Synthesis and Biological Evaluation of Coumarin Derivatives as Inhibitors of Mycobacterium bovis (BCG)

Authors

  • Ali Hossein Rezayan,

    1. Department of Life Science Engineering, Faculty of New Science and Technology, University of Tehran, Tehran, Iran
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  • Parisa Azerang,

    1. Drug Design and Bioinformatics Unit, Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute, Tehran, Iran
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  • Soroush Sardari,

    Corresponding author
    1. Drug Design and Bioinformatics Unit, Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute, Tehran, Iran
      Corresponding author: Soroush Sardari,ssardari@hotmail.com; sardari@pasteur.ac.ir
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  • Afshin Sarvary

    1. Department of Chemistry, Faculty of Basic Science, Babol University of Technology, Babol, Iran
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Corresponding author: Soroush Sardari,ssardari@hotmail.com; sardari@pasteur.ac.ir

Abstract

The coumarin compounds are an important class of biologically active molecules, which have attractive caught the attention of many organic and medicinal chemists, due to potential pharmaceutical implications and industrial applications. We herein report the one-pot procedure for the efficient synthesis of coumarin derivatives using commercially available substrates via isocyanide-based multicomponent condensation reactions. These compounds were evaluated for anti-mycobacterium activity against Mycobacterium bovis (Bacillus Calmette–Guerin). The preliminary results indicated that all of the tested compounds showed relatively good activity against the test organism. The compounds 7e, 7l, and 7m showed high anti-tuberculosis activity.

Infectious diseases remain the largest cause of the death in the world today, greater than cardiovascular disease or cancer (1). Tuberculosis (TB) is one of the deadly infectious diseases caused by Mycobacterium tuberculosis, within the Mycobacterium spp. This notorious pathogen infects about one-third of the world’s population and is responsible for approximately 2 million deaths worldwide per year (2–4). In 2005, mortality and morbidity statistics included 14.6 million chronic active TB cases, 8.9 million new cases, and 1.6 million deaths, mostly in developing countries (5). The World Health Organization (WHO) Fact Sheet on TB estimates that between 2000 and 2020, nearly one billion people will get sick and 35 million will die from TB (6). On the other hand, Bacillus Calmette–Guerin (BCG) is an attenuated strain of Mycobacterium bovis, a non-virulent tubercle bacillus very closely related to M. tuberculosis (7,8). Therefore, M. bovis is simpler to use and in less strict biosafety regulations in the laboratory; hence, it can be used in earlier screenings instead of M. tuberculosis.

Serious challenges associated with the rising epidemic are multidrug resistance and the growing number of people co-infected with M. tuberculosis and human immunodeficiency virus (HIV) (9). Classically, two intervention strategies exist for infectious diseases: (I) chemotherapy, which aims at curing diseased individuals using chemical substances that preferentially eliminate the pathogen, and (II) vaccination, which aims at preventing infection in healthy individuals by priming their protective immune response. In the case of TB, both have existed since last century but recent escalation in cases has prompted research aimed at improving them.

The chemotherapy agents for TB can be divided into two classes: First-line drugs are including isoniazid (INH), pyrazinamide (PZA), ethambutol (EMB), rifampin (RIF), and streptomycin given for 6 months. If the treatment fails as a result of bacterial drug resistance, or intolerance to one or more drugs, second-line drugs are used, such as para-aminosalicylate, kanamycin, fluoroquinolones, capreomycin, ethionamide, and cycloserine, that are generally either less effective or more toxic with serious side-effects (10,11).

Hence, it is clear that there is an urgent need to develop novel anti-TB drugs with improved properties such as enhanced activity against multidrug resistance, reduced toxicity, shortened duration of therapy, rapid mycobactericidal mechanism of action, ability to penetrate host cells, and exert anti-mycobacterial effect in the intracellular environment. There are various sources for providing molecules with the desired profile of biologic activity, among which natural products and synthetic reactions are two important ones. Although many natural products present the scaffolds with different applications, the synthetic routes have the advantage of diversity of variation in derivative functionalization.

Multicomponent reactions (MCRs) are special types of synthetically useful organic reactions in which three or more different starting materials react to give a final product in a one-pot protocol. Most of these reactions are atom-efficient processes by incorporating the essential parts of the starting materials into the final product. Major applications of MCRs described until today arise from the area of drug discovery. Potentially, the ease of performance, the time-saving aspect, the versatility and diversity of scaffolds, and the very large chemical space will attract chemists in pharmaceutical companies to use MCRs for their projects. Recently, the pharmaceutical industries have focused more and more on diversity-oriented combinatorial libraries (12–15).

Coumarin and their derivatives are very important biological active compounds for organic and medicinal chemists. They are widely used as additives in food, perfumes, cosmetics, pharmaceuticals, optical brighteners, dispersed fluorescent, and laser dyes (16). The antifungal and anti-TB activities of some coumarin derivatives as a novel pharmacophore have also been investigated (1,17–19). Our literature survey showed that there are few examples in which the coumarin with different substitutions was investigated against M. bovis and M. tuberculosis (18,19). Very recently, a series of coumarins with carbohydrazides substitution only in position 3 was synthesized and evaluated against M. bovis. These compounds exhibited a significant activity (50–100 μg/mL) when compared with the first-line drugs (18).

In continuation of our research on the development of synthetic methods in heterocyclic chemistry via MCRs (20–22), specifically in coumarin derivatives (17,23–25) and also our drug discovery program (26–28), here we report synthesis of different coumarin derivatives and evaluation of their anti-TB activity.

Methods and Materials

General

Melting points were measured on an Electrothermal 9100 apparatus and are uncorrected. IR spectra were recorded on a Shimadzu IR-470 spectrometer. 1H and 13C NMR spectra were recorded on a BRUKER DRX-300 AVANCE spectrometer at 300.13 and 75.4 MHz. The NMR spectra were obtained on solution in DMSO using TMS as internal standard. The chemical shifts were referenced to the solvent peak, namely δ = 2.50 ppm for (CD3)2SO/DMSO-d6 using TMS as an internal standard. All the chemicals and reagents were purchased from commercial suppliers like Merck (Whitehouse Station, NJ, USA), Fluka, and Sigma-Aldrich (St Louis, MO, USA).

General procedure for preparation of products 5a–f

In a round-bottomed flask, the selected salicylaldehyde (1 mmol) and Meldrum’s acid (0.14 g, 1.2 mmol) in water (20 mL) were heated at reflux under stirring for 12 h; then, the reaction mixture was cooled and filtered on Büchner funnel. The products were purified, if necessary, by crystallization from ethyl acetate or methanol. All products were known compounds (except 5d and 5f, Table 1), which were identified by 1H NMR and 13C NMR spectral data, and their melting points were compared with literature reports.

Table 1.   The structure of coumarin derivatives and their MIC (μg/mL) against Mycobacterium bovis BCG in microbroth dilution method assay Thumbnail image of

6-Chloro-2-oxo-2H-chromene-3-carboxylic acid 5d

White powder (0.3 g, yield 75%), mp 197–202 °C. 1H NMR (300 MHz, DMSO-d6): δH (ppm) 7.45 (1H, d, 3J = 8.9 Hz, CH arom), 7.73 (1H, dd, 3J = 6.2, and 2.7 Hz, CH arom), 8.00 (1H, d, 3J = 2.7 Hz, CH arom), 8.66 (1H, s, CH=C-CO2H)). 13C NMR (75.4 MHz, DMSO-d6): δC (ppm) 118.3, 119.5, 119.8, 128.6, 129.1, 133.8 (6C arom), 147.1 (=C-Cl), 153.2 (=C-O), 156.4 (C=O), 164.0 (C=O).

6-Nitro-2-oxo-2H-chromene-3-carboxylic acid 5f

Cream powder (0.19 g, yield 80%), mp 235–238 °C. 1H NMR (300 MHz, DMSO-d6): δH (ppm) 7.64 (1H, d, 3J = 9.1 Hz, CH arom), 8.50 (1H, dd, 3J = 6.2, and 2.7 Hz, CH arom), 8.91 (1H, d, 3J = 2.7 Hz, CH arom), 8.90 (1H, s, CH=C-CO2H)). 13C NMR (75.4 MHz, DMSO-d6): δC (ppm) 117.7, 118.4, 120.3, 126.0, 126.3, 143.6 (6C arom), 147.2 (=C-NO2), 155.4 (=C-O), 158.1 (C=O), 163.05 (C=O).

General procedure for preparation of products 6a–h

To a magnetically stirred solution of Meldrum’s acid (0.14 g, 1 mmol), salicylaldehyde (1 mmol) in dichloromethane (5 mL) was added isocyanide (1 mmol). The reaction mixture was stirred for 24–36 h at room temperature. After completion of the reaction, as indicated by TLC (ethyl acetate/n-hexane, 2:1), the solvent was removed under vacuum and the solid residue was washed with ether, and the product 6a–h was obtained as a white powder (23).

N-tert-Butyl-3,4-dihydro-2-oxo-2H-chromene-4-carboxamide 6a

White powder; mp 219–221 °C. IR (KBr) (νmax/cm−1): 3296 (NH), 2976, 2941, 1772, 1641, 1558. MS, m/z (%): 247 (M+, 15), 191 (5), 148 (100), 147 (100), 131 (25), 120 (25), 91 (50), 57 (90), 41 (65). 1H NMR (300 MHz, DMSO-d6): δH (ppm) 1.20 (9H, s, C(CH3)3), 2.69 (1H, dd, 2JHH = 16.1 Hz, 3JHH = 2.4 Hz, CH2-CH), 2.93 (1H, dd, 2JHH = 16.1 Hz, 3JHH = 6.0 Hz, CH2-CH), 3.85 (1H, dd, 2JHH = 6.0 Hz, 3JHH = 2.4 Hz, CH2-CH), 7.01–7.46 (4H, m, H-Ar), 7.97 (1H, s, NH). 13C NMR (75 MHz, DMSO-d6): δC (ppm) 28.77 (C(CH3)3), 31.79 (CH2-CH), 42.22 (CH2-CH), 50.73 (C(CH3)3), 117.03, 122.93, 124.52, 128.48, 129.15, 151.99 (C-Ar), 167.55, 171.23 (2C=O).

N-Benzyl-3,4-dihydro-2-oxo-2H-chromene-4-carboxamide 6b

White powder; mp 169–172 °C. 1H NMR (300 MHz, DMSO-d6): δH (ppm) 2.80 (1H, d, 2JHH = 15.9 Hz, CH2-CH), 3.00 (1H, dd, 2JHH = 15.9 Hz, 3JHH = 5.5 Hz, CH2-CH), 3.95 (1H, d, 3JHH = 5.5 Hz, CH2-CH), 4.28 (2H, brs, CH2 of benzyl), 7.04–7.49 (9H, m, H-Ar), 8.86 (1H, s, NH). 13C NMR (75.4 MHz, DMSO-d6): δC (ppm) 31.71 (CH2-CH), 41.77 (CH2-CH), 42.57 (CH2 of benzyl), 117.13, 122.54, 124.59, 127.37, 127.59, 128.71, 128.78, 129.42, 139.30, 151.98 (C-Ar), 167.43, 171.68 (2C=O).

N-tert-Butyl-6-bromo-3,4-dihydro-2-oxo-2H-chromene-4-carboxamide 6c

White powder; mp 225–228 °C (dec). IR (KBr) (νmax/cm): 3297(NH), 3084, 2975, 2928, 1774, 1642, 1557. 1H NMR (300 MHz, DMSO-d6): δH (ppm) 1.20 (9H, s, C(CH3)3), 2.70 (1H, d, 2JHH = 16.1 Hz, CH2-CH), 2.93 (1H, dd, 2JHH = 16.1 Hz, 3JHH = 5.9 Hz, CH2-CH), 3.84 (1H, d, 3JHH = 5.9 Hz, CH2-CH), 7.02 (1H, d, 3JHH = 8.6 Hz, H-Ar), 7.46 (1H, d, 3JHH = 8.6 Hz, H-Ar), 7.70 (1H, s, H-Ar), 7.96 (1H, s, NH).

6-Bromo-N-cyclohexyl-3,4-dihydro-2-oxo-2H-chromene-4-carboxamide 6d

White powder; mp 203–206 °C. IR (KBr) (νmax/cm): 3317 (NH), 2928, 2853, 1781, 1641, 1551. MS, m/z (%): 353 (M+, 81Br, 15), 351 (M+, 79Br, 15), 272 (10), 270 (10), 227 (40), 226 (75), 225 (40), 200 (50), 198 (50), 147 (5), 118 (40), 102 (10), 83 (100), 55 (95). 1H NMR (300 MHz, DMSO-d6): δH (ppm) 1.03–1.74 (10H, m, 5CH2 of cyclohexyl), 2.72 (1H, d, 2JHH = 16.0 Hz, CH2-CH), 2.91 (1H, dd, 2JHH = 16.0 Hz, 3JHH = 5.6 Hz, CH2-CH), 3.33 (H, m, CH-N of cyclohexyl), 3.83 (1H, d, 2JHH = 5.6 Hz, CH2-CH), 6.98 (1H, d, 3JHH = 8.6 Hz, H-Ar), 7.43 (1H, d, 3JHH = 8.6 Hz, H-Ar), 7.64 (1H, s, H-Ar), 8.16 (1H, d, 3JHH = 7.2 Hz, NH). 13C NMR (75 MHz, DMSO-d6): δC (ppm) 24.65, 24.76, 25.52, 32.49, 32.59 (C-cyclohexyl), 31.26 (CH2-CH), 41.53 (CH2-CH), 48.12 (CH-N of cyclohexyl), 115.97, 119.31, 125.25, 130.96, 131.92, 151.28 (C-Ar), 166.93, 170.12 (2C=O).

N-Benzyl-6-bromo-3,4-dihydro-2-oxo-2H-chromene-4-carboxamide 6e

White powder; mp 165–168 °C (dec). IR (KBr) (νmax/cm): 3334 (NH), 3033, 2920, 1763, 1641, 1528. 1H NMR (300 MHz, DMSO-d6): δH (ppm) 2.83 (1H, d, 2JHH = 16.2 Hz, CH2-CH), 3.01 (1H, dd, 2JHH = 16.2 Hz, 3JHH = 5.9 Hz, CH2-CH), 3.96 (1H, d, 3JHH = 5.9 Hz, CH2-CH), 4.26 (2H, brs, CH2 of benzyl), 7.04 (1H, d, 3JHH = 8.6 Hz, H-Ar), 7.16–7.31 (5H, m, H-Ar), 7.50 (1H, d, 3JHH = 8.6 Hz, H-Ar), 7.74 (1H, s, H-Ar), 8.84 (1H, s, NH).

6-Bromo-3,4-dihydro-2-oxo-N-(tosylmethyl)-2H-chromene-4-carboxamide 6f

White powder; mp 198–203 °C (dec). IR (KBr) (νmax/cm): 3314 (NH), 3057, 2935, 1797, 1781, 1685, 1597. MS, m/z (%): 439 (M+, 81Br, 30), 437 (M+, 79Br, 30), 358 (20), 227 (50), 226 (70), 225 (50), 200 (25), 198 (25), 118 (30), 102 (5), 91 (100), 55 (55). 1H NMR (300 MHz, DMSO-d6): δH (ppm) 2.32 (CH3), 2.52 (1H, d, 2JHH = 16.2 Hz, CH2-CH), 2.90 (1H, dd, 2JHH = 16.2 Hz, 3JHH = 5.9 Hz, CH2-CH), 3.88 (1H, d, 3JHH = 5.9 Hz, CH2-CH), 4.56 (1H, dd, 2JHH = 14.1 Hz, 3JHH = 5.5 Hz, CH2-NH), 4.82 (1H, dd, 2JHH = 14.1 Hz, 3JHH = 7.6 Hz, CH2-NH), 6.96–7.73 (7H, m, H-Ar), 9.41 (1H, dd, 3JHH = 7.6 Hz, 3JHH = 5.5 Hz, CH2-NH). 13C NMR (75 MHz, DMSO-d6): δC (ppm) 21.54 (CH3), 31.13 (CH2-CH), 40.86 (CH2-CH), 60.03 (CH2-NH), 116.16, 119.29, 123.00, 128.67, 129.89, 131.46, 132.28, 134.28, 144.93, 151.19 (C-Ar), 166.44, 171.10 (2C=O).

N-Cyclohexyl-3,4-dihydro-7-methoxy-2-oxo-2H-chromene-4-carboxamide 6g

White powder; mp 146–147 °C (dec). IR (KBr) (νmax/cm): 3278 (NH), 3093, 2935, 2852, 1774, 1623, 1562. 1H NMR (300 MHz, DMSO-d6): δH (ppm) 1.04–1.77 (10H, m, 5CH2 of cyclohexyl), 2.69 (1H, dd, 2JHH = 16.0 Hz, 3JHH = 2.1 Hz, CH2-CH), 2.90 (1H, dd, 2JHH = 16.0 Hz, 3JHH = 6.0 Hz, CH2-CH), 3.37 (1H, dd, 2JHH = 6.0 Hz, 3JHH = 2.1 Hz, CH2-CH), 3.40 (H, m, CH-N of cyclohexyl), 3.73 (3H, s, OCH3), 6.62 (1H, d, 4JHH = 2.3 Hz, H-Ar), 6.71 (1H, dd, 3JHH = 8.4 Hz, 4JHH = 2.3 Hz, H-Ar), 7.32 (1H, d, 3JHH = 8.4 Hz, H-Ar), 8.13 (1H, d, 3JHH = 7.6 Hz, NH).

N-Cyclohexyl-3,4-dihydro-8-methoxy-2-oxo-2H-chromene-4-carboxamide 6h

White powder; mp 185–187 °C (dec). IR (KBr) (νmax/cm): 3329 (NH), 2936, 2852, 1772, 1640, 1537. 1H NMR (300 MHz, DMSO-d6): δH (ppm) 1.18–1.65 (10H, m, 5CH2 of cyclohexyl), 2.69 (1H, d, 2JHH = 16.0 Hz, CH2-CH), 2.91 (1H, dd, 2JHH = 16.0 Hz, 3JHH = 5.8 Hz, CH2-CH), 3.37 (H, m, CH-N of cyclohexyl), 3.78 (3H, s, OCH3), 3.80 (1H, d, 3JHH = 5.8 Hz, CH2-CH), 6.99–7.03 (3H, m, H-Ar), 8.16 (1H, d, 3JHH = 6.7 Hz, NH).

General procedure for preparation of products 7a–p

To a magnetically stirred solution of Meldrum’s acid (0.14 g, 1 mmol), salicylaldehyde (1 mmol) in ethanol or methanol (5 mL) was added isocyanide (1 mmol), and the reaction mixture stirred for 8 h at room temperature. After completion of the reaction, as indicated by TLC (ethyl acetate/n-hexane, 2:1), the precipitate was washed with ethanol and the product 7a–n was obtained as a white powder (24).

Methyl-4-(tert-butylcarbamoyl)-3,4-dihydro-2-oxo-2H-chromene-3-carboxylate 7a

White powder; mp 151−152 °C. IR (KBr) (νmax/cm): 3321, 3070, 2988, 1778, 1743, 1638, 1546. MS, m/z (%): 306 (M+, 15), 220 (25), 190 (20), 173 (35), 140 (100), 118 (50), 89 (30), 58 (90), 32 (40). 1H NMR (300 MHz, DMSO-d6) cis and trans: δH (ppm) 1.20 (18H, s, 2C(CH3)3, cis, and trans), 3.58 (3H, s, OCH3, trans), 3.69 (3H, s, OCH3, cis), 4.19 (2H, d, 3JHH = 4.1 Hz, 2CH-4, cis, and trans), 4.28 (2H, d, 3JHH = 4.1 Hz, 2CH-3, cis, and trans), 7.06–7.44 (8H, m, H-Ar, cis, and trans), 7.96 (1H, brs, NH, cis), 8.19 (1H, brs, NH, trans). 13C NMR (75 MHz, DMSO-d6) Cis: δC (ppm) 28.70 (C(CH3)3), 45.55 (CH-4), 48.47 (CH-3), 50.97 (C(CH3)3), 53.39 (OCH3), 117.10, 121.14, 125.17, 128.38, 129.69, 151.12 (C-Ar), 163.75, 167.77, 169.18 (3C=O). 13C NMR (75 MHz, DMSO-d6) Trans: δC (ppm) 28.77 (C(CH3)3), 45.13 (CH-4), 47.54 (CH-3), 50.63 (C(CH3)3), 52.67 (OCH3), 117.02, 121.93, 125.04, 128.34, 129.58, 151.15 (C-Ar), 163.92, 167.70, 170.14 (3C=O).

Ethyl-4-(tert-butylcarbamoyl)-3,4-dihydro-2-oxo-2H-chromene-3-carboxylate 7b

White powder; mp 178–180 °C. IR (KBr) (νmax/cm): 3401, 3345, 2981, 2929, 1769, 1746, 1689, 1543. MS, m/z (%): 320 (M+ + 1, 10), 274 (4), 246 (6), 220 (20), 190 (20), 173 (50), 147 (100), 118 (45), 91 (30), 58 (90), 32 (90). 1H NMR (300 MHz, DMSO-d6) cis and trans: δH (ppm) 1.03 (6H, t, 3JHH = 7.06, 2OCH2CH3, cis, and trans), 1.17 (9H, s, C(CH3)3, trans), 1.19 (9H, s, C(CH3)3, cis), 4.04 (4H, q, 3JHH = 7.06 Hz, 2OCH2CH3, cis, and trans), 4.15 (2H, d, 3JHH = 4.27 Hz, 2CH-4, cis, and trans), 4.27 (2H, d, 3JHH = 4.27 Hz, 2CH-3, cis, and trans), 7.04–7.46 (8H, m, H-Ar, cis, and trans), 7.95 (1H, brs, NH, trans), 8.19 (1H, brs, NH, cis). 13C NMR (75 MHz, DMSO-d6) cis: δC (ppm) 14.23 (OCH2CH3), 28.71 (C(CH3)3), 45.69 (CH-4), 48.64 (CH-3), 50.98 (C(CH3)3), 62.13 (OCH2CH3), 117.06, 121.18, 125.12, 128.34, 129.66, 151.20 (C-Ar), 163.90, 167.27, 169.13 (3C=O). 13C NMR (75 MHz, DMSO-d6) trans: δC (ppm) 14.52 (OCH2CH3), 28.77 (C(CH3)3), 45.14 (CH-4), 48.60 (CH-3), 50.33 (C(CH3)3), 61.62 (OCH2CH3), 117.00, 121.98, 124.05, 128.24, 129.60, 151.24 (C-Ar), 163.92, 167.14, 170.11 (3C=O).

Methyl-4-((ethoxycarbonyl)methylcarbamoyl)-3,4-dihydro-2-oxo-2H-chromene-3-carboxylate 7c

White powder; mp 183–185 °C. IR (KBr) (νmax/cm): 3355, 2931, 2853, 17.72, 16.50, 1541. MS, m/z (%): 332 (M+ + 1, 5), 331 (M+, 5), 300 (5), 272 (20), 190 (20), 173 (25), 147 (100), 118 (30), 83 (20), 55 (50), 41 (30).

Ethyl-4-(cyclohexylcarbamoyl)-3,4-dihydro-2-oxo-2H-chromene-3-carboxylate 7d

White powder; mp 167–170 °C. IR (KBr) (νmax/cm): 3356, 2933, 2851, 1759, 1649, 1543. MS, m/z (%): 345 (M+, 10), 299 (20), 272 (20), 218 (35), 190 (30), 173 (35), 147 (100), 118 (60), 91 (30), 56 (35), 32 (52).

Sec-butyl-4-(cyclohexylcarbamoyl)-3,4-dihydro-2-oxo-2H-chromene-3-carboxylate 7e

White powder (0.27 g, yield 73%); mp 108–110 °C. IR (KBr) (νmax/cm): 3306, 2932, 2885, 1801, 1725, 1645, 1548. MS, m/z (%): 373 (M+, 5), 300 (5), 272 (25), 190 (20), 173 (25), 147 (100), 118 (25), 83 (30), 57 (50), 41 (70).

Ethyl-4-(2,6-dimethylphenylcarbamoyl)-3,4-dihydro-2-oxo-2H-chromene-3-carboxylate 7f

White powder; mp 166–170°C. IR (KBr) (νmax/cm): 3356, 3066, 2952, 2917, 1782, 1747, 1655, 1544. MS, m/z (%): 353 (M+, 20), 321 (70), 294 (30), 275 (20), 173 (20), 147 (100), 118 (75), 91 (50), 83 (20), 51 (20), 31 (40).

Cycloheptyl-4-(2,6-dimethylphenylcarbamoyl)-3,4-dihydro-2-oxo-2H-chromene-3-carboxylate 7g

White powder; mp 159–163 °C. IR (KBr) (νmax/cm): 3316, 2922, 2855, 1800, 1735, 1615, 1555. MS, m/z (%): 435 (M+, 2), 339 (5), 321 (40), 295 (10), 147 (100), 118 (50), 81 (20), 57 (70), 41 (25). 1H NMR (300 MHz, DMSO-d6) cis and trans: δH (ppm) 1.45–1.90 (24H, m, 12CH2 of two cycloheptanes, cis, and trans), 2.10 (6H, brs, 2CH3, cis), 2.17 (6H, brs, 2CH3, trans), 4.08–4.98 (6H, m, 2OCH of cycloheptans, 2CH-4, 2CH-3, cis, and trans), 6.79–7.67 (14H, m, H-Ar, cis, and trans), 9.95 (1H, brs, NH, cis), 10.29 (1H, brs, NH, trans). 13C NMR (75 MHz, DMSO-d6) cis: δC (ppm) 17.58, 17.83 (2CH3), 22.40, 22.65, 28.03, 28.20, 33.01, 33.42 (C-cycloheptans), 48.68 (CH-4), 54.10 (CH-3), 77.06 (OCH), 115.79, 116.65, 122.71, 128.20, 128.66, 128.86, 129.75, 130.04, 132.19, 136.05, 136.99, 155.39 (C-Ar), 167.67, 171.24, 175.70 (3C=O). 13C NMR (75 MHz, DMSO-d6) trans: δC (ppm) 18.14, 18.50 (2CH3), 22.01, 22.40, 27.81, 28.09, 32.90, 33.12 (C-cycloheptans), 47.51 (CH-4), 48.73 (CH-3), 76.91 (OCH), 115.98, 120.11, 125.21, 126.18, 128.69, 130.48, 132.03, 130.94, 134.50, 135.72, 136.24, 154.47 (C-Ar), 166.14, 170.46, 175.25 (3C=O).

Ethyl-4-(benzylcarbamoyl)-3,4-dihydro-2-oxo-2H-chromene-3-carboxylate 7h

White powder; mp 136–138 °C. IR (KBr) (νmax/cm): 3372, 2975, 2925, 1782, 1735, 16.51, 1540. MS, m/z (%): 353 (M+, 20), 307 (20), 216 (15), 173 (30), 147 (50), 118 (55), 91 (100), 65 (20), 31 (20).

Ethyl-4-((ethoxycarbonyl)methylcarbamoyl)-3,4-dihydro-2-oxo-2H-chromene-3-carboxylate 7i

White powder; mp 126–129 °C. IR (KBr) (νmax/cm): 3317, 3105, 2976, 2941, 1784, 1737, 1646, 1614, 1565. MS, m/z (%): 349 (M+, 40), 303 (60), 276 (20), 246 (18), 229 (40), 202 (70), 173 (85), 146 (100), 118 (85), 91 (60), 57 (65), 32 (95).

Ethyl-4-(tosylmethylcarbamoyl)-3,4-dihydro-2-oxo-2H-chromene-3-carboxylate 7j

White powder; mp 145–148 °C. IR (KBr) (νmax/cm): 3313, 29.88, 2929, 1780, 1743, 1690, 1541. MS, m/z (%): 385 (M+-EtOH, 2), 321 (2), 230 (30), 202 (20), 159 (25), 118 (20), 91 (2), 56 (100), 31 (50). 1H NMR (300 MHz, DMSO-d6): δH (ppm) 1.16 (3H, brs, OCH2CH3), 2.35 (3H, s, CH3), 3.88–4.34 (4H, m, OCH2CH3, CH-4, CH-3), 5.03 (2H, brs, SO2-CH2-NH), 6.78–7.74 (8H, m, H-Ar), 10.04 (1H, brs, NH). 13C NMR (75 MHz, DMSO-d6): δC (ppm) 14.31 (OCH2CH3), 21.58 (CH3), 47.39 (CH-4), 53.24 (CH-3), 59.19 (SO2-CH2-NH), 62.34 (OCH2CH3), 115.70, 119.56, 122.33, 128.94, 129.97, 130.34, 131.65, 135.49, 145.62, 155.42 (C-Ar), 167.32, 170.44, 174.49 (3C=O).

Ethyl-4-(tert-butylcarbamoyl)-6-bromo-3,4-dihydro-2-oxo-2H-chromene-3-carboxylate 7k

White powder; mp 153–157 °C. IR (KBr) (νmax/cm): 3350, 2973, 2931, 1780, 1743, 1672, 1531. MS, m/z (%): 399 (M+, 81Br, 5), 397 (M+, 79Br, 5), 327 (5), 325 (5), 297 (10), 253 (15), 251 (15), 227 (50), 225 (50), 118 (20), 89 (20), 57 (100), 41 (40).

Ethyl-4-(benzylcarbamoyl)-6-bromo-3,4-dihydro-2-oxo-2H-chromene-3-carboxylate 7l

White powder; mp 160–165 °C. IR (KBr) (νmax/cm): 3401, 2976, 2917, 1784, 1774, 1660, 1526. MS, m/z (%): 411 (M+, 81Br, 5), 409 (M+, 79Br, 5), 397 (10), 377 (10), 352 (10), 350 (10), 298 (25), 296 (25), 253 (25), 227 (100), 225 (100), 198 (30), 196 (30), 147 (10), 118 (20), 83 (50), 55 (90), 41 (75).

4-Fluorobenzyl-4-(cyclohexylcarbamoyl)-6-bromo-3,4-dihydro-2-oxo-2H-chromene-3-carboxylate 7m

White powder; mp 162–165 °C. IR (KBr) (νmax/cm): 3306, 2932, 2858, 1801, 1725, 1645, 1548. MS, m/z (%): 461 (M+-44 (CO2), 10), 459 (M+-44 (CO2) 10), 381 (10), 379 (210), 352 (5), 296 (10), 227 (50), 225 (50), 198 (40), 126 (60), 109 (100), 83 (50), 55 (80), 31 (40).

Ethyl-4-(cyclohexylcarbamoyl)-3,4-dihydro-6-methoxy-2-oxo-2H-chromene-3-carboxylate 7n

White powder; mp 153–156 °C. IR (KBr) (νmax/cm): 3350, 2933, 2852, 1781, 1748, 1648, 1545. MS, m/z (%): 375 (M+, 5), 329 (10), 302 (10), 248 (10), 220 (10), 203 (10), 177 (100), 148 (20), 121 (10), 91 (5), 55 (20), 41 (10).

Compounds 8a and 8b were purchased from Merck chemical company.

In vitroevaluation of anti-mycobacterial activity

In vitro anti-mycobacterial activity evaluations of the compounds were carried out by the broth microtiter dilution method against BCG (1173P2). Ethambutol was used as a positive drug control.

The test compounds were initially dissolved in DMSO to give a concentration of 1 or 2 mg/L. All wells of micro plates received 100 μL of freshly prepared Middle broke 7H9 medium (Himedia, Mumbai, India), except first column. Two hundred microliters of distilled water was added to the first column of 96-well plates to minimize evaporation of the medium in the test wells during incubation. Then, 100 μL of test compounds with desired concentrations (1000 or 2000 μL) was added to the wells of the first row (each concentration was assayed in duplicate), and serial dilution was made from the first row to the last. Microbial suspension of BCG (1173P2) (100 μL), which had been prepared with standard concentration of 0.5 McFarland and diluted with 1:10 proportion by the distilled water, was added to all test wells. Plates were then sealed and incubated for 4 days at 37 °C. After that, 12 μL Tween 80 10% and 20 μL Alamar blue 0.01% (Himedia) were added to each test well. The results were assessed after 24 and 48 h. A blue color was interpreted as no bacterial growth, and color change to pink was scored as bacterial growth. Wells with a well-defined pink color were scored as positive for growth. The minimal inhibition concentration (MIC) was defined as the lowest drug concentration, which prevented a color change from blue to pink. Ethambutol (Irandaru, Tehran, Iran) was used as positive control and DMSO as negative control.

In silico calculation of CLogP

The CLogP value was obtained by Chem Draw (V. 9.0, 1996).

Results and Discussion

The reactions carried out are indicated in Scheme 1. Treatment of various 2-hydroxybenzaldehydes 2 with Meldrum’s acid 1 in water led to products 5a–f (29), and also, isocyanide-based, one-pot three- or four-component reactions between commercially available 2-hydroxybenzaldehyde (2), Meldrum’s acid (1), and an isocyanide (3) in the absence or presence of an aromatic or an aliphatic alcohol (4) (as reagent or solvent) in dichloromethane efficiently provide 3,4-dihydrocoumarin derivatives (6a–h) and (7a–n), respectively (23,24). Coumarins 8a and 8b are commercially available and were purchased from Sigma-Aldrich. All reaction products were characterized by IR, 1H, and 13C NMR spectra and melting point values as compared with those obtained from authentic samples (23,24,29). All the synthesized and purchased compounds were evaluated for anti-mycobacterial activity, and the results are summarized in Table 1.

Figure 
                Scheme 1:
              .

 Synthesis of coumarin derivatives examined for anti-mycobacterial activity in this study.

As can be seen in Table 1, in compounds 5, various derivatives were made and only the 6-nitro substituted compound (5f) showed more activity at 24 h. Among these molecules, an electron donating substitution (OMe) at positions 7 or 8 has little improving benefit compared with parent structure (5a) without substitution. Comparing the activity of 5d and 5e with that of 5f, it is evident that the electron withdrawal from the ring through resonance movement of π electrons has a more potent effect on improving the activity. Considering lipophilicity also, among the members of group 5, the biological activity seems to depend on the effect of substitution at position 6 by the electronegativity of that functional group, and this has been reflected in the CLogP value. For example, compound 5f has the highest activity and lowest lipophilicity among the group members.

In compounds 6, two positions (R1 in position 4 and X in position 6, 7, 8) are changed, and various derivatives (6a6h) were prepared among which compounds 6c and 6h have more activity than others. Comparing the activity of 6c, 6d, 6e, and 6f that have Br in position 6 and only differ in position 4 shows that tert-butyl (6c) is more and 4-toluene-methylsulfonyl (6f) is less active than the others. This difference in activity might be because of substitution size and lipophilicity. Products 6g and 6h have the same substitution (-OMe) but in different positions 7 and 8; the results show -OMe in position 8 exerts more effectivity. Among the compounds of group 6, there are patterns in lipophilicity values that can be attributed to the bioactivity. Comparing compound 6a and 6c reveals that bromine substitution at position 6 would enhance the bioactivity, while due to replacement of H with Br, the CLogP value is increasing. The nature of amide functional group is important as well, which can be observed by comparing compounds 6c to 6d, 6e and 6f, among them the tertiary butyl functional group exerts the highest activity, and lowest CLogP value.

For compounds of group 7, it is important to note that compound 7a–n has two stereogenic centers, and therefore, two pairs of diastereoisomers are expected. The 1H NMR and 13C NMR spectra of the reaction mixture show that the products are diastereoisomeric mixtures. Compounds 7 have three variable positions (X, R1, and R2), were prepared, and were tested as anti-mycobacterial agents. The results in Table 1 show compounds 7e, 7l, and 7m were almost equally active against M. bovis and more active than the other derivatives. Comparing these three active compounds (7e, 7l, and 7m) relative to remaining compounds shows that the size or type of substitutions (aliphatic, alicyclic, and or aromatic) in position 4 (R1) is more important than the two other positions (X and R2). On the other hand, by comparing 7f with 7g and 7l with 7m, it is evident that substitution on position 3 is not very important because the mentioned compounds in spite of having different substitutions in position 3 have same MIC [7f and 7g = 62.5 (μg/mL), 7l and 7m = 15.6 (μg/mL)]. Therefore, among different substitutions in position 4, cyclohexyl is more active and 4-toluene-methylsulfonyl and ethylacetate are less active groups. However, it is necessary to examine the mechanism of action in detail to clear the reason for the increase and decrease of activity. Comparing lipophilicity values for 7c, 7d, and 7e, a trend of increasing lipophilicity and bioactivity due to alkyl group enlargement at position 3 is observed. Bromination of compound 7c at position 6 would further increase the lipophilicity and bioactivity. Correlation of lipophilicity and activity is seen in sub-classes of compounds 7d and 7p also among 7b, 7j, and 7k as a comparable group and among the 6-Br derivatives 7m, 7n, and 7o.

Compounds 8a and 8b are only different in position of OH group. The results in Table 1 show OH group in position 7 contribute to more activity than position 4.

Our literature survey showed that very recently a series of coumarin with substitution only in position 3 were synthesized and evaluated against M. bovis. In comparison with reported papers (18,19), our results show promising activity (15.6–125 μg/mL versus 50–100 μg/mL), and also, synthesis of variety of coumarin derivatives with different substitution in different position via MCRs has advantages such as good yield, facile purification method, and high diversity.

In conclusion, all synthesized and purchased compounds were evaluated for anti-tubercular activities against M. bovis. Among them, compounds 7e, 7l, and 7m were almost equally active against M. bovis and more active than the other derivatives. The obtained results show that the size or type of substitutions (aliphatic, alicyclic, and or aromatic) in position 4 (R1) is more important than the two other positions (X and R2). Also, among different substitutions in position 4, cyclohexyl is more active and 4-toluene-methylsulfonyl and ethylacetate are less active groups that could be due to general lipophilicity produced by the functional group. Finally, these results make novel coumarin derivatives, alone or in combination of other agents, interesting molecule for more synthetic and biological evaluation.

Acknowledgment

We gratefully acknowledge the Pasteur institute of Iran and Ministry of Health and Medical Education in Iran for funding of this project as part of grant number 456.

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