Synthesis and Biological Screening of 5-{[(4,6-Disubstituted pyrimidine-2-yl)thio]methyl}-N-phenyl-1,3,4-thiadiazol-2-amines

A number of substituted-α,β-unsaturated carbonyl compounds (1a-i) were prepared by Claisen-Schmidt condensation of substituted acetophenone with selected araldehydes, which on cycloaddition with thiourea furnished 4,6-disubstituted pyrimidine-2-thiols (2a-i). Reaction of (2a-i) with ethyl chloroacetate followed by condensation with hydrazine hydrate yielded 2-[(4,6-disubstituted pyrimidine-2-yl) thio] acetohydrazides (4a-c). Condensation of compounds (4a-c) with phenyl isothiocyanate gave 2-{[(4,6-disubstituted pyrimidine-2-yl) thio] acetyl}-N-phenylhydrazinecarbothioamides (5a-c) which on treatment with concentrated sulphuric acid afforded titled compounds 5-{(4,6-disubstituted pyrimidine-2-yl) thio] methyl}-N-phenyl-1,3,4-thiadiazole-2-amines (6a-c). These compounds have been characterized on the basis of elemental analysis, IR, 1H NMR and MS. Compounds have been evaluated for their anticancer and antioxidant activities. Compounds 2b, 2c and 6b exhibited significant antitumor activity against human breast cancer MCF 7 cell line. However, moderate antioxidant activity was observed with compounds 2c, 2d, 2g and 6b.

Melting points were taken in open capillary tubes and are uncorrected.The IR spectra (KBr, cm -1 ) were recorded on a Shimadzu FTIR 800 series spectrophotometer and 1 H NMR spectra (CDCl 3 ) on Varian EM 390 MHz spectrometer using TMS as internal standard.Mass spectra were recorded on Shimadzu 2010A LC-MS system.The reactions were monitored by thin layer chromatography using silica gel plates and detected by UV chamber and iodine as visualizing agent.The purity of the compounds was checked on silica gel precoated plates.All the solvents used were purifi ed according to the standard methods 16 .Phenyl isothiocyanate was prepared according to the standard method 17 .
For the preparation of 4, 6-disubstituted pyrimidine-2-thiols (2a-i) a mixture of appropriate chalcones (1a-i, Scheme 1) (0.01 mol) and thiourea (0.01 mol) in ethanol (50 ml) and sodium hydroxide (0.01 mol) dissolved in minimum quantity of water was refl uxed on a water bath for 12 h and poured into 250 ml of cold water.The solid that separated in each case was filtered, washed with water and recrystallized from ethyl acetate (   18 and nitric oxide 19 free radical scavenging methods.The methods were used to screen compounds (2a-i) and (6a-c) for the antioxidant activity.Ascorbic acid and rutin were used as reference standards at a concentration level of 100 g/ml.Results are presented in Table 2.
Antitumor activity of the compounds was evaluated by tryphan blue dye exclusion technique 20 against human breast cancer MCF-7 cell line at 20 M concentration.Primary screening of the compounds was done to indicate whether a substance possessed enough activity at this concentration to inhibit cell growth by 50%.Results are presented in Table 2.
Chalcones (1a-i) required as starting material were prepared 21 by stirring equimolar solution of various substituted acetophenones and araldehydes in the presence of sodium hydroxide in ethanol at room temperature (Scheme 1).Solution in ethanol of chalcones (1a-i) and thiourea in the presence of  1.In the IR spectrum of 2b, the presence of band at 2840 cm -1 (SH) and the absence of band due to >C=O confi rmed the formation of pyrimidine-2-thiol moiety.
The appearance of singlet at δ 9.72 due to SH also confi rmed the formation of 2b.The IR spectrum of 5b exhibited band at 1681 cm -1 (>C=O), 1667 cm -1 (>C=N) and 3120-3218 cm -1 due to N-H.The 1 H NMR spectrum of 5b exhibited the aromatic and heterocyclic protons as a multiplet integrating for 15 protons from δ 7.21-7.92and a multiplet integrating for 3 protons from δ 8.20-10.12due to NH.NH.CS.NH.In the IR spectrum of 6b, the disappearance of bands at 1681 cm -1 (>C=O) and 1453 cm -1 (>C=S) and the appearance of band at 746 cm -1 (C-S-C) confi rmed the formation of thiadiazole ring.The 1 H NMR spectrum of 6b exhibited the aromatic and heterocyclic protons as a multiplet integrating for 15 protons from δ 7.21-8.32and a singlet at δ 9.3 integrating for one proton due to NH.In the mass spectra, the molecular ion peak at 483 (M + ) also confi rmed the formation of titled compound 6b.
Compound 2c, 2d, 2g and 6b showed moderate DPPH free radical scavenging activity while all other compounds were found to be less active.Compounds 2c, 2d and 6b showed moderate nitric oxide free radical scavenging activity and all other compounds were found to be less active.As shown in Table 2 compounds 2b, 2c and 6b exhibited signifi cant activity against human breast cancer MCF-7 cell line, while compounds 2e and 2h showed moderate cytotoxicity.
Barringtonia acutangula (L.) Gaertn belonging to family Barringtoniaceae is a medium size glabrous tree found throughout India in decidous and evergreen forests, mostly along the bank of rivers and streams 1 .It is used in the folklore in vitiated conditions of kapha and pitta, leprosy, arthralgia, dysmenorrhea, plumbago, skin diseases, diarrhea, inflammation, fl atulence, hemorrhoids, as an anthelmintic 2 .The plant has been reported to have antiimplantation activity in female albino rats 3 .In Ayurveda, its preparations include powder and pastes.The present study is intended to determine the antibacterial activity of the plant against selected urinary tract pathogens by disc diffusion assay 4,5 .
The plant material (twig) was collected from Keonjhar district of Orissa and authentificated by the taxonomist of Department of Botany, Utkal University, Bhubaneswar.One voucher specimen (No.UDB/B-912) was deposited in the herbarium of the department for future reference.After authentifi cation the seeds were collected in bulk, shade dried for 2 d and then dried in the hot air oven at 50 0 for 12 h.Dried seeds were pulverized by a mechanical grinder and the coarse powder obtained was taken for extraction in petroleum ether followed by chloroform, ethanol (95%) and water by using Soxhlet assembly for 48 h each.The extracts were dried under reduced pressure and the percentage of yield was calculated on the dried weight of extract.The in vitro screening for antimicrobial was carried out using selected urinary tract infection (UTI) causing pathogens which includes two gram positive bacteria (Staphylococcus aureus and Enterococcus faecalis) and three gram

TABLE 2 : IN VITRO ANTICANCER AND ANTIOXIDANT ACTIVITIES OF COMPOUNDS (2A-I) AND (6A-C)
*Average of three determinations, both test compounds and standard were tested at 100 g/ml, IC 50 concentration of the test compound causing 50% decrease of activity against control.**Mean of two determinations, a zero indicates that no cells have died.