Thiazolyl-pyrazole derivatives as potential antimycobacterial agents

Bioorganic & Medicinal Chemistry Letters 29 (2019) 1199–1202

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Thiazolyl-pyrazole derivatives as potential antimycobacterial agents a,e,⁎

b,e

c,e

c,e

Sushma J. Takate , Abhijit D. Shinde , Bhausaheb K. Karale , Hemant Akolkar , ⁎ Laxman Nawaled, Dhiman Sarkard, Pravin C. Mhaskeb,e,

T

a

Department of Chemistry and Research Centre, New Arts, Commerce and Science College, Ahmednagar 414001, India Department of Chemistry, S. P. Mandali’s Sir Parashurambhau College, Tilak Road, Pune 411 030, India Department of Chemistry, Rayat Shikshan Santhsa’s Radhabai Kale Mahila Mahavidyalya, Ahmednagar 414001, India d CombiChemBio Resource Centre, National Chemical Laboratory, Pune 411 008, India b c

ARTICLE INFO

ABSTRACT

Keywords: Thiazoles Pyrazole Antimycobacterial activity Cytotoxicity

Mycobacterium tuberculosis (Mtb) is an obligate aerobe that is capable of long-term persistence under conditions of low oxygen tension. A series of thiazolyl-pyrazole derivatives (6a–f, 7a–f, 8c, 8e) were screened for antimycobacterial activity against dormant M. tuberculosis H37Ra (D-MTB) and M. bovis BCG (D-BCG). Nine thiazolyl-pyrazole analogs, 6c, 6e, 7a, 7b, 7c, 7e, 7f, 8c and 8e exhibited promissing minimum inhibitory concentration (MIC) values (0.20–28.25 µg/mL) against D-MTB and D-BCG strains of Mtb. Importantly, six compounds (7a, 7b, 7e, 7f, 8c and 8e) exhibited excellent antimycobacterial activity and low cytotoxicity at the maximum evaluated concentration of > 250 µg/mL. Finally, the promising antimycobacterial activity and lower cytotoxicity profile suggested that, these compounds could be further subjected for optimization and development as a lead, which could have the potential to treat tuberculosis.

Mycobacterium tuberculosis (Mtb) is among the most challenging bacterial infections declared by World Health Organization (WHO).1 In 2016, WHO projected that globally 10.4 million people were diagnosed by TB and 1.7 million died from the disease. Amongst these, > 95% of TB deaths occur in low- and middle-income countries.1 Even with the availability of first line and second line anti-TB drugs and the BacilleCalmette-Guérin (BCG) vaccine, WHO estimated that there were 6 lakhs new cases with resistance to Rifampicin (the most effective first-line drug), of which 4.9 lakhs had MDR-TB.1,2 Due to spontaneous mutations in genes of the pathogenic strains, there are increasing reports of multi-drug resistant tuberculosis (MDR-TB) and the extensively drug resistant tuberculosis (XDR-TB).3,4 This increase in antibiotic resistance has encouraged the search for new compounds, which can be termed as active against both the acute as well as chronic forms of tuberculosis.5–8 The hybrid architecture of two or more bioactive pharmacophore scaffolds is one of the powerful tools used in the new drug discovery.9 In recent years, the thiazole ring clubbed with other azole rings are privileged scaffolds for the generation of new lead molecules.10 The synthesis of thiazole and pyrazole nucleus containing heterocycles have received considerable notice due to their promising antimycobacterial activity (Fig. 1). The large number of natural products and synthetic compounds containing thiazole ring11 are reported for promising

antibacterial, antitumor, antimalarial and antiviral activities.12,13 Thiazole and its derivatives are important structures in medicinal chemistry that are capable of producing a rich spectrum of biological activities such as antimycobacterial,14–21 antimicrobial,22–29 anti-inflammatory,30–33 antiviral,34 CNS active agents,35 and anticancer activities.36,37 Pyrazole nucleus containing compounds are reported for antitubercular, anticancer, anti-inflammatory antipyretic and antimicrobial activities.38–42 Further chalcones derived from thiazole and pyrazole are also known for their antitubercular and antimicrobial activity.43–46 Thiazole clubbed with pyrazole have displayed antitubercular,10,47,48 anti-inflammatory and antimicrobial49–51 activities. Chromone derivatives are reported for potential antimycobacterial activity.52–54 These reports facilitated the structural diversity and biological importance of thiazole and pyrazole, which have made them attractive targets for synthesis. Keeping in mind, the biological significance of thiazole and pyrazole derivatives and in continuation of our search for new antitubercular agents,14–17,31 we report herein the clubbed thiazolyl-pyrazole derivatives as potential antimycobacterial agents. The synthesis of 1-(substituted phenyl)-3-(3-(2,4-dimethylthiazol-5yl)-1-(4-fluorophenyl)-1H-pyrazol-4-yl)prop-2-en-1-one, 6a–f and 2-(3′(2,4-dimethylthiazol-5-yl)-1′-(4-fluorophenyl)-3,4-dihydro-1′H,2H-

Corresponding authors. E-mail addresses: [email protected] (S.J. Takate), [email protected] (P.C. Mhaske). e Affiliated to Savirtibai Phule Pune University. ⁎

https://doi.org/10.1016/j.bmcl.2019.03.020 Received 31 January 2019; Received in revised form 17 March 2019; Accepted 18 March 2019 Available online 19 March 2019 0960-894X/ © 2019 Elsevier Ltd. All rights reserved.

Bioorganic & Medicinal Chemistry Letters 29 (2019) 1199–1202

S.J. Takate, et al.

Fig. 1. Thiazole and pyrazole derivatives as antitubercular agents.

Scheme 1. Synthetic route of compounds 6a–f and 7a–f.

[3,4′-bipyrazol]-5-yl)phenol, 7a–f (Scheme 1) were achieved as per our previous report.51 1-(2,4-Dimethylthiazol-5-yl)ethanone, 3 on condensation with 4-fluorophenyl hydrazine in EtOH followed by reaction with DMF-POCl3 in DMF resulted the formation of 3-(2,4-dimethylthiazol-5-yl)-1-(4-fluorophenyl)-1H-pyrazole-4-carbaldehyde, 4. Aldehyde 4 on condensation with 2-hydroxyacetophenones, 5a–f furnished 3-(3-(2,4-dimethylthiazol-5-yl)-1-(4-fluorophenyl)-1H-pyrazol-4-yl)-1-(2-hydroxyphenyl)prop-2-en-1-one derivatives, 6a–f. Chalcones 6a–f on cyclization reaction with hydrazine hydrate in absolute ethanol furnished 2-(3′-(2,4-dimethylthiazol-5-yl)-1′-(4-fluorophenyl)-3,4-dihydro-1′H,2H-[3,4′-bipyrazol]-5-yl)phenols, 7a–f. The thiazolyl-pyrazole chalcones 6c and 6e showed good anti-tubercular activity and were further cyclized to chromen-4-one derivatives 8c and 8e, respectively (Scheme 2). The synthesized thiazolyl-pyrazole derivatives were screened for in vitro antimycobacterial activity against avirulent strain of D-MTB (ATCC 25177) and D-BCG (ATCC 35743) in liquid medium. The convincing antimycobacterial activity of thiazolyl-pyrazole compounds in

our preliminary screen studies (Table S1) leads us to determine minimum inhibition concentration (MIC). To investigate this class of compounds further, we screened thirteen thiazolyl-pyrazole analogs for inhibition of D-MTB and D-BCG growth in MIC by using an established XTT Reduction Menadione assay (XRMA) method.55–58 The MIC of antiTB drug, Rifampicin was determined by using the XRMA method and this was used as a reference. The result of antimycobacterial activity is presented in Tables 1 and S2 (IC50 and MIC). The analysis of antimycobacterial activity data revealed that the thiazolyl-pyrazole anagoges 6c, 6e, 7a, 7b, 7c, 7e, 7f, 8c and 8e exert a statistically significant 90% decrease in growth of D-MTB and D-BCG. From the structure activity relationship of compounds 6a–f, 7a–f, 8c and 8e, it has noticed that substituent like Br, Cl, and CH3 on phenyl ring significantly influence the antimycobacterial activity. The structure activity relationship reported that, among the thiazolyl-pyrazole chalcone derivatives 6a–f, compounds 6a (R3 = Br) and 6b (R3 = Cl) were inactive. Substitution at R3 by methyl group in compound 6c exhibited excellent activity against D-MTB (MIC: 14.13 μg/mL) and D-BCG (MIC:7.01 μg/mL). Further it was noted that substitution at R1 and R3 by chlorine in compound 6d, decreased the activity. Substitution at R2 by methyl and R3 by chlorine in compound 6e reported good activity against D-MTB (MIC: 23.24 μg/mL) and excellent activity against D-BCG (MIC: 3.21 μg/mL) whereas substitution of one more methyl group at R4 in compound 6f, decreases the activity. It was noticed that thiazolyl-bispyrazol derivatives, 7a–f were found to be more potent against both strains of Mtb. Compound 7a (R3 = Br) has shown excellent activity against D-MTB with MIC 2.96 μg/mL. In addition, against D-BCG the compound 7a was found to be fourfold

Scheme 2. Synthetic route of chromen-4-one derivatives 8c and 8e. 1200

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Table 1 In vitro-ex vivo antimycobacterial activity expressed by MIC in µg/mL (µM) of compounds 6a–f, 7a–f, 8c and 8e against two strains of Mtb.

Comp.

R1

R2

R3

R4

Ex vivo (D-MTB) MICc

In vitro (D-MTB) MICc

In vitro (D-BCG) MICc

6a 6b 6c 6d 6e 6f 7a 7b 7c 7d 7e 7f 8c 8e RPa

H H H Cl H H H H H Cl H H H H

H H H H Me Me H H H H Me Me H Me

Br Cl Me Cl Cl Cl Br Cl Me Cl Cl Cl Me Cl

H H H H H Me H H H H H Me H H

> 30 > 30 7.43(17.14) > 30 3.54(7.56) > 30 1.51(2.95) 0.7(1.50) 14.95(33.41) ndb 4.14(8.59) 0.61(1.23) 0.53(1.23) 1.37(2.94)

> 30 > 30 14.13(32.69) > 30 23.24(49.66) > 30 2.96(5.79) 1.16(2.48) 20.76(46.38) nd 4.65(9.65) 2.5(5.04) 2.54(5.89) 1.72(3.69) 0.75(0.91)

> 30 > 30 7.01(16.17) > 30 3.21(6.85) > 30 0.20(0.39) 0.72(1.54) 28.25(63.12) nd 2.3(4.77) 2.26(4.55) 0.51(1.18) 1.33(2.85) 0.81(0.98)

The bold value indicates MIC value of active compounds. a RP: Rifampicin; b nd: Not determined; c MIC: minimum inhibitory concentration.

more activity than Rifampicin with MIC 0.20 μg/mL. It was also noticed that when R3 was substituted by chlorine in compound 7b, it showed comparable activity against D-MTB (MIC: 1.16 μg/mL) and D-BCG (MIC: 0.72 μg/mL) with respect to the standard drug. Substitution at R3 by methyl group in compound 7c showed moderate to good activity against both Mtb strains. Compound 7e (R2 = CH3, R3 = Cl) reported an excellent activity against D-MTB (MIC 4.65 μg/mL) and D-BCG (MIC 2.3 μg/mL). Also, it was noticed that, on substitution of one more methyl group at R4 in compound 7f, the activity increased against D-MTB by two fold (MIC 2.5 μg/mL) and retained against D-BCG (MIC 2.26 μg/ mL). From the chromen-4-one derivatives 8c and 8e, compound 8c reported excellent activity against D-MTB (MIC: 2.54 μg/mL) and D-BCG (MIC: 0.51 μg/mL). Similarly, compound 8e reported excellent activity against D-MTB (MIC: 1.72 μg/mL) and D-BCG (MIC: 1.33 μg/mL). From compounds 8c and 8e it was observed that in chromen-4-one derivatives, methyl group at R3 is more potent against D-BCG than the methyl group at R2 and chlorine at R3 position. In sharp contrast, it is worth noting that, cyclization of compounds 6a–f into compounds 7a–f and compounds 8c, 8e enhanced the antitubercular activity; particularly compounds 7a, 7b, 7e, 7f, 8c and 8e were found to be the most active derivatives. The efficacy of thiazolyl-bispyrazol derivatives was also examined with an intracellular model of Mtb infection as per protocols reported in literature (Khan et al, 2012).57 The results gathered in Table 1 show that lead compounds 7a, 7b, 7e, 7f, 8c and 8e exhibits interesting minimum inhibitory concentration (MIC) values in the range 0.53–4.14 μg/mL in human THP-1 macrophages and was devoid of cytotoxicity in human THP-1 cells (GI50: > 30 μg/mL) (Table S3). Interestingly, all lead compounds exert a similar minimum inhibitory effect (D-MTB-ex vivo) on the antimycobacterial activity (in vitro) of the D-MTB and D-BCG, which also suggests these compounds showed

excellent anti-mycobacterial potential against dormant strains of Mtb. All compounds were also examined for in vitro cytotoxicity in human cancer cell lines (HeLa, A549 and PANC-1) by the MTT colorimetric assay.59 The results, expressed as GI50/GI90 in the presence of at different concentrations of each compound, are summarized in the Table 2. The result of GI50 and GI90 (> 250 µg/mL) of compounds 7b, 7c, 7e, 8c and 8e confirmed that these molecules did not exhibit any significant toxicity effect on the selected cell line. Table 2 In vitro cytotoxicity activity of compounds 6a–f, 7a–f, 8c and 8e against three human cancer cell lines. Compound

6a 6b 6c 6d 6e 6f 7a 7b 7c 7d 7e 7f 8c 8e Rifampicin Paclitaxel

1201

HeLa (Cervix) Cell line

A549 (Lung) Cell line

PANC-1 (Pancreas) Cell line

GI50 (µg/ mL)

GI90 (µg/ mL)

GI50 (µg/ mL)

GI90 (µg/ mL)

GI50 (µg/ mL)

GI90 (µg/ mL)

4.88 > 250 29.88 > 250 34.87 59.29 21.62 > 250 > 250 nd > 250 25.21 > 250 16.68 > 100 0.0048

44.94 > 250 77.32 > 250 87.29 128.28 43.11 > 250 > 250 nd > 250 46.87 > 250 53.48 > 100 0.075

> 250 > 250 47.77 > 250 > 250 33.45 81.44 > 250 > 250 nd 145.68 43.36 > 250 > 250 > 100 0.0035

> 250 > 250 136.87 > 250 > 250 84.31 154.68 > 250 > 250 nd > 250 88.98 > 250 > 250 > 100 0.0706

> 250 > 250 52.41 > 250 > 250 35.22 76.41 > 250 > 250 nd 127.44 41.64 > 250 > 250 > 100 0.1279

> 250 > 250 114.87 > 250 > 250 97.41 115.65 > 250 > 250 nd > 250 77.64 > 250 > 250 > 100 5.715

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In this study, the synthesis and the antimycobacterial evaluations of 1-(substituted phenyl)-3-(3-(2,4-dimethylthiazol-5-yl)-1-(4-fluorophenyl)-1H-pyrazol-4-yl)prop-2-en-1-ones, 6a–f, 2-(3′-(2,4-dimethylthiazol-5-yl)-1′-(4-fluorophenyl)-3,4-dihydro-1′H,2H-[3,4′-bipyrazol]-5-yl)phenols, 7a–f, and 2-(3-(2,4-dimethylthiazol-5-yl)-1-(4fluorophenyl)-1H-pyrazol-4-yl)-4H-substituted chromen-4-ones, 8c and 8e were achieved. The cyclization of chalcones 6a–f to pyrazole 7a–f and chromene-4-ones 8c and 8e enhanced the antimycobacterial activity. These active thiazolyl-pyrazole derivatives were found to display no manifest cytotoxicity toward HeLa, A549 and PANC-1 human cells and among them, six compounds (7a, 7b, 7e, 7f, 8c and 8e) exhibited attractive inhibitory activities against D-MTB and D-BCG strains of Mtb.

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Acknowledgments Authors are thankful to CSIR-NCL, Pune for lending support with their biological activities. Central Instrumentation facility, Savitribai Phule Pune University, Pune is also acknowledged for spectral analysis. A D Shinde is grateful to CSIR-for award of JRF, Award No 08/ 319(0004/)17-EMR-1. Conflicts of interest There are no conflicts to declare Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bmcl.2019.03.020. References 1. World Health Organization, Tuberculosis Fact Sheet; 2016. http://www.who.int/ news-room/fact-sheets/detail/tuberculosis. 2. Wang Q, Song F, Xiao X, et al. Angew Chem Int Ed. 2013;52:1231. 3. Martinelli KBL, Rotta M, Villela AD, et al. Sci Rep. 2017;7:e46696. 4. Wang X, Dai Z, Chen Y, et al. Eur J Med Chem. 2017;126:171. 5. Shenoi S, Friedland G. Annu Rev Med. 2009;60:307. 6. Tantry SJ, Markad SD, Shinde V, et al. J Med Chem. 2017;60:1379. 7. Jeankumar VU, Rudraraju SR, Vats R, et al. Eur J Med Chem. 2016;122:216. 8. Holas O, Ondrejcek P, Dolezal M. J Enzyme Inhib Med Chem. 2015;30:629. 9. Beruve G. Expert Opinion Drug Discovery. 2016;11:281. 10. Azzali E, Machado D, Kaushik A, et al. J Med Chem. 2017;60:7108. 11. Rouf A, Tanyeli C. Eur J Med Chem. 2015;97:911. 12. Ayati A, Emami S, Asadipour A, Shafiee A, Foroumadi A. Eur J Med Chem. 2015;97:699. 13. Das D, Sikdar P, Bairagi M. Eur J Med Chem. 2016;109:89. 14. Abhale YK, Sasane AV, Chavan AP, et al. Eur J Med Chem. 2015;94:340.

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