Synthesis, Anticancer and Antioxidant Activity of Novel 2,4-Disubstituted Thiazoles

Abstract

A new series of carbazole based 2,4-disubstituted thiazole derivatives were synthesized. All the synthesized compounds were tested for their cytotoxicity against three different cancer cell lines A549, MCF-7, and HT29. Some of these compounds showed good cytotoxicity. These compounds were also evaluated for antioxidant activity. Compounds 3a, 3b, 3d-f and 3i showed higher antioxidant activity than standard BHT.

Introduction

Nitrogen and sulfur heterocyles have been under investi-gation for a long time because of their significant medicinal properties. Among the wide range of heterocycles explored in the recent years, thiazole derivatives have attracted medi-cinal chemists because of their varied biological activities.1-9 Thiazole ring is an interesting building block in a variety of natural products and many potent biologically active mole-cules such as vitamine B1, epothilones, nizatidine, ritonavir, sulfathiazole, abafungin and tiazofurin. Thiazole derivatives have been extensively studied and so far, a variety of bio-logical activities have been reported, for a large number of their derivatives, such as antihypertensive, anti-inflammatory, antischizophrenia, antibacterial, anti-HIV, hypnotics, anti-allergic, analgesic, fibrinogen receptor antagonist, bacterial DNA gyrase B inhibitor and antitumor activities.10-22 Also they have wide range of applications in organic functional materials such as fluorescent dyes23 and liquid crystals.24

On the other hand, carbazole and its derivatives have attracted considerable attention from both synthetic organic and medicinal chemists due to their potential biological activity covering a wide range of medicinal applications. Carbazoles belong to an unusual class of DNA binding agents. These molecules contain a planar chromophore, which is the characteristic of DNA intercalators.25 Carbazole derivatives exhibit diverse biological activities such as antimalarial, antimicrobial, anti-tuberculosis, anti-HIV, anti-inflammatory, antihistaminic, and antitumor activities.26-32

Keeping in view of the importance of thiazole and carba-zole derivatives and in continuation of our search on bio-logically active molecules,33-36 we herein report the syn-thesis, anticancer and antioxidant activity of a new series of carbazole based 2,4-disubstituted thiazole derivatives.

 

Experimental

All reagents were obtained from Aldrich Chemical Com-pany Company and used as supplied. The 6-bromo-9-ethyl-9H-carba-zole- 3-carbaldehyde, was prepared by the ethylation37 follow-ed by formylation38 and bromination of carbazole.39 Melting points were determined in open capillaries using Electro-thermal (IA 9100) digital melting point apparatus and are uncorrected. IR spectra were recorded on Bruker (Tensor 37) FT-IR spectrometer using KBr pellets. 1H, 13C NMR spectra were recorded on a Bruker AM500 FT-NMR spectrometer. Mass spectral data were obtained from the Korea Basic Science Institute (Daegu) on JEOL JMS-700 high resolution mass spectrometer.

2-((6-Bromo-9-ethyl-9H-carbazol-3-yl)methylene)hydra-zinecarbothioamide (2): A mixture of equimolar quantity of 6-bromo-9-ethyl-9H-carbazole-3-carbaldehyde (1) (25 mmol) and thiosemi-carbazide (25 mmol) in 70 mL of ethanol and catalytic amount of acetic acid was heated under reflux in an oil bath for 3-5 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the solid product was filtered and washed with water. The solid was then dried, and recrystallized from EtOH.

mp 214-215 °C; Yield 90%; IR (KBr): cm−1 3429, 3285, 3150, 2974, 1627, 1592, 1532, 1481, 1233, 1087, 800; 1H NMR (500 MHz, DMSO-d6) δ 1.28 (t, J = 6.8 Hz, 3H), 4.42 (q, J = 6.5 Hz, 2H), 7.57-7.58 (m, 2H), 7.63 (d, J = 8.5 Hz, 1H), 7.96 (d, J = 8.5 Hz, 1H), 8.00 (s, 1H), 8.22 (s, 2H), 8.42 (s, 1H), 8.65 (s, 1H), 11.44 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 13.7, 37.3, 109.6, 111.4, 111.5, 120.6, 121.4, 123.1, 124.2, 125.7, 125.9, 128.4, 138.7, 140.8, 143.2, 177.6.

General Procedure for the Synthesis of Compounds 3a-j. A mixture of equimolar quantity of thiosemicarbazone (2) (2 mmol) and α-bromoketone (2 mmol) in 10 mL of absolute ethanol was heated under reflux in an oil bath for 2-3 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the solution was cooled and the resulting solid was filtered and dried. Finally, the product was recrystallized from ethanol.

2-(2-((6-Bromo-9-ethyl-9H-carbazol-3-yl)methylene)-hydrazinyl)-4-phenylthiazole (3a): mp 250-251 °C; Yield 88%; IR (KBr): cm−1 3393, 3051, 2973, 1622, 1480, 1233, 802, 738; 1H NMR (500 MHz, DMSO-d6) δ 1.30 (t, J = 7.0 Hz, 3H), 4.45 (q, J = 6.8 Hz, 2H), 7.31-7.33 (m, 2H), 7.42 (t, J = 7.5 Hz, 2H), 7.58-7.63 (m, 2H), 7.70 (d, J = 9.0 Hz, 1H), 7.86 (d, J = 7.5 Hz, 2H), 7.91 (d, J = 8.5 Hz, 1H), 8.26 (s, 1H), 8.44 (s, 1H), 8.48 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 13.7, 37.4, 103.5, 110.1, 111.4, 111.5, 120.5, 121.3, 123.2, 124.0, 124.2, 125.6, 125.7, 127.7, 128.4, 128.6, 134.0, 138.7, 140.6, 143.5, 168.4. HRMS (EI) m/z: Calcd for C24H19BrN4S: 474.0514 (M+), Found: 474.0516.

2-(2-((6-Bromo-9-ethyl-9H-carbazol-3-yl)methylene)-hydrazinyl)-4-p-tolylthiazole (3b): mp 279-280 °C; Yield 90%; IR (KBr): cm−1 3416, 3053, 2975, 1620, 1483, 1235, 801; 1H NMR (500 MHz, DMSO-d6) δ 1.30 (t, J = 7.0 Hz, 3H), 2.32 (s, 3H), 4.45 (q, J = 6.8 Hz, 2H), 7.23 (d, J = 8.0 Hz, 2H), 7.26 (s, 1H), 7.58-7.63 (m, 2H), 7.70 (d, J = 9.0, 1H), 7.73 (d, J = 8.0 Hz, 2H), 7.91 (d, J = 8.5 Hz, 1H), 8.28 (s, 1H), 8.44 (s, 1H), 8.47 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 13.7, 20.8, 37.4, 102.7, 110.1, 111.4, 111.5, 120.6, 121.3, 123.2, 124.0, 124.3, 125.6, 128.4, 129.2, 137.2, 138.7, 140.6, 144.0, 148.6, 148.7, 168.3; HRMS (EI) m/z: Calcd for C25H21BrN4S: 488.0670 (M+), Found: 488.0669.

2-(2-((6-Bromo-9-ethyl-9H-carbazol-3-yl)methylene)-hydrazinyl)-4-(4-methoxyphenyl)thiazole (3c): mp 243-244 °C; Yield 90%; IR (KBr): cm−1 3400, 3062, 1621, 1510, 1255, 1020, 804; 1H NMR (500 MHz, DMSO-d6) δ 1.30 (t, J = 7.0 Hz, 3H), 3.79 (s, 3H), 4.45 (q, J = 7.0 Hz, 2H), 6.99 (d, J = 8.5 Hz, 2H), 7.17 (s, 1H), 7.58-7.63 (m, 2H), 7.70 (d, J = 8.5 Hz, 1H), 7.77 (d, J = 8.5 Hz, 2H), 7.92 (d, J = 8.5 Hz, 1H), 8.30 (s, 1H), 8.45 (s, 1H), 8.47 (d, J = 1.5 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ 13.7, 37.4, 55.2, 101.5, 110.1, 111.4, 111.5, 114.0, 120.7, 121.3, 123.2, 124.0, 124.3, 125.5, 126.1, 127.1, 128.4, 138.7, 140.7, 144.3, 159.1, 168.3; HRMS (EI) m/z: Calcd for C25H21BrN4OS: 504.0619 (M+), Found: 504.0623.

2-(2-((6-Bromo-9-ethyl-9H-carbazol-3-yl)methylene)-hydrazinyl)-4-(4-(trifluoromethoxy) phenyl)thiazole (3d): mp 267-268 °C; Yield 80%; IR (KBr): cm−1 3398, 3056, 2977, 1623, 1485, 1266, 1018, 803; 1H NMR (500 MHz, DMSO-d6) δ 1.29 (t, J = 7.3 Hz, 3H), 4.43 (q, J = 6.8 Hz, 2H), 7.40 (t, J = 4.0 Hz, 3H), 7.57-7.61 (m, 2H), 7.67 (d, J = 8.5 Hz, 1H), 7.90 (d, J = 8.5 Hz, 1H), 7.96 (d, J = 9.0 Hz, 2H), 8.26 (s, 1H), 8.42 (s, 1H), 8.46 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 13.7, 37.4, 104.5, 110.0, 111.4, 111.5, 119.1, 120.5, 121.2, 121.3, 123.2, 124.0, 124.2, 125.7, 127.4, 128.4, 133.4, 138.7, 140.6, 143.5, 147.6, 148.1, 168.6; HRMS (EI) m/z: Calcd for C25H18BrF3N4OS: 558.0337 (M+), Found: 558.0335.

2-(2-((6-Bromo-9-ethyl-9H-carbazol-3-yl)methylene)-hydrazinyl)-4-(4-chlorophenyl)thiazole (3e): mp 275-276 ℃; Yield 89%; IR (KBr): cm−1 3386, 3049, 2974, 1622, 1489, 1234, 1092, 797; 1H NMR (500 MHz, DMSO-d6) δ 1.28 (t, J = 7.0 Hz, 3H), 4.41 (q, J = 6.8 Hz, 2H), 7.36 (s, 1H), 7.46 (d, J = 8.5 Hz, 2H), 7.55-7.59 (m, 2H), 7.65 (d, J = 8.5 Hz, 1H), 7.85 (d, J = 8.5 Hz, 2H), 7.89 (d, J = 8.5 Hz, 1H), 8.28 (s, 1H), 8.41 (s, 1H), 8.44 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 13.7, 37.4, 104.3, 110.0, 111.5, 120.6, 121.3, 123.2, 124.0, 124.3, 125.7, 127.4, 128.5, 128.6, 132.2, 132.8, 138.7, 140.6, 143.9, 147.9, 168.5; HRMS (EI) m/z: Calcd for C24H18BrClN4S: 508.0124 (M+), Found: 508.0128.

2-(2-((6-Bromo-9-ethyl-9H-carbazol-3-yl)methylene)-hydrazinyl)-4-(4-bromophenyl)thiazole (3f): mp 288-289 °C; Yield 88%; IR (KBr): cm−1 3410, 3050, 2974, 1621, 1487, 1235, 1007, 751; 1H NMR (500 MHz, DMSO-d6) δ 1.30 (t, J = 7.0 Hz, 3H), 4.45 (q, J = 6.8 Hz, 2H), 7.40 (s, 1H), 7.58-7.63 (m, 4H), 7.69 (d, J = 8.5 Hz, 1H), 7.81 (d, J = 8.0 Hz, 2H), 7.90 (d, J = 8.5 Hz, 1H), 8.24 (s, 1H), 8.42 (s, 1H), 8.47 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 13.7, 37.3, 104.3, 110.0, 111.4, 111.5, 120.4, 120.6, 121.3, 123.2, 124.0, 124.2, 125.8, 127.6, 128.4, 131.5, 133.6, 138.7, 140.5, 143.2, 148.7, 168.5; HRMS (EI) m/z: Calcd for C24H18Br2N4S: 551.9619 (M+), Found: 551.9620.

2-(2-((6-Bromo-9-ethyl-9H-carbazol-3-yl)methylene)-hydrazinyl)-4-(4-nitrophenyl)thiazole (3g): mp 283-284 ℃; Yield 83%; IR (KBr): cm−1 3387, 3083, 2976, 1627, 1517, 1344, 1236, 853; 1H NMR (500 MHz, DMSO-d6) δ 1.30 (t, J = 7.0 Hz, 3H), 4.43 (q, J = 7.0 Hz, 2H), 7.57-7.62 (m, 2H), 7.67 (d, J = 8.5, 1H), 7.70 (s, 1H), 7.88 (d, J = 8.5, 1H), 8.10 (d, J = 8.5 Hz, 2H), 8.25 (t, J = 8.3 Hz, 3H), 8.41 (s, 1H), 8.46 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 13.7, 37.4, 108.2, 110.0, 111.4, 111.5, 120.4, 121.3, 123.2, 124.0, 124.2, 125.8, 126.3, 128.4, 138.7, 140.5, 140.6, 143.2, 146.2, 148.3, 168.8; HRMS (EI) m/z: Calcd for C24H18BrN5O2S: 519.0365 (M+), Found: 519.0364.

2-(2-((6-Bromo-9-ethyl-9H-carbazol-3-yl)methylene)-hydrazinyl)-4-(3-nitrophenyl)thiazole (3h): mp 281-282 °C; Yield 82%; IR (KBr): cm−1 3422, 3129, 2976, 1625, 1527, 1347, 1236, 805; 1H NMR (500 MHz, DMSO-d6) δ 1.30 (t, J = 6.8 Hz, 3H), 4.44 (q, J = 7.0 Hz, 2H), 7.57-7.62 (m, 2H), 7.64 (s, 1H), 7.67-7.71 (m, 2H), 7.89 (d, J = 8.5, 1H), 8.13 (d, J = 7.5 Hz, 1H), 8.22 (s, 1H), 8.30 (d, J = 7.5 Hz, 1H), 8.42 (s, 1H), 8.46 (s, 1H), 8.67 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 13.7, 37.3, 106.0, 110.0, 111.4, 111.5, 119.9, 120.4, 121.3, 121.9, 123.2, 124.0, 124.2, 125.8, 128.4, 130.2, 131.5, 136.1, 138.7, 140.5, 143.0, 147.9, 148.2, 168.7; HRMS (EI) m/z: Calcd for C24H18BrN5O2S: 519.0365 (M+), Found: 519.0367.

4-(2-(2-((6-Bromo-9-ethyl-9H-carbazol-3-yl)methylene)-hydrazinyl)thiazol-4-yl)benzonitrile (3i): mp 276-277 °C; Yield 83%; IR (KBr): cm−1 3402, 3055, 2974, 2227, 1626, 1484, 1235, 805; 1H NMR (500 MHz, DMSO-d6) δ 1.29 (t, J = 7.0 Hz, 3H), 4.42 (q, J = 6.8 Hz, 2H), 7.55-7.60 (m, 2H), 7.61 (s, 1H), 7.66 (d, J = 9.0 Hz, 1H), 7.85 (d, J = 8.0 Hz, 2H), 7.88 (d, J = 8.5 Hz, 1H), 8.02 (d, J = 8.5 Hz, 2H), 8.24 (s, 1H), 8.40 (s, 1H), 8.44 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 13.7, 37.4, 107.2, 109.6, 110.0, 111.4, 111.5, 119.0, 120.4, 121.3, 123.2, 124.0, 124.2, 125.7, 126.1, 128.4, 132.6, 138.6, 138.7, 140.5, 143.3, 148.4, 168.7; HRMS (EI) m/z: Calcd for C25H18BrN5S: 499.0466 (M+), Found: 499.0470.

2-(2-((6-Bromo-9-ethyl-9H-carbazol-3-yl)methylene)-hydrazinyl)-4-(naphthalen-1-yl) thiazole (3j): mp 255-256 ℃; Yield 88%; IR (KBr): cm−1 3399, 3051, 2973, 1620, 1480, 1234, 807; 1H NMR (500 MHz, DMSO-d6) δ 1.26 (t, J = 7.0 Hz, 3H), 4.39 (q, J = 6.7 Hz, 2H), 7.48-7.54 (m, 3H), 7.56 (s, 2H), 7.65 (d, J = 8.5 Hz, 1H), 7.89-7.98 (m, 5H), 8.34 (s, 1H), 8.37 (s, 1H), 8.44 (d, J = 5.5 Hz, 2H); 13C NMR (125 MHz, DMSO-d6) δ 13.7, 37.4, 104.4, 110.0, 111.5, 120.7, 121.3, 123.2, 123.9, 124.0, 124.3, 124.4, 125.5, 126.2, 126.6, 127.6, 128.2, 128.5, 130.8, 132.5, 133.0, 138.7, 140.7, 144.5, 148.2, 168.5; HRMS (EI) m/z: Calcd for C28H21BrN4S: 524.0670 (M+), Found: 524.0672.

Biological Assay.

Cell Lines and Culture: A549 (human lung cancer cell line), MCF-7 (human breast cancer cell line) and HT-29 (human colon cancer cell line) cells were obtained from American Type Culture Collection (ATCC, USA). The cells were cultured in standard growth medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin/strepto-mycin and incubated at 37 °C in a 5% CO2 atmosphere.

Cytotoxicity Assay: Cytotoxicity of the compounds was assessed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) staining method. Briefly, cells were seeded in 96-well plate (Falcon, USA) at a density of 2 × 104 cells/well. Next day, cells were incubated with different concentrations of each compound for 24 h. Then, 10 μL of MTT solution was added to the well and further incubated for 4 h at 37 °C. At the end of the incubation, the media were removed, and 200 μL of dimethyl sulfoxide (DMSO) was added into each well to solubilize the form-azan crystals. Finally, absorbance was measured at 540 nm using a microplate reader (Molecular Devices, Versa MAX Sunnyvale, CA, USA).

Antioxidant Activity: 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity of the samples was assessed by reported method40 with minor modifications. Briefly, 0.1 mM ethanolic DPPH solution (100 μL) was added to a sample solution (100 μL) of various concen-trations (500 μM - 62.5 μM). After 30 min of incubation in dark at room temperature, the absorbance was measured at 517 nm. All measurements were made in triplicate. A lower absorbance of the compound mixtures indicates the higher DPPH radical scavenging activity. The percentage of inhibition was calculated as follows:

Radical scavenging activity (%) = [(Acontrol − Asample)/Acontrol] × 100

Where Acontrol is the absorbance of negative control (contain-ing all reagents except test compounds) and Asample is the absorbance of the test compounds and all the reagents. IC50 (50% inhibitory concentration of compound) was calculated from the curve drawn by plotting the inhibition percentage against sample concentration. The antioxidant activity of the synthesized compounds was compared with a synthetic antioxidant Butylhydroxytoluene (BHT) as the reference standard.

 

Results and Discussion

A new series of carbazole based 2,4-disubstituted thiazole derivatives were synthesized in a two step synthetic process (Scheme 1). In the first step 2-((6-bromo-9-ethyl-9H-carbazol-3-yl)methylene)hydrazinecarbothioamide (2) was synthesized by the condensation of 6-bromo-9-ethyl-9H-carbazole-3-carbaldehyde (1) with thiosemicarbazide in ethanol in the presence of catalytic amount of acetic acid. In the second step, the compound 2 was reacted with varies α-bromoketones (a-j) in refuxing ethanol to yield the corre-sponding 2,4-disubstituted thiazoles (3a-j, Table 1). The chemical structures of all the synthesized compounds were characterized by IR, 1H, 13C NMR and Mass spectrometry.

FT-IR spectra of compounds 3a-j showed the expected absorption bands at 3422-3386 and 1627-1620 cm−1 for NH and C=N (azomethine) groups respectively. The absence of the absorption band corresponding to carbonyl stretching frequency of the α-bromoketones clearly confirmed the formation of thiazole ring. In the 1H NMR spectra of compounds 3a-j the azomethine proton (HC=N) resonated as a singlet in the region of 8.34-8.22 ppm. The C-5 proton of the thiazole ring showed a singlet in the region of 7.70-7.17 ppm. The remaining proton signals are observed in the expected regions. 13C NMR spectra of compounds 3a-j showed signals in the range of 168.8-168.3 and 108.2-101.5 ppm corresponding to carbon atom of thiazole-C-2 and C-5 respectively. The high resolution mass spectral data of compounds 3a-j are provided in the experimental section.

Cytotoxic Activity. All the synthesized molecules 3a-j were evaluated for their cytotoxicity against three different cancer cell lines A549 (human lung cancer), MCF-7 (human breast cancer) and HT29 (human colon cancer) by MTT assay. The IC50 values of compounds are listed in Table 2. As shown in Table 2, compounds 3b (IC50 22.8 μM), 3e (IC50 23.9 μM), 3f (IC50 31.6 μM), 3h (IC50 9.7 μM) and 3i (IC50 8.5 μM) were found to exhibit good cytotoxic activity against A549 cells. It was observed that, compounds 3f (IC50 19.1 μM), 3g (IC50 44.7 μM), and 3i (IC50 24.5 μM) dis-played good activity on MCF-7 cells. In the case of HT29 cells, compounds 3b (IC50 7.8 μM) and 3e (IC50 23.9 μM) showed good cytotoxicity and other compounds showed negligible activity.

Scheme 1.Synthesis of carbazole based 2,4-disubstituted thiazoles.

Antioxidant Activity. Compounds 3a-j were tested for antioxidant activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method at different concentrations 500 μM-62.5 μM. The results on the antioxidant activity are given in Table 3. The radical scavenging activity of compounds 3a-j was compared with that of reference compound Butylhydroxytoluene (BHT). Compounds 3a (IC50 110.74 μM), 3b (IC50 86.33μM), 3d (IC50 139.18 μM), 3e (IC50 140.67), 3f (IC50 171.70 μM) and 3i (IC50 158.54 μM) showed higher activity than BHT (IC50 207.45 μM). Particularly compound 3b (IC50 86.33 μM) displayed the highest activity of all the synthesized compounds (Fig. 1).

Table 1.Synthesis of carbazole based 2,4-disubstituted thiazole derivatives

 

Conclusion

In conclusion, we have synthesized a new class of carba-zole based 2,4-disubstituted thiazoles and their cytotoxicity was evaluated. Among all the thiazole derivatives, compounds 3h and 3i against A549 cells, compound 3f against MCF-7 cells and compound 3b against HT29 cells, showed good cytotoxicity. These compounds were also tested for anti-oxidant activity by DPPH method and compounds 3a, 3b, 3d-f and 3i showed higher activity than BHT.

Table 2.Cytotoxic effects of compounds 3a-j on three cancer cell lines

Table 3.Antioxidant activity of compounds 3a-j

Figure 1.Antioxidant activity (IC50) of compound 3a-j.