Synthesis and Biological Evaluation of Some Novel Thiazole-Based Heterocycles as Potential Anticancer and Antimicrobial Agents

A novel series of thiazole-based heterocycles was synthesized using 1,3-dipolar cycloaddition reactions in the presence of chitosan-grafted-poly(vinylpyridine) as an eco-friendly biopolymeric basic catalyst. The molecular structure of the synthesized compounds was illustrated by spectroscopic and elemental analysis. Various in vitro biological assays were performed to explore the potential antitumor, antimicrobial and hepatoprotective activities of the newly synthesized compounds. The cytotoxic activities were assessed against human hepatocellular carcinoma (HepG-2), colorectal carcinoma (HCT-116) and breast cancer (MCF-7) cell lines and results revealed that all compounds displayed antitumor activities with the chlorine-containing derivatives, 11c and 6g, being the most potent. The majority of the tested thiazole derivatives exhibited satisfactory antibacterial activity towards the used gram positive and gram-negative bacterial species. Moreover, many derivatives showed weak hepatoprotective activity against CCl4-induced hepatotoxicity.

Cancer is regarded as one of the dominant causes of mortality nowadays. The development of new antitumor agents represents an urgent need due to the increasing problems of various, sometimes, intolerable toxic side effects of the currently marketed drugs and the evolution of resistance to their actions [17,18]. Furthermore, liver diseases are viewed as one of the highly serious health issues globally [19]. The lack of satisfactory treatment strategies for these diseases with the occurrence of different side effects upon long term therapy, raise the demand for finding out new chemical entities that offer more efficient hepatoprotection and considerable safety. Moreover, there is a continuous compelling need for the development of new antibiotics to replace the current medications that are losing their efficacy and that could have higher efficiency or a wider spectrum.
In view of these precedents and together with our research concerns of developing new convenient approaches for the synthesis of different heterocyclic systems with auspicious pharmacological activities [20][21][22][23][24][25], we present in this report an efficient synthesis of some new series of novel thiazole derivatives using chitosan-grafted-poly(vinyl pyridine) as an eco-friendly biopolymeric basic catalyst. Additionally, we have assessed a variety of biological activities for the newly synthesized compounds that demonstrated their potential antitumor, antimicrobial and hepatoprotective effectiveness.

Chemistry
Refluxing of 5-acetyl-4-methyl-2-phenyl-thiazole (1) [26] and 2-cyanoacetohydrazide (2) [27] afforded a single product identified as 2-cyano-N′-(1-(4-methyl-2-phenylthiazol-5-yl)ethylidene)acetohydrazide ( methyl, CH 2, and NH protons, respectively. Moreover, its mass spectrum showed a molecular ion peak at m/z = 298. Treatment of hydrazone derivative 3 with the appropriate hydrazonoyl halides 4a-h [28][29][30][31][32] using triethylamine or chitosan as a basic catalyst and under the same experimental conditions, afforded in each case the same products which are identified as the thiazole derivatives 6a-h rather than the other possible product 7 based on the spectral data (IR, MS and 1 H-NMR) of the isolated products (Scheme 1, see Supporting Information). The distinction between the two possible products 6 and 7 was done based on the results of the spectral analysis. The IR spectra showed the absence of nitrile absorption band. Also, their 1 H-NMR spectra revealed the presence of signals corresponds to NH 2 protons. Moreover, their mass spectrum showed peaks corresponding to their molecular ions. The results of Table 1 indicated that high yield was obtained using chitosan as a basic catalyst. Heating a mixture of hydrazonoyl halides 8a or 8b [23] and the appropriate arylidine malononitriles 9a-c [33] in ethanol containing piperidine under irradiation by MW led to the formation of the thiazolyl pyrazoles 11a-f (Scheme 2). The structure of the latter products was established based on their elemental analysis and spectral data (cf. Experimental, see Supporting Information). Its mass spectrum was compatible with the molecular formula C15H14N4OS and its IR spectrum showed absorption bands at 1643, 2338, and 3430 cm −1 due to amido carbonyl group, cyano and NH functions, respectively. Also, its 1 H-NMR revealed signals at δ 2.49, 2.72, 3.30 and 10.6 due to two methyl, CH2, and NH protons, respectively. Moreover, its mass spectrum showed a molecular ion peak at m/z = 298.
Treatment of hydrazone derivative 3 with the appropriate hydrazonoyl halides 4a-h [28][29][30][31][32] using triethylamine or chitosan as a basic catalyst and under the same experimental conditions, afforded in each case the same products which are identified as the thiazole derivatives 6a-h rather than the other possible product 7 based on the spectral data (IR, MS and 1 H-NMR) of the isolated products (Scheme 1, see Supporting Information). The distinction between the two possible products 6 and 7 was done based on the results of the spectral analysis. The IR spectra showed the absence of nitrile absorption band. Also, their 1 H-NMR spectra revealed the presence of signals corresponds to NH2 protons. Moreover, their mass spectrum showed peaks corresponding to their molecular ions. The results of Table 1 indicated that high yield was obtained using chitosan as a basic catalyst. Heating a mixture of hydrazonoyl halides 8a or 8b [23] and the appropriate arylidine malononitriles 9a-c [33] in ethanol containing piperidine under irradiation by MW led to the formation of the thiazolyl pyrazoles 11a-f (Scheme 2). The structure of the latter products was established based on their elemental analysis and spectral data (cf. Experimental, see Supporting Information).  When the above reaction was repeated in presence of grafted-chitosan as a catalyst and under typical reaction conditions, the same products which are identical in all aspects (m.p., mixed m.p. and IR spectra) were obtained in good yields (Table 2). To account for the formation of the product 11, it is suggested that the 1,3-dipolar cycloaddition of nitrile imine 8' generated in situ from hydrazonoyl halides 8 in the presence of base) to the arylidine derivative 9 to give the intermediate 10, followed by aromatization via losing of HCN molecule to give the final product 11 as illustrated in Scheme 2.

Cytotoxic Activity
The in vitro antitumor activity of the newly-synthesized compounds 6a-h and 11a-f and the reference drug, Doxorubicin was investigated against three cancer cell lines, human hepatocellular carcinoma cell line (HepG-2), colon carcinoma cells (HCT-116), and human breast carcinoma cells (MCF-7 cell line). The cytotoxic potential was determined using the MTT (methyl thiazolyl tetrazolium) assay after 24 h of incubation [34]. The concentration of the tested compounds needed to inhibit 50% of the cells (IC 50 ) was calculated and presented in Table 3 and Figures 1-3. Results of the MTT assay indicated that most of investigated compounds exhibited inhibitory activity against the tested cell lines, with some derivatives showing prominent antitumor activity. Thiazole derivatives 11c and 6g displayed the highest cytotoxic activities against the tested cell lines with IC 50 values of about 4 µg/mL and 7 µg/mL for HepG-2, 3 µg/mL and 4 µg/mL for MCF-7, and 7 µg/mL and 12 µg/mL, for HCT-116 cells, respectively.
According to these results, we can suggest the following structure activity relationships: A-In the thiazolylpyrazoles 6a-h: (1) Attachment of chlorine (6d) or methoxy group (6c) at position 4 in the aryl moiety of the pyrazole ring is important for cytotoxic activity with chlorine having the higher impact in compound (6d). (2) Addition of another chlorine atom in position 2 in the aryl moiety of compound (6g) increases the activity which reaches the double against MCF-7 cells.
B-In the thiazolylpyrazoles 11a-f: (1) Substitution on only one of the aryl moieties of the pyrazole ring in compounds (11c,d) induces cytotoxic activity, most prominently by chlorine in compound (11c). (2) Substitution on the second aryl moiety of the pyrazole ring by methyl group as in compounds (11b,f) induces great reduction (nearly abolishes) the cytotoxic activity. with IC50 values of about 4 µg/mL and 7 µg/mL for HepG-2, 3 µg/mL and 4 µg/mL for MCF-7, and 7 µg/mL and 12 µg/mL, for HCT-116 cells, respectively. According to these results, we can suggest the following structure activity relationships: A-In the thiazolylpyrazoles 6a-h: (1) Attachment of chlorine (6d) or methoxy group (6c) at position 4 in the aryl moiety of the pyrazole ring is important for cytotoxic activity with chlorine having the higher impact in compound (6d). (2) Addition of another chlorine atom in position 2 in the aryl moiety of compound (6g) increases the activity which reaches the double against MCF-7 cells.
B-In the thiazolylpyrazoles 11a-f: (1) Substitution on only one of the aryl moieties of the pyrazole ring in compounds (11c,d) induces cytotoxic activity, most prominently by chlorine in compound (11c). (2) Substitution on the second aryl moiety of the pyrazole ring by methyl group as in compounds (11b,f) induces great reduction (nearly abolishes) the cytotoxic activity. Dox: doxorubicin, the standard drug. The analysis was performed using the MTT assay after 24h of incubation. Values are shown as mean ± SD of three replicates. All compounds exhibited cytotoxic effects and, 11c and 6g were the most potent. Compound 6a has IC50 > 500 µg/mL. Dox: doxorubicin, the standard drug. The analysis was performed using the MTT assay after 24 h of incubation. Values are shown as mean ± SD of three replicates. All compounds exhibited cytotoxic effects and, 11c and 6g were the most potent. Compound 6a has IC 50 > 500 µg/mL. with IC50 values of about 4 µg/mL and 7 µg/mL for HepG-2, 3 µg/mL and 4 µg/mL for MCF-7, and 7 µg/mL and 12 µg/mL, for HCT-116 cells, respectively. According to these results, we can suggest the following structure activity relationships: A-In the thiazolylpyrazoles 6a-h: (1) Attachment of chlorine (6d) or methoxy group (6c) at position 4 in the aryl moiety of the pyrazole ring is important for cytotoxic activity with chlorine having the higher impact in compound (6d). (2) Addition of another chlorine atom in position 2 in the aryl moiety of compound (6g) increases the activity which reaches the double against MCF-7 cells.
B-In the thiazolylpyrazoles 11a-f: (1) Substitution on only one of the aryl moieties of the pyrazole ring in compounds (11c,d) induces cytotoxic activity, most prominently by chlorine in compound (11c). (2) Substitution on the second aryl moiety of the pyrazole ring by methyl group as in compounds (11b,f) induces great reduction (nearly abolishes) the cytotoxic activity. Dox: doxorubicin, the standard drug. The analysis was performed using the MTT assay after 24h of incubation. Values are shown as mean ± SD of three replicates. All compounds exhibited cytotoxic effects and, 11c and 6g were the most potent. Compound 6a has IC50 > 500 µg/mL.  Dox: doxorubicin, the standard drug. The analysis was performed using the MTT assay after 24h of incubation. Values are shown as mean ± SD of three replicates. All compounds exhibited cytotoxic effects and in particular, 11c and 6g were the most potent. Compound 6a has IC50 > 500 µg/mL.

Evaluation of the Antimicrobial Activity
The in vitro antimicrobial effectiveness of the newly synthesized thiazolyl pyrazoles 6a-h, 11af, and standard drugs were investigated using the inhibition zone technique and minimum inhibitory concentration (MIC) [35,36]. The antibacterial activities were tested against the gram-positive bacteria, Staphylococcus aureus (CMB010010) and Bacillus subtilis (RCMB 010067), and the gramnegative bacteria: Escherichia coli (RCMB 010052) and Proteus vulgaris (RCMB 004 (1) ATCC 13315), while the antifungal activities were tested against Aspergillus fumigatus (RCMB 002008 (4)) and Candida albicans (RCMB 05036). Gentamycin was used as the standard antibacterial drug while ketoconazole was used as the standard antifungal drug. The results are presented in Tables 4 and 5 and Supplementary Figures S1-S6.  1 1 a 1 1 b 1 1 c 1 1 d 1 1 e 1 1  Values are shown as mean ± SD of three replicates. All compounds exhibited cytotoxic effects and in particular, 11c and 6g were the most potent. Compound 6a has IC 50 > 500 µg/mL.

Evaluation of the Antimicrobial Activity
The in vitro antimicrobial effectiveness of the newly synthesized thiazolyl pyrazoles 6a-h, 11a-f, and standard drugs were investigated using the inhibition zone technique and minimum inhibitory concentration (MIC) [35,36]. The antibacterial activities were tested against the gram-positive bacteria, Staphylococcus aureus (CMB010010) and Bacillus subtilis (RCMB 010067), and the gram-negative bacteria: Escherichia coli (RCMB 010052) and Proteus vulgaris (RCMB 004 (1) ATCC 13315), while the antifungal activities were tested against Aspergillus fumigatus (RCMB 002008 (4)) and Candida albicans (RCMB 05036). Gentamycin was used as the standard antibacterial drug while ketoconazole was used as the standard antifungal drug. The results are presented in Tables 4 and 5 and Supplementary Figures S1-S6.  The results of the antimicrobial evaluation demonstrated that all the newly synthesized thiazoles exhibited good antibacterial effect towards the gram-positive bacteria Staphylococcus aureus (except 11b), and Bacillus subtilis (except 11f). With regards to the gram-negative bacteria, all compounds had antibacterial activity against Escherichia coli, while only 6f, 6h and 11a-e were effective against Proteus vulgaris. Of notice, the thiazole derivative 6f possessed the highest antibacterial activity compared to all other tested thiazoles against Staphylococcus aureus, Bacillus subtilis, and Escherichia coli. Interestingly, the antimicrobial activity of this derivative approaches the potency of gentamicin, against the tested gram-negative bacteria. However, the derivative 6h exerted the most prominent antibacterial activity against Proteus vulgaris. On the contrary, all synthesized compounds had no antifungal activity against Aspergillus fumigatus or Candida albicans. From these data, we can conclude that the presence of ethoxy carbonyl group and p-tolyl as substituents on the pyrazole ring increased the antimicrobial activity of compound 6f.

In Vitro Hepatoprotective Activity
The hepatoprotective potential of the newly synthesized thiazole derivatives was studied using an in vitro model of CCl 4 -induced hepatotoxicity. In vitro hepatoprotective activity was performed by assessing the viability of isolated rat hepatocytes treated with CCl 4 in the presence and absence of the tested compounds [37]. Rat hepatocytes were isolated as previously described [38], and their viability was evaluated by the MTT reduction assay method [34,39] using silymarin as the reference standard drug. The concentration required to cure 50% of CCl 4 -exposed hepatocytes, EC 50 was calculated and presented in Table 6. Results declared that compounds 6c, 6d, 6f, 6g, 6h, 11c, 11d, and 11e offered protection against CCl 4 -induced liver damage but lower than the standard drug. These results would suggest that these thiazole derivatives could be a candidate starting materials for the synthesis of more potent hepatoprotective drugs.
Method A. A mixture of hydrazone 3 (0.298 g, 1 mmol) and the appropriate hydrazonoyl halides 4 (1 mmol) in dioxane (20 mL) containing TEA (0.07 mL) was irradiated by MW at 400 Watt in a closed Teflon vessel until all the starting material was consumed (6-10 min as monitored by TLC). The hot reaction mixture was allowed to cool to room temperature and the precipitated solid was filtered off, washed with EtOH, dried and recrystallized from the suitable solvent to give the corresponding thiazole derivatives 6a-h.
Method B. A mixture of hydrazone 3 (0.298 g, 1 mmol) and the appropriate hydrazonoyl halides 4 (1 mmol) in dioxane (20 mL) containing grafted-chitosan (0.1 g) was irradiated by MW at 400 Watt in a closed Teflon vessel until all the starting material was consumed (6-10 min as monitored by TLC). The hot solution was filtered to remove grafted-chitosan and excess solvent was removed under reduced pressure. The reaction mixture was triturated with methanol and the product separated was filtered, washed with methanol, dried and recrystallized from the proper solvent to give the corresponding products, 6a-h which were identical in all aspects (m.p., mixed m.p. and IR spectra) with those obtained from method A. The physical constants of products 6a-h are provided below:     Method A: Equimolecular mixture of 2-(2-(4-methyl-2-phenylthiazole-5-carbonyl)hydrazono)-N -arylpropane hydrazonoyl chlorides 8a,b (l mmol) and the appropriate arylidine malononitriles 9a-c (1 mmol) in absolute EtOH (10 mL) containing catalytic amounts of piperidine (0.50 mL) was irradiated by MW at 400 Watt in a closed Teflon vessel until all the starting material was consumed (4-8 min as monitored by TLC), then cooled to room temperature. The solid product was filtered off, washed with ethanol and recrystallized from the proper solvent to give the thiazole derivatives 11a-f, respectively.
Method B: A mixture of 8a,b (1 mmol) and the appropriate arylidine malononitriles 9a-c (1 mmol) in absolute EtOH (10 mL) containing grafted-chitosan (0.1 g) was irradiated by MW at 400 Watt in a closed Teflon vessel until all the starting material was consumed (4-8 min as monitored by TLC). The hot solution was filtered to remove grafted-chitosan and excess solvent was removed under reduced pressure. The reaction mixture was triturated with MeOH and the product separated was filtered, washed with MeOH, dried and recrystallized from the proper solvent to give the corresponding products, 11a-f which were identical in all aspects (m.p., mixed m.p. and IR spectra) with those obtained from method A. The physical constants of the products 11a-f are listed below.

In Vitro Cytotoxic Activity
The cytotoxic potential of the newly synthesized compounds was examined against three cancer cell lines HepG2, HCT-116, and MCF-7 using the MTT assay after 24 h of incubation [34]. For more details, see the Supporting Information file.

Antimicrobial Evaluation
Antifungal and antibacterial activities of the synthesized thiazoles were assessed towards different microbes using the agar diffusion method and were compared to standard reference drugs [35,36]. Refer to the Supporting Information file for more details.

Hepatoprotective Activity
In vitro hepatoprotective activity was done by assessing the viability of isolated rat hepatocytes exposed to 1% CCl 4 along with or without the tested compounds [37]. Rat hepatocytes were isolated as described [38] and cell viability was evaluated by the MTT reduction assay [34,39] using silymarin as the reference standard drug. For further details, refer to the Supporting Information file.

Conclusions
We have efficiently synthesized a new series of thiazolylpyrazoles using hydrazonoyl halides and 2-cyano-N -(1-(4-methyl-2-phenylthiazol-5-yl)ethylidene)acetohydrazide in the presence of chitosan-grafted-poly(vinylpyridine) as an eco-friendly biopolymeric basic catalyst. The structures of these novel compounds were determined using spectroscopic analyses (IR, NMR, and MS). All compounds were evaluated for their cytotoxic effectiveness against HepG-2, MCF-7, and HCT-116 cell lines. Our results indicated that most compounds exhibited a good anticancer activity and importantly, the thiazole derivatives 11c and 6g exhibited the greatest cytotoxic potential against the examined cell lines. In addition, antibacterial evaluation experiments illustrated that the thiazole derivative 6f has the most potent activity towards Staphylococcus aureus, Bacillus subtilis, and Escherichia coli. The antibacterial potency of 6f against the tested gram-positive bacteria even approaches that of gentamicin. However, the derivative 6h exerted the highest antibacterial activity against Proteus vulgaris. Some of the tested compounds showed a weak hepatoprotective effect against CCl 4 -induced liver damage suggesting their usage as a candidate starting materials for the synthesis of more potent hepatoprotective drugs. Taken together, the current work presents an eco-friendly approach for the synthesis of novel thiazole derivatives that have potential values in protection against cancer cells and bacterial infections and could be used to develop effective agents to guard against liver toxicity.

Conflicts of Interest:
The authors declare that there is no conflict of interests regarding the publication of this paper.