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.

1. Introduction

Thiophene and their bis-heterocycles have received considerable attention during the last two decades, as they are endowed with a wide range of therapeutic properties, such as analgesic, antibacterial, antioxidant, anti-inflammatory, antifungal, antin class="Disease">cancer, and local anesthetic activities [1,2,3,4,5,6]. On the other hand, pyrazolines have been reported to possess a variety of significant and diverse pharmacological activities such as antibacterial [7], antifungal [8], anticonvulsant [9], DPPH radical scavenging and anti-diabetic [10], antitubercular [11], antidepressant [12], anti-inflammatory [13], antiamebic [14], analgesic [15], and anticancer [16] activities. Moreover, thiazoles are a familiar group of heterocyclic compounds possessing a wide variety of biological activities such as antimicrobial [17], antioxidant [18], antitubercular [19], anticonvulsant [20], anticancer [21,22,23,24,25,26,27], and anti-inflammatory [28] agents.

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 n class="Chemical">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 IC50 for seven compounds.

2. Results

2.1. Synthesis

The reactivity of 5,5′-(1,4-phenylene)bis(3-(thiophen-2-yl)-4,5-dihydro-1H-n class="Chemical">pyrazole-1-carbothio-amide) (1) [32] was investigated towards hydrazonoyl halides aiming to synthesize new bis-heterocyclic compounds containing 1,3-thiazole ring. Thus, refluxing compound 1 with two moles of each of the hydrazonoyl halides 2a–i in dioxane in the presence of triethylamine gave, in each case, a single product, as indicated by TLC analysis of the crude product, with 68%–76% yield. The structure of the product was identified as 1,4-bis(1-(5-(aryldiazenyl)thiazol-2-yl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-5-yl)benzene 3 as outlined in Scheme 1 based on its elemental analysis and spectral (IR, 1H NMR and MS) data [21,24,33]. For example, the 1H NMR spectra of compound 3a, taken as a typical example of the series, showed six signals at δ 2.59 (s, 6H, 2CH3), 3.15–3.19 (m, 2H, 2CH-pyrazoline), 3.77–3.82 (m, 2H, 2CH-pyrazoline), 5.92–5.96 (m, 2H, 2CH-pyrazoline), 7.02–7.82 (m, 16H, Ar-H and thiophene-H), and 7.99 (s, 4H, Ar-H). The mass spectrum of 3a showed a peak corresponding to its molecular ion at m/z 780 (see Material and Methods).

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 n class="Chemical">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, 1H NMR, mass) data. 1H 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).

Scheme 2

Alternate synthesis of bis-arylazothiazole derivatives 3a,b,e,f.

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.

Finally, compound 1 reacted with ethyl chloroacetate (7) in n class="Chemical">ethanol in the presence of fused sodium acetate as a basic catalyst to give the bis-thiazolone derivative 8 (77% yield) (Scheme 3). Condensation of the latter product 8 with methyl glyoxalate (9) in absolute ethanol containing catalytic amounts of glacial acetic acid afforded dimethyl (2Z,2′Z)-2,2′-(2,2′-(5,5′-(1,4-phenylene)bis(3-(thiophen-2yl)-4,5-dihydro-1H-pyrazole-3,1-diyl))bis(4-oxothiazole-2(4H)-yl-5(4H)-ylidene))diacetate (10) as the end product in 66% yield. Compound 10 was also prepared by a one-step reaction of compound 1 with dimethyl acetylenedicarboxylate (DMAD) (11) in refluxing methanol as outlined in Scheme 3. The structure of compounds 10 was confirmed by elemental analyses and spectral data. For example, the 1H NMR spectrum of product 10 revealed the presence of a singlet at δ 6.68 ppm assigned to the olefinic CH proton of =CH–COOCH3 group, in addition to the signals of the aromatic, methyl, and ester protons. Its mass spectrum revealed a peak corresponding to its molecular ion at m/z 716 (see Material and Methods). The stereochemistry of the methylidene proton of compound 10 is Z-configuration according to literature reports [35,36,37].

Scheme 3

Synthesis of bis-thiazolone derivative 10.

2.2. Anti-Cancer Activity

The anticancer activity of some newly synthesized compounds was determined against a n class="Disease">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.

Table 1

The in vitro inhibitory activity of tested compounds against a liver carcinoma cell line HepG2 expressed as IC50 values (μM) ± standard deviation from six replicates.

Compound No.	R	Ar	IC50 (μM)
1	-	-	4.05 ± 0.17
3a	Me	C6H5	6.29 ± 0.22
3b	Me	4-MeC6H4	18.2 ± 0.14
3c	Me	3-MeC6H4	17.6 ± 0.09
3d	Me	4-MeOC6H4	52.4 ± 0.17
3e	Me	4-ClC6H4	1.62 ± 0.20
3f	Me	4-BrC6H4	2.09 ± 0.19
3g	Me	4-NO2C6H4	1.37 ± 0.15
3h	C6H5	C6H5	10.49 ± 0.22
3i	C4H4S	C6H5	2.03 ± 0.25
5a	Me	C6H5	5.13 ± 0.09
5b	Me	4-MeC6H4	14.29 ± 0.22
5e	Me	4-ClC6H4	1.41 ± 0.17
5f	Me	4-BrC6H4	1.93 ± 0.08
8	-	-	12.37 ± 0.14
10	-	-	1.86 ± 0.20
Doxorubicin	-	-	0.72 ± 0.18

The results are represented in Table 1 showed the following:

The in vitro inhibitory activities of tested compounds against the human liver carcinoma (HepG2) have the descending order as follow: 3g > 5e > 3e > 10 > 5f > 3i > 3f > 3a > 1 > 3a > 3h > 8 > 5b > 3c > 3b > 3d.

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).

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.

The introduction of electron-withdrawing group (nitro group > n class="Chemical">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.

3. Materials and Methods

3.1. 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 n class="Chemical">potassium bromide discs. The 1H NMR and 13C NMR spectra were recorded on a Varian Mercury (Varian, Inc., Karlsruhe, Germany, 300 MHz for 1H NMR and 75 MHz for 13C NMR) and the chemical shifts were related to that of the solvent DMSO-d6. 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].

3.2. Synthetic Procedures (See Supplementary Material 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.

1,4-Bis(1-(4-methyl-5-((E)-phenyldiazenyl)thiazol-2-yl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-5-yl)-benzene (3a). Orange solid; yield (71%); m.p. 192–194 °C (from DMF); IR (KBr) νmax: 3050, 2935 (CH), 1602 (C=N) cm−1; 1H NMR (DMSO-d6) δ: 2.59 (s, 6H, 2CH3), 3.15–3.19 (m, 2H, 2CH-pyrazoline), 3.77–3.82 (m, 2H, 2CH-pyrazoline), 5.92–5.96 (m, 2H, 2CH-pyrazoline), 7.02–7.82 (m, 16H, ArH and thiophene-H), 7.99 (s, 4H, ArH); 13C NMR (DMSO-d6) δ: 8.5 (CH3), 43.3 (CH2), 62.2 (CH), 121.2, 125.3, 125.8, 129.5, 130.4, 131.2, 131.7, 134.8, 136.2, 140.5, 141.8, 144.8 (Ar-C), 151.1, 158.3 (C=N); MS m/z (%): 780 (M+, 9), 341 (13), 185 (51), 119 (66), 78 (42), 51 (100). Anal. Calcd. For C40H32N10S4 (780.17): C, 61.51; H, 4.13; N, 17.93; Found: C, 61.39; H, 4.08; N, 17.72%.

1,4-Bis(1-(4-methyl-5-((E)-p-tolyldiazenyl)thiazol-2-yl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-5-yl)benzene (3b). Red solid; yield (74%); m.p. 180–182 °C (from DMF); IR (KBr) νmax: 3036, 2931 (CH), 1600 (C=N) cm−1; 1H NMR (DMSO-d6) δ: 2.35 (s, 6H, 2CH3), 2.57 (s, 6H, 2CH3), 3.04–3.09 (m, 2H, 2CH-pyrazoline), 3.56–3.60 (m, 2H, 2CH-pyrazoline), 5.92–5.94 (m, 2H, 2CH-pyrazoline), 7.15–7.86 (m, 14H, ArH and thiophene-H), 7.97 (s, 4H, ArH); 13C NMR (DMSO-d6) δ: 8.8, 20.4 (CH3), 45.3 (CH2), 67.2 (CH), 116.3, 125.6, 125.7, 129.0, 129.5, 129.6, 129.7, 131.6, 133.1, 133.4, 148.4, 149.9 (Ar-C), 160.5, 164.2 (C=N); MS m/z (%): 808 (M+, 5), 522 (11), 185 (44), 106 (23), 78 (80), 51 (100); Anal. Calcd. For C42H36N10S4 (808.20): C, 62.35; H, 4.48; N, 17.31; Found: C, 62.27; H, 4.41; N, 17.24%.

1,4-Bis(1-(4-methyl-5-((E)-m-tolyldiazenyl)thiazol-2-yl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-5-yl)-benzene (3c). Red solid; yield (68%); m.p. 164–166 °C (from DMF); IR (KBr) νmax: 3052, 2939 (CH), 1600 (C=N) cm−1; 1H NMR (DMSO-d6) δ: 2.32 (s, 6H, 2CH3), 2.62 (s, 6H, 2CH3), 3.03–3.07 (m, 2H, 2CH-pyrazoline), 3.53–3.57 (m, 2H, 2CH-pyrazoline), 5.90–5.94 (m, 2H, 2CH-pyrazoline), 7.06–7.82 (m, 14H, ArH and thiophene-H), 7.99 (s, 4H, ArH); 13C NMR (DMSO-d6) δ: 8.5, 20.0 (CH3), 45.6 (CH2), 66.3 (CH), 121.3, 125.7, 125.8, 127.7, 128.4, 128.6, 133.3, 138.5, 143.7, 144.5, 148.4, 148.8, 151.6 (Ar-C), 157.8, 163.6 (C=N); MS m/z (%): 808 (M+, 9), 631 (6), 512 (30), 464 (35), 185 (32), 92 (80), 87 (100), 51 (50); Anal. Calcd. For C42H36N10S4 (808.20): C, 62.35; H, 4.48; N, 17.31; Found C, 62.26; H, 4.40; N, 17.22%.

1,4-Bis(1-(5-((E)-(4-methoxyphenyl)diazenyl)-4-methylthiazol-2-yl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-5-yl)benzene (3d). Red solid; yield (70%); m.p. 190–192 °C (from DMF); IR (KBr) νmax: 3052, 2936 (CH), 1601 (C=N) cm−1; 1H NMR (DMSO-d6) δ: 2.59 (s, 6H, 2CH3), 3.04–3.07 (m, 2H, 2CH-pyrazoline), 3.79 (s, 6H, 2OCH3), 3.54–3.59 (m, 2H, 2CH-pyrazoline), 5.92–5.94 (m, 2H, 2CH-pyrazoline), 6.99–7.51 (m, 14H, ArH and thiophene-H), 7.90 (s, 4H, Ar-H); MS m/z (%): 840 (M+, 13), 631 (4), 348 (10), 220 (36), 185 (25), 109 (37), 78 (86), 51 (100); Anal. Calcd. For C42H36N10O2S4 (840.19): C, 59.98; H, 4.31; N, 16.65; Found C, 59.79; H, 4.26; N, 16.53%.

1,4-Bis(1-(5-((E)-(4-chlorophenyl)diazenyl)-4-methylthiazol-2-yl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-5-yl)benzene (3e). Red solid; yield (74%); m.p. 225–227 °C (from DMF); IR (KBr) νmax: 3052, 2948 (CH), 1602 (C=N) cm−1; 1H NMR (DMSO-d6) δ: 2.61 (s, 6H, 2CH3), 3.03–3.07 (m, 2H, 2CH-pyrazoline), 3.55–3.59 (m, 2H, 2CH-pyrazoline), 5.91–5.96 (m, 2H, 2CH-pyrazoline), 7.11–7.80 (m, 14H, ArH and thiophene-H), 7.98 (s, 4H, ArH); 13C NMR (DMSO-d6) δ: 8.4, 20.4 (CH3), 45.5 (CH2), 66.3 (CH), 115.5, 115.7, 125.2, 126.2, 126.3, 129.8, 131.9, 132.6, 135.2, 146.6, 155.7, 156.1 (ArC), 159.4, 162.6 (C=N); MS m/z (%): 850 (M++2, 2), 848 (M+, 7), 631 (13), 404 (22), 243 (15), 185 (67), 117 (26), 78 (80), 51 (100); Anal. Calcd. For C40H30Cl2N10S4 (848.09): C, 56.53; H, 3.56; N, 16.48; Found: C, 56.44; H, 3.51; N, 16.36%.

1,4-Bis(1-(5-((E)-(4-bromophenyl)diazenyl)-4-methylthiazol-2-yl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-5-yl)benzene (3f). Red solid; yield (72%); m.p. 203–205 °C (from DMF); IR (KBr) νmax: 3050, 2933 (CH), 1601 (C=N) cm−1; 1H NMR (DMSO-d6) δ: 2.60 (s, 6H, 2CH3), 3.04–3.07 (m, 2H, 2CH-pyrazoline), 3.54–3.57 (m, 2H, 2CH-pyrazoline), 5.91–5.95 (m, 2H, 2CH-pyrazoline),7.30–7.86 (m, 14H, ArH and thiophene-H), 7.98 (s, 4H, ArH); 13C NMR (DMSO-d6) δ: 8.3 (CH3), 45.2 (CH2), 66.3 (CH), 116.1, 123.4, 124.2, 129.1, 130.9, 131.9, 132.2, 133.0, 141.3, 142.5, 142.8, 146.8, (Ar-C), 151.2, 162.1 (C=N); MS m/z (%): 937 (M++2, 3), 935 (M+, 3), 657 (83), 592 (86), 490 (75), 414 (99), 185 (91), 78 (72), 51 (100); Anal. Calcd. For C40H30Br2N10S4 (935.99): C, 51.17; H, 3.22; N, 14.92; Found: C, 51.05; H, 3.13; N, 14.75%.

1,4-Bis(1-(5-((E)-(4-nitrophenyl)diazenyl)-4-methylthiazol-2-yl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-5-yl)benzene (3g). Red solid; yield (74%); m.p. 271–273 °C (from DMF); IR (KBr) νmax: 3056, 2033 (CH), 1602 (C=N) cm−1; 1H NMR (DMSO-d6) δ: 2.62 (s, 6H, 2CH3), 3.04–3.07 (m, 2H, 2CH-pyrazoline), 3.57–3.59 (m, 2H, 2CH-pyrazoline), 5.90–5.94 (m, 2H, 2CH-pyrazoline),7.08–7.79 (m, 14H, ArH and thiophene-H), 7.99 (s, 4H, ArH); 13C NMR (DMSO-d6) δ: 8.5 (CH3), 45.4 (CH2), 62.7 (CH), 114.8, 116.2, 123.5, 127.2, 128.6, 129.2, 139.4, 140.3, 141.0, 142.1, 142.8, 144.5, (ArC), 151.5, 162.1 (C=N); MS m/z (%): 870 (M+, 8), 657 (64), 530 (77), 442 (40), 334 (57), 185 (64), 78 (50), 51 (100); Anal. Calcd. For C40H30N12O4S4 (870.14): C, 55.16; H, 3.47; N, 19.30; Found: C, 55.07; H, 3.41; N, 19.21%.

1,4-Bis(1-(4-phenyl-5-((E)-phenyldiazenyl)thiazol-2-yl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-5-yl)benzene (3h). Orange solid; yield (77%); m.p. 260–262 °C (from DMF); IR (KBr) νmax: 3051, 2941 (CH), 1598 (C=N) cm−1; 1H NMR (DMSO-d6) δ: 3.09–3.10 (m, 2H, 2CH-pyrazoline), 3.57–3.59 (m, 2H, 2CH-pyrazoline), 5.93–5.96 (m, 2H, 2CH-pyrazoline), 7.47–7.94 (m, 26H, ArH and thiophene-H), 8.27 (s, 4H, ArH); MS m/z (%): 904 (M+, 3), 657 (64), 631 (7), 380 (12), 252 (37), 185 (42), 78 (77), 51 (100); Anal. Calcd. For C50H36N10S4 (904.20): C, 66.35; H, 4.01; N, 15.47; Found: C, 66.30; H, 4.13; N, 15.36%.

1,4-Bis(1-(5-((E)-phenyldiazenyl)-4-(thiophen-2-yl)thiazol-2-yl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-5-yl)benzene (3i). Orange solid; yield (76%); m.p. 248–250 °C (from DMF); IR (KBr) νmax: 3057, 2942 (CH), 1602 (C=N) cm−1; 1H NMR (DMSO-d6) δ: 3.05–3.08 (m, 2H, 2CH-pyrazoline), 3.55–3.58 (m, 2H, 2CH-pyrazoline), 5.92–5.95 (m, 2H, 2CH-pyrazoline), 7.05–7.94 (m, 22H, ArH and thiophene-H), 8.10 (s, 4H, ArH); 13C NMR (DMSO-d6) δ: 43.6 (CH2), 66.5 (CH), 119.0, 125.8, 126.0, 128.4, 128.5, 128.6, 128.9, 129.0, 129.4, 129.5, 130.8, 130.9, 131.1, 132.1, 132.3, 143.8 (ArC), 148.8, 162.8 (C=N); MS m/z (%): 916 (M+, 13), 522 (20), 409 (48), 285 (40), 111 (100), 78 (65), 51 (84); Anal. Calcd. For C46H32N10S6 (916.11): C, 60.24; H, 3.52; N, 15.27; Found: C, 60.15; H, 3.40; N, 15.19%.

3.2.2. Synthesis of 4-Methyl-5-((E)-aryldiazenyl)-2-(2-(1-(thiophen-2-yl)ethylidene)hydrazinyl) Thiazole Derivatives 5a,b,e,f

A mixture of thiosemicarbazone 4 (0.199 g, 1 mmol) and the appropriate hydrazonoyl halides 2 (1 mmol) in dioxane (20 mL) containing TEA (0.5 mL) was refluxed for 3–6 h and allowed to cool, and the solid formed was filtered off, washed with EtOH, dried, and recrystallized from DMF to give the corresponding arylazothiazoles 5a,b,e,f. The products together with their physical constants are listed below.

4-Methyl-5-((E)-phenyldiazenyl)-2-(2-(1-(thiophen-2-yl)ethylidene)hydrazinyl)thiazole (5a). Red solid; yield (72%); m.p. 183–185 °C (from DMF); IR (KBr) νmax: 3427 (NH), 3043 (=C–H), 2931 (–C–H), 1603 (C=N) cm−1; 1H NMR (DMSO-d6) δ: 2.50 (s, 3H, CH3), 2.58 (s, 3H, CH3), 6.99–7.75 (m, 8H, ArH and thiophene-H), 10.67 (s, 1H, D2O-exchangeable, NH); 13C NMR (75 MHz, DMSO-d6) δ: 8.5, 15.5 (CH3), 114.2, 122.1, 128.0, 128.3, 129.2, 130.2, 130.8, 137.9, 143.4 (ArC), 160.4, 170.6, 177.6 (C=N); MS m/z (%): 341 (M+, 44), 238 (71), 106 (53), 78 (82), 51 (100); Anal. Calcd. for C16H15N5S2 (341.08): C, 56.28; H, 4.43; N, 20.51; Found: C, 56.22; H, 4.36; N, 20.40%.

4-Methyl-2-(2-(1-(thiophen-2-yl)ethylidene)hydrazinyl)-5-((E)-p-tolyldiazenyl)thiazole (5b). Red solid; yield (74%); m.p. 169–170 °C (from DMF); IR (KBr) νmax: 3427 (NH), 3042 (=C–H), 2928 (–C–H), 1602 (C=N) cm−1; 1H NMR (DMSO-d6) δ: 2.25 (s, 3H, CH3), 2.50 (s, 3H, CH3), 2.56 (s, 3H, CH3), 7.12–7.73 (m, 7H, ArH and thiophene-H), 10.60 (s, 1H, D2O-exchangeable, NH); 13C NMR (75 MHz, DMSO-d6) δ: 8.5, 15.5, 20.3 (CH3), 114.2, 128.0, 129.6, 130.0, 130.7, 131.1, 137.3, 141.1, 142.7 (ArC), 160.1, 177.3, 170.8 (C=N); MS m/z (%): 355 (M+, 20), 238 (53), 185 (77), 78 (69), 51 (100); Anal. Calcd. for C17H17N5S2 (355.09): C, 57.44; H, 4.82; N, 19.70; Found: C, 57.36; H, 4.77; N, 19.63%.

5-((E)-(4-Chlorophenyl)diazenyl)-4-methyl-2-(2-(1-(thiophen-2-yl)ethylidene)hydrazinyl) thiazole (5e). Red solid; yield (76%); m.p. 216–218 °C (from DMF); IR (KBr) νmax: 3427 (NH), 3043 (=C–H), 2931 (–C–H), 1603 (C=N) cm−1; 1H NMR (DMSO-d6) δ: 2.51 (s, 3H, CH3), 2.57 (s, 3H, CH3), 7.17–7.74 (m, 7H, ArH and thiophene-H), 10.71 (s, 1H, D2O-exchangeable, NH); 13C NMR (75 MHz, DMSO-d6) δ: 8.5, 15.4 (CH3), 115.6, 125.6, 128.0, 128.3, 129.1, 129.3, 138.7, 142.4, 142.5 (ArC), 160.6, 170.4, 177.6 (C=N); MS m/z (%): 377 (M++2, 32), 375 (M+, 100), 222 (54), 186 (72), 78 (69), 51 (80); Anal. Calcd. for C16H14ClN5S2 (375.04): C, 51.12; H, 3.75; N, 18.63; Found: C, 51.03; H, 3.66; N, 18.51%.

5-((E)-(4-Bromophenyl)diazenyl)-4-methyl-2-(2-(1-(thiophen-2-yl)ethylidene)hydrazinyl) thiazole (5f). Red solid; yield (75%); m.p. 203–205 °C (from DMF); IR (KBr) νmax: 3441 (NH), 3043 (=C–H), 2937 (–C–H), 1601 (C=N) cm−1; 1H NMR (DMSO-d6) δ: 2.51 (s, 3H, CH3), 2.57 (s, 3H, CH3), 7.17–7.75 (m, 7H, ArH and thiophene-H), 10.72 (s, 1H, D2O-exchangeable, NH); 13C NMR (75 MHz, DMSO-d6) δ: 8.5, 15.5 (CH3), 116.1, 128.0, 130.3, 130.9, 131.9, 132.1, 138.8, 142.5, 142.8 (ArC), 160.6, 170.4, 177.6 (C=N); MS m/z (%): 421 (M+ + 2, 18), 419 (M+, 19), 302 (100), 185 (83), 78 (82), 51 (74); Anal. Calcd. for C16H14BrN5S2 (418.99): C, 45.72; H, 3.36; N, 16.66; Found: C, 45.55; H, 3.30; N, 16.42%.

3.2.3. 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.

3.2.4. Synthesis of 2,2′-(5,5′-(1,4-phenylene)bis(3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-3,1-diyl))bis(thiazol-4(5H)-one) (8)

A mixture of bis-pyrazolylcarbothioamide 1 (0.336 g, 1 mmol) and ethyl chloroacetate (7) (0.244 g, 2 mmol) in EtOH (20 mL) containing fused sodium acetate (1 g) was refluxed for 8 h and cooled to room temperature. The solid product was filtered off, washed with ethanol, and recrystallized from ethanol to afford the bis-thiazolone derivative 8 as yellow solid; yield (77%); m.p. 188–190 °C (from ethanol); IR (KBr) νmax: 3066, 2951 (CH), 1682 (C=O), 1600 (C=N) cm−1; 1H NMR (DMSO-d6) δ: 3.21–3.27 (m, 2H, 2CH-pyrazoline), 3.65–3.72 (m, 2H, 2CH-pyrazoline), 4.10 (s, 4H, 2CH2), 5.94–5.97 (m, 2H, 2CH-pyrazoline), 7.16–7.70 (m, 6H, thiophene-H), 7.97 (s, 4H, ArH); MS m/z (%): 576 (M+, 15), 445 (39), 283 (31), 185 (64), 78 (79), 51 (100); Anal. Calcd. for C26H20N6O2S4 (576.05): C, 54.15; H, 3.50; N, 14.57; Found: C, 54.15; H, 3.50; N, 14.57%.

3.2.5. Synthesis of (2Z,2′Z)-dimethyl 2,2′-(2,2′-(5,5′-(1,4-phenylene)bis(5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-3,1-diyl))bis(4-oxothiazole-2(4H)-yl-5(4H)-ylidene))diacetate (10)

A mixture of bis-thiazolone derivative 8 (0.580 g, 1 mmol), methyl glyoxalate (9) (0.176 g, 2 mmol), and glacial acetic acid (0.5 mL) in ethanol (20 mL) was refluxed for 6 h, left to cool, then poured gradually with stirring onto cold water. The solid formed was filtered off, washed with water, and crystallized from DMF to give compound 10 as canary yellow solid; yield (66%), m.p. 217–219 °C (from DMF); IR (KBr) νmax: 3061, 2936 (CH), 1742, 1682 (C=O), 1601 (C=N) cm−1; 1H NMR (DMSO-d6) δ: 3.54–3.57 (m, 2H, 2CH-pyrazoline), 3.71 (s, 6H, 2CH3), 3.84–3.87 (m, 2H, 2CH-pyrazoline), 5.97–6.01 (m, 2H, 2CH-pyrazoline), 6.68 (s, 2H, 2 =CHCOOCH3), 7.23–7.72 (m, 6H, thiophene-H), 7.99 (s, 4H, ArH); 13C NMR (DMSO-d6) δ: 44.0 (CH2), 52.4 (CH3), 63.9 (CH), 114.2, 116.3, 126.5, 128.4, 131.9, 133.5, 142.4, 146.5 (ArC), 158.0, 165.8, (C=N), 171.5, 177.1 (C=O); MS m/z (%): 716 (M+, 84), 631 (40), 314 (48), 185 (72), 116 (79), 51 (100). Anal. Calcd. For C32H24N6O6S4 (716.06): C, 53.62; H, 3.37; N, 11.72; Found: C, 53.53; H, 3.31; N, 11.59%.

3.2.6. 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.

3.3. 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% CO2 and were sub-cultured two to three times a week.

For antitumor assays, the n class="Disease">tumor cell lines were suspended in medium at concentration 5 × 104 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% CO2 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 (IC50), 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].

4. Conclusions

In our present work, a new series of 2-ethylidenehydrazono-5-arylazothiazoles and n class="Chemical">2-ethylidene-hydrazono-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 IC50 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.