Introduction

Identification of novel structure leads that may be of use in designing new, potent, selective and less toxic anticancer agents remains a major challenge for medicinal chemistry researchers. Compounds containing thiazole core have diverse biological activities as antihypertension, antifungal, antimicrobial, anti-inflammatory, antioxidant, antitubercular [1,2,3,4,5,6,7], and anticancer [8,9,10,11,12]. Also, thiazole ring present in many drugs such as Nizatidine, Abafungin, and Amiphenazole (Fig. 1).

Fig. 1
figure 1

Some marketed drugs containing thiazole ring

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Many biological activities were reported for the compounds containing 1,3,4-thiadiazole ring such as antituberculosis, anti-inflammatory, antidepressant and anxiolytic, antioxidant, anticonvulsants [13,14,15,16,17] and anticancer activities [18,19,20]. In addition, many drugs containing 1,3,4-thiadiazole ring are available in the market such as acetazolamide, methazolamide, and megazol (Fig. 2).

Fig. 2
figure 2

Examples of drugs containing a 1,3,4-thiadiazole ring

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In continuation of our studies dealing with the utility of hydrazonoyl halides for synthesis of various bioactive bridgehead nitrogen polyheterocycles [21,22,23,24,25,26,27,28,29,30], we wish to report herein a new facile synthesis of new heterocycles containing thiazole and 1,3,4-thiadiazole or two thiazole rings in one molecular frame. We anticipated that the synthesized compounds would have potent pharmacological activities.

Results and discussion

Chemistry

2-(4-Methyl-2-phenylthiazole-5-carbonyl)-N-phenylhydrazinecarbothioamide (3) [31] was prepared via reaction of 4-methyl-2-phenylthiazole-5-carbohydrazide (2) with phenyl isothiocyanate in EtOH (Scheme 1).

Scheme 1
scheme 1

Synthesis of thiazoles 2,3

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The reaction of compound 2 with the appropriate hydrazonoyl chlorides 4a–c [32,33,] in refluxing ethanol yielded the corresponding condensation product 5 (Scheme 2). The IR spectra of the latter products revealed a carbonyl and two NH absorption bands (see “Experimental” part). Their 1HNMR showed two D2O exchangeable signals of two NH protons in the regions δ 10.03–10.06 and δ 10.57–10.59 ppm. Also, their mass spectra confirmed the assigned structure 5 (Scheme 2). Treatment of thioamide derivative 3 with the appropriate hydrazonoyl halides of type 5a–c in refluxing EtOH containing TEA gave the corresponding thiadiazole derivatives 7a–c (Scheme 2). Their structures were elucidated on the basis of their spectral data and elemental analysis (see “Experimental”).

Scheme 2
scheme 2

Synthesis of thiadiazole derivatives 7a–c

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Next, refluxing of compound 3 with 3-chloropentane-2,4-dione (8) or 2-chloro-3-oxo-N-phenyl butanamide (10) in EtOH in the presence of triethylamine afforded the thiazole derivatives 9 and 11, respectively (Scheme 3).The structure of compounds 9 and 11 were elucidated based on their elemental analysis and spectral data (see “Experimental”).

Scheme 3
scheme 3

Synthesis of thiazole derivatives 9, 11, 13 and 15

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In a similar manner, thioamide 3 reacted with phenacyl bromide 12 under the same experimental condition to afford one isolable product 13 named as N′-(3,4-diphenylthiazol- 2(3H)-ylidene)-4-methyl-2-phenyl thiazole-5-carbohydrazide (Scheme 3). The structure of thiazole 13 was established based on its elemental analysis and spectral data (see “Experimental”).

Finally, thioamide derivative 3 reacted with ethyl chloroacetate (14) to afford thiazole 15 as showed in Scheme 3. Its IR spectrum showed absorption bands at v 3331 (NH), and 1726, 1648 (2C=O) cm−1. In addition, its 1HNMR spectrum showed singlet signal at δ 4.23 ppm due to the thiazolidinone (CH2) group.

Anticancer activity

The synthesized compounds were tested as anticancer agents against human Hepatocellular carcinoma cell line (HepG-2) using colorimetric MTT assay. We also included the well-known anticancer standard drug (Cisplatin) in the same assay to compare the potency of the synthesized compounds. The IC50 (the concentration of test compounds required to kill 50% of cell population) was determined (Table 1, Fig. 3).

Table 1 The in vitro inhibitory activity of the tested compounds against tumor cell lines expressed as IC50 values (μg/mL) ± standard deviation from three replicates
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Fig. 3
figure 3

Comparison of the IC50 of the new synthesized compounds against Cisplatin

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The results of Table 1 revealed that the ascending order of the cytotoxic activity of the newly synthesized compounds towards the human Hepatocellular carcinoma cell line (HepG-2) were as follow: 5c < 13 < 5a < 5b < 9 < 7c < 15 < 7a < 11 < 7b (Fig. 4).

Fig. 4
figure 4

The ascending order of the cytotoxic activity

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From the data of Table 1, we concluded the following structure–activity relationships (SARs):

  • The thiazole ring is essential for the activity.

  • Less number of thiazole ring as in compounds 5a–c lead to drastic drop in activity.

  • 1,3,4-Thiadiazole ring is crucial for the cytotoxic activity.

  • Presence of methyl group (electron donating group) at position 4 of the phenyl ring in compound 7b increase its activity more than compound 7a.

  • The presence of the N-phenylcarboxamide group in compound 11 leads to increasing of its cytotoxic activity.

Experimental

Chemistry

General

Melting points were measured on an Electrothermal IA 9000 series digital melting point apparatus (Bibby Sci. Lim. Stone, Staffordshire, UK). IR spectra were measured on PyeUnicamSP 3300 and Shimadzu FTIR 8101 PC infrared spectrophotometers (Shimadzu, Tokyo, Japan) in potassium bromide discs. NMR spectra were measured on a Varian Mercury VX-300 NMR spectrometer (Varian, Inc., Karlsruhe, Germany) operating at 300 MHz (1HNMR) and run in deuterated dimethylsulfoxide (DMSO-d 6 ). Chemical shifts were related to that of the solvent. Mass spectra were recorded on a Shimadzu GCMS-QP1000 EX mass spectrometer (Tokyo, Japan) at 70 eV. Elemental analyses were measured by using a German made Elementarvario LIII CHNS analyzer. 2-(4-Methyl-2-phenylthiazole-5-carbonyl)-N-phenylhydrazinecarbothioamide (3) [31], and hydrazonoyl halides 4a–c [32] were prepared as reported in the respective literature.

Synthetic procedures

Synthesis of hydrazonoyl chlorides 5a–c

A mixture of 4-methyl-2-phenylthiazole-5-carbohydrazide (2) (2.33 g, 10 mmol) and the appropriate hydrazonoyl chlorides 4a–c (10 mmol) in ethanol (30 mL) was refluxed for 3–5 h (monitored through TLC).The resulting solid product was collected and recrystallized from the proper solvent to give the corresponding products 5a–c.

2-(2-(4-Methyl-2-phenylthiazole-5-carbonyl)hydrazono)-N′-phenylpropane hydrazonoyl chloride (5a)

Yellow solid; yield (84%); m.p. 188–190 °C (EtOH); IR (KBr) v 3440, 3316 (2NH), 3036, 2922 (CH), 1640 (C=O), 1599 (C=N) cm−1; 1H NMR (DMSO-d 6 ) δ 2.36 (s, 3H, CH3), 2.76 (s, 3H, CH3), 7.06–7.86 (m, 10H, ArH), 10.03 (s, br, 1H, D2O-exchangeable NH), 10.57 (s, br, 1H, D2O-exchangeable NH); MS m/z (%): 413 (M++2, 12), 411 (M+, 40), 375 (48), 202 (100), 174 (45), 71 (26). Anal. Calcd for C20H18ClN5OS (411.91): C, 58.32; H, 4.40; N, 17.00. Found: C, 58.19; H, 4.37; N, 16.88%.

2-(2-(4-Methyl-2-phenylthiazole-5-carbonyl)hydrazono)-N′-(p-tolyl)propane- hydrazonoylchloride (5b)

Yellow solid; yield (86%); m.p. 172–174 °C (EtOH); IR (KBr) v 3437, 3313 (2NH), 3041, 2917 (CH), 1679 (C=O), 1598 (C=N) cm−1; 1H NMR (DMSO-d 6 ) δ 2.24 (s, 3H, CH3), 2.34 (s, 3H, CH3), 2.77 (s, 3H, CH3), 7.08–7.99 (m, 9H, ArH), 10.06 (s, br, 1H, D2O-exchangeable NH), 10.59 (s, br, 1H, D2O-exchangeable NH); MS m/z (%) 427 (M++2, 10), 425 (M+, 33), 389 (26), 202 (81), 106 (100), 64 (66). Anal. Calcd for C21H20ClN5OS (425.93): C, 59.22; H, 4.73; N, 16.44. Found: C, 59.18; H, 4.65; N, 16.37%.

N′-(4-Chlorophenyl)-2-(2-(4-methyl-2-phenylthiazole-5-carbonyl)hydrazono) propane hydrazonoyl chloride (5c)

Yellow solid; yield (87%); m.p. 194–196 °C (DMF); IR (KBr) v 3434, 3319 (2NH), 3044, 2926 (CH), 1682 (C=O), 1593 (C=N) cm−1; 1H NMR (DMSO-d 6 ) δ 2.37 (s, 3H, CH3), 2.77 (s, 3H, CH3), 7.08–7.99 (m, 9H, Ar–H), 10.06 (s, br, 1H, D2O-exchangeable NH), 10.57 (s, br, 1H, D2O-exchangeable NH); MS m/z (%) 446 (M+, 8), 283 (14), 202 (39), 104 (46), 80 (100), 64 (90). Anal. Calcd for C20H17Cl2N5OS (446.35): C, 53.82; H, 3.84; N, 15.69. Found: C, 53.75; H, 3.79; N, 15.58%.

Synthesis of 1,3,4-thiadiazole derivatives 7a–c

A mixture of compound 3 (0.368 g, 1 mmol) and the appropriate hydrazonoyl chlorides 5a–c (1 mmol) in ethanol (20 mL) containing triethylamine (0.1 g, 1 mmol) was refluxed for 6 h. The formed solid product was filtered, washed with methanol, dried and recrystallized from the suitable solvents to give corresponding products 7a–c.

4-Methyl-N′-(1-(-5-(2-(4-methyl-2-phenylthiazole-5-carbonyl)hydrazono)-4-phenyl-4,5-dihydro-1,3,4-thiadiazol-2-yl)ethylidene)-2-phenylthiazole-5-carbohydrazide(7a)

Yellow solid; yield (74%); m.p. 162–164 °C (EtOH); IR (KBr) v 3421, 3307 (2NH), 3031, 2951 (CH), 1649 (C=O), 1596 (C=N) cm−1; 1H NMR (DMSO-d 6 ) δ 2.34 (s, 3H, CH3), 2.66 (s, 3H, CH3), 2.76(s, 3H, CH3), 6.97-8.14 (m, 15H, ArH), 10.18 (s, br, 1H, D2O-exchangeable NH), 11.17 (s, br, 1H, D2O-exchangeable NH); MS m/z (%) 650 (M+, 34), 526 (30), 416 (60), 358 (28), 104 (55), 64 (100). Anal. Calcd for C32H26N8O2S3 (650.80): C, 59.06; H, 4.03; N, 17.22. Found C, 58.94; H, 4.01; N, 17.07%.

4-Methyl-N′-(1-(5-(2-(4-methyl-2-phenylthiazole-5-carbonyl)hydrazono)-4-(p-tolyl)-4,5-dihydro-1,3,4-thiadiazol-2-yl)ethylidene)-2-phenylthiazole-5-carbohydrazide (7b)

Yellow solid; yield (72%); m.p. 149–151 °C (EtOH); IR (KBr) v 3422, 3328 (2NH), 3053, 2929 (CH), 1647 (C=O), 1597 (C=N) cm−1; 1H NMR (DMSO-d 6 ) δ 2.26 (s, 3H, CH3),2.35 (s, 3H, CH3), 2.65 (s, 3H, CH3), 2.76(s, 3H, CH3), 6.91–8.03 (m, 14H, ArH), 10.18 (s, br, 1H, D2O-exchangeable NH), 11.14 (s, br, 1H, D2O-exchangeable NH); MS m/z (%) 664 (M+, 35), 553 (60), 334 (19), 202 (65), 104 (85), 64 (100). Anal. Calcd for C33H28N8O2S3 (664.82): C, 59.62; H, 4.25; N, 16.85. Found C, 59.47; H, 4.17; N, 16.79%.

N′-(3-(4-Chlorophenyl)-5-(1-(2-(4-methyl-2-phenylthiazole-5-carbonyl)hydrazono)eth-yl)-1,3,4-thiadiazol-2(3H)-ylidene)-4-methyl-2-phenylthiazole-5-carbohydrazide (7c)

Yellow solid; yield (76%); m.p. 191–193 °C (Dioxane); IR (KBr) v 3424, 3312 (2NH), 3047, 2932 (CH), 1649 (C=O), 1599 (C=N) cm−1; 1H NMR (DMSO-d 6 ) δ 2.33 (s, 3H, CH3), 2.66 (s, 3H, CH3), 2.77(s, 3H, CH3), 6.90–8.11 (m, 14H, ArH), 10.13 (s, br, 1H, D2O-exchangeable NH), 11.19 (s, br, 1H, D2O-exchangeable NH); MS m/z (%) 686 (M++2, 8), 684 (M+, 26), 513 (53), 368 (39), 257 (17), 104 (25), 64 (100). Anal.Calcd for C32H25ClN8O2S3 (685.24): C, 56.09; H, 3.68; N, 16.35. Found C, 56.02; H, 3.58; N, 16.22%.

General procedure for the synthesis of thiazole derivatives 9, 11, 13, and 15

A mixture of compound 3 (0.368 g, 1 mmol) and the appropriate α-halo-compounds namely, 3-chloropentane-2,4-dione (8), 2-chloro-3-oxo-N-phenylbutanamide (10), 2-bromo-1-phenyl ethanone (12) and ethyl 2-chloroacetate (14) (1 mmol for each) in ethanol (20 mL) containing triethylamine (0.1 g, 1 mmol) was refluxed for 4–6 h. (monitored by TLC The solid product was filtered, washed with water, dried and recrystallized from the proper solvent to give the corresponding thiazole derivatives 9, 11, 13, and 15, respectively.

N′-(5-Acetyl-4-methyl-3-phenylthiazol-2(3H)-ylidene)-4-methyl-2-phenylthiazole-5-carbohydrazide (9)

Yellow solid; yield (78%); m.p. 155–157 °C (EtOH); IR (KBr) v 3432 (NH), 3036, 2993 (CH), 1695, 1648 (2C=O), 1590 (C=N) cm−1; 1H NMR(DMSO-d 6 ) δ 2.32 (s, 3H, CH3),2.46 (s, 3H, CH3), 2.77 (s, 3H, CH3), 6.91–7.86 (m, 10H, ArH), 10.61 (s, br, 1H, D2O-exchangeable NH); MS m/z (%) 448 (M+, 57), 246 (60), 176 (35), 104 (80), 77 (100). Anal.Calcd for C23H20N4O2S2 (448.56): C, 61.59; H, 4.49; N, 12.49. Found C, 61.48; H, 4.36; N, 12.37%.

4-Methyl-2-(2-(4-methyl-2-phenylthiazole-5-carbonyl)hydrazono)-N-3-diphenyl-2,3-dihydrothiazole-5-carboxamide (11)

Yellow solid; yield (79%); m.p. 182–84 °C (DMF); IR (KBr): v 3435, 3176 (2NH), 3030, 2928(CH), 1671, 1649 (2C=O), 1594 (C=N) cm−1; 1H NMR (DMSO-d 6 ) δ 2.36 (s, 3H, CH3),2.76(s, 3H, CH3), 6.97–7.73 (m, 15H, ArH), 10.46 (s, br, 1H, D2O-exchangeable NH), 11.72 (s, br, 1H, D2O-exchangeable NH); MS m/z (%) 525 (M+, 7), 447 (16), 334 (100), 200 (59), 77 (89). Anal.Calcd for C28H23N5O2S2 (525.64): C, 63.98; H, 4.41; N, 13.32. Found C, 63.84; H, 4.30; N, 13.28%.

N′-(3,4-Diphenylthiazol-2(3H)-ylidene)-4-methyl-2-phenylthiazole-5-carbohydrazide (13)

Yellow solid; yield (70%); m.p. 174–178 °C (EtOH); IR (KBr) v 3369 (NH), 3047, 2926(CH), 1648 (C=O), 1594 (C=N) cm−1; 1H NMR (DMSO-d 6 ) δ 2.75 (s, 3H, CH3), 7.03 (s, 1H, thiazole-H5), 7.35–8.02 (m, 15H, ArH), 10.73 (s, br, 1H, D2O-exchangeable NH); MS m/z (%) 468 (M+, 25), 334 (100), 200 (40), 104 (69), 64(65). Anal.Calcd for C26H20N4OS2 (468.59): C, 66.64; H, 4.30; N, 11.96. Found C, 66.55; H, 4.21; N, 11.79%.

4-Methyl-N′-(4-oxo-3-phenylthiazolidin-2-ylidene)-2-phenylthiazole-5-carbo- hydrazide (15)

Yellowish-white solid; yield (72%); m.p. 192–194 °C (Dioxane); IR (KBr) v 3331(NH), 3036, 2926 (CH), 1726, 1648 (2C=O), 1596 (C=N) cm−1; 1H NMR (DMSO-d 6 ) δ 2.65 (s, 3H, CH3), 4.23 (s, 2H, thiazolone-CH2), 7.40–7.88 (m, 10H, ArH), 10.82 (s, br, 1H, D2O-exchangeable NH); MS m/z (%) 408 (M+, 65), 334 (18), 202 (100), 104 (86), 64 (69). Anal.Calcd for C20H16N4O2S2 (408.50): C, 58.80; H, 3.95; N, 13.72. Found C, 58.68; H, 3.84; N, 13.64%.

Anticancer activity

The cytotoxic evaluation of the synthesized compounds was carried out at the Regional Center for Mycology and Biotechnology at Al-Azhar University, Cairo, Egypt according to the reported method [33].

Conclusions

We successfully synthesized a series of novel heterocycles containing thiazole and 1,3,4-thiadiazole rings by a facile and convenient method. The structure of the newly prepared compounds was established based on both elemental analysis and spectroscopic data. The anticancer activity of the synthesized compounds was measured and showed promising activity.