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

Article abstract of DOI:10.1111/cbdd.12044

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 t

Full text of DOI:10.1111/cbdd.12044

ª 2012 John Wiley & Sons A/S
Chem Biol Drug Des 2012; 80: 929–936
doi: 10.1111/cbdd.12044
Research Article
Synthesis and Biological Evaluation of Coumarin
Derivatives as Inhibitors of Mycobacterium bovis
(BCG)
Ali Hossein Rezayan1, Parisa Azerang2,
Soroush Sardari2,* and Afshin Sarvary3
hand, Bacillus Calmette–Guerin (BCG) is an attenuated strain of Myco-
bacterium 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
1Department of Life Science Engineering, Faculty of New Science
and Technology, University of Tehran, Tehran, Iran
2Drug Design and Bioinformatics Unit, Department of Medical
Biotechnology, Biotechnology Research Center, Pasteur Institute,
Tehran, Iran
used in earlier screenings instead of M. tuberculosis.
Serious challenges associated with the rising epidemic are multi-
drug resistance and the growing number of people co-infected with
M. tuberculosis and human immunodeficiency virus (HIV) (9). Classi-
cally, 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 indi-
viduals 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.
3Department of Chemistry, Faculty of Basic Science, Babol
University of Technology, Babol, Iran
*Corresponding author: Soroush Sardari, ssardari@hotmail.com;
sardari@pasteur.ac.ir
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 pharma-
ceutical 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-myco-
bacterium 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.
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 resis-
tance, 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 pen-
etrate host cells, and exert anti-mycobacterial effect in the intracel-
lular 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.
Key words: coumarin derivatives, multicomponent reaction, Myco-
bacterium bovis
Received 2 May 2012, revised 18 July 2012 and accepted for publica-
tion 19 August 2012
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
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
929

Rezayan et al.
time-saving aspect, the versatility and diversity of scaffolds, and
the very large chemical space will attract chemists in pharmaceuti-
cal companies to use MCRs for their projects. Recently, the pharma-
ceutical industries have focused more and more on diversity-
oriented combinatorial libraries (12–15).
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): dC (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).
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 lit-
erature 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 cou-
marins with carbohydrazides substitution only in position 3 was syn-
thesized and evaluated against M. bovis. These compounds
exhibited a significant activity (50–100 lg ⁄ mL) when compared
with the first-line drugs (18).
6-Nitro-2-oxo-2H-chromene-3-carboxylic acid 5f
Cream powder (0.19 g, yield 80%), mp 235–238 ꢀC. 1H NMR
3
(300 MHz, DMSO-d6): dH (ppm) 7.64 (1H, d, J = 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):
dC (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).
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 pro-
gram (26–28), here we report synthesis of different coumarin deriv-
atives and evaluation of their anti-TB activity.
Methods and Materials
N-tert-Butyl-3,4-dihydro-2-oxo-2H-chromene-4-
carboxamide 6a
General
White powder; mp 219–221 ꢀC. IR (KBr) (mmax ⁄ cm)1): 3296 (NH),
Melting points were measured on an Electrothermal 9100 apparatus
and are uncorrected. IR spectra were recorded on a Shimadzu IR-470
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). H
NMR (300 MHz, DMSO-d6): dH (ppm) 1.20 (9H, s, C(CH3)3), 2.69 (1H,
1
1
spectrometer. H and 13C NMR spectra were recorded on a BRUKER
2
3
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 d = 2.50 ppm for (CD3)2SO ⁄ DMSO-d6 using TMS as an inter-
nal 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).
dd, JHH = 16.1 Hz, JHH = 2.4 Hz, CH2-CH), 2.93 (1H, dd,
2JHH = 16.1 Hz, JHH = 6.0 Hz, CH2-CH), 3.85 (1H, dd, JHH = 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): dC (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).
3
2
N-Benzyl-3,4-dihydro-2-oxo-2H-chromene-4-
General procedure for preparation of products
5a–f
carboxamide 6b
White powder; mp 169–172 ꢀC. H NMR (300 MHz, DMSO-d6): dH
1
2
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 Bꢀchner funnel. The products were puri-
fied, if necessary, by crystallization from ethyl acetate or methanol.
All products were known compounds (except 5d and 5f, Table 1),
(ppm) 2.80 (1H, d, JHH = 15.9 Hz, CH2-CH), 3.00 (1H, dd,
3
3
2JHH = 15.9 Hz, JHH = 5.5 Hz, CH2-CH), 3.95 (1H, d, JHH = 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): dC (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).
1
which were identified by H NMR and 13C NMR spectral data, and
their melting points were compared with literature reports.
N-tert-Butyl-6-bromo-3,4-dihydro-2-oxo-2H-
chromene-4-carboxamide 6c
6-Chloro-2-oxo-2H-chromene-3-carboxylic acid
5d
White powder; mp 225–228 ꢀC (dec). IR (KBr) (mmax ⁄ cm): 3297(NH),
1
White powder (0.3 g, yield 75%), mp 197–202 ꢀC. 1H NMR
3084, 2975, 2928, 1774, 1642, 1557. H NMR (300 MHz, DMSO-d6):
3
2
(300 MHz, DMSO-d6): dH (ppm) 7.45 (1H, d, J = 8.9 Hz, CH arom),
dH (ppm) 1.20 (9H, s, C(CH3)3), 2.70 (1H, d, JHH = 16.1 Hz, CH2-CH),
930
Chem Biol Drug Des 2012; 80: 929–936

Synthesis and Biological Evaluation of Coumarin Derivatives
2
3
4
2.93 (1H, dd, JHH = 16.1 Hz, JHH = 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).
OCH3), 6.62 (1H, d, JHH = 2.3 Hz, H-Ar), 6.71 (1H, dd,
3JHH = 8.4 Hz, JHH = 2.3 Hz, H-Ar), 7.32 (1H, d, JHH = 8.4 Hz, H-
Ar), 8.13 (1H, d, JHH = 7.6 Hz, NH).
4
3
3
6-Bromo-N-cyclohexyl-3,4-dihydro-2-oxo-2H-
chromene-4-carboxamide 6d
N-Cyclohexyl-3,4-dihydro-8-methoxy-2-oxo-2H-
chromene-4-carboxamide 6h
White powder; mp 203–206 ꢀC. IR (KBr) (mmax ⁄ 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
White powder; mp 185–187 ꢀC (dec). IR (KBr) (mmax ⁄ cm): 3329 (NH),
2936, 2852, 1772, 1640, 1537. 1H NMR (300 MHz, DMSO-d6): dH
(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,
1
(50), 198 (50), 147 (5), 118 (40), 102 (10), 83 (100), 55 (95). H NMR
(300 MHz, DMSO-d6): dH (ppm) 1.03–1.74 (10H, m, 5CH2 of cyclo-
2
3
hexyl), 2.72 (1H, d, JHH = 16.0 Hz, CH2-CH), 2.91 (1H, dd,
OCH3), 3.80 (1H, d, JHH = 5.8 Hz, CH2-CH), 6.99–7.03 (3H, m, H-Ar),
3
3
2JHH = 16.0 Hz, JHH = 5.6 Hz, CH2-CH), 3.33 (H, m, CH-N of cyclo-
8.16 (1H, d, JHH = 6.7 Hz, NH).
2
hexyl), 3.83 (1H, d, JHH = 5.6 Hz, CH2-CH), 6.98 (1H, d,
3
3JHH = 8.6 Hz, H-Ar), 7.43 (1H, d, JHH = 8.6 Hz, H-Ar), 7.64 (1H, s,
3
H-Ar), 8.16 (1H, d, JHH = 7.2 Hz, NH). 13C NMR (75 MHz, DMSO-
General procedure for preparation of products
7a–p
d6): dC (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).
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 is-
ocyanide (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 etha-
nol and the product 7a–n was obtained as a white powder (24).
N-Benzyl-6-bromo-3,4-dihydro-2-oxo-2H-
chromene-4-carboxamide 6e
White powder; mp 165–168 ꢀC (dec). IR (KBr) (mmax ⁄ cm): 3334 (NH),
3033, 2920, 1763, 1641, 1528. 1H NMR (300 MHz, DMSO-d6): dH
Methyl-4-(tert-butylcarbamoyl)-3,4-dihydro-2-
oxo-2H-chromene-3-carboxylate 7a
2
(ppm) 2.83 (1H, d, JHH = 16.2 Hz, CH2-CH), 3.01 (1H, dd,
3
3
2JHH = 16.2 Hz, JHH = 5.9 Hz, CH2-CH), 3.96 (1H, d, JHH = 5.9 Hz,
White powder; mp 151)152 ꢀC. IR (KBr) (mmax ⁄ cm): 3321, 3070,
3
CH2-CH), 4.26 (2H, brs, CH2 of benzyl), 7.04 (1H, d, JHH = 8.6 Hz,
H-Ar), 7.16–7.31 (5H, m, H-Ar), 7.50 (1H, d, JHH = 8.6 Hz, H-Ar),
2988, 1778, 1743, 1638, 1546. MS, m ⁄ z (%): 306 (M+, 15), 220 (25),
3
1
190 (20), 173 (35), 140 (100), 118 (50), 89 (30), 58 (90), 32 (40). H
7.74 (1H, s, H-Ar), 8.84 (1H, s, NH).
NMR (300 MHz, DMSO-d6) cis and trans: dH (ppm) 1.20 (18H, s,
2C(CH3)3, cis, and trans), 3.58 (3H, s, OCH3, trans), 3.69 (3H, s,
3
OCH3, cis), 4.19 (2H, d, JHH = 4.1 Hz, 2CH-4, cis, and trans), 4.28
3
6-Bromo-3,4-dihydro-2-oxo-N-(tosylmethyl)-2H-
chromene-4-carboxamide 6f
(2H, d, JHH = 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: dC (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: dC (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).
White powder; mp 198–203 ꢀC (dec). IR (KBr) (mmax ⁄ 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):
dH (ppm) 2.32 (CH3), 2.52 (1H, d, 2JHH = 16.2 Hz, CH2-CH), 2.90 (1H, dd,
3
3
2JHH = 16.2 Hz, JHH = 5.9 Hz, CH2-CH), 3.88 (1H, d, JHH = 5.9 Hz,
2
3
CH2-CH), 4.56 (1H, dd, JHH = 14.1 Hz, JHH = 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, JHH = 7.6 Hz, JHH = 5.5 Hz, CH2-NH). 13C NMR
(75 MHz, DMSO-d6): dC (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).
3
3
Ethyl-4-(tert-butylcarbamoyl)-3,4-dihydro-2-oxo-
2H-chromene-3-carboxylate 7b
White powder; mp 178–180 ꢀC. IR (KBr) (mmax ⁄ 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). H NMR (300 MHz, DMSO-d6) cis and trans: dH (ppm)
1.03 (6H, t, JHH = 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, JHH = 7.06 Hz,
2OCH2CH3, cis, and trans), 4.15 (2H, d, JHH = 4.27 Hz, 2CH-4, cis,
1
N-Cyclohexyl-3,4-dihydro-7-methoxy-2-oxo-2H-
chromene-4-carboxamide 6g
3
3
White powder; mp 146–147 ꢀC (dec). IR (KBr) (mmax ⁄ cm): 3278 (NH),
1
3
3093, 2935, 2852, 1774, 1623, 1562. H NMR (300 MHz, DMSO-d6):
3
dH (ppm) 1.04–1.77 (10H, m, 5CH2 of cyclohexyl), 2.69 (1H, dd,
and trans), 4.27 (2H, d, JHH = 4.27 Hz, 2CH-3, cis, and trans), 7.04–
2JHH = 16.0 Hz, 3JHH = 2.1 Hz, CH2-CH), 2.90 (1H, dd,
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: dC (ppm) 14.23
(OCH2CH3), 28.71 (C(CH3)3), 45.69 (CH-4), 48.64 (CH-3), 50.98 (C(CH3)3),
3
2
2JHH = 16.0 Hz, JHH = 6.0 Hz, CH2-CH), 3.37 (1H, dd, JHH = 6.0 Hz,
3JHH = 2.1 Hz, CH2-CH), 3.40 (H, m, CH-N of cyclohexyl), 3.73 (3H, s,
Chem Biol Drug Des 2012; 80: 929–936
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Rezayan et al.
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: dC (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).
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
Methyl-4-((ethoxycarbonyl)methylcarbamoyl)-
3,4-dihydro-2-oxo-2H-chromene-3-carboxylate
7c
White powder; mp 136–138 ꢀC. IR (KBr) (mmax ⁄ 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).
White powder; mp 183–185 ꢀC. IR (KBr) (mmax ⁄ 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-((ethoxycarbonyl)methylcarbamoyl)-3,4-
dihydro-2-oxo-2H-chromene-3-carboxylate 7i
White powder; mp 126–129 ꢀC. IR (KBr) (mmax ⁄ 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-(cyclohexylcarbamoyl)-3,4-dihydro-2-
oxo-2H-chromene-3-carboxylate 7d
White powder; mp 167–170 ꢀC. IR (KBr) (mmax ⁄ 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).
Ethyl-4-(tosylmethylcarbamoyl)-3,4-dihydro-2-
oxo-2H-chromene-3-carboxylate 7j
White powder; mp 145–148 ꢀC. IR (KBr) (mmax ⁄ 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): dH (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): dC (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).
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)
(mmax ⁄ 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 166–170ꢀC. IR (KBr) (mmax ⁄ 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).
Ethyl-4-(tert-butylcarbamoyl)-6-bromo-3,4-
dihydro-2-oxo-2H-chromene-3-carboxylate 7k
White powder; mp 153–157 ꢀC. IR (KBr) (mmax ⁄ 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).
Cycloheptyl-4-(2,6-dimethylphenylcarbamoyl)-
3,4-dihydro-2-oxo-2H-chromene-3-carboxylate
7g
Ethyl-4-(benzylcarbamoyl)-6-bromo-3,4-dihydro-
2-oxo-2H-chromene-3-carboxylate 7l
White powder; mp 159–163 ꢀC. IR (KBr) (mmax ⁄ cm): 3316, 2922,
2855, 1800, 1735, 1615, 1555. MS, m ⁄ z (%): 435 (M+, 2), 339 (5),
White powder; mp 160–165 ꢀC. IR (KBr) (mmax ⁄ 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).
1
321 (40), 295 (10), 147 (100), 118 (50), 81 (20), 57 (70), 41 (25). H
NMR (300 MHz, DMSO-d6) cis and trans: dH (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 cyclo-
heptans, 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: dC (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:
dC (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),
4-Fluorobenzyl-4-(cyclohexylcarbamoyl)-6-
bromo-3,4-dihydro-2-oxo-2H-chromene-3-
carboxylate 7m
White powder; mp 162–165 ꢀC. IR (KBr) (mmax ⁄ 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).
932
Chem Biol Drug Des 2012; 80: 929–936

Synthesis and Biological Evaluation of Coumarin Derivatives
Ethyl-4-(cyclohexylcarbamoyl)-3,4-dihydro-6-
1H, and 13C NMR spectra and melting point values as compared with
methoxy-2-oxo-2H-chromene-3-carboxylate 7n
White powder; mp 153–156 ꢀC. IR (KBr) (mmax ⁄ 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).
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.
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 substi-
tution (OMe) at positions 7 or 8 has little improving benefit compared
with parent structure (5a) without substitution. Comparing the activ-
ity of 5d and 5e with that of 5f, it is evident that the electron with-
drawal from the ring through resonance movement of p electrons has
a more potent effect on improving the activity. Considering lipophilici-
ty also, among the members of group 5, the biological activity seems
to depend on the effect of substitution at position 6 by the electro-
negativity 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.
Compounds 8a and 8b were purchased from Merck chemical com-
pany.
In vitro evaluation 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 con-
centration of 1 or 2 mg ⁄ L. All wells of micro plates received
100 lL of freshly prepared Middle broke 7H9 medium (Himedia,
Mumbai, India), except first column. Two hundred microliters of dis-
tilled water was added to the first column of 96-well plates to min-
imize evaporation of the medium in the test wells during
incubation. Then, 100 lL of test compounds with desired concentra-
tions (1000 or 2000 lL) 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 lL), 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 lL Tween
80 10% and 20 lL 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 inhibi-
tion concentration (MIC) was defined as the lowest drug concentra-
tion, which prevented a color change from blue to pink. Ethambutol
(Irandaru, Tehran, Iran) was used as positive control and DMSO as
negative control.
In compounds 6, two positions (R1 in position 4 and X in position
6, 7, 8) are changed, and various derivatives (6a–6h) were pre-
pared 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 substitu-
tion (-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 attrib-
uted to the bioactivity. Comparing compound 6a and 6c reveals
that bromine substitution at position 6 would enhance the bioactiv-
ity, 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 dia-
1
stereoisomers are expected. The H 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 sub-
stitutions in position 3 have same MIC [7f and 7g = 62.5 (lg ⁄ mL),
7l and 7m = 15.6 (lg ⁄ mL)]. Therefore, among different substitu-
tions in position 4, cyclohexyl is more active and 4-toluene-meth-
ylsulfonyl and ethylacetate are less active groups. However, it is
necessary to examine the mechanism of action in detail to clear
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-hy-
droxybenzaldehyde (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-dihydro-
coumarin derivatives (6a–h) and (7a–n), respectively (23,24). Cou-
marins 8a and 8b are commercially available and were purchased
from Sigma-Aldrich. All reaction products were characterized by IR,
Chem Biol Drug Des 2012; 80: 929–936
933

Rezayan et al.
Table 1: The structure of coumarin derivatives and their MIC (lg ⁄ mL) against Mycobacterium bovis BCG in microbroth dilution method
assay
R1
R1
O
NH
O
NH
R1
O
COOH
O
OR2
4
5
3
2
6
7
X
X
X
X
8
1
O
O
O
O
O
O
O
8a,b
7a-n
5a-f
6a-h
Entry
X
R1
R2
MIC (lg ⁄ mL) 24 ⁄ 48 (h)
CLogP
5a
H
–
–
–
–
–
–
–
–
–
–
–
–
–
–
125 ⁄ 125
1.5417
1.5433
1.5433
2.2637
2.41
5b
5c
7-OMe
8-OMe
6-Cl
6-Br
6-NO2
–
62.5 ⁄ 62.5
62.5 ⁄ 62.5
125 ⁄ 250
62.5 ⁄ 125
31.25 ⁄ 62.5
125 ⁄ 125
5d
5e
5f
6a
1.30
tert-Butyl
Benzyl
tert-Butyl
Cyclohexyl
Benzyl
4-Toluene-methylsulfonyl
Cyclohexyl
Cyclohexyl
tert-Butyl
tert-Butyl
Cyclohexyl
Cyclohexyl
1.5107
2.1959
2.5137
3.307
6b
6c
–
62.5 ⁄ 62.5
31.25 ⁄ 31.25
62.5 ⁄ 62.5
62.5 ⁄ 62.5
125 ⁄ 125
62.5 ⁄ 62.5
31.25 ⁄ 62.5
62.5 ⁄ 62.5
46.8 ⁄ 46.8
62.5 ⁄ 62.5
62.5 ⁄ 62.5
15.6 ⁄ 15.6
62.5 ⁄ 62.5
62.5 ⁄ 62.5
62.5 ⁄ 62.5
125 ⁄ 125
6-Br
6-Br
6-Br
6-Br
7-OMe
8-OMe
–
–
–
–
–
6d
6e
–
–
–
–
–
3.1989
3.2205
2.3937
2.3937
1.3017
1.83075
2.0957
2.6247
3.4627
1.5341
4.1241
2.5090
1.3489
1.8683
2.8337
3.0987
4.9259
2.7137
1.9163
1.632
6f
6g
6h
7a
Methyl
Ethyl
Methyl
Ethyl
Isobutyl
Methyl
Cycloheptyl
Ethyl
Ethyl
Ethyl
Ethyl
Methyl
7b
7c
7d
7e
Cyclohexyl
7f
–
–
–
–
2,6-Dimethyl-phenyl
2,6-Dimethyl-phenyl
Benzyl
Ethylacetate
4-Toluene-methylsulfonyl
tert-Butyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
4-OH
7g
7h
7i
7j
7k
–
125 ⁄ 125
6-Br
6-Br
6-Br
7-OMe
H
62.5 ⁄ 62.5
15.6 ⁄ 15.6
15.6 ⁄ 15.6
31.25 ⁄ 31.25
125 ⁄ 125
7l
7m
7n
8a
4-Fluorobenzyl
Ethyl
–
–
8b
Ethambutol
DMSO
7-OH
H
62.5 ⁄ 62.5
0.75 ⁄ 0.75
6.5 ⁄ 6.5% (v ⁄ v)
BCG, Bacillus Calmette–Guerin.
the reason for the increase and decrease of activity. Comparing
lipophilicity values for 7c, 7d, and 7e, a trend of increasing lipo-
philicity and bioactivity due to alkyl group enlargement at position
3 is observed. Bromination of compound 7c at position 6 would fur-
ther increase the lipophilicity and bioactivity. Correlation of lipophi-
licity 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.
against M. bovis. In comparison with reported papers (18,19), our
results show promising activity (15.6–125 lg ⁄ mL versus 50–
100 lg ⁄ mL), and also, synthesis of variety of coumarin derivatives
with different substitution in different position via MCRs has advanta-
ges such as good yield, facile purification method, and high diversity.
In conclusion, all synthesized and purchased compounds were eval-
uated 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, alicy-
clic, 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 gen-
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
934
Chem Biol Drug Des 2012; 80: 929–936

Synthesis and Biological Evaluation of Coumarin Derivatives
COOH
4
5
6
7
3
X
2
8
1
O
O
5a-f
H2O, Reflux
X
Me
O
Me
O
CHO
OH
+
O
O
1
2
R1
R1
R2 OH, r.t., 24 h
R1
N C
3
R1
N
C
CH2Cl2, r.t., 36 h
4
O
NH
O
NH
3
O
O
OR2
X
X
O
O
O
7a-n
6a-h
Scheme 1: Synthesis of coumarin derivatives examined for anti-mycobacterial activity in this study.
eral 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.
bovis, Mycobacterium bovis BCG, Mycobacterium microti, and
Mycobacterium africanum. Int J Syst Bacteriol;35:147–150.
8. Mahairas G.G., Sabo P.J., Hickey M.J., Singh D.C., Stover C.K.J.
(1996) Molecular analysis of genetic differences between Myco-
bacterium bovis BCG and virulent M. bovis.
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Acknowledgment
9. Nurbo J., Roos A.K., Muthas D., Wahlstrom E., Ericsson D.J.,
Lundstedt T., Unge T., Karlen A. (2007) Design, synthesis and
evaluation of peptide inhibitors of Mycobacterium tuberculosis
ribonucleotide reductase. J Pept Sci;13:822–832.
10. Nathan C., Gold B., Lin G., Stegman M., de Carvalho L.P., Vandal
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We gratefully acknowledge the Pasteur institute of Iran and Minis-
try of Health and Medical Education in Iran for funding of this pro-
ject as part of grant number 456.
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