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Synthesis, characterization and anti cancer activity of some fluorinated 3,6-diaryl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles
⁎Corresponding author. Tel.: +91 755 2678883; fax: +91 755 2742001. piyush.trivedi@rgtu.net (Piyush Trivedi)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
A series of fluorinated 3,6-diaryl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles (2a–2i) was synthesized by condensation of various substituted 4-amino-5-phenyl-4H-1,2,4-triazole-3-thiols (1a–1i) with penta fluoro benzoic acid in good yields (60–80%). The synthesized compounds were screened for anticancer activity against three cancerous cell lines MCF7 (human breast cancer), SaOS-2 (human osteosarcoma) and K562 (human myeloid leukemia). The compounds showed moderate to good antiproliferative potency against the studied cell lines. Among these, compound 2b showed higher antiproliferative activity (IC50 22.1, 19 and 15 μM against MCF7, SaOS-2 and K562, respectively) while 2a exhibited least antiproliferative activity (IC50 30.2, 39 and 29.4 μM against MCF7, SaOS-2 and K562 cells, respectively). Therefore, the present study demonstrates that fluorine substituted 3,6-diaryl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles would be a better prospective in the development of anticancer drugs.
Keywords
Cancer
Triazolothiadiazoles
Fluoro compounds
Antiproliferative agents
MTT assay
1 Introduction
Cancer remains to be the leading cause of death in humans second only to cardiovascular diseases and more than 70% of all cancer deaths occur in developing and under-developed countries (Fadeyi et al., 2008; Sherif and Rostom, 2006; Moorthy et al., 2009). There is a continuous rise of deaths from various cancers worldwide, with an estimated 12 million deaths in 2030 (WHO website). Despite the advancement in the knowledge of biochemical processes associated with carcinogenesis, the successful treatment of cancer remains a significant challenge because of the general toxicity associated with the clinical use of traditional cancer chemotherapeutic agents. Hence, the design and development of new drugs for cancer therapeutics remains to be an important and challenging task for medicinal chemists worldwide.
The substituted-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole ring represents an interesting class of hetero compounds with a broad spectrum of pharmacological activities which include antifungal (Karabasanagouda et al., 2007), anti-inflammatory (Chawla et al., 2012), antiviral (Kritsanida et al., 2002), analgesic (Chawla et al., 2012), anthelmintic (el-Khawass et al., 1989), antibacterial (Holla et al., 1996) and antitumor agents (Ibrahim, 2009). Of particular interest is the impressive anticancer activity exhibited by this class of compounds. As a result, the literature is abundant with reports on the cytotoxic potency of 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazoles against various cancer cell lines (Holla et al., 2002; Bhat et al., 2009; Zhao et al., 2012; Rashid et al., 2013; Sunil et al., 2010).
Fluorine-containing compounds have attracted much interest since the introduction of fluorine atoms or fluoroalkyl moieties to an organic compound can bring about remarkable changes in the physical, chemical, and biological properties (Wang et al., 2010). Fluoro compounds have also been traditionally associated with potent antitumor properties. Other than the well established fluoronucleosides such as 5-fluoro uracil, the fluorine containing anticancer molecules include flutamide, an anti-androgen which was launched in 1983 for the treatment of prostate cancer and fluorinated anthracycline antibiotics, steroids, Vitamin D3 analogs and fluorine containing taxoids have been shown to be much more effective than their parent analogs (Filler and Saha, 2009).
In view of the above mentioned findings and in continuation of our interest in exploration of novel heterocyclic scaffolds for anticancer activity (Moorthy et al., 2009, 2010; Patel et al., 2011), we report herein the synthesis and evaluation of fluorinated diaryl-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazoles as potential anticancer agents. Our design strategy was to fix up a penta fluoro substitution in aryl ring (B) attached to the thiadiazole scaffold and vary the substitution pattern in the second aryl ring (A) attached to the triazole moiety as shown in Fig. 1.![Design strategy of 6-(perfluorophenyl)-3-phenyl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles.](/content/184/2017/10/2_suppl/img/10.1016_j.arabjc.2013.08.026-fig1.png)
2 Materials and methods
2.1 Measurements
All commercial chemicals and solvents used are of reagent grade and were used without further treatment unless otherwise noted. 1H NMR spectra were recorded with a Bruker Avance II 300 NMR spectrometer. Chemical shifts were recorded in parts per million (ppm) and were reported relative to the TMS. Mass spectral data were recorded on an Applied Biosystem Qtrap 3200 LC-MS/MS system in ESI mode. The FT-IR spectra of the synthesized compounds were recorded on Schimadzu IRPrestige-21 in KBr. Melting points of the compounds were determined using a Veego digital melting point apparatus and are reported uncorrected. The purity of the compounds was confirmed by thin layer chromatography using Merck silica gel 60 F254 coated alumina plates.
2.2 Synthesis of 3,6-diphenyl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives (2a–2i)
An equimolar mixture (0.10 M) of 4-amino-5-substituted-3-mercapto-(4H)-1,2,4-triazoles (3a,b) and aromatic acids in phosphorus oxychloride (10 mL) was refluxed for 5 h. The reaction mixture was cooled to room temperature and then gradually poured onto crushed ice with stirring. The mixture was allowed to stand overnight and the solid separated out was filtered, treated with dilute sodium hydroxide solution and washed thoroughly with cold water. The compound so obtained was dried and crystallized from ethanol.
2.2.1 3,6-Diphenyl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (2a)
Yield 82 (%), m.p. 176–178 °C, IR (KBr) ν cm−1: 3064 (Ar C–H str), 2923 (methyl C–H str), 1517 (C⚌N str), 1466 (C⚌C str), 971 (Ar–H bend); 1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.49–7.71 (m, 6H of Ar–H), 8.06 (d, 2H of Ar–H, J = 7.2), 8.33 (d, 2H of Ar–H, J = 7.65); MS (ESI): m/z 278.9 (M+H).
2.2.2 6-(Perfluorophenyl)-3-phenyl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (2b)
Yield 73 (%), m.p. 207–209 °C, IR (KBr) v (cm−1): 3053 (Ar C–H str), 2922 (methyl C–H str), 1511 (C⚌N str), 1473 (C⚌C str), 1232 (Ar C–F str), 990 (Ar C–H bend); 1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.55–7.65 (m, 3H of Ar–H), 8.25(d, 2H of Ar–H, J = 7.8); MS (ESI): m/z 368.9 (M+H).
2.2.3 3-(3-Methoxyphenyl)-6-(perfluorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (2c)
Yield 76 (%), m.p. 236–238 °C, IR (KBr) ν cm−1: 3078 (Ar C–H str), 2922 (methyl C–H str), 1513 (C⚌N str), 1479 (C⚌C str), 1231 (C–O–C str Ar C–F str), 1178 (Ar C–F str), 991 (Ar C–H bend); 1H NMR (400 MHz, DMSO-d6) δ (ppm): 3.84 (s, 3H of OCH3), 7.13 (d, 1H of Ar–H, J = 8.1), 7.52 (m, 1H of Ar–H, J = 8.1), 7.80 (t, 1H of Ar–H, J = 7.5); MS (ESI): m/z 298.7 (M+H).
2.2.4 3-(3,4-Dimethoxyphenyl)-6-(perfluorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (2d)
Yield 65 (%), m.p. 249–251 °C, IR (KBr) ν cm−1: 2922 (Ar C–H str), 2848 (methyl C–H str), 1490 (C⚌N str), 1463 (C⚌C str), 1266 (C–O–C str), 1181 (Ar C–F str), 990 (Ar–H bend); 1H NMR (400 MHz, DMSO-d6) δ (ppm): 3.82 (d, 6H of OCH3), 7.18 (d, 1H of Ar–H, J = 7.8), 7.80 (d, 2H of Ar–H, J = 9.6); MS (ESI): m/z 428.7 (M+H).
2.2.5 6-(Perfluorophenyl)-3-(3,4,5-trimethoxyphenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (2e)
Yield 69 (%), m.p. 283–285 °C, IR (KBr) ν cm−1: 2935 (Ar C–H str), 2850 (methyl C–H str), 1485 (C⚌N str), 1465 (C⚌C str), 1255 (C–O–C str), 1184 (Ar C–F str), 991 (Ar–H bend); 1H NMR (400 MHz, DMSO-d6) δ (ppm): 3.76 (s, 3H of OCH3), 3.86 (s, 6H of OCH3), 7.12 (s, 2H of Ar–H); MS (ESI): m/z 458.6 (M+H).
2.2.6 6-(Perfluorophenyl)-3-p-tolyl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (2f)
Yield 71 (%), m.p. 175–177 °C, IR (KBr) ν cm−1: 2924 (Ar C–H str), 2851 (methyl C–H str), 1485 (C⚌N str), 1462 (C⚌C str), 1185 (Ar C–F str), 990 (Ar–H bend); 1H NMR (400 MHz, DMSO-d6) δ (ppm): 2.40 (s, 3H of CH3), 7.43 (d, 2H of Ar–H, J = 7.5), 8.13 (d, 1H of Ar–H, J = 7.5); MS (ESI): m/z 382.8 (M+H).
2.2.7 3-(4-Nitrophenyl)-6-(perfluorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (2g)
Yield 46 (%), m.p. 212–214 °C, IR (KBr) ν cm−1: 2921 (Ar C–H str), 2851 (methyl C–H str), 1602 (nitro N⚌O str), 1510 (C⚌N str), 1492 (C⚌C str), 1185(Ar C–F str), 990 (Ar–H bend); 1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.36(d, 1H of Ar–H, J = 8.1), 8.36 (d, 2H of Ar–H, J = 8.4); MS (ESI): m/z 413 (M+H).
2.2.8 4-(6-(Perfluorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)phenol (2h)
Yield 87 (%), m.p. 235–237 °C, IR (KBr) ν cm−1: 2921 (Ar C–H str), 2853 (methyl C–H str), 1513 (C⚌N str), 1483 (C⚌C str), 1187 (Ar C–F str), 992.56 (Ar–H bend); 1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.41 (d, 1H of Ar–H, J = 10.8), 8.27 (d, 2H of Ar–H, J = 31.5), MS (ESI): m/z 381 (M+H).
2.2.9 6-(Perfluorophenyl)-3-(pyridin-4-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (2i)
Yield 68 (%), m.p. 164–166 °C, IR (KBr) ν cm−1: 2925 (Ar C–H str), 2853 (methyl C–H str), 1511 (C⚌N str), 1479 (C⚌C str), 1184 (Ar C–F str), 988 (Ar–H bend); 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.28 (s, 2H of Ar–H), 8.850 (s, 2H of Ar–H); MS (ESI): m/z 396.7 (M+H).
2.3 Biological activity
2.3.1 Cell lines and culture
The human cancerous cell lines viz MCF-7 (breast cancer), SaOS-2 (osteosarcoma), and K562 (myelogenous leukemia) were obtained from cell repository-NCCS, Pune, India. The cells were maintained in Eagle’s minimal essential medium (MEM, Himedia), McCoy’s 5a medium (Himedia) and RPMI-1640 (Himedia) respectively, and supplemented with NaHCO3, sodium pyruvate, 10% fetal calf serum (Himedia), and 1% penicillin and streptomycin. Cells were grown at 37 °C, 5% CO2 in humidified air.
2.3.2 In vitro MTT assay for antiproliferative activity
The antiproliferative activities of the compounds were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sharma et al., 2010). MTT is a quantitative colorimetric assay which is based on the phenomenon of enzymatic reduction of MTT dye. The assay provides a direct relationship between the cell viability and color formation (absorbance). The cells (MCF-7, SaOS-2 and K562) were seeded at an initial density of 1 × 104 cells/100 μL complete culture media in 96-well cultured plates and incubated for 24 h at 37 °C in humidified, 5% CO2 atmosphere. Different concentrations of test compounds were added with respective vehicle control (DMSO). The cell viability was determined after 48 h of treatment, with the help of a microplate reader (BIO RAD Model 680) at an absorbance of 540 nm. After 45 h of incubation, media were removed and to each well 10 μL MTT (5 mg/mL of media without phenol red and serum) was added and the plates were further incubated for 3 h at 37 °C. Supernatant from each well was carefully removed and formazan crystals thus formed were solubilized by mixing in 100 μL of DMSO. Subsequently for suspended cell line K562 plate was centrifuged at 1500 rpm for 10 min after addition of MTT and absorbance was recorded at 540 nm by a microplate reader (BIO RAD Model 680).
The percentage cytotoxicity was calculated as per the formula given below The plot of % cytotoxicity versus sample concentration was used to calculate the concentration lethal to 50% of the cells (IC50).
3 Results and discussions
3.1 Chemistry
The strategy adopted for the synthesis of the target compounds is depicted in Scheme 1. The intermediates 4-amino-5-phenyl-4H-1,2,4-triazole-3-thiol (1a–1i) were prepared by the fusion of the benzoic acids with thiocarbohydrazide according to the reported procedure (Bhat et al., 2009). The obtained 4-amino-5-phenyl-4H-1,2,4-triazole-3-thiols (1a–1i) were then treated with pentafluoro benzoic acids in the presence of phosphorous oxychloride to get the desired diaryl-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazoles (2a–2i). The structures of all synthesized compounds were confirmed on the basis of IR, 1HNMR and mass spectral data.![Synthetic route for fluorinated 3,6-diaryl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles.](/content/184/2017/10/2_suppl/img/10.1016_j.arabjc.2013.08.026-fig2.png)
3.2 Biological activity
The synthesized diaryl-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazoles were tested for in vitro antiproliferative activity against MCF7 (human breast cancer), SaOS-2 (osteosarcoma) and K562 (myeloid leukemia) cells using MTT reduction assay. The anticancer drug tamoxifen was used as reference standard. All the nine compounds exhibited moderate antiproliferative activity (<40 μM) against the three cancer cells used (Table 1). Compound 2 was found to be the most potent compound in the series with IC50 values of 22.1, 19 and 15 against MCF7, SaOS-2 and K562 cells, respectively. In comparison to the unsubstituted derivative 2a, compound 2b showed twofold greater antiproliferative potency against SaOS-2 and K562 cells and a moderate improvement in MCF7 cell growth inhibition clearly indicating that the fluoro substitution benefits antiproliferative activity shown by the title compounds. In order to improve the antiproliferative potency of compound 2b, the aryl ring attached to triazole moiety was substituted with electron releasing groups (2c–2f), electron withdrawing groups (2g), Hydrogen bond donors and acceptors (2c–2f, 2h) and replaced with a heteroaryl pyridinyl ring (2i). In case of the MCF7 cell growth inhibition, the structural manipulation at the aryl ring resulted in a modest decrease in antiproliferative activity with an exception of compound 2c which shows almost comparable cell growth inhibition as that of compound 2b. A similar phenomenon was also observed in the antiproliferative activity against SaOS-2 and K562 cells. Overall, the findings suggest that structural variation at the phenyl ring attached to triazole core is not tolerated for the antiproliferative activity exhibited by diaryl-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazoles.
| Compound code | R | Cell growth inhibition (IC50 μM) | ||
|---|---|---|---|---|
| MCF-7 | SaOS-2 | K562 | ||
| 2a | – | 30.2 | 39 | 29.4 |
| 2b | H | 22.1 | 19 | 15 |
| 2c | 3-OCH3 | 23 | 23.2 | 18.5 |
| 2d | 3-OCH3, 4-OCH3 | 25.2 | 28.5 | 21.5 |
| 2f | 3-OCH3, 4-OCH3, 5-OCH3 | 29.1 | 36.5 | 28 |
| 2f | 4-CH3 | 28.8 | 36 | 26 |
| 2g | 4-NO2 | 27.5 | 25 | 20 |
| 2h | 4-OH | 29.4 | 31 | 28.5 |
| 2i | – | 26.5 | 34.5 | 24.5 |
| Tamoxifen | _ | 5.8 | 10.5 | 28.6 |
4 Conclusion
A series of fluorinated diaryl-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazoles was synthesized with an objective to study the effect of fluoro substitution in the aryl ring attached to thiadiazole core and various substitutions in the aryl ring attached to the triazole moiety on antitumor activity exhibited by these compounds. The synthesized compounds were tested in vitro against human breast adenocarcinoma (MCF7), osteosarcoma (SaOS-2) and myelogenous leukemia (K562) cell lines. The results of anticancer screening suggest that the penta fluoro substitution in aryl ring A favors the antiproliferative activity against all the three cancer cells whereas structural manipulation at aryl ring B is detrimental to the antiproliferative potency.
Acknowledgments
The authors gratefully acknowledge the Sophisticated Analytical Instrumentation Facility; Indian Institute of Technology, Delhi, India, for the NMR spectral analysis of the compounds used in this study. The authors Deepak Chowrasia and Lokesh Choure are the recipients of GPAT scholarship offered by All India Council of Technical Education, New Delhi. The authors are thankful to Dr. Rituraj Konwar, Division of Endocrinology, CDRI, Lucknow for providing tamoxifen.
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