Synthesis and preliminary biological evaluation of novel N-(3-aryl-1,2,4-triazol-5-yl) cinnamamide derivatives as potential antimycobacterial agents: An operational Topliss Tree approach

Article abstract of DOI:10.1016/j.bmcl.2011.08.076

A series of novel N-(3-aryl-1,2,4-triazol-5-yl) cinnamamide derivatives were designed on basis of structural similarity to the known FAS II inhibitors. Topliss operational method was used to optimize the potency of molecules. The minimum inhibitory concen

Full text of DOI:10.1016/j.bmcl.2011.08.076

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6523–6526
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and preliminary biological evaluation of novel
N-(3-aryl-1,2,4-triazol-5-yl) cinnamamide derivatives as potential
antimycobacterial agents: An operational Topliss Tree approach
Manoj D. Kakwani a, Nutan H. Palsule Desai a, Arundhati C. Lele a, Muktikant Ray b,
M.G.R. Rajan b, Mariam S. Degani a,
⇑
a Institute of Chemical Technology, Nathalal Parekh Marg, Matunga, Mumbai 400 019, India
b Radiation Medicine Centre, Tata Memorial Hospital, Parel, Mumbai, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 February 2011
Revised 10 June 2011
Accepted 13 August 2011
Available online 22 August 2011
A series of novel N-(3-aryl-1,2,4-triazol-5-yl) cinnamamide derivatives were designed on basis of struc-
tural similarity to the known FAS II inhibitors. Topliss operational method was used to optimize the
potency of molecules. The minimum inhibitory concentration (MIC) of all synthesized compounds was
determined against Mycobacterium tuberculosis H37Rv using resazurin microtitre assay (REMA) plate
method. The synthesized compounds exhibit antimycobacterial activity in the range of 5–95
a good safety profile.
lM with
Keywords:
Mycobacterium tuberculosis
FAS II
Ó 2011 Elsevier Ltd. All rights reserved.
Triazoles
Topliss tree
The recurrence of tuberculosis (TB) as a global health problem
over the past few decades, accompanied by the rise of drug-
resistant strains of Mycobacterium tuberculosis, emphasizes the
need for discovery of new therapeutic drugs against this disease.
Mycobacteria construct a wide range of complex fatty acids
1,2,4-triazole ring systems exhibits antibacterial, analgesic,
anti-inflammatory, anticancer, anticonvulsant, antiviral, insecti-
cide, antidepressant, central nervous system (CNS) activities.7
Triazoles are also well known for their antimycobacterial as well
as antifungal action via inhibiting the cell wall synthesis.8
such as mycolic acids, very long chain (C-60 to C-90)
a
-branched
Cinnamamide scaffold containing compounds are known to pos-
sess antituberculosis activity. Additionally, cinnamamide deriva-
tives act at the mycolic acid biosynthesis level.6,9 Recently, we
have reported the synergistic effect of cinnamamide derivatives
with rifampicin.9 More recently, the synthesis and biological evalu-
ation of new and very potent drug candidates, an extended family of
cinnamyl-thioesters, amides, hydrazides, and triazolo-phthalazides
has been reported wherein, compounds with enoyl amide function-
ality act at mycolic acid biosynthesis level. Replacement of this enoyl
backbone by a styryl moiety results in compound with different
mechanism of action and that by cyclopropyl causes deteriorating
effect on the antituberculosis activity, further highlighting the
importance of enoyl functionality, and hence the cinnamamide scaf-
fold.10 The presence of both, cinnamamide and triazole moieties
would make the compounds more attractive as cell wall biosynthe-
sis inhibitors and also because of the structural similarities of these
compounds with the previously reported inhibitors, they are likely
to act on the biosynthesis of mycolic acids and more specifically
and b-hydroxylated fatty acids, which are not found in mammalian
cells. Their biosynthesis involves the fatty acid synthase type II
(FAS-II) system.1 The FAS-II system is however unique in M. tuber-
culosis (MTb), in that it elongates long chain fatty acids produced
by the FAS-I system to the very long meromycolic acyl chain of
mycolates. One of the four steps of the elongation cycles, of the
growing long chain fatty acids (Scheme 1) is catalyzed by the enoyl
reductase InhA, an NADH-dependent enzyme. This enzyme has al-
ready been proved to be the target of two of the front-line antitu-
bercular drugs, Isoniazid and Ethionamide.2 Therefore, the FAS-II
biosynthesis pathway represents a validated and promising target
for drug discovery.3 Families of compounds that have been studied
as inhibitors of the FAS-II system are diphenyl ethers, indoles, cin-
namic acid derivatives etc.4–6
Additionally, 1,2,4-triazoles and their derivatives represent an
attractive class of compounds possessing a broad spectrum of
biological activities. A large number of compounds containing
on FAS-II, which uses
a,b-enoyl systems as substrates (Scheme
1).11 In continuation of our research in the direction of combating
with tuberculosis via different strategies like analog based drug
design,9 siderophore mimics12 and molecular hybridization13,14
⇑
Corresponding author. Tel.: +91 22 3361 2213; fax: +91 22 3361 1020.
E-mail
addresses:
ms.degani@ictmumbai.edu.in,
m_degani@yahoo.com
(M.S. Degani).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.076

6524
M. D. Kakwani et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6523–6526
NADP+
NADPH
CoA+ CO2
FabH
O
O
O
OH
O
R
S-CoA
R
S-AcpM
O
R
S-AcpM
O
Dehydrase
H2O
R
S-AcpM
AcpM
+ CO2
KasA
KasB
O
O
-O
S-AcpM
O
O
CoA
FabD
InhA
R
S-AcpM
R
S-AcpM
NAD+
ACP
-O
NADH
O
O
S-CoA
R= C12- C16 carbon chain fatty acids
Scheme 1. General scheme of FAS II system involved in mycolic acid biosynthesis.
Step I
N
O
H
N
O
NH2
N
NH2
H
toluene
MW
H2O
N
N
N
NH2
Cl
H
.HCO3
H
H2N
NH
R.T. 12 h
NH
A
Yield 75%
Yield 95%
Step II
O
O
O
O
0°C DCM
TEA, methylchloroformate
0°C DCM
O
OH
R
R
N
O
N
N
N
H
H
R
Overall yield 60-70%
R= -H, 4-Cl, 4-OH, 4-NH2, 3,4-dioxymethylene, 3,4-diOCH3,
4-CH3, 3-Cl, 3-NO2, 4-OCF3, 4-(2-methylallyl)oxy
Ar-R= 2-Furyl, 5-nitrothiophen-2-yl
Scheme 2. Synthetic route for N-(3-aryl-1,2,4-triazol-5-yl) cinnamamide derivatives.
herein, we report, the synthesis and preliminary biological evalua-
tion of the novel N-(3-aryl-1,2,4-triazol-5-yl) cinnamamide deriva-
tives (Fig. 1) against M. tuberculosis H37Rv.
The designed compounds possess cinnamoyl frame work with
the aromatic moiety adjacent to enoyl function attached to the tri-
azole ring system. The effect of substituents of cinnamoyl aromatic
ring on the antituberculosis activity was studied using systematic
Topliss operational method.
Various novel N-(3-phenyl-1,2,4-triazol-5-yl) cinnamamide
derivatives were synthesized (yield 60–70%) according to Scheme
2. The required 5-amino-3-phenyl-1,2,4-triazole (yield 75%) was
synthesized by reaction of benzoyl chloride with amino guanidine

M. D. Kakwani et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6523–6526
6525
Table 1
Designed molecules
O
Synthesized N-(3-aryl-1,2,4-triazol-5-yl) cinnamamide derivatives with their MIC and
CC50 values
N
N
MICa
(lM)
CC50
(l
M)
b
Code
MW
R1
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1NH
290
324
320
304
306
305
332
350
324
335
280
341
360
374
137
86.20
92.6
39.1
82.2
40.8
20.5
18.8
4.6
38.6
9.7
11.6
4.8
1565.52
1620.37
1281.25
1759.87
1846.41
1911.48
1822.29
1214.29
1466.05
1985.07
2178.57
1668.62
1416.67
1564.17
1459.85
N
N
H
H
R
Figure 1. Designed novel N-(3-aryl-1,2,4-triazol-5-yl) cinnamamide derivatives.
9.0
8.6
1.8
N
O
N
a
Compounds were tested against Mtb H37Rv strains using REMA method.
Cytotoxicity studies were done on VERO Cell line C1008 using MTT assay.
B
b
N
N
H
H
A
bicarbonate using toluene as solvent and subsequent cyclization
of the intermediate in water under microwave irradiation.15
Mixed anhydride of cinnamic acid, synthesized using triethylamine
Figure 2. N-(3-Phenyl-1,2,4-triazol-5-yl) cinnamamide.
Figure 3. Topliss operational method adopted (only highlighted molecules were synthesized).

6526
M. D. Kakwani et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6523–6526
and methyl chloroformate was then reacted with 5-amino-3-
phenyl-1,2,4-triazole to obtain desired cinnamamide derivatives.
The structure of synthesized compounds was confirmed by spectral
analysis (IR, 1H NMR, 13C NMR and EIMS).
bacterial agents. The structural similarity of the molecules with the
reported FASII inhibitors, suggests that compounds may act by
inhibiting the FASII. Topliss operational method was used success-
fully to optimize the molecules with regards to activity and signif-
All the synthesized compounds, 1a–1n were screened against
Mtb H37Rv using two fold dilution technique, in order to determine
the actual minimum inhibitory concentration (MIC) using resazu-
rin microtiter assay (REMA).16 MIC is the lowest concentration at
which complete inhibition was observed and was determined by
visual inspection (color change from blue to pink in case of growth)
(Table 1). Isoniazid (INH) was used as the reference drug. The com-
pounds were also evaluated for toxicity on mammalian VERO cell
line (C1008) using 96-well microtitre plate and the CC50 values
were determined.17
icant improvement was observed from >90 lM to <5 lM. Taking
into account our preliminary biological results, our efforts are
now focused on understanding the mechanistic details of the mol-
ecules along with series expansion.
Acknowledgments
M.D.K. and A.C.L. are thankful to Department of Biotechnology,
Delhi and N.H.P.D. is thankful to University Grants Commission for
financial assistance.
Initially, compound (1a) with unsubstituted phenyl ring
was synthesized which showed moderate activity with MIC of
86.20 lM against M. tuberculosis. Subsequently, systematic synthe-
Supplementary data
sis of cinnamamide derivatives was carried out using Topliss oper-
ational method, since designed compounds possess two aromatic
rings as depicted in Figure 2.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.08.076.
At the beginning, ring B was kept constant as unsubstituted
phenyl ring, while ring A was optimized for its best substitution
with regards to activity. According to Topliss stepwise approach
(Fig. 3), the first substitution to aromatic ring has to be an electro-
negative group, chlorine. The compound with para-Cl substituent
on aromatic ring (1b) was synthesized and tested for its antimyco-
bacterial activity. para-Cl substitution resulted in slight decline in
References and notes
1. Daffé, M.; Reyrat, J.-M. The Mycobacterial Cell Envelope; ASM: Washington, D.C,
2008. Oxford Blackwell [distributor].
2. Banerjee, A.; Dubnau, E.; Quemard, A.; Balasubramanian, V.; Um, K. S.; Wilson,
T.; Collins, D.; de Lisle, G.; Jacobs, W. R., Jr. Science 1994, 263, 227.
3. Payne, D. Drug Discovery Today 2001, 6, 537.
4. Lu, H.; Tonge, P. J. Acc. Chem. Res. 2008, 41, 11.
5. Carvalho, S. A.; da Silva, E. F.; de Souza, M. V. N.; Lourenço, M. C. S.; Vicente, F. R.
Bioorg. Med. Chem. Lett. 2008, 18, 538.
activity (MIC 92.6
lM) from parent molecule (1a) with unsubsti-
tuted phenyl ring (MIC 86.20
l
M). Following the Topliss tree, both
6. Yoya, G. K.; Bedos-Belval, F.; Constant, P.; Duran, H.; Daffé, M.; Baltas, M. Bioorg.
Med. Chem. Lett. 2009, 19, 341.
7. Güzeldemirci, N. U.; Küçükbasmacı, Ö. Eur. J. Med. Chem. 2010, 45, 63. and
references cited in.
8. Bechet, K. H.; Draber, W.; Regal, K. Drugs Germ. 1972, 15, 79.
9. Kakwani, M. D.; Suryavanshi, P.; Ray, M.; Rajan, M. G. R.; Majee, S.; Samad, A.;
Devarajan, P.; Degani, M. S. Bioorg. Med. Chem. Lett. 2011. doi:10.1016/
j.bmcl.2011.02.022.
branches, least (L) as well as equal (E) were explored and com-
pounds 1c and 1d having para-OCH3 and para-CH3 substituents,
respectively, were synthesized. Significant improvement in activity
was observed with compound 1c (MIC 39.1
1d showed slight decline in activity (MIC 82.2
lM) while compound
lM). Moving ahead,
derivatives with substituents like para-OH (1e), para-NH2 (1f) and
3,4-dimethoxy (1h) were synthesized following the more (M)
branch directed by better activity of 1c. Compounds 1e, 1f and
1h exhibited MIC’s 40.8 lM, 20.5 lM and 4.6 lM, respectively.
Also, compound 1i with meta-Cl substituent was synthesized fol-
lowing the (L and E) branch. Compound 1i exhibited MIC of
38.6 lM. Following the (M) branch directed by compound 1i and
10. De, P.; Yoya, G. K.; Constant, P.; Bedos-Belval, F.; Duran, H.; Saffon, N.; Daff’e,
M.; Baltas, M. J. Med. Chem. 2011, 54, 1449.
11. Rawat, R.; Whitty, A.; Tonge, P. J. Proc. Nat. Acad. Sci. 2003, 100, 13881.
12. Bairwa, R.; Kakwani, M.; Tawari, N. R.; Lalchandani, J.; Ray, M. K.; Rajan, M. G.
R.; Degani, M. S. Bioorg. Med. Chem. Lett. 2010, 20, 1623.
13. Bairwa, R.; Tawari, N. R.; Alate, A.; Anam, S.; Degani, M. S.; Ray, M. K.; Rajan, M.
G. Med. Chem. 2010. [Epub ahead of print].
14. Tawari, N. R.; Bairwa, R.; Ray, M. K.; Rajan, M. G. R.; Degani, M. S. Bioorg. Med.
Chem. Lett. 2010, 20, 6175.
15. General procedure for the synthesis of 5-amino-3-(aryl)-1,2,4-triazoles
Step I: (a) Aminoguanidine hydrogen carbonate (6.0 g) in dry toluene (50 mL)
was cooled to 0 °C while freshly distilled benzoyl chloride (6.0 mL) was added
with stirring, during 0–5 h. After 12 h stirring at room temperature, the formed
precipitate was removed by filtration, and the residue was treated with water
(50 mL) and made alkaline with sodium carbonate. The solid was collected (3–
4 g; m p 180–182 °C) and recrystallised from water to obtain large colorless
prisms, mp 185 °C (dec.) Lit 183 °C.
1j was synthesized.
The compound (1j) with nitro substituent at meta position of
aromatic ring B showed MIC of <10
l
M while compound (1h) with
dimethoxy substituent showed MIC <5
l
M. To study the effect of
more bulkier substituent on ring A, compounds 1g, 1m and 1n
were synthesized with 3,4-di-oxo-methylene, 4-((2-methylal-
lyl)oxy) and 4-(trifluoromethoxy) substituents, respectively. Com-
Step I: (b) Arylamidoguanidine and water 20 mL were subjected to microwave
irradiation for 3 min at 90 °C and 100 W. After cooling, the solid obtained were
filtered, washed with ice-cold water and dried. Pure triazole was obtained after
recrystallization from water or aq EtOH.
pounds 1m and 1n showed activity <10
shows decline in activity (MIC-18.8 M) compared to compound
1h (MIC 4.6 M). Compound 1k and 1l were synthesized to study
lM, while compound 1g
l
l
Step II: (c) In a three-necked round-bottomed flask containing substituted
cinnamic acid 0.05 mol, 50 mL dichloromethane and triethylamine 0.1 mol,
methyl chloroformate 0.1 mol was added dropwise at 0 °C with stirring, the
mixture was stirred for 2 h at room temperature. Amine 0.025 mol was added
dropwise at 0 °C with stirring, the mixture was stirred 6–8 h at room
temperature. The solvents were removed under reduced pressure. The
residue was poured into 100 mL ice water and stirred for 10 min. The solid
obtained was filtered and further purified either by recrystallization in hot
methanol or by chromatographic purification using flash chromatography
(SiO2, 5 g, 35% EtOAc/hexanes).
the effect of heterocyclic ring system on antituberculosis activity.
Compound 1k with furan ring exhibit moderate activity of
11.6
1h with 3,4-dimethoxy showed MIC 4.6
(MIC 4.6 M) and compound 1j (MIC 9.7
l
M while compound 1l showed best activity of 4.8
M. Thus, compound 1h
M) were successfully ob-
lM and
l
l
l
tained with reasonably good antituberculosis activity by systemat-
ically optimizing the substituents on the aromatic ring A using
operational Topliss tree.
In conclusion, a novel series of N-(3-aryl-1,2,4-triazol-5-yl)
cinnamamide derivatives were synthesized as potential antimyco-
16. Palomino, J. C.; Martin, A.; Camacho, M.; Guerra, H.; Swings, J.; Portaels, F.
Antimicrob. Agents Chemother. 2002, 46, 2720.
17. Freshney, R. L. Culture of Animal Cells: A Manual of Basic Techniques, 4th ed.;
Willy-LISS, 2000.