Synthesis and Characterization of Some New Bis-Pyrazolyl-Thiazoles Incorporating the Thiophene Moiety as Potent Anti-Tumor Agents

A new series of 1,4-bis(1-(5-(aryldiazenyl)thiazol-2-yl)-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-3-yl)benzenes 3a–i were synthesized via reaction of 5,5′-(1,4-phenylene)bis(3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide) (1) with hydrazonoyl halides 2a–i. In addition, reaction of 1 with ethyl chloroacetate afforded bis-thiazolone derivative 8 as the end product. Reaction of compound 8 with methyl glyoxalate gave bis-thiazolone derivative 10. The structures of the newly synthesized compounds were established on the basis of spectroscopic evidences and their alternative syntheses. All the synthesized compounds were evaluated for their anti-tumor activities against hepatocellular carcinoma (HepG2) cell lines, and the results revealed promising activities of compounds 3g, 5e, 3e, 10, 5f, 3i, and 3f with IC50 equal 1.37 ± 0.15, 1.41 ± 0.17, 1.62 ± 0.20, 1.86 ± 0.20, 1.93 ± 0.08, 2.03 ± 0.25, and 2.09 ± 0.19 μM, respectively.

In view of these reports and in continuation of our previous works in synthesis of bioactive bis-heterocyclic compounds [29][30][31], we are herein interested in synthesis of bis-pyrazolyl-thiazoles incorporating the thiophene moiety using the hitherto unreported 5,5 -(1,4-phenylene)bis(3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide) as versatile building blocks for the synthesis of the title compounds. All the newly synthesized products were screened for their anti-tumor activities against hepatocellular carcinoma (HepG2) cell lines and showed activities with good IC 50 for seven compounds.
Next, the reaction of 2-(1-(thiophen-2-yl)ethylidene)hydrazinecarbothioamide (4) [34] with 2-oxo-N′-arypropanehydrazonoyl chlorides 2a,b,e,f in dioxane under reflux in the presence of TEA as a basic catalyst afforded one isolable product (as evidenced by TLC analysis of the crude product), which were identified to be arylazothiazole derivatives 5a,b,e,f (72%-76% yield) as outlined in Scheme 2. The structure of compounds 5a,b,e,f was elucidated by elemental and spectral (IR, 1 H NMR, mass) data. 1 H NMR spectra of compounds 7 revealed in addition to the expected signals of the aromatic protons, and the protons of the two methyl groups, a singlet at δ 10.60-10.72 ppm, assigned to the -NH proton. The mass spectra of all products 5 exhibited, in each case, a molecular ion peak at the correct molecular weight for the respective compound (see Material and Methods).
Refluxing equimolar amounts of the appropriate arylazothiazole (5) and terephthalaldehyde (6) in EtOH containing catalytic amounts of glacial acetic acid gave the corresponding products, 3a,b,e,f,which were identical in all respects (melting point (m.p.), mixed m.p. and IR spectra) with those obtained from reaction of compounds 1 with 2. Scheme 1. Synthesis of bis-arylazothiazole derivatives 3a-i.
Next, the reaction of 2-(1-(thiophen-2-yl)ethylidene)hydrazinecarbothioamide (4) [34] with 2-oxo-N -arypropanehydrazonoyl chlorides 2a,b,e,f in dioxane under reflux in the presence of TEA as a basic catalyst afforded one isolable product (as evidenced by TLC analysis of the crude product), which were identified to be arylazothiazole derivatives 5a,b,e,f (72%-76% yield) as outlined in Scheme 2. The structure of compounds 5a,b,e,f was elucidated by elemental and spectral (IR, 1 H NMR, mass) data. 1 H NMR spectra of compounds 7 revealed in addition to the expected signals of the aromatic protons, and the protons of the two methyl groups, a singlet at δ 10.60-10.72 ppm, assigned to the -NH proton. The mass spectra of all products 5 exhibited, in each case, a molecular ion peak at the correct molecular weight for the respective compound (see Material and Methods).

Anti-Cancer Activity
The anticancer activity of some newly synthesized compounds was determined against a liver carcinoma cell line HepG2, using doxorubicin as a reference drug. 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 (IC 50 ) was determined. The cytotoxic activity was expressed as the mean IC 50 of three independent experiments (Table 1), and the results revealed that all the tested compounds showed inhibitory activity to the tumor cell lines in a concentration dependent manner.
The results are represented in Table 1 showed the following: 1.
The in vitro inhibitory activities of tested compounds against the human liver carcinoma (HepG2) have the descending order as follow: The thiazole derivatives 5a,b,e,f have in vitro inhibitory activity greater than the bis-thiazole derivatives 3a,b,e,f (5a > 3a, 5b > 3b, 5e > 3e, and 5f > 3f).

3.
The substituent at position 4 in the thiazole ring affect the in vitro inhibitory activity, while the thienyl ring has a greater effect than the methyl group, which has a greater effect than the phenyl group.

4.
The introduction of electron-withdrawing group (nitro group > chlorine atom > bromine atom) at the fourth position of the phenyl group at position 5 in the thiazole ring enhances the antitumor activity. In contrast, the introduction of electron-donating group (methoxy group > methyl group) decreases the antitumor activity.

Anti-Cancer Activity
The anticancer activity of some newly synthesized compounds was determined against a liver carcinoma cell line HepG2, using doxorubicin as a reference drug. 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. The cytotoxic activity was expressed as the mean IC50 of three independent experiments (Table 1), and the results revealed that all the tested compounds showed inhibitory activity to the tumor cell lines in a concentration dependent manner.
The results are represented in Table 1 showed the following: 1. The in vitro inhibitory activities of tested compounds against the human liver carcinoma (HepG2) have the descending order as follow: The thiazole derivatives 5a,b,e,f have in vitro inhibitory activity greater than the bis-thiazole derivatives 3a,b,e,f (5a > 3a, 5b > 3b, 5e > 3e, and 5f > 3f). 3. The substituent at position 4 in the thiazole ring affect the in vitro inhibitory activity, while the thienyl ring has a greater effect than the methyl group, which has a greater effect than the phenyl group. 4. The introduction of electron-withdrawing group (nitro group > chlorine atom > bromine atom) at the fourth position of the phenyl group at position 5 in the thiazole ring enhances the antitumor activity. In contrast, the introduction of electron-donating group (methoxy group > methyl group) decreases the antitumor activity.

General Experimental Procedures
All melting points were determined on an electrothermal Gallenkamp apparatus (Bibby Sci. Lim. Stone, Staffordshire, UK). Solvents were distilled and dried by standard literature procedures prior to use. The IR spectra were measured on a Pye-Unicam SP300 instrument (Shimadzu, Tokyo, Japan) in potassium bromide discs. The 1 H NMR and 13 C NMR spectra were recorded on a Varian Mercury (Varian, Inc., Karlsruhe, Germany, 300 MHz for 1 H NMR and 75 MHz for 13 C NMR) and the chemical shifts were related to that of the solvent DMSO-d 6 . The mass spectra were recorded on GCMS-Q1000-EX Shimadzu (Tokyo, Japan), and the ionizing voltage was 70 eV. Elemental analyses were carried out by the Microanalytical Center of Cairo University, Giza, Egypt. Hydrazonoyl halides 2a-i were prepared following a method from the literature [38]. Figures S1-S16) 3.2.1. General Method for the Synthsis of 1,4-Bis(1-(4-substituted-5-((E)-aryldiazenyl)thiazol-2-yl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-5-yl)benzene 3a-i A mixture of bis-pyrazolylcarbothioamide 1 (0.336 g, 1 mmol) and the appropriate hydrazonoyl halides 2a-c (2 mmol) in dioxane (20 mL) containing TEA (1 mL) was refluxed for 2-6 h (monitored by TLC) and allowed to cool, and the solid formed was filtered off, washed with EtOH, dried, and recrystallized from DMF to give 3a-i. The products 3a-i together with their physical constants is listed below.

Alternate Synthesis of Compounds 3a,b,e,f
A mixture of terephthalaldehyde (6) (0.134 g, 1 mmol) and the appropriate thiazole 5 (2 mmol) in EtOH (10 mL) containing 0.5 mL of glacial acetic acid was refluxed for 8 h and then cooled to room temperature. The solid precipitated was filtered off, washed with water, dried, and recrystallized from DMF to give the corresponding products, 3a,b,e,f, which were identical in all respects (m.p., mixed m.p., and IR spectra) with those obtained from reaction of 1 with 2.

Alternate Synthesis of Compound 10
To a solution of bis-pyrazolylcarbothioamide 1 (0.336 g, 1 mmol) in dry methanol (20 mL) was added dimethylacetylenedicarboxylate (11) (0.284 g, 2 mmol). The solution was refluxed for 4 h. The precipitated product after cooling was filtered, washed with methanol, and recrystallized from DMF to give product 10, which was identical in all aspects (m.p., mixed m.p., and IR spectra) with that obtained from the reaction of 8 with 9.

Anti-Tumor Activity
Human liver carcinoma (HepG2) cell lines were 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 of gentamicin. The cells were maintained at 37 • C in a humidified atmosphere with 5% CO 2 and were sub-cultured two to three times a week.
For antitumor assays, the tumor cell lines were suspended in medium at concentration 5 × 10 4 cell/well in corning ® 96-well plates (six replicates) to achieve eight concentrations for each compound. Six vehicle controls with media or 0.5% DMSO were run for each 96-well plate as a control. After incubating for 24 h, the numbers of viable cells were determined by the MTT test. Briefly, the media were removed from the 96-well plates and replaced with 100 µL of fresh culture RPMI 1640 medium without phenol red, followed by 10 µL of the 12 Mm MTT stock solutions (5 mg of MTT in 1 mL of PBS) to each well, including the untreated controls. The 96-well plates were then incubated at 37 • C and 5% CO 2 for 4 h. An 85-µL aliquot of the media was removed from the wells, and 50 µL of DMSO was added to each well and mixed thoroughly with the pipette and incubated at 37 • C for 10 min. Then, the optical density was measured at 590 nm with the microplate reader (SunRise, TECAN, Inc, Männedorf, Switzerland) to determine the number of viable cells and the percentage of viability was calculated as (1 − (ODt/ODc)) × 100%, where ODt is the mean optical density of untreated cells. The relation between surviving cells and drug concentration is plotted to obtain the survival curve of each tumor cell line after treatment with the specified compound. The 50% inhibitory concentration (IC 50 ), the concentration required to cause toxic effects in 50% of intact cells, was estimated from graphic plots of the dose-response curve for each concentration using Graphed Prism software (San Diego, CA, USA) [39][40][41].

Conclusions
In our present work, a new series of 2-ethylidenehydrazono-5-arylazothiazoles and 2-ethylidenehydrazono-5-arylazothiazolones were synthesized from a reaction of ethylidene-thiosemicarbazide with various hydrazonoyl halides. The structures of the newly synthesized compounds were established on the basis of spectroscopic evidences and their synthesis by alternative methods. The in-vitro growth inhibitory activity of the synthesized compounds against hepatocellular carcinoma (HepG2) cell lines was investigated in comparison with doxorubicin as a standard drug using an MTT assay, and the results revealed promising activities of compounds 3g, 5e, 3e, 10, 5f, 3i, and 3f with an IC 50 equal to 1.37 ± 0.15, 1.41 ± 0.17, 1.62 ± 0.20, 1.86 ± 0.20, 1.93 ± 0.08, 2.03 ± 0.25, and 2.09 ± 0.19 µM, respectively.