Antitrypanosomatid Pharmacomodulation at Position 3 of the 8-Nitroquinolin-2(1H)-one Scaffold Using Palladium-Catalysed Cross-Coupling Reactions

Article abstract of DOI:10.1002/cmdc.201800456

An antikinetoplastid pharmacomodulation study at position 3 of the recently described hit molecule 3-bromo-8-nitroquinolin-2(1H)-one was conducted. Twenty-four derivatives were synthesised using the Suzuki–Miyaura cross-coupling reaction and evaluated in vitro on both Leishmania infantum axenic amastigotes and Trypanosoma brucei brucei trypomastigotes. Introduction of a para-carboxyphenyl group at position 3 of the scaffold led to the selective antitrypanosomal hit molecule 3-(4-carboxyphenyl)-8-nitroquinolin-2(1H)-one (21) with a lower reduction potential (?0.56 V) than the initial hit (?0.45 V). Compound 21 displays micromolar antitrypanosomal activity (IC₅₀=1.5 μm) and low cytotoxicity on the human HepG2 cell line (CC₅₀=120 μm), having a higher selectivity index (SI=80) than the reference drug eflornithine. Contrary to results previously obtained in this series, hit compound 21 is inactive toward L. infantum and is not efficiently bioactivated by T. brucei brucei type I nitroreductase, which suggests the existence of an alternative mechanism of action.

Full text of DOI:10.1002/cmdc.201800456

DOI: 10.1002/cmdc.201800456
Full Papers
Antitrypanosomatid Pharmacomodulation at Position 3 of
the 8-Nitroquinolin-2(1H)-one Scaffold Using Palladium-
Catalysed Cross-Coupling Reactions
Julien Pedron+,[a] Clotilde Boudot+,[b] Sandra Bourgeade-Delmas,[c] Alix Sournia-Saquet,[a]
Lucie Paloque,[a] Maryam Rastegari,[a] Mansour Abdoulaye,[a] Hussein El-Kashef,[d]
Colin Bonduelle,[a] Geneviꢀve Pratviel,[a] Susan Wyllie,[e] Alan H. Fairlamb,[e]
Bertrand Courtioux,[b] Pierre Verhaeghe,*[a] and Alexis Valentin[c]
An antikinetoplastid pharmacomodulation study at position 3
of the recently described hit molecule 3-bromo-8-nitroquino-
lin-2(1H)-one was conducted. Twenty-four derivatives were syn-
thesised using the Suzuki–Miyaura cross-coupling reaction and
evaluated in vitro on both Leishmania infantum axenic amasti-
gotes and Trypanosoma brucei brucei trypomastigotes. Intro-
duction of a para-carboxyphenyl group at position 3 of the
scaffold led to the selective antitrypanosomal hit molecule 3-
(4-carboxyphenyl)-8-nitroquinolin-2(1H)-one (21) with a lower
reduction potential (ꢀ0.56 V) than the initial hit (ꢀ0.45 V).
Compound 21 displays micromolar antitrypanosomal activity
(IC50 =1.5 mm) and low cytotoxicity on the human HepG2 cell
line (CC50 =120 mm), having a higher selectivity index (SI=80)
than the reference drug eflornithine. Contrary to results previ-
ously obtained in this series, hit compound 21 is inactive
toward L. infantum and is not efficiently bioactivated by
T. brucei brucei type I nitroreductase, which suggests the exis-
tence of an alternative mechanism of action.
Introduction
Kinetoplastids are flagellated protozoan parasites responsible
for lethal neglected tropical diseases such as human African
trypanosomiasis (HAT) and visceral leishmaniasis (VL). They are
characterised by the presence of a circular piece of DNA, called
a kinetoplast, adjacent to the flagellar basal body. Trypanosoma
parasites are transmitted by the bite of an infected sandfly and
are the causative agents of HAT, also known as sleeping sick-
ness. There are many species of Trypanosoma, but only
T. brucei gambiense and T. brucei rhodesiense are responsible for
HAT.[1] During the process of infection, metacylic trypomasti-
gotes enter the blood circulation from the bite of the tsetse fly
and spread throughout the whole organism, where they differ-
entiate into bloodstream trypomastigotes; this is the hemo-
lymphatic stage. After several weeks or months of symptoms
such as headaches, anaemia and hepatosplenomegaly, trypo-
mastigotes cross the blood–brain barrier and cause damage to
the central nervous system, leading to sleeping disorders, be-
havioural disorders, seizures, coma and finally death: this is the
meningoencephalic stage.[2] Leishmania, which is responsible
for leishmaniasis, is another protozoan parasite and is transmit-
ted by the bite of an infected sandfly. L. donovani and L. infan-
tum are the two major species causing the most severe form
of the disease: VL.[3] In brief, metacyclic promastigotes pene-
trate the skin during the blood meal of an infected sandfly.
They are internalised by mononuclear phagocytic cells such as
macrophages, in which they differentiate into amastigotes. The
parasites continue to multiply until the destruction of these
cells and disseminate into many organs, such as liver and
spleen, leading to death.[4]
[a] J. Pedron,+ Dr. A. Sournia-Saquet, Dr. L. Paloque, M. Rastegari,
M. Abdoulaye, Dr. C. Bonduelle, Dr. G. Pratviel, Prof. P. Verhaeghe
LCC-CNRS, Universitꢀ de Toulouse, CNRS, UPS, 205 route de Narbonne,
31077 Toulouse (France)
E-mail: pierre.verhaeghe@lcc-toulouse.fr
[b] C. Boudot,+ Dr. B. Courtioux
Universitꢀ de Limoges, UMR INSERM 1094, Neuroꢀpidꢀmiologie Tropicale,
Facultꢀ de Pharmacie, 2 rue du Dr Marcland, 87025 Limoges (France)
[c] Dr. S. Bourgeade-Delmas, Prof. A. Valentin
UMR 152 PharmaDev, Universitꢀ de Toulouse, IRD, UPS, 35 Chemin des Mar-
aꢁchers, 31400 Toulouse (France)
It is estimated that more than 1 billion people are at risk of
contracting HAT and VL and that each year there are 55000
new cases with more than 25000 deaths from these diseas-
es.[5,6] These numbers are likely underestimated because of the
difficulty in accessing rural areas and the unspecific symptoms
in the early stages of the diseases. Currently, there are few effi-
cient and safe drugs available on the market against these ne-
glected tropical diseases. Pentamidine and suramin are used
for the treatment of the first stage of HAT but these drugs are
highly toxic and require the patient to be hospitalised for i.v.
[d] Prof. H. El-Kashef
Assiut University, Faculty of Science, Department of Chemistry, 71516 Assiut
(Egypt)
[e] Dr. S. Wyllie, Prof. A. H. Fairlamb
University of Dundee, School of Life Sciences, Division of Biological Chemis-
try and Drug Discovery, Dow Street, Dundee DD1 5EH (UK)
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under:
https://doi.org/10.1002/cmdc.201800456.
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Figure 1. Structure, indication and route of administration of antitrypanosomal drugs on the market.
administration.[7] Melarsoprol, an arsenic-containing drug, and
a combination of eflornithine and nifurtimox are suitable for
the second stage of HAT (Figure 1).[8] The same is true of VL,
for which only amphotericin B, miltefosine, antimonial deriva-
tives, pentamidine and paromomycin are available. Among
these drugs, miltefosine is the only orally available drug. These
drugs are either expensive (e.g., liposomal amphotericin B),
present severe side effects (e.g., the nephrotoxicity of ampho-
tericin B, the teratogenicity of miltefosine) or show an increas-
ing lack of efficacy due to the emergence of resistant parasites
(antimony derivatives and miltefosine).[9] This global issue calls
for the discovery of new antikinetoplastid molecules.
sulfoxide and a sulfone derivative) which are active against the
Trypanosoma parasites. Fexinidazole is selectively bioactivated
by type I parasitic nitroreductases (NTRs) leading, through a
successive two-electron reduction, to cytotoxic electrophilic
metabolites such as nitroso and hydroxylamine derivatives.[14]
Two NTRs have been identified in Leishmania (NTR1 and
NTR2)[15,16] and only one in Trypanosoma.[17] These nitroreduc-
tases are absent from mammalian cells. Therefore, substrates
of these enzymes could be selective antikinetoplastid candi-
dates. Unfortunately, no X-ray structure of these parasitic NTRs
is available, which restricts the use of most classical rational
medicinal chemistry approaches, such as docking, for the
design of new substrates of these enzymes.
Unfortunately, today, there are only two new chemical enti-
ties in clinical trials against HAT and none against VL
(Figure 2).[10] Acoziborole is an orally active benzoxaborole in
phase IIb/III of clinical trials; it is active against both stages of
HAT.[11] Fexinidazole, a 5-nitroimidazole, was recently in phase II
of clinical trials against VL but showed a lack of efficacy, where-
as it progressed to a phase IIIb study against HAT.[12,13] This
molecule is rapidly metabolised in vivo into two metabolites (a
Our research team has been working on the synthesis of
new antikinetoplastid molecules for several years. Starting
from a chemical study on 2-substituted nitroquinoline deriva-
tives with antiparasitic potential,[18] we identified a new anti-
leishmanial hit: 8-nitroquinolin-2(1H)-one.[19] Pharmacomodula-
tion studies at position 4 of this scaffold were then real-
ised.[20,21] Recently, we described a comprehensive electro-
chemistry-guided study of this pharmacophore and the devel-
opment of a computational model able to predict the redox
potentials of each molecule in the series.[22] Thus, a new antiki-
netoplastid hit molecule was identified (Figure 3). This mole-
cule was not genotoxic in a comet assay and was selectively
bioactivated by type 1 NTRs of L. donovani and T. brucei
brucei.[22]
Figure 2. Molecular structure of drug candidates acoziborole and fexinida-
zole (with its active metabolites).
Figure 3. Structure and biological profile of the previously identified antiki-
netoplastid hit 3-bromo-8-nitroquinolin-2(1H)-one.
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Here, we present a pharmacomodulation study at position 3
of the scaffold, using Suzuki–Miyaura or Sonogashira cross-cou-
pling reactions. To explore structure–activity relationships, 26
molecules were synthesised and evaluated on both L. infantum
axenic amastigotes and T. brucei brucei trypomastigotes. These
molecules were also assessed for their cytotoxicity on the
HepG2 human cell line.
position 3 by heating in HBr (48% aqueous solution) at reflux
in the presence of sodium bromate, as reported by O’Brien
and co-workers.[24]
Then, the microwave-assisted Suzuki–Miyaura cross-coupling
reaction between 3-bromo-8-nitroquinolin-2(1H)-one and p-
methoxyphenylboronic acid was studied to find optimal condi-
tions (Table 1). Several parameters were investigated such as
the nature of the solvent, base, Pd catalyst and the reaction
temperature.
The first Suzuki–Miyaura reaction conditions (Table 1,
entry 1) were inspired from a previously described protocol for
introducing an aryl moiety at position 3 of the quinolinone
ring.[25] This reaction was achieved in DMF with microwave
(MW) heating, using 3 equivalents of Na2CO3 as a base, 0.1
equivalents of Pd(OAc)2 as a catalyst and 1.2 equivalents of p-
methoxyphenylboronic acid in a sealed tube, to afford the de-
sired compound in 43% yield. The first parameter studied was
the solvent (Table 1, entries 1–7). By using toluene, dioxane,
THF, 1,2-dimethoxyethane (DME) or DMSO, partial conversion
or degradation of the substrate was observed, whereas the use
of a DMF/H2O mixture led to a 46% yield. Then, two others
bases were studied (Table 1, entries 8–9) but in each case led
to the degradation of the substrate within 1 h. In DMF, the re-
placement of Pd(OAc)2 with Pd(PPh3)4 decreased the yield of
the reaction from 43% to 26% (Table 1, entry 10). Then, adapt-
ing another previously described protocol using DME and
Pd(PPh3)4 (Table 1, entry 11), a slightly better yield was ob-
tained.[26] This result was then improved by replacing Na2CO3
with K2CO3, which afforded a yield of 84%. The nature of the
Results and Discussion
Chemistry
3-Bromo-8-nitroquinolin-2(1H)-one was prepared in three
steps, as presented in Scheme 1.[22] Nitration of 2-chloroquino-
line mainly led to the intermediate 2-chloro-8-nitroquinoline,
which was transformed in a second step into the correspond-
ing lactam, according to a previously reported procedure.[23] 8-
Nitroquinolin-2(1H)-one was finally selectively halogenated at
Scheme 1. Synthesis of the initial antikinetoplastid hit. a) H2SO4, HNO3, RT,
2 h, 50%; b) CH3CN, HClO4, 1008C, 72 h, 96%; c) NaBrO3, HBr (48% in H2O),
1008C, 5 h, 68%.
Table 1. Optimisation of the Suzuki–Miyaura cross-coupling reaction between 3-bromo-8-nitroquinolin-2(1H)-one and p-methoxyphenylboronic acid.
Entry
Solvent
Base, 3 equiv
Catalyst
Temperature [8C]
Time [h]
Yield [%][a]
43
1
2
3
4
5
6
7
8
DMF
Na2CO3
Na2CO3
Na2CO3
Na2CO3
Na2CO3
Na2CO3
Na2CO3
K2CO3
Cs2CO3
Na2CO3
Na2CO3
K2CO3
K2CO3
K2CO3
K2CO3
Cs2CO3
CsF
Pd(OAc)2, 0.1 equiv
Pd(OAc)2, 0.1 equiv
Pd(OAc)2, 0.1 equiv
Pd(OAc), 0.1 equiv
Pd(OAc)2, 0.1 equiv
Pd(OAc)2, 0.1 equiv
Pd(OAc)2, 0.1 equiv
Pd(OAc)2, 0.1 equiv
Pd(OAc)2, 0.1 equiv
Pd(PPh3)4, 0.1 equiv
Pd(PPh3)4, 0.1 equiv
Pd(PPh3)4, 0.1 equiv
Pd(PPh2)Cl2, 0.1 equiv
Pd(dppf)Cl2, 0.1 equiv
Pd(OAc)2, 0.1 equiv
Pd(PPh3)4, 0.1 equiv
Pd(PPh3)4, 0.1 equiv
Pd(PPh3)4, 0.05 equiv
150
150
110
100
66
1
1
8
8
8
8
1
1
1
1
3
4
8
8
8
2
2
4
[b]
DMSO
Toluene
Dioxane
THF
–
–
–
–
[c]
[c]
[c]
[c]
DME
85
–
DMF/H2O (8:2)
DMF/H2O (8:2)
DMF/H2O (8:2)
DMF
DME
DME
DME
DME
DME
DME
100
100
100
150
85
85
85
85
85
46
[b]
–
–
[b]
9
10
11
12
13
14
15
16
17
18
26
52
84
[c]
–
–
–
[c]
[c]
85
85
85
88
88
56
DME
DME
Cs2CO3
[a] The yield was calculated after purification by chromatography on silica gel. [b] Degradation of the substrate was observed by TLC. [c] Only partial con-
version of the substrate was observed by TLC.
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catalyst was then studied (Table 1, entries 13–15), but none of
these reactions led to an improvement in the yield, and only a
few percent conversion was observed. Finally, the best results
were obtained by using Cs2CO3 or CsF as a base (Table 1, en-
tries 16 and 17), which afforded efficient Suzuki–Miyaura cross-
coupling reactions of this substrate. Decreasing of the amount
of catalyst to 0.05 equivalents led to a lower reaction yield
(56% Table 1, entry 18) in comparison with the reaction condi-
tions of entry 16, which were chosen as the most suitable.
Next, the procedure was extended to 19 other arylboronic
acids, to afford new derivatives bearing phenyl, thiophene,
furan or pyridine moieties at position 3 of the scaffold
(Scheme 2). The reactions yields were generally greater than
65% (for 14 derivatives) but were lower for 4-hydroxyphenyl-
boronic acid and 4-aminophenylboronic acid with 37% and
41% yields, respectively.
Scheme 3. Preparation of compounds 21–23.
that temperature had a strong influence on the reaction. At
08C in DME, the reaction did not take place, whereas it pro-
ceeded at 15 and 258C, affording compound 24. Surprisingly,
the unexpected 8-nitro-2-phenylfuro[2,3-b]quinoline (25)
formed with heating at 408C in DME or DMF. Compound 25
was also obtained upon attempting to recrystallise 24 from
acetonitrile, underlining the instability of this product. Such a
consecutive Sonogashira coupling and cyclisation reaction,
leading to furo[2,3-b]pyridine derivatives, has been reported in
refs. [28] and [29].
The optimisation of both Suzuki–Miyaura and Sonogashira
cross-coupling reactions led to the synthesis of 25 new mole-
cules with either an aryl or an alkynyl group at position 3 of
the scaffold. With the exception of compound 24, which was
considered too unstable, all these molecules were evaluated in
vitro to determine their antikinetoplastid potential. Compound
25 could not be evaluated because of low aqueous solubility.
Scheme 2. General procedure for the Suzuki–Miyaura cross-coupling reac-
tion between 3-bromo-8-nitroquinolin-2(1H)-one and various arylboronic
acids.
Three additional compounds (21–23) were synthesised by
the saponification of compounds 18–20 into the carboxylic
acid derivatives, using an excess of sodium hydroxide in an
ethanol/water mixture (Scheme 3).
Compound evaluation
Initially, the cytotoxicity of these molecules was assessed in
vitro on the HepG2 human cell line, and the corresponding
CC50 values were compared with that of the reference drug
doxorubicin (Table 3). The biological results showed that com-
pounds 1–11 were not soluble enough in aqueous medium to
be tested. The water solubility was improved with compounds
10, 12–17 and 21–23 bearing either a pyridin-3-yl or a phenyl
moiety at position 3 of the scaffold; the phenyl group being
substituted by a hydrophilic group such as hydroxymethyl or
aldehyde or an ionised carboxyl group. These compounds dis-
In parallel, with the aim of broadening the chemical diversity
at position 3 of the scaffold and introduce alkynyl moieties, a
Sonogashira cross-coupling reaction was devised. The initial
conditions were adapted from a previously described proto-
col.[27] As presented in Table 2, after brief optimisation of the
reaction between 3-bromo-8-nitroquinolinone and phenylace-
tylene, DME was chosen as the solvent, and the reaction was
carried out at 158C (Table 2, entry 6). Interestingly, it was noted
Table 2. Optimisation of the Sonogashira cross-coupling reactions of 3-bromo-8-nitroquinolin-2(1H)-one.
Entry
Solvent
Temperature [8C]
Yield 24 [%][a]
Yield 25 [%][a]
Time [h]
[b]
[b]
1
2
3
4
5
6
7
8
DMF
THF
25
25
25
25
0
15
40
40
–
–
–
–
–
–
48
48
48
0.25
48
1.5
0.5
36
[b]
[b]
[b]
[b]
Et3N
DME
DME
DME
DME
DMF
56
0
[b]
[b]
–
–
72
0
0
0
51
56
[a] The yield was calculated after purification by chromatography on silica gel. [b] Only partial conversion of the substrate was observed by TLC.
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Table 3. In vitro antileishmanial, antitrypanosomal and cytotoxic activities of compounds 1–25 and reference standards.
Compound
R (% yield)
IC50 [mm]
L. infantum axenic
HepG2 CC50 [mm]
Antitrypanosomal
selectivity index[g]
T. brucei brucei
amastigotes
trypomastigotes
1
2
3
4
5
6
7
8
Phenyl (90)
>12[a]
>6[a]
4.7ꢂ2.7
>12[a]
>6[a]
>2
–
–
–
–
–
–
–
4-OCH3-phenyl (88)
4-OH-phenyl (37)
4-NH2-phenyl (41)
4-Cl-phenyl (73)
4-F-phenyl (70)
4-CF3-phenyl (60)
3-thienyl (92)
2-furyl (65)
3-pyridyl (62)
4-pyridyl (71)
–
>3[a]
–
–
–
–
–
–
–
>3[a]
>3[a]
>3[a]
>3[a]
>3[a]
>6[a]
>6[a]
>6[a]
>6[a]
>6[a]
>6[a]
9
10
11
>3[a]
>3[a]
–
>9
–
>50[a]
NS[b]
2.8ꢂ0.8
–
>25[a]
NS[b]
12
13
14
15
16
17
18
19
20
21
22
23
4-CH2OH-phenyl (77)
3-CH2OH-phenyl (87)
2-CH2OH-phenyl (72)
4-CHO-phenyl (51)
3-CHO-phenyl (55)
2-CHO-phenyl (65)
4-COOCH3-phenyl (72)
3-COOCH3-phenyl (71)
2-COOCH3-phenyl (71)
4-COOH-phenyl (65)
3-COOH-phenyl (60)
2- COOH-phenyl (66)
Br
>25[a]
29.3ꢂ4.2
22ꢂ2.0
35ꢂ1.7
10.2ꢂ0.6
9.8ꢂ1.2
>12.5[a]
NS[b]
1.9ꢂ0.3
1.5ꢂ0.3
7.2ꢂ0.6
0.5ꢂ0.1
5.6ꢂ0.4
7.5ꢂ0.4
–
>25[a]
>25[a]
>100[c]
30ꢂ3.7
>100[c]
>50[a]
>12.5[a]
NS[b]
>13
>17
>14
60
>18
>7
–
–
–
–
–
NS[b]
NS[b]
>100[c]
>100[c]
>100[c]
7.1ꢂ1.5
15.5ꢂ0.5
–
0.06ꢂ0.001
0.8ꢂ0.2
3.3ꢂ0.7
–
1.5ꢂ0.2
7.5ꢂ1.0
>50[a]
1.9ꢂ0.44
23.4ꢂ5.7
–
120ꢂ7
>100[c]
>100[c]
92ꢂ13.0
164ꢂ28
0.2ꢂ0.02
7.0ꢂ0.25
84.5ꢂ8.8
>100[c]
>100[c]
>100[c]
80
>13
-
48
7
–
–
–
Initial hit[22]
8-Nitroquinolinone[22]
Doxorubicin[d]
Amphotericin B[e]
Miltefosine[e]
Fexinidazole[e,f]
Suramin[f]
Eflornithine[f]
H
–
–
0.4ꢂ0.18
0.03ꢂ0.009
15.8ꢂ2.1
>250
>3333
>6
–
[a] The product could not be tested at higher concentrations due to low solubility in aqueous media. [b] The product was not soluble at any test concen-
tration. [c] The IC50 or CC50 value was not reached at the highest tested concentration. [d] Doxorubicin was used as a cytotoxic reference drug. [e] Ampho-
tericin B, miltefosine and fexinidazole were used as antileishmanial reference drugs or drug candidates. [f] Fexinidazole, suramin and eflornithine were
used as antitrypanosomal reference drugs or drug candidates. [g] The antitrypanosomal selectivity index was calculated according to the following formu-
la: SI=CC50 HepG2/IC50 T. brucei brucei.
played low cytotoxicity on the HepG2 human cell line with
CC50 values >25 mm. The compound bearing an aldehyde
group in the para position of the phenyl ring (15) was the
most cytotoxic of this series with a CC50 value of 30 mm.
Then, all synthesised compounds were tested in vitro
against L. infantum axenic amastigotes. Their IC50 values were
determined and compared with those of two antileishmanial
reference drugs (amphotericin B and miltefosine) and to the
drug candidate fexinidazole.
In a second assay, only the compounds with appropriate
aqueous solubility were tested in vitro against T. brucei brucei
trypomastigotes and compared with reference antitrypanoso-
mal drugs (suramin and eflornithine) and the drug candidate
fexinidazole. All tested molecules displayed good antitrypano-
somal activity (0.5 mmꢁIC50 ꢁ7.5 mm), better than that of eflor-
nithine (IC50 =15.8 mm) and similar to that of fexinidazole
(IC50 =0.4 mm). Interestingly, compounds substituted at the
ortho position of the phenyl ring (14, 17, 23) displayed higher
IC50 values than their meta- and para-substituted analogues.
Compounds with a para-substituted phenyl ring generally
showed lower IC50 values than their meta-substituted ana-
logues, for example, 15 being 11 times more potent than 16,
and 21 five times more potent than 22. Compounds 15 and
21 appeared to be the most promising antitrypanosomal mol-
ecules in this series with respective IC50 values of 0.5 and
With respect to antileishmanial activity, apart for aldehyde-
containing compounds 16 and 17, the tested series appeared
either weakly active (IC50 =22–35 mm) or even inactive (IC50 >
100 mm for carboxylic acid derivatives) toward L. infantum in
comparison with the reference drugs. Thus, introducing an aryl
moiety at position 3 of the pharmacophore did not seem to
favour antileishmanial activity.
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1.5 mm. Compound 21 emerged as a new antitrypanosomal hit,
with an activity against T. brucei brucei close to that of the
parent compound (IC50 =1.9 mm) but with a better cytotoxicity
profile, leading to a better selectivity index (SI=80 versus 48
for the initial hit). By comparison with reference drugs, 21 ap-
pears less active than suramin but more active than eflorni-
thine. With respect to fexinidazole, another nitroheterocycle,
compound 21 presents the same cytotoxic profile with CC50 =
120 mm, and is three times less active than the drug candidate.
Indeed, unlike fexinidazole and 3-bromo-8-nitroquinolin-2(1H)-
one, compound 21 displayed a selective antitrypanosomal ac-
tivity, being inactive (IC50 >100 mm) against L. infantum. This is
a first indicator that molecule 21 shows a specific antiparasitic
profile in the studied series.
Table 4. Study of the bioactivation of 21 by trypanosomal nitroreduc-
tase.
Compound
T. brucei brucei trypomastigotes IC50 [mm]
Fold change
Wild-type strain NTR-overexpressing strain
21
5.4ꢂ0.12
17.7ꢂ1.0
1.9ꢂ0.05
4.2ꢂ0.2
3.9ꢂ0.1
0.6ꢂ0.05
1.3
4.5
3.1
Initial hit
Nifurtimox
than the wild type, whereas 21 is only 1.3 times more active
on the strain overexpressing the NTR. This result is a third
piece of evidence that suggests that 21, which is less inten-
sively bioactivated by the trypanosomal NTR than the initial
hit, could act on another parasite target. This assay also
showed that compound 21 presents the same level of activity
toward T. brucei brucei than the drug nifurtimox, which was
used as a bioactivation control.
Finally, the O-methylated analogue of hit compound 21 (26)
was synthesised in two steps: O-methylation of 18, using
methyl iodide in DMF under an inert atmosphere, followed by
saponification by the same procedure as described for com-
pounds 21–23 (Scheme 4). Compound 26 was then tested
in vitro against both L. infantum and T. brucei brucei (Figure 4).
In parallel, an electrochemistry study was carried out by
measuring in DMSO the reduction potentials of five 8-nitroqui-
nolin-2(1H)-one derivatives bearing an aryl group at
position 3, using cyclic voltammetry (Table 5). For all
compounds, a reversible single-electron reduction
was observed (formation of an anion radical). The
redox potentials of the new compounds bearing an
aryl group at position 3 ranged between ꢀ0.53 and
ꢀ0.59 V, higher than for the initial hit (ꢀ0.45 V) but
similar to that of 8-nitroquinolin-2(1H)-one (ꢀ0.54 V).
As previously noted in the studied series,[22] the O-
Scheme 4. Preparation of compound 26.
methylation of compound 21 to give 24, is responsi-
ble for an important decrease in the redox potential
value from ꢀ0.56 to ꢀ0.93 V. This shift is mainly due to the re-
moval of the intramolecular hydrogen bond between the
lactam function and the nitro group. It can be concluded that
the introduction of a phenyl ring at the position 3 of the scaf-
fold has no significant impact on the redox potential of the
studied series but that it allows access to novel antitrypanoso-
mal molecules that display lower reduction potential than the
initial hit with the same level of efficacy.
Figure 4. In vitro antiparasitic activities and cytotoxicity of compound 26.
It was as active against T. brucei brucei as 21 (IC50 =2.2 mm).
Compound 26 was also active against L. infantum (IC50 =
12.8 mm), whereas 21 was not (IC50 >100 mm). These results are
surprising, considering that the hydrogen bond between the
lactam function and the nitro group appeared mandatory for
conferring antileishmanial activity on the 8-nitroquinolin-2(1H)-
one series by increasing the reduction potential.[22] This is a
second indicator that the introduction of an aryl group at posi-
tion 3 of the 8-nitroquinolin-2(1H)-one scaffold might lead to a
new antikinetoplasid mechanism of action, different from that
of 3-bromo-8-nitroquinolin-2(1H)-one.
Thus, to understand the selective antitrypanosomal activity
of 21, other parasitic targets should be investigated. Among
the parasitic targets that were recently reported in literature
concerning the antitrypanosomal activity of new diverse nitro-
hetrocycles, S-adenosylmethionine decarboxylase was identi-
fied by a high-throughput mass spectrometry-based assay con-
ducted on 400000 molecules,[30] and should be considered.
Conclusion
An optimised Suzuki–Miyaura reaction at position 3 of 3-
bromo-8-nitroquinolin-2(1H)-one led to the synthesis of 24
new derivatives. These molecules were screened in vitro
against L. infantum and T. brucei brucei to evaluate their antiki-
netoplastid potential. Among these molecules, a new selective
antitrypanosomal hit 21, bearing a para-carboxyphenyl group,
was identified. Compound 21 was not cytotoxic on the HepG2
human cell line (CC50 =120 mm), displayed good antitrypanoso-
To assess if the antitrypanosomal nitroheterocycle 21 was bi-
oactivated by the NTR of T. brucei brucei, its IC50 value was
measured on both a wild-type T. brucei brucei trypomastigote
strain and a NTR-overexpressing strain (Table 4). The results
were compared with those obtained for the initial hit.[22] The
latter is clearly bioactivated by the trypanosomal NTR, being
4.5-fold more active against the strain overexpressing the NTR
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The H and 13C NMR spectra were recorded on Bruker UltraShield
300 MHz, Bruker IconNMR 400 MHz or Bruker Avance NEO 600 MHz
instruments at the Laboratoire de Chimie de Coordination, and
data are presented as follows: chemical shift d in parts per million
(ppm) using tetramethylsilane (TMS) as reference, coupling con-
stant J in Hertz (Hz), and multiplicity: s, singlet; d, doublet; t, trip-
let; q, quartet; dd, doublets of doublets; m, multiplet; brs, broad
singlet. Melting points were measured on a Stuart Melting Point
SMP3 instrument. High-resolution mass measurements were re-
corded on a GCT Premier Spectrometer (DCI, CH4) or Xevo G2
QTOF (Waters, ESI+) instrument at the Universitꢂ Paul Sabatier,
Toulouse (France). Microwave reactions were performed in a CEM
Discover microwave reactor.
1
Table 5. Effects of substitution of the phenyl ring on reduction potentials
E8 in the Suzuki–Miyaura series.
Compound
Structure
E8 [V][a]
1
ꢀ0.59
7
ꢀ0.53
ꢀ0.53
ꢀ0.56
ꢀ0.93
3-Bromo-8-nitroquinolin-2(1H)-one was prepared according to a
previously reported procedure.[22]
13
21
26
General procedure for the preparation of compounds 1–20
and 25
3-Bromo-8-nitroquinolin-2(1H)-one (300 mg, 1.1 mmol, 1 equiv),
cesium carbonate (1.1 g, 3.3 mmol, 3 equiv), Pd(PPh3)4 (127 mg,
0.12 mmol, 0.1 equiv) and the appropriate phenylboronic acid
(1.2 equiv) were added in a sealed 25 mL flask. Under an argon at-
mosphere, anhydrous DME (10 mL) was then added. The reaction
mixture was heated at 858C in a microwave reactor for 2 h. The re-
action mixture was poured into water and extracted with dichloro-
methane (3ꢃ100 mL). The organic layers were combined, washed
with water, dried over anhydrous MgSO4 and evaporated in vacuo.
The crude residue was purified by chromatography on silica gel
and recrystallised if necessary to give compounds 1–20 or 25.
8-Nitro-3-phenylquinolin-2(1H)-one (1) was purified by chroma-
tography on silica gel using dichloromethane/ethyl acetate (97:3)
as eluent, and recrystallised from acetonitrile to yield a yellow solid
Initial hit
ꢀ0.45
ꢀ0.54
1
(264 mg, 0.99 mmol, 90%). Tdec =1778C; H NMR (CDCl3, 400 MHz):
d=7.31–7.35 (m, 1H; H6), 7.41–7.50 (m, 3H; H3’, H4’ and H5’),
7.75–7.77 (m, 2H; H2’ and H6’), 7.91 (s, 1H; H4), 7.94 (dd, J=7.6,
1.4 Hz, 1H; H5), 8.49 (dd, J=8.4, 1.4 Hz, 1H; H7), 11.40 ppm (brs,
1H; NH); 13C NMR (100 MHz, CDCl3): d=121.4 (CH), 122.7 (C), 127.3
(CH), 128.5 (2ꢃCH), 128.7 (2ꢃCH), 129.1 (CH), 132.7 (C), 133.1 (C),
134.5 (C), 134.6 (C), 135.6 (CH), 136.7 (CH), 161.0 ppm (C); HRMS
(DCI, CH4): m/z calcd for C15H11N2O3: 267.0770 [M+H]+; found:
267.0762.
8-Nitroquinolin-2(1H)-one
[a] Cyclic voltammetry conditions: DMSO/TBAPF6, SCE/GC; one-electron
reversible reduction; values are expressed in V versus NHE.
mal activity (IC50 =1.5 mm, SI=80), which is better than that of
the drug eflornithine and similar to that of fexinidazole, a 5-ni-
troimidazole in phase III of clinical trials against HAT. Interest-
ingly, 21 was inactive against L. infantum, showing a parasitic
selectivity among kinetoplastids. Unlike fexinidazole and the
previously identified hit molecule in the series, this molecule
was not efficiently metabolised by the type I trypanosomal
NTR, suggesting a probable alternative mechanism of action in
this series.
3-(4-Methoxyphenyl)-8-nitroquinolin-2(1H)-one (2) was purified
by chromatography on silica gel using dichloromethane/ethyl ace-
tate (95:5) as eluent, and recrystallised from acetonitrile to yield an
orange solid (287 mg, 0.97 mmol, 88%); mp: 2298C; 1H NMR
(CDCl3, 400 MHz): d=3.87 (s, 3H; CH3), 6.99–7.02 (m, 2H; H3’ and
H5’), 7.29–7.33 (m, 1H; H6), 7.73–7.77 (m, 2H; H2’ and H6’), 7.87 (s,
1H; H4), 7.91 (dd, J=7.6, 1.4 Hz, 1H; H5), 8.47 (dd, J=8.3, 1.4 Hz,
1H; H7), 11.38 ppm (brs, 1H; NH); 13C NMR (CDCl3, 75 MHz): d=
55.4 (CH3), 114.0 (2ꢃCH), 121.3 (CH), 122.9 (C), 125.6 (C), 126.9
(CH), 130.0 (2ꢃCH), 132.7 (C), 132.9 (C), 134.0 (C), 135.3 (CH), 135.4
(CH), 160.3 (C), 161.2 ppm (C); HRMS (DCI, CH4): m/z calcd for
C16H13N2O4: 297.0875 [M+H]+; found: 297.0864.
Experimental Section
3-(4-Hydroxyphenyl)-8-nitroquinolin-2(1H)-one (3) was purified
by chromatography on silica gel using dichloromethane/ethyl ace-
tate (75:25) as eluent to yield an orange solid (115 mg, 0.41 mmol,
Chemistry
All reagents and solvents were obtained from commercial sources
(Fluorochem, Sigma–Aldrich or Alfa Aesar) and used as received.
The progress of the reactions was monitored on ALUGRAM SIL G/
UV254 (Macherey–Nagel) pre-coated thin layer chromatography
(TLC) sheets and were visualised under ultraviolet light at 254 nm.
1
37%); mp: 2668C; H NMR ([D6]DMSO, 400 MHz): d=6.84–6.88 (m,
2H; H3’ and H5’), 7.40–7.44 (m, 1H; H6), 7.67–7.71 (m, 2H; H2’ and
H6’), 8.20 (dd, J=7.7, 1.4 Hz, 1H; H5), 8.26 (s, 1H; H4), 8.40 (dd, J=
8.3, 1.4 Hz, 1H; H7), 9.74 (s, 1H; OH), 11.12 ppm (brs, 1H; NH);
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13C NMR ([D6]DMSO, 75 MHz): d=115.4 (2ꢃCH), 122.0 (CH), 122.8
(C), 125.8 (C), 127.1 (CH), 130.4 (2ꢃCH), 132.3 (C), 132.5 (C), 133.5
(C), 136.0 (CH), 136.1 (CH), 158.5 (C), 160.8 ppm (C); HRMS (DCI,
CH4): m/z calcd for C15H11N2O4: 283.0719 [M+H]+; found: 283.0706.
3-(2-Furanyl)-8-nitroquinolin-2(1H)-one (9) was purified by chro-
matography on silica gel using dichloromethane as eluent to yield
a dark red solid (183 mg, 0.71 mmol, 65%); mp: 2578C; 1H NMR
(CDCl3, 300 MHz): d=6.58 (dd, J=3.3 Hz, 1.9 Hz, 1H; H4’), 7.30–
7.35 (m, 1H; H6), 7.55–7.56 (m, 2H; H3’ and H5’), 7.96 (dd, J=7.6,
1.4 Hz, 1H; H5), 8.23 (s, 1H; H4), 8.46 (dd, J=8.3, 1.4 Hz, 1H; H7),
11.42 ppm (brs, 1H; NH); 13C NMR (CDCl3, 100 MHz): d=112.6 (CH),
114.2 (CH), 121.6 (CH), 122.5 (C), 123.6 (C), 126.9 (CH), 130.7 (CH),
132.0 (C), 132.8 (C), 135.5 (CH), 143.3 (CH), 147.7 (C), 158.7 ppm (C);
HRMS (DCI, CH4): m/z calcd for C13H9N2O4: 257.0562 [M+H]+;
found: 257.0558.
3-(4-Aminophenyl)-8-nitroquinolin-2(1H)-one (4) was purified by
chromatography on silica gel using dichloromethane/ethyl acetate
(95:5) as eluent to yield an orange solid (128 mg, 0.45 mmol, 41%);
mp: 2808C; 1H NMR ([D6]DMSO, 400 MHz): d=5.44 (s, 2H; NH2),
6.61–6.65 (m, 2H; H3’ and H5’), 7.37–7.41 (m, 1H; H6), 7.58–7.62
(m, 2H; H2’ and H6’), 8.15–8.18 (m, 2H; H4 and H5), 8.36 (dd, J=
8.3, 1.4 Hz, 1H; H7), 11.07 ppm (brs, 1H; NH); 13C NMR ([D6]DMSO,
100 MHz): d=113.6 (2ꢃCH), 121.9 (CH), 122.1 (C), 123.1 (C), 126.5
(C), 129.9 (2ꢃCH), 131.9 (C), 132.9 (C), 133.4 (C), 134.1 (CH), 135.8
(CH), 150.0 (CH), 161.0 ppm (C); HRMS (DCI, CH4): m/z calcd for
C15H12N3O3:282.0879 [M+H]+; found: 282.0874.
8-Nitro-3-(pyridin-3-yl)quinolin-2(1H)-one (10) was purified by
chromatography on silica gel using dichloromethane/ethyl acetate
as eluent to yield a yellow solid (182 mg, 0.68 mmol, 62%); mp:
1
2378C; H NMR (CDCl3, 400 MHz): d=7.35–7.39 (m, 1H; H6), 7.40–
7.43 (m, 1H; H5’), 7.97–7.99 (m, 2H; H4 and H5), 8.20–8.23 (m, 1H;
H6’), 8.53 (dd, J=8.4, 1.4 Hz, 1H; H7), 8.66 (dd, J=4.9, 1.7 Hz; 1H;
H4’), 8.90 (dd, J=2.3, 0.9 Hz, 1H; H2’), 11.45 ppm (brs, 1H; NH);
13C NMR (CDCl3, 100 MHz): d=121.7 (CH), 122.3 (C), 123.1 (CH),
127.9 (CH), 130.5 (C), 131.4 (C), 132.8 (C), 133.3 (C), 135.8 (CH),
136.4 (CH), 137.2 (CH), 149.0 (CH), 150.0 (CH), 160.6 ppm (C); HRMS
(DCI, CH4): m/z calcd for C14H10N3O3: 268.0722 [M+H]+; found:
268.0712.
3-(4-Chlorophenyl)-8-nitroquinolin-2(1H)-one (5) was purified by
chromatography on silica gel using dichloromethane/ethyl acetate
(97:3) as eluent to yield a yellow solid (241 mg, 0.80 mmol, 73%);
mp: 2228C; 1H NMR (CDCl3, 400 MHz): d=7.33–7.37 (m, 1H; H6),
7.40–7.42 (m, 2H; H3’ and H5’), 7.66–7.75 (m, 2H; H2’ and H6’),
7.92 (s, 1H; H4), 7.95 (dd, J=7.6, 1.5 Hz, 1H; H5), 8.51 (dd, J=8.4,
1.5 Hz, 1H; H7), 11.42 ppm (brs, 1H; NH); 13C NMR (CDCl3,
100 MHz): d=121.6 (CH), 122.4 (C), 126.9 (CH), 127.7 (CH), 128.7
(CH), 129.1 (CH), 129.7 (CH), 132.8 (C), 133.1 (C), 133.2 (C), 134.4 (C),
135.7 (CH), 136.2 (C), 137.3 (CH), 160.6 ppm (C); HRMS (DCI, CH4):
m/z calcd for C15H10ClN2O3: 301.0380 [M+H]+; found: 301.0373.
8-Nitro-3-(pyridin-4-yl)quinolin-2(1H)-one (11) was purified by
chromatography on silica gel using dichloromethane/acetone
(80:20) as eluent to yield a yellow solid (209 mg, 0.78 mmol, 71%);
mp: 2978C; 1H NMR (CDCl3, 400 MHz): d=7.36–7.40 (m, 1H; H6),
7.70–7.72 (m, 2H; H2’ and H6’), 7.98 (dd, J=7.9, 1.4 Hz, 1H; H5),
8.03 (s, 1H; H4), 8.55 (dd, J=8.4, 1.4 Hz, 1H; H7), 8.73–8.74 (m, 2H;
H3’ and H5’), 11.46 ppm (brs, 1H; NH); 13C NMR (CF3CO2D,
100 MHz): d=121.6 (C), 123.7 (CH), 126.1 (C), 126.7 (2ꢃCH), 130.7
(CH), 132.4 (C), 133.4 (C), 137.5 (CH), 140.6 (2ꢃCH), 144.1 (CH),
153.5 (C), 161.1 ppm (C); HRMS (DCI, CH4): m/z calcd for
C14H10N3O3: 268.0722 [M+H]+; found: 268.0713.
3-(4-Fluorophenyl)-8-nitroquinolin-2(1H)-one (6) was purified by
chromatography on silica gel using dichloromethane/ethyl acetate
(98:2) as eluent to yield a yellow solid (219 mg, 0.77 mmol, 70%);
mp: 2178C; 1H NMR (CDCl3, 400 MHz): d=7.12–7.20 (m, 2H; H2’
and H6’), 7.31–7.36 (m, 1H; H6), 7.73–7.80 (m, 2H; H3’ and H5’),
7.89 (s, 1H; H4), 7.94 (dd, J=7.6, 1.5 Hz, 1H; H5), 8.50 (dd, J=8.4,
1.5 Hz, 1H; H7), 11.41 ppm (brs, 1H; NH); 13C NMR (CDCl3,
100 MHz): d=115.5 (d, J=21.6 Hz; 2ꢃCH), 121.5 (CH), 122.6 (C),
124,4 (C), 127.4 (CH), 130.6 (d, J=8.3 Hz; 2ꢃCH), 132.7 (C), 133.1
(C), 133.5 (C), 135.5 (CH), 136.5 (CH), 160.9 (C), 163.2 ppm (d, J=
249.2 Hz, C); HRMS (DCI, CH4): m/z calcd for C15H10FN2O3: 285.0675
[M+H]+; found: 285.0676.
3-(4-Hydroxymethylphenyl)-8-nitroquinolin-2(1H)-one (12) was
purified by chromatography on silica gel using dichloromethane/
acetone (75:25) as eluent to yield
a yellow solid (251 mg,
0.85 mmol, 77%); mp: 2198C; 1H NMR ([D6]DMSO, 400 MHz): d=
4.56 (d, J=5.6 Hz, 2H; CH2), 5.26 (t, J=5.6 Hz, 1H; OH), 7.41–7.46
(m, 3H; H6, H3’ and H5’), 7.76–7.79 (m, 2H; H2’ and H6’), 8.23 (dd,
J=7.6, 1.4 Hz, 1H; H5), 8.36 (s, 1H; H4), 8.43 (dd, J=8.3, 1.4 Hz,
1H; H7), 11.17 ppm (brs, 1H; NH); 13C NMR ([D6]DMSO, 100 MHz):
d=63.1 (CH2), 122.1 (CH), 122.6 (C), 126.6 (2ꢃCH), 127.6 (CH),
128.8 (2ꢃCH), 132.6 (C), 132.7 (C), 133.6 (C), 134.0 (C), 136.4 (CH),
137.6 (CH), 143.6 (C), 160.8 ppm (C); HRMS (DCI, CH4): m/z calcd for
C16H13N2O4: 297.0875 [M+H]+; found: 297.0878.
8-Nitro-3-(4-trifluoromethylphenyl)quinolin-2(1H)-one (7) was
purified by chromatography on silica gel using dichloromethane/
ethyl acetate (98:2) as eluent to yield a yellow solid (220 mg,
1
0.66 mmol, 60%); mp: 1688C; H NMR (CDCl3, 400 MHz): d=7.34–
7.38 (m, 1H; H6), 7.72–7.75 (m, 2H; H2’ and H6’), 7.88 (m, 2H; H3’
and H5’), 7.95–7.98 (m, 2H; H4 and H5), 8.53 (dd, J=8.4, 1.4 Hz,
1H; H7), 11.45 ppm (brs, 1H; NH); 13C NMR (CDCl3, 150 MHz): d=
121.7 (CH), 122.4 (C), 124.0 (q, J=272.3 Hz, C), 125.4 (q, J=3.7 Hz,
2ꢃCH), 127.9 (CH), 129.1 (2ꢃCH), 130.9 (q, J=32.5 Hz, C), 132.8
(C), 133.2 (C), 133.4 (C), 135.8 (CH), 137.6 (CH), 138.0 (C), 160.5 ppm
(C); HRMS (DCI, CH4): m/z calcd for C16H10F3N2O3: 335.0644 [M+H]+
; found: 335.0629.
3-(3-Hydroxymethylphenyl)-8-nitroquinolin-2(1H)-one (13) was
purified by chromatography on silica gel using dichloromethane/
acetone (75:25) as eluent to yield
a yellow solid (267 mg,
0.90 mmol, 82%); mp: 1558C; 1H NMR ([D6]DMSO, 300 MHz): d=
4.58 (d, J=5.6 Hz, 2H; CH2), 5.27 (t, J=5.6 Hz, 1H; OH), 7.37–7.47
(m, 3H; H6, H4’ and H5’), 7.66–7.68 (m, 1H; H6’), 7.73–7.75 (m, 1H;
H2’), 8.25 (dd, J=7.8, 1.4 Hz, 1H; H5), 8.35 (s, 1H; H4), 8.44 (dd, J=
8.3 and 1.4 Hz, 1H; H7), 11.17 ppm (brs, 1H; NH); 13C NMR
([D6]DMSO, 75 MHz): d=63.3 (CH2), 122.1 (CH), 122.6 (C), 127.1
(CH), 127.2 (CH), 127.5 (CH), 127.7 (CH), 128.4 (CH), 132.7 (C), 133.0
(C), 133.7 (C), 135.1 (C), 136.5 (CH), 138.0 (CH), 142.9 (C), 160.6 ppm
(C); HRMS (DCI, CH4): m/z calcd for C16H13N2O4: 297.0875 [M+H]+;
found: 297.0861.
8-Nitro-3-(thiophen-3-yl)quinolin-2(1H)-one (8) was purified by
chromatography on silica gel using dichloromethane/ethyl acetate
(98:2) as eluent to yield a yellow solid (275 mg, 1.01 mmol, 92%);
mp: 1998C; 1H NMR (CDCl3, 400 MHz): d=7.31–7.35 (m, 1H; H6),
7.41–7.43 (m, 1H; H4’), 7.59 (dd, J=5.1, 1.3 Hz, 1H; H5’), 7.94 (dd,
J=7.7, 1.4 Hz, 1H; H5), 8.05 (s, 1H; H4), 8.39 (dd, J=3.0, 1.3 Hz,
1H; H2’), 8.48 (dd, J=8.4, 1.4 Hz, 1H; H7), 11.42 ppm (brs, 1H;
NH); 13C NMR (CDCl3, 75 MHz): d=121.4 (CH), 122.6 (C), 123.6 (C),
125.6 (CH), 126.2 (CH), 127.0 (CH), 127.1 (CH), 128.6 (C), 132.4 (C),
134.1 (CH), 134.3 (C), 135.4 (CH), 160.6 ppm (C); HRMS (DCI, CH4):
m/z calcd for C13H9N2O3S: 273.0334 [M+H]+; found: 273.0330.
3-(2-Hydroxymethylphenyl)-8-nitroquinolin-2(1H)-one (14) was
purified by chromatography on silica gel using dichloromethane/
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ethyl acetate (50:50) as eluent to yield a yellow solid (235 mg,
0.79 mmol, 72%); mp: 2088C; 1H NMR ([D6]DMSO, 400 MHz): d=
4.44 (d, J=5.5 Hz, 2H; CH2), 5.09 (s, 1H; OH), 7.28 (dd, J=7.5,
1.4 Hz, 1H; H3’), 7.33–7.37 (m, 1H; H6), 7.42–7.46 (m, 2H; H4’ and
H5’), 7.57 (dd, J=7.5, 1.4 Hz, 1H; H6’), 8.12 (s, 1H; H4), 8.19 (dd,
J=7.6, 1.4 Hz, 1H; H5), 8.45 (dd, J=8.3, 1.4 Hz, 1H; H7), 11.19 ppm
(brs, 1H; NH); 13C NMR (CDCl3, 100 MHz): d=63.7 (CH2), 121.9 (CH),
122.5 (C), 127.8 (CH), 128.4 (CH), 129.8 (CH), 130.1 (CH), 130.6 (CH),
132.7 (C), 133.1 (C), 133.9 (C), 135.7 (CH), 135.8 (C), 139.2 (CH),
139.9 (C), 162.2 ppm (C); HRMS (DCI, CH4): m/z calcd for
C16H13N2O4: 297.0875 [M+H]+; found: 297.0869.
cyclohexane (90:10) as eluent to yield a yellow solid (253 mg,
0.78 mmol, 71%); mp: 2128C; 1H NMR (CDCl3, 400 MHz): d=3.95
(s, 3H; CH3); 7.33–7.37 (m, 1H; H6), 7.53–7.58 (m, 1H; H5’), 7.96
(dd, J=7.7 Hz, 1.4 Hz, 1H; H5), 7.98 (s, 1H; H4), 8.05–8.11 (m, 2H;
H4’ and H6’), 8.36–8.37 (m, 1H; H2’), 8.51 (dd, J=8.4, 1.4 Hz, 1H;
H7), 11.42 ppm (brs, 1H; NH); 13C NMR (CDCl3, 100 MHz): d=52.3
(CH3), 121.6 (CH), 122.5 (C), 127.7 (CH), 128.6 (CH), 129.6 (CH), 130.0
(CH), 130.5 (C), 132.8 (C), 133.2 (C), 133.4 (CH), 133.5 (C), 134.8 (C),
135.7 (CH), 137.3 (CH), 160.7 (C), 166.7 ppm (C); HRMS (DCI, CH4):
m/z calcd for C17H13N2O5: 325.0824 [M+H]+; found: 325.0809.
3-(2-Methoxycarbonylphenyl)-8-nitroquinolin-2(1H)-one (20) was
purified by chromatography on silica gel using cyclohexane/ace-
tone (70:30) as eluent to yield a yellow solid (257 mg, 0.79 mmol,
3-(4-Formylphenyl)-8-nitroquinolin-2(1H)-one (15) was purified
by chromatography on silica gel using dichloromethane/cyclohex-
ane (80:20) as eluent, and recrystallised from acetonitrile to yield a
yellow solid (165 mg, 0.56 mmol, 51%); mp: 2438C; 1H NMR
(CDCl3, 300 MHz): d=7.34–7.39 (m, 1H; H6), 7.93–8.00 (m, 6H; H4,
H5, H2’, H3’, H5’ and H6’), 8.53 (dd, J=8.5, 1.4 Hz, 1H; H7), 10.08
(s, 1H; CHO), 11.45 ppm (brs, 1H; NH); 13C NMR (CDCl3, 75 MHz):
d=121.7 (CH), 122.3 (C), 128.0 (CH), 129.4 (2ꢃCH), 129.8 (2ꢃCH),
132.8 (C), 133.2 (C), 133.4 (C), 135.9 (CH), 136.4 (C), 137.9 (CH),
140.5 (C), 160.5 (C), 191.7 ppm (CH); HRMS (DCI, CH4): m/z calcd for
C16H11N2O4: 295.0719 [M+H]+; found: 295.0710.
1
72%); mp: 2128C; H NMR (CDCl3, 400 MHz): d=3.82 (s, 3H; CH3),
7.29–7.33 (m, 1H; H6), 7.37–7.39 (m, 1H; H6’), 7.49–7.53 (m, 1H;
H4’), 7.60–7.64 (m, 1H; H5’), 7.77 (s, 1H; H4), 7.91 (d, J=7.65 Hz,
1H; H3’), 8.02 (d, J=7.8 Hz, 1H; H5), 8.48 (d, J=8.4 Hz, 1H; H7),
11.34 ppm (brs, 1H; NH); 13C NMR (CDCl3, 100 MHz): d=52.3 (CH3),
121.4 (CH), 122.7 (C), 127.2 (CH), 129.0 (CH), 130.1 (CH), 130.6 (CH),
131.0 (C), 132.4 (CH), 132.9 (C), 133.4 (C), 135.5 (CH), 135.6 (CH),
135.8 (C), 137.0 (C), 160.9 (C), 167.5 ppm (C); HRMS (DCI, CH4): m/z
calcd for C17H13N2O5: 325.0824 [M+H]+; found: 325.0819.
3-(3-Formylphenyl)-8-nitroquinolin-2(1H)-one (16) was purified
by chromatography on silica gel using dichloromethane/acetone
(75:25) as eluent to yield a yellow solid (104 mg, 0.35 mmol, 32%);
mp: 2098C; 1H NMR (CDCl3, 300 MHz): d=7.34–7.39 (m, 1H; H6),
7.63–7.69 (m, 1H; H5’), 7.94–7.99 (m, 2H; H5 and H6’), 8.01 (s, 1H;
H4), 8.10–8.13 (m, 1H; H4’), 8.25–8.26 (m, 1H; H2’), 8.53 (dd, J=
8.4, 1.4 Hz, 1H; H7), 10.10 (s, 1H; CHO), 11.5 ppm (brs, 1H; NH);
13C NMR (CDCl3, 100 MHz): d=121.7 (CH), 122.4 (C), 127.8 (CH),
129.2 (CH), 129.7 (CH), 130.2 (CH), 132.8 (C), 133.1 (C), 133.3 (C),
134.7 (CH), 135.6 (C), 135.8 (CH), 136.6 (C), 137.5 (CH), 160.7 (C),
192.0 ppm (CH); HRMS (DCI, CH4): m/z calcd for C16H11N2O4:
295.0719 [M+H]+; found: 295.0713.
General procedure for the preparation of compounds 21–23
A mixture of H2O/ethanol (2:8, 40 mL) was added to 1 equiv
(200 mg) of the 3-(methoxycarbonylphenyl)-8-nitroquinolin-2(1H)-
one derivative (18–20). Then, NaOH (5 equiv) was added and the
reaction mixture was stirred at 808C for 3 h. The reaction mixture
was then poured into water, acidified to pH 1 with 37% HCl, and
extracted with dichloromethane (2ꢃ50 mL) and ethyl acetate (2ꢃ
50 mL). The combined organic layers were washed with water,
dried over anhydrous MgSO4 and evaporated in vacuo. The crude
residue was purified by chromatography on silica gel and recrystal-
lised if necessary to give compound 21, 22 or 23.
3-(2-Formylphenyl)-8-nitroquinolin-2(1H)-one (17) was purified
by chromatography on silica gel using cyclohexane/acetone
(60:40) as eluent to yield a yellow solid (210 mg, 0.71 mmol, 65%);
mp: 230–2318C; 1H NMR (CDCl3, 400 MHz): d=7.33–7.37 (m, 1H;
H6), 7.44 (dd, J=7.5, 1.3 Hz, 1H; H6’), 7.61–7.65 (m, 1H; H4’), 7.68–
7.72 (m, 1H; H5’), 7.82 (s, 1H; H4), 7.93 (dd, J=7.6, 1.4 Hz, 1H;
H3’), 7.99 (dd, J=7.8, 1.4 Hz, 1H; H5), 8.53 (dd, J=8.4, 1.4 Hz, 1H;
H7), 10.03 (s, 1H; CHO), 11.42 ppm (brs, 1H; NH); 13C NMR (CDCl3,
100 MHz): d=121.6 (CH), 122.3 (C), 127.8 (CH), 129.5 (CH), 130.8
(CH), 131.0 (CH), 133.0 (C), 133.7 (C), 133.9 (CH), 134.5 (C), 135.0 (C),
135.7 (CH), 136.1 (C), 138.0 (CH), 160.8 (C), 191.2 ppm (CH); HRMS
(DCI, CH4): m/z calcd for C16H11N2O4: 295.0719 [M+H]+; found:
295.0718.
3-(4-Carboxyphenyl)-8-nitroquinolin-2(1H)-one (21) was washed
with dilute aqueous hydrochloric acid and recrystallised from ace-
tonitrile to yield a yellow solid (124 mg, 0.40 mmol, 65%); mp:
1
>3108C; H NMR ([D6]DMSO, 400 MHz): d=7.45–7.49 (m, 1H; H6),
7.93–7.96 (m, 2H; H2’ and H6’), 8.03–8.06 (m, 2H; H3’ and H5’),
8.26 (dd, J=7.9, 1.4 Hz, 1H; H5), 8.46 (dd, J=8.3, 1.4 Hz, 1H; H7),
8.49 (s, 1H; H4), 11.23 (brs, 1H; NH), 13.07 ppm (brs, 1H; COOH);
13C NMR ([D6]DMSO, 100 MHz): d=122.3 (CH), 122.35 (C), 128.1
(CH), 129.2 (2ꢃCH), 129.6 (2ꢃCH), 131.0 (C), 131.7 (C), 132.8 (C),
133.8 (C), 136.7 (CH), 139.1 (CH), 139.5 (C), 160.4 (C), 167.5 ppm (C);
HRMS (ESI+): m/z calcd for C16H11N2O5: 311.0668 [M+H]+; found:
311.0669.
3-(4-Methoxycarbonylphenyl)-8-nitroquinolin-2(1H)-one (18) was
purified by chromatography on silica gel using dichloromethane as
eluent, and recrystallised from acetonitrile to yield a yellow solid
3-(3-Carboxyphenyl)-8-nitroquinolin-2(1H)-one (22) was washed
with dilute aqueous hydrochloric acid and recrystallised from ace-
tonitrile to yield a brown solid (88 mg, 0.28 mmol, 42%). Tdec
=
1
305–3068C; 1H NMR (CDCl3, 400 MHz): d=7.44–7.48 (m, 1H; H6),
7.60–7.64 (m, 1H; H5’), 8.00–8.02 (m, 1H; H6’), 8.03–8.06 (m, 1H;
H4’), 8.27 (dd, J=7.8, 1.4 Hz, 1H; H5), 8.39–8.40 (m, 1H; H2’), 8.46
(dd, J=8.3, 1.4 Hz, 1H; H7), 8.47 (s, 1H; H4), 11.22 (brs, 1H; NH),
13.05 ppm (brs, 1H; COOH); 13C NMR ([D6]DMSO, 100 MHz): d=
122.2 (CH), 122.5 (C), 127.9 (CH), 129.0 (CH), 129.8 (CH), 129.9 (CH),
131.3 (C), 131.8 (C), 132.8 (C), 133.4 (CH), 133.7 (C), 135.6 (C), 136.7
(CH), 138.6 (CH), 160.5 (C), 167.6 ppm (C); HRMS (DCI, CH4): m/z
calcd for C16H11N2O4: 311.0668 [M+H]+; found: 311.0672.
(257 mg, 0.79 mmol, 72%); mp: 2458C; H NMR (CDCl3, 300 MHz):
d=3.95 (s, 3H; CH3), 7.33–7.38 (m, 1H; H6), 7.84–7.87 (m, 2H; H2’
and H6’), 7.95–7.97 (m, 2H; H4 and H5), 8.12–8.15 (m, 2H; H3’ and
H5’), 8.52 (dd, J=8.3, 1.4 Hz, 1H; H7), 11.43 ppm (brs, 1H; NH);
13C NMR (CDCl3, 100 MHz): d=52.3 (CH3), 121.6 (CH), 122.4 (C),
127.8 (CH), 128.7 (2ꢃCH), 129.7 (2ꢃCH), 130.4 (C), 132.8 (C), 133.3
(C), 133.5 (C), 135.8 (CH), 137.6 (CH), 139.0 (C), 160.6 (C), 166.7 ppm
(C); HRMS (DCI, CH4): m/z calcd for C17H13N2O5: 325.0824 [M+H]+;
found: 325.0818.
3-(3-Methoxycarbonylphenyl)-8-nitroquinolin-2(1H)-one (19) was
3-(2-Carboxyphenyl)-8-nitroquinolin-2(1H)-one (23) was purified
purified by chromatography on silica gel using dichloromethane/
by chromatography on silica gel using dichloromethane/ethyl ace-
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tate (50:50) as eluent to yield a yellow solid (109 mg, 0.35 mmol,
57%). Tdec =282–2858C; 1H NMR ([D6]DMSO, 400 MHz): d=7.43–
7.48 (m, 2H; H6 and H6’), 7.54–7.58 (m, 1H; H4’), 7.66–7.70 (m, 1H;
H5’), 7.91 (dd, J=7.7, 1.4 Hz, 1H; H5), 8.13 (s, 1H; H4), 8.20 (dd, J=
7.8, 1.4 Hz, 1H; H3’), 8.42 (dd, J=8.3, 1.4 Hz, 1H; H7); 11.08 (brs,
1H; NH), 12.72 ppm (brs, 1H; COOH); 13C NMR ([D6]DMSO,
100 MHz): d=122.1 (CH), 122.6 (C), 127.4 (CH), 129.1 (CH), 129.8
(CH), 131.3 (CH), 132.4 (CH), 132.5 (C), 132.7 (C), 133.9 (C), 136.1
(CH), 136.2 (C), 136.3 (CH), 136.4 (C), 160.7 (C), 168.4 ppm (C);
HRMS (DCI, CH4): m/z calcd for C16H11N2O5: 311.0668 [M+H]+;
found: 311.0673.
Preparation of 3-(4-carboxyphenyl)-2-methoxy-8-nitroquino-
line (26)
Under an argon atmosphere, 3-(4-methoxycarbonylphenyl)-8-nitro-
quinolin-2(1H)-one (260 mg, 0.80 mmol, 1 equiv) was dissolved in
anhydrous DMF (5 mL). This solution was then added to 60%
sodium hydride (64 mg, 1.6 mmol, 2 equiv) in DMF (5 mL). After
10 min of stirring at RT, methyl iodide (100 mL, 1.6 mmol, 2 equiv)
was added dropwise. The reaction mixture was stirred at RT over-
night, before being poured over ice. A precipitate formed and was
filtered off and washed with water. The precipitate was dissolved
in dichloromethane and this solution was dried over anhydrous
MgSO4 and evaporated in vacuo. The crude residue was purified
by chromatography on silica gel using dichloromethane as eluent.
The resulting white solid intermediate was dissolved in a mixture
of H2O/ethanol (2:8, 40 mL). NaOH (excess, solid) was then added,
and the reaction mixture was stirred at 808C for 2 h. The reaction
mixture was then poured into water, acidified to pH 1 with 37%
HCl, and extracted with dichloromethane (2ꢃ50 mL) and ethyl ace-
tate (2ꢃ50 mL). The combined organic layers were washed with
water, dried over anhydrous MgSO4 and evaporated in vacuo. The
residue was washed with alkaline water (pH 8) to yield 26 as a
white solid (87 mg, 0.27 mmol, 61%). Tdec =270–2748C; 1H NMR
([D6]DMSO, 400 MHz): d=4.00 (s, 3H; CH3), 7.61–7.65 (m, 1H; H6),
7.80–7.82 (m, 2H; H2’ and H6’), 8.05–8.07 (m, 2H; H3’ and H5’),
8.23 (dd, J=7.8, 1.4 Hz, 1H; H5), 8.27 (dd, J=8.1, 1.4 Hz, 1H; H7),
8.56 (s, 1H; H4), 13.07 ppm (brs, 1H; NH); 13C NMR ([D6]DMSO,
100 MHz): d=54.5 (CH3), 124.1 (CH), 124.7 (C), 126.6 (CH), 127.0 (C),
129.7 (2ꢃCH), 130.0 (2ꢃCH), 130.9 (C), 132.7 (CH), 136.7 (C), 139.3
(CH), 140.0 (C), 146.2 (C), 160.8 (C), 167.5 ppm (C); HRMS (DCI, CH4):
m/z calcd for C17H13N2O5: 325.0824 [M+H]+; found: 325.0822.
Preparation of 8-nitro-3-phenylethynylquinolin-2(1H)-one (24)
3-Bromo-8-nitroquinolin-2(1H)-one (100 mg, 0.37 mmol, 1 equiv)
previously synthesised in our team, CuI (7 mg, 0.037 mmol,
0.1 equiv) and Pd(PPh3)4, (43 mg, 0.037 mmol, 0.1 equiv) were
added to a sealed 10 mL flask. Under an argon atmosphere, anhy-
drous dimethoxyethane (5 mL), Et3N (155 mL, 1.11 mmol, 3 equiv)
and phenylacetylene (61 mL, 0.56 mmol, 1.5 equiv) were successive-
ly added. The reaction mixture was cooled at 158C for 1.5 h. The
reaction mixture was poured into water and extracted three times
with dichloromethane. The organic layers were combined, washed
with water, dried over anhydrous MgSO4 and evaporated in vacuo.
The crude residue was purified by chromatography on silica gel
using dichloromethane as an eluent to yield 24 as a yellow solid
(78 mg, 0.27 mmol, 72%); mp: 186–1878C; 1H NMR (CDCl3,
400 MHz): d=7.31–7.35 (m, 1H; H6), 7.37–7.38 (m, 2H; H3’, H4’
and H5’), 7.59–7.63 (m, 2H; H2’ and H6’), 7.87 (dd, J=7.6, 1.5 Hz,
1H; H5), 8.06 (s, 1H; H4), 8.49 (dd, J=8.4, 1.4 Hz, 1H; H7),
11.40 ppm (brs, 1H; NH); 13C NMR (CDCl3, 100 MHz): d=83.4 (C),
97.3 (C), 119.6 (C), 121.8 (CH), 122.0 (C), 122.1 (C), 128.0 (CH), 128.4
(2ꢃCH), 129.2 (CH), 132.1 (2ꢃCH), 133.0 (C), 133.0 (C), 135.3 (CH),
141.6 (CH), 159.9 ppm (C); HRMS (DCI, CH4): m/z calcd for
C17H13N2O5: 291.0770 [M+H]+; found: 291.0776.
Electrochemistry
Voltammetric measurements were carried out with a potentiostat
Autolab PGSTAT100 (ECO Chemie, The Netherlands) controlled by
GPES 4.09 software. Experiments were performed at room temper-
ature in a homemade airtight three-electrode cell connected to a
vacuum/argon line. The reference electrode consisted of a saturat-
ed calomel electrode (SCE) separated from the solution by a
bridge compartment. The counter electrode was a platinum wire
with an apparent surface area of approximately 1 cm2. The working
electrode was a GC microdisk (1.0 mm diameter; Bio-logic SAS).
The supporting electrolyte [nBu4N][PF6] (99% puriss electrochemi-
cal grade, Fluka) and the solvent DMSO (Sigma–Aldrich puriss p.a.,
dried, <0.02% water) were used as received and simply degassed
under argon. The solutions used during the electrochemical studies
were typically 10ꢀ3 and 0.1m with respect to test compound and
supporting electrolyte, respectively. Before each measurement, the
solutions were degassed by bubbling argon, and the working elec-
trode was polished with a polishing machine (Presi P230). Under
these experimental conditions, the half-wave potential (E1/2) of the
ferrocene Fc+/Fc couple in DMSO was E1/2 =0.45 V versus SCE. Ex-
perimental peak potentials were measured versus SCE and convert-
ed to NHE by adding 0.241 V.
Preparation of 8-nitro-2-phenyl-furo[2,3-b]quinoline (25)
3-Bromo-8-nitroquinolin-2(1H)-one (100 mg 0.37 mmol, 1 equiv)
previously synthesised in our team, CuI (7 mg, 0.037 mmol,
0.1 equiv) and Pd(PPh3)4 (43 mg, 0.037 mmol, 0.1 equiv) were
added to a sealed 10 mL flask. Under an argon atmosphere, anhy-
drous dimethoxyethane (5 mL), Et3N (155 mL, 1.11 mmol, 3 equiv)
and phenylacetylene (61 mL, 0.56 mmol, 1.5 equiv) were successive-
ly added. The reaction mixture was heated at 408C for 30 min. The
reaction mixture was then poured into water and extracted three
times with dichloromethane. The organic layer was washed with
water, dried over anhydrous MgSO4 and evaporated in vacuo. The
crude residue was purified by chromatography on silica gel using
dichloromethane as an eluent and isolated to yield 25 as a grey
solid (55 mg, 0.19 mmol, 51%); mp: 2268C; 1H NMR (CDCl3,
400 MHz): d=7.16 (s, 1H; H3), 7.45–7.55 (m, 3H; H3’, H4’ and H5’),
7.56–7.59 (m, 1H; H6), 7.99–8.02 (m, 2H; H2’ and H6’), 8.08 (dd, J=
7.5, 1.4 Hz, 1H; H5), 8.14 (dd, J=8.3, 1.4 Hz, 1H; H7), 8.41 ppm (s,
1H; H4); 13C NMR (CDCl3, 100 MHz): d=99.2 (CH), 123.3 (CH), 123.4
(CH), 124.0 (C), 125.9 (2ꢃCH), 127.7 (C), 128.2 (CH), 128.6 (C), 129.1
(2ꢃCH), 130.5 (CH), 132.0 (CH), 136.0 (C), 147.7 (C), 159.6 (C),
162.4 ppm (C); HRMS (DCI, CH4): m/z calcd for C17H11N2O3: 291.0770
[M+H]+; found: 291.0768.
Biology
Antileishmanial activity on L. infantum axenic amastigotes[31]
L. infantum promastigotes (MHOM/MA/67/ITMAP-263, CNR Leish-
mania, Montpellier, France, expressing luciferase activity) in the
logarithmic phase, cultivated in RPMI 1640 medium supplemented
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with 5% foetal calf serum, l-glutamine (2 mm) and antibiotics
(100 UmLꢀ1 penicillin and 100 mgmLꢀ1 streptomycin), were centri-
fuged at 900 g for 10 min. The supernatant was removed carefully
and was replaced by the same volume of RPMI 1640 complete
medium at pH 5.4 and incubated for 24 h at 248C. The acidified
promastigotes were incubated for 24 h at 378C in a ventilated
flask. Promastigotes were then transformed into axenic amasti-
gotes. The effects of the test compounds on the growth of L. infan-
tum axenic amastigotes were assessed as follows. L. infantum
amastigotes were incubated at a density of 2ꢃ106 parasitesmLꢀ1
in sterile 96-well plates with various concentrations of compounds
dissolved in DMSO (final concentration <0.5% v/v), in duplicate.
The appropriate controls DMSO, amphotericin B, miltefosine and
fexinidazole (reference drugs purchased from Sigma–Aldrich) were
added to each set of experiments. After a 48 h incubation period
at 378C, each well was examined by microscope for precipitate for-
mation. To estimate the luciferase activity of axenic amastigotes,
80 mL of each well were transferred to white 96-well plates. Steady
Glow reagent (Promega) was added according to the manufactur-
er’s instructions, and plates were incubated for 2 min. The lumines-
cence was measured using a Microbeta luminescence counter (Per-
kinElmer). IC50 was defined as the concentration of drug required
to inhibit by 50% the metabolic activity of L. infantum amastigotes
relative to the control. IC50 values were calculated by nonlinear re-
gression analysis of dose–response curves, using TableCurve 2D V5
software. IC50 values represent the mean of three independent ex-
periments.
Melford) to maintain expression of T7 RNA polymerase and the tet-
racycline repressor protein. Bloodstream trypanosomes overex-
pressing the T. brucei nitroreductase (NTR1)[36] were grown in
medium supplemented with phleomycin (2.5 mgmLꢀ1) and expres-
sion of NTR was induced by the addition of tetracycline
(1 mgmLꢀ1). Cultures were initiated with 1ꢃ105 cells per mL and
sub-cultured if the cell density approached 1ꢃ106–2ꢃ106 cells per
mL. To examine the effects of inhibitors on the growth of these
parasites, triplicate cultures containing the inhibitor were seeded
at 1ꢃ105 trypanosomes per mL. Cells overexpressing NTR were in-
duced with tetracycline 48 h prior to EC50 analysis. Cell densities
were determined after culture for 72 h, as previously described.[37]
EC50 values were determined using the following two-parameter
equation by nonlinear regression using GraFit [Eq. (1)]:
100
ꢀ
ꢁ
y ¼
m
ð1Þ
½Iꢃ
1 þ
EC50
The experimental data were corrected for background cell density
and expressed as a percentage of the uninhibited control cell den-
sity. In this equation, [I] represents inhibitor concentration and m is
the slope factor.
Cytotoxicity evaluation on HepG2 cells
The evaluation of the cytotoxicity of the test molecules on HepG2
cells (hepatocarcinoma cell line from ECACC purchased from
Sigma–Aldrich, ref. 85011430-1VL, certificated without mycoplas-
ma) was performed according to the method of Mosmann with
slight modifications.[38] In brief, cells (1ꢃ105 cells per mL) in com-
plete medium [100 mL, Alpha MEM Eagle from PAN BIOTECH sup-
plemented with 10% foetal bovine serum, l-glutamine (2 mm) and
antibiotics (100 UmLꢀ1 penicillin and 100 mgmLꢀ1 streptomycin)]
were seeded into each well of 96-well plates and incubated at
378C and 5% CO2. After a 24 h incubation, medium (100 mL) with
various product concentrations and appropriate controls was
added and the plates were incubated for 72 h at 378C and 5%
CO2. Each well was then examined by microscope for possible pre-
cipitate formation before the medium was aspirated from the
wells. MTT solution (100 mL, 0.5 mgmLꢀ1 in Alpha MEM Eagle) was
then added to each well. Cells were incubated for 2 h at 378C and
5% CO2. After this time, the MTT solution was removed and DMSO
(100 mL) was added to dissolve the resulting formazan crystals.
Plates were shaken vigorously (300 rpm) for 5 min. The absorbance
was measured at 570 nm with a microplate spectrophotometer
(Eon BioTek). DMSO was used as a blank and doxorubicin (Sigma–
Aldrich) as a positive control. CC50 values were calculated by non-
linear regression analysis of dose–response curves using Table-
Curve 2D V5 software. CC50 values represent the mean value calcu-
lated from three independent experiments.
Antitrypanosomal activity on T. brucei brucei trypomastigotes
Assays were performed on T. brucei brucei AnTat 1.9 strain (IMTA,
Antwerp, Belgium). Trypanosomes were cultured in MEM contain-
ing Earle’s salts, supplemented according to the protocol of Baltz
et al.[32] with the following additions: mercaptoethanol (0.5 mm,
Sigma–Aldrich, France), l-cysteine (1.5 mm, Sigma–Aldrich), batho-
cuproine disulfonate (0.05 mm, Sigma–Aldrich) and 20% heat-inac-
tivated horse serum (Gibco, France), at 378C in an atmosphere
containing 5% CO2. The parasites were incubated at an average
density of 2000 parasites per well in sterile 96-wells plates (Mc2,
France) with various concentrations of compounds dissolved in
DMSO (Sigma–Aldrich), in duplicate. Reference drugs suramin,
eflornithine, and fexinidazole (purchased from Sigma–Aldrich,
France and Fluorochem, UK) suspended in 0.9% NaCl or DMSO,
were added to each set of experiments. The effects of the test
compounds were assessed using the viability marker Alamar Blue
assay described by Rꢄz et al.[33] After a 69 h incubation period at
378C, Alamar Blue (10 mL, Fisher, France) was added to each well,
and the plates were incubated for 5 h.[34] The plates were read
using a PerkinElmer (Germany) ENSPIRE microplate reader using an
excitation wavelength of 530 nm and an emission wavelength of
590 nm. IC50 values were calculated by nonlinear regression analy-
sis of dose–response curves using GraphPad Prism software. IC50
was defined as the concentration of drug required to inhibit by
50% the viability of T. brucei brucei relative to the control. IC50
values were calculated from three independent experiments in du-
plicate.
Acknowledgements
The authors thank the Universitꢀ Paul Sabatier de Toulouse and
the Rꢀgion Occitanie/Pyrꢀnꢀes-Mꢀditerranꢀe for funding this
work. A.H.F. and S.W. are supported by funding from the Well-
come Trust (WT105021). Christian Bijani from the NMR facility
Laboratoire de Chimie de Coordination de Toulouse, Catherine
Claparols, Nathalie Martins-Froment, Valꢀrie Bourdon, and Eric
Leroy from the mass spectrometry service of Institut de Chimie de
Antitrypanosomal activity on T. brucei trypomastigotes over-
expressing the nitroreductase NTR1
Trypanosoma brucei bloodstream-form “single marker” S427
(T7RPOL TETR NEO) and drug-resistant cell lines were cultured at
378C in HMI9-T medium[35] supplemented with G418 (2.5 mgmLꢀ1
,
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[17] B. S. Hall, X. Wu, L. Hu, S. R. Wilkinson, Antimicrob. Agents Chemother.
2010, 54, 1193–1199.
[18] P. Verhaeghe, P. Rathelot, S. Rault, P. Vanelle, Lett. Org. Chem. 2006, 3,
891–897.
Toulouse are also acknowledged for their support of NMR and
HRMS experiments, respectively.
[19] L. Paloque, P. Verhaeghe, M. Casanova, C. Castera-Ducros, A. Dumꢀtre,
L. Mbatchi, S. Hutter, M. Kraiem-M’Rabet, M. Laget, V. Remusat, P. Rathe-
lot, N. Azas, P. Vanelle, Eur. J. Med. Chem. 2012, 54, 75–86.
[20] C. Kieffer, A. Cohen, P. Verhaeghe, S. Hutter, C. Castera-Ducros, M. Laget,
V. Remusat, M. Kraiem-M’Rabet, S. Rault, P. Rathelot, N. Azas, P. Vanelle,
Eur. J. Med. Chem. 2015, 92, 282–294.
Conflict of interest
The authors declare no conflict of interest.
Keywords: 8-nitroquinolin-2(1H)-ones
pharmacomodulation · palladium-catalysed cross-coupling ·
parasitic nitroreductases · trypanosomatids
·
antikinetoplastid
[21] C. Kieffer, A. Cohen, P. Verhaeghe, L. Paloque, S. Hutter, C. Castera-
Ducros, M. Laget, S. Rault, A. Valentin, P. Rathelot, N. Azas, P. Vanelle,
Bioorg. Med. Chem. 2015, 23, 2377–2386.
[22] J. Pedron, C. Boudot, S. Hutter, S. Bourgeade-Delmas, J.-L. Stigliani, A.
Sournia-Saquet, A. Moreau, E. Boutet-Robinet, L. Paloque, E. Mothes, M.
Laget, L. Vendier, G. Pratviel, S. Wyllie, A. H. Fairlamb, N. Azas, B. Cour-
tioux, A. Valentin, P. Verhaeghe, Eur. J. Med. Chem. 2018, 155, 135–152.
[23] V. P. Andreev, Y. P. Nizhnnik, Russ. J. Org. Chem. 2002, 38, 137–138.
[24] N. J. O’Brien, M. Brzozowski, D. J. D. Wilson, L. W. Deady, B. M. Abbott,
Bioorg. Med. Chem. 2014, 22, 3781–3790.
[1] P. Capewell, N. J. Veitch, C. M. Turner, C. M. R. Turner, J. Raper, M. Berri-
man, S. L. Hadjuk, A. MacLeod, PLoS Neglected Trop. Dis. 2011, 5, e1287.
[2] P. Bꢅscher, G. Cecchi, V. Jamonneau, G. Priotto, Lancet 2017, 390, 2397–
2405.
[3] D. Pace, J. Infect. 2014, 69, S10–S18.
[4] a) M. M. Ashkan, K. M. Rahim, Trop. Doct. 2008, 38, 186–188; b) Neglect-
ed Tropical Diseases, World Health Organization: http://www.who.int/ne-
glected_diseases/diseases/en/ (accessed September 7, 2018).
[5] Leishmaniasis, WHO Fact Sheet, World Health Organization: http://
www.who.int/news-room/fact-sheets/detail/leishmaniasis,
March 14, 2018.
[6] Trypanosomiasis, human African (sleeping sickness), WHO Fact Sheet,
World Health Organization: http://www.who.int/news-room/fact-sheets/
detail/trypanosomiasis-human-african-(sleeping-sickness), updated Feb-
ruary 16, 2018.
[7] M. C. Field, D. Horn, A. H. Fairlamb, M. A. J. Ferguson, D. W. Gray, K. D.
Read, M. De Rycker, L. S. Torrie, P. G. Wyatt, S. Wyllie, I. H. Gilbert, Nat.
Rev. Microbiol. 2017, 15, 217–231.
[8] G. Eperon, M. Balasegaram, J. Potet, C. Mowbray, O. Valverde, F. Chap-
puis, Expert Rev. Anti-Infect. Ther. 2014, 12, 1407–1417.
[9] B. Zulfiqar, T. B. Shelper, V. M. Avery, Drug Discovery Today 2017, 22,
1516–1531.
[10] S. Patterson, A. H. Fairlamb, Curr. Med. Chem. 2018, DOI: https://
doi.org/10.2174/0929867325666180426164352.
[11] R. T. Jacobs, B. Nare, S. A. Wring, M. D. Orr, D. Chen, J. M. Sligar, M. X.
Jenks, R. A. Noe, T. S. Bowling, L. T. Mercer, C. Rewerts, E. Gaukel, J.
Owens, R. Parham, R. Randolph, B. Beaudet, C. J. Bacchi, N. Yarlett, J. J.
Plattner, Y. Freund, C. Ding, T. Akama, Y. K. Zhang, R. Brun, M. Kaiser, I.
Scandale, R. Don, PLoS Neglected Trop. Dis. 2011, 5, e1151.
[12] S. Wyllie, S. Patterson, L. Stojanovski, F. R. C. Simeons, S. Norval, R. Kime,
K. D. Read, A. H. Fairlamb, Sci. Transl. Med. 2012, 4, e119.
[13] V. K. B. K. Mesu, W. M. Kalonji, C. Bardonneau, O. V. Mordt, S. Blesson, F.
Simon, S. Delhomme, S. Bernhard, W. Kuziena, J. F. Lubaki, S. L. Vuvu,
P. N. Ngima, H. M. Mbembo, M. Ilunga, A. K. Bonama, J. A. Heradi, J. L. L.
Salomo, G. Mandula, L. K. Badibabi, F. R. Dama, P. K. Lukula, D. N. Tete, C.
Lumbala, B. Scherrer, N. Strub-Wourgaft, A. Tarral, Lancet 2018, 391,
144–154.
[25] M. S. Tremblay, M. Halim, D. Sames, J. Am. Chem. Soc. 2007, 129, 7570–
7577.
[26] E. Chong, L. L. Schafer, Org. Lett. 2013, 15, 6002–6005.
[27] S. D. Kuduk, J. W. Skudlarek, C. N. Di Marco, J. G. Bruno, M. A. Pausch,
J. A. O’Brien, T. D. Cabalu, J. Stevens, J. Brunner, P. L. Tannenbaum, A. L.
Gotter, C. J. Winrow, J. J. Renger, P. J. Coleman, Bioorg. Med. Chem. Lett.
2014, 24, 1784–1789.
[28] R. Zhou, W. Wang, Z.-j. Jiang, K. Wang, X.-l. Zheng, H.-y. Fu, H. Chen, R.-
x. Li, Chem. Commun. 2014, 50, 6023–6026.
[29] H. Van de Poꢆl, G. Guillaumet, M. C. Viaux-Massuard, Heterocycles 2002,
57, 55–71.
[30] O. A. Volkov, C. C. Cosner, A. J. Brockway, M. Kramer, M. Booker, S.
Zhong, A. Ketcherside, S. Wei, J. Longgood, M. K. McCoy, T. E. Richard-
son, S. A. Wring, M. Peel, J. D. Klinger, B. A. Posner, J. K. De Brabander,
M. A. Phillips, ACS Infect. Dis. 2017, 3, 512–526.
[31] C. Zhang, S. Bourgeade-Delmas, A. F. Alvarez, A. Valentin, C. Hemmert,
H. Gornitzka, Eur. J. Med. Chem. 2018, 143, 1635–1643.
[32] T. Baltz, D. Baltz, C. Giroud, J. Crockett, EMBO J. 1985, 4, 1273–1277.
[33] B. Rꢄz, M. Iten, Y. Grether-Bꢅhler, R. Kaminsky, Acta. Trop. 1997, 68, 139–
147.
[34] J. Guillon, A. Cohen, R. N. Das, C. Boudot, N. M. Gueddouda, S. Moreau,
L. Ronga, S. Savrimoutou, L. Basmaciyan, C. Tisnerat, S. Mestanier, S.
Rubio, S. Amaziane, A. Dassonville-Klimpt, N. Azas, B. Courtioux, J.-L.
Mergny, C. Mulliꢂ, P. Sonnet, Chem. Biol. Drug Des. 2018, 91, 974–995.
[35] N. Greig, S. Wyllie, S. Patterson, A. H. Fairlamb, FEBS J. 2009, 276, 376–
386.
updated
[36] S. Wyllie, B. J. Foth, A. Kelner, A. Y. Sokolova, M. Berriman, A. H. Fairlamb,
Antimicrob. Agents Chemother. 2016, 71, 625–634.
[37] D. C. Jones, I. Hallyburton, L. Stojanovski, K. D. Read, J. A. Frearson, A. H.
Fairlamb, Biochem. Pharmacol. 2010, 80, 1478–1486.
[38] T. Mosmann, J. Immunol. Methods 1983, 65, 55–63.
[14] S. Patterson, S. Wyllie, Trends Parasitol. 2014, 30, 289–298.
[15] S. Wyllie, S. Patterson, A. H. Fairlamb, Antimicrob. Agents Chemother.
2013, 57, 901–906.
Manuscript received: July 8, 2018
Revised manuscript received: August 29, 2018
[16] S. Wyllie, A. J. Roberts, S. Norval, S. Patterson, B. Foth, M. Berriman, K.
Read, A. H. Fairlamb, PLoS Pathog. 2016, 12, e1005971/22.
Accepted manuscript online: && &&, 0000
Version of record online: && &&, 0000
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FULL PAPERS
Para-sites against parasites: Antikine-
toplastid pharmacomodulation at posi-
tion 3 of an 8-nitroquinolin-2(1H)-one
scaffold was achieved using an opti-
mised Suzuki–Miyaura cross-coupling re-
action. Among the 24 molecules syn-
thesised, a para-carboxyphenyl deriva-
tive was identified as a new selective
antitrypanosomal hit with a lower re-
duction potential. Unlike the initial hit,
this compound is not efficiently bioacti-
vated by type I trypanosomal nitrore-
ductase, which suggests an alternative
mechanism of action.
J. Pedron, C. Boudot,
S. Bourgeade-Delmas, A. Sournia-Saquet,
L. Paloque, M. Rastegari, M. Abdoulaye,
H. El-Kashef, C. Bonduelle, G. Pratviel,
S. Wyllie, A. H. Fairlamb, B. Courtioux,
P. Verhaeghe,* A. Valentin
&& – &&
Antitrypanosomatid
Pharmacomodulation at Position 3 of
the 8-Nitroquinolin-2(1H)-one Scaffold
Using Palladium-Catalysed Cross-
Coupling Reactions
ChemMedChem 2018, 13, 1 – 13
www.chemmedchem.org
13
ꢁ 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ