Antitubercular activity of new coumarins

Article abstract of DOI:10.1111/j.1747-0285.2011.01120.x

The present article describes a series of 21 N'-benzylidene-2-oxo-2H-chromene-3-carbohydrazides 4a-4v, which were synthesized and evaluated for their cell viabilities in non-infected and Mycobacterium bovis Bacillus Calmette-Guerin-infected macrophages. Subsequently, the non-cytotoxic compounds 4c, 4g, 4h, 4j, 4l and 4t were assessed against Mycobacterium tuberculosis ATCC 27294 using the microplate Alamar Blue assay and the activity expressed as the minimum inhibitory concentration in μg/mL. These compounds exhibited a significant activity (50-100μg/mL) when compared to the first-line drugs, such as pyrazinamide (PZA >100μg/mL). These results could be considered a good starting point for further studies to develop new lead compounds to treat multidrug-resistant tuberculosis. The present article describes a series of twenty-one coumarine derivatives, which were synthesized and evaluated against Mycobacterium tuberculosis.

Full text of DOI:10.1111/j.1747-0285.2011.01120.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01120.x
Chem Biol Drug Des 2011; 77: 489–493
Research Letter
Antitubercular Activity of New Coumarins
Silvia H. Cardoso1, Milena B. Barreto2,
being AIDS pandemic and the emergence of bacterial resistance
(MDR and XDR-TB) the most important (1,2).a
Maria C. S. Lourenc¸o3, Maria das Grac¸as
4
4
´
´
M. de O. Henriques , Andre L. P. Candea ,
Carlos R. Kaiser2 and Marcus V. N. de
Souza4,*
AIDS Epidemic, in the mid-1980s, has led to an outbreak of coinfec-
tion with TB in seropositive patients. As an example, the coinfec-
tion of TB and HIV has led to a fourfold increase in TB cases in
several African and Asian countries. According to statistics, pres-
ently, about 10–12 million people are coinfected by these two
micro-organisms, and TB is the most common opportunist infection
in HIV-positive patients, being responsible for the morbidity and
mortality of one in every three patients. Nowadays, there is no
optimum standard anti-TB therapy for AIDS patients or any agent
that is active against infection caused by both HIV and M. tubercu-
losis. Consequently, because of the high impact of TB in global
society, new drugs and strategies are urgently needed to fight this
disease, particularly new agents with activity against both TB and
HIV infection and MDR strain. In this context, the development of
potent new antitubercular agents with low-toxicity profiles, a rapid
mycobactericidal mechanism of action and the ability to penetrate
host cells and shortened duration of therapy are in great interest
(2).
1Universidade Federal de Alagoas, Campus Arapiraca, Rua Manoel
Severino Barbosa s ⁄ n, Bonsucesso, 57309-005 Arapiraca, AL, Brazil
2Departamento de Quꢀmica Orgꢁnica, Universidade Federal do Rio
de Janeiro, Instituto de Quꢀmica, CP 68563, 21945-970 Rio de
Janeiro, RJ, Brazil
3Instituto de Pesquisas Clꢀnica Evandro Chagas – IPEC – Av. Brasil,
4365 Manguinhos, Rio de Janeiro, RJ, Brazil
4Fundażo Oswaldo Cruz, Instituto de Tecnologia em Fꢂrmacos-
Far-Manguinhos, Rua Sizenando Nabuco, 100, Manguinhos,
21041-250 Rio de Janeiro, RJ, Brazil
*Corresponding author: Marcus V. N. de Souza,
marcos_souza@far.fiocruz.br
The present article describes a series of 21 N ¢-
benzylidene-2-oxo-2H-chromene-3-carbohydrazides
4a–4v, which were synthesized and evaluated for
their cell viabilities in non-infected and Mycobac-
terium bovis Bacillus Calmette–Guerin-infected
macrophages. Subsequently, the non-cytotoxic
compounds 4c, 4g, 4h, 4j, 4l and 4t were assessed
against Mycobacterium tuberculosis ATCC 27294
using the microplate Alamar Blue assay and the
activity expressed as the minimum inhibitory con-
centration in lg/mL. These compounds exhibited a
significant activity (50–100 lg/mL) when compared
to the first-line drugs, such as pyrazinamide (PZA
>100 lg/mL). These results could be considered a
good starting point for further studies to develop
new lead compounds to treat multidrug-resistant
tuberculosis.
Naturally occurring coumarins are endowed with different types of
biological applications. The 1,2-benzopyrone structure consists of
structural units of several natural products and is widely present in
pharmacologically and biologically active compounds, such as novo-
biocin (3), warfarin (4), 677 cumate (5) and psoralen (6). In addition
to functionalized coumarins, the calanolides isolated from Calophyl-
lum genus showed potent anti-HIV activity. Specifically, (+)-calano-
lide A was found to inhibit not only the wild type of HIV-1 but also
clinically isolated resistant strains, such as A17 (7). Recently, it has
been demonstrated that (+)-calanolide A was active against all of
the strains of M. tuberculosis tested, including those resistant to
the standard antitubercular drugs. Efficacy evaluations in macro-
phages revealed that (+)-calanolide A significantly inhibited intracel-
lular replication of M. tuberculosis H37Rv in vitro at a 3.13 lg ⁄ mL
concentration (8). (+)-Calanolide A and its related coumarins are the
first class of compounds identified to possess antimycobacterial
and antiretroviral activities, representing a new pharmacophore for
antitubercular activity.
Key words: coumarins, drugs, N¢-acylhydrazones, tuberculosis
Received 7 February 2010, revised 19 September 2010 and accepted for
publication 27 February 2011
Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a conta-
gious disease which reappeared in the mid-1980s and has become
a serious global public health problem. Currently, it is estimated
that TB kills 2 million people per year of which 450 000 are
children. Globally, the number of TB cases is rising 2% annually. It
is estimated that 32% of the world population is infected by latent
TB. Many factors contribute to the increase in tuberculosis cases,
Inspired in the antitubercular activity of (+)-calanolide A, we have
proposed the synthesis of some N¢-acylhydrazones that contained
the coumarin nucleus. According to literature, many N¢-acylhydraz-
ones are described with a wide range of pharmacological activities
(9), such as antibacterial agents. For example, hydrazone deriva-
tives, prepared by our group, exhibit promising anti-TB activity com-
parable to the first-line anti-TB drugs as isoniazid, rifampicin and
489

Cardoso et al.
O
O
pyrazinamide (10–18). This work is part of our continuous pro-
gramme in the search of new antitubercular agent candidates.
O
O
O
CHO
OH
EtO
OEt
OEt
Piperidine, 120 °C
42 h
81 %
1
2
The design concept of these compounds explored the introduction
of mono- and disubstituted benzaldehydes moieties (B) into couma-
rins core structure (A) to obtain N¢-acylhydrazones groups (C)
(Scheme 1). This modification aims to evaluate their selectivity
against M. tuberculosis, their mechanism of action and the cyto-
toxic effects of these compounds. Furthermore, we report the syn-
thesis (Scheme 2) and preliminary in vitro antitubercular and
cytotoxic activities of 21 N¢-acylhydrazones coumarins derivatives,
of which fourteen are novels.
NH2NH2
EtOH reflux, 2 h
CHO
R1
O
O
O
O
O
O
NH
N
NHNH2
R1
EtOH reflux
2–12 h
3
4a-v
(25–90 %)
Scheme 2: Synthetic route for the preparation of N¢-benzylid-
ene-coumarins-3-carbohydrazides derivatives.
General procedure for the synthesis of
N¢-benzylidene-2-oxo-2H-chromene-3-
carbohydrazides derivatives
cores in the 1570–1320 cm)1 region and in the 900–686 cm)1
region, originated in the angular deformation outside the rings
plane and C-H. The stretching vibrations of N–H binding were
observed in the 3588–3331 cm)1 region while stretching vibrations
of C=O (lactone and amide) in the 1720–1620 cm)1 region. In the
1275–1271 cm)1 region, stretching vibrations of C–O binding were
observed.
The strategy for the synthesis of N¢-benzylidene-2-oxo-2H-chrom-
ene-3-carbohydrazides derivatives 4a–4v from salicylaldehyde 1 is
described in Scheme 2. All target compounds 4a–4v were
obtained, with moderate to good yields (Table 1), by the nucleo-
philic attack of the most basic nitrogen of hydrazine 3 to the car-
bonyl group of the aldehyde derivative. The synthetic route
employed for the preparation of 4a–4v is outlined in Scheme 2.
Primarily, salicylaldehyde 1, diethyl malonate and piperidine catalyst
were refluxed at 120 ꢀC for 42 h to produce ethyl 2-oxo-2H-chrom-
ene-3-carboxylate derivatives 2 under Knoevenagel reaction condi-
tions (19). In the second step, 2-oxo-2H-chromene-3-carbohydrazide
derivative 3 was synthesized by refluxing the coumarin-3-carboxyl-
ate ethyl ester with 80% hydrazine solution in ethanol. Finally,
reflux with aldehyde in ethanol leads to the target compounds 4a–
4v. The properties of N¢-benzylidene-2-oxo-2H-chromene-3-carbo-
hydrazides derivatives are summarized in Table 1.
1
The analysis of H NMR spectrum showed the signal of imine pro-
ton (H–C=N–) as singlet at 9.14–8.70 ppm, the hydrazide proton
(N–H) as a singlet at 11.96–11.30 ppm and five signals correspond-
ing to the protons of 1,2-benzopyrone moiety at 9.00–7.44 ppm.
The 13C NMR spectrum showed the nine coumarin carbon signals
at the region of 168.0–108.9 ppm and the hydrazide carbon
()N=CH–R) signals at 168–162 ppm.
In the case of each of the 4b (2-Cl), 4f (4-Br), 4n (2-NO2), 4o (3-
NO2), 4q (2,3-Cl), 4s (2,6-Cl) derivatives, the occurrence of stereo-
isomerism at the N=CH bond results in the formation of a pair of
(E) and (Z)-diastereomers. The presence of these diastereomers was
Compounds 4a–4v were identified by spectral data. In general, IR
spectra showed the strong bands of aromatic and heteroaromatic
1
confirmed by the H NMR spectra results, which in each case dis-
plays two separate sets of signals. The assignment of the relative
configuration of (E) and (Z)-diastereomers of these N¢-acylhydraz-
ones derivatives was made in agreement with previous results
obtained by Karabatsos et al. (20–22), which describes that imine-
attached hydrogen signal of the (E)-diastereomer is downfielded by
0.2–0.3 ppm from the corresponding hydrogen atom signal in the
(Z)-diastereomer. Therefore, after careful analysis of the 1H NMR
spectra of the mixture of diastereomers, we were able to evidence
that the main one presents (E) configuration similar to that found
for other N¢-acylhydrazones in literature (23,24).
OH
CHO
O
O
O
R
B
A
O
Molecular
hybridization
(+) - Calanolide A
The antimycobacterial activities of these compounds were assessed
against M. tuberculosis ATCC 27294 (29) using the microplate
Alamar Blue assay made in triplicate (30) (Table 1). This methodol-
ogy is non-toxic, uses a thermally stable reagent and shows good
correlation with proportional and BACTEC radiometric methods (31,
32). These results showed that compounds 4c, 4h, 4j, 4l, 4p and
4t (Entry 3, 8, 10, 11, 15 and 19, respectively) exhibit an antimyco-
bacterial activity of 100 lg ⁄ mL, while the compound 4g (Entry 7)
presented an activity of 50 lg ⁄ mL. These compounds were more
active than the first-line drug reference, pyrazinamide (Entry 23).
O
O
H
N
N
R
B
O
C
A
Scheme 1: Design concept of N-acylhydrazones coumarin deriv-
atives.
490
Chem Biol Drug Des 2011; 77: 489–493

Antitubercular Activity of New Coumarins
Table 1: Antimycobacterial activities, melting points, clogP measurements and yields of N¢-benzylidene-2-oxo-2H-chromene-3-carbohydraz-
ides derivatives
Substituents
Entry
Compound
R
R1
Yield (%)
cLogPa
mp (ꢀC)
MICb (lg ⁄ mL)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
4a
4b
4c
4d
4e
4f
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
–
H
2-Cl
3-Cl
4-Cl
3-Br
4-Br
30
82
88
80
73
76
45
25
48
53
40
62
65
68
58
57
68
73
35
45
55
–
3.10
3.74
3.76
3.78
3.89
3.91
2.60
2.62
3.11
3.14
3.16
3.21
3.01
3.04
3.06
4.36
4.39
4.36
2.54
2.54
2.75
)0.71
133–137 (25)
86–88 (26)
70–71
121–122 (27)
140–141
143–144
111–113 (25)
173–176
133–136
84–85
Res.
Res.
100.0
Res.
Res.
Res.
50.0
100.0
Res.
100.0
100.0
Res.
Res.
Res.
100.0
Res.
Res.
Res.
100.0
Res.
Res.
>100.
4g
4h
4i
3-OH
4-OH
2-OMe
3-OMe
4-OMe
4-N(CH3)2
2-NO2
3-NO2
4-NO2
2,3-Cl
2,4-Cl
2,6-Cl
2,4-OH
2,5-OH
3,4-OMe
–
4j
4l
85–86
155–156
87–88
4m
4n
4o
4p
4q
4r
123–125 (26)
135–138 (26)
111–112
124–125
83–85
131–134
121–124
123–124 (28)
–
4s
4t
4u
4v
PZA
PZA, pyrazinamide; MIC, minimum inhibitory concentration.
aCalculated using http://www.molinspiration.com.
bRes. indicates that the strain is resistant to the tested substance.
Furthermore, these derivatives that showed antitubercular activity
were submitted to the cellular viability in non-infected or Mycobac-
terium bovis Bacillus Calmette–Guerin (BCG)-infected macrophages.
The test was determined by Mosman's MTT (3-(4,5-dim-
ethylthylthiazol-2-yl)-2,5-dimethyl tetrazolium bromide; Merck) micro-
cultured tetrazolium assay (33, 34). The results were represented as
cell viability percentage (Table 2). This table shows that only the
compound 4p (4-NO2) was cytotoxic (70% cell viability) in its
respective minimum inhibitory concentration (100 lg ⁄ mL); therefore,
the derivatives 4c, 4g, 4h, 4j, 4l and 4t (Entries 1, 2, 3, 4, 5
and 7) were selected to be tested on macrophages infected with
M. bovis BCG (Table 3).
The purpose was to evaluate the action of these compounds
against macrophages that show metabolism change after infection.
Nevertheless, if we analyse Tables 2 and 3, it can be verified that
the derivatives 4c, 4h, 4j, 4l and 4t were not cytotoxic, because
<5% of the cells were killed at the minimum concentration tested.
The synthesis of 21 N¢-benzylidene-2-oxo-2H-chromene-3-carbohyd-
razides derivatives 4a–4v was performed with moderate to good
yields (25–90%), among which fourteen are new compounds (4c,
4e, 4f, 4h, 4i, 4j, 4l, 4m, 4n, 4q, 4r, 4s, 4t, 4u). Only the
derivatives 4c, 4g, 4h, 4j, 4l, 4p and 4t that showed antituber-
cular activity were submitted to the cellular viability in non-infected
Table 2: Cellular viability data for a macrophage cell line J774
(ATTC TIB-67ꢁ) by Mosmans's assay
Table 3: Cellular viability data for a macrophage cell line J774
infected (ATTC TIB-67ꢁ) with BCG by Mosmans's assay
% Cell viability ⁄ dose (lg ⁄ mL)
% Cell viability ⁄ dose (lg ⁄ mL)
Entry
Compound
50
100
150
Entry
Compound
50
100
150
1
2
3
4
5
6
7
8
4c
4g
4h
4j
100
100
95
100
100
69
100
100
80
95
89
62
100
100
98
75
100
98
100
57
100
100
1
2
3
4
5
6
4c
4g
4h
4j
100
60
99
100
100
100
100
74
80
89
100
100
93
84
85
100
100
100
4l
4l
4p
4t
4t
100
100
PZA
BCG, Bacillus Calmette–Guerin.
Chem Biol Drug Des 2011; 77: 489–493
491

Cardoso et al.
or M. bovis BCG-infected macrophages. In relation to the antimyco-
bacterial activity, it was found that the compounds 4c, 4g, 4h, 4j,
4l, 4p and 4t (50–100 lg ⁄ mL) exhibited better activities than PZA
(>100 mg ⁄ mL), when Alamar Blue assay was used and that the
derivatives 4c, 4h, 4j, 4l, 4p and 4t were not cytotoxic. These
results suggest promising perspectives for MDR ⁄ XDR-TB. However,
it is necessary to examine the action mechanism in detail to ascer-
tain the reason for the increased activity of the N¢-acylhydrazones.
anti-mycobacterial activity of (E)-N-(monosubstituted-benzyl-
idene)isonicotinohydrazide derivatives. Eur J Med Chem;43:
1344–1347.
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Vicente F.R.C., Souza M.V.N.de. (2009) Synthesis and Antituber-
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Acknowledgments
substituted-benzylidene)]-2-pyrazinecarbohydrazide
derivatives.
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We gratefully acknowledge the financial support of Fiocruz, UFRJ,
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