Ethionamide and Prothionamide Based Coumarinyl-Thiazole Derivatives: Synthesis, Antitubercular Activity, Toxicity Investigations and Molecular Docking Studies

Abstract

The goal of this research work was to prepare and evaluate the antitubercular (anti-TB) activity of ethionamide (ETH) and prothionamide (PTH) based coumarinyl-thiazole derivatives. ETH and PTH were reacted with coumarin intermediates (3a-3e) to provide the target compounds (4a-4e and 4f-4j, respectively). Spectral studies confirmed the assigned structures of 4a-4j. The Microplate Alamar Blue Assay was utilized to evaluate the anti-TB activity of compounds 4a-4j against Mycobacterium tuberculosis H37Rv strain in comparison to ETH, PTH, isoniazid (INH), and pyrazinamide (PYZ) as standard drugs. The cytotoxicity studies were carried out versus HepG2 and Vero cell lines. In addition. molecular docking studies of 4a-4j concerning the DprE1 enzyme and the in-silico evaluation of physicochemical and pharmacokinetic parameters were performed. Compounds 4a, 4b, 4f, and 4g displayed equal minimum inhibitory concentration (MIC) values in comparison to INH (3.125 μg/ml) and PYZ (3.125 μg/ml), whereas 4c-4e and 4h-4j displayed better MIC values (1.562 μg/mL) than INH and PYZ. All compounds presented better anti-TB potential than ETH (6.25 μg/mL) and PTH (6.25 μg/mL). The studies of toxicity revealed that 4a-4j were safe up to 300 μg/mL concentration versus Vero and HepG2 cell lines. The molecular docking studies suggested that 4a-4j could possess anti-TB activity through the inhibition of the DprE1 enzyme. The in silico studies showed that 4a-4j followed Lipinski’s rule (drug-likeliness) and exhibited better gastrointestinal absorption than BTZ043 and macozinone. In conclusion, the ETH and PTH-based coumarinyl-thiazole template can help developing selective DprE1 enzyme inhibitors as potent anti-TB agents.

Introduction

Tuberculosis (TB), a global concern, is instigated through Mycobacterium tuberculosis (Mtb) bacteria. This communicable illness is among the top ten bases of mortality, and the principal reason of mortality from this particular contagious microorganism. The World Health Organization (WHO) global tuberculosis report of 2020 declared that about ten million people suffered from TB in 2019, and about 1.2 million and 0.208 million HIV-negative and HIV-positive people, respectively, died in 2019. The recent report also states that the number of TB patients is expected to increase by 0.2 – 0.4 million in 2020 due to COVID-19 [1]. Conventional treatment of TB comprises the use of isoniazid (INH), rifampin, ethambutol, pyrazinamide (PYZ), pretomanid, and bedaquiline along with other anti-tubercular (anti-TB) drugs for at least 6 – 9 months. However, many TB treatments can cause hepatotoxicity in chronic use. The rise of multi-drug resistant TB (MDR-TB), totally drug-resistant TB (TDR-TB), and extensive drug-resistant TB (XDR-TB) is also posing questions to TB treatment. This situation necessitates additional research into new molecular frameworks that may tackle these difficulties with insignificant adverse impacts [1, 2].

In the pursuit of innovative and safe anti-TB treatment, researchers from all over the world have experimented with several molecular scaffolds of various designs [3]. The research work on coumarin [4] and thiazole [5] scaffolds has provided some new promising anti-TB agents. Coumarinthiazole based compounds (Fig. 1) have also been reported as potent anti-TB agents [6, 7]. Ethionamide (ETH) and prothioanamide (PTH) are thioamide groups containing second-line anti-TB drugs, which are utilized as a replacement for INH and rifampin in the case of MDR-TB. The structure of ETH and PTH is analogous to INH (Fig. 2). However, ETH and PTH are relatively weak anti-TB agents as compared to INH. This leads to an increase in the treatment duration (up to two years) and the probability of increased side effects [8]. The literature teaches that the thioamide group of ETH and PTH can cyclize to thiazole derivatives after reaction with phenyl acyl bromide, for example, with 3-coumarinylacetyl bromide [6, 7, 9]. Therefore, in continuation of our endeavor to develop anti-TB compounds [10, 11] and because of the challenges associated with TB [1, 2], the author decided to develop ETH and PTH-based coumarinylthiazole derivatives as anti-TB agents.

Fig. 1.

Design of the compounds A and B: The structure of coumarin-thiazole based compounds as anti-TB agents reported in reference [6, 7], respectively. 4a-4j: The structure of the synthesized compounds as anti-TB agents.

Fig. 2.

Chemical structures of isoniazid (INH), ethionamide (ETH), and prothionamide (PTH).

Experimental Chemical Part

Chemicals and Instruments

Compounds ETH, PTH, INH, and PYZ were purchased from Sigma Aldrich (USA). Analytical grade solvents (Sigma, Spectrochem, and Merck) were used during the synthesis of target compounds. A mixture of formic acid, ethyl acetate, and toluene (1:4:5) was utilized to establish the R_f values. The uncorrected melting points (m.p.) in °C (Gallenkamp apparatus), FTIR spectra in KBr (Shimadzu 440 spectrometer), ¹H and ¹³C NMR spectra in DMSO-d₆ (Varian Gemini 500/125 MHz spectrometer), mass spectra in m/z (GCMS/QP 1000 Ex mass spectrometer, 70 eV), and elemental analyses (Vario El Elementar apparatus) were recorded on the instruments stated in parentheses.

Synthesis of Intermediates (3a-3e)

The preparation of intermediates (3a-3e) from salicyldehydes (1a-1e) and ethyl acetoacetate (2) has been reported in our earlier publication [9] (Scheme 1).

Scheme 1.

Synthesis of intermediates (3a-3e)

Synthesis of 3-(2-(2-Ethylpyridin-4-yl)thiazol-4-yl)-2H-chromen-2-one (4a)

Equimolar amounts of 3a (0.01 mol) and ETH (0.01 mol) in absolute ethanol (40 mL) were mixed and refluxed for 2 h. A solid precipitate formed during reflux was filtered without cooling to obtain intermediate 4a (yellowish-brown crystal). Yield: 80%; m.p. 185 – 187°C; R_f 0.79; IR (ν_max, cm^-1): 1718 (C=O), 1568 (C=N), 1527 (C=C), 1258 (C–O–C), 1111 (C–S); ¹H NMR (, ppm): 1.28 (t, J = 7 Hz, 3H, -CH₃), 3.38 (q, J = 4.5 Hz, 2H, -CH₂-), 7.36 – 7.42 (m, 2H, Ar-H), 7.58 – 7.61 (dd, 1H, Ar-H), 7.84 (d, J = 8 Hz, 1H, Ar-H), 8.11 (s, 1H, C4-coumarin), 8.23 (d, J = 8 Hz, 1H, C5-pyridine), 8.31 (s, 1H, C3-pyridine), 8.40 (s, 1H, C5-thiazole), 8.78 (d, J = 8 Hz, 1H, C6-pyridine); ¹³C-NMR (DMSO-d₆, 125 MHz, , ppm): 13.2 (-CH₃), 30.1 (-CH₂-), 107.2, 111.6, 115.0, 119.8, 123.1, 124.3, 126.8, 127.2, 128.3, 136.7, 143.1, 145.1, 148.3, 150.5, 152.5, 159.2, 160.8 (C=O); Mass (m/z): 334 (M⁺); Elemental analysis (EA) for C₁₉H₁₄N₂O₂S [Calcd. (Found)]: C, 68.25 (68.14); H, 4.22 (4.18); N, 8.38 (8.32).

Compounds 4b-4j were prepared in a similar manner (Scheme 2).

Scheme 2.

Synthesis of ethionamide and prothionamide based coumarinyl-thiazole compounds (4a-4j)

3-(2-(2-Ethylpyridin-4-yl)thiazol-4-yl)-6-fluoro-2Hchromen-2-one (4b). The reaction of 3b with ETH provided 4b (yellow crystals). Yield:80%; m.p. 188 – 189°C; R_f 0.82; IR (ν_max, cm^-1): 1716, 1562, 1525, 1255, 1112; ¹H NMR (δ, ppm): 1.27 (t, J = 7 Hz, 3H, -CH₃), 3.37 (q, J = 4.5 Hz, 2H, -CH₂-), 7.13 (d, J = 8 Hz, 1H, Ar-H), 7.31 (dd, 2H, Ar-H), 8.10 (s, 1H, C4-coumarin), 8.21 (d, J = 8.5 Hz, 1H, C5-pyridine), 8.30 (s, 1H, C3-pyridine), 8.40 (s, 1H, C5-thiazole), 8.78 (d, J = 8 Hz, 1H, C6-pyridine); ¹³C-NMR: 13.2 (-CH₃), 30.1 (-CH₂-), 107.2, 111.6, 113.5, 114.1, 122.7, 123.3, 124.0, 128.3, 136.7, 143.0, 145.1, 147.5, 148.3, 152.3, 158.5, 159.3, 160.7 (C=O); Mass: 352 (M⁺); EA for C₁₉H₁₃FN₂O₂S: C, 64.76 (64.66); H, 3.72 (3.68); N, 7.95 (7.88).

6-Chloro-3-(2-(2-ethylpyridin-4-yl)thiazol-4-yl)-2Hchromen-2-one (4c). The reaction of 3c with ETH provided 4c(golden crystals). Yield: 85%; m.p. 174 – 176°C; R_f 0.75; IR (ν_max, cm^-1): 1718, 1567, 1529, 1255, 1110; ¹H-NMR: 1.26 (t, J = 7 Hz, 3H, -CH₃), 3.35 (q, J = 4.5 Hz, 2H, -CH₂-), 7.27 (d, J = 7.5 Hz, 1H, Ar-H), 7.42 (d, J = 7.5 Hz, 1H, Ar-H), 7.99 (s, 1H, Ar-H), 8.09 (s, 1H, C4-coumarin), 8.19 (d, J = 7.5 Hz, 1H, C5-pyridine), 8.28 (s, 1H, C3-pyridine), 8.39 (s, 1H, C5-thiazole), 8.77 (d, J = 7.5 Hz, 1H, C6-pyridine); ¹³C NMR: 13.2 (-CH₃), 30.3 (-CH₂-), 107.1, 111.5, 117.1, 122.5, 123.0, 125.7, 128.3, 128.4, 132.0, 136.7, 143.0, 145.1, 148.3, 150.1, 152.3, 159.2, 160.8 (C=O); Mass: 368 (M⁺), 369 (M⁺+1), 370 (M⁺+1); EA for C₁₉H₁₃ClN₂O₂S: C, 61.87 (61.79); H, 3.55 (3.49); N, 7.60 (7.54).

6-Bromo-3-(2-(2-ethylpyridin-4-yl)thiazol-4-yl)-2Hchromen-2-one (4d). The reaction of 3d with ETH provided 4d (amber crystals).Yield: 85%; m.p. 195 – 197°C; R_f 0.82; IR (ν_max, cm^-1): 1717, 1566, 1528, 1258, 1113; ¹H-NMR: 1.28 (t, J = 7 Hz, 3H, -CH₃), 3.36 (q, J = 4 Hz, 2H, -CH₂-), 7.20 (d, J = 8 Hz, 1H, Ar-H), 7.55 (d, J = 8 Hz, 1H, Ar-H), 8.01 (s, 1H, Ar-H), 8.10 (s, 1H, C4-coumarin), 8.19 (d, J = 8 Hz, 1H, C5-pyridine), 8.28 (s, 1H, C3-pyridine), 8.40 (s, 1H, C5-thiazole), 8.78 (d, J = 8 Hz, 1H, C6-pyridine); ¹³C NMR: 13.2 (-CH₃), 30.1 (-CH₂-), 107.3, 111.7, 117.2, 118.7, 123.0, 123.4, 128.2, 129.2, 133.1, 136.7, 143.0, 145.0, 148.3, 151.0, 152.4, 159.3, 160.9 (C=O); Mass: 412 (M⁺), 413 (M⁺+1), 414 (M⁺+2); EA for C₁₉H₁₃BrN₂O₂S: C, 55.22 (55.15); H, 3.17 (3.10); N, 6.78 (6.75).

3-(2-(2-Ethylpyridin-4-yl)thiazol-4-yl)-6-iodo-2H-chromen-2-one (4e). The reaction of 3e with ETH provided 4e (brown crystals). Yield: 80%; m.p. 169 – 171°C; R_f 0.81; IR (ν_max, cm^-1): 1719, 1566, 1527, 1256, 1112; ¹H NMR: 1.27 (t, J = 7 Hz, 3H, -CH₃), 3.38 (q, J = 4.5 Hz, 2H, -CH₂-), 7.08 (d, J = 7.5 Hz, 1H, Ar-H), 7.79 (d, J = 7.5 Hz, 1H, Ar-H), 7.99 (s, 1H, Ar-H), 8.11 (s, 1H, C4-coumarin), 8.21 (d, J = 7.5 Hz, 1H, C5-pyridine), 8.30 (s, 1H, C3-pyridine), 8.42 (s, 1H, C5-thiazole), 8.79 (d, J = 7.5 Hz, 1H, C6-pyridine); ¹³C NMR: 13.3 (-CH₃), 30.3 (-CH₂-), 91.8, 107.1, 111.4, 119.2, 122.6, 123.0, 128.2, 133.1, 136.1, 136.7, 143.0, 145.0, 148.3, 150.8, 152.3, 159.2, 160.8 (C=O); Mass: 460 (M⁺), 461 (M⁺+1), 462 (M⁺+1); EA for C₁₉H₁₃IN₂O₂S: C, 49.58 (49.52); H, 2.85 (2.81); N, 6.09 (6.03).

3-(2-(2-Propylpyridin-4-yl)thiazol-4-yl)-2H-chromen-2-one (4f). The reaction of 3a with PTH provided 4f (yellow crystals). Yield: 75%; m.p. 181 – 183°C; R_f 0.77; IR (ν_max, cm^-1): 1715, 1565, 1525, 1256, 1115; ¹H NMR: 0.95 (t, J = 7 Hz, 3H, -CH₃), 1.79 (m, 2H, -CH₂-Me), 3.03 (t, J = 7 Hz, 2H, -CH₂-), 7.36 – 7.42 (m, 2H, Ar-H), 7.58 (dd, 1H, Ar-H), 7.83 (dd, 1H, Ar-H), 8.08 (s, 1H, C4-coumarin), 8.21 (d, J = 8 Hz, 1H, C5-pyridine), 8.28 (s, 1H, C3-pyridine), 8.41 (s, 1H, C5-thiazole), 8.79 (d, J = 8 Hz, 1H, C6-pyridine); ¹³C NMR: 13.3 (-CH₃), 22.4 (-CH₂-Me), 41.1 (-CH₂-), 108.2, 113.3, 115.1, 119.8, 123.0, 124.3, 126.8, 127.2, 128.3, 136.6, 143.1, 145.0, 148.3, 152.0, 152.6, 159.5, 160.7 (C=O); Mass: 348 (M⁺); EA for C₂₀H₁₆N₂O₂S: C, 68.95 (68.88); H, 4.63 (4.55); N, 8.04 (8.01).

6-Fluoro-3-(2-(2-propylpyridin-4-yl)thiazol-4-yl)-2Hchromen-2-one (4g). The reaction of 3b with PTH provided 4g (reddish yellow crystals). Yield: 85%; m.p. 173 – 175°C; R_f 0.80; IR (ν_max, cm^-1): 1718, 1564, 1526, 1257, 1110; ¹H NMR: 0.96 (t, J = 7 Hz, 3H, -CH₃), 1.81 (m, 2H, -CH₂-Me), 3.03 (t, J = 7 Hz, 2H, -CH₂-), 7.10 (d, J = 8 Hz, 1H, Ar-H), 7.31 (dd, 2H, Ar-H), 8.08 (s, 1H, C4-coumarin), 8.22 (d, J = 8 Hz, 1H, C5-pyridine), 8.29 (s, 1H, C3-pyridine), 8.41 (s, 1H, C5-thiazole), 8.79 (d, J = 8 Hz, 1H, C6-pyridine); ¹³C NMR: 13.2 (-CH₃), 22.5 (-CH₂-Me), 41.0 (-CH₂-), 108.1, 113.3, 113.8, 114.5, 122.7, 123.0, 124.0, 128.3, 136.6, 143.2, 145.1, 147.5, 148.3, 152.3, 158.1, 159.4, 160.7 (C=O); Mass: 366 (M⁺); EA for C₂₀H₁₅FN₂O₂S: C, 65.56 (65.50); H, 4.13 (4.08); N, 7.65 (7.60).

6-Chloro-3-(2-(2-propylpyridin-4-yl)thiazol-4-yl)-2Hchromen-2-one (4h). The reaction of 3c with PTH provided 4h (orange crystals). Yield: 70%; m.p. 193 – 195°C; R_f 0.77; IR (ν_max, cm^-1): 1721, 1566, 1529, 1256, 1111; ¹H NMR: 0.96 (t, J = 7 Hz, 3H, -CH₃), 1.79 (m, 2H, -CH₂-Me), 3.04 (t, J = 7 Hz, 2H, -CH₂-), 7.27 (d, J = 8 Hz, 1H, Ar-H), 7.40 (d, J = 8 Hz, 1H, Ar-H), 7.98 (s, 1H, Ar-H), 8.09 (s, 1H, C4-coumarin), 8.19 (d, J = 8 Hz, 1H, C5-pyridine), 8.28 (s, 1H, C3-pyridine), 8.39 (s, 1H, C5-thiazole), 8.78 (d, J = 8 Hz, 1H, C6-pyridine); ¹³C NMR: 13.4 (-CH₃), 22.6 (-CH₂-Me), 41.1 (-CH₂-), 108.1, 113.2, 117.1, 122.4, 123.2, 126.7, 128.2, 128.5, 130.0, 136.6, 143.2, 145.1, 148.3, 150.0, 152.3, 159.5, 160.7 (C=O); Mass: 382 (M⁺), 383 (M⁺+1), 384 (M⁺+2); EA for C₂₀H₁₅ClN₂O₂S: C, 62.74 (62.68); H, 3.95 (3.92); N, 7.32 (7.30).

6-Bromo-3-(2-(2-propylpyridin-4-yl)thiazol-4-yl)-2Hchromen-2-one (4i). The reaction of 3d with PTH provided 4i (creamy crystals). Yield: 80%; m.p. 178 – 180°C; R_f 0.75; IR (ν_max, cm^-1): 1720, 1566, 1530, 1256, 1112; ¹H NMR: 0.95 (t, J = 7 Hz, 3H, -CH₃), 1.79 (m, 2H, -CH₂-Me), 3.03 (t, J = 7 Hz, 2H, -CH₂-), 7.19 (d, J = 8 Hz, 1H, Ar-H), 7.55 (d, J = 8 Hz, 1H, Ar-H), 8.01 (s, 1H, Ar-H), 8.08 (s, 1H, C4-coumarin), 8.18 (d, J = 8 Hz, 1H, C5-pyridine), 8.29 (s, 1H, C3-pyridine), 8.39 (s, 1H, C5-thiazole), 8.77 (d, J = 8 Hz, 1H, C6-pyridine); ¹³C NMR: 13.3 (-CH₃), 22.4 (-CH₂-Me), 41.0 (-CH₂-), 108.1, 113.3, 117.1, 118.7, 123.1, 123.5, 128.4, 129.2, 133.1, 136.6, 143.2, 145.1, 148.3, 151.0, 152.3, 159.5, 160.8 (C=O); Mass: 426 (M⁺), 427 (M⁺+1), 428 (M⁺+2); EA for C₂₀H₁₅BrN₂O₂S: C, 56.22 (56.15); H, 3.54 (3.50); N, 6.56 (6.53).

6-Iodo-3-(2-(2-propylpyridin-4-yl)thiazol-4-yl)-2Hchromen-2-one (4j). The reaction of 3e with PTH provided 4j (yellow crystals). Yield: 85%; m.p. 195 – 197°C; R_f 0.84; IR (ν_max, cm^-1): 1721, 1566, 1529, 1256, 1112; ¹H NMR: 0.95 (t, J = 7 Hz, 3H, -CH₃), 1.81 (m, 2H, -CH₂-Me), 3.02 (t, J = 7 Hz, 2H, -CH₂-), 7.08 (d, J = 8 Hz, 1H, Ar-H), 7.77 (d, J = 8 Hz, 1H, Ar-H), 7.96 (s, 1H, Ar-H), 8.10 (s, 1H, C4-coumarin), 8.19 (d, J = 8 Hz, 1H, C5-pyridine), 8.28 (s, 1H, C3-pyridine), 8.40 (s, 1H, C5-thiazole), 8.76 (d, J = 8 Hz, 1H, C6-pyridine); ¹³C NMR: 13.4 (-CH₃), 22.6 (-CH₂-Me), 41.0 (-CH₂-), 91.9, 108.4, 115.2, 119.5, 122.7, 123.1, 128.2, 133.1, 136.1, 136.7, 143.2, 145.1, 148.3, 150.8, 152.5, 159.6, 160.7 (C=O); Mass: 474 (M⁺), 475 (M⁺+1), 476 (M⁺+2); EA for C₂₀H₁₅IN₂O₂S: C, 50.65 (50.60); H, 3.19 (3.15); N, 5.91 (5.88).

Experimental Biological Activity Part

In-Vitro Anti-TB Activity Study

The study was accomplished by employing the Microplate Alamar Blue Assay (MABA) method versus the Mycobacterium tuberculosis (Mtb H37Rv) strain [10, 12]. This assay is based on the color change of resazurin (blue), wherein the blue color changes to pink with microbial growth. All the chemicals and reagents were made corresponding to the detailed method specified in the literature [12]. The dilutions (0.781 – 50 μg/ml) of 4a-4j and standard drugs (ETH, PTH, INH, and PYZ) were prepared in sterile DMSO. The microplate absorbance was read at 530 and 590 nm (excitation and emission wavelengths, respectively), and the minimum inhibitory concentration (MIC) of the compounds was calculated (see Table 1 below).

Table 1.

Anti-TB Activity and Cytotoxicity Data and In-Silico Studies (PC and PK parameters) of Compounds 4a-4j

Compound	MIC (μg/mL) against Mtb H37Rv [CI 99%]	MTT assay data (CC50 in μg/mL)		Selectivity Index	TPSA	Log P	Aqueous solubility	Druglikeness (Lipinski’s rule violations)	Calculated absorption (%)	BBB permeant	P-gp substrate	Mutagenicity
		HCL	VCL
4a	3.125 ± 0.0* [3.125 ± 0.0]	> 300	> 300	> 96	84.23	4.01	Poor	Yes (0)	79.94	No	No	No
4b	3.125 ± 0.0* [3.125 ± 0.0]	> 300	> 300	> 96	84.23	4.35	Poor	Yes (0)	79.94	No	No	Yes
4c	1.562 ± 0.0* [1.562 ± 0.0]	> 300	> 300	> 192	84.23	4.61	Poor	Yes (0)	79.94	No	No	No
4d	1.562 ± 0.0* [1.562 ± 0.0]	> 300	> 300	> 192	84.23	4.61	Poor	Yes (0)	79.94	No	No	No
4e	1.562 ± 0.0* [1.562 ± 0.0]	> 300	> 300	> 192	84.23	4.65	Poor	Yes (0)	79.94	No	No	-
4f	3.125 ± 0.0* [3.125 ± 0.0]	> 300	> 300	> 96	84.23	4.35	Poor	Yes (0)	79.94	No	No	No
4g	3.125 ± 0.0* [3.125 ± 0.0]	> 300	> 300	> 96	84.23	4.62	Poor	Yes (0)	79.94	No	No	No
4h	1.562 ± 0.0* [1.562 ± 0.0]	> 300	> 300	> 192	84.23	4.84	Poor	Yes (0)	79.94	No	No	No
4i	1.562 ± 0.0* [1.562 ± 0.0]	> 300	> 300	> 192	84.23	4.93	Poor	Yes (0)	79.94	No	No	No
4j	1.562 ± 0.0* [1.562 ± 0.0]	300	300	> 192	84.23	4.95	Poor	Yes (0)	79.94	No	No	-
BTZ043	ND	ND	ND	ND	125.72	2.89	Moderate	Yes (0)	65.62	No	Yes	Yes
Macozinone	ND	ND	ND	ND	110.50	3.86	Moderate	Yes (0)	70.87	No	No	Yes
ETH	6.25 ± 0.0* [6.25 ± 0.0]	> 150	> 150	> 24	71.0	1.47	Soluble	Yes (0)	84.50	No	No	-
PTH	6.25 ± 0.0* [6.25 ± 0.0]	> 150	> 150	> 24	71.0	1.84	Soluble	Yes (0)	84.50	Yes	No	-
INH	3.125 ± 0.0* [3.125 ± 0.0]	> 200	> 200	> 64	68.01	-0.35	Soluble	Yes (0)	85.53	No	No	Yes
PYZ	3.125 ± 0.0* 3.125 ± 0.0]	> 200	> 200	> 64	68.87	-0.37	Soluble	Yes (0)	85.23	No	No	No

ND: not determined; * p < 0.05 (SPSS, version 20; n = 3); CI 99% = confidence interval 99% (SPSS, version 2.0; n = 3).

MTT Assay of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide

The toxicity profile of 4a-4j was evaluated against HepG2 cell (HCL) and Vero cell (VCL) lines by the MTT assay [13, 14]. The test is based on living cells (HCL, VCL, etc.) exhibiting dehydrogenase enzyme activity that converts MTT to formazan crystals. The intensity of formazan color (purple) is measured colorimetrically, and the %viability of cells is calculated. The HCL (5 × 103 cells/well) and VCL (104 cells/well) were placed in well plates and incubated (37°C) for 24 hours. The working solutions of the sample and standard (50, 100, 150, 200, 250, and 300 μg/ml) were prepared in Dulbecco’s Modified Eagle’s Medium (DMEM). The blank solution without standard/test compounds was also prepared. The working/standard/blank solutions were added to wells comprising HCL and VCL. The HCL and VCL well plates were incubated for 24 hours and 72 hours, respectively. The MTT reagent (50 μl, 2 mg/ml) was added to well plates and incubated for 4 hours. The sterile dimethyl sulfoxide (50 μl) was added to each well to dissolve the formed crystals of formazan. The optical density (OD) of the wells was measured at 540 nm utilizing an Elisa reader. The %cell viability (OD of test × 100 / OD of blank), and %cell inhibition (100 - %cell viability) were calculated. The CC₅₀ values (minimum concentration needed for 50% cell death) were determined by the curve fitting program, and the selectivity index (SI = CC₅₀/MI) was also determined (Table 1).

Determination of Physicochemical (PC) and Pharmacokinetic (PK) Parameters

The Swiss web server [15] was used to determine the PC and PK parameters of the compounds, whereas the mutagenicity was found by the test software [16]. The SMILES of the compounds were generated, incorporated into the software, and the data were collected (Table 1). The percentage absorption was calculated according to the following formula [17]:

$$\%\mathrm{ABS}=109-\left(0.345\times \text{tPSA}\right),$$

where tPSA is the topological polar surface area.

Molecular Docking Studies

It was performed by Molecular Operating Environment (MOE) 2019.0102 (Chemical Computing Group Inc., Canada). The chain A of various MtB proteins (PDB IDs: 6HEZ, 4NCR, and 4F4Q) were used for the docking purpose [18 – 20]. Chain A was purified by employing the Quickprep functionality of the software. The ligand structures (4a-4j and the standard drugs) were also prepared and stored as mdb files. The docking was done by the default docking setting of the software with 10 poses. The docking score (DS) and the root mean square deviation (RMSD) of the docked molecules are provided in Table 2.

Table 2.

Molecular Docking Results and Anti-TB Activity of Compounds 4a-4j against Mtb

Compound	6HEZ (Chain A)			4NCR (Chain A)			4F4Q (Chain A)
	DS [CI 99%]	RMSD [CI 99%]	Main interacting amino acids	DS [CI 99%]	RMSD [CI 99%]	Main interacting amino acids	DS [CI 99%]	RMSD [CI 99%]	Main interacting amino acids
4a	-6.46 ± 0.03* [-6.505 to -6.415]	0.99 ± 0.01* [0.975 to 1.005]	Lys418	-6.22 ± 0.04* [-6.279 to -6.161]	0.78 ± 0.03* [0.735 to 0.825]	Lys418, Tyr415, Lys134	-6.63 ± 0.04* [-6.689 to -6.571]	1.24 ± 0.05* [1.166 to 1.314]	Val372, Gly124
4b	-6.94 ± 0.02* [-6.970 to -6.910]	1.20 ± 0.03* [1.155 to 1.245]	Lys418, Tyr60	-7.08 ± 0.05* [-7.154 to -7.006]	1.40 ± 0.04* [1.341 to 1.459]	Tyr415	-6.41 ± 0.01* [-6.425 to -6.395]	1.16 ± 0.04* [1.101 to 1.219]	Lys425
4c	-7.01 ± 0.01* [-7.025 to -6.995]	0.87 ± 0.02* [0.840 to 0.900]	Lys418, Tyr60	-6.65 ± 0.05* [-6.724 to -6.576]	0.82 ± 0.04* [0.761 to 0.879]	Lys134	-6.74 ± 0.01* [-6.755 to -6.725]	0.95 ± 0.04* [0.891 to 1.009]	Val372, Gly124, Lys425
4d	-6.86 ± 0.03* [-6.905 to -6.815]	1.27 ± 0.03* [1.225 to 1.315]	Tyr415	-6.75 ± 0.02* [-6.780 to -6.720]	1.14 ± 0.03* [1.095 to 1.185]	Leu363, Gly117	-6.60 ± 0.03* [-6.645 to -6.555]	1.48 ± 0.03* [1.435 to 1.525]	-
4e	-6.36 ± 0.05* [-6.434 to -6.286]	0.83 ± 0.04* [0.771 to 0.889]	Lys418, Val365	-6.57 ± 0.04* [-6.629 to -6.511]	1.31 ± 0.05* [1.236 to 1.384]	Leu363, Gly117, Lys134	-7.02 ± 0.04* [-7.079 to -6.961]	1.08 ± 0.05* [1.006 to 1.154]	-
4f	-7.07 ± 0.04* [-7.129 to -7.011]	1.03 ± 0.05* [0.956 to 1.104]	-	-6.62 ± 0.05* [-6.694 to -6.546]	1.29 ± 0.01* [1.275 to 1.305]	His132, Gly117	-6.73 ± 0.05* [-6.804 to -6.656]	1.33 ± 0.02* [1.300 to 1.360]	Gly124
4g	-7.03 ± 0.02* [-7.060 to -7.000]	1.30 ± 0.03* [1.255 to 1.345]	Lys418	-6.76 ± 0.03* [-6.805 to -6.715]	1.33 ± 0.04* [1.271 to 1.389]	Lys418, Gly117, Lys134	-6.11 ± 0.03* [-6.155 to -6.065]	1.25 ± 0.04* [1.191 to 1.309]	Pro123
4h	-7.21 ± 0.01* [-7.225 to -7.195]	0.95 ± 0.04* [0.891 to 1.009]	-	-6.57 ± 0.02* [-6.600 to -6.540]	1.02 ± 0.01* [1.005 to 1.035]	His132	-6.95 ± 0.04* [-7.009 to -6.891]	1.43 ± 0.03* [1.385 to 1.475]	His139, Gly124
4i	-6.27 ± 0.03* [-6.315 to -6.225]	1.29 ± 0.04* [1.231 to 1.349]	Lys418, Arg58, Gly117	-6.90 ± 0.03* [-6.945 to -6.855]	1.29 ± 0.02* [1.260 to 1.320]	-	-7.02 ± 0.04* [-7.079 to -6.961]	0.64 ± 0.03* [0.595 to 0.685]	Gly124
4j	-7.53 ± 0.04* [-7.589 to -7.471]	1.36 ± 0.03* [1.315 to 1.405]	Lys418, Tyr60	-6.94 ± 0.02* [-6.970 to -6.910]	0.99 ± 0.02* [0.960 to 1.020]	Ly418	-7.10 ± 0.04* [-7.159 to -7.041]	1.33 ± 0.04* [1.271 to 1.389]	Gly124
BTZ043	-6.82 ± 0.03* [-6.865 to -6.775]	1.11 ± 0.05* [1.036 to 1.184]	Cy387, Val365, Lys134	-6.70 ± 0.03* [-6.745 to -6.655]	1.17 ± 0.01* [1.155 to 1.185]	Gly133	-5.90 ± 0.03* [-5.945 to -5.855]	1.27 ± 0.04* [1.211 to 1.329]	Cys394, Val372, Lys141
Macozinone	-6.76 ± 0.03* [-6.805 to -6.715]	1.23 ± 0.02* [1.200 to 1.260]	-	-7.14 ± 0.04* [-7.199 to -7.081]	1.44 ± 0.04* [1.381 to 1.499]	Cys387, Lys134	-7.26 ± 0.04* [-7.319 to -7.201]	1.24 ± 0.05* [1.166 to 1.314]	Gly124
ETH	-4.71 ± 0.03* [-4.755 to -4.665]	1.22 ± 0.03* [1.175 to 1.265]	Lys418, Lys134	-4.98 ± 0.04* [-5.039 to -4.921]	1.36 ± 0.04* [1.301 to 1.419]	Lys134	-5.09 ± 0.01* [-5.105 to -5.075]	1.27 ± 0.05* [1.196 to 1.344]	His139
PTH	-4.78 ± 0.02* [-4.810 to -4.750]	0.78 ± 0.01* [0.765 to 0.795]	-	-5.13 ± 0.03* [-5.175 to -5.085]	0.62 ± 0.05* [0.546 to 0.694]	Lys134	-5.09 ± 0.02* [-5.120 to -5.060]	1.30 ± 0.04* [1.241 to 1.359]	-
INH	-4.38 ± 0.01* [-4.395 to -4.365]	1.25 ± 0.02* [1.220 to 1.280]	Lys134, Leu115	-4.63 ± 0.04* [-4.689 to -4.571]	1.49 ± 0.04* [1.431 to 1.549]	Lys134, Leu115	-4.37 ± 0.03* [-4.415 to -4.325]	1.34 ± 0.04* [1.281 to 1.399]	Leu122, Gly124
PYZ	-4.23 ± 0.03* [-4.275 to -4.185]	1.42 ± 0.04* [1.361 to 1.479]	Lys418, His132	-4.04 ± 0.02* [-4.070 to -4.010]	1.07 ± 0.01* [1.055 to 1.085]	Leu115, Gly117	-4.18 ± 0.04* [-4.239 to -4.121]	1.38 ± 0.03* [1.335 to 1.425]	-

*p < 0.05 (SPSS, version 2.0; n = 3); CI 99% = confidence interval 95% (SPSS, version 2.0; n = 3).

Statistical Analysis

All results were statistically processed. The statistical analysis of results was done utilizing SPSS software (version 20, Chicago, IL, USA). The data is represented as mean±standard deviation. The p-value < 0.05 (number of experiments = 3) denotes statistically significant result, whereas CI 99% represents confidence interval 99%.

Results and Discussion

Chemistry

Compounds (4a-4j) were synthesized corresponding to Schemes 1 and 2. The substituted salicyl aldehydes (1a-1e) were reacted with ethyl acetoacetate (2) to get 6-substituted-3-acetylcoumarins, which were treated with bromine to provide compounds 3a-3e (Scheme 1). The intermediates (3a-3e) were reacted with ETH and PTH to obtain 4a-4e and 4f-4j, respectively (Scheme 2). The novelty of 4a-4j was revealed by an accurate structural search in the Sci-Finder database. The IR spectrum of 4a-4j showed characteristic peaks for C=O (1715 – 1721 cm^-1), C=N (1562 – 1568 cm^-1), C=C (1525 – 1530 cm^-1), C–O–C (1255 – 1258 cm^-1), and C–S (1110 – 1115 cm^-1). The ¹H NMR spectra of 4a-4j displayed characteristic singlets for the proton of C4-coumarin (ä 8.08 – 8.11 ppm), C3-pyridine (δ 8.28 – 8.31), and C5-thiazole (δ 8.39 – 8.42 ppm). The proton at C6-pyridine was the most deshielded and appeared as a doublet at δ 8.76 – 8.79 ppm. The methyl proton of 4a-4e appeared as a triplet at δ 1.26 – 128 ppm, whereas the methyl proton of 4f-4j appeared at δ 0.95 – 0.96 ppm due to the shielding effect. Similar effects were observed for the –CH₂- group adjacent to the -CH₃ group (quartet at δ 3.35 – 3.38 ppm for 4a-4e and multiplet at δ 1.79 – 1.81 ppm for 4f-4j). The -CH₂- group of 4f-4j attached to the pyridine ring appeared at δ 3.02 – 3.04 ppm. The ¹³C NMR spectra of 4a-4j displayed a characteristic peak for C=O (δ 160.7 – 160.9) ppm, and the methyl carbon (δ 13.2 – 13.4 ppm). The –CH₂- group carbon adjacent to the –CH₃ group appeared at δ 30.1 – 30.3 ppm for 4a-4e, whereas it appeared at δ 22.4 – 22.6 ppm for 4f-4j due to the shielding effect. The -CH₂- group of 4f-4j attached to the pyridine ring appeared at δ 41.0 – 41.1 ppm due to the deshielding effect. The mass spectra and the elemental analysis were in concurrence with the assigned structures of 4a-4j.

Anti-TB Activity and Toxicity Studies

The anti-TB activity of compounds 4a-4j unveiled the potency of synthesized derivatives (Table 1). It was surprising to observe that all these compounds displayed equal or better anti-TB activity than INH and PYZ against Mtb. All these compounds also displayed better anti-TB activity than ETH and PTH. Compounds 4a, 4b, 4f, and 4g displayed equal MIC values in comparison to INH and PYZ, whereas 4c-4e and 4h-4j displayed better MIC values than INH and PYZ (Table 1, Fig. 3). The MIC values were statistically significant (p-values and CI 99% values). This observation indicates that the synthesized coumarin-thiazole-pyridine nucleus is a promising pharmacophore to develop potent anti-TB drugs. The presence of -Cl, -Br, and -I (4c-4e and 4h-4j) in this nucleus provides more potent compounds than the fluorine substituted nucleus (4b and 4f). This effect might be because of the higher lipophilic character of 4c-4e and 4h-4j (Table 1). The literature has reported molecules similar to 4a-4j (compounds A and B of Fig. 1) [6, 7]. Compound A of Fig. 1 stated MIC values in the range of 15 to >663 μg/ml against Mtb H37Rv, whereas compound B testified MIC between 6.25 – 25 μg/mL. The MIC values expressed by compounds A and B were more than the MIC values of 4a-4j. The 4a-4j possess a pyridine ring at C-2 of the thiazole ring, whereas the pyridine ring is not the structural part of compounds A and B. Therefore, the author trusts that 4a-4j displayed higher potency than compounds A and B because of the presence of the pyridine ring in the structure of 4a-4j. The MTT assay of 4a-4j against HCL and VCL demonstrated non-toxic behavior of 4a-4j up to 300 μg/ml concentration. The selectivity index of 4a-4j was also higher than that of clinically used drugs (ETH, PTH, INH, and PYZ) (Table 1, Fig. 3).

Fig. 3.

MIC and selectivity index values of compounds 4a-4j, ETH, PTH, INH, and PYZ.

Molecular Docking Studies

The author was surprised to observe the potent anti-TB activity displayed by compounds 4a-4j. Accordingly, the author also performed the molecular docking of compounds using various proteins of Mtb (Table 2) to identify the possible mechanism of action and the reason behind the potency of the synthesized compounds. For this purpose 6HEZ protein [18] and 4F4Q protein [19] of DprE1 enzyme complexed with BTZ043, and 4NCR protein [20] of DprE1 enzyme complexed with macozinone were employed. The DprE1 enzyme is a new validated target to develop novel anti-TB agents [2]. BTZ043 and macozinone are DprE1 inhibitors, which are in a clinical trial [2, 21]. Like MIC value data, the DS and the RMSD values were also statistically significant (p-values and CI 99% values). The docking results revealed that the interacting pattern of BTZ043 and macozinone with 6HEZ/4F4Q(Cy387 and Lys134) and 4NCR (Cy394, Lys141) proteins were as per the reported literature [2, 18 – 20] (Figs. 4–6). A higher negative value of the docking score (DS) is an indicator of the potency of a compound, whereas the RMSD value < 2 represents good binding with the protein receptor. Compounds 4b-4d, 4f-4h, and 4j displayed a higher negative DS than BTZ043 and maozinone with 6HEZ. Compounds 4b, 4c, 4g, and 4j displayed interaction with Lys418 and other amino acids of 6HEZ. This interaction might be the reason for the better DS of these compounds. Compounds 4b, 4d, 4g, 4i, and 4j displayed a higher negative DS than BTZ043 with 4NCR. However, the interaction patterns of these compounds with 4NCR were different from each other. Compounds 4a-4j displayed a higher negative DS than BTZ043 with 4F4Q. The interaction of 4a, 4c, 4f, and 4h-4j with Gly124 of 4F4Q might be responsible for their higher negative DS. Compound 4j displayed the highest negative DS with 6HEZ (Fig. 7) in addition to 4F4Q (Fig. 8), and the second maximum negative DS with 4NCR (Fig. 9). The molecular docking data of compounds 4a-4j suggest that they are inhibitors of DprE1 (Fig. 10).

Fig. 5.

Interaction of BTZ043 with Chain A of 4F4Q.

Fig. 4.

Interaction of BTZ043 with Chain A of 6HEZ.

Fig. 6.

Interaction of macozinone with Chain A of 4NCR.

Fig. 7.

Interaction of 4j with Chain A of 6HEZ.

Fig. 8.

Interaction of 4j with Chain A of 4F4Q.

Fig. 9.

Interaction of 4j with Chain A of 4NCR.

Fig. 10.

The docking scores of compounds 4a-4j and other drugs involving DprE1 proteins (6HEZ, 4NCR, and 4F4Q).

PC and PK Parameters

The physicochemical (PC) properties of a compound are determinants of its pharmacokinetic (PK) parameters and behavior [22]. According to the PC values of compounds 4a-4j and standard drugs, the LogP values of compounds were higher than those of standard drugs. Compounds 4a-4j, BTZ043, macozinone, ETH, INH, and PYZ did not exhibit BBB permeation property (except for PTH). All compounds obeyed Lipinski’s rule (drug likeliness). The calculated gastrointestinal absorption of 4a-4j was higher than that of BTZ043 and macozinone. None of the compounds displayed P-gp substrate inhibitory property. This indicates that their PK behavior will not be affected by drugs that induce or inhibit these enzymes. Further, only compound 4b displayed mutagenicity potential. All these parameters of 4a-4j indicate their promising PK and safety profile in comparison to the existing anti-TB drugs [2, 23].

In conclusion, the ETH and PTH-based coumarinyl-thiazole derivatives (4a-4j) displayed outstanding anti-TB activity against Mtb H37Rv and did not demonstrate any toxicity against HCL and VCL. The molecular docking studies demonstrate that 4a-4j are DprE1 inhibitors. The in silico studies revealed that 4a-4j followed Lipinski’s rule (drug-likeliness) and exhibited higher gastrointestinal absorption than BTZ043 and macozinone. These observations indicate that the synthesized nucleus is a good template for developing selective DprE1 enzyme inhibitors and potent anti-TB agents. Accordingly, further structure-activity relationship studies are recommended.

Funding

The author extends his appreciation to the Deputyship for Research & Innovation, Ministry of Education, Saudi Arabia, for funding this research work through the project number IF-2020-NBU-209.