Synthesis and biological evaluation of 2-acetamidothiophene-3-carboxamide derivatives against Leishmania donovani

Article abstract of DOI:10.1039/c3md00299c

A high-throughput (HTS) and high-content screening (HCS) campaign of a commercial library identified 2-acetamidothophen-3-carboxamide as a novel scaffold for developing new anti-leishmanial agents. A series of chemical modifications were performed to study the structure-activity relationship (SAR) and in vitro anti-leishmanial activities were evaluated using biological assays of not only extracellular promastigotes but also intracellular amastigotes. Compound 6a showed promising anti-amastigote activity (EC₅₀ = 6.41 μM) against L. donovani without any cytotoxicity (CC₅₀ > 50 μM) towards human macrophages.

Full text of DOI:10.1039/c3md00299c

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Synthesis and biological evaluation of
2-acetamidothiophene-3-carboxamide derivatives
against Leishmania donovani†
Cite this: Med. Chem. Commun., 2014,
5, 142
Sangmi Oh,a Bosung Kwon,a Sunju Kong,a Gyongseon Yang,b Nakyung Lee,b
Dawoon Han,b Junghyun Goo,b Jair L. Siqueira-Neto,‡b Lucio H. Freitas-Junior,§b
c
d
a
*
Michel Liuzzi, Jinhwa Lee and Rita Song
A high-throughput (HTS) and high-content screening (HCS) campaign of a commercial library identified
2-acetamidothophen-3-carboxamide as a novel scaffold for developing new anti-leishmanial agents.
A series of chemical modifications were performed to study the structure–activity relationship (SAR) and in
vitro anti-leishmanial activities were evaluated using biological assays of not only extracellular
promastigotes but also intracellular amastigotes. Compound 6a showed promising anti-amastigote activity
(EC50 ¼ 6.41 mM) against L. donovani without any cytotoxicity (CC50 > 50 mM) towards human macrophages.
Received 8th October 2013
Accepted 15th November 2013
DOI: 10.1039/c3md00299c
www.rsc.org/medchemcomm
in clinical development are mostly either reformulations or
combination therapies based on old drugs.5 Therefore, there is
Introduction
an urgent unmet medical need for the development of novel
anti-leishmanial drugs with improved efficacy and safety. Drug
discovery for leishmaniasis has been complicated by the lack of
validated drug targets and relevant drug assays. Target free cell-
based assays have been widely used, as they bypass the need for
a known target. In this context, we previously reported a high-
content screening (HCS) assay for nding novel chemical scaf-
folds targeting intracellular L. donovani amastigotes in human
macrophages.6 Most of the compounds reported as potential
anti-leishmanial agents such as pentamidine-like molecules,7,8
natural product-inspired hybrid compounds,9,10 and a novel
series of N-benzoyl-2-hydroxybenzamide have been validated
from target-free cell-based assays.11 Here we report the synthesis
and anti-leishmanial activity of 2-acetamidothiophene-3-
carboxamide derivatives which were tested against both L.
donovani promastigotes and intracellular amastigotes.
Leishmaniasis is one of the most neglected tropical diseases
caused by Leishmania, a protozoan parasite transmitted by the
bite of female phlebotomine sandies.1 More than 1.2 million
people worldwide are estimated to be infected by Leishmania
and more than 50 000 deaths are reported every year.2 Three
different types of leishmaniasis are characterized: visceral
leishmaniasis (VL), cutaneous leishmaniasis (CL), and muco-
cutaneous leishmaniasis (MCL). VL caused by Leishmania
donovani (L. donovani) is the most severe form of the disease,
being fatal if le untreated.
Most of the current drugs to treat VL, such as pentavalent
antimonials, amphotericin B, and miltefosine, are decades-old
and have many limitations. Both pentavalent antimonials and
amphotericin B are administered parenterally and their high
toxicity along with the emergence of drug resistance has posed
serious problems. Miltefosine is the only oral drug for the
treatment of VL; however, this drug is teratogenic and causes
extremely severe side effects.3,4 In addition, the drugs currently
Results and discussion
A high-throughput (HTS) and high-content screening (HCS)
campaign of a 200 000 chemical library utilizing amastigotes
infected cell-based assay identied the 2-acetamidothophen-3-
carboxamide scaffold. In search of compounds with improved
aMedicinal Chemistry Group, Institut Pasteur Korea, 696 Sampyeong-dong,
Bundang-gu, Seongnam-si, Gyeonggi-do 463-400, Korea. E-mail: rsong@ip-korea.org
bCenter for Neglected Diseases Drug Discovery (CND3), Institut Pasteur Korea, 696
Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do 463-400, Korea
cEarly Discovery Program, Institut Pasteur Korea, 696 Sampyeong-dong, Bundang-gu, activity, a series of 2-acetamidothiophene-3-carboxamide deriva-
Seongnam-si, Gyeonggi-do 463-400, Korea
tives were prepared by introducing diverse substitutions in three
parts: change of the aliphatic ring fused with thiophene, modi-
cation of both the 2-amino functional group and 3-carboxamide
part with different substituents. A general synthesis scheme for
dLate Discovery Program, Institut Pasteur Korea, 696 Sampyeong-dong, Bundang-gu,
Seongnam-si, Gyeonggi-do 463-400, Korea
† Electronic supplementary information (ESI) available: Experimental details in
biology and chemistry, melting point and NMR spectra of all new compounds.
2-acetamidothiophene-3-carboxamide derivatives is depicted in
Scheme 1. 2-Aminothiophenecarboxylates 1a–e were obtained
using Gewald cyclization as previously reported, and the reaction
See DOI: 10.1039/c3md00299c
‡ Present address: University of California, San Francisco, USA.
´
ˆ
§ Present address: Laboratorio Nacional de Biociencias (LNBio), Brazil.
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Scheme 1 Reagents and conditions: (A) sulfur 1.2 eq., morpholine 1.5 eq., EtOH, 45 ꢀC, 24 h; (B) TFAA or Ac2O 2.0 eq., DIPEA 2.0 eq., MC, 0 ꢀC, 24
h; (C) TFA 5.0 eq., MC, RT, 4 h; (D) R2H 1.2 eq., EDC 2.0 eq., HOBt 2.0 eq., TEA 3.0 eq., DMF, 50 ꢀC, 24 h; (E) TEA 4.0 eq., DMF, 45 ꢀC, 3 h.
of acylation of the 2-amino group followed by de-esterication promastigotes or axenic amastigotes; the latter usually in vitro
and EDC coupling reaction yielded the desired nal compounds cultured under conditions of acidic pH and elevated tempera-
6a–e and 7a–17a.12,13
ture.14 Ease of mass production of the parasites facilitated the
Due to the fact that the nal coupling reaction did not yield adaption of both species to a high-throughput assay format.
compound 18a as shown in Scheme 1, the synthesis of 18a was To evaluate the anti-leishmanial activity of the compounds
rst started from an amide coupling reaction (Scheme 2). In the under more physiologically relevant conditions, we measured the
case of compound 19a with a phenyl moiety at the R1 position, in vitro activity of the compounds against amastigotes in human
an unexpected cyclocondensation reaction occurred during macrophages and extracellular promastigotes.6 The intracellular
EDC coupling resulting in thienopyrimidinone ring formation. amastigote cell-based assay was developed by infecting differen-
Opening the ring by the reaction of LiHMDS in the presence of tiated THP-1 macrophages with L. donovani metacyclic promas-
p-anisidine nally yielded compound 19a (Scheme 3).
tigotes to lead to amastigote transformation.6 Each compound
For assessing the anti-leishmanial activity of the was added to the plate 24 hours aer infection and the quanti-
compounds, two different stages in the Leishmania life cycle cation of the infection ratio was done 3 days aer compound
were tested. One, the amastigote form, is intracellular and treatment. Fluorescent image analysis using automated confocal
specically found in the host macrophage. The other, the pro- microscopy and customized soware allowed for the quantitative
mastigote form, is predominately found in the insect vector. assessment of diverse information such as number of cells per
Even though amastigotes and promastigotes are substantially well, number of parasites per cell, infection ratio, and cell cyto-
different from a molecular perspective, drug discovery for plasmic area.6 Cytotoxicity against host cells was also quantied
leishmaniasis has traditionally been performed using either
Scheme 3 Reagents and conditions: (A) BzOH 1.2 eq., EDC 2.0 eq.,
Scheme 2 Reagents and conditions: (A) EDC 2.0 eq., HOBt 1.5 eq., HOBt 1.5 eq., TEA 1.5 eq., DMF, 50 ꢀC, 6 h, 50%; (B) TFA, RT, 1 h, 45%;
TEA 2.0 eq., DMF, 55 ꢀC, 22 h, 62%; (B) cycloheptanone 1.0 eq., sulfur (C) p-anisidine 1.2 eq., EDC 2.0 eq., HOBt 1.5 eq., TEA 1.5 eq., DMF, 50
1.2 eq., morpholine 3.0 eq., EtOH, 55 ꢀC, 30 h, 5%; (C) MeOCOCl 1.1 ꢀC, 6 h, 80%; (D) p-anisidine 1.0 eq., LiHMDS 1.0 eq., THF, RT, 15 min,
eq., In 0.7 eq., ACN, RT, 12 h, 18%.
15%.
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by ascertaining the cell number. The viability of promastigotes, retaining anti-amastigote activity and in reducing cytotoxicity. It
on the other hand, was analysed by resazurin assay aer is believed that this is an electronic effect because other
compound treatment.15,16 The in vitro anti-leishmanial activity of compounds containing an electron withdrawing group at the
the compounds is summarized in Table 1.
para-position were totally inactive (11a) or showed high cyto-
Compound 6a which contained cycloheptane fused with a toxicity against host macrophages (12a–14a). And also, it can be
thiophene moiety exhibited comparable anti-amastigote activity inferred that a hydrogen bond acceptor is essential at this
(6.41 mM) to that of the reference drug, miltefosine (7.10 mM). In position (6a), because methyl-substituted compound (10a) lost
the case of cytotoxicity, compound 6a did not show any toxicity their activity. The aminophenyl moiety at the R2 position is also
against host macrophages (CC50 > 50 mM), but miltefosine was important for anti-amastigote activity, as one carbon elongated-
found to be toxic at lower concentrations (CC50 ¼ 22.7 mM), aminobenzyl compound (15a) was found to be inactive. Other
resulting in a lower selectivity index, 3.19. Considering that substituents at the R1 position such as methyl (17a), methoxy
miltefosine was originally developed as an antineoplastic agent (18a), or phenyl (19a) instead of triuoromethyl groups were not
for topical use, a lower selectivity index was expected.
effective in improving the anti-amastigote activity. It is inter-
Compounds incorporating sterically hindered aliphatic esting to note that most of the compounds described here were
cycles such as cycloheptyl (6a) exhibited the best activity within inactive in the L. donovani promastigote assay although
the series, while compounds with cyclohexyl (6b) or cyclopentyl compounds 6a and 6d showed considerable activity in the
(6c) showed gradually decreasing activity (14.6 and >50 mM, amastigote cell-based assay. As described above, traditional
respectively). Likewise, substitution with two methyl groups drug screening has been carried out using promastigotes or
(6d) enhanced anti-amastigote activity compared to compounds axenic amastigotes. However, it has been known that promas-
with a less hindered cyclopentyl (6c) or without a substituent tigotes and axenic amastigotes are not fully representative of
(6e) (10.1 and >50 mM, respectively). Given that the sterically human leishmaniasis because amastigotes are exclusively
hindered aliphatic substitution improved the activity, lip- intracellular in vivo.6 The nding that 2-acetamidothiophene-
ophilicity of the compound seems to be an important factor for 3-carboxamide derivatives show anti-leishmanial activity only
exhibiting anti-leishmanial activity. A signicant decrease of against intracellular amastigotes may imply that the target of
anti-amastigote potency was observed when the para-methoxy the compound in this series is amastigote-specic or that the
group in R2 was changed to hydrogen (9a) or ortho-methoxy (8a). compound acts through a novel mechanism of action; for
The methoxy group at the para-position plays a critical role in example, by interacting with one or more cellular factors.
Table 1 In vitro activity of 2-acetamidothiophene-3-carboxamide derivatives against the amastigote and promastigote forms of L. donovani
Chemical structure
Activity against L. donovani
Promastigote form
Amastigote form
Compoundsa
R
R1
R2
EC50 (mM)b
CC50 (mM)c
SId
EC50 (mM)e
—
1a
3a
6a
6b
6c
6d
6e
7a
8a
9a
10a
11a
12a
13a
14a
15a
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)4–
–(CH2)3–
Me
tertButoxy
>50
>50
6.41
14.6
>50
10.1
>50
10.1
>50
>50
>50
>50
17.6
9.85
8.53
>50
>50
>50
>50
>50
0.25
7.10
>50
>50
>50
>50
>50
>50
>50
37.6
>50
>50
>50
>50
17.2
11.9
9.11
>50
>50
>50
>50
>50
7.57
22.7
N/Af
N/A
>7.80
>3.42
N/A
>4.95
N/A
3.71
N/A
N/A
N/A
N/A
0.90
1.20
1.06
N/A
N/A
N/A
N/Tg
N/T
>50
>50
N/T
>50
>50
21.5
42.0
N/T
N/T
N/T
>50
>50
>50
>50
N/T
>50
N/T
N/T
0.18
7.40
Methyl
tertButoxy
Triuoromethyl
Triuoromethyl
Triuoromethyl
Triuoromethyl
Triuoromethyl
Triuoromethyl
Triuoromethyl
Triuoromethyl
Triuoromethyl
Triuoromethyl
Triuoromethyl
Triuoromethyl
Triuoromethyl
Triuoromethyl
Triuoromethyl
Methyl
4-Methoxyphenylamino
4-Methoxyphenylamino
4-Methoxyphenylamino
4-Methoxyphenylamino
4-Methoxyphenylamino
3-Methoxyphenylamino
2-Methoxyphenylamino
Phenylamino
4-Methylphenylamino
4-Chlorophenylamino
4-Triuoromethylphenylamino
4-Triuoromethoxyphenylamino
4-Cyanophenylamino
4-Methoxybenzylamino
tertButylamino
H
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
—
16a
17a
18a
19a
4-Methoxyphenylamino
4-Methoxyphenylamino
4-Methoxyphenylamino
—
Methoxy
Phenyl
—
—
N/A
N/A
30.2
3.19
Amphotericin B
Miltefosine
—
—
a
Compounds 1a, 6a–6c, 8a–12a, 17a, 19a are already known molecules. b EC50 indicates half maximal effective concentration against intracellular L.
donovani (2-fold dilution with 10 point dose response). The average of duplicate determinations. c CC50 indicates cytotoxicity against THP-1 cells,
which was determined by THP-1 cell number. The average of duplicate determinations. d SI is the selectivity index (EC50/CC50). e EC50 indicates half
maximal effective concentration against extracellular L. donovani. The average of duplicate determinations. f N/A: not available. g N/T: not tested.
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Fig. 1 Confocal microscopy images of compounds 6a and 17a. (A) Infected THP-1 cells in the presence of 50 mM of compound 6a (upper left)
showed complete parasite clearance from the host cells while 50 mM of compound 17a (upper right) showed no effect. Amphotericin B (4 mM)
was used as a positive control (lower left), and DMSO (1%) was used as a negative control (lower right). Fluorescent DNA dye Draq5 was used to
visualize DNA within host cells and parasites. The solid arrow indicates nucleus and the dotted arrow indicates parasites in cytosol. (B) Dose
response curves of compounds 6a and 17a plotting the normalized anti-leishmanial activity (C) and the relative percentage of cell number
compared to DMSO represented cytotoxicity control (-).
The images from automated confocal microscopy and the
corresponding dose response curves of the representative
Acknowledgements
compounds 6a and 17a are shown in Fig. 1. In the case of
compound 6a, which showed comparable anti-leishmanial
activity to that of the marketed drug miltefosine, no parasite
was observed inside of host THP-1 cells at a concentration of 50
mM, while most of the parasites still remained using the same
concentration of the inactive compound 17a. In addition to the
promising anti-leishmanial activity, no signicant decrease in
cell number was observed when host THP-1 cells were treated
with compound 6a. The calculated percentage infection based
on the EC100 (effective concentration showing 100% activity as a
minimum measured infection ratio) of amphotericin B and 1%
DMSO (0% activity as a maximum measured infection ratio) was
normalized to percentage activity. Cytotoxicity was determined
by the percentage of cell number normalized against DMSO
control.
This work was supported by the National Research foundation
of Korea (NRF) grant funded by the Korea government (MSIP,
no. 2007-00559), Gyeonggi-do (no. K204EA000001-09E0100-
00110), and KISTI.
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A series of compounds based on the 2-acetamidothophen-3-
carboxamide scaffold were synthesized and tested using amas-
tigote infected cell-based assay and extracellular promastigote
assay. Compound 6a showed promising anti-amastigote activity
against L. donovani without any cytotoxicity toward human
macrophages. Further studies to improve the anti-leishmanial
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