H-pyrzole 8f 3-(chloromethyl)-5-ethyl-1-methyl-1H-pyrzole 8g 3-(chloromethyl)-5-isopropyl-1-methyl-1H-pyrzole (C 8 H S, 59%, C (29.67); H 2.14 (2.42); (32.04); (16.71); 9.71 (9.25), IR ν mx in cm 1 (Group): 3225 (>H); 2980 (liphtic - -); 1654 (>C= of CH); 1134 (C=S), 1 HMR (300 MHz, DMS-d 6 ) δ ppm: 9.1 (s, 1H, CH); 8.83 (s, 1H, pyridine ring); 5.2 (s, 2H, C ); 2.2 (s, H, HCS); 8.32 (s, 2H, CSH 2 Chrcteriztion of 5-((3,5,6-trichloropyridin-2-yloxy)-methyl)- 2H-1,2,4-trizole-3(4H)-thione (5) Moleculr formul, Yield, Element culted% (Found%): C 8 H S, 71%, C (29.79); H 1.62 (1.82); (34.32); (17.61); 5.13 (4.91), IR ν mx in cm 1 (Group): 3225 (>H); 2980 (liphtic - -); 1654 (>C= of CH), 1605 (>C=); 1134 (>C=S), 1 HMR (300 MHz, DMS-d 6 ) δ ppm: 8.15 (s, H, H-- of 1,2,4-trizole-thione ring), 2.3 (s, H, -H- of 1,2,4-trizole thione ring), 5.2 (s, 2H, - - trizole), 8.83 (s, H, pyridine ring Chrcteriztion dt of 2-((5-(substituted)-1H-pyrzol-3-yl)- methylthio)-4h-[1,2,4]trizol-3-yl)-methoxy)-3,5,6-trichloropyridine 6(-g) [Compound: Moleculr Formul, Yield (%), m.p( 0 c), Element:culted% (Found%); IR ν mx in cm 1 (Group); 1 HMR (300 MHz, DMS-d 6 ) δ ppm (Group)] 6: C 16 H S, 62, , C:41.71(42.92); H:3.53(3.83); :19.30(18.77); :24.19(23.75); :3.10(3.57); 708 (>C-S); 3229 (>H); 1610 (>C=-); 9.1 (s, 1H, pyridine ring); 5.35 (s, 2H, -- -trizole); 2.35 (s, 1H, H- of 1,2,4-trizole); 4.3 (s, 2H, ); 6.12 (s, 1H, pyrzole =CH); 3.8 (q, 2H, >- ); 2.43 (q, 2H, C- ); (m, 6H, two -CH 3 S 6b: C 17 H S 60, , C:44.00(44.21); H:4.60(4.15); :17.87(18.20); :23.25(23.03); :3.56(3.46); 715 (>C-S); 3231 (>H); 1620 (>C=-); 9.1 (s, 1H, Pyridine ring); 5.35 (s, 2H, -- -Trizole); 2.35 (s, 1H, H- of 1,2,4-Trizole); 4.3 (s, 2H, ); 6.14 (s, 1H, pyrzole -CH); 3.92 (s, 3H, -CH 3 6c: C13H1136S 55, , C:38.49(37.59); H:2.73(2.51); :20.7(20.67) :26.22(25.91); :3.94(4.39); 710 (>C-S); 3234 (>H); 1615 (>C=-); 9.1(s, 1H, pyridine ring); 5.35 (s, 2H, -- -trizole); 2.35 (s, 1H, H- of 1,2,4-trizole); 4.3 (s, 2H, ); 6.12 (d, 1H, pyrzole -CH); 7.42 (d, 1H, pyrzole -CH); 3.9 (s, 3H, -CH 3 6d: C 14 H S 60, , C:40.06(40.65); H:3.12(3.58); :20.02(19.72); :25.34(25.87); :3.81(4.21); 714 (>C-S); 3236 (>H); 1610 (>C=-); 9.1 (s, 1H, pyridine ring); 5.35 (s, 2H, -- -trizole); 2.35 (s, 1H, H- of 1,2,4-trizole); 4.3 (s, 2H, ); 6.12 (s, 1H, pyrzole -CH); 7.42 (d, 1H, pyrzole -CH); 3.79 (q, 2H, - ); 1.32 (t, 3H, -CH 3 of ethyl group 6e: C 14 H S, 63, , C:40.06(40.45); H:3.12(3.52); :20.02(20.43); :25.34(24.83); :3.81(4.25); 706 (>C-S); 3230 (>H); 1608 (>C=-); 9.1 (s, 1H, pyridine ring); 5.35 (s, 2H, -- -trizole); 2.35 (s, 1H, -Hof 1,2,4-trizole); 4.3 (s, 2H, ); 6.12 (s, 1H, pyrzole -CH); 3.79 (s, 3H, -CH 3 ); 2.84 (s, 3H, -CH 3 6f: C 15 H S, 63, , C:41.54(42.01); H:3.49(3.84); :19.38(18.82); :24.52(24.08); :3.69(3.92); 715 (>C-S); 3228 (>H); 1615 (>C=-); 9.1 (s, 1H, pyrdine ring); 5.35 (s, 2H, -- -trizole); 2.35 (s, 1H, H of 1,2,4-trizole); 4.3 (s, 2H, ); 6.12 (s, 1H, pyrzole -CH); 3.81 (s, 3H, -CH 3 ); 2.63 (q, 2H, ); 1.32 (t, 3H, CH 3 of ethyl group 6g: C 16 H S, 60, , C:42.92(42.21); H:3.83(3.59); :18.77(19.24); :23.75(22.98); :3.57(3.89); 710 (>C-S); Journl of inicl nd Anlyticl Medicine

4 1,2,4,-Trizoller / 1,2,4-Trizoles (>H); 1610 (>C=-); 9.1 (s, 1H, pyridine ring); 5.35 (s, 2H, -- -trizole); 2.35 (s, 1H, H- of 1,2,4-trizole); 4.3 (s, 2H, ); 6.12 (s, 1H, pyrzole -CH); 3.82 (s, 3H, -CH 3 ); 3.04 (m, 1H, CH(CH 3 ); 1.32 (d, 6H, CH-(CH 3 Chrcteriztion dt of 4 (3 ((substituted) 1H pyrzol 3 yl-methylthio 5 ((3,5,6 trichloro-pyridine 2 yl-oxy)-methyl)-[1,2,4]trizol 4 yl)-methyl)-morpholine (7-g) [Compound: Moleculr Formul, Yield (%), m.p ( 0 c), Element:culted% (Found%) IR ν mx in cm 1 (Group); 1HMR (300 MHz, DMS-d 6 ) δ ppm (Group)] 7: C S, 68, , C:46.12(45.71); H:4.79(4.92); :17.93(17.49); :19.45(19.76); :5.85(6.19); 710 (C-S); 1615 (C=); 3040 (Aromtic pyridine ring); 9.1 (s,1h, pyridine ring); 5.35 (s, 2H, -- -trizole); 4.3 (s, 2H, ); 6.12 (s, 1H, pyrzole=ch); 3.8 (q, 2H, - ); 2.43 (q, 2H, C- ); (m, 6H, 2 -CH 3 groups); 4.12 (s, 2H, - -); 3.2(t, 4H, -- ring); 2.45 (t, 4H, -- ring 7b: C 22 H S, 56, , C:47.11(47.29); H:5.03(4.85); :17.48(18.88); :18.96(18.22); :5.70(6.62); 708 (C-S); 1610 (C=); 3050 (Aromtic pyridine ring); 9.1 (s,1h, pyridine ring); 5.35 (s, 2H, -- -trizole); 4.3 (s, 2H, ); 6.14 (s, 1H, pyrzole CH); 3.92 (s, 3H, -CH 3 ); 1.42 (s, 9H, CH 3 ); 4.12 (s, 2H, - -); 3.2 (t, 4H, -- ring); 2.45 (t, 4H, -- ring 7c: C 18 H S, 64, , C:42.83(43.27); H:3.99(4.19); :19.42(18.76); :21.07(21.68); :6.34(6.59); 712 (C-S); 1608 (C=); 3044 (Aromtic pyridine ring); 9.1 (s, 1H, pyrdine ring); 5.35 (s, 2H, -- -trizole); 4.3 (s, 2H, ); 6.12 (d, 1H, pyrzole CH); 7.42 (d, 1H, pyrzole-ch); 3.9 (s, 3H, -CH 3 ); 4.12 (s, 2H, - -); 3.2 (t, 4H, -- ring); 2.45 (t, 4H, -- ring 7d: C S, 59, , C:43.98(44.25); H:4.27(4.88); :18.90(18.64); :20.50(21.04); :6.17(6.72); 708 (C-S); 1615 (C=); 3040 (Aromtic pyridine ring); 9.1 (s, 1H, pyridine ring); 5.35 (s, 2H,-- -trizole); 4.3 (s, 2H, ); 6.12 (d, 1H, pyrzole-ch); 7.42 (d, 1H, pyrzole CH); 3.79 (q, 2H, -- ); 1.32 (t, 3H, -CH 3 of ethyl group); 4.12 (s, 2H, - -); 3.2 (t, 4H, -- ring); 2.45 (t, 4H, -- ring 7e: C S, 69, , C:43.98(43.81); H:4.27(4.59); :18.90(18.46); :20.50(21.09); :6.17(6.84); 710 (C-S); 1612 (C=); 3044 (Aromtic pyridine ring); 9.1 (s, 1H, pyridine ring); 5.35 (s, 2H, -- -trizole); 4.3 (s, 2H, ); 6.12 (d, 1H, pyrzole CH); 3.79 (s, 3H, -CH 3 ); 2.84 (s, 3H, CH 3 ); 4.12 (s, 2H, - -); 3.21 (t, 4H, -- ring); 2.45 (t, 4H, -- ring 7f: C 20 H S, 58, , C:45.08(45.73); H:4.54(4.75); :18.40(18.59); :19.96(20.18); :6.00(6.75); 712 (C-S); 1615 (C=); 3050 (Aromtic pyridine ring); 9.1(s,1H, pyridine ring); 5.35 (s, 2H, -- -trizole); 4.3 (s, 2H, ); 6.12 (s, 1H, pyrzole CH); 3.81 (s, 3H, -CH 3 ); 2.63 (s, 2H, ); 1.32(t, 3H, CH 3 ); 4.12 (s, 2H, - -); 3.21(t, 4H, -- ring); 2.45 (t, 4H, -- ring 7g: C S, 66, , C:46.12(46.81); H:4.79(5.18); :17.93(17.49); :19.45(20.18); :5.85(6.31); 710 (C-S); 1608 (C=); 3044 (Aromtic pyridine ring); 9.1 (s, 1H, pyridine ring); 5.35 (s, 2H, -- -trizole); 4.3 (s, 2H, ); 6.12 (d, 1H, pyrzole CH); 3.82 (s, 3H, -CH 3 ); 3.04 (m, 1H, CH(CH 3 )2); 1.32 (d, 6H, (CH(CH 3 ); 4.12 (s, 2H, - -); 3.21 (t, 4H, -- of morpholine ring); 2.45 (t, 4H, -- ring Chrcteriztion dt of 1,2,4-trizole thiones (8-g) [Compound: Moleculr Formul, Yield (%), m.p( 0c), Element:culted% (Found%) IR ν mx in cm 1 (Group); 1HMR (300 MHz, DMS-d 6 ) δ ppm (Group)] 8: C S, 57, , C:43.57(43.19); H:4.53(5.05); :16.94(16.21); :18.37(17.65); :11.06(11.92); 715 (C-S); 1615 (C=); 1375, 1160 (=S=); 9.1 (s,1h, Pyridine ring); 5.35 (s, 2H, -- -trizole) 4.67 (s, 2H, S 2 - ); 6.12 (s, 1H, pyrzole=ch); 3.8 (q, 2H, - ); 2.43 (q, 2H, C- ); (m, 6H, 2 CH 3 groups); 4.12 (s, 2H, - -), 3.2 (t, 4H, -- ring); 2.45 (t, 4H, ring 8b: C 22 H S, 59, , C:44.56 (44.21) H:4.76(4.94); :16.54(16.71); :17.94(17.62); :10.79(11.01); 712 (C-S); 1610 (C=); 1380, 1165 (=S=); 9.1 (s, 1H, pyridine ring); 5.35 (s, 2H, trizole); 4.68 (s, 2H, S 2 - ); 6.14 (s, 1H, pyrzole CH); 3.92 (s, 3H, -CH 3 ); 1.42 (s, 9H, CH 3 ); 4.12 (s, 2H, - -), 3.2 (t, 4H, -- ring); 2.45 (t, 4H, -- ring 8c: C 18 H S, 63, , C:40.27(4.10); H:3.76(3.45); :18.26(18.59); :19.81(20.71); :11.9 (12.64); 710 (C-S); 1608 (C=); 1370, 1160 (=S=); 9.1 (s, 1H, pyridine ring); 5.35 (s, 2H, -- -trizole); 4.65 (s, 2H, S 2 - ); 6.12 (d, 1H, pyrzole CH); 7.42 (d, 1H, pyrzole -CH); 3.9 (s, 3H, -CH 3 ); 4.12 (s, 2H, - -); 3.2 (t, 4H, -- ring); 2.45 (t, 4H, -- ring 8d: C S, 54, , C:41.43(41.08); H:4.03(4.95); :17.80(17.15); :19.31(18.29); :11.62(12.45); 708 (C-S); 1615 (C=); 1377, 1167 (=S=); 9.1 (s, 1H, pyridine ring); 5.35 (s, 2H, -- -trizole); 4.69 (s, 2H, S 2 - ); 6.12 (d, 1H, pyrzole-ch); 7.42 (d, 1H, pyrzole CH); 3.79 (q, 2H, -- ); 1.32(t, 3H, -CH 3 of ethyl group); 4.12 (s, 2H, - -); 3.2 (t, 4H, ring); 2.45 (t, 4H, -- of morpholine ring 8e: C S, 59, , C:41.43 (41.22); H:4.03(4.16); :17.80(17.74); :19.31(19.57); :11.62(11.81); 714 (C-S); 1612 (C=); 1380, 1170 (=S=); 9.1 (s, 1H, pyridine ring); 5.35 (s, 2H, -- -trizole); 4.68 (s, 2H, S 2 - ); 6.12 (d, 1H, pyrzole CH); 3.79 (s, 3H, -CH 3 ); 2.84 (s, 3H, CH 3 ); 4.12 (s, 2H, - -); 3.21 (t, 4H, -- ring); 2.45 (t, 4H, -- ring 8f: C 20 H S, 68, , C:42.53(42.28) H:4.28(4.07); :17.36(16.87); :18.83(18.29); :11.33(12.05); 710 (C-S); 1615 (C=); 1385, 1175 (=S=); 9.1 (s, 1H, pyridine ring); 5.35 (s, 2H,-- -trizole); 4.7 (s, 2H, S 2 - ); 6.12 (s, 1H, pyrzole CH); 3.81 (s, 3H, -CH 3 ); 2.63 (s, 2H, ); 1.32 (t, 3H, -CH 3 ); 4.12 (s, 2H, ); 3.21 (t, 4H, -- ring); 2.45 (t, 4H, -- ring 8g: C S, 69, , C:43.57(43.71); H:4.53(4.66); :16.94(16.25); :18.37(18.88); :11.06(11.28); 712 (C-S); 1608 (C=); 1370, 1155 (=S=); 9.1 (s, 1H, pyrdine ring); 5.35 (s, 2H, -- -trizole); 4.68 (s, 2H, S 2 ); 6.12 (d, 1H, pyrzole CH); 3.82(s, 3H, CH 3 ); 3.04 (m, 1H, CH(CH 3 ); 1.32 (d, 6H, (C(CH 3 ), 4.12 (s, 2H, ); 3.21 (t, 4H, -- of morpholine ring); 2.45 (t, 4H, -- ring Journl of inicl nd Anlyticl Medicine 593

5 1,2,4,-Trizoller / 1,2,4-Trizoles Mss spectrl detils Mss spectrum of 4-(3-((1,5-diethyl)-1H-pyrzol-3-ylmethylsulphonyl)-5-((3,5,6-trichloro-pyridin-2-yl-oxymethyl)-[1,2,4]trizol 4 yl-methyl)-morpholine (8) exhibits moleculr ion (M + ) pek t m/z 551 (19.3% The bse pek ws observed t m/z 354 (100% ther prominent peks were ppered t m/z 86 (24.7%), 160 (15.7%), 245 (27.1%), 269 (19.3%), 354 (16.6%) nd 464 (27.8% Antimicrobil ctivity The preliminry investigtions pertining to ntimicrobil ctivity ws performed by the disc diffusion method [26]. The grm positive bcteri screened were Stphylococcus ureus CCS 2079 nd Bcillus cereus CCS The grm negtive bcteril screened were Escherichi coli CCS 265 nd Pseudomons eruginos CCS2200. The fungi screened were Aspergillus niger nccs 1196 nd Cndid lbicns CCS Minimum inhibitory concentrtion ws estimted by broth dilution method [27]. It cn be seen from Tble 2 nd Tble 3 tht ll compounds were ctive ginst tested microbes. However none of them were superior to the stndrds tested. It is lso worth mentioning tht ntifungl ctivity of the synthesized compounds ws comprble to tht of stndrds. As mentioned supr, 1,2,4-trizoles once gin proved to be better ntifungl gents. Tble 2. Antimicrobil ctivity of 1,2,4-trizole thiones (8-g) Compound (40 μg/ml) Stphylococus ureus CCS 2079 Bcillus Cereus CCS 2106 Zone inhibition (mm) Escherichi coli CCS 2065 Pseudomns eruginos CCS 2200 Conclusion It cn be concluded tht ll the seven compounds synthesized nmely, 4-(3-((substituted)-1H-pyrzol-3-yl-methylsulphonyl)- 5-((3,5,6-trichloro-pyridin-2-yl-oxymethyl)-[1,2,4]trizol 4 ylmethyl)-morpholines (8-g) demonstrted ntibcteril nd ntifungl ctivity. Among the vrious 1,2,4-trizole thiones studied, 4-(3-((1-methyl)-1H-pyrzol-3-yl-methylsulphonyl)- 5-((3,5,6-trichloro-pyridin-2-yl-oxymethyl)-[1,2,4]trizol 4 ylmethyl)-morpholine (8C) ws found to exhibit better ntibcteril nd ntifungl ctivity thn the other compounds of the series. 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