A facile synthesis and anticancer activity of some novel thiazoles carrying 1,3,4-thiadiazole moiety

Background Thiazoles and 1,3,4-thiadiazoles have been reported to possess various pharmacological activities. Results A novel series of thiazoles carrying 1,3,4-thiadiazole core were designed and prepared via the reaction of the 2-(4-methyl-2-phenylthiazole-5-carbonyl)-N-phenylhydrazinecarbo-thioamide with the appropriate hydrazonoyl chlorides. The structures of the newly synthesized compounds were confirmed based on elemental and spectral analysis as well as their alternative syntheses. The cytotoxic potency of the newly synthesized thiadiazoles was evaluated by their growth inhibitory potency in liver HepG2 cancer cell line. Also, the structure activity relationship was studied. Conclusions All the newly synthesized compounds were evaluated for their anticancer activity against liver carcinoma cell line (HepG2) using MTT assay. The results revealed that the compounds 12d, 12c, 6g, 18b, 6c, and 6f (IC50 = 0.82, 0.91, 1.06, 1.25, 1.29 and 1.88 µM, respectively) had good antitumor activity against liver carcinoma cell line (HepG2) when compared with the standard drug Doxorubicin (IC50 = 0.72 µM).Graphical abstract A facile synthesis and anticancer activity of some novel thiazoles carrying 1,3,4-thiadiazole moiety. Electronic supplementary material The online version of this article (doi:10.1186/s13065-017-0255-7) contains supplementary material, which is available to authorized users.


Background
Cancer is the 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, it is often limited by the severity of the side effects and the development of tumor cell resistance to 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 thioamide hydrazine moiety as a side chain in compound 3 prompted us to utilize it for constructing 1,3,4-thiadiazole ring through its reaction with many hydrazonoyl chlorides. Thus, treatment of compound 3 with the appropriate hydrazonoyl chlorides 4a-g [36] led to the 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 spectral data are consistent 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 showed in each case the presence of broad singlet signals assigned for the NH proton near δ 11.19 ppm, in addition to the expected signals for the two CH 3 , and the aryl protons. Also, the mass spectrum of each of products 6 revealed the presence of a molecular ion peak (see materials and methods). A suggested mechanism for the synthesis of 1,3,4-thiadiazole derivatives 6 is outlined in Scheme 1.
To explain the synthesis of 1,3,4-thiadiazole 6a-g, we assumed that the reaction started with S-alkylation to afford the non-isolable intermediate 5 followed by intramolecular cyclization and elimination of aniline molecule to give the respective thiadiazole derivatives 6a-g (Scheme 1). The structure of 6 was proved chemically via an alternative method (Scheme 1). Thus, the reaction of 5-(4-methyl-2-phenylthiazol-5-yl)-1,3,4-oxadiazole-2(3H)-thione (9) [37] with 4a in ethanol in the presence of triethylamine under reflux led to the formation of a Scheme 1 Synthesis of thiadiazoles 6a-g product which is identical in all respects (mp, mixed mp, and IR) with compound 6a.
Next, to test of the biological activities of a vast array of these compounds, we reacted compound 3 with the appropriate hydrazonoyl chlorides 10a-d [38], 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, the reaction of ethyl 4-methyl-2-phenyl thiazole-5-carboxylate (1) with 1,3,4-thiadiazole 15 [37] in ethanol under reflux, gave a product which was 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 cyclization via losing of aniline molecule (route a) to give the final product 12. The other routes (b) and (c) outlined in Scheme 2 were excluded since they led to the formation of products 13 and 14, which were completely different in all respects (IR, 1 H-NMR, mass spectra) from products 12.
Moreover, the reaction of compound 3 with hydrazonoyl halide of type 16 was studied. Thus refluxing compound 3 with the hydrazonoyl chloride 16a or 16b [38] under the same experimental conditions, afforded the corresponding 1,3,4-thiadiazole derivatives 18a, b (Scheme 3). The 1 H-NMR spectrum of compound 18a revealed two D 2 O-exchangeable signals at δ 10.18 and 11.72 corresponding to two NH protons, in addition to an aromatic multiplet in the region 7.02-7.78 ppm. Also, its mass spectrum revealed a molecular ion peak at m/z = 512. 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.
In view of these facts, the antitumor activity of the synthesized compounds was determined against liver carcinoma cell line HepG2. Doxorubicin was used as a reference standard. Data generated were used to plot a dose-response curve of which the concentration (μM) of test compounds required to kill 50% of the cell population (IC 50 ) was determined. Cytotoxic activity was expressed as the mean IC 50 of three independent experiments. The results depicted in Table 1. Gomha  The results of Table 1 revealed that most of the tested compounds showed a great variable activity compared to reference drug. The order of their antitumor activity and the influence of the substituents were shown in Fig. 2.
From the results of Table 1 and Fig. 2, we can suggest the following points.
• The ester group (CO 2 Et) at position 2 of the thiadiazole ring is necessary to have higher antitumor activity than the acetyl and the N-phenyl carboxamide (CONHPh) groups.
• The presence of chlorine group (electron-withdrawing group) at the position 2, 4 or 4 in the aryl moiety of the thiadiazole ring increased the cytotoxic activity. • Chlorine at positions 2, 4 or 4 in the aryl moiety had high cytotoxic activity than halogen at position 3. • The compounds containing chlorine had high cytotoxic activity than the compounds containing bromine. • The presence of electron-donating groups such as methyl or methoxy at the position 4 in the aryl moiety as in the compounds 12b, 6b and 6d decreased the cytotoxic activity.

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 detailed procedure for the in vitro antitumor assay is presented in Additional file 1.

Conclusions
A series of novel thiazoles carrying 1,3,4-thiadiazole ring 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. All the synthesized compounds were evaluated for their anti-cancer activity against the human hepatocellular carcinoma (HepG2) cell line. The results showed that the thiazole derivatives 12d, 12c, 6g,18b, 6c and 6f having IC 50 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.