Novel molecular hybrids of cinnamic acids and guanylhydrazones as potential antitubercular agents

Article abstract of DOI:10.1016/j.bmcl.2010.01.031

In an attempt to identify potential new agents active against tuberculosis, 20 novel phenylacrylamide derivatives incorporating cinnamic acids and guanylhydrazones were synthesized using microwave assisted synthesis. Activity of the synthesized compounds was evaluated using resazurin microtitre plate assay (REMA) against Mycobacterium tuberculosis H37Rv. Based on empirical structure-activity relationship data it was observed that both steric and electronic parameters play major role in the activity of this series of compounds. Compound 7s (2E)-N-((-2-(3,4-dimethoxybenzylidene) hydrazinyl) (imino) methyl)-3-(4-methoxyphenyl) acrylamide showed MIC of 6.49 μM along with good safety profile of >50-fold in VERO cell line. Thus, this compound could act as a potential lead for further antitubercular studies.

Full text of DOI:10.1016/j.bmcl.2010.01.031

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1623–1625
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel molecular hybrids of cinnamic acids and guanylhydrazones
as potential antitubercular agents
Ranjeet Bairwa a, Manoj Kakwani a, Nilesh R. Tawari a, Jaya Lalchandani a, M. K. Ray b, M. G. R. Rajan b,
Mariam S. Degani a,
*
a Institute of Chemical Technology, Nathalal Parikh Marg, Matunga, Mumbai 400 019, India
b Radiation Medicine Center, Bhabha Atomic Research Centre, Tata Memorial Hospital Annex, Parel, Mumbai 400 012, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In an attempt to identify potential new agents active against tuberculosis, 20 novel phenylacrylamide
derivatives incorporating cinnamic acids and guanylhydrazones were synthesized using microwave
assisted synthesis. Activity of the synthesized compounds was evaluated using resazurin microtitre plate
assay (REMA) against Mycobacterium tuberculosis H37Rv. Based on empirical structure–activity relation-
ship data it was observed that both steric and electronic parameters play major role in the activity of this
series of compounds. Compound 7s (2E)-N-((-2-(3,4-dimethoxybenzylidene) hydrazinyl) (imino)
methyl)-3-(4-methoxyphenyl) acrylamide showed MIC of 6.49 lM along with good safety profile of
>50-fold in VERO cell line. Thus, this compound could act as a potential lead for further antitubercular
studies.
Received 14 September 2009
Revised 18 December 2009
Accepted 13 January 2010
Available online 20 January 2010
Keywords:
Cinnamic acid
Guanylhydrazone
Phenylacrylamide
Antitubercular
Ó 2010 Elsevier Ltd. All rights reserved.
Mycobacterium tuberculosis, a deadly obligate pathogen, is the
causative agent of tuberculosis (TB), which remains a leading cause
of death worldwide.1–3 Tuberculosis is difficult to treat due to res-
idence of bacteria within the macrophages and its unusual cell wall
barrier. Moreover, multi-drug resistant strains of TB (MDR-TB) and
extensively drug resistant tuberculosis (XDR-TB) have emerged
recently.4,5 Hence, there is an urgent need to develop newer
anti-mycobacterial agents acting by novel mode of action and with
minimal chances of cross resistance to existing drugs. Several
researchers continue to work for the identification of newer anti-
mycobacterial agents.6–11
In an attempt to synthesize and evaluate novel compounds
active against TB, herein we report synthesis and evaluation of a
series of phenylacrylamides designed by molecular hybridization
of E-cinnamic acids and guanylhydrazones. Cinnamic acid deriva-
tives are known to have antimicrobial activity. They also show
synergism to some first line antitubercular agents.12–15 Recently,
while the current work was under progress, two reports on deriv-
atives of cinnamic acid as antitubercular agents have appeared.16,17
Guanylhydrazones have been shown to have antimicrobial activity
including an interesting Gram-negative bacterial endotoxin lipo-
polysaccharide (LPS) sequestering activity owing to their cationic
nature.18–20 M. tuberculosis contains lipoarabinomannan (LAM), a
complex lipid glycoprotein anchored to the cell membrane by
phosphatidylinositol which has structural and functional similarity
to LPS, including the presence of anionic phosphate groups.21
Biosynthesis of LAM is known to be a target for several antituber-
cular agents, including the first line antitubercular agent,
ethambutol.22,23
In this light, molecules were designed by molecular hybridiza-
tion of guanylhydrazones and cinnamic acids. The design principle
was aimed at combining the synergistic property of cinnamic acid
with sequestering activity of guanylhydrazone moiety to get com-
pounds with better antitubercular activity. Yet another objective of
the study was to evaluate the effect of steric and electronic param-
eters on antitubercular activity and to optimize the activity
through systematic modification of the substituents on the phenyl-
acrylamide core.
In the present work, the target compounds were synthesized
utilizing the reaction sequence as shown in Scheme 1. For the syn-
thesis of desired phenylacrylamide derivatives, guanylhydrazones
3, required as starting materials were prepared by the microwave
assisted reaction of substituted ketones or aldehydes 1, with
guanyl hydrazine hydrochloride 2. The phenylcinnamates 6, were
prepared by treating phenol 4, with thionyl chloride and the appro-
priate cinnamic acid 5.16 The reaction of equimolar quantities of
guanylhydrazones 3, with phenylcinnamates 6, under microwave
irradiation in the presence of triethylamine and ethanol as solvent
resulted in the formation of the target phenylacrylamide deriva-
tives 7, Table 1. The microwave assisted synthesis was advanta-
geous over conventional reaction.24,25 Spectral data (IR, 1H NMR,
13C NMR and MS) of all synthesized compounds were in agreement
with the proposed structures.
* Corresponding author. Tel.: +91 22 24145616.
E-mail address: ms.degani@ictmumbai.edu.in (M.S. Degani).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.031

1624
R. Bairwa et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1623–1625
Scheme 1. Synthetic route for the synthesis of phenylacrylamide derivatives (7a–7t). Reagents and conditions: (a) EtOH, microwave irradiation 60 W, 15 min, target
temperature 100 °C; (b) SOCl2, phenol, reflux; (c) EtOH, triethylamine, microwave irradiation 60 W, 30 min, target temperature 100 °C.
Table 1
change from blue to pink) (Table 1). Isoniazid was used as the ref-
erence drug.
In vitro antituberculosis activity of phenylacrylamide derivatives
Compounds 7a–7t were synthesized with modifications on R1,
R2 and R3 positions on phenylacrylamide core (Table 1). Com-
pounds 7a–7e were synthesized in an attempt to find out optimal
substitution at R2 position. According to biological activity results,
the aldehyde derivative 7a (R2 = –H) and the acetophenone deriva-
tive 7b (R2 = –CH3) were significantly more potent than the benzo-
phenone derivative 7c (R2 = –Ph). The increase in volume or
different electronic nature of –H, –CH3 and –Ph substitutions in
7a, 7b and 7c could be the possible reason for this behavior. The
substitution –R2 directly affects the electronic nature of guan-
ylhydrazone bridge which according to our design postulate, is
important for activity. Hence, to confirm the reason of this behav-
ior, compounds 7d (R1 = H; R2 = 4-ClPh) and 7e (R1 = 4-Cl; R2 = 4-
ClPh) with activating group at para-position of phenyl ring were
synthesized. Interestingly, compound 7d was more potent than
7c. Furthermore, compound 7e was slightly more potent than 7d.
Thus, at this stage, it can be postulated that decrease in activity
in 7c compared to 7a can be attributed to combination of steric
and electronic factors. Compounds 7f–7p were synthesized with
various substitutions at –R1 position keeping –R2 as hydrogen or
O
HN
R2
R1
N
N
H
N
R3
H
7 a-t
Compound
R1
R2
R3
MIC (lM)
CC50 (lM)
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
7s
7t
Isoniazid
H
H
H
H
H
CH3
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
130.0
160.0
532.0
273.0
219.5
40.5
124.1
8.9
38.3
30.6
189.5
36.4
254.4
46.3
18.3
17.0
9.2
359.2
365.6
312.1
327.6
311.0
454.1
465.3
368.9
367.2
413.1
194.7
193.2
201.6
354.6
249.4
199.9
399.0
405.5
340.0
308.0
4-ClPh
4-ClPh
H
4-Cl
4-OH
4-OH
CH3
H
H
3,4-DiOCH3
3-Cl
2-Cl
4-Br
4-OCH3
2-OH
4-F
4-Cl
2,4-DiCl
4-OH
H
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
—
methyl. One compound, 7h exhibited good MIC of 8.88 lM. Fur-
OCH3
OCH3
OCH3
OCH3
—
thermore, the aldehyde derivative 7f again was more potent than
the corresponding ketone derivative 7g, thus confirming our previ-
ous findings and reinforcing the direction of our research. To fur-
ther improve the activity compounds incorporating a methoxy
substitution at cinnamoyl moiety in aldehyde (R2 = H) derivatives
7q–7t were synthesized. All the four compounds 7q–7t showed
4-F
13.8
6.5
3,4-DiOCH3
3,4,5-TriOCH33
—
7.6
1.8
5833.5
The synthesized compounds (7a–7t) were screened against M.
tuberculosis H37Rv in order to determine the minimum inhibitory
concentration (MIC) with Resazurin microtiter assay (REMA).6–
good antitubercular activity with MIC <15
est one compound 7s showed MIC of 6.49
l
M. Of particular inter-
M. The compounds
l
were also evaluated for toxicity in a mammalian VERO cell line
(C1008) in 96-well microtitre plates and the CC50 values were
determined (Table 1).27 Complete data of percentage cell viability
at various concentrations is given in Table 1, Supplementary data.
Interestingly, the most potent compound 7s exhibited good safety
profile with selectivity index >50.
To further confirm our design postulate, the MIC’s of an guan-
ylhydrazone 3f ((2)-2-(4-hydroxybenzylidene)hydrazinecarboxi-
midamide), and phenylcinnamate 6a, were determined. The
11,26
Homogenous mycobacterial (H37Rv) culture suspension was
seeded in microtitre plates at density of 105 cells per well in
100 lL of the Middlebrook 7H9 broth (Difco laboratories, Detroit,
MI, USA) and the test compounds were serially diluted directly
on the plate. The control received equivalent amount of DMSO.
The plates were incubated at 37 °C for 7 days. Freshly prepared res-
azurin dye (0.02%) was added and plates were again incubated for
48 h. MIC is the lowest concentration at which complete inhibition
was observed and was determined by visual inspection (color
MIC’s obtained for guanylhydrazone 3f; 1122.4 lM and phenylcin-

R. Bairwa et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1623–1625
1625
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has significantly enhanced activity as compared to cinnamic acid
(MIC: 675.0 l
M15). Thus, these additional evaluation indicate the
value of chemical hybridization in this instance.
In summary, using systematic iteration of design, synthesis and
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of cinnamic acids and guanylhydrazones were synthesized and
evaluated for their antitubercular activity against M. tuberculosis
H37Rv. Based on empirical structure–activity relationship data, it
was observed that both the steric and the electronic parameters
play major role in the activity of this series of compounds. Starting
from the initial compounds with MIC of >100
lM, a compound, 7s,
with MIC of 6.49 M was successfully identified. This compound
l
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Supplementary data
Supplementary data (detailed experimental procedures and
complete data of percentage cell viability at various concentra-
tions) associated with this article can be found, in the online ver-
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