Synthesis and antimycobacterial evaluation of new trans-cinnamic acid hydrazide derivatives

Article abstract of DOI:10.1016/j.bmcl.2007.11.091

In this work, we report the synthesis and the antimycobacterial evaluation of new trans-cinnamic acid derivatives of isonicotinic acid series (5) and benzoic acid series (6), designed by exploring the molecular hybridization approach between isoniazid (1)

Full text of DOI:10.1016/j.bmcl.2007.11.091

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 538–541
Synthesis and antimycobacterial evaluation of new
trans-cinnamic acid hydrazide derivatives
Samir A. Carvalho,a,* Edson F. da Silva,a Marcus V. N. de Souza,a
Maria C. S. Lourenc¸ob and Felipe R. Vicenteb
aFioCruz-Fundac¸a˜o Oswaldo Cruz, Instituto de Tecnologia em Fa´rmacos-Far Manguinhos, Rua Sizenando Nabuco, 100,
Manguinhos, 21041-250 Rio de Janeiro, RJ, Brazil
b
´
Instituto de Pesquisas Clınica Evandro Chagas – IPEC – Av. Brasil, 4365, Manguinhos, Rio de Janeiro, RJ, Brazil
Received 18 September 2007; revised 21 November 2007; accepted 21 November 2007
Available online 28 November 2007
Abstract—In this work, we report the synthesis and the antimycobacterial evaluation of new trans-cinnamic acid derivatives of isoni-
cotinic acid series (5) and benzoic acid series (6), designed by exploring the molecular hybridization approach between isoniazid (1)
and trans-cinnamic acid derivative (3). The minimum inhibitory concentration (MIC) of the compounds 5a–d and 6c exhibited activ-
ity between 3.12 and 12.5 lg/mL and could be a good start point to find new lead compounds against multi-drug resistant
tuberculosis.
Ó 2007 Elsevier Ltd. All rights reserved.
O
O
Tuberculosis (TB) remains among the world’s great
public health challenges. Worldwide resurgence of TB
is due to two major problems: the AIDS epidemic,
which started in the mid-1980s, and the outbreak of
multi-drug resistant (MDR) TB. For example, the
deadly combination of TB and HIV has led to a quadru-
pling of TB cases in several African and Asian coun-
tries.1 MDR-TB, defined as resistance to at least
isoniazid (1) and rifamycin (RIF) (2), two current first-
line drugs, has increased morbidity and mortality with
an overall increase in healthcare costs. It is estimate that
4% of all worldwide TB patients are resistant to at least
one of the current first-line drugs (Fig. 1).
NH2
N
H
OH
N
Isoniazid (1)
trans-cinnamic acid (3)
AcO
OH
OH OH
OH
H
N
H3CO
O
O
TB treatment is long, possesses important side-effects,
and patients often interrupt it. The first-line treatment
has some disadvantages such as important side effects
and weak sterility problems and must be administered
for 6–9 months. When standard treatments fail, sec-
ond-line TB drugs are used, but these drugs have a far
lower efficacy and require even longer administration
periods (18–24 months) with higher cost (US $2500–
OH
O
N
N
O
N
R
R= CH3 (2)
R =C(=O)-CH=CH-C6H5 (4)
Figure 1. Chemical structures of compounds 1–4.
3000), higher rates of adverse effects, and low cure rates
(around 60%).1
Keywords: trans-Cinnamic acid; Isoniazid; Tuberculosis; Antimyco-
bacterial activity.
*
TB is responsible for 20% of all deaths in adults, and
each year there are about nine millions of new cases,
Corresponding author. Tel.: +55 2139772461; fax: +55 2125602518;
e-mail: scarvalho@far.fiocruz.br
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.091

S. A. Carvalho et al. / Bioorg. Med. Chem. Lett. 18 (2008) 538–541
539
of which 15% are children, and two millions of deaths,
of which 450.000 are children. Globally, the number of
TB cases is currently rising at 2% per year with the esti-
mative of 32% of the world population, which have
about two billion people, infected by latent TB. In the
case of patients with AIDS, TB is the most common
opportunistic infection and cause of death killing one
of every three patients. Due to the increase of MDR-
TB and AIDS cases worldwide and the lack of new
drugs nowadays, there is an urgent need for new drugs
to fight against this disease.
In our continuous program in the search of new potent
and safe isoniazid derivatives, we decided to construct a
new class of isonicotinic (5) and benzoic (6) acid N0-(3-
phenyl-acryloyl)-hydrazide derivatives (Fig. 2) as attrac-
tive antitubercular agents, designed by molecular
hybridization between isoniazid (1) and trans-cinnamic
acid (3). The design concept of these compounds ex-
plored the introduction of the trans-cinnamic moiety
(A) into isoniazid core structure aiming to potencialize
its activity, possible by the incorporation of a different
mode of action through a different target. Our second
goal was to investigate the effects of the isosteric substi-
tution of pyridine ring (B, Fig. 2), present in the more
active derivatives of series (5), to a simple phenyl group,
producing the corresponding benzoic derivatives of ser-
ies (6).
The goals of tuberculosis control are to cure active dis-
ease, prevent relapse, reduce transmission, and avert the
emergence of drug resistance.
The literature indicates that many isoniazid deriva-
tives2,3 have shown important antimycobacterial activ-
ity. In addition trans-cinnamic acid (3) derivatives
have a wide range of therapeutical importance, such
as, antitumor activity,4 antioxidant activity,5 and anti-
bacterial activity.6 Promising results have been shown
by Rastogi and coworkers7 which reported the synergis-
tic activity of trans-cinnamic acid (3) in drug combina-
tions with isoniazid (1), RIF (2), and other known
antimycobacterial agents against Mycobacterium tuber-
culosis. The increase of activity was even observed with
drug resistant isolates.
The synthetic route used for the preparation of the title
compounds is outlined in Scheme 1. Cinnamic acid
derivatives were employed as starting material. In order
to obtain more stable intermediates, we utilized 4-nitro-
phenol esters as general substrates. The 4-nitro-phenyl
esters were prepared by treating the appropriate cin-
namic acid with thionyl chloride and 4-nitro-phenol9
resulting in a stoichiometric amount of the 4-nitro-phe-
nyl esters. The target hydrazides were obtained, in good
yields, by the nucleophilic substitution of the 4-nitro-
phenol moiety for the acyl hydrazide, as described in
1
the general procedure.10 The analysis of the H NMR
Additionally, a cinnamyl rifamycin derivative (4) exhib-
its 2- to 8-fold lower MICs than those of (RIF) (1) for
most of the 20 susceptible and multi-drug resistant M.
tuberculosis strains tested, superior intracellular and
in vivo activities compared with those of RIF (1).8
showed the two hydrazine protons as two singlets at
10.21–10.62 ppm and 10.70–10.90 ppm, and the 13C
NMR spectra were consistent with the presence of the
two C@O signals at 163.91–166.02 ppm and 162.12–
163.89 ppm.
The antimycobacterial activities of compounds (5a–e
and 6a–e) were assessed against M. tuberculosis ATTC
2729411, using the microplate Alamar Blue assay
(MABA)11,12 (Table 1). This methodology is nontoxic,
uses thermally stable reagent, and shows good correla-
tion with proportional and BACTEC radiometric meth-
ods.13,14 Briefly, 200 ll of sterile deionized water was
added to all outer-perimeter wells of sterile 96-well
plates (falcon, 3072: Becton Dickinson, Lincoln Park,
NJ) to minimize evaporation of the medium in the test
wells during incubation. The 96-well plates received
100 lL of the Middlebrook 7H9 broth (Difco laborato-
ries, Detroit, MI, USA) and a serial dilution of the com-
pounds 9–16 was made directly on the plate. The final
drug concentrations tested were 0.01–10.0 lL/mL.
Plates were covered and sealed with paraffin and incu-
bated at 37 °C for 5 days. After this time, 25 ll of a
freshly prepared 1:1 mixture of Alamar Blue (Accumed
International, Westlake Ohio) reagent and 10% Tween
80 was added to the plate and incubated for 24 h. A blue
color in the well was interpreted as no bacterial growth,
and a pink color was scored as growth. The MIC (Min-
imal Inhibition Concentration) was defined as the lowest
drug concentration, which prevented a color change
from blue to pink.
O
O
A
NH2
N
H
HO
N
1
3
Molecular
Hybridization
R4
R3
R2
O
H
N
N
H
A
R1
N
O
5
B
Classical Bioisosterism
R4
R3
R2
O
H
N
N
H
R1
O
B
6
Four of the five isonicotinic derivatives (Table 1) were
sensitive in the minimum concentration tested
Figure 2. Design concept of new isonicotinic (5) and benzoic (6) acid
N0-(3-phenyl-acryloyl)-hydrazides.

540
S. A. Carvalho et al. / Bioorg. Med. Chem. Lett. 18 (2008) 538–541
NO2
R1
O
R1
O
R2
R3
R2
R3
a
O
OH
7a-e
b
a: SOCl2, 4-NO2-phenol, Toluene, Reflux
b: Acylhidrazide, Pyridin, 100 oC
R1
O
X
H
N
R2
R3
N
H
O
X = N - 5a-e
X = C - 6a-e
Scheme 1. Synthetic route for the preparation of the new isonicotinic (5) and benzoic (6) acid N0-(3-phenyl-acryloyl)-hydrazides.
Table 1. Antimycobacterial activities, melting points, logP measurements, and yields of isonicotinic (5) and benzoic (6) acid N0-(3-phenyl-acryloyl)-
hydrazides
R1
O
X
H
N
R2
R3
N
H
O
Entry
Compound
R1
R2
R3
X
Yield (%)
Mp (°C)
logPa
MICb
1
5a
6a
H
H
H
H
H
H
H
H
Cl
Cl
—
H
H
H
H
H
H
H
N
C
82
80
87
83
85
80
83
83
85
84
—
221.6
209.7
257.1
278.2
243.2
234.5
252.2
225.6
238.3
260.8
—
2.12
2.84
1.86
2.42
1.96
2.96
2.26
2.76
2.57
3.04
—
3.12
50
2
H
3
5b
6b
NO2
NO2
OCH3
OCH3
N
C
3.12
>100
3.12
12.5
3.12
25
4
5
5c
6c
N
C
6
7
5d
6d
O–CH2-O
O–CH2-O
N
C
8
9
5e
6e
H
H
—
Cl
Cl
—
N
C
>100
>100
0.2
10
11
Isoniazid
—
a Calculated by www.logp.com.
b Minimal Inhibition Concentration (MIC) is expressed in lg/mL.
(MIC = 3.12 lg/mL). All of the benzoic acid series (6)
were less active than the corresponding ones of the series
(5), reinforcing the pharmacophoric contribution of
isonicotinic moiety to the mechanism of action against
the M. tuberculosis. On the other hand, we are able to
identify the interesting profile of 4-methoxy derivative
(entry 6), which in spite of the absence of the pyridine
framework was able to inhibit bacterial growth with a
MIC = 12.5 lg/mL, indicating clearly the compensatory
effect promoted by introduction of the trans-cinnamic
subunit.
Acknowledgments
Thanks are due to CNPq (BR.) for financial support and
fellowships (to M.V.N.S., M.C.S.L., and F.R.V.).
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In conclusion, the antimicrobial activity of the two new
series described here suggests that they may be selec-
tively targeted to M. tuberculosis growths. They were
effective in inhibiting M. tuberculosis infection at 3.12,
12.5, 25.0, and 50.0 lg/mL concentrations, and could
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