Discovery of new thienopyrimidinone derivatives displaying antimalarial properties toward both erythrocytic and hepatic stages of Plasmodium

Article abstract of DOI:10.1016/j.ejmech.2015.03.011

A preliminary in vitro screening of compounds belonging to various chemical families from our library revealed the thieno[3,2-d]pyrimidin-4(3H)-one scaffold displayed a promising profile against Plasmodium falciparum. Then, 120 new derivatives were synthesized and evaluated in vitro; compared to drug references, 40 showed good activity toward chloroquine sensitive (IC₅₀ 35-344 nM) and resistant (IC₅₀ 45-800 nM) P. falciparum strains. They were neither cytotoxic (CC₅₀ 15-50 μM) toward HepG2 and CHO cells, nor mutagenic. Structure-activity relationships were defined. The lead-compound also appeared active against the Plasmodium liver stages (Plasmodium yoelii IC₅₀ Combining double low line 35 nM) and a preliminary in vivo evaluation indicated the in vitro activity was preserved (45% reduction in parasitemia compared to untreated infected mice). A mechanistic study demonstrated these molecules do not involve any of the pathways described for commercial drugs and exert a specific activity on the ring and trophozoite stages.

Full text of DOI:10.1016/j.ejmech.2015.03.011

European Journal of Medicinal Chemistry 95 (2015) 16e28
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of new thienopyrimidinone derivatives displaying
antimalarial properties toward both erythrocytic and hepatic stages of
Plasmodium
,
Anita Cohen a *, Peggy Suzanne b, Jean-Charles Lancelot b, Pierre Verhaeghe c,
b
a
a
a
ꢀ
ꢀ
ꢀ
ꢁ
Aurelien Lesnard , Louise Basmaciyan , Sebastien Hutter , Michele Laget ,
a
d
d
e
e
ꢁ
Aurelien Dumetre , Lucie Paloque , Eric Deharo , Maxime D. Crozet , Pascal Rathelot ,
Patrick Dallemagne b, Audrey Lorthiois f, Carol Hopkins Sibley g, Patrice Vanelle e,
Alexis Valentin d, Dominique Mazier f *, Sylvain Rault b *, Nadine Azas a
,
,
, *
a
ꢀ
ꢀ
ꢀ
Aix-Marseille Universite, MD, Infections Parasitaires, Transmission, Pharmacologie et Therapeutique IP-TPT UMR MD3, Faculte de Pharmacie, 27 Boulevard
Jean Moulin e CS30064, 13385 Marseille Cedex 05, France
b
ꢀ
ꢀ
UNICAEN, CERMN (Centre d'Etudes et de Recherche sur le Medicament de Normandie-FR CNRS INC3M e SF ICORE, Universite de Caen Basse-Normandie,
UFR des Sciences Pharmaceutiques, Bd Becquerel), CS14032, F-14032 Caen, France
c
ꢀ
ꢀ
Universite Paul Sabatier, CNRS, UPR-CNRS 8241 Laboratoire de Chimie de Coordination, Faculte des Sciences Pharmaceutiques, BP 44099, 205 Route de
Narbonne, 31077 Toulouse Cedex 4, France
d
ꢀ
ꢀ
Universite Paul Sabatier, IRD, UPS PHARMA-DEV, Equipe BIOCID UMR 152, Faculte des Sciences Pharmaceutiques, 35 Chemin des Maraîchers, 31400
Toulouse, France
e
ꢀ
ꢀ
Aix-Marseille Universite, CNRS, ICR UMR 7273, Laboratoire de PharmacoChimie Radicalaire, Faculte de Pharmacie, 27 Boulevard Jean Moulin e CS30064,
13385 Marseille Cedex 05, France
f
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢁ
^
Universite Pierre et Marie Curie, INSERM, Immunite et Infection, UMR S945, Faculte de Medecine: CHU Pitie-Salpetriere, 91 Boulevard de l'Hopital, 75013
Paris, France
g WorldWide Antimalarial Resistance Network (WWARN) and Department of Genome Sciences, University of Washington, Foege Building S-250,
Box 355065, 3720 15th Avenue NE, Seattle, WA 98195-5065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A preliminary in vitro screening of compounds belonging to various chemical families from our library
revealed the thieno[3,2-d]pyrimidin-4(3H)-one scaffold displayed a promising profile against Plasmo-
dium falciparum. Then, 120 new derivatives were synthesized and evaluated in vitro; compared to drug
references, 40 showed good activity toward chloroquine sensitive (IC50 35e344 nM) and resistant (IC50
Received 28 November 2014
Received in revised form
3 March 2015
Accepted 4 March 2015
Available online 10 March 2015
45e800 nM) P. falciparum strains. They were neither cytotoxic (CC50 15e50 mM) toward HepG2 and CHO
cells, nor mutagenic. Structureeactivity relationships were defined. The lead-compound also appeared
active against the Plasmodium liver stages (Plasmodium yoelii IC50 ¼ 35 nM) and a preliminary in vivo
evaluation indicated the in vitro activity was preserved (45% reduction in parasitemia compared to un-
treated infected mice). A mechanistic study demonstrated these molecules do not involve any of the
pathways described for commercial drugs and exert a specific activity on the ring and trophozoite stages.
© 2015 Elsevier Masson SAS. All rights reserved.
Keywords:
Thieno[3,2-d]pyrimidin-4(3H)-one
Pharmacomodulation
In vitro antiplasmodial profile
Genotoxicity
Activity against Plasmodium liver stages
Antiplasmodial mechanism of action
1. Introduction
627,000 deaths (range 473,000e789,000) [1]. This infection,
transmitted via the bite of the female Anopheles mosquito, is caused
Malaria is a devastating pathology, which in 2012 affected 207
million people worldwide (range 135e287 million) and caused
by five species of protozoan parasites belonging to the Plasmodium
genus, namely falciparum, malariae, vivax, ovale and knowlesi.
Plasmodium falciparum is the most virulent [2], causing more than
95% of malaria-related morbidity and mortality. According to the
WHO [1], important and durable progress has been recorded in
recent years, with the estimated incidence of malaria globally
* Corresponding authors.
E-mail address: anita.cohen@univ-amu.fr (A. Cohen).
http://dx.doi.org/10.1016/j.ejmech.2015.03.011
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

A. Cohen et al. / European Journal of Medicinal Chemistry 95 (2015) 16e28
17
reduced by 25% since 2000. Moreover, malaria-specific mortality
rates were also reduced by 42% between 2000 and 2012. These
encouraging statistics indicate that malaria programs are having a
good impact, the combined result of vector control, chemopre-
vention, diagnostic testing and effective malaria treatment. None-
theless, the growing drug resistance of parasites around the world
[3] remains a real and ever-present danger, attributable mainly to
P. falciparum. Currently, the only fully effective antimalarial drugs
utilize artemisinin and its derivatives in combination with several
different partner drugs in artemisinin-based combination therapies
(ACTs), now recommended as the first line of treatment in endemic
areas. However, reduced susceptibility of P. falciparum to treatment
with artemisinin derivatives identified and confirmed on the
CambodiaeThailand border in 2009 [4,5]. This emerging resistance
could lead to a resurgence of more virulent levels of malaria unless
new chemical classes of effective drugs be rapidly found. In this
context, all innovative practices [6] and encouraging results are
being sponsored and shared [7,8], so as to accelerate the develop-
ment and licensing of new antimalarial drugs. Particularly, as the
currently available 8-aminoquinolines (primaquine, tafenoquine)
can lead to severe side effects such as acute intravascular hemolysis
in individuals with severe glucose-6-phosphate deficiency, the
search for original compounds capable of eliminating hepatic
stages of the parasite, including Plasmodium vivax hypnozoites, is
becoming essential [9,10].
tolylthieno[3,2-d]pyrimidin-4(3H)-one 1 in 70% yield (Scheme 1).
Compound 1 was initially evaluated in vitro for its anti-
plasmodial profile against a chloroquine sensitive (3D7) and a
multi-drug resistant (K1) P. falciparum strains. The evaluation of
in vitro cytotoxicity was performed on two complementary
adherent cancer cell lines: HepG2 and CHO. HepG2 is a commonly
used human-derived hepatocarcinoma cell line expressing many of
the hepatocyte-specific metabolic enzymes. The aim of this assay
using HepG2 in addition to CHO cells was to evaluate the impact of
metabolic activation of the tested compounds on cell viability [18]
(Table 1).
Based on these experimental results, compound 1 was used for
SAR studies and three regions were probed within this chemical
structure: the amine region A, the substitution of the phenyl moiety
B and the nature of the cycle C (Fig. 2). All derivatives described
below were prepared by using the general route presented for the
preparation of compound 1 in Scheme 1.
From the structure of hit-compound 1, primary, secondary and
tertiary amine substituents were explored in region A (Table 2).
Compounds including a secondary amine at position 2 were
selected as the best derivatives since others (compounds 2, 14e18)
showed poor solubility in the cell culture medium, a high toxicity
toward the HepG2 cells and/or no antiplasmodial activity. However,
although the compounds including a secondary amine at position 2
(compounds 3, 8e13) did not appear to be cytotoxic, the substitu-
tion of the nitrogen atom of the amine group by a longer aliphatic
chain notably increased the cytotoxicity (compounds 4e7). Among
all tested substituents, tert-butylamine (compound 1) and iso-
propylamine (compound 8) provided the best antiplasmodial
profiles.
A SAR study was also conducted for region B (Table 3). The
suppression of the methyl substituent of the phenyl ring led to a
reduction of the potency (compound 27). The substitution of the
same methyl group by an electron-withdrawing group at the para
position, such as chlorine (compounds 20, 24), bromine (com-
pounds 19, 23), fluorine (compounds 21, 25), or by an electron-
donating group, such as methoxy (compounds 22, 26), were
tolerated mainly with isopropylamine at region A. However, a sig-
nificant loss in potency was observed if the phenyl group was
suppressed (compounds 28, 29) or moved at position 7 of the
thieno[3,2-d]pyrimidin-4(3H)-one ring (compounds 32e38).
Finally, two analogs derived from compound 1, 2-(tert-butyla-
mino)-6-m-tolylthieno[3,2-d]pyrimidin-4(3H)-one 30 and 2-(tert-
butylamino)-6-o-tolylthieno[3,2-d]pyrimidin-4(3H)-one 31 were
3e7 fold less potent than the hit-compound and also more
cytotoxic.
This work began by an in vitro screening of numerous molecules
belonging to our chemical library (part of the CNRS French National
Chemical Library), toward P. falciparum. This library contained
numerous human kinase inhibitor-candidates which had previ-
ously been synthesized for a research program centered on the
design of new anti-cancer agents [11e13]. Among tested com-
pounds, a thieno[3,2-d]pyrimidin-4(3H)-one derivative appeared
as a hit-compound (IC50 < 10 mM against the K1 strain). A closely
related scaffold had already been proposed by GlaxoSmithKline as a
potential antimalarial structure [7] (Fig. 1). In this context, we
investigated the antiplasmodial properties of a series of 120 new
derivatives [14]. The very recent work reported by Gonzalez Cab-
rera et al. also demonstrated the great potential of the 2-aminated-
6-arylthienopyrimidine scaffold (Fig. 1) in the research for new
antimalarial products [15].
2. Results and discussion
Original thieno[3,2-d]pyrimidin-4(3H)-one derivatives were
synthesized by a one pot procedure including a condensation and a
cyclization of methyl-3-amino-5-p-tolylthiophene-2-carboxylate
with ethoxycarbonyle isothiocyanate in DMF [14]. During this re-
action, an intermediary thiourea carbamate was formed. This spe-
cies then reacted with an alkylamine, added to the crude mixture
with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, HCl)
[16], and triethylamine [17]. As an example, using terbutylamine,
this process led to the formation of 2-(tert-butylamino)-6-p-
We finally explored the SAR based on region C by modifying the
heterocyclic core (Table 4). As various substituted quinazolines
have already been described as antiplasmodial agents [19e23], we
particularly studied the replacement of the thiophene ring by a
benzene one, resulting in quinazoline analogs (Table 4). This
modification led to a significant loss of activity and widely
Fig. 1. Thieno[3,2-d]pyrimidine derivatives described as antiplasmodial compound by GlaxoSmithKline [7] and Gonzalez Cabrera et al. [15].

18
A. Cohen et al. / European Journal of Medicinal Chemistry 95 (2015) 16e28
Scheme 1. Synthesis of 2-(tert-butylamino)-6-p-tolylthieno[3,2-d]pyrimidin-4(3H)-one 1.
Table 1
pyrimidin-4(3H)-one, carrying either a tert-butylamine substituent
Antiplasmodial profile of compound 1.
at position 2 and a Me or Cl substituent at the para position of the
phenyl moiety, or an isopropylamine substituent at position 2 and a
Me, MeO, Br, Cl or F substituent at the para position of the phenyl
moiety (Fig. 3).
Drug
Cytotoxicitya
(
mM)
Antiplasmodial
activitya
M)
3D7
K1
(
m
strain SIb strain SIb
HepG2 CHO CC50 3D7 IC50 K1 IC50
CC50
In an attempt to improve the solubility of this series, various
salts were prepared and evaluated (Fig. 4). It then appeared that
hydrochloride or fumarate salts were favorable for the anti-
plasmodial activity, the corresponding salts displaying K1 IC50
values ranging from 45 nM (1a) to 800 nM (8a and 20a), compared
with chloroquine (K1 IC50 ¼ 500 nM) and atovaquone (K1
IC50 ¼ 1.3 nM) used as reference drugs [14] (Fig. 4).
1
25.6
3.1)
27.5
0.14
0.2
183
128
(
(
2.8)
(
0.01)
(
0.02)
Doxorubicinc 0.2
Chloroquined 30
Doxycyclined 20
Atovaquoned >15.6e
0.6
155
e
e
e
e
e
0.04
e
0.5
5.0
750
e
60
4
>12,000
e
e
0.0013
e
a
The values are mean SD of three independent experiments.
Selectivity index (SI) was calculated according to the following formula:
Exploring the antiparasitic spectrum of activity of this molecular
scaffold toward other protozoa, compounds 1, 8 and 20 were then
evaluated in vitro against Toxoplasma gondii and the promastigote
stage of Leishmania donovani. No activity was revealed against these
b
SI ¼ CC50 (HepG2)/IC50
.
c
Doxorubicin was used as reference drug compound for human cell toxicity.
Chloroquine, doxycycline and atovaquone were used as reference anti-
d
plasmodial drug compounds.
parasites (IC50 > 10
mM) in comparison with reference drugs
e
Compound could not be tested at a higher concentration because of its lack of
(Table 4). Therefore, contrary to doxycycline [24] or atovaquone
[25,26], this series exhibits a selective antiplasmodial profile.
Then, to further investigate the toxicological profile of this
promising series, compounds 1, 8 and 20 were evaluated for their
mutagenicity via the Ames test [27,28], at a concentration of
2.5 mM on two distinct Salmonella typhimurium strains (TA97A and
TA100), in two different conditions, with and without metabolic
activation (S9 mix). These compounds were also evaluated for their
genotoxicity by the DNA-methyl green test [29]. These molecules
were neither mutagenic nor genotoxic (Table 5).
solubility in the cell culture medium.
increased the cytotoxicity, indicating the importance of the thieno
[3,2-d]pyrimidin-4(3H)-one core for antiplasmodial profile; varia-
tion in the position of the tolyle group on the quinazoline core
(compounds 39 and 40) had no significant influence.
Global conclusion of the SAR studies from compound 1 was that
the precise structure of our in vitro antiplasmodial pharmacophore
corresponded to the 2-amino-6-p-substituted phenylthieno[3,2-d]
Fig. 2. SARs from compound 1.

A. Cohen et al. / European Journal of Medicinal Chemistry 95 (2015) 16e28
19
Table 2
SARs of region A.
The most active antiplasmodial compound 1a in the series was
also tested in vivo against a chloroquine sensitive strain of Plas-
modium berghei (NK65). This preliminary evaluation (one concen-
tration of compound 1a, one administration route, groups of 5
animals) of the antimalarial potential was determined by the
classical Peters four-day curative standard test [30e32]. Thus, 3
groups of five infected mice were compared: untreated (control
group) and treated by intra peritoneal route with 2.5 mg/kg of
either compound 1a or chloroquine (reference drug, Sigma), twice a
day, for four consecutive days beginning on the day of infection. On
day 4, Giemsa-stained thin blood smears were made for each
mouse, and parasitemia was estimated by visual numeration. These
preliminary results are encouraging, compound 1a being respon-
sible for a 45% reduction in parasitemia in comparison with the
chloroquine control at the same dose: 85% (Table 6).
The effect of compound 1a on the erythrocytic life cycle of the
K1 P. falciparum strain was also evaluated from synchronized
parasite cultures (Fig. 5) [33]. During the erythrocytic cycle, the
highest activity of 1a was noted between 0 and 8 h for the highest
concentration (5 ꢀ IC50), with 41% reduction in parasitemia
compared to negative control. This result suggests that compound
1a is active against ring stage parasites, and may inhibit the inva-
sion of new erythrocytes. We noticed that this activity remained
until the 32nd hour of the erythrocytic life cycle with 30% reduction
in parasitemia for the highest tested concentration. This period of
activity corresponds to the ring and trophozoïte forms of the par-
asites. All these observations suggest that compound 1a does not
act on a single target during the erythrocytic life cycle, and may
simultaneously employ several mechanisms of action.
In the development of new antimalarial compounds, the
assessment of the effect toward the hepatic stage of the parasite
remains a key point, since new agents that act at stages of the
parasite life cycle in addition to the erythrocytic stage are sorely
needed. To investigate the efficacy of our molecules toward the
liver stage of Plasmodium, we performed an in vitro assay in which
hepatic HepG2-CD81 cells are infected by Plasmodium yoelii 17X NL
sporozoites [34] (Table 7). In this assay, atovaquone and primaquine
(as a member of the 8-aminoquinoline family), two of the very few
molecules showing good hepatic activity, were used as reference-
drugs. Indeed, atovaquone and primaquine are known to be
active against the blood and liver stages of malaria [35], even if
primaquine also has described stage activity against mature ga-
metocytes of P. falciparum [36]. Very interestingly, lead-compounds
1a, 8a and 20a (more especially 1a) displayed IC50 values
(35e120 nM) close to the excellent level of atovaquone (9 nM) and
much better than that of primaquine (1160 nM).
Compound
NR1R2
Cytotoxicitya Antiplasmodial SIb
HepG2
activitya K1
CC50 IC50 M)
(
m
M)
(m
1
25.6 ( 3.1)
0.2 ( 0.02)
128
H3C
N
CH3
CH3
H
2
3
ee
ee
e
e
H
N
H
>62.5f
>5f
CH3
N
H
4
5
6.0 ( 1.1)
4.2 ( 1.8)
>5f
>5f
e
e
N
H
CH3
CH3
N
H
6
7
10.6 ( 3.9)
6.8 ( 1.4)
>5f
>5f
e
e
N
H
CH3
CH3
N
H
8
9
49.4 ( 1.0)
0.8 ( 0.5)
62
CH3
CH3
N
H
101.3 ( 10.1) >5f
e
CH3
CH3
CH3
N
H
10
11
21.6 ( 6.1)
19.4 ( 0.3)
>5f
>5f
e
e
N
H
CH3
CH3
CH3
N
H
12
13
9.2 ( 2.8)
>5f
>5f
e
e
CH3
N
H
32.6 ( 3.8)
Cl
N
H
14
15
3.0 ( 1.1)
>5f
e
e
N
CH3
CH3
ee
ee
N
CH3
CH3
Furthermore, an investigation was carried out on the lead-
compounds in an attempt to elucidate their mechanism(s) of ac-
tion. The mechanisms studied were those known for the most
important commercial antimalarial drugs: chloroquine [38], ato-
vaquone [39], pyrimethamine [40] and artesunate [41]. Inhibition
of hemozoin polymerization is an important target for 4-
aminoquinoline-based antimalarials such as chloroquine and
amodiaquine, two well-known blood schizonticidals. Using the
microtitre-base assay [38], out of the tested compounds, only 1
16
17
ee
ee
ee
ee
e
e
CH3
CH3
N
N
CH3
CH3
18
ee
ee
e
CH3
CH3
N
Doxorubicinc
Chloroquined
Doxycyclined
Atovaquoned
0.2
30
e
e
showed some b-hematin inhibition activity (hemozoin polymeri-
0.5
5
0.0013
60
4
>12,000
zation IC50 ¼ 21.6 mM), compared with chloroquine (hemozoin
polymerization IC50 ¼ 1.46 mM) used as a reference drug (Table 8).
The antimalarial mechanism of action of atovaquone, an active
drug against blood and liver stages that targets Plasmodium respi-
ration, consists in the depolarization of the mitochondrial mem-
brane. By using a very low concentration (2 nM) of the cationic,
lipophilic and fluorescent probe, 3,30-dihexyloxacarbocyanide io-
dide (DiOC6), a measurement of the mitochondrial membrane po-
tential in infected erythrocytes by flow cytometry is defined as an
20
>15.6f
a
The values are means SD of three independent experiments.
b
c
Selectivity index (SI) was calculated according to the following formula: SI ¼ CC50/IC50
.
Doxorubicin was used as reference drug compound for human cell toxicity.
Chloroquine, doxycycline and atovaquone were used as reference anti-
d
plasmodial drug compounds.
e
Compound could not be tested because of its lack of solubility in the cell culture
medium.
f
No activity was observed at the highest concentration tested.

20
A. Cohen et al. / European Journal of Medicinal Chemistry 95 (2015) 16e28
Table 3
SAR of region B.
Compound
Region A
Region B
Cytotoxicitya HepG2 CC50
25.6 ( 3.1)
(
mM)
Antiplasmodial activitya K1 IC50
0.2 ( 0.02)
(
m
M)
SIb
1
128
6
H3C
CH3
CH3
H3C
N
H
19
1.4 ( 0.6)
>5f
e
6
H3C
CH3
Br
Cl
F
N
H
CH3
20
21
15.0 ( 2.7)
5.1 ( 1.0)
0.8
19
6
6
H3C
CH3
CH3
N
H
>5f
e
H3C
CH3
CH3
N
H
22
32.3 ( 2.2)
>5f
e
H3C
H3C
O
6
CH3
CH3
N
H
23
24
13.4 ( 1.9)
1.7
0.6
9
6
CH3
Br
Cl
F
N
H
CH3
>15.6
>26
6
CH3
CH3
N
H
25
26
>25f
0.5
0.5
>66
6
CH3
CH3
N
H
27.1 ( 3.9)
136
CH3
H3C
O
6
N
H
CH3
27
28
29
12.5 ( 2.5)
8.4 ( 3.5)
23.9 ( 2.1)
1.0
>5f
>5f
10
e
6
H3C
CH3
N
H
CH3
6
H3C
CH3
CH3
H
N
H
e
6
CH3
H
N
H
CH3
30
31
14.1 ( 1.4)
4.0 ( 1.1)
3.6
1.7
3
2
H3C
H3C
CH3
CH3
6
N
H
H3C
CH3
CH3
CH3
6
N
H
32
33
1.4 ( 0.4)
2.3 ( 0.2)
>5f
>5f
e
e
7
H3C
CH3
CH3
H3C
N
H
7
H3C
CH3
CH3
F
N
H
34
35
8.8 ( 0.8)
2.7
3
7
CH3
H3C
N
H
CH3
>15.6f
>5f
e
7
CH3
CH3
F
N
H

A. Cohen et al. / European Journal of Medicinal Chemistry 95 (2015) 16e28
21
Table 3 (continued )
Compound
36
Region A
Region B
Cytotoxicitya HepG2 CC50
6.5 ( 0.1)
(
m
M)
Antiplasmodial activitya K1 IC50
(m
M)
SIb
>5f
e
CH3
7
7
Br
N
H
CH3
37
38
7.1 ( 0.6)
>5f
>5f
e
e
CH3
CH3
Cl
N
H
27.8 ( 4.1)
CH3
CH3
H3C
O
7
N
H
Doxorubicinc
Chloroquined
Doxycyclined
Atovaquoned
0.2
30
e
e
60
4
0.5
5
0.0013
20
>15.6e
>12,000
a
The values are means SD of three independent experiments.
b
c
d
e
f
Selectivity index (SI) was calculated according to the following formula: SI ¼ CC50/IC50
.
Doxorubicin was used as reference drug compound for human cell toxicity.
Chloroquine, doxycycline and atovaquone were used as reference antiplasmodial drug compounds.
Compound could not be tested because of its lack of solubility in the cell culture medium.
No activity was observed at the highest concentration tested.
Table 4
SAR of region C.
Drug
1
Chemical structure
Cytotoxicitya HepG2 CC50
25.6 ( 3.1)
(
m
M)
Antiplasmodial activitya K1 IC50
0.2 ( 0.02)
(
mM)
SIb
128
O
N
S
NH
H3C
N
H3C
CH3
H
CH3
39
40
0.49 ( 0.9)
0.98 ( 0.6)
>5f
<10.2
<5.1
H3C
O
HCl
NH
H3C
CH3
N
N
H
CH3
>5f
O
NH
H3C
N
CH3
N
H
CH3
H3C
Doxorubicinc
Chloroquined
Doxycyclined
Atovaquoned
0.2
30
e
e
0.5
5
0.0013
60
4
>12,000
20
>15.6e
a
The values are means SD of three independent experiments.
b
c
d
e
f
Selectivity index (SI) was calculated according to the following formula: SI ¼ CC50/IC50
.
Doxorubicin was used as reference drug compound for human cell toxicity.
Chloroquine, doxycycline and atovaquone were used as reference antiplasmodial drug compounds.
Compound could not be tested because of its lack of solubility in the cell culture medium.
No activity was observed at the highest concentration tested.
alternative in vitro assay to study the mitochondrial function in
malarial parasites [39]. Atovaquone, used as reference drug, caused
a depolarization of the plasmodial mitochondria membrane, as
seen in the flow cytometric profile. At K1 IC50, none of the tested
compounds (1, 8, 20) had any effect on malarial mitochondrial
membrane potential (Fig. 6).
We also evaluated in vitro the effect of lead-compounds 1, 8 and
20 toward their P. falciparum dihydrofolate reductase (PfDHFR)
inhibitory activity by using two antifolinic erythrocytic schizo-
nticidals, pyrimethamine and cycloguanil, as reference drugs. None
of tested compounds revealed any inhibition of this parasitic target
by using both the spoke assay and the 96-well microtiter plates
assay [40] (Table 9).
Finally, we tested the ability of these compounds to generate
free radicals, using artesunate as reference, an antimalarial drug
that targets pathogenic blood stage parasites and immature ga-
metocytes [36], proposed to utilize that molecular mechanism of
action. The general procedure for this assay was similar to the
Fig. 3. Antiplasmodial pharmacophore in 6-phenylthieno[3,2-d]pyrimidin-4(3H)-one
series.

22
A. Cohen et al. / European Journal of Medicinal Chemistry 95 (2015) 16e28
Fig. 4. Promising compounds 1, 8 and 20 and their salts 1a, 8a and 20a.
Table 5
Complementary biological results displayed by lead-compounds 1, 8 and 20.
Drug
Antileishmanial activity
Antitoxoplasmic
Mutagenicity at 2.5 mM (Ames test)
Genotoxicity (DNA-methyl
green test) DC50 (mM)
g
promastigotes IC50
(m
M)
activity IC50
(
m
M)
Without S9 mixf
With S9 mixf
1
8
20
>10h
>10h
>10h
0.08
e
>10h
>10h
>10h
e
Negative
Negative
Negative
e
Negative
Negative
Negative
e
>25i
>25i
>25i
e
Amphotericine Ba
Pyrimethamineb
ICR170c
2
e
e
e
e
e
Positive
Positive
e
e
e
c
NaN3
e
e
e
e
Benzo[a]pyrened
e
e
Positive
e
e
Daunorubicinee
e
e
e
0.001
a
Amphotericine B was used as reference antileishmanial drug compound.
Pyrimethamine was used as reference antitoxoplasmic drug compound.
ICR170 (TA97A S. typhimurium strain) and NaN3 (TA100 S. typhimurium strain) were used as reference mutagenic drug compounds without metabolic activation.
Benzo[a]pyrene (TA97A and TA100 S. typhimurium strains) was used as reference mutagenic drug compound with metabolic activation.
Daunorubicine was used as reference genotoxic drug compound.
S9 mix was used to test mutagenicity after metabolic activation.
DC50 refers to amount of compound required to displace 50% of the methyl green from DNA-methyl green complex.
No activity was observed at the highest concentration tested.
b
c
d
e
f
g
h
i
No toxicity was observed at the highest concentration tested.
in vitro determination of the antiplasmodial activity with addition
of a radical scavenger, ascorbic acid, in the parasite culture medium.
None of the tested compounds seemed to produce free radicals,
their respective K1 IC50 being similar in the presence or in absence
of ascorbic acid (Table 10), compared with artesunate.
mechanisms of action of commercialized antimalarial drugs, which
suggests that they might be appropriate partner drugs in a com-
bination. Further work will be necessary to identify the mecha-
nism(s) of action of these newly identified antimalarial
thienopyrimidines. Among possible targets which have to be seri-
ously considered, regarding the thienopyrimidine core of these
molecules, we will focus on the P. falciparum dihydroorotate de-
hydrogenase (PfDHOD) [42] and also on several P. falciparum ki-
nases which have already been reported as relevant targets
[7,43e46] remembering that these novel compounds were
initially identified by screening a chemical library containing
molecules which had been designed as human kinase inhibitors.
In summary of all these mechanistic experiments, we conclude
that the tested compounds do not use the already described
Table 6
Parasitemia of Plasmodium berghei infected mice not treated and intraperitoneally
treated with 5 mg/kg of chloroquine or compound 1a in a classic four-day sup-
pressive test.
Untreated mice
Chloroquinea
2 ꢀ 2.5 mg/kg
1.5%
1a
3. Conclusion
2 ꢀ 2.5 mg/kg
The present work globally demonstrated that 2-aminated-6-
phenylthieno[3,2-d]pyrimidin-4(3H)-one derivatives present
Parasitemia
9%
4.9%
a
a
Chloroquine was used as reference antimalarial drug compound.
real promising selective antimalarial profile, targeting both the

A. Cohen et al. / European Journal of Medicinal Chemistry 95 (2015) 16e28
23
Fig. 5. Effect of compound 1a on the erythrocytic life cycle of P. falciparum K1 strain.
erythrocytic and hepatic stages of the parasite through an original
mechanism of action which remains to be elucidated. Comple-
mentary investigations including more complete in vivo and
mechanistic studies, will then be carried out in a view to address
the pharmaceutical development of such novel antimalarial
scaffold.
carboxylate in DMF (40 mL) was added ethoxycarbonyle isothio-
cyanate (1 eq). The solution was left stirring at room temperature
overnight. During this reaction, a thiourea carbamate intermediate
was formed and was not isolated. To the reaction mixture were
then added triethylamine (3 eq), terbutylamine (2 eq) and 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide, hydrochloride (EDCI,
HCl) (1.2 eq). The solution was then heated under reflux for 2 h. The
solvent was removed under reduced pressure. Water (10 mL) was
then added to the reaction mixture and the precipitated solid ob-
tained was filtered, washed with diethyl ether and recrystallized
from acetonitrile to give the corresponding 2-alkyl(dialkyl)aryl-2-
aminothieno[3,2-d]pyrimidin-4(3H)-one derivative.
4. Experimental
4.1. Chemistry
4.1.1. General material and methods
All chemical reagents and solvents were purchased from com-
mercial sources and used without further purification. Melting
points were determined on a Kofler melting point apparatus.1H and
13C NMR spectra were recorded on a JEOL Lambda 400 spectrom-
eter and BRUKER AVANCE III 500 MHz with chemical shifts
expressed in parts per million (in DMSO-d6) downfield from tet-
ramethylsilane as an internal standard and coupling in Hertz.
Elemental analyses were performed at the “Institut de Recherche
en Chimie Organique Fine” (Rouen, France).
4.1.3. General procedure for the synthesis of 2-tert-butylamino-6-p-
tolyl-3H-quinazolin-4-one, hydrochloride (39)
To a solution of methyl 2-amino-5-(4-methylphenyl)benzoate
(1 g, 4.14 mmol) in DMF (8 mL) were added ethoxycarbonyl iso-
thiocyanate (0.49 mL, 1 eq). The solution was left stirring at room
temperature overnight. During this reaction, a thiourea carbamate
intermediate was formed and was not isolated. To the reaction
mixture was then added triethylamine (1.73 mL, 3 eq), terbutyl-
amine (0.87 mL, 2 eq), and 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide, hydrochloride (EDCI, HCl) (952 mg, 1.2 eq). The so-
lution was then heated under reflux for 2 h. The solvent was
removed under reduced pressure. Water (5 mL) was then added to
the reaction mixture and the precipitate obtained was filtered,
washed with diethyl ether and recrystallized from acetonitrile. The
residue obtained (600 mg) was dissolved into ethanol (35 mL) and
hydrochloric acid (0.2 mL, 1.2 eq) was added. The reaction was then
heated under reflux for 1 h. The precipitate obtained was filtered
and recrystallized from acetonitrile to give 39, as a white solid
(320 mg, 48%).
4.1.2. General procedure for the synthesis of 2-alkyl (dialkyl)aryl-2-
aminothieno[3,2-d]pyrimidin-4(3H)-one derivatives (1e40)
To
a
solution of methyl-3-amino-5-p-tolylthiophene-2-
Table 7
Liver stage activity displayed by the most promising compounds 1a, 8a and 20a.
Drug
P. yoelii IC50 (nM)
HepG2-CD81
CC50 M)
Selective indexa
(
m
1a
8a
20a
35
120
>143c
>111c
>120c
9.5d
>4086
>925
>2000
1056d
5.3d
60
Atovaquoneb
Primaquineb
Chloroquine
Artemisinin
9d
Table 8
1160d
1650d
>10,000d
6,1d
Inhibition of hemozoin polymerization in vitro displayed by compounds 1, 8 and 20.
1.6d
0.97d
e
Drug
Hemozoin polymerization IC50 (mM)
>10d
1
8
20
21.6
>25b
>25b
1.46
a
Therapeutic index was calculated according to the following formula: HepG2-
CD81 CC50/P. yoelii IC50
.
b
Atovaquone and primaquine were used as reference drug compounds active
Chloroquinea
during the Plasmodium liver stage.
c
a
No toxicity was observed at the highest concentration tested.
Data already published [37].
Chloroquine was used as reference drug inhibiting hemozoin polymerization.
No inhibition was observed at the highest concentration tested.
d
b

24
A. Cohen et al. / European Journal of Medicinal Chemistry 95 (2015) 16e28
Fig. 6. Cytometric profiles in the depolarization of the mitochondrial membrane (red curve: without compound; blue curve: A ¼ Atovaquone used as reference drug compound
depolarizing mitochondrial membrane or B ¼ Compound 1 at K1 IC50). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article.)
Table 9
filtered and recrystallized from acetonitrile to give 8a as a white
solid (430 mg, 77%).
P. falciparum dihydrofolate reductase (PfDHFR) inhibitory activity in vitro assay.
Compound
P. falciparum DHFR inhibitory activity IC50 (mM)
4.2. Biological evaluation
1
8
20
>500b
>250b
>250b
2.8
4.2.1. In vitro cytotoxicity evaluation on CHO and HepG2 cell lines
CHO and HepG2 cell lines were maintained at 37 ꢁC, 6% CO2, 14%
O2, 80% N2, with 90% humidity in RPMI supplemented with 10%
Pyrimethaminea
Cycloguanila
5
a
Pyrimethamine and cycloguanil were used as reference drugs inhibiting PfDHFR.
No inhibitory activity was observed at the highest tested concentration.
fœtal bovine serum,1%
L-glutamine (200 mM) and penicillin (100 U/
b
mL)/streptomycin (100
m
g/mL) (complete RPMI medium). In vitro
cytotoxicity evaluation on CHO and HepG2 cell lines was performed
according to the method described by Mosman [47] with slight
4.1.4. General procedure for the synthesis of 2-tert-butylamino-6-
(p-tolyl)-3H-thieno[3,2-d]pyrimidin-4-one, hydrochloride (1a)
To a solution of 1 (2.5 g, 7.97 mmol) in ethanol (150 mL) was
added hydrochloric acid (0.8 mL, 1.2 eq). The reaction was then
heated under reflux for 1 h. The precipitate obtained was filtered
and recrystallized from acetonitrile to give 1a as a yellow solid
(2.4 g, 85%).
modifications. Briefly, 5.103 cells in 100
mL of culture medium
(RPMI þ 10% CO2) were inoculated into each well of 96-well plates
and incubated at 37 ꢁC in a humidified 6% CO2, 14% O2, 80% N2 at-
mosphere. After 24 h incubation, 100 mL of medium with various
product concentrations dissolved in DMSO (final concentration less
than 0.5% v/v) were added and the plates were incubated from 24 h
(CHO) to 72 h (HepG2). Duplicate assays were performed for each
sample. At the end of the treatment and incubation, each plate-well
was microscope-examined for detecting possible precipitate for-
mation before the medium was aspirated from the wells. Then,
4.1.5. General procedure for the synthesis of 2-tert-butylamino-6-
(p-chlorophenyl)-3H-thieno[3,2-d]pyrimidin-4-one, fumarate (20a)
To a solution of 20 (500 mg, 1.49 mmol) in isopropanol (30 mL)
was added fumaric acid (208 mg, 1.2 eq). The reaction mixture was
then heated under reflux for 3 h. The precipitate obtained was
filtered and recrystallized from acetonitrile to give 20a as a white
solid (500 mg, 74%).
10
mL yellow MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazolium bromide) solution (5 mg MTT/mL in PBS) was added to
each well with 100 mL of medium without fœtal bovine serum. Cells
were incubated for 2 h at 37 ꢁC to allow MTT oxidation by mito-
chondrial dehydrogenase in the viable cells. After 2 h, the MTT
solution was aspirated and DMSO (100 mL) was added to each well
4.1.6. General procedure for the synthesis of 2-isopropylamino-6-
(p-tolyl)-3H-thieno[3,2-d]pyrimidin-4-one, fumarate (8a)
To a solution of 8 (400 mg, 1.33 mmol) in isopropanol (25 mL)
was added fumaric acid (186 mg, 1.2 eq). The reaction mixture was
then heated under reflux for 3 h. The precipitate obtained was
to dissolve the resulting blue formazan crystals. Plates were then
shaken vigorously (300 rpm) for a few minutes. The absorbance
was measured at 570 nm with 630 nm as reference wavelength,
using a BIO-TEK ELx808 Absorbance Microplate Reader. DMSO was
used as blank and doxorubicin as positive control. Cell viability was
calculated as percentage of control (cells incubated without com-
pound). The 50% cytotoxic concentrations (CHO CC50 and HepG2
CC50) were determined by non-linear regression analysis processed
on doseeresponse curves, using the Table Curve software 2D v.5.0.
CC50 values represent the mean value calculated from three inde-
pendent experiments.
Table 10
Free radical production assay.
Compound
Antiplasmodial
activity K1 IC50
Antiplasmodial activity in presence
(
mM)
of ascorbic acid (800
mM)
Ascorbic acid K1 IC50
(
m
M)
1
8
20
0.2
0.8
0.8
0.2
0.9
0.8
0.7
4.2.2. Antiplasmodial activity in vitro assay [48,49]
The evaluation of compounds' antiplasmodial activity was
conducted in vitro on one to three culture-adapted P. falciparum
strains (3D7 sensible; K1 resistant to chloroquine, pyrimethamine
Artesunatea
<0.1
a
Artesunate was used as reference drug compound generating radicals.

A. Cohen et al. / European Journal of Medicinal Chemistry 95 (2015) 16e28
25
and proguanil). P. falciparum strains were maintained in fresh Aþ
human erythrocytes at 2.5% haematocrit in complete medium
(RPMI 1640 with 25 mM HEPES, 25 mM NaHCO3, 10% of Aþ human
serum) at 37 ꢁC under reduced O2 atmosphere (gas mixture 6% CO2,
14% O2, 80% N2). Parasitaemia was maintained daily between 1%
and 6%. The P. falciparum drug susceptibility test was carried out by
comparing quantities of DNA in treated and control cultures of
parasite in human erythrocytes according to an SYBR Green I
fluorescence-based method using a 96-well fluorescence plate
reader. Parasite culture was synchronized at ring stage with 5%
sorbitol. Compounds dissolved in DMSO (final concentration less
software 2D v.5.0. Ld IC50 values represent the mean value calcu-
lated from three independent experiments.
4.2.4. Antitoxoplasmic activity in vitro assay
The effects of the tested compounds on the growth of T. gondii
tachyzoites (PRU-b-Gal strain, kindly provided by Pr. I. Villena,
Reims, France) were assessed by a colorimetric microtiter assay
according to the method of McFadden et al. [50]. Briefly, tachyzoites
were maintained by serial passage in confluent human foreskin
fibroblast (HFF) monolayer (a gift of Pr. I. Dimier-Poisson, Tours,
France). For assay, 96-well microtiter plates were seeded with 1.104
HFF cells and allowed to grow to confluence in complete RPMI
medium at 37 ꢁC with 6% CO2, 14% O2, 80% N2 and 90% humidity.
Cell monolayers were infected with 2.103 parasites per well and
incubated at 37 ꢁC in a humidified 6% CO2, 14% O2, 80% N2 atmo-
sphere for 3 h. Then, various concentrations of compounds dis-
solved in DMSO (final concentration less than 0.5% v/v) were
incorporated in triplicate. Pyrimethamine was used as the refer-
ence drug compound. Appropriate controls treated by DMSO or
pyrimethamine were added to each set of experiments. Negative
control consisted in cell monolayers incubated without parasite
and drug. After a 96-h incubation period at 37 ꢁC in a humidified 6%
than 0.5% v/v) were incubated in a total assay volume of 200 mL
(RPMI, 2% haematocrit and 1% parasitaemia) for 72 h in a humidi-
fied atmosphere (6% CO2, 14% O2, 80% N2) at 37 ꢁC, in 96-well flat-
bottom plates. Duplicate assays were performed for each sample.
After incubation, 125
washed twice with 150
transferred to 96-well flat bottom non-sterile black plates (Greiner
Bio-one). 15 L of the SYBR Green lysis buffer (2XSYBR Green,
mL supernatant was discarded and cells were
m
L 1X PBS. 15 L re-suspended cells were
m
m
20 mM Tris base pH 7.5, 20 mM EDTA, 0.008% w/v saponin, 0.08% w/
v Triton X-100) was added to each well. Negative control (treated
by DMSO) and positive controls (doxycycline and chloroquine)
were added to each set of experiments. Plates were incubated for
0.25 h at 37 ꢁC and then read on a TECAN Infinite F-200 spectro-
photometer with excitation and emission wavelengths at 497 and
520 nm respectively. The concentrations of compounds required to
induce a 50% decrease in parasite growth (IC50) were calculated
from three independent experiments. The selectivity indexes pre-
sented correspond to the ratios between, respectively, the cyto-
toxicity and the antiplasmodial activity. They are calculated as
follows: SI ¼ CC50 (HepG2)/IC50 (3D7 or K1).
CO2, 14% O2, 80% N2 atmosphere, 20
galactopyranoside (CPRG) were added to each well to give a final
concentration of 500
M. The plates were incubated at 37 ꢁC for an
mL of chlorophenol red-b-D-
m
additional 24 h. Then, the absorbance was measured at 570 nm
with 630 nm as reference wavelength, using a BIO-TEK ELx808
Absorbance Microplate Reader. DMSO was used as blank and
reference drugs as positive controls. The T. gondii drug suscepti-
bility was calculated as percentage of control (cells incubated
without compound). The 50% inhibitory concentration (Tg IC50
)
represents the concentration of compound required to induce a 50%
decrease of parasite growth. It was determined by non-linear
regression analysis processed on doseeresponse curve, using the
Table Curve software 2D v.5.0. Tg IC50 values represent the mean
value calculated from three independent experiments.
4.2.3. Antileishmanial activity in vitro assay
L. donovani strain was maintained at 27 ꢁC, 6% CO2, 14% O2, 80%
N2, with 90% humidity in Schneider's medium supplemented with
10% fœtal bovine serum, 1%
L-glutamine (200 mM) and penicillin
(100 U/mL)/streptomycin (100
mg/mL) (complete Schneider's me-
dium). The L. donovani promastigotes drug susceptibility test was
4.2.5. Ames test
carried out by using the method described by Mosman [47] with
Mutagenicity test was carried out by using a modified version of
the liquid incubation assay of the classical Ames test [27,28].
S. typhimurium tester strains (TA97a and TA100) were grown
overnight in an Oxoid nutrient broth. After this period, 2.5e25 mM
DMSO solutions of the tested drugs were added to 0.1 mL of culture
and incubated with and without 4% S9 mix for 1 h at 37 ꢁC with
shaking. After this period, 2 mL of molten top agar were added and
the mixture was transferred onto Vogel-Bonner agar plates. After
48 h at 37 ꢁC in the dark, the number of spontaneous and drug
induced revertants per plate was determined for each dose with a
laser bacterial colony counter. A product is considered mutagenic
when it induces a twofold increase of the number of revertants
compared with the spontaneous frequency.
slight modifications. Briefly, 1.106 cells in log-phase in 100
m
L of
culture medium (complete Schneider's medium) were inoculated
into each well of 96-well plates. 100 L of cells in culture medium
m
with various product concentrations dissolved in DMSO (final
concentration less than 0.5% v/v) were added and the plates were
incubated at 27 ꢁC in a humidified 6% CO2, 14% O2, 80% N2 atmo-
sphere for 72 h. Amphotericin B and pentamidine were used as
reference drugs. Duplicate assays were performed for each sample.
At the end of the treatment and incubation, the medium was
aspirated from wells and 10
mL yellow MTT (3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) solution (5 mg
MTT/mL in PBS) was added to each well with 100 mL of medium
without fœtal bovine serum. Cells were incubated for 6 h at 27 ꢁC to
allow MTT oxidation by mitochondrial dehydrogenase in the viable
4.2.6. Microtiter DNA-methyl green assay
cells. After 6 h, the MTT solution was aspirated and DMSO (100
m
L)
Microtiter DNA-methyl green assay was carried out by using the
method described by Burres [29]. Before use in an assay, DNA-
methyl green solution was prepared as follows: 4 mg of DNA-
methyl green was first dissolved in a 20 mL TriseHCl buffer
0.05 M, pH 7.5 with 7.5 mmol/L of magnesium sulfate. This DNA-
was added to each well to dissolve the resulting blue formazan
crystals. Plates were then shaken vigorously (500 rpm) for a few
minutes. The absorbance was measured at 570 nm with 630 nm as
reference wavelength, using a BIO-TEK ELx808 Absorbance Micro-
plate Reader. DMSO was used as blank and reference drugs as
positive controls. The L. donovani promastigotes drug susceptibility
was calculated as percentage of control (cells incubated without
compound). The 50% inhibitory concentration (Ld IC50) represents
the concentration of compound required to induce a 50% decrease
of parasite growth. It was determined by non-linear regression
analysis processed on doseeresponse curve, using the Table Curve
solution was then shaken at 37 ꢁC for 24 h. 2
mL various concen-
trations of compounds dissolved in DMSO (final concentration less
than 0.5% v/v) was incorporated into the wells of 96-well microtiter
plate. Daunorubicin was used as the reference drug compound.
200 mL DNA-methyl green solution was added to each well and
plate was shaken vigorously (300 rpm) for a few minutes. Absor-
bance was determined at 630 nm, using a BIO-TEK ELx808

26
A. Cohen et al. / European Journal of Medicinal Chemistry 95 (2015) 16e28
Absorbance Microplate Reader. DMSO was used as blank, DNA-
methyl green as negative control and daunorubicin as positive
one. Plate was incubated at room temperature in darkness for 24 h
before a new absorbance measurement. Agents that displace
methyl green from DNA are detected spectrophotometrically by a
decrease in absorbance at 630 nm. The 50% displacement concen-
tration (DC50) represents the concentration of compound required
to induce a 50% displacement of methyl green from DNA.
enhance the infection rate. The treatment was renewed three
hours later, after sporozoites penetration into cells, and at 24 h
post-infection. At 48 h post-infection cultures were fixed 10 min
with cold methanol. Parasite quantification was done by immu-
nofluorescence analysis. Intracellular parasites were stained with
a mouse polyclonal serum raised against Plasmodium heat shock
protein 70 [53], followed by goat anti-mouse Alexa Fluor 488
conjugate (Invitrogen, Molecular Probes) and cell nuclei were
stained with 1
mg/mL of diamidino-phenylindole (DAPI; Invi-
trogen). Fluorescence intensity of DAPI in the different conditions
was quantified on a FlexStation (Molecular devices Ltd., UK) at
4.2.7. In vivo preliminary study
The in vivo antimalarial activity was determined by the classical
four-day suppressive test [30] against P. berghei NK65. Swiss male
l
ex ¼ 350 nm and em ¼ 470 nm. The 50% cytotoxic concentra-
l
tion (CC50) values correspond to the drug concentration at which a
50% reduction of DAPI fluorescence was observed as compared to
that observed in the control cultures (control wells consisted on
cells treated with the drug solvent final concentration, i.e. 0.5%
DMSO). Parasite numbers in the different conditions were counted
under a fluorescence microscope with a 200ꢀꢀ magnification.
IC50 values correspond to the drug concentration at which a 50%
reduction in the number of parasites was observed as compared to
the number in the control wells.
mice (Janvier, France), of a mean body weight 20
2 g, were
infected with 107 parasitized cells in 0.9% saline, on day 0.
Groups of 5 mice were treated by intra peritoneal route from day
0 to day 3 with 2.5 mg/kg twice a day of tested compound. Chlo-
roquine was used as control at 2.5 mg/kg twice a day. The sup-
pressive effects was estimated on day 4, examining Giemsa-stained
thin blood smears made from the tail of the treated mice and
compared with a control group of mice treated with saline. The
stained thin blood smears were examined under ꢀ 1000 magnifi-
cation, and the percentage of parasitized red blood cells was
counted on at least 9000 red blood cells observed for each con-
centrations. Percent growth inhibition of the parasite was calcu-
lated by the following formula:
4.2.10. Inhibition of heme polymerization in vitro assay [38]
100
mL of a 4 mM solution of hemozoin, previously dissolved in
0.1 M NaOH, were distributed in 96-well microtiter plates. 50
m
L of
different concentrations of tested compounds at a drug:heme ratio
of between 1:1 to 8:1 were added to triplicate test wells. Hemozoin
polymerization was initiated by adding 0.8 mmol of acetic acid
½ðparasitaemia in control ꢂ parasitaemia with drugÞ ꢀ 100ꢃ=
parasitaemia in control
(50 m
L) at a final pH of 3 and the suspension was incubated at 37 ꢁC
for 24 h to allow complete polymerization. Plates were then
centrifuged at 3300 g for 15 min and the soluble fraction of
unprecipitated material collected (fraction 1). The remaining pellet
All experimental animal procedures respect French legislation
on laboratory animal use and care (Nꢁ2001-464).
was resuspended with 200
hemozoin. Plates were then centrifuged at 3300 g for 15 min. The
DMSO-soluble fraction (fraction 2) was collected and the pellet,
consisting of a pure precipitate of
0.1 M NaOH (fraction 3) for spectroscopic quantification. A 150
aliquot of each fraction was transferred on to a new plate and serial
four-fold dilutions in 0.1 M NaOH were performed. The amount of
hemozoin was determined by measuring the absorbance at 405 nm
using a BIO-TEK ELx808 Absorbance Microplate Reader. A standard
curve for hemozoin dissolved in 0.1 M NaOH was used to calculate
the amount of porphyrin present in each fraction. The 50% inhibi-
tory concentration (hemozoin polymerization IC50) represents the
concentration of compound required to induce a 50% decrease of
hemozoin polymerization.
mL of DMSO to remove unreacted
4.2.8. Stage of action in the erythrocytic life cycle
The K1 P. falciparum strain was synchronized to an 8-h period.
The method consisted of synchronizing young forms with 5% D-
sorbitol [51]. Different concentrations of compound dissolved in
DMSO (final concentration less than 0.5% v/v), negative control
(treated by DMSO) and positive control (chloroquine) were tested
in 24-well plate with culture at 0.8% parasitemia (hematocrit 4%).
Cultures were subjected to 8-h pulses (corresponding to 1/6 of
the erythrocytic cycle time) with products (0.2 K1 IC50; K1 IC50; 5
K1 IC50). After being pulsed, the cultures were washed three
times with culture medium and then returned to normal condi-
tions until the next cycle [33]. At the end of the experiment (the
ring stage of the next erythrocytic cycle), parasitemia was
determined on a TECAN Infinite F-200 spectrophotometer with
excitation and emission wavelengths at 497 and 520 nm
respectively. The results were expressed as an inhibition per-
centage of parasitic growth.
b-hemozoin, was dissolved in
mL
4.2.11. Depolarization of mitochondrial membrane potential in vitro
assay [39]
Parasitized cells at a concentration of 5.106/mL in RPMI 1640
medium containing 1% fœtal bovine serum were incubated with a
2 nM final concentration of DiOC6 for 20 min at 37 ꢁC. At the end of
the incubation period the suspension was aliquoted into different
4.2.9. Plasmodium liver stages activity assay
HepG2/CD81 cells [52], were cultured in Dulbecco modified
Eagle medium (D-MEM), supplemented with 10% fetal bovine
tubes of 250 mL each. Different concentrations of tested compounds
serum heat inactivated, 2 mM
penicillin and 100 g/mL of streptomycin (all from Invitrogen,
France). Cells were seeded in 96 well plates coated with 5 g/mL
L
-glutamine, 100 units/mL of
were added, and the mixture was incubated for an additional
20 min. At the end of the incubation period, each sample was
subjected to flow cytometric analysis (Gallios, Beckman Coulter).
For each sample 10 000 events were counted at the same flow
cytometric setting. Measurements of fluorescence in infected
erythrocytes in the presence or in absence of dye were carried out
to establish baselines.
m
m
collagen (rat tail type I, BD Bioscience) at a density of 25.103 cells
per well. After 24 h of incubation at 37 ꢁC, cells were treated with
the various concentrations of molecule 1a, 8a or 20a added in the
culture medium and in the same time infected with 5000 spo-
rozoites of P. yoelii yoelii (17X NL strain, obtained from dissection
of infected Anopheles stephensi mosquito salivary glands 14 days
after their infective blood meal). Sporozoite-inoculated culture
plates were centrifuged for 10 min at 900 ꢀ g at 4 ꢁC in order to
4.2.12. P. falciparum DHFR inhibitory activity in vitro assay
The drug sensitivity tests used a recombinant Saccharomyces
cerevisiae culture: the DHFR gene from the budding yeast, S.

A. Cohen et al. / European Journal of Medicinal Chemistry 95 (2015) 16e28
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