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European Journal of Medicinal Chemistry 45 (2010) 5200e5207

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

Synthesis, antioxidant activities and urease inhibition of some new 1,2,4-triazoleand 1,3,4-thiadiazole derivatives

Imtiaz Khan a, Sajid Ali a, Shahid Hameed a, Nasim Hasan Rama a,*, Muhammad Tahir Hussain b,Abdul Wadood c, Reaz Uddin c, Zaheer Ul-Haq c, Ajmal Khan c, Sajjad Ali c, M. Iqbal Choudhary c

aDepartment of Chemistry, Quaid-i-Azam University, Islamabad 45320, PakistanbDepartment of Applied Sciences, National Textile University, Faisalabad 37610, PakistancDr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan

a r t i c l e i n f o

Article history:Received 3 June 2010Received in revised form9 August 2010Accepted 12 August 2010Available online 18 August 2010

Keywords:1,2,4-Triazoles1,3,4-ThiadiazolesAntioxidant activitiesUrease inhibition

* Corresponding author: Tel.: þ92 51 90642135; faE-mail address: nhrama@qau.edu.pk (N.H. Rama).

0223-5234/$ e see front matter � 2010 Elsevier Masdoi:10.1016/j.ejmech.2010.08.034

a b s t r a c t

New series of 4,5-disubstituted-2,4-dihydro-3H-1,2,4-triazole-3-thiones (8aej) and 2,5-disubstituted-1,3,4-thiadiazoles (9aeh) were synthesized by dehydrative cyclization of hydrazinecarbothioamidederivatives (7aek) by refluxing in 4 N aqueous sodium hydroxide and by overnight stirring with poly-phosphoric acid, respectively. The structures of the newly synthesized compounds were characterized byIR, 1H NMR, 13C NMR, elemental analysis and mass spectroscopic studies and the synthesized compoundswere screened for their antioxidant and urease inhibition activities. N-(2,4-Dimethylphenyl)-5-(4-nitrophenyl)-1,3,4-thiadiazol-2-amine (9h) showed excellent antioxidant activity more than the stan-dard drug whereas 4-(2,4-dimethylphenyl)-5-(3-nitrophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione(8d) and 4-(2,3-dimethylphenyl)-5-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (8e) exhibited potenturease inhibitory activities.

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1. Introduction

Reactive oxygen species (ROS), capable of causing damage toDNA, has been associated with carcinogenesis, coronary heartdisease, and many other health problems related to advancing age[1,2]. In low concentrations, synthetic antioxidants are also in usefor many industrial processes e.g. inhibition of radical formation forpreventing premature polymerization during processing, storageand transportation of unsaturated monomers. They exert theireffects by scavenging or preventing the generation of ROS [3] whichcan protect the formation of free radicals and retard the progress ofmany chronic diseases [4] including cancer, neurodegenerative,inflammation and cardiovascular diseases [5].

Urease (urea amidohydrolase, E.C. 3.5.1.5) catalyzes the hydro-lysis of urea resulting to the formation of ammonia and carbamicacid. Spontaneous decomposition of carbamic acid producesa second molecule of ammonia followed by its transformation toammonium, which may be up taken by plants under ideal condi-tions (soil pH <6e6.5) [6]. However, when the conditions are otherthan ideal, ammonia is escaped to the surroundings which maylead to environmental and economic problems [7]. Ureolyticactivity of several microorganisms, e.g. Proteus mirabilis, Proteus

x: þ92 51 90642241.

son SAS. All rights reserved.

vulgaris, and Ureaplsma urealyticum, is involved in the formation ofurinary tract stones, whichmay lead to the chronic inflammation ofkidney and its pelvis. Moreover, urinary catheter obstruction inpatients causes colonization of urease-producing microorganisms,essentially P. mirabilis. Overproduction of ammonia by infectiousmicroorganisms may contribute to ammonia encephalopathy orhepatic coma. Another mechanism of urease involvement inpathogenic bacteria infection relies on the formation of a micro-environment appropriate for the pathogen existence. Helicobacterpylori infection of stomach is possible only after local neutralizationof gastric acid by released ammonia [8]. Furthermore highconcentration of ammonia disturbs mucosal permeability, inparticular hydrogen ions passage through mucosal surface andcauses formation of peptic ulcers. In this respect, increasingattention has been focused on the search for compounds that willinhibit urease activity for the possible development of highlyneeded therapy for urease mediated bacterial infections [8e12].

Azoles, a versatile bunch of heterocycles are known chemo-therapeutic agents. Among these, 1,2,4-triazole and 1,3,4-thiadia-zoles possess urease inhibition and antioxidant activities [13,14]. Inthe present study, we have synthesized some new 1,2,4-triazoleand 1,3,4-thiadiazole derivatives which were tested against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and superoxide anionscavenging activities. Moreover, the synthesized compounds werescreened as urease inhibitors.

I. Khan et al. / European Journal of Medicinal Chemistry 45 (2010) 5200e5207 5201

2. Results and discussion

2.1. Chemistry

The synthetic route for the newly synthesized compounds,1,2,4-triazole and 1,3,4-thiadiazole derivatives (8aej and 9aeh), is illus-trated and outlined in Scheme 1. Substituted aromatic esters (2aeh)were synthesized by the reaction of corresponding acids (1a-h)withmethanol in the presence of catalytic amount of sulfuric acid. Esters(2aeh) were converted to the corresponding acid hydrazides(3aeh) by refluxing with hydrazine hydrate (80%) in methanol[15e18]. Dimethylphenylanilines (4aec) were converted into cor-responding dithiocarbamate salts (5aec) by stirring with concen-trated ammonia solution and pure carbon disulphide in methanol.The intermediate dithiocarbamateswerenot isolated and converteddirectly to substituted phenylisothiocyanates (6aec) by overnightstirringwith lead nitrate solution [19]. The compounds (6aec) wereisolated by steamdistillation. Hydrazinecarbothioamide derivatives(7aek)were synthesized by the condensation of corresponding acidhydrazides (3aeh) and phenylisothiocyanates (6aec) in methanol.Due to some unavoidable conditions, only 11 carbothioamides(7aek) could be prepared. These compounds were used as the key

OH

O

R

1a-h

R'(d)'

N

S

SNH4

H

R

+

5a-c

RNH2

'

(c)

4a-c

R'

N

NHN

SR

R S

NN

NH

(f)

8a-j

9a-h

A

B

4-6a R′ = 2,3-diCH3 8a R = 4-CH3

b R′ = 2,4-diCH3 b R = 2-Cl c R′ = 2,6-diCH3 c R = 4-Cl

7a R = 4-CH3 R′ = 2,6-diCH3 d R = 3-NO2

b R = 2-Cl R′ = 2,6-diCH3 e R = H c R = 4-Cl R′ = 2,3-diCH3 f R = 4-Cl d R = 3-NO2 R′ = 2,4-diCH3 g R = 3-Cl e R = H R′ = 2,3-diCH3 h R = 2-CH3

f R = 4-Cl R′ = 2,6-diCH3 i R = 3-Cl g R = 3-Cl R′ = 2,4-diCH3 j R = 4-Cl h R = 4-NO2 R′ = 2,4-diCH3

i R = 2-CH3 R′ = 2,3-diCH3

j R = 3-Cl R′ = 2,6-diCH3

k R = 4-Cl R′ = 2,4-diCH3

Scheme 1. Synthesis of 1,2,4-triazole and 1,3,4-thiadiazole derivatives. (a) Dry methanol, c(33%), dry methanol; (d) Pb(NO3)2; (e) dry methanol, reflux; (f) 4 N NaOH, reflux; and (g) P

intermediates for the synthesis of 1,2,4-triazole and 1,3,4-thiadia-zole derivatives. The 4,5-disubstituted-2,4-dihydro-3H-1,2,4-tri-azole-3-thiones (8aej) were synthesized by intramoleculardehydrative cyclization of carbothioamides (7aek) when refluxedin 4 N sodium hydroxide solution, followed by neutralization withconcentrated HCl [20], while 2,5-disubstituted-1,3,4-thiadiazoles(9aeh) were synthesized by overnight stirring of carbothioamides(7aek) at 70 �C in polyphosphoric acid [21]. All the reactions weremonitored by TLC and the newly synthesized compounds werepurified by column chromatography, if necessary. The structureelucidations of the newly synthesized compounds were carried outby modern spectroscopic techniques like IR, 1H NMR and 13C NMR.Further confirmations of the compounds were carried out by massspectrometry and microanalysis.

2.2. Pharmacology

2.2.1. Antioxidant activitiesCompounds 8d, 8j, and 9f with the IC50 values 242.49� 3.64,

292.38� 1.60 and 319.62� 2.04, respectively, have shownhydrogen donating ability on reaction with DPPH radical. It is clearfrom the results that the antioxidant potential of compounds is

(a)R O

OCH3

(b)

N

ONH2

HR

3a-h

2a-h

NCS+

6a-c(e)

R N

ON

H S

N

H H

'R

7a-k

'R (g)

R′ = 2,6-diCH3 9a R = 4-CH3 R′ = 2,6-diCH3

R′ = 2,6-diCH3 b R = 2-Cl R′ = 2,6-diCH3

R′ = 2,3-diCH3 c R = 4-Cl R′ = 2,3-diCH3

R′ = 2,4-diCH3 d R = 3-NO2 R′ = 2,4-diCH3

R′ = 2,3-diCH3 e R = H R′ = 2,3-diCH3

R′ = 2,6-diCH3 f R = 4-Cl R′ = 2,6-diCH3

R′ = 2,4-diCH3 g R = 3-Cl R′ = 2,4-diCH3

R′ = 2,3-diCH3 h R = 4-NO2 R′ = 2,4-diCH3

R′ = 2,6-diCH3

R′ = 2,4-diCH3

onc. H2SO4, reflux; (b) dry methanol, hydrazine hydrate (80%), reflux; (c) CS2, NH4OHPA. Stirring.

I. Khan et al. / European Journal of Medicinal Chemistry 45 (2010) 5200e52075202

associated with the position of the substituents on both the rings Aand B. In compounds 8d, 8j and 9f, meta-nitro and para-chlorosubstituents are present at aryl ring A while in 8d and 8j, thedimethyl substituents are present at ortho and para positions onaryl ring B which contributed in the radical scavenging ability. Thethiadiazole class of compounds was found to be significantly activeagainst superoxide anion radical. The compound 9h with an IC50value 94.84� 0.34 was found to be more active than the standardantioxidant propyl gallate. Since themode of action of superoxide isdifferent from DPPH radical, it adds to labile site of the compoundand creates a negative charge on the resulting adduct [22]. Thuselectron withdrawing groups could stabilize the formed negativelycharged adduct. The activity of 9h is enhanced in this class due tothe presence of nitro group which is a strong electron withdrawingin nature. The compounds 9c, 9d and 9g have shown comparableantioxidant potential with the standard, whereas the compound 9fshowed a moderate superoxide anion scavenging activity.

2.2.2. Urease inhibition bioassayAlmost all compounds showed moderate to good activities.

Potent compounds have their activities in the range of 45.60� 0.04to 483.55�1.99 mM. Among the two series of compounds, triazolederivatives are relatively more active than the derivatives of thia-diazole (Table 3). The triazole derivatives may be regarded assubstrate like inhibitors on the basis of their structural similaritywith the natural substrate of urease i.e. urea. The Compounds 8dand 8e were found to be the most active with IC50 values of45.60� 0.04 and 86.0� 0.04 mM, respectively. In compound 8e, thearomatic ring at position 5 of the triazole nucleus is unsubstitutedwhereas in 8d this has a meta-nitro group. This indicates thata nitro group on the aryl ring enhances the activity. Among thechlorosubstituted groups, compound 8j with the para-chlorosubstituent is the most active, while compounds 8c and 8f alsohaving a para-chlorosubstituent were found inactive. In thecompound 8j, 2,4-dimethyl substituents are present at ortho andpara positions of the aryl ring which are perhaps more suited to fitthe enzyme active site as compared to 2,3-dimethyl and 2,6-dimethyl substituents at aryl ring in compounds 8e and 8f. Thisactivity trend indicates that the triazole moiety with small lipo-philic methyl substituent at para position of the aryl ring anda relatively large and polarizable group like NO2 at meta position isfavorable in terms of activity enhancement against urease.

2.2.3. ConclusionNew series of triazole (8aej) and thiadiazole (9aeh) derivatives

were synthesized. The antioxidant ability of these compounds wasevaluated. Some of the compounds were found to be significantscavengers of free radicals. The compound (9h) showed excellentantioxidant activity more than the standard drug. Therefore, thediscovered scavengers with curative values should be furtherinvestigated, especially for their efficacy to prevent or control theoxidative stress in diseases whose tangible and beneficial alterna-tives are still insufficient.

The synthesized compounds were also evaluated for ureaseinhibition activity which demonstrates that some compounds inthe series are most promising. The identified compounds can beutilized in further optimization of bioactivity using structuralvariations in the parent skeleton. The compounds 8d, 8e and 8j arepotential lead candidates with respect to the urease inhibitionactivity whereas compounds 9d and 9f show moderate activity.

3. Experimental

All the common chemicals and solvents were of analytical gradeor dry distilled. Air or moisture sensitive processes were conducted

under the inert atmosphere of dry nitrogen. Melting points weredetermined on Stuart melting point apparatus (SMP3) and areuncorrected. The IR spectra were recorded on Bruker Optics AlphaFT-IR Spectrometer. 1H NMR (300 MHz) and 13C NMR (75 MHz)were recorded on a Bruker AV-300 as DMSO-d6 and CDCl3 solutionsusing TMS as an internal standard. Electron impact mass spectra(EIMS) were recorded on a Finnigan MAT-311A (Germany) massspectrometer operating at an ionization potential of 70 eV. CHNanalyses were performed on a Carlo Erba Strumentazion-Mod-1106Italy. Thin layer chromatography was performed on pre-coatedsilica gel plates (Kieselgel 60, 254, E. Merck, Germany) and thechromatograms were visualized by UV at 254 and 365 nm.

3.1. Synthesis of substituted aromatic esters (2aeh) and aromaticacid hydrazides (3aeh)

Substituted aromatic acids (1aeh) were esterified (2aeh) andthe later were converted into their corresponding acid hydrazides(3aeh) by the standard procedure [15e18].

3.2. Synthesis of substituted phenylisothiocyanates (6aec)

Substituted anilines (4aec) were converted to their dithiocar-bamates (5aec) which were oxidized to respective phenyl-isothiocyanates (6aec) by the standard procedure [15e18].

3.3. General procedure for the synthesis ofhydrazinecarbothioamide derivatives (7aek)

The corresponding acid hydrazide (3) (0.0068 moles) was dis-solved in methanol (30 mL) and added dropwise to the solution ofsubstituted isothiocyanate (0.0066 moles) in methanol (10 mL).The reaction mixture was refluxed for 10e12 h and monitored byTLC. After consumption of the starting materials, the mixture wascooled to room temperature. Evaporation of solvent under reducedpressure left crude hydrazinecarbothioamide (7) as an oil whichwas solidified on cooling. It was purified by recrystallization froma mixture of ethyl acetate and petroleum ether to yield hydrazi-necarbothioamide (7).

3.3.1. N-(2,6-Dimethylphenyl)-2-(4-methylbenzoyl)hydrazinecarbothioamide (7a)

White solid (57%, 1.18 g): m.p 148e149 �C; Rf: 0.53 (n-hex-ane:ethyl acetate, 8:2); IR (pure, cm�1): 3341, 3223 (NH), 1677 (C]O), 1515, 1498 (C]C), 1245 (C]S); 1H NMR (300 MHz, DMSO-d6):d 10.47 (s, 1H, NHeC]O), 9.54 (s, 1H, NHeC]S), 9.49 (s, 1H,NHeC]S), 7.86 (d, 2H, J¼ 8.1 Hz, AreH), 7.19 (d, 2H, J¼ 8.1 Hz,AreH), 7.08e7.01 (m, 3H, AreH), 2.36 (s, 3H, CH3), 2.15 (s, 6H, 2�CH3); 13C NMR (75 MHz, DMSO-d6): d 181.80, 166.31, 142.14, 141.35,137.52,130.00, 129.07, 128.50, 127.86,127.11, 22.29, 18.45; EI-MS:m/z (rel. abund. %), 313.14 (M�þ, 13.6), 279.17(41.8), 164.07(38.9),163.02(93.6), 150.11(52.4), 148.06(33.6), 131.09(39.7), 120.09(41.5),119.04(100), 105.09(37.7), 91.06(88.0), 77.07(32.9), 65.07(39.8).Analysis. Calcd for C17H19N3SO: C, 65.15; H, 6.11; N, 13.41; S, 10.23.Found: C, 65.09; H, 6.07; N, 13.25; S, 10.02.

3.3.2. 2-(2-Chlorobenzoyl)-N-(2,6-dimethylphenyl)hydrazinecarbothioamide (7b)

White solid (73%, 1.61 g): m.p 155e156 �C; Rf: 0.39 (n-hex-ane:ethyl acetate, 8:2); IR (pure, cm�1): 3326, 3281 (NH), 1644(C]O), 1593, 1520, 1469 (C]C), 1235 (C]S); 1H NMR (300 MHz,DMSO-d6): d 10.50 (s, 1H, NHeC]O), 9.75 (s, 1H, NHeC]S), 9.19 (s,1H, NHC]S), 7.55e7.38 (m, 4H, AreH), 7.13e7.05 (m, 3H, AreH),2.17 (s, 6H, 2� CH3); 13C NMR (75 MHz, DMSO-d6): d 182.25, 166.13,137.49, 132.07, 131.38, 131.29, 130.79, 130.09, 129.63, 127.95, 127.52,

I. Khan et al. / European Journal of Medicinal Chemistry 45 (2010) 5200e5207 5203

127.30, 18.41; EI-MS: m/z (rel. abund. %), 334.95 (M� þ 2, 2.3),332.99 (M�þ, 4.5), 298.99(25.7), 211.91(29.3), 162.94(100), 151.97(33.7), 147.99(24.4), 145.03(17.5), 140.95(77.8), 138.90(100), 130.03(68.1), 121.05(28.0), 105.03(24.5), 91.01(10.5), 77.02(24.9), 75.00(34.0). Analysis. Calcd for C16H16ClN3SO: C, 57.56; H, 4.83; N, 12.59;S, 9.61. Found: C, 57.41; H, 4.67; N, 12.43; S, 9.52.

3.3.3. 2-(4-Chlorobenzoyl)-N-(2,3-dimethylphenyl)hydrazinecarbothioamide (7c)

White solid (75%, 1.66 g): m.p 165e166 �C; Rf: 0.44 (n-hex-ane:ethyl acetate, 8:2); IR (pure, cm�1): 3296, 3163 (NH), 1673 (C]O), 1596, 1512 (C]C), 1268 (C]S); 1H NMR (300 MHz, DMSO-d6):d 10.63 (s, 1H, NHeC]O), 9.62 (s, 2H, 2NHeC]S), 7.96 (d, 2H,J¼ 8.4 Hz, AreH), 7.58 (d, 2H, J¼ 8.4 Hz, AreH), 7.07e7.06 (m, 3H,AreH), 2.24 (s, 3H, CH3), 2.06 (s, 3H, CH3); 13C NMR (75 MHz,DMSO-d6): d 182.06, 165.06, 137.25, 137.03, 131.91, 130.35, 129.37,129.33, 128.72, 128.48, 127.09, 126.71, 125.47, 20.52, 14.56; EI-MS:m/z (rel. abund. %), 335.01 (M�þ þ 2, 5.7), 332.98 (M�þ, 18.4), 324.12(72.9), 291.12(42.1), 237.03(38.9), 163.00(100), 148.03(24.9), 145.09(42.9), 131.07(32.9), 130.07(83.7), 121.09(51.2), 105.08(39.5), 91.06(21.4), 77.06(42.1). Analysis. Calcd for C16H16ClN3SO: C, 57.56; H,4.83; N, 12.59; S, 9.61. Found: C, 57.51; H, 4.69; N, 12.50; S, 9.49.

3.3.4. N-(2,4-Dimethylphenyl)-2-(3-nitrobenzoyl)hydrazinecarbothioamide (7d)

White solid (59%, 1.27 g): m.p 143e145 �C; Rf: 0.40 (n-hex-ane:ethyl acetate, 8:2); IR (pure, cm�1): 3354, 3130 (NH), 1681 (C]O),1586, 1555,1530 (C]C), 1242 (C]S); 1H NMR (300 MHz, DMSO-d6): d 10.92 (s, 1H, NHeC]O), 9.71 (s, 1H, NHeC]S), 9.61 (s, 1H,NHeC]S), 8.78 (s, 1H, AreH), 8.43 (dd, 1H, J¼ 8.1,1.8 Hz, AreH),8.35 (d, 1H, J¼ 8.1 Hz, AreH), 7.81 (t, 1H, J¼ 7.8 Hz, AreH),7.03e6.98 (m, 3H, AreH), 2.26 (s, 3H, CH3), 2.14 (s, 3H, CH3); 13CNMR (75 MHz, DMSO-d6): d 182.06, 164.65, 148.00, 136.31, 135.85,134.73, 131.09, 129.10, 126.88, 125.53, 125.41, 125.11, 123.89, 123.15,21.08, 18.09; EI-MS: m/z (rel. abund. %), 344.27 (M�þ, 2.5), 181.03(15.3), 163.00(100), 150.00(94.1), 148.01(24.1), 145.07(8.5), 130.05(37.7), 105.05(25.3), 77.03(15.9), 63.00(11.1). Analysis. Calcd forC16H16N4SO3: C, 55.80; H, 4.68; N, 16.27; S, 9.31. Found: C, 55.64; H,4.53; N, 16.14; S, 9.20.

3.3.5. N-(2,3-Dimethylphenyl)-2-(benzoyl)hydrazinecarbothioamide (7e)

White solid (61%, 1.20 g): m.p 135e136 �C; Rf: 0.37 (n-hex-ane:ethyl acetate, 8:2); IR (pure, cm�1): 3458, 3183 (NH), 1669(C]O), 1601, 1580, 1531 (C]C), 1237 (C]S); 1H NMR (300 MHz,DMSO-d6): d 10.54 (s, 1H, NHeC]O), 9.60 (s, 2H, 2NHeC]S), 7.95(d,1H, J¼ 7.5 Hz, AreH), 7.60e7.55 (m, 2H, AreH), 7.51e7.46 (m, 3H,AreH), 7.06 (d, 2H, J¼ 4.5 Hz, AreH), 2.24 (s, 3H, CH3), 2.07 (s, 3H,CH3); 13C NMR (75 MHz, DMSO-d6): d 182.32,166.54,138.43,137.28,135.03, 133.08, 132.21, 128.61, 128.43, 127.08, 126.43, 125.44, 20.50,19.03; EI-MS: m/z (rel. abund. %), 299.18 (M�þ, 3.1), 265.17(66.2),195.14(13.5), 163.03(99.8), 148.08(46.6), 145.12(28.9), 130.08(94.3),105.02(100), 91.08(15.7), 77.05(95.4), 63.03(41.4), 51.04(34.0).Analysis. Calcd for C16H17N3SO: C, 64.09; H, 5.72; N, 14.04; S, 10.71.Found: C, 63.94; H, 5.62; N, 13.91; S, 10.56.

3.3.6. 2-(4-Chlorobenzoyl)-N-(2,6-dimethylphenyl)hydrazinecarbothioamide (7f)

White solid (72%, 1.59 g): m.p 185e186 �C; Rf: 0.36 (n-hex-ane:ethyl acetate, 8:2); IR (pure, cm�1): 3319, 3121 (NH), 1681(C]O), 1594, 1525, 1492 (C]C), 1251 (C]S); 1H NMR (300 MHz,DMSO-d6): d 10.64 (s, 1H, NHeC]O), 9.61 (s, 1H, NHeC]S), 9.43 (s,1H, NH-C]S), 7.97 (d, 2H, J¼ 8.4 Hz, AreH), 7.58 (d, 2H, J¼ 8.7 Hz,AreH), 7.07e7.02 (m, 3H, AreH), 2.15 (s, 6H, 2� CH3); 13C NMR(75 MHz, DMSO-d6): d 181.78, 165.74, 137.42, 136.99, 131.95, 130.41,

129.32, 128.68, 127.89, 127.18, 21.08; EI-MS: m/z (rel. abund. %),335.00 (M�þ þ 2, 3.2), 332.99 (M�þ, 5.4), 298.96(98.2), 178.98(14.5),171.00(21.6), 162.99(86.4), 152.01(39.7), 148.03(31.9), 145.06(91.3),140.97(71.7), 138.94(100), 130.05(66.6), 121.08(26.2), 110.98(77.1),105.06(42.4), 77.05(37.8), 75.03(41.5), 51.03(16.5). Analysis. Calcdfor C16H16ClN3SO: C, 57.56; H, 4.83; N, 12.59; S, 9.61. Found: C,57.41; H, 4.59; N, 12.38; S, 9.47.

3.3.7. 2-(3-Chlorobenzoyl)-N-(2,4-dimethylphenyl)hydrazinecarbothioamide (7g)

White solid (69%, 1.52 g): m.p 167e169 �C; Rf: 0.38 (n-hex-ane:ethyl acetate, 8:2); IR (pure, cm�1): 3345, 3165 (NH), 1673 (C]O), 1555, 1533 (C]C), 1239 (C]S); 1H NMR (300 MHz, DMSO-d6):d 10.66 (s, 1H, NHeC]O), 9.63 (s, 1H, NHeC]S), 9.54 (s, 1H,NHeC]S), 8.01 (s,1H, AreH), 7.87 (d,1H, J¼ 7.8 Hz, AreH), 7.65 (dd,1H, J¼ 8.1, 1.8 Hz, AreH), 7.53 (t, 1H, J¼ 7.8 Hz, AreH), 7.03e6.97(m, 3H, AreH), 2.26 (s, 3H, CH3), 2.13 (s, 3H, CH3); 13C NMR (75 MHz,DMSO-d6): d 182.18, 165.29, 136.25, 135.91 135.12, 133.42, 132.02,131.06, 130.69, 129.11, 128.29, 127.13, 126.83, 125.60, 21.08, 18.09;EI-MS:m/z (rel. abund. %), 335.01 (M�þ þ 2, 1.0), 332.90 (M�þ þ 2.9),298.94(30.0), 170.92(21.7), 169.93(52.3), 162.88(99.1), 151.95(14.0),147.95(53.2), 145.01(19.3), 140.93(92.6), 138.86(100), 129.99(96.9),121.02(19.5), 110.91(98.7), 105.01(60.3), 77.00(35.7), 62.99(14.2),50.99(20.6) Analysis. Calcd for C16H16ClN3SO: C, 57.56; H, 4.83; N,12.59; S, 9.61. Found: C, 57.44; H, 4.65; N, 12.42; S, 9.41.

3.3.8. N-(2,4-Dimethylphenyl)-2-(4-nitrobenzoyl)hydrazinecarbothioamide (7h)

Yellow solid (63%, 1.35 g): m.p 160e161 �C; Rf: 0.49 (n-hex-ane:ethyl acetate, 8:2); IR (pure, cm�1): 3309, 3154 (NH), 1668 (C]O), 1596, 1499 (C]C), 1257 (C]S); 1H NMR (300 MHz, DMSO-d6):d 10.87 (s, 1H, NHeC]O), 9.70 (s, H, NHeC]S), 9.60 (s, 1H, NHeC]S), 8.35 (d, 2H, J¼ 9.0 Hz, AreH), 8.16 (d, 2H, J¼ 8.7 Hz, AreH),7.03e6.98 (m, 3H, AreH), 2.26 (s, 3H, CH3), 2.14 (s, 3H, CH3); 13CNMR (75 MHz, DMSO-d6): d 182.18, 165.06, 149.75, 138.86, 136.30,135.85, 131.10, 129.93, 129.14, 126.86, 126.63, 123.80, 21.07, 18.08;EI-MS: m/z (rel. abund. %), 344.17 (M�þ, 2.8), 310.05(23.7), 182.04(38.0), 181.02(34.6), 162.97(100), 151.03(53.0), 149.98(95.3), 148.02(49.7), 145.07(11.6), 131.06(21.7), 130.05(78.2), 121.07(27.8), 105.06(64.9), 104.01(80.6), 91.05(12.0), 77.05(35.1), 63.02(27.4). Analysis.Calcd for C16H16N4SO3: C, 55.80; H, 4.68; N, 16.27; S, 9.31. Found: C,55.61; H, 4.55; N, 16.19; S, 9.17.

3.3.9. N-(2,3-Dimethylphenyl)-2-(2-methylbenzoyl)hydrazinecarbothioamide (7i)

White solid (53%, 1.09 g): m.p 150e151 �C; Rf: 0.47 (n-hex-ane:ethyl acetate, 8:2); IR (pure, cm�1): 3250, 3154 (NH), 1665 (C]O),1555,1526, 1491 (C]C), 1237 (C]S); 1H NMR (300 MHz, DMSO-d6): d 10.20 (s, 1H, NHeC]O), 9.48 (s, 1H, NHeC]S), 9.40 (s, 1H,NHeC]S), 7.68 (bs, 1H, AreH), 7.36 (dt, 1H, J¼ 8.4,0.9 Hz, AreH),7.27e7.21 (m, 3H, AreH), 7.06 (d, 2H, J¼ 8.1 Hz, AreH), 2.41 (s, 3H,CH3), 2.26 (s, 3H, CH3), 2.09 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6): d 182.18, 168.94, 138.46, 137.19, 136.13, 136.10, 134.95, 130.99,130.85, 129.81, 127.70, 125.92, 125.68, 125.57, 21.63, 20.12, 14.51; EI-MS:m/z (rel. abund. %), 313.11 (M�þ, 3.4), 280.15(44.5), 279.08(96.3),164.05(19.1), 163.02(96.4), 159.05(32.8), 148.05(28.9), 131.07(52.6),120.07(35.5), 119.01(100), 105.06(28.1), 91.04(99.8), 77.06(36.5),65.06(36.0), 63.02(25.4). Analysis. Calcd for C17H19N3SO: C, 65.15; H,6.11; N, 13.41; S, 10.23. Found: C, 64.99; H, 6.01; N, 13.21; S, 10.08.

3.3.10. 2-(3-Chlorobenzoyl)-N-(2,6-dimethylphenyl)hydrazinecarbothioamide (7j)

White solid (71%, 1.57 g): m.p 175e176 �C; Rf: 0.41 (n-hex-ane:ethyl acetate, 8:2); IR (pure, cm�1): 3300, 3143 (NH), 1674(C]O), 1522, 1497 (C]C), 1236 (C]S); 1H NMR (300 MHz, DMSO-

I. Khan et al. / European Journal of Medicinal Chemistry 45 (2010) 5200e52075204

d6): d 10.69 (s, 1H, NHeC]O), 9.62 (s, 1H, NHeC]S), 9.44 (s, 1H,NHeC]S), 8.03 (s, 1H, AreH), 7.88 (d, 1H, J¼ 7.8 Hz, AreH), 7.65(td, 1H, J¼ 7.2, 0.9 Hz, AreH), 7.53 (t, 1H, J¼ 8.1 Hz, AreH),7.08e7.04 (m, 3H, AreH), 2.16 (s, 6H, 2� CH3); 13C NMR (75 MHz,DMSO-d6): d 181.78, 165.50, 137.39, 136.99, 135.72, 135.19, 133.38,131.98, 130.65, 128.34, 127.90, 126.11, 18.41; EI-MS: m/z (rel.abund. %), 334.98 (M�þ þ 2, 7.7), 332.98 (M�þ, 14.4), 299.01(18.2),269.03(50.2), 170.98(53.0), 162.96(98.0), 148.01(30.2), 145.06(19.1), 140.95(86.8), 138.92(100), 130.04(85.8), 121.08(52.0),110.95(93.1), 77.04(34.3), 75.02(58.6), 51.02(17.7). Analysis. Calcdfor C16H16ClN3SO: C, 57.56; H, 4.83; N, 12.59; S, 9.61. Found: C,57.49; H, 4.71; N, 12.47; S, 9.48.

3.3.11. 2-(4-Chlorobenzoyl)-N-(2,4-dimethylphenyl)hydrazinecarbothioamide (7k)

White solid (66%, 1.46 g): m.p 159e160 �C; Rf: 0.33 (n-hex-ane:ethyl acetate, 8:2); IR (pure, cm�1): 3253, 3140 (NH), 1679(C]O), 1594, 1509, 1476 (C]C), 1238 (C]S); 1H NMR (300 MHz,DMSO-d6): d 10.61 (s, 1H, NHeC]O), 9.60 (s, 1H, NHeC]S), 9.52(s, 1H, NHeC]S), 7.95 (d, 2H, J¼ 8.4 Hz, AreH), 7.58 (d, 2H,J¼ 8.7 Hz, AreH), 7.02e6.97 (m, 3H, AreH), 2.26 (s, 3H, CH3), 2.14(s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6): d 182.25, 165.56,137.03, 136.19, 131.91, 131.09, 130.34, 129.35, 128.90, 128.72,128.11, 126.81, 21.07, 18.09; EI-MS: m/z (rel. abund. %), 334.98(M�þ þ 2, 1.3), 332.98 (M�þ, 3.7), 300.06(29.1), 299.05(65.3),171.00(36.0), 162.95(100), 148.02(44.9), 145.07(29.9), 140.97(89.0), 138.92(99.0), 130.05(83.7), 121.07(31.7), 110.97(79.0),105.05(51.3), 77.03(28.0), 75.01(35.1), 51.01(13.1). Analysis. Calcdfor C16H16ClN3SO: C, 57.56; H, 4.83; N, 12.59; S, 9.61. Found: C,57.47; H, 4.63; N, 12.50; S, 9.46.

3.4. General procedure for the synthesis of 1,2,4-triazol-3-thiones(8aej)

The corresponding carbothioamide (7) (0.0014 moles) wasrefluxed in aqueous sodium hydroxide solution (4 N, 25 mL). Thereaction was monitored by TLC. After completion of reaction(12e14 h), the reaction mixture was cooled to room temperatureand filtered. The filtrate was neutralized with hydrochloric acid(4 N) to precipitate the 1,2,4-triazol-3-thione (8) which wasrecrystalized from aqueous ethanol.

3.4.1. 4-(2,6-Dimethylphenyl)-5-(4-methylphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (8a)

White crystalline solid (69%, 0.28 g): m.p 262e265 �C; Rf: 0.66(n-hexane:ethyl acetate, 8:2); IR (pure, cm�1): 3355 (NH), 2910,2846 (sp3 CH), 1533 (C]N), 1489, 1472 (C]C), 1281 (C]S); 1H NMR(300 MHz, DMSO-d6): d 14.37 (s, 1H, NH), 7.36e7.31 (m, 1H, AreH),7.23 (d, 2H, J¼ 7.8 Hz, AreH), 7.14 (bs, 4H, AreH), 2.25 (s, 3H, CH3),1.95 (s, 6H, 2� CH3); 13C NMR (75 MHz, DMSO-d6): d 167.82, 150.26,141.13, 136.40, 133.34, 130.33, 129.97, 129.20, 126.91, 123.28, 21.29,17.99; EI-MS: m/z (rel. abund. %), 295.12 (M�þ, 56.9), 262.17(100),145.13(24.8), 105.10(6.9), 78.05(57.1), 63.03(62.3); Analysis. Calcdfor C17H17N3S: C, 69.12; H, 5.80; N, 14.22; S, 10.85. Found: C, 69.05;H, 5.63; N, 14.09; S, 10.63.

3.4.2. 5-(2-Chlorophenyl)-4-(2,6-dimethylphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (8b)

White crystalline solid (63%, 0.28 g): m.p 282e284 �C; Rf: 0.64(n-hexane:ethyl acetate, 8:2); IR (pure, cm�1): 3352 (NH), 2939 (sp3

CH), 1552 (C]N), 1498, 1475 (C]C), 1233 (C]S); 1H NMR(300 MHz, DMSO-d6): d 14.40 (s, 1H, NH), 7.60 (d, 1H, J¼ 7.5 Hz,AreH), 7.46 (dt, 1H, J¼ 7.8,1.5 Hz, AreH), 7.30e7.20 (m, 2H, AreH),7.14e7.09 (m, 3H, AreH) 2.05 (s, 6H, 2� CH3); 13C NMR (75 MHz,DMSO-d6): d 167.00, 148.20, 136.61, 133.34, 133.03, 131.96, 131.72,

131.02, 130.14, 128.96, 127.77, 124.69, 18.23; EI-MS: m/z (rel. abund.%), 317.00 (M�þ þ 2, 14.4), 315.00 (M�þ, 37.9), 284.10(32.9), 282.01(100), 145.05(26.3), 136.90(12.1), 105.02(13.7), 91.08(13.9), 77.00(26.4), 69.10(43.4). Analysis. Calcd for C16H14ClN3S: C, 60.85; H,4.47; N, 13.31; S, 10.15. Found: C, 60.69; H, 4.22; N, 13.24; S, 10.05.

3.4.3. 5-(4-Chlorophenyl)-4-(2,3-dimethylphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (8c)

White crystalline solid (61%, 0.27 g): m.p 290e291 �C; Rf: 0.55(n-hexane:ethyl acetate, 8:2); IR (pure, cm�1): 3262 (NH), 2961 (sp3

CH), 1555 (C]N), 1501, 1477 (C]C), 1253 (C]S); 1H NMR(300 MHz, DMSO-d6): d 14.53 (s, 1H, NH), 7.40 (d, 2H, J¼ 7.2 Hz,AreH), 7.28 (d, 1H, J¼ 7.5 Hz, AreH), 7.24 (d, 2H, J¼ 8.7 Hz, AreH),7.18 (t, 1H, J¼ 7.8 Hz, AreH), 7.02 (d, 1H, J¼ 7.5 Hz, AreH), 2.26 (s,3H, CH3), 1.81 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6): d 167.33,148.57, 138.48, 135.00, 131.27, 129.21, 129.16, 128.10, 127.78, 127.19,126.78, 126.06, 20.34, 14.39; EI-MS: m/z (rel. abund. %), 317.07(M�þ þ 2, 18.6), 315.05 (M�þ, 50.5), 300.05(11.5), 284.10(35.5),282.11(100), 163.07(10.5), 145.11(23.2), 103.07(7.3), 78.05(27.2),77.08(15.2), 63.03(25.0). Analysis. Calcd for C16H14ClN3S: C, 60.85;H, 4.47; N, 13.31; S, 10.15. Found: C, 60.75; H, 4.29; N, 13.13; S, 9.98.

3.4.4. 4-(2,4-Dimethylphenyl)-5-(3-nitrophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (8d)

Yellow solid (47%, 0.21 g): m.p 278e281 �C; Rf: 0.57 (n-hex-ane:ethyl acetate, 8:2); IR (pure, cm�1): 3373 (NH), 2955 (sp3 CH),1529 (C]N), 1478 (C]C), 1261 (C]S); 1H NMR (300 MHz, DMSO-d6): d 14.38 (s, 1H, NH), 8.08 (s, 1H, AreH), 8.26 (d, 1H, J¼ 7.8 Hz,AreH), 7.73 (d, 1H, J¼ 7.5 Hz, AreH), 7.66 (t, 1H, J¼ 8.1 Hz, AreH),7.25 (d, 2H, J¼ 8.1 Hz, AreH), 7.16 (d, 1H, J¼ 9.0 Hz, AreH), 2.34 (s,3H, CH3), 2.00 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6): d 167.16,148.87, 148.11, 140.28, 136.01, 133.86, 132.25, 131.24, 131.09, 129.39,128.40, 127.27, 125.44, 122.43, 21.21, 17.73; EI-MS: m/z (rel. abund.%), 326.00 (M�þ, 62.8), 293.00(100), 247.01(38.0), 162.90(12.1),145.06(14.6), 105.03(19.3), 91.07(21.4), 77.02(30.7), 65.10(10.3),51.11(11.9). Analysis. Calcd for C16H14N4O2S: C, 58.88; H, 4.32; N,17.17; S, 9.82. Found: C, 58.72; H, 4.21; N, 16.98; S, 9.64.

3.4.5. 4-(2,3-Dimethylphenyl)-5-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (8e)

White crystalline solid (53%, 0.20 g): m.p 207e209 �C; Rf: 0.59(n-hexane:ethyl acetate, 8:2); IR (pure, cm�1): 3268 (NH), 2963(sp3 CH), 1550 (C]N), 1489, 1475 (C]C), 1240 (C]S); 1H NMR(300 MHz, DMSO-d6): d 14.19 (s, 1H, NH), 7.41e7.27 (m, 6H, AreH),7.21 (t, 1H, J¼ 7.8 Hz, AreH), 7.12 (d, 1H, J¼ 7.5 Hz, AreH), 2.27 (s,3H, CH3), 1.87 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6): d 168.83,150.87, 138.64, 135.03, 134.03, 131.64, 130.96, 129.19, 127.96, 127.27,126.93, 126.21, 20.13, 14.38; EI-MS:m/z (rel. abund. %), 281.00 (M�þ,72.7), 266.01(10.6), 248.12(100), 163.03(7.1), 145.09(13.1), 103.07(26.3), 77.00(33.4), 51.10(13.5). Analysis. Calcd for C16H15N3S: C,68.30; H, 5.37; N, 14.93; S, 11.40. Found: C, 68.11; H, 5.15; N, 14.86;S, 11.25.

3.4.6. 5-(4-Chlorophenyl)-4-(2,6-dimethylphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (8f)

White crystalline solid (72%, 0.31 g): m.p 257e260 �C; Rf: 0.62(n-hexane:ethyl acetate, 8:2); IR (pure, cm�1): 3206 (NH), 2984 (sp3

CH), 1565 (C]N), 1503, 1481 (C]C), 1222 (C]S);1H NMR(300 MHz, DMSO-d6): d 14.43 (s, 1H, NH), 7.40 (d, 2H, J¼ 9.3 Hz,AreH), 7.33 (dd, 1H, J¼ 9.0,9.0 Hz, AreH), 7.24 (d, 2H, J¼ 8.7 Hz,AreH), 7.23 (d, 2H, J¼ 7.2 Hz, AreH), 1.93 (s, 6H, 2� CH3); 13C NMR(75 MHz, DMSO-d6): d 167.87, 148.12, 136.38, 135.51, 133.51, 130.21,129.54, 128.47, 125.46, 124.21, 18.05; EI-MS: m/z (rel. abund. %),317.00 (M�þ þ 2, 15.3), 315.00 (M�þ, 40.7), 284.10(25.5), 282.16(80.2), 145.05(30.3), 91.12(9.1), 78.02(82.4), 63.10(100). Analysis.

I. Khan et al. / European Journal of Medicinal Chemistry 45 (2010) 5200e5207 5205

Calcd for C16H14ClN3S: C, 60.85; H, 4.47; N, 13.31; S, 10.15. Found: C,60.71; H, 4.38; N, 13.25; S, 9.91.

3.4.7. 5-(3-Chlorophenyl)-4-(2,4-dimethylphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (8g)

White crystalline solid (59%, 0.26 g): m.p 260e261 �C; Rf: 0.51(n-hexane:ethyl acetate, 8:2); IR (pure, cm�1): 3201 (NH), 2943(sp3 CH), 1515 (C]N), 1496, 1486 (C]C), 1274 (C]S); 1H NMR(300 MHz, DMSO-d6): d 14.30 (s, 1H, NH), 7.48 (dd, 1H,J¼ 2.1,0.9 Hz, AreH), 7.38 (t, 1H, J¼ 8.1 Hz, AreH), 7.36 (d, 1H,J¼ 2.1 Hz, AreH), 7.23e7.20 (m, 3H, AreH), 7.14 (d, 1H, J¼ 7.8 Hz,AreH), 2.33 (s, 3H, CH3), 1.96 (s, 3H, CH3); 13C NMR (75 MHz,DMSO-d6): d 167.90, 148.21, 135.89, 135.41, 133.32, 132.52, 131.48,130.51, 130.24, 130.19, 129.68, 127.40, 126.56, 126.55, 20.91, 17.73;EI-MS: m/z (rel. abund. %), 317.00 (M�þ þ 2, 21.4), 315.01 (M�þ,57.9), 284.06(35.3), 282.03(100), 162.90(8.2), 145.05(24.2), 105.01(16.4), 91.00(17.4), 77.00(26.2), 51.10(12.8). Analysis. Calcd forC16H14ClN3S: C, 60.85; H, 4.47; N, 13.31; S, 10.15. Found: C, 60.77; H,4.36; N, 13.25; S, 9.97.

3.4.8. 4-(2,3-Dimethylphenyl)-5-(2-methylphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (8h)

White crystalline solid (57%, 0.23 g): m.p 244e247 �C; Rf: 0.63(n-hexane:ethyl acetate, 8:2); IR (pure, cm�1): 3277 (NH), 2905(sp3 CH), 1563 (C]N), 1500, 1482 (C]C), 1255 (C]S); 1H NMR(300 MHz, DMSO-d6): d 14.18 (s, 1H, NH), 7.31e7.21 (m, 2H,AreH), 7.18 (dd, 2H, J¼ 6.3,2.4 Hz, AreH), 7.12e7.05 (m, 3H,AreH), 2.24 (s, 3H, CH3), 2.20 (s, 3H, CH3), 1.90 (s, 3H, CH3); 13CNMR (75 MHz, DMSO-d6): d 168.14, 150.86, 138.32, 138.07, 134.96,133.42, 131.29, 130.97, 130.82, 130.64, 127.29, 126.44, 126.04,125.74, 20.24, 20.06, 14.84; EI-MS: m/z (rel. abund. %), 295.10(M�þ, 81.1), 262.00(100), 163.02(10.5), 144.90(17.7), 131.05(8.5),117.04(16.3), 91.00(13.7), 77.01(19.8). Analysis. Calcd forC17H17N3S: C, 69.12; H, 5.80; N, 14.22; S, 10.85. Found: C, 68.99; H,5.54; N, 14.17; S, 10.66.

3.4.9. 5-(3-Chlorophenyl)-4-(2,6-dimethylphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (8i)

White crystalline solid (66%, 0.29 g): m.p 224e226 �C; Rf: 0.51(n-hexane:ethyl acetate, 8:2); IR (pure, cm�1): 3296 (NH), 2922(sp3CH), 1576 (C]N), 1541, 1473 (C]C), 1235 (C]S); 1H NMR(300 MHz, DMSO-d6): d 14.42 (s, 1H, NH), 7.53 (dd, 1H, J¼ 2.7,1.8 Hz, AreH), 7.41e7.35 (m, 2H, AreH), 7.28e7.19 (m, 4H, AreH),1.97 (s, 6H, 2� CH3); 13C NMR (75 MHz, DMSO-d6): d 168.20, 148.77,136.42, 133.80, 133.35, 132.84, 131.48, 131.19, 130.65, 129.34, 125.65,125.56, 17.95; EI-MS: m/z (rel. abund. %), 317.00 (M�þ þ 2, 24.7),314.90 (M�þ, 62.1), 283.91(36.3), 282.02(100), 144.90(26.7), 136.92(10.8), 105.00(17.6), 91.10(15.8), 77.00(27.9), 51.10(10.5). Analysis.Calcd for C16H14ClN3S: C, 60.85; H, 4.47; N, 13.31; S, 10.15. Found: C,60.74; H, 4.26; N, 13.16; S, 9.96.

3.4.10. 5-(4-Chlorophenyl)-4-(2,4-dimethylphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (8j)

White crystalline solid (75%, 0.33 g): m.p 285e286 �C; Rf: 0.48(n-hexane:ethyl acetate, 8:2); IR (pure, cm�1): 3346 (NH), 2986 (sp3

CH), 1555 (C]N), 1485 (C]C), 1276 (C]S); 1H NMR (300 MHz,DMSO-d6): d 14.24 (s, 1H, NH), 7.44 (d, 2H, J¼ 8.4 Hz, AreH), 7.30 (d,2H, J¼ 8.7 Hz, AreH), 7.20e7.11 (m, 3H, AreH), 2.32 (s, 3H, CH3),1.96 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6): d 168.85, 149.94,140.11, 135.94, 135.84, 132.19, 131.30, 129.78, 129.41, 128.33, 125.49,125.09, 21.21, 17.71; EI-MS: m/z (rel. abund. %), 317.18 (M�þ þ 2,37.3), 315.19 (M�þ, 93.7), 300.16(9.2), 284.21(51.5), 282.14(100),163.11(7.8), 145.17(26.3), 105.13(8.3), 91.10(7.1), 78.08(13.4), 77.09(9.4), 63.05(13.1). Analysis. Calcd for C16H14ClN3S: C, 60.85; H, 4.47;N, 13.31; S, 10.15. Found: C, 60.66; H, 4.41; N, 13.11; S, 9.87.

3.5. General procedure for the synthesis of 2,5-disubstitutedthiadiazoles (9aeh)

The corresponding carbothioamide (7) (0.0014 moles) in poly-phosphoric acid was stirred overnight at 70 �C. After completion ofthe reaction, the cooled solution was poured onto the crushed ice.The reaction mixture was extracted with ethyl acetate (3� 20 mL)and the combined extracts were washed with sodium bicarbonate(5%) and water until the washings were neutral. The organic layerwas dried with anhydrous sodium sulphate and concentratedunder reduced pressure to yield 2,5-disubstituted-1,3,4-thiadiazole(9) which was purified by recrystallization from ethanol.

3.5.1. N-(2,6-Dimethylphenyl)-5-(4-methylphenyl)-1,3,4-thiadiazol-2-amine (9a)

White solid (58%, 0.24 g): m.p 245e248 �C; Rf: 0.48 (n-hex-ane:ethyl acetate, 8:2); IR (pure, cm�1): 3124 (NH), 2845 (sp3 CH),1593 (C]N), 1551 (C]C); 1H NMR (300 MHz, DMSO-d6): d 9.54 (s,1H, NH), 7.63 (d, 2H, J¼ 8.1 Hz, AreH), 7.25 (d, 2H, J¼ 8.1 Hz, AreH),7.16 (bs, 3H, AreH), 2.32 (s, 3H, CH3), 2.22 (s, 6H, 2� CH3); 13C NMR(75 MHz, DMSO-d6): d 168.83, 157.04, 140.01, 138.66, 135.95, 130.14,128.99, 128.46, 126.78, 21.36, 18.36; EI-MS: m/z (rel. abund. %),295.32 (M�þ, 100), 280.31(39.6), 178.20(93.6), 163.17(16.5), 145.21(66.5), 135.14(23.0), 119.17(26.6), 91.14(26.6), 77.12(11.7). Analysis.Calcd for C17H17N3S: C, 69.12; H, 5.80; N, 14.22; S, 10.85. Found: C,69.08; H, 5.59; N, 14.11; S, 10.69.

3.5.2. 5-(2-Chlorophenyl)-N-(2,6-dimethylphenyl)-1,3,4-thiadiazol-2-amine (9b)

White solid (56%, 0.25 g): m.p 240e241 �C; Rf: 0.45 (n-hex-ane:ethyl acetate, 8:2); IR (pure, cm�1): 3134 (NH), 2846 (sp3 CH),1592 (C]N), 1554 (C]C); 1H NMR (300 MHz, DMSO-d6): d 9.65 (s,1H, NH), 8.05e8.02 (m, 1H, AreH), 7.60e7.57 (m, 1H, AreH),7.497.45 (m, 2H, AreH), 7.16 (bs, 3H, AreH), 2.22 (s, 6H, 2�CH3); 13CNMR (75 MHz, DMSO-d6): d 170.64, 152.19, 138.28, 135.95, 131.62,130.98, 130.82, 130.68, 129.63, 129.05, 128.23, 127.80, 18.34; EI-MS:m/z (rel. abund. %), 317.04 (M�þ þ 2, 33.1), 315.05 (M�þ, 78.4), 302.02(15.7), 300.02(36.3), 178.00(100), 163.02(29.1), 154.97(26.5), 145.09(97.3), 136.99(13.0), 119.06(40.8), 77.03(16.6). Analysis. Calcd forC16H14ClN3S: C, 60.85; H, 4.47; N, 13.31; S, 10.15. Found: C, 60.59; H,4.41; N, 13.12; S, 9.88.

3.5.3. 5-(4-Chlorophenyl)-N-(2,3-dimethylphenyl)-1,3,4-thiadiazol-2-amine (9c)

Yellow solid (39%, 0.17 g): m.p 252e254 �C; Rf: 0.47 (n-hex-ane:ethyl acetate, 8:2); IR (pure, cm�1): 3212 (NH), 2907 (sp3 CH),1600 (C]N), 1561, 1501 (C]C); 1H NMR (300 MHz, DMSO-d6):d 9.80 (s, 1H, NH), 7.82e7.79 (m, 2H, AreH), 7.55e7.47 (m, 3H,AreH), 7.12 (t, 1H, J¼ 7.8 Hz, AreH), 7.03 (d, 1H, J¼ 7.5 Hz, AreH),2.27 (s, 3H, CH3), 2.16 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6):d 168.48, 156.14, 139.60, 138.13, 134.82, 130.28, 129.89, 129.70,127.28, 126.49, 121.57, 20.70, 14.34; EI-MS: m/z (rel. abund. %),317.24 (M�þ þ 2, 29.4), 315.22 (M�þ, 57.7), 302.22(10.4), 300.19(26.7), 282.24(10.7), 178.15(59.1), 163.13(23.8), 155.07(28.2), 145.17(100), 137.07(18.3), 119.15(25.4), 111.05(11.5), 91.10(14.1), 77.09(18.4). Analysis. Calcd for C16H14ClN3S: C, 60.85; H, 4.47; N, 13.31; S,10.15. Found: C, 60.62; H, 4.35; N, 12.89; S, 10.02.

3.5.4. N-(2,4-Dimethylphenyl)-5-(3-nitrophenyl)-1,3,4-thiadiazol-2-amine (9d)

Yellow solid (45%, 0.20 g): m.p 240e243 �C; Rf: 0.40 (n-hex-ane:ethyl acetate, 8:2); IR (pure, cm�1): 3287 (NH), 2953 (sp3 CH),1602 (C]N), 1580, 1556 (C]C); 1H NMR (300 MHz, DMSO-d6):d 9.81 (s, 1H, NH), 8.54 (s, 1H, AreH), 8.28 (d, 1H, J¼ 8.4 Hz, AreH),8.19 (d, 1H, J¼ 7.8 Hz, AreH), 7.79e7.59 (m, 2H, AreH), 7.76 (t, 1H,

Table 1DPPH radical scavenging activities of the synthesized compounds (8aej) and(9aeh).

Compounds Conc. (mM) % RSA IC50� SEM (mM)

8a 0.5 24.55 e

8b 0.5 23.64 e

8c 0.5 20.29 e

8d 0.5 72.81 242.49� 3.648e 0.5 41.57 e

8f 0.5 16.82 e

8g 0.5 11.03 e

8h 0.5 15.14 e

8i 0.5 24.46 e

8j 0.5 63.27 292.38� 1.609a 0.5 46.32 e

9b 0.5 38.59 e

9c 0.5 47.95 e

9d 0.5 45.37 e

9e 0.5 15.28 e

9f 0.5 58.06 319.62� 2.049g 0.5 34.97 e

9h 0.5 38.42 e

Std (n-propyl gallate) e 90.31 30.46� 0.27

e, Not done.

Table 2Superoxide ion scavenging activities of the synthesized compounds (8aej) and(9aeh).

Compounds Conc. (mM) % RSA IC50� SEM (mM)

8a 0.5 e e

8b 0.5 e e

8c 0.5 e e

8d 0.5 e e

8e 0.5 e e

8f 0.5 e e

8g 0.5 e e

8h 0.5 e e

8i 0.5 e e

8j 0.5 e e

9a 0.5 e e

9b 0.5 18.35 e

9c 0.5 94.65 138.64� 1.749d 0.5 98.58 130.76� 2.579e 0.5 3.24 e

9f 0.5 87.21 255.21� 3.899g 0.5 95.37 124.30� 1.469h 0.5 99.32 94.84� 0.34Std (n-propyl gallate) e 90.31 106.34� 1.70

e, Not done.

I. Khan et al. / European Journal of Medicinal Chemistry 45 (2010) 5200e52075206

J¼ 8.1 Hz, AreH), 7.09e7.04 (m, 1H, AreH), 2.27 (s, 3H, CH3), 2.24(s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6): d 168.83, 150.87,148.69, 136.98, 134.79, 133.25, 132.43, 131.91, 131.44, 130.99,127.77, 124.63, 123.22, 120.75, 20.90, 14.35; EI-MS: m/z (rel.abund. %), 326.10 (M�þ, 4.8), 163.10(10.3), 148.00(25.9), 118.02(23.2), 102.06(58.5), 77.07(13.3), 75.00(23.0), 64.02(100). Anal-ysis. Calcd for C16H14N4O2S: C, 58.88; H, 4.32; N, 17.17; S, 9.82.Found: C, 58.68; H, 4.18; N, 16.93; S, 9.67.

3.5.5. N-(2,3-Dimethylphenyl)-5-phenyl-1,3,4-thiadiazol-2-amine(9e)

White solid (43%, 0.17 g): m.p 235e238 �C; Rf: 0.36 (n-hex-ane:ethyl acetate, 8:2); IR (pure, cm�1): 3178 (NH), 2917 (sp3 CH),1601 (C]N), 1576, 1556 (C]C); 1H NMR (300 MHz, DMSO-d6):d 9.74 (s, 1H, NH), 7.81e7.76 (m, 2H, AreH), 7.51e7.44 (m, 4H,AreH), 7.13 (t, 1H, J¼ 7.8 Hz, AreH), 7.03 (d, 1H, J¼ 7.2 Hz, AreH),2.27 (s, 3H, CH3), 2.17 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6):d 168.12, 157.36, 139.70, 138.08, 131.03, 130.40, 130.19, 129.67,127.17, 126.96, 126.47, 121.48, 20.72, 14.35; EI-MS: m/z (rel. abund.%), 280.99 (M�þ, 35.3), 265.95(19.6), 248.04(14.8), 204.00(10.9),177.98(43.1), 162.97(36.3), 145.03(92.4), 131.02(11.0), 120.98(60.3),103.00(74.2), 91.02(43.7), 77.05(100), 51.01(27.3). Analysis. Calcdfor C16H15N3S: C, 68.30; H, 5.37; N, 14.93; S, 11.40. Found: C, 68.17;H, 5.13; N, 14.72; S, 11.19.

3.5.6. 5-(4-Chlorophenyl)-N-(2,6-dimethylphenyl)-1,3,4-thiadiazol-2-amine (9f)

White solid (54%, 0.24 g): m.p 231e234 �C; Rf: 0.43 (n-hex-ane:ethyl acetate, 8:2); IR (pure, cm�1): 3412 (NH), 2857 (sp3 CH),1577 (C]N), 1484 (C]C); 1H NMR (300 MHz, DMSO-d6): d 9.69 (s,1H, NH), 7.77 (d, 2H, J¼ 8.7 Hz, AreH), 7.51 (d, 2H, J¼ 8.7 Hz, AreH),7.16 (bs, 3H, AreH), 2.22 (s, 6H, 2� CH3); 13C NMR (75 MHz, DMSO-d6): d 169.50, 155.73, 138.51, 135.95, 134.66, 129.99, 129.65, 129.02,128.46, 127.77, 18.32; EI-MS: m/z (rel. abund. %), 317.21 (M�þ þ 2,15.8), 315.19 (M�þ, 52.8), 302.14(7.0), 300.19(26.5), 282.22(11.6),178.15(97.3), 163.13(25.2), 155.05(34.8), 137.07(100), 119.13(27.7),91.10(15.1), 77.09(18.2), 64.01(60.8), 47.96(19.0); Analysis. Calcd forC16H14ClN3S: C, 60.85; H, 4.47; N, 13.31; S, 10.15. Found: C, 60.61; H,4.33; N, 13.14; S, 9.90.

3.5.7. 5-(3-Chlorophenyl)-N-(2,4-dimethylphenyl)e1,3,4-thiadiazol-2-amine (9g)

White solid (67%, 0.30 g): m.p 274e277 �C; Rf: 0.57 (n-hex-ane:ethyl acetate, 8:2); IR (pure, cm�1): 3352 (NH), 2952 (sp3 CH),1594 (C]N), 1567, 1485 (C]C); 1H NMR (300 MHz, DMSO-d6):d 9.71 (s, 1H, NH), 7.84 (s, 1H, AreH), 7.76e7.72 (m, 1H, AreH), 7.61(d,1H, J¼ 8.1 Hz, AreH), 7.51e7.49 (m, 2H, AreH), 7.07e7.03 (m, 2H,AreH), 2.26 (s, 3H, CH3), 2.24 (s, 3H, CH3); 13C NMR (75 MHz,DMSO-d6): d 168.31, 155.79, 137.09, 134.53, 134.33, 132.96, 131.86,131.60, 130.80, 130.09, 127.70, 126.08, 125.78, 123.07, 20.90, 18.25;EI-MS: m/z (rel. abund. %), 317.00 (M�þ þ 2, 2.2), 315.00 (M�þ, 6.5),178.02(14.1), 163.10(10.0), 154.90(61.2), 136.90(100), 118.05(41.3),110.91(34.0), 91.10(36.0), 77.00(38.6), 75.06(40.0), 64.01(83.0),51.10(28.2). Analysis. Calcd for C16H14ClN3S: C, 60.85; H, 4.47; N,13.31; S, 10.15. Found: C, 60.81; H, 4.27; N, 13.05; S, 10.01.

3.5.8. N-(2,4-Dimethylphenyl)e5e(4-nitrophenyl)e1,3,4-thiadiazol-2-amine (9h)

Yellow solid (41%, 0.19 g): m.p 263e267 �C; Rf: 0.42 (n-hex-ane:ethyl acetate, 8:2); IR (pure, cm�1): 3460 (NH), 2937 (sp3 CH),1585 (C]N), 1557, 1540 (C]C); 1H NMR (300 MHz, DMSO-d6):d 9.80 (s, 1H, NH), 7.81 (d, 2H, J¼ 8.4 Hz, AreH), 7.54e7.48 (m, 1H,AreH), 7.53 (d, 2H, J¼ 8.4 Hz, AreH), 7.15e7.07 (m, 1H, AreH), 7.03(d,1H, J¼ 7.2 Hz, AreH), 2.27 (s, 3H, CH3), 2.17 (s, 3H, CH3); 13C NMR(75 MHz, DMSO-d6): d 168.45, 159.54, 156.11, 138.21, 138.11, 134.81,

130.25, 129.90, 129.70, 129.35, 126.23, 121.54, 20.71, 14.35; EI-MS:m/z (rel. abund. %), 326.10 (M�þ, 20.6), 178.00(17.3), 163.02(21.5),148.06(22.9), 145.11(27.2), 119.90(17.7), 119.00(20.5), 118.08(33.2),102.07(51.6), 77.11(18.5), 75.05(19.3), 64.00(100), 51.10(20.4).Analysis. Calcd for C16H14N4O2S: C, 58.88; H, 4.32; N, 17.17; S, 9.82.Found: C, 58.63; H, 4.14; N, 17.01; S, 9.69.

3.6. Pharmacological assays

The antioxidant activities against DPPH radical and superoxideanion scavenging activities were measured for new triazole (8aej)and thiadiazole (9aeh) derivatives which are shown in Tables 1and 2, respectively. The synthesized compounds were also screenedfor their urease inhibition activity which is shown in Table 3. Thecompounds were found inhibiting the urease in variable concentra-tions. The standard thiourea was used as a reference material.

3.6.1. Antioxidant assay (DPPH scavenging assay)The free radical scavenging capacity of the compounds was

measured by modified 1,1-diphenyl-2-picrylhydrazyl (DPPH)

Table 3Urease inhibition activities of the synthesized compounds (8aej) and (9aeh).

Compounds Conc. (mM) % Inhibition IC50� SEM (mM)

8a 0.5 6.8 e

8b 0.5 48.3 e

8c 0.5 48.3 e

8d 0.5 97.0 45.60� 0.048e 0.5 93.4 86.0� 0.048f 0.5 7.5 e

8g 0.5 53.1 459.56� 2.268h 0.5 43.4 e

8i 0.5 64.8 357.03� 1.908j 0.5 88.6 170.76� 0.239a 0.5 27.1 e

9b 0.5 82.8 313.2� 0.099c 0.5 52.8 483.55� 1.999d 0.5 73.4 398.2� 0.409e 0.5 56.8 426.1� 0.009f 0.5 91.1 214.7� 0.169g 0.5 12.4 e

9h 0.5 7.2 e

Std (thiourea) e e 21� 0.11

e, Not done.

I. Khan et al. / European Journal of Medicinal Chemistry 45 (2010) 5200e5207 5207

methods described by M. I. Choudhary et al [23]. Test compoundswere allowed to react with stable free radical, 1,1-diphenyl-2-pic-rylhydrazyl radical (DPPH) for half an hour at 37 �C. The concen-tration of DPPH was kept as 300 mM. The test samples weredissolved in DMSO while the DPPH solution was prepared inethanol. After incubation, decrease in absorption was measured at515 nm using multiplate reader (Spectra MAX-384). Percent radicalscavenging activity (RSA) of samples was determined in compar-isonwith a DMSO treated control group [24,25] using the followingformula (Table 1):

%RSA ¼ 100� fðODtestcompound=ODcontrolÞ � 100g:

3.6.2. Superoxide anion scavenging assayMeasurement of superoxide radical scavenging activity (Table 2)

was carried out by the modified method used by Ferda [26]. Thereaction mixture comprised of 40 mL of 280 mM b-nicotinamideadenine dinucleotide reduced form (NADH), 40 mL of 80 mM nitroblue tetrazolium (NBT), 20 mL phenazine methosulphate (PMS)10 mL of 1 mM sample and 90 mL of 0.1 M phosphate buffer (pH 7.4).Reagents were prepared in buffer solution and the sample in DMSO.The reaction was performed in 96-well microtitre plate at roomtemperature and absorbance was measured at 560 nm. Theformation of superoxide was monitored by measuring the forma-tion of water soluble blue formazan dye. A lower absorbance ofreaction mixture indicates a higher scavenging activity of thesample. Percent Radical Scavenging Activity (% RSA) by samples canbe determined in comparison with a control [26].

%RSA ¼ 100� fðODtestcompound=ODcontrolÞ � 100g:

3.6.3. Urease inhibition assayReaction mixtures comprising 25 mL of enzyme (jack bean

urease) solution and 55 mL of buffers containing 100 mM urea were

incubated with 5 mL of the test synthesized compounds (0.5 mMconcentration) at 30 �C for 15 min in 96-well plates. Urease activitywas determined by measuring ammonia production using theindophenol method as described by weatherburn [27]. Briefly,45 mL each of phenol reagent (1% w/v phenol and 0.005% w/vsodium nitroprusside) and 70 mL of alkali reagent (0.5% w/v NaOHand 0.1% active chloride NaOCl) were added to each well. Theincreasing absorbance at 630 nmwas measured after 50 min, usinga microplate reader (Molecular Device, USA). All the reactions wereperformed in triplicate in a final volume of 200 mL. The results(change in absorbance per min) were processed by using softMaxPro software (Molecular Device, USA). The entire assays were per-formed at pH 6.8. Percentage inhibitions were calculated from theformula (Table 3):

100� ðODtestwell=ODcontrolÞ � 100:

Thiourea was used as the standard inhibitor of urease [28].

Acknowledgment

Financial support from Higher Education Commission (HEC)Islamabad-45320, Pakistan is gratefully acknowledged.

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