A Novel Series of Thiazoles and 1 , 3 , 4-Thiadiazoles Bearing Thiazole Moiety as Anticancer Agents : Synthesis , Spectral Studies , Biological Evaluation and Structure Activity relationship

A novel series of thiazole based-1,3,4-thiadiazoles were designed and prepared via the reaction of the 2-(4-methyl-2-phenylthiazole-5-carbonyl)-N-phenylhydrazinecarbothioamide with the appropriate hydrazonoyl chlorides. The structures of the newly synthesized compounds were established based on spectroscopic evidences and their alternative syntheses. Thirteen new 1,3,4-thiadiazoles have been evaluated for their anticancer activity against liver carcinoma cell line (HepG2). Also, their structure activity relationship (SAR) was studied. The 1,3,4-thiadiazoles 12d, 12c, 6g,18b, 6c, and 6f(IC50 = 0.82, 0.91, 1.06, 1.25, 1.29 and 1.88 μM, respectively) have promising antitumor activity against liver carcinoma cell line (HepG2).


Introduction
Cancer is a devastating and most common life-threatening disease representing a major health problem for many decades.The clinical application of chemotherapy still considered as a major compartment in treating cancer, however, is often limited by the severity of the side effects and the development of tumor cell resistance against these cytotoxic agents.Clinical administration of high doses of anticancer drugs to overcome resistance leads to severe toxicities [1].Therefore, the development of novel effective anticancer drugs and strategies is eagerly being pursued.
The presence of the thioamidehydrazine moiety as a side chain in compound 3 prompted us to utilize it for constructing 1,3,4-thiadiazole ring via its reaction with hydrazonoyl chlorides.Thus, reaction of compound 3 with the appropriate 2-oxo-N'-arylpropanehydrazonoyl chlorides 4a-g [36] under reflux in ethanol in the presence of triethylamine as a basic catalyst led to formation of the respective 1,3,4-thiadiazoles 6a-g, rather than thiadiazines 7a-g or 1,3-thiazoles 8a-g (Scheme 1).The elemental analysis together with the data derived from IR, 1 HNMR and mass spectra are in agreement with the proposed structure 6.The IR spectra of products 6 showed in each case the presence of two absorption bands around 1700, 1650 cm -1 for the two carbonyl groups, in addition to another band near v 3350 cm -1 for the NH function.The 1 HNMR spectra of 6 revealed the presence of broad singlet signals assigned for the NH proton near δ 11.19 ppm, in addition to the expected signals for the protons of the CH3 group, the acetyl group at position-2 of the 1,3,4-thiadiazole ring and the aryl protons.The mass spectrum of each of products 6 revealed the presence of a molecular ion peak (see experimental section).Scheme 1. Synthesis of thiadiazoles 6a-g 1,3,4-Thiadiazole 6a-g was assumed to be formed through the intramolecluar cyclization of NH group in the hydrazone moiety with the imino group in the non-isolable intermediates 5a-g, followed by elimination of aniline molecule to give the respective thiadiazole derivatives 6a-g (Scheme 1).
Next, in order to test of the biological activities of a vast array of these compounds, we reacted compound 3 with the appropriate hydrazonoyl chlorides 10a-d [36], under the same experimental conditions, which gave the corresponding 1,3,4-thiadiazole derivatives 12a-d(Scheme 2).
The IR, and 1 H-NMR spectra of 12a taken as an example of the prepared series, revealed the presence of the ester group and the disappearance of the hydrazone-NH function.Also, the mass spectrum of the reaction products 12a-d showed, in each case, a peak corresponding to their molecular ions.
The structure assigned for product 12 was further evidenced via an alternative method.Thus, reaction of ethyl 4-methyl-2-phenylthiazole-5-carboxylate (1) with 1,3,4-thiadiazole 15 [37] in ethanol under reflux, afforded a product which is typical in all respects (mp, mixed mp and IR) with that obtained from the reaction of 3 with 10a (Scheme 2).To account for the formation of the product 12, it was suggested that the reaction of compound 3 with hydrazonoyl chloride 10 initially gave the intermediate 11, which underwent nucleophilic addition, followed by in situ cyclization via losing of one molecule of aniline (route a ) to give the final product 12.The other routes (b) and (c) outlined in Scheme 2 were excluded since they led to formation of products 13 and 14, which were completely different in all respects (IR, 1 HNMR, mass spectra) from products 12. Scheme 2. Synthesis of thiadiazole derivatives 12a-d Also, The reaction of compound 3 with hydrazonoyl halide of type 16 was studied.Thus refluxing compound 3 with the hydrazonoyl chloride 16a or 16b [36] under the same experimental conditions, afforded the corresponding 1,3,4-thiadiazole derivatives 18a,b (Scheme 3).The 1 HNMR spectrum of compound 18a, revealed two D2O-exchangeable signals at δ 10.18 and 11.72 corresponding to two NH protons, in addition to an aromatic multiplet in the region 7.02-7.78ppm.Also, its mass spectrum of revealed a molecular ion peak at m/z = 512 which is in complete agreement with the proposed structure (see Experimental).In addition, compound 18a was proved chemically via an alternative method from the reaction of compound 9 with 16a which gave a product identical in all respects (mp, mixed mp and IR) with compound 18a.

Scheme 3. Synthesis of thiadiazole derivatives 18a,b
The reaction of 4-methyl-2-phenylthiazole-5-carbohydrazide (2) with 2-oxo-N-arylpropane hydrazonoyl chlorides 4a-c in refluxing ethanol gave, in each case, the corresponding condensation product whose elemental analysis and spectra data(see Experimental) were consistent with structure 19 (Scheme 4).The IR spectra of the latter products exhibited a carbonyl and two NH absorption bands (see experimental part).Their 1 HNMR showed two D2O exchangeable signals of two NH groups in the regions δ 10.03-10.06 and δ 10.57-10.59ppm.Also, the mass spectra of the latter products confirmed the assigned structure 19 (Scheme 4).
Treatment of thioamide derivative 3 with the appropriate hydrazonoyl halides of type 19a-c in refluxing EtOH in the presence of TEA gave the corresponding thiadiazole derivatives 21a-c (Scheme 4).The structures of the isolated products 21a-c were elucidated on the basis of their spectral data and elemental analysis (see Experimental section).The latter products 21a-c were alternatively prepared by condensing 6a-c each with 4-methyl-2-phenylthiazole-5-carbohydrazide (2) in refluxing ethanol in quantitative yields (Scheme 4).

Scheme 4. Synthesis of thiadiazolederivatives 21a-c
Finally, the starting compound 3 was used for preparation of thiazole derivatives.Thus, refluxing of compound 3 with 3-chloropentane-2,4-dione (22) or 2-chloro-3-oxo-Nphenylbutanamide (24) in EtOH in the presence of triethylamine afforded the thiazole derivatives 23 and 25, respectively, as outlined in Scheme 5.The structure of compounds 23 and 25 were elucidated based on their elemental analysis and spectral data (see Experimental).

Cytotoxic activity
The Literature survey showed that many derivatives of thiazole and 1,3,4-thiadiazole have antitumor activity with excellent IG50 as depicted in Figure 1 [38][39][40][41][42].In view of these facts, the antitumor activity of the synthesized compounds was determined against a liver carcinoma cell line HepG2.Doxorubicin was used as a reference standard and showed IC50 = 0.72 µM against a liver carcinoma cell line.Data generated were used to plot a dose-response curve of which the concentration (µM) 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 depicted in table 1 The results revealed that most of the tested compounds showed a great variable activity compared to reference drug as shown in Table 1.The order of activity of the newly synthesized compounds was as follow: 6d< 6b< 12b< 6a< 18a< 12a< 6e< 6f< 6c< 18b< 6g< 12c< 12d These results lead to the following conclusions.
• The thiadiazole derivatives 12d,12c, 6g, 18b, 6c and 6f showed high antitumor activity, and the thiadiazole derivatives 6e, 12a, 18a and 6a revealed moderate antitumor activity, while the thiadiazole derivatives 12b, 6b and 6d exhibited poor antitumor activity.• The ester group (CO2Et) at position 2 of the thiadiazole ring is necessary to have higher antitumor activity than the acetyl and the N-phenylcarboxamide (CONHPh) groups.• The presence of chlorine or bromine group (electron-withdrawing groups) at the position 2 or 4 in the aryl moiety of the thiadiazole ring as in the compounds 12d, 12c, 6g, 18b, 6c and 6f increased the cytotoxic activity.Also, halogen at positions 2 or 4 had more cytotoxic activity than halogen at position 3. • While presence of electron-donating groups such as methyl or methoxy at the position 4 as in the compounds 12b, 6b and 6d decresed the cytotoxic activity.
The influence of substituents on the observed biological activity:

Chemistry 3.1.1. General
Melting points were measured on an Electrothermal IA 9000 series digital melting point apparatus.IR spectra were measured on PyeUnicam SP 3300 and Shimadzu FTIR 8101 PC infrared spectrophotometers in potassium bromide discs.NMR spectra were meaured on a Varian Mercury VX-300 NMR spectrometer operating at 300 MHz ( 1 H-NMR) and run in deuterated dimethylsulfoxide (DMSO-d6).Chemical shifts were related to that of the solvent.Mass spectra were recorded on a Shimadzu GCMS-QP1000 EX mass spectrometer at 70 eV.Elemental analyses were measured by using a German made Elementarvario LIII CHNS analyzer.Antitumor activity of the products was measured at the Regional Center for Mycology and Biotechnology at Al-Azhar University, Cairo, Egypt.

Synthetic Procedures 3.2.1. Synthesis of 1,3,4-thiadiazole derivatives (6a
General procedure.A mixture of compound 3 (0.368 g, 1 mmol) and the appropriate hydrazonoyl chlorides 4a-g or 10a-d or 16a,b (1 mmol) in ethanol (20 mL) containing triethylamine (0.1 g, 1 mmol) was refluxed for 4-6 h.(monitored by TLC).The formed solid product was filtered, washed with methanol, dried and recrystallized from the proper solvents to afford products 6a-g, 10a-d and 18a,b, respectively.The physical constants and spectral data of the obtained products are listed below:

Method B:
A mixture of 4-methyl-2-phenylthiazole-5-carbohydrazide (2) (0.233 g, 1 mmol) and the appropriate 1,3,4-thiadiazoles 6a-c (1 mmol) in ethanol (10 mL) was refluxed for 4h, allowed to cool and the solid product that formed was filtered off, washed with EtOH, dried and recrystallized from the proper solvent to give the corresponding product, 21a-c which were identical in all aspects (m.p., mixed m.p. and IR spectra) with those obtained from reaction of 3 with 19a-c.

Evaluation of the antitumor activity using Viability assay:
Human hepatocellular carcinoma (HepG2) cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD).The cells were grown on RPMI-1640 medium supplemented with 10% inactivated fetal calf serum and 50 µg/mL gentamycin.The cells were maintained at 37 o C in a humidified atmosphere with 5% carbon dioxide and were subcultured 2 to 3 times a week.Potential cytotoxicity of the tested compounds was evaluated on tumor cells using the reported method of Gangadevi and Muthumary [43].The cells were grown as monolayers in growth RPMI-1640.The monolayers of 10 4 cells adhered at the bottom of the wells in a 96-well microtiter plate incubated for 24 h at 37 o C in a humidified incubator with 5% carbon dioxide.The monolayers were then washed with sterile phosphate buffered saline (0.01 M pH 7.2) and simultaneously the cells were treated with 100 µL from different dilutions of tested sample in fresh maintenance medium and incubated at 37 o C. A control of untreated cells was made in the absence of tested sample.Positive controls containing doxroubcin drug was also tested as reference drug for comparison.Six wells were used for each concentration of the test sample.Every 24 h the observation under the inverted microscope was made.The number of the surviving cells was determined by staining the cells with crystal violet [44] followed by cell lysing using 33% glacial acetic acid and read the absorbance at 590 nm using microplate reader (SunRise, TECAN, Inc, USA) after well mixing.The absorbance values from untreated cells were considered as 100% proliferation.The number of viable cells was determined using microplate reader as previously mentioned before and the percentage of viability was calculated as [1-(ODt/ODc)] x 100% where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cells.The relation between surviving cells and drug concentration is plotted to get the survival curve of each tumor cell line after treatment with the specified compound.The 50% inhibitory concentration (IC50), the concentration required to cause toxic effects in 50% of intact cells, was estimated from graphic plots. .

Conclusions
In this context, a series of novel thiazoles and 1,3,4-thiadiazoles bearing thiazole were synthesized.The structure of the newly prepared compounds was established based on both elemental analysis and spectroscopic data and by an alternative method wherever possible.Moreover, the mechanisms of formation of the title compounds were discussed.Some of the synthesized compounds were evaluated for their anti-cancer activity against the human hepatocellular carcinoma (HepG2) cell line.The results showed that the thiadiazole derivatives 12d, 12c, 6g,18b, 6c and 6f having IC50 values 0.82, 0.91, 1.06, 1.25, 1.29 and 1.88 µM, respectively, were found to be the highly active compounds of the prepared series.Based on the experimental results of the antitumor activity, the structure-activity relationships were discussed.

Figure 1 .
Figure 1.Lead compounds among thiazole and thiadiazole derivatives with anticancer activityIn view of these facts, the antitumor activity of the synthesized compounds was determined against a liver carcinoma cell line HepG2.Doxorubicin was used as a reference standard and showed IC50 = 0.72 µM against a liver carcinoma cell line.Data generated were used to plot a dose-response