Synthesis and evaluation of a novel series of pseudo-cinnamic derivatives as antituberculosis agents

Article abstract of DOI:10.1016/j.bmcl.2008.11.082

In an effort to develop potent new antituberculous drugs effective against Mycobacterium tuberculosis, we have prepared series of cinnamic derivatives (thioesters and amides) with 4-hydroxy and 4-alkoxy groups and investigated the in vitro activities of these compounds. Among them some displayed a good in vitro antibacterial activity, such as (E)-N-(2-acetamidoethyl)-3-{4-[(E)-3,7-dimethylocta-2,6-dienyloxy]phenyl}acrylamide 4b that showed a minimum inhibitory concentration of 0.1 μg/mL (0.26 μM) against M. tuberculosis H37Rv.

Full text of DOI:10.1016/j.bmcl.2008.11.082

Bioorganic & Medicinal Chemistry Letters 19 (2009) 341–343
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of a novel series of pseudo-cinnamic derivatives
as antituberculosis agents
Georges Koumba Yoya a,c, Florence Bedos-Belval a,c, Patricia Constant b,d, Hubert Duran a,c
,
Mamadou Daffé b,d, Michel Baltas a,c,
*
a Université de Toulouse, UPS, LSPCMIB (Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt Biologique), 118, Route de Narbonne, F-31062 Toulouse Cedex 9, France
b Université de Toulouse, UPS, IPBS (Institut de Pharmacologie et Biologie Structurale), Département Mécanismes Moléculaires des Infections Mycobactériennes,
205 route de Narbonne, F-31077 Toulouse Cedex 04, France
c CNRS, LSPCMIB (Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt Biologique), 118, Route de Narbonne, F-31062 Toulouse Cedex 9, France
d CNRS, IPBS (Institut de Pharmacologie et Biologie Structurale), Département Mécanismes Moléculaires des Infections Mycobactériennes, 205 route de Narbonne,
F-31077 Toulouse Cedex 04, France
a r t i c l e i n f o
a b s t r a c t
Article history:
In an effort to develop potent new antituberculous drugs effective against Mycobacterium tuberculosis, we
have prepared series of cinnamic derivatives (thioesters and amides) with 4-hydroxy and 4-alkoxy
groups and investigated the in vitro activities of these compounds. Among them some displayed a good
in vitro antibacterial activity, such as (E)-N-(2-acetamidoethyl)-3-{4-[(E)-3,7-dimethylocta-2,6-dienyl-
Received 23 October 2008
Revised 19 November 2008
Accepted 21 November 2008
Available online 27 November 2008
oxy]phenyl}acrylamide 4b that showed a minimum inhibitory concentration of 0.1
against M. tuberculosis H37Rv.
lg/mL (0.26 lM)
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
Cinnamic derivatives
Antituberculosis agents
Mycobacterium tuberculosis
TB MIC
Mycobacterium tuberculosis, the causative agent of tuberculosis,
is the greatest single infectious cause of mortality worldwide, kill-
ing roughly two million people annually.1 In addition, human
immunodeficiency virus infection has been a major contributing
factor in the actual resurgence of tuberculosis.2 The emergence of
multidrug-resistant (MDR) strains of M. tuberculosis that are resis-
tant to the two most effective drugs, isonicotinic acid hydrazide
(INH)3 and rifampicin4 have reaffirmed tuberculosis as a primary
public health threat. Moreover, strains that are even more resistant
than MDR, the so-called extensively drug resistant (XDR), have re-
cently been described.5 So there is a pressing need for new chemo-
therapeutic agents to combat the emergence of resistance and
shorten the duration of treatment to improve patient compliance.6
Mycobacteria produce a wide array of complex fatty acids such
fatty acids produced by the FAS-I system to yield the very long
meromycolic acyl chain of mycolates. Genetic knockout and knock
down experiments have shown that the individual enzymes of the
FAS-II pathway are essential for mycobacterial cell survival.8–11
One of the four steps of the elongation cycles of the growing long
chain fatty acids (Scheme 1) catalyzed by the enoyl reductase InhA,
an NADH-dependent enzyme, has proven to be the target of both
NADPH NADP+
O
O
O
O
O
OH
O
CoA + CO2
FabH
R
S-CoA
R
S-AcpM
R
S-AcpM
R
S-AcpM
Dehydrase
H2O
as mycolic acids, very long chain (C-60-90)
a-branched and b-
AcpM
CO2
hydroxylated fatty acids, not found in mammalian cells, whose bio-
synthesis involved the fatty acid synthase type II (FAS-II) system.7
FAS-II is an acyl carrier protein (ACP)-dependent fatty acid syn-
thase system found in plants, bacteria, parasites, and mitochondria
where it usually performs de novo biosynthesis.7 The FAS-II system
is however unique in mycobacteria in that it elongates long chain
FAS II
+
KasA
KasB
O
O
-O
S-AcpM
R
O
O
CoA
FabD
ACP
InhA
NAD+ NADH
S-AcpM
R
S-AcpM
O
O
-O
S-CoA
* Corresponding author. Tel.: +33 (0)5 61 55 62 89; fax: +33 (0)5 61 55 60 11.
E-mail address: baltas@chimie.ups-tlse.fr (M. Baltas).
Scheme 1. General scheme of FAS-II system involved in mycolic acids biosynthesis.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.082

342
G. K. Yoya et al. / Bioorg. Med. Chem. Lett. 19 (2009) 341–343
the front-line antituberculous drug INH and ethionamide.12 There-
fore, the FAS-II biosynthesis pathway represents a validated and
yet promising target for drug discovery.13 Among families of com-
pounds that have been explored as inhibitors of the FAS-II system,
we can mention diphenyl ether systems interacting with the en-
zyme-cofactor binary complex, the compounds that covalently
bind to the cofactor enzyme site, but also indols, benzofuran and
more recently cinnamic acid based derivatives.14,15 Herein we
present our preliminary results on the synthesis and evaluation
of the antimycobacterial activities of some cinnamic acid deriva-
tives. It is envisaged from structural similarities, that these com-
pounds may act on the biosynthesis of mycolic acids and more
tion. The corresponding phenoxy ethers obtained in good yield
(86–95%) after silica gel purification, were then converted quanti-
tatively into the corresponding acids 1a–c under reflux and basic
conditions (K2CO3 in methanol).
Acids 1a–c thus prepared and the commercially available ones
were used as starting materials for the preparation of all thioesters
and amides mentioned in Scheme 2. Coupling of acid 1a–g with
thiols (N-acetylcysteamine) or various amines (2-aminopyridine,
acetylethylenediamine, or tryptamine) using EDC in the presence
of DMAP, in CH2Cl2 for 24 h afforded the respective thioesters
2a–g and amides derivatives 4a, 4b, 4d–f, 5a–b, 5d, 5f, 6a–b, 6d
in good to excellent yield (62–96%).19 Only thiophenyl esters 3e–
f were synthesized differently through coupling reaction between
specifically on FAS-II, which uses
a,b-enoyl systems as substrates
(Scheme 1).16
The compounds synthesized, present the following general char-
acteristics. They possess all cinnamoyl main frames namely the aro-
matic moiety adjacent to the enoyl function. The aromatic group
possesses the different substitutions of the cinnamic acids namely
ferulic acid (4-OH, 3-OMe), sinapic acid (4-OH, 3,5-OMe), or couma-
ricacid(4-OH). Compoundslipophilicitywas modulatedby different
4-alkoxy substitutions, such as methoxy, isopentenyloxy, geranyl-
oxy, or farnesyloxy groups introduced only on para-coumaric deriv-
atives. The carbonyl function of the enoyl system was functionalized
withN-acetylcysteamine, thesmallestframeof ACPorits amideana-
logue (first family of compounds). Aromatic systems like thiophenyl
Table 1
Structure, MIC, and solubility data of cinnamic acid derivatives
R1
RO
R2
O
Compound
R
R1
R2
TB MIC (
l
g/mL)
CLogPa
2a
2b
2c
2d
2e
2f
2g
3e
3f
4a
4b
4d
5a
5b
5d
5f
6a
6b
6d
6h
Isopentenyl
Geranyl
Farnesyl
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
32
0.6
0.6
63
63
125
125
8
3.46
5.75
8.54
1.65
1.09
0.91
0.74
4.10
3.92
2.78
4.81
1.08
5.43
7.54
3.63
2.89
4.34
6.65
2.54
2.10
or aminopyridine have also been introduced into the
a-position
H
from the carbonyl (second family). Finally, tryptamine, a frame with
linear side chain and aromatic ring has been used as coupling re-
agent. These different patterns have been introduced in order to esti-
mate the impact on activity of functionalised carbonyl group
addressed to the substrate’s or cofactor’s active site.
All synthesis started from acid derivatives. 4-Methoxycinnamic,
coumaric, ferulic, and sinapic acids are commercially available;
acids 1a–c were obtained in three steps starting from the coumaric
acid. Firstly the corresponding methyl ester was obtained by
refluxing the acid for 24 h in methanol in presence of H2SO4 and
3 Å molecular sieves (86% yield after silica gel purification).17 Then
we proceeded to alkylation of the 4-OH phenolic group. All reac-
tions were carried out by refluxing the convenient alkyl bromide
(isopentenyl, geranyl, or farnesyl) in dry acetone in presence of
K2CO3 and KI.18 The reactions were monitored by TLC until comple-
OCH3
OCH3
H
OCH3
H
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
8
Isopentenyl
Geranyl
CH3
Isopentenyl
Geranyl
CH3
63
0.1
>500
63
32
500
8
16
1
63
125
H
H
H
H
H
H
OCH3
H
Isopentenyl
Geranyl
CH3
H
H
CH3
OCH3
a
CLogP was calculated using the ChemDraw Ultra, version 10.0, software by
Cambridge Soft.
R1
R1
RO
R2
RO
O
S
S
N
H
R2
O
O
ii)
2a-g
i)
3e, 3f
R1
R1
RO
R2
RO
RO
O
H
N
i)
i)
H
N
OH
N
N
H
O
O
O
6a, 6b, 6d, 6h
4a, 4b, 4d
1a-g
i)
R1
RO
H
N
NH
O
5a, 5b, 5d, 5f
Scheme 2. Reagents and conditions: (i) DMAP, EDC, N-acetylcysteamine, 4-acetylethylenediamine, tryptamine, or 2-aminopyridine CH2Cl2, rt, 24 h; (ii) (COCl)2, DMF, CH2Cl2,
0 °C, 1 h, then thiophenol, BuLi, THF, À30 °C, 2 h.

G. K. Yoya et al. / Bioorg. Med. Chem. Lett. 19 (2009) 341–343
343
the thiophenate lithium salt and the corresponding carboxymeth-
ylene iminium chlorides prepared by the reaction of carboxylic
acids with N,N-dimethylchloromethylene iminium chloride.20
Compounds were purified by flash silica gel chromatography
(60–70%) before being tested on M. tuberculosis.
Susceptibility of M. tuberculosis (strain H37Rv) to the synthetic
compounds was tested by determining the minimal inhibitory con-
centration (MIC) (Table 1). We used a colorimetric microassay
based on the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, Sigma) to formazan by metaboli-
cally active cells.21,22 Briefly, serial twofold dilutions of each com-
pound were prepared in 7H9 broth (Middlebrook 7H9 broth base,
behave as weaker inhibitors in comparison to the corresponding
aromatic and linear ones.
In conclusion, this preliminary study shows that cinnamic
derivatives may be regarded as interesting leads in the design
and synthesis of M. tuberculosis inhibitors. While the 2-aminopyr-
idine derivative 6b presented a good MIC value against M. tubercu-
losis, the linear thioester 2b and especially the amide 4b showed
the best inhibitory activity of this explored series. Taking into ac-
count our preliminary results on the families of compounds evalu-
ated, our efforts are now focused on the understanding of their
activities on the FAS-II system. Efforts are also undertaken towards
elaboration of new families possessing the functionalized cinnam-
oyl frames.
Difco) using 96-well microtiter plates and 100
suspension in 7H9 broth were added to each well. After 6 days
incubation, MTT was added (50 L, 1 mg/mL). After one day incu-
lL of M. tuberculosis
l
Acknowledgments
bation, solubilization buffer was added to each well. The optical
densities were measured at 570 nm. The MIC was determined as
the lowest concentration of compound that inhibited bacterial
growth (absorbance from untreated bacilli was taken as a control
for growth). In these conditions, ethambutol, an effective and clin-
We thank Gabon Government for GK grant. Thanks are also due
to the CNRS, and the ‘Université Paul Sabatier’ for financial support.
This project has also been supported by the European Commission
Contract No. LSHP-CT-2005-018923 (NM4TB).
ically used drug, was also tested exhibited a MIC of 2–4 lg/mL, in
agreement with published data, validating thus the method used in
the present study.
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geranyl chain (2b, 0.6
was increased 100-fold in comparison with the compound 2d
(0.6 vs 63 g/mL, respectively).
lg/mL, 1.5 lM) was introduced. Activity
l
The same trends were also observed when N-acetylethylenedi-
amine was used as coupling partner of the enoyl system of amides
possessing only 4-alkoxy substitution (compounds 4a, 4b, 4d).
While amides 4a, 4d, are less active than the corresponding thioes-
ter counterparts, the derivative 4b showed a very potent activity
(0.1 lg/mL). It is noteworthy, that 4b has CLogP less than 5, which
means that it is likely to have greater oral bioavailability by Lipin-
ski’s rules23 than 2b (as well as MIC, Table 1).
Regarding the second family of compounds tested, two aro-
matic systems are reported here: thiophenyl and aminopyridine
derivatives. While 3e, 3f aromatic thioesters, present weak activi-
ties against M. tuberculosis, their MIC values are found to be eight-
fold lower than those of the corresponding linear thioesters 2e and
2f, indicating a better activity for aromatic thioesters. Their high
lipophilicity, and the fragility of the C(O)–SAr bond towards hydro-
lysis will probably prevent these compounds from being investi-
gated by further modification.
The pyridyl amides 6a, 6d synthesized showed the same trends
as the corresponding linear amides in terms of activity vs substitu-
tion pattern on the 4-hydroxy position. The best result was ob-
tained for the geranylated derivative 6b (1 lg/mL, 2.6 lM).
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However, 6b in comparison to 4b showed higher lipophilicity as
well as MIC.
Finally, amides possessing the mixed linear and aromatic frame
found in tryptamine were tested. Tryptamine derivatives seem to