HETEROCYCLES

Paper | Regular issue | Vol. 87, No. 2, 2013, pp. 341-356
Received, 12th November, 2012, Accepted, 6th December, 2012, Published online, 12th December, 2012.
DOI: 10.3987/COM-12-12625

■ Synthetic Utility of Ethylidenethiosemicarbazide: Synthesis and Anticancer Activity of 1,3-Thiazines and Thiazoles with Imidazole Moiety

Sobhi M. Gomha, Sayed M. Riyadh,* Ikhlass M. Abbas, and Mohammed A. Bauomi

Department of Chemistry, Faculty of Science, Cairo University, Giza 12613, Egypt

Abstract

Reactions of ethylidenethiosemicarbazide 3 with DMAD 4 or substituted methylenemalononitriles 8 gave thiazolidin-4-one 6 or 1,3-thiazine derivatives (10, 11), respectively. Also, treatment of 3 with hydrazonoyl halides 12a-i, α-haloketones 15a-d, and chloroacetic acid 18 afforded the corresponding arylazothiazoles 14a-i, thiazoles 17a-d, and thiazolin-4-one derivative 20, respectively. The structures of the synthesized products were confirmed by IR, 1H NMR, 13C NMR and mass spectral techniques. The anticancer activity of the selected products against the colon carcinoma cell line (HCT-116) was determined and the results revealed promising activity of compound 6.

INTRODUCTION
Recent literature is enriched with synthesis and biological activities of alkylidenethiosemicarbazides. 1-(1-Arylethylidene)thiosemicarbazides exhibited potent inhibitory activities against mushroom tyrosinase1,2 (multifunctional copper-containing enzyme cause dermatological disorders). Also, 1-[1-(heterocyclic)ethylidene]thiosemicarbazides and their metal complexes have been investigated as potential anticancer agents.3-5 The cytotoxic activity of thiosemicarbazones against human tumor cell lines has been attributed to their ability to inhibit ribonucleoside diphosphate reductase (RDR), a rate-limiting enzyme in DNA syntheses that catalyzes the conversion of ribonucleotides into deoxyribonucleotides.4 On the other hands, alkylidenethiosemicarbazides are reactive building blocks for construction of bioactive heterocycles such as 1,3,4-thiadiazoles,6 imidazolinones,7 thiazoles,8-11 and thiazolidin-4-one.9 As a part of our research interest towards developing new routes for the synthesis of a variety of heterocyclic systems with promising biological and pharmacological activities,12-16 we report in the present work the synthesis of a new series of 1,3-thiazines and thiazoles bearing imidazole moiety.

RESULTS AND DISCUSSION
1-[1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene]thiosemicarbazide (3) was prepared via condensation of 5-acetyl-2-mercapto-4-methyl-1-phenyl-1H-imidazole (1)17 with thiosemicarbazide (2) in absolute ethanol in the presence of a catalytic amount of HCl as depicted in Scheme 1.

The structure elucidation of the product 3 was substantiated through spectral data. Product 3 can be existed in two geometric structures (E, and Z). In 1H NMR, when N-H signal is revealed at δ = 10.79-10.90 ppm suggesting the E configuration, in which N–H is hydrogen bonded to the solvent.18–20 On the other hand, if N-H signal is revealed at δ = 14.69–14.66 ppm indicates the presence of the Z configuration, in which N–H is hydrogen bonded to the nitrogen of the ring.18–20 in our case, NH signal was revealed at δ = 10.98 ppm which confirms the E-form. Also another signal was observed at δ = 10.45 ppm characteristic to mercapto (SH)21 group of imidazole ring.
We commenced our study on the reaction of ethylidenethiosemicarbazide with activated triple bond. It was reported that, reactions of alkylidenethiosemicarbazides with dimethyl acetylenedicarboxylate (DMAD) afforded either thiazolidin-4-ones22-24 or 1,3-thiazin-4-ones22,23,25 according to the reaction conditions. Thus, treatment of 1-[1-(2-mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene]- thiosemicarbazide (3) with DMAD in methanol gave the corresponding thiazolidin-4-one derivative 6 or its isomeric structure 1,3-thiazin-4-one derivative 7 (Scheme 2). The structure of the isolated product was inferred from its elemental analysis and spectral data [IR and 1H NMR]. Its IR spectrum showed absorption bands at v = 3247 (NH), 1704, 1694 (2C=O), and 1606 (C=N) cm-1, its 1H NMR spectrum revealed singlet signal at δ 12.77 ppm (D2O-exchangeable) assignable to (NH) group and another singlet signal at δ 6.64 ppm due to vinylic-H.23,26 Revealing of the latter signal of vinylic-H excludes 1,3-thiazin-4-one structure 7. To account for the formation of product 6 we assumed that the reaction initially proceeds via addition of thiol group in thiosemicarbazone moiety into triple bond to give the non-isolable intermediate 5. Elimination of methanol molecule from the latter intermediate afforded the final product 6 (Scheme 2).

Next, we investigated the behavior of ethylidenethiosemicarbazide 3 towards substituted methylenemalononitriles, as activated double bond. Thus, treatment of compound 3 with benzylidenemalononitrile 8a or ethoxymethylenemalononitrile 8b in refluxing methanol afforded the respective 1,3-thiazines (10 or 11) (Scheme 3). The reactions proceeded through addition of thiol group in thiosemicarbazone moiety into activated double bond to give the non-isolable intermediates 9a,b. Intramolecluar cyclization of intermediate 9a via addition of amino group into nitrile group27 afforded 6H-1,3-thiazine derivative 10. On the other hand, intramolecular addition of intermediate 9b followed by elimination of ethanol molecule gave the respective 2H-1,3-thiazine derivative 11 (cf. Scheme 3). The structures of products 10, 11 were established based on analytical and spectral data. The structures of 1,3-thiazines in agreement with literature reports28,29 concerning the reactions of thiosemicarbazides with substituted methylenemalononitriles.

We extended our study on the reactivity of ethylidenethiosemicarbazide 3 towards halogenated compounds, such as hydrazonoyl halides, α-halocarbonyl compounds and chloroacetic acid. Reaction of 3 with each of the hydrazonoyl halides 12a-i in dioxane under thermal condition in the presence of triethylamine for 6-8 hrs yielded in each case one isolable product as evidenced by TLC analysis. The structures of isolated products were evidenced by spectral data together with elemental analyses. For instance, the IR spectra of products display in each case the absorption bands in the region 3213-3171 and 1604-1584 cm-1 due to the (NH) and (C=N) groups, respectively. In 1H NMR spectra all the products have characteristic singlet signals in the region δ 10.83-10.54 ppm (D2O exchangeable) assignable to the (NH) protons. On the basis of the foregoing results, the isolated products from the reactions of 3 with 12a-i can be assigned 2-[2-{1-(2-mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-substituted-5-arylazothiazole (14a-i) (Scheme 4).

Products 14a-i can be present in three tautomeric forms 14A, 14B, and 14C as shown in Figure 1. To elucidate the actual tautomeric form of these compounds, their electronic absorption was measured. The spectra of these compounds (14a, 14g, and 14i) in dioxane showed in each case pair of absorption bands in the regions 442-447 and 292-296 nm, analogous for the azo chromophore30 and another pair in the regions 366-373 and 298-306 nm analogous for the hydrazone chromophore31 (Table 1). These finding excluded tautomeric form 14B. Furthermore, the aromatic stability of thiazole ring excluded tautomeric form 14C.

Finally, ethylidenethiosemicarbazide 3 reacted with α-halocarbonyl compounds, namely chloroacetone 15a, phenacyl bromide 15b, 3-chloro-2,4-pentanedione 15c, N-phenyl 2-chloro-3-oxobutanamide 15d, and chloroacetic acid 18 under thermal conditions in dioxane to afford 2-[2-{1-(2-mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-substituted thiazoles (17a-d), and 2-[2-{1-(2-mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-oxo-4,5-dihydrothiazole 20, respectively (Scheme 5). Analytical and spectroscopic data were consistent with the final products 17a-d and 20.

ANTICANCER EVALUATION
The cytotoxicity of synthesized products 6, 10, 11,14a, 14g and 20 was evaluated against human colon carcinoma cell line (HCT-116) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and doxorubicin was used as a reference drug (IC50 value of doxorubicin = 0.469 ± 0.03 µg/mL). Data generated were used to plot a dose response curve of which the concentration of test compounds required to kill 50% of cell population (IC50) was determined. Cytotoxic activity was expressed as the mean IC50 of three independent experiments. The results are represented in Tables 2, 3 and Figure 2.
The results revealed that thiazolidin-4-one derivative 6 (IC50 = 1.9 µg/mL) has promising antitumor activity against colon carcinoma (HCT-116) while 1,3-thiazines (10 and 11) and arylazothiazoles (14a and 14g) have moderate activity (IC50 = 3.9-4.6 µg/mL). On the other hand, thiazolin-4-one 20 has poor inhibitory activity against (HCT-116) (IC50 = 33 µg/mL).

CONCLUSION
New thiazolidin-4-one, 1,3-thiazines, arylazothiazoles, thiazoles, and thiazolin-4-one have been synthesized using ethylidenethiosemicarbazide as starting material under thermal conditions.

EXPERIMENTAL
Melting points were measured on an Electrothermal IA 9000 series digital melting point apparatus. IR spectra were recorded in potassium bromide discs on Pye Unicam SP 3300 and Shimadzu FTIR 8101 PC infrared spectrophotometers. NMR spectra were recorded on a Varian Mercury VX-300 NMR spectrometer operating at 300 MHz (1H NMR) or 75 MHz (13C NMR) and run in deuterated dimethylsulfoxide (DMSO-d6). Chemical shifts were related to that of the solvent. Mass spectra were recorded on a Shimadzu GCeMS-QP1000 EX mass spectrometer at 70 eV. Elemental analyzes were measured by using a German made Elementar vario LIII CHNS analyzer. Antitumor activity was evaluated by the Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt. Hydrazonoyl halides32,33 were prepared as previously reported in the respective literature.

Synthesis of 1-[1-(2-mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene]thiosemicarbazide (3).
5-Acetyl-2-mercapto-4-methyl-1-phenyl-1H-imidazole (1) (11.5 g, 50 mmol) was dissolved in 100 mL of EtOH and stirred with an equimolar quantity of thiosemicarbazide for 24 h at room temperature with catalytic amounts of HCl. The desired thiosemicarbazone precipitated from reaction mixture was filtered, washed with EtOH and recrystallized from acetic acid to give pure product of compound 3.
Yellow solid (12.2 g, 80%); mp 229 °C; IR (KBr): v 3439, 3236 (NH2), 3155 (NH), 1587 (C=N) cm-1; 1H-NMR (DMSO-d6): δ 2.35 (s, 3H, CH3), 2.96 (s, 3H, CH3), 4.31 (s, br, 2H, NH2), 6.99-7.66 (m, 5H, Ar-H), 10.45 (s, 1H, SH), 10.98 (s, 1H, NH); MS m/z (%): 305 (M+, 100), 230 (45), 91 (82), 77 (27). Anal. Calcd for C13H15N5S2 (305.08): C, 51.12; H, 4.95; N, 22.93; S, 21.00. Found C, 51.31; H, 5.18; N, 23.12; S, 21.09%.

Synthesis of methyl 2-[2-{1-(2-mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-oxo-thiazolidin-5-ylidenethanoate (6).
To a solution of 1-[1-(2-mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene]thiosemicarbazide (3) (0.305 g, 1 mmol) in dry MeOH (20 mL) was added dimethyl acetylenedicarboxylate (0.142 g, 1 mmol). The solution was refluxed for 2 h. The precipitate was filtered, washed with MeOH, and recrystallized from EtOH to give product 6.
Canary yellow solid (0.31 g, 75%); mp 352 °C; IR (KBr): v 3247 (NH), 1704, 1694 (2C=O), 1606 (C=N) cm-1; 1H-NMR (DMSO-d6): δ 2.53 (s, 3H, CH3), 3.35 (s, 3H, CH3), 3.77 (s, 3H, COOCH3), 6.64 (s, 1H, C=CH), 6.96-7.66 (m, 5H, Ar-H), 10.41 (s, 1H, SH), 12.77 (s, 1H, NH); MS m/z (%): 415 (M+, 68), 229 (68), 118 (68), 77 (100). Anal. Calcd for C18H17N5O3S2 (415.08): C, 52.03; H, 4.12; N, 16.86; S, 15.43. Found C, 52.21; H, 4.18; N, 16.72; S, 15.29%.

Reactions of 1-[1-(2-mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene]thiosemicarbazide (3) with benzylidenemalononitrile (8a) and ethoxymethylenemalononitrile (8b).
To a solution of 1-[1-(2-mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene]thiosemicarbazide (3) (0.305 g, 1 mmol) in dry MeOH (20 mL) was added benzylidenemalononitrile (8a) or ethoxymethylenemalononitrile (8b) (1 mmol). The solution was refluxed for 2 h. The precipitate was filtered, washed with methanol, and recrystallized from EtOH to give product 10 or 11, respectively.

2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-amino-6-phenyl-6H-1,3-thiazine-5-carbonitrile (10).
Yellow solid (0.32 g, 70%); mp 214 °C; IR (KBr): v 3446-3196 (NH2 + NH), 2264 (C≡N), 1596 (C=N) cm-1; 1H-NMR (DMSO-d6): δ 2.32 (s, 3H, CH3), 3.35 (s, 3H, CH3), 3.77 (s, 1H, thiazine-H), 4.65 (s, br, 2H, NH2), 7.04-7.75 (m, 10H, Ar-H), 10.41 (s, 1H, SH), 11.37 (s, 1H, NH); 13C-NMR (DMSO-d6): δ 9.31 (CH3), 16.38 (CH3), 52.17 (CH), 108.51 (CN), 117.19, 118.63, 120.62, 120.91, 122.82, 123.45, 123.88, 124.02, 126.18, 128.79, 128.96, 129.52, 139.81, 142.32, 143.45 (Ar-C); MS m/z (%): 459 (M+, 52), 229 (100), 118 (31), 77 (83). Anal. Calcd for C23H21N7S2 (459.13): C, 60.11; H, 4.61; N, 21.33; S, 13.95. Found C, 60.21; H, 4.48; N, 21.42; S, 13.99%.

2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-amino-1,3-thiazine-5-carbonitrile (11).
Canary yellow solid (0.27 g, 72%); mp 212 °C; IR (KBr): v 3429, 3238 (NH2), 2258 (C≡N), 1598 (C=N) cm-1; 1H-NMR (DMSO-d6): δ 2.38 (s, 3H, CH3), 3.05 (s, 3H, CH3), 4.64 (s, br, 2H, NH2), 6.99 (s, 1H, thiazine-H), 7.04-7.68 (m, 5H, Ar-H), 10.63 (s, 1H, SH); 13C-NMR (DMSO-d6): δ 9.29 (CH3), 16.34 (CH3), 108.41 (CN), 117.09, 118.32, 120.61, 120.66, 122.82, 126.22, 128.89, 128.99, 129.32, 139.84, 142.11, 143.75 (Ar-C); MS m/z (%): 381 (M+, 6), 288 (97), 230 (63), 127 (58), 77 (100). Anal. Calcd for C17H15N7S2 (381.08): C, 53.52; H, 3.96; N, 25.70; S, 16.81. Found C, 53.41; H, 4.08; N, 25.62; S, 16.91%.

Synthesis of 2-[2-{1-(2-mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-substituted-5-arylazothiazoles (14a-i).
General procedure:
A mixture of 1-[1-(2-mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene]thiosemicarbazide (3)
(0.305 g, 1 mmol) and appropriate hydrazonoyl halides 12a-i (1 mmol) in dioxane (30 mL) containing triethylamine (0.1 g, 1 mmol) was refluxed for 6-8 h. (monitored by TLC). The formed precipitate was isolated by filtration, washed with MeOH, dried and recrystallized from appropriate solvent to give products 14a–i.

2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-methyl-5-phenylazothiazole (14a).
Red solid (0.36 g, 80%); mp 266 °C; IR (KBr): v 3213 (NH), 1594 (C=N) cm-1; 1H-NMR (DMSO-d6): δ 2.48 (s, 3H, CH3), 3.29 (s, 3H, CH3), 3.57 (s, 3H, CH3), 6.97-7.63 (m, 10H, Ar-H), 10.50 (s, 1H, SH), 10.54 (s, 1H, NH); 13C-NMR (DMSO-d6): δ 9.42 (CH3), 13.77 (CH3), 15.01 (CH3), 114.15, 118.45, 120.22, 121.15, 121.85, 123.25, 124.33, 125.75, 127.16, 127.31, 129.73, 131.61, 133.73, 138.61, 143.13 (Ar-C); MS m/z (%): 447 (M+, 41), 229 (61), 77 (100). Anal. Calcd for C22H21N7S2 (447.13): C, 59.04; H, 4.73; N, 21.91; S, 14.33. Found C, 59.21; H, 4.88; N, 21.82; S, 14.19%.

2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-methyl-5-(4-methylphenylazo)thiazole (14b).
Red solid (0.38 g, 82%); mp 254 °C; IR (KBr): v 3281 (NH), 1601 (C=N) cm-1; 1H-NMR (DMSO-d6): δ 2.25 (s, 3H, CH3), 2.57 (s, 3H, CH3), 3.29 (s, 3H, CH3), 3.56 (s, 3H, CH3), 7.03-7.62 (m, 9H, Ar-H), 10.47 (s, 1H, SH), 10.58 (s, 1H, NH); MS m/z (%): 461 (M+, 12), 229 (77), 91 (100). Anal. Calcd for C23H23N7S2 (461.15): C, 59.84; H, 5.02; N, 21.24; S, 13.89. Found C, 59.71; H, 4.98; N, 21.32; S, 13.74%.

2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-methyl-5-(4-methoxy-phenylazo)thiazole (14c).
Red solid (0.37 g, 78%); mp 248 °C; IR (KBr): v 3194 (NH), 1603 (C=N) cm-1; 1H-NMR (DMSO-d6): δ 2.45 (s, 3H, CH3), 3.29 (s, 3H, CH3), 3.56 (s, 3H, CH3), 3.87 (s, 3H, OCH3), 7.06-7.72 (m, 9H, Ar-H), 10.48 (s, 1H, SH), 10.62 (s, 1H, NH); MS m/z (%): 477 (M+, 19), 229 (71), 91 (100). Anal. Calcd for C23H23N7OS2 (477.14): C, 57.84; H, 4.85; N, 20.53; S, 13.43. Found C, 57.72; H, 4.91; N, 20.33; S, 13.24%.

2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-methyl-5-(4-chlorophenylazo)-thiazole (14d).
Red solid (0.39 g, 75%); mp 262 °C; IR (KBr): v 3191 (NH), 1590 (C=N) cm-1; 1H-NMR (DMSO-d6): δ 2.47 (s, 3H, CH3), 3.30 (s, 3H, CH3), 3.62 (s, 3H, CH3), 7.09-7.83 (m, 9H, Ar-H), 10.51 (s, 1H, SH), 10.80 (s, 1H, NH); MS m/z (%): 483 (M++2, 12), 481 (M+, 30), 229 (100), 111 (50). Anal. Calcd for C22H20ClN7S2 (481.09): C, 54.82; H, 4.18; N, 20.34; S, 13.30. Found C, 54.78; H, 4.28; N, 20.52; S, 13.19%.

2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-methyl-5-(4-bromo-phenylazo)thiazole (14e).
Red solid (0.39 g, 75%); mp 270 °C; IR (KBr): v 3171 (NH), 1584 (C=N) cm-1; 1H-NMR (DMSO-d6): δ 2.48 (s, 3H, CH3), 3.31 (s, 3H, CH3), 3.62 (s, 3H, CH3), 7.07-7.84 (m, 9H, Ar-H), 10.50 (s, 1H, SH), 10.83 (s, 1H, NH); MS m/z (%): 527 (M++2, 12), 525 (M+, 13), 229 (65), 77 (100). Anal. Calcd for C22H20BrN7S2 (525.04): C, 50.19; H, 3.83; N, 18.62; S, 12.18. Found C, 50.28; H, 3.78; N, 18.82; S, 12.29%.

2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-methyl-5-(4-nitro-phenylazo)thiazole (14f).
Dark red solid (0.34 g, 70%); mp 224 °C; IR (KBr): v 3190 (NH), 1590 (C=N) cm-1; 1H-NMR (DMSO-d6): δ 2.48 (s, 3H, CH3), 3.33 (s, 3H, CH3), 3.71 (s, 3H, CH3), 7.12-7.93 (m, 9H, Ar-H), 10.51 (s, 1H, SH), 10.92 (s, 1H, NH); MS m/z (%): 492 (M+, 35), 229 (97), 118 (76), 77 (100). Anal. Calcd for C22H20N8O2S2 (492.12): C, 53.64; H, 4.09; N, 22.75; S, 13.02. Found C, 53.78; H, 4.18; N, 22.62; S, 13.15%.

2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-phenyl-5-phenyl-azothiazole (14g).
Red solid (0.36 g, 70%); mp 352 °C; IR (KBr): v 3192 (NH), 1602 (C=N) cm-1; 1H-NMR (DMSO-d6): δ 3.24 (s, 3H, CH3), 3.29 (s, 3H, CH3), 6.99-8.29 (m, 15H, Ar-H), 10.53 (s, 1H, SH), 10.65 (s, 1H, NH); 13C-NMR (DMSO-d6): δ 13.31 (CH3), 15.77 (CH3), 114.17, 115.93, 118.15, 119.33, 120.42, 121.17, 122.15, 123.25, 123.87, 124.36, 125.79, 126.11, 127.26, 127.41, 129.72, 131.57, 133.83, 137.62, 142.13 (Ar-C); MS m/z (%): 509 (M+, 9), 231 (31), 77 (100). Anal. Calcd for C27H23N7S2 (509.15): C, 63.63; H, 4.55; N, 19.24; S, 12.58. Found C, 63.51; H, 4.68; N, 19.12; S, 12.49%.

2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-phenyl-5-(4-chloro-phenylazo)thiazole (14h).
Red solid (0.38 g, 70%); mp 316 °C; IR (KBr): v 3193 (NH), 1604 (C=N) cm-1; 1H-NMR (DMSO-d6): δ 3.26 (s, 3H, CH3), 3.32 (s, 3H, CH3), 7.03-8.29 (m, 14H, Ar-H), 10.54 (s, 1H, SH), 10.68 (s, 1H, NH); MS m/z (%): 545 (M++2, 3), 543 (M+, 9), 231 (54), 111 (50), 77 (100). Anal. Calcd for C27H22ClN7S2 (543.11): C, 59.60; H, 4.08; N, 18.02; S, 11.79. Found C, 59.41; H, 4.18; N, 18.12; S, 11.89%.

2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-(2-thienyl)-5-phenylazothiazole (14i).
Red solid (0.36 g, 70%); mp 268 °C; IR (KBr): v 3198 (NH), 1597 (C=N) cm-1; 1H-NMR (DMSO-d6): δ 3.28 (s, 3H, CH3), 3.31 (s, 3H, CH3), 7.01-8.03 (m, 13H, Ar-H), 10.50 (s, 1H, SH), 10.62 (s, 1H, NH); 13C-NMR (DMSO-d6): δ 13.33 (CH3), 15.87 (CH3), 113.77, 114.93, 118.15, 119.35, 120.72, 121.13, 122.19, 123.35, 123.82, 124.36, 125.77, 126.18, 127.26, 127.91, 129.79, 132.57, 133.81, 136.61, 143.63 (Ar-C); MS m/z (%): 515 (M+, 9), 231 (31), 77 (100). Anal. Calcd for C25H21N7S3 (515.10): C, 58.23; H, 4.10; N, 19.01; S, 18.65. Found C, 58.41; H, 4.18; N, 19.16; S, 18.49%.

Reactions of 1-[1-(2-mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene]thiosemicarbazide (3) with α-haloketones and α-haloacid.
General procedure:
A mixture of 3 (0.305 g, 1 mmol) and α-haloketones (15a-d) or chloroacetic acid (18) (1 mmol) in dioxane (30 mL) was refluxed for 4-6 h. (monitored by TLC). The product started to separate out during the course of reaction. The solid product was filtered, washed with water, dried and recrystallized from DMF to give the corresponding compounds 17a-d or 20, respectively.

2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-methylthiazole(17a).
Yellow solid (0.28 g, 82%); mp 154 °C; IR (KBr): v 3197 (NH), 1606 (C=N) cm-1; 1H-NMR (DMSO-d6): δ 2.28 (s, 3H, CH3), 3.31 (s, 3H, CH3), 3.57 (s, 3H, CH3), 6.18 (s, 1H, thiazole-H), 6.92-7.62 (m, 5H, Ar-H), 10.30 (s, 1H, SH), 10.94 (s, 1H, NH); 13C-NMR (DMSO-d6): δ 9.45 (CH3), 13.47 (CH3), 15.33 (CH3), 118.48, 120.53, 121.23, 123.15, 124.33, 127.21, 129.73, 131.61, 133.63, 138.65, 143.43 (Ar-C); MS m/z (%): 343 (M+, 53), 231 (100), 113 (90), 77 (94). Anal. Calcd for C16H17N5S2 (343.09): C, 55.95; H, 4.99; N, 20.39; S, 18.67. Found C, 55.81; H, 4.88; N, 20.22; S, 18.59%.

2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-phenylthiazole (17b).
Yellow solid (0.30 g, 75%); mp 192 °C; IR (KBr): v 3296 (NH), 1616 (C=N) cm-1; 1H-NMR (DMSO-d6): δ 3.34 (s, 3H, CH3), 3.61 (s, 3H, CH3), 6.22 (s, 1H, thiazole-H), 7.12-7.92 (m, 10H, Ar-H), 10.34 (s, 1H, SH), 10.84 (s, 1H, NH); 13C-NMR (DMSO-d6): δ 13.33 (CH3), 15.81 (CH3), 117.15, 119.33, 120.41, 121.17, 122.15, 123.25, 123.87, 124.36, 126.15, 127.24, 127.25, 131.59, 133.73, 137.52, 142.33 (Ar-C); MS m/z (%): 405 (M+, 52), 231 (78), 176 (91), 134 (100), 113 (55), 77 (96). Anal. Calcd for C21H19N5S2 (405.11): C, 62.19; H, 4.72; N, 17.27; S, 15.81. Found C, 62.11; H, 4.88; N, 17.42; S, 15.69%.

2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-methyl-5-acetylthiazole (17c).
Yellow solid (0.29 g, 75%); mp 112 °C; IR (KBr): v 3214 (NH), 1708 (C=O), 1602 (C=N) cm-1; 1H-NMR (DMSO-d6): δ 2.31 (s, 3H, CH3), 2.42 (s, 3H, COCH3), 3.31 (s, 3H, CH3), 3.57 (s, 3H, CH3), 6.96-7.64 (m, 5H, Ar-H), 10.30 (s, 1H, SH), 10.90 (s, 1H, NH); MS m/z (%): 385 (M+, 38), 230 (45), 189 (45), 148 (42), 112 (51), 77 (100). Anal. Calcd for C18H19N5OS2 (385.10): C, 56.08; H, 4.97; N, 18.17; S, 16.64. Found C, 55.91; H, 4.88; N, 18.22; S, 16.59%.

2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-methyl-5-(N-phenyl-carbamoyl)thiazole (17d).
Yellow solid (0.32 g, 70%); mp 185 °C; IR (KBr): v 3233 (NH), 1671 (C=O), 1600 (C=N) cm-1; 1H-NMR (DMSO-d6): δ 2.31 (s, 3H, CH3), 3.29 (s, 3H, CH3), 3.49 (s, 3H, CH3), 7.06-7.81 (m, 10H, Ar-H), 10.30 (s, 1H, SH), 10.90 (s, 1H, NH), 11.08 (s, 1H, NH); MS m/z (%): 462 (M+, 13), 343 (26), 252 (98), 141 (100), 77 (69). Anal. Calcd for C23H22N6OS2 (462.13): C, 59.72; H, 4.79; N, 18.17; S, 13.86. Found C, 59.91; H, 4.85; N, 18.12; S, 13.79%.

2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-oxo-4,5-dihydrothiazole (20).
Yellow solid (0.26 g, 75%); mp 278 °C; IR (KBr): v 3246 (NH), 1697 (C=O), 1614 (C=N) cm-1; 1H-NMR (DMSO-d6): δ 2.35 (s, 3H, CH3), 3.54 (s, 3H, CH3), 3.68 (s, 2H, CH2), 6.93-7.63 (m, 5H, Ar-H), 10.35 (s, 1H, SH), 10.84 (s, 1H, NH); MS m/z (%): 345 (M+, 20), 344 (100), 229 (47), 118 (44), 77 (78). Anal. Calcd for C15H15N5OS2 (345.07): C, 52.15; H, 4.38; N, 20.27; S, 18.56. Found C, 52.21; H, 4.28; N, 20.15; S, 18.49%.

CYTOTOXIC ACTIVITY
Potential cytotoxicity of the compounds was tested using the method of Skehan et al.,34 using Sulfo-Rhodamine-B stain (SRB). Cells were plated in 96-multiwill plates (104 cells/well) for 24 h before treatment with the tested compound to allow attachment of cell to the wall of the plate. Different concentrations of the compound under test (0, 1.56, 3.125, 6.25, 12.5, 25, and 50 µg/mL) were added to the cell monolayer in triplicate wells individual dose, monolayer cells were incubated with the compounds for 48 h at 37 °C and in atmosphere of 5% CO2. After 48 h, cells were fixed, washed and stained with SRB stain, excess stain was washed with acetic acid and attached stain was recovered with tris-EDTA buffer, color intensity was measured in an ELISA reader. The relation between surviving fraction and drug concentration is plotted (Figure 2).

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