Targeting the human malaria parasite Plasmodium falciparum: In vitro identification of a new antiplasmodial hit in 4-phenoxy-2- trichloromethylquinazoline series

Article abstract of DOI:10.1016/j.ejmech.2011.06.021

From the promising results we previously obtained in quinazoline series and to complete the evaluation of the in vitro antiplasmodial activity of original 2-trichloromethylquinazolines, we synthesized new quinazolines possessing a variously substituted phenoxy group at position 4 through a simple and efficient two-step-synthesis approach. The studies of their activity toward the multi-resistant W2 Plasmodium falciparum strain and of their cytotoxicity on the human hepatocyte HepG2 cell line highlighted a hit compound (molecule 7) displaying a W2 IC₅₀ value of 1.1 μM and a HepG2 CC₅₀ value of 50 μM, comparable to chloroquine and doxycycline. Structure-activity- and toxicity relationships indicate that the trichloromethyl group plays a key role in the antiplasmodial activity of such chemical scaffold and also that the phenoxy group substitution as a direct influence on the molecules selectivity. Moreover, molecule 7 displays significant specific activity against the Plasmodium genus in comparison with Toxoplasma and does not show any mutagenic property at the Ames test.

Full text of DOI:10.1016/j.ejmech.2011.06.021

European Journal of Medicinal Chemistry 46 (2011) 4184e4191
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Targeting the human malaria parasite Plasmodium falciparum: In vitro
identification of a new antiplasmodial hit in 4-phenoxy-2-
trichloromethylquinazoline series
,
a
b
a
Caroline Castera-Ducros a, Nadine Azas b , Pierre Verhaeghe , Sébastien Hutter , Philippe Garrigue ,
**
Aurélien Dumètre b, Litaty Mbatchi a, Michèle Laget b, Vincent Remusat a, France Sifredi a, Sylvain Rault c,
Pascal Rathelot a, Patrice Vanelle a
,
*
a Laboratoire de Pharmaco-Chimie Radicalaire, Faculté de Pharmacie, Universités d’Aix-Marseille I, II et III e UMR CNRS 6264, Laboratoire Chimie Provence, 27 Boulevard Jean Moulin,
13385 Marseille cedex 05, France
b Relation Hôte-Parasites, Pharmacologie et Thérapeutique, UMR MD3, Faculté de Pharmacie, Université de la Méditerranée (Aix-Marseille II), 27 Boulevard Jean Moulin,
13385 Marseille cedex 05, France
c Centre d’Etudes et de Recherche sur le Médicament de Normandie (CERMN), UPRES EA 4258, FR CNRS 3038, UFR des Sciences Pharmaceutiques, Université de Caen Basse
Normandie, Boulevard Becquerel, 14032 Caen cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
From the promising results we previously obtained in quinazoline series and to complete the evaluation
of the in vitro antiplasmodial activity of original 2-trichloromethylquinazolines, we synthesized new
quinazolines possessing a variously substituted phenoxy group at position 4 through a simple and
efficient two-step-synthesis approach. The studies of their activity toward the multi-resistant W2 Plas-
modium falciparum strain and of their cytotoxicity on the human hepatocyte HepG2 cell line highlighted
Received 9 March 2011
Received in revised form
11 June 2011
Accepted 16 June 2011
Available online 23 June 2011
a hit compound (molecule 7) displaying a W2 IC50 value of 1.1 mM and a HepG2 CC50 value of 50 mM,
comparable to chloroquine and doxycycline. Structure-activity- and toxicity relationships indicate that
the trichloromethyl group plays a key role in the antiplasmodial activity of such chemical scaffold and
also that the phenoxy group substitution as a direct influence on the molecules selectivity. Moreover,
molecule 7 displays significant specific activity against the Plasmodium genus in comparison with
Toxoplasma and does not show any mutagenic property at the Ames test.
Keywords:
Malaria
Plasmodium falciparum
Phenoxquinazolines
Trichloromethyl group
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
Since more than 40 years, it is well known that the quinazoline
ring can display antiplasmodial properties, as many 2,4-
Plasmodium falciparum is a protozoan parasite belonging to the
Apicomplexa phylum and is the causative agent of the most severe
type of malaria: cerebral malaria. According to the W.H.O., 225
million people were suffering from malaria in 2009 among which
about 800.000 died, especially African children aged less than 5
years [1]. Because of the emergence of multi-drug-resistant
P. falciparum strains, new antimalarial drug-compounds present-
ing original mechanisms of action are needed in order to contribute
to solving such alarming public health problem [2].
diaminoquinazolines were known to inhibit strongly P. falciparum
dihydrofolate reductase (PfD.H.F.R.), an essential enzyme to the
parasite DNA synthesis [3]. Quite recently, new molecules
belonging to the quinazoline series, substituted at position 2, 4 or 6,
were identified as exerting promising antiplasmodial activities
[4e8]. Among them, three different series were issued from our
research team (Fig. 1) specialized on the design and the synthesis of
original molecules with anti-infectious properties [9e11].
In order to carry on with the investigation of the antiplasmodial
potential of new quinazoline derivatives, we focused on the eval-
uation of the influence of the substitution of position 4 of the
quinazoline ring toward this activity by preparing a new series of 2-
trichloromethylquinazolines bearing a phenoxy group at position 4.
Then, their in vitro antiplasmodial properties on the W2 multi-
resistant P. falciparum strain were evaluated. In parallel, a cytotox-
icity study was conducted on the human HepG2 cell line, in order to
* Corresponding author. Tel.: þ33 491 835 528.
** Corresponding author. Tel.: þ33 491 835 564.
E-mail addresses: nadine.azas@univmed.fr (N. Azas), patrice.vanelle@univmed.
fr (P. Vanelle).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.06.021

C. Castera-Ducros et al. / European Journal of Medicinal Chemistry 46 (2011) 4184e4191
4185
F
R1
Cl
Cl
H
N
N
CF3
OH
H
O
N
1) NaH 95% (2 eq.)
N
O2N
N
O
S
O
DMSO, 20 min, N2, RT
N
25-80%
CCl3
N
R1
N
Cl
N
CCl3
W2 IC50 = 0.4 µM
Fig. 1. Quinazolines displaying W2 P. falciparum antiplasmodial properties.
N
CCl3
2)
1 eq.
CH3
N
3-24
W2 IC50 = 0.95 µM
W2 IC50 = 2.5 µM
N
CCl3
1
1 eq., 24 h, RT, N2
Scheme 2. Preparation of phenoxyquinazolines 3e24.
assess their selectivity. These two studies permitted us to identify
a new antiplasmodial hit (molecule 7) which biological profile was
finally refined with an antitoxoplasmic evaluation and an Ames
mutagenic test.
2.1.4. Preparation of compound 26
The trifluoromethylquinazoline 26, a close structural analog of
hit compound 7 was synthesized by applying the same protocol as
for molecules 3e24. The reaction between 4-chloro-2-
trifluoromethylquinazoline and 4-chlorophenate, formed as
above, afforded 26 in 78% yield (Scheme 4).
2. Results and discussion
2.1. Chemistry
2.2. Biology
2.1.1. Preparation of substrate 1 and compound 2
2.2.1. Antiplasmodial activity
Key starting material 4-chloro-2-trichloromethylquinazoline 1
was prepared from commercial 2-methyl-4-(3H)quinazolinone by
a microwave-assisted chlorination protocol we previously described
[12]. This substrate reacts easily with sodium hydroxide in DMSO, at
RT, to afford 4-hydroxyquinazoline 2 which exhibits in solution
a double lactam-lactim prototropic tautomerism (Scheme 1).
Molecules were tested in vitro on the W2 multi-resistant strain
of P. falciparum by using the SYBR green I assay with an incubation
time of 72 h. This method appears to be one of the most relevant
methods for the in vitro screening of antiplasmodial candidates
nowadays [13]. Chloroquine and doxycycline were used as positive
controls (Table 1). Despite the presence of a trichloromethyl group
on molecules 1 and 2, none of them displayed any antiplasmodial
activity which indicates that such substituent does not confer
antiplasmodial properties alone. By considering the activities of
molecules 3e5, it appears that the substitution of position 4 of the
quinazoline ring by aryl alcoholate derivatives improves very
2.1.2. Preparation of molecules 3e24
4-Chloroquinazolines and especially molecules such as
compound 1, bearing a strong electro-attracting group at position 2,
display a good reactivity at position 4 toward most of nucleophiles.
Thus, substrate 1 was involved in SNAr reactions with a series of aryl
alcoholates, extemporaneously generated by the action of an excess
of 95% sodium hydride on the corresponding phenols in anhydrous
DMSO, at RT under nitrogen atmosphere, to afford phenox-
yquinazolines 3e24 (Scheme 2). Sodium hydride was chosen as
a base displaying weak nucleophilic properties in order to avoid the
formation of undesirable substitution products. Depending on the
phenol reagent used, reaction yields varied from 25 to 80% (mean
yield ¼ 63%). In order to define a single general operating proce-
dure, the reaction time was fixed to 24 h even if most reactions
were much faster.
significantly the IC50 values. Among such derivatives,
a- and b-
naphtols were not retained for further investigations as regards of
their pronounced toxic profile. Then, a series of 19 compounds
(6e24) including a substituted phenol ring were prepared and
evaluated. Their antiplasmodial activities were rather good, ranging
from 1.1 to 6
compound, displaying an IC50 value of 1.1
ones of the reference-drugs chloroquine (0.4
(4.8 M). The most active molecules were the ones including
m
M. Among them, molecule 7 appears to be a hit
M, intermediate to the
M) and doxycycline
m
m
m
electro-attracting groups on the phenoxy moiety, apart with ortho-
substituted ones which were always less active. The evaluation of
nitro-substituted molecules 16 and 18 revealed that such functional
group is responsible for a poor solubility of the corresponding
molecules. Compounds bearing electrodonating substituents
(19e24) were either globally less active or toxic. In order to set up
the importance of the trichloromethyl group to the antiplasmodial
activity of this quinazoline scaffold, a methyl and a trifluoromethyl
2.1.3. Preparation of compound 25
Reduction of the trichloromethyl hit molecule 7 was operated in
refluxing acetic acid with a large excess of iron and permitted to
isolate the methylquinazoline 25 in a fast and efficient way
(Scheme 3).
O
NH
O
N
PCl5 6 eq.
POCl3
N
O
CCl3
OH
Cl
N
NaOH 1 eq.
NH
CH3
N
N
83%
DMSO
MW 800 W
15 min
N
CCl3
CCl3
RT, 12 h
2
1
75%
N
N
H
CCl3
Scheme 1. Preparation of compounds 1 and 2 from 2-methyl-4-(3H)-quinazolinone.

4186
C. Castera-Ducros et al. / European Journal of Medicinal Chemistry 46 (2011) 4184e4191
Cl
required for performing the in vitro antitoxoplasmic evaluation
Cl
which we applied. In comparison with pyrimethamine used as
a reference-drug, molecule 7 displays a weak antitoxoplasmic
activity which appears in favor of its selective antiplasmodial
profile. Moreover, in addition to the HepG2 cell line, molecule 7
remains non toxic on the HFF cell line. Finally, to insure the in vitro
toxicological evaluation of hit compound 7, its mutagenic potential
was evaluated by using the Ames Test [16]. No significant number
of revertants was noted for molecule 7, in comparison with nega-
tive controls, indicating that this trichloromethylated compound is
not mutagenic in vitro.
O
N
O
N
90%
Fe (15 eq.)
N
N
AcOH
CCl3
CH3
Reflux, 1 h
7
25
Scheme 3. Preparation of methylquinazoline 25.
analog (25 and 26) were prepared and their activities where
compared to the one of hit compound 7. In both cases, anti-
plasmodial properties were not maintained which demonstrates
that the trichloromethyl group, even if not bringing antiplasmodial
activity alone (see molecules 1 and 2), plays a key role in the anti-
infectious activity of such a series.
3. Conclusion
A new series of 4-phenoxy-2-trichloromethylquinazolines was
prepared in two steps through a simple and efficient synthesis
pathway. Among these molecules, compound 7 was identified as an
2.2.2. Cytotoxicity and selectivity index
in vitro antiplasmodial hit, displaying an IC50 value of 1.1
W2 multi-resistant strain of P. falciparum and being safe as regards
both of its 50 M CC50 value on the human HepG2 cell line and its
>50 M value on the HFF cell line, in comparison with chloroquine
mM on the
In parallel, all compounds were assessed regarding their in vitro
cytotoxicity so as to be able to determine their selectivity indexes
and validate their real potential as selective antiplasmodial agents
(Table 1). Because of its metabolic activation potential, the HepG2
human cell line is one of the most frequently used cell line for
conducting in vitro toxicological screenings [14]. By using these
cells, we wanted to investigate both the toxic potential of each
molecule and the ones of some of their eventual metabolites.
m
m
and doxycycline chosen as reference-drugs. Moreover, molecule 7
appears rather selective toward the Plasmodium genus, in
comparison with the Toxoplasma one and does not exert in vitro
mutagenic properties. Structureeactivity and etoxicity relation-
ships show that the trichloromethyl group plays a key role in the
antiplasmodial activity which can be significantly improved by
modifying the substitution of the phenoxy moiety, especially with
electro-attracting groups in para or meta positions (except nitro
group because of poor solubility of corresponding nitrated deriva-
tives). The ortho substitution of the phenoxy moiety appears as
unfavorable to the activity while the presence of electrodonating
substituents in position ortho and essentially meta are responsible
for cytotoxicity increase. These medicinal chemistry data support
the idea that some 4-substituted-2-trichloromethylquinazolines
constitute attractive chemical scaffolds for the conception of new
molecular entities with antimalarial potential.
Cytotoxicity concentrations 50% (CC50
) were measured and
compared to the one of doxorubicin, used as positive control.
Among the 26 tested molecules, only 5 appeared as significantly
toxic. As already mentioned above, naphtol-substituted quinazo-
lines 4 and 5 were quite toxic. Among other molecules, 4-phenoxy-
2-trichloromethylquinazolines including a methyl group at posi-
tion meta (20) or a methoxy group at position ortho or meta (23 and
24) were also cytotoxic. As already noted in oxazolidine series [15],
although being nitated, molecules 16 and 17 did not exhibit in vitro
cytotoxicity. Most of compounds bearing a trichloromethyl group at
position 2 of the quinazoline ring did not display cytotoxic prop-
erties, in comparison with both reference drug-compounds and
methyl (25) or trifluoromethyl (26) analogs of molecule 7. Thus,
selectivity indexes (CC50/IC50 ratio) calculated in the whole 4-
phenoxy series are ranging from 0.3 to 45, indicating that the
type of substitution of the phenoxy moiety has a major influence on
the biological profile of the series and can be responsible for its
cytotoxicity.
4. Experimental section
4.1. Chemistry
4.1.1. General
Melting points were determined in open capillary tubes with
a Büchi apparatus and are uncorrected. 1H and 13C NMR spectra
were determined on a Bruker Avance 200 MHz instrument, at the
Faculté de Pharmacie de Marseille. Chemical shifts are given in
2.2.3. Complementary investigations
Then, in order to get more informations about the biological
profile of molecule 7, complementary investigations were carried
out (Table 2). P. falciparum belonging to the Apicomplexa phylum,
we decided to evaluate the in vitro biological activity of hit
compound 7 on an other closely related Apicomplexa protozoan,
Toxoplasma gondii. In parallel, the cytotoxicity of the tested mole-
cules was assessed on the HFF fibroblast cell line which is the one
d
values referenced to the solvent. Elemental analyses were carried
out with a Thermo Finnigan EA 1112 apparatus at the Spectropôle
department of the Faculté des Sciences et Techniques de St Jérôme.
Silica Gel 60 (Merck 70-230) was used for column chromatography.
The progress of the reactions was monitored by thin layer chro-
matography using Kieselgel 60 F254 (Merck) plates.
Cl
4.1.2. Preparation of 4-chloro-2-trichloromethylquinazoline 1 [12]
Compound 1 was prepared as described previously [12] and was
obtained after purification by silica gel column chromatography,
eluting with dichloromethane/petroleum ether (1:1), as a white
OH
1) NaH 95% (2 eq.)
O
N
DMSO, 20 min, N2, RT
N
78%
CF3
Cl
N
2)
1 eq.
solid in 75% yield; mp 127 ꢀC 1H NMR (200 MHz, CDCl3)
d:
Cl
7.82e7.90 (m, 1H), 8.03e8.12 (m, 1H), 8.20e8.24 (m, 1H), 8.33e8.38
26
(m, 1H). 13C NMR (50 MHz, CDCl3)
d
: 95.9 (C), 122.9 (C), 126.0 (CH),
N
CF3
129.7 (CH), 130.6 (CH), 135.9 (CH), 150.2 (C), 159.9 (C), 164.0 (C).
Anal. Calcd for C9H4Cl4N2: C, 38.34; H,1.43; N, 9.94. Found: C, 38.25;
H, 1.14; N, 9.60.
1 eq., 24 h, RT, N2
Scheme 4. Preparation of trifluoromethylquinazoline 26.

C. Castera-Ducros et al. / European Journal of Medicinal Chemistry 46 (2011) 4184e4191
4187
Table 1
Antiplasmodial activity and human cell toxicity of the studied series.
R1
N
N
R2
Entry
R1e
eR2
W2 P. falciparum antiplasmodiala
HepG2 human cell toxicitya
Selectivity indexb
activity IC50
(
m
M)
CC50 (mM)
1[12]
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Ref.
Ref.
Ref.
Cle
HOe
PheOe
1-NaphthyleOe
2-NaphthyleOe
3,4-DieCH3ePheOe
4-ClePheOe
3-ClePheOe
2-ClePheOe
4-FePheOe
eCCl3
eCCl3
eCCl3
eCCl3
eCCl3
eCCl3
eCCl3
eCCl3
eCCl3
eCCl3
eCCl3
eCCl3
eCCl3
eCCl3
eCCl3
eCCl3
eCCl3
eCCl3
eCCl3
eCCl3
eCCl3
eCCl3
eCCl3
eCCl3
eCH3
eCF3
54.5
>50e
3.1
3.9
2.3
3.3
1.1
2.4
4.4
2.4
2.3
5.0
1.8
2.3
3.3
3.5
3.0
4.9
3.2
2.9
5.5
2.9
1.9
6.0
28
>125e
21
50
2.4
3.6
29
>2.3
<0.42
16
0.6
1.6
8.8
45
50
26.1
33.6
29.5
41.7
38.3
29.6
25.1
34.9
10.9
7.6
12.3
18.1
7.7
16.4
10.9
10.6
>4.6
12.1
>1.6
13.4
2.6
4.6
11.8
0.3
1.1
0.9
1.1
75
4.2
e
3-FePheOe
2-FePheOe
4-CF3ePheOe
3-CF3ePheOe
2eCF3e PheOe
4-NO2ePheOe
3-NO2ePheOe
2-NO2ePheOe
4-CH3ePheOe
3-CH3ePheOe
2-CH3ePheOe
4-OCH3ePheOe
3-OCH3ePheOe
2-OCH3ePheOe
4-ClePheOe
4-ClePheOe
Chloroquinec
Doxycyclinec
Doxorubicind
>16f
36.3
>8f
42.8
7.6
25.1
34.1
0.6
6.7
26
72
30
68
0.4
4.8
e
20
0.2
a
Mean of three independent experiments.
b
c
d
e
f
Selectivity index was calculated according to the following formula: SIW2Plasmodium ¼ HepG2 CC50/W2 P. falciparum IC50
Chloroquine and doxycycline were used as antiplasmodial drugecompounds of reference.
Doxorubicin was used as a drugecompound of reference for human cell toxicity.
No significant antiplasmodial activity or toxicity noted at highest concentration tested.
Molecule could not be tested at higher concentration because of poor solubility.
.
4.1.3. Preparation of 4-hydroxy-2-trichloromethylquinazoline 2 [17]
Compound 1 (1 eq.) was dissolved in DMSO. 1 eq. of sodium
hydroxide was then added and the reaction mixture was stirred at
RT for 12 h. Water was then added to the reaction mixture which
was extracted three times with dichloromethane. The organic layer
was washed several times with water, dried over anhydrous Na2SO4
and concentrated in vacuo. Compound 2 was finally isolated, after
purification by silica gel column chromatography, eluting with
dichloromethane, as a white solid in 83% yield; mp 215 ꢀC 1H NMR
in vacuo. The crude product was purified by column chromatog-
raphy on silica gel with an appropriate solvent to afford the cor-
responding nucleophilic aromatic substitution product.
4.1.4.1. 4-Phenoxy-2-trichloromethylquinazoline (3) [18]. Compound
3 was obtained, after purification by column chromatography
(eluent: cyclohexane-ethyl acetate 8:2) as a white solid in 76% yield;
mp 106 ꢀC 1H NMR (200 MHz, CDCl3)
d
: 7.27e7.51 (m, 5H), 7.72e7.80
(m, 1H), 7.94e8.03 (m, 1H), 8.12e8.17 (m, 1H), 8.41e8.45 (m, 1H). 13C
NMR (50 MHz, CDCl3) : 96.7 (C), 115.3 (C), 121.4 (CH*2), 123.6 (CH),
(200 MHz, CDCl3)
d
: 7.58e7.66 (m, 1H), 7.80e7.90 (m, 2H),
d
8.33e8.37 (m, 1H), 11.01 (br s, 1H). 13C NMR (50 MHz, CDCl3)
d
: 92.2
125.8 (CH),128.9 (CH),129.0 (CH),129.4 (CH*2),134.8 (CH),151.0 (C),
152.2 (C), 160.1 (C), 167.2 (C). Anal. Calcd for C15H9Cl3N2O: C, 53.05;
H, 2.67; N, 8.25. Found: C, 53.13; H, 2.69; N, 8.16.
(C), 121.2 (C), 126.8 (CH), 128.9 (CH), 129.0 (CH), 135.4 (CH), 146.6
(C), 149.3 (C), 162.1 (C). Anal. Calcd for C9H5Cl3N2O: C, 41,02; H, 1.91;
N, 10.63. Found: C, 41.12; H, 1.89; N, 10.49.
4.1.4.2. 4-(Naphthalen-1-yloxy)-2-trichloromethylquinazoline
(4). Compound 4 was obtained, after purification by column
chromatography (eluent: petroleum ether-ethyl acetate 9:1) as
a pale beige solid in 60% yield; mp 126 ꢀC 1H NMR (200 MHz,
4.1.4. General procedure for the preparation of compounds 3e24
1 equivalent of the appropriate aryl alcohol was dissolved in
dimethylsulfoxyde (DMSO), then added onto 2 eq. of NaH 95%, and
stirred under N2 for 20 min. 1 eq. of 4-chloro-2-trichloromethyl-
quinazoline was dissolved in DMSO, added to the mixture and
stirred at RT for 24 h under nitrogen atmosphere. The reaction was
stopped by addition of water and the reaction mixture was then
extracted three times with dichloromethane. The organic layer was
washed with water, dried over anhydrous Na2SO4 and concentrated
CDCl3) d: 7.43e7.63 (m, 4H), 7.79e8.08 (m, 5H), 8.18e8.22 (m,
1H), 8.62 (dd, J ¼ 0.7 Hz and 8.2 Hz, 1H). 13C NMR (50 MHz, CDCl3)
d
: 96.6 (C), 115.3 (C), 118.0 (CH), 121.3 (CH), 123.6 (CH), 125.4 (CH),
126.0 (CH), 126.3 (CH), 126.4 (CH), 126.8 (C), 128.1 (CH), 129.1
(CH), 129.2 (CH), 134.8 (C), 134.9 (CH), 148.0 (C), 151.2 (C), 160.3

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C. Castera-Ducros et al. / European Journal of Medicinal Chemistry 46 (2011) 4184e4191
Table 2
Comparison of anti-apicomplexa activities and mutagenicity evaluation of hit compound 7.
Entry
W2 P. falc. antiplasmodial activitya T. gondii antitoxoplasmic activitya HFF fibroblast toxicitya
HepG2 human cell toxicitya
CC50 M)
Ames test
resultb
IC50
(
m
M)
IC50
(
mM)
CC50
(m
M)
(m
Cl
O
N
1.1
8.8
>50
50
Negative
N
CCl3
7
Chloroquinec
Doxycyclinec
0.4
4.8
e
e
e
30
20
7.1
0.2
e
e
e
e
Pyrimethamined
Doxorubicine
1.1
e
>50
0.03
e
e
Positive
a
Mean of three independent experiments.
b
c
A product is considered mutagenic when it induces, at least, a two-fold increase of the number of revertants in comparison with negative control.
Chloroquine and doxycycline were used as antiplasmodial drugecompounds of reference.
Pyrimethamine was used as an antitoxoplasmic drugecompound of reference.
d
e
Doxorubicin was used as a drugecompound of reference for cytotoxicity.
(C), 167.7 (C). Anal. Calcd for C19H11Cl3N2O: C, 58.56; H, 2.85; N,
7.19. Found: C, 59.03; H, 3.02; N, 7.06.
152.5 (C), 160.0 (C), 166.8 (C). Anal. Calcd for C15H8Cl4N2O: C,
48.16; H, 2.16; N, 7.49. Found: C, 48.04; H, 2.22; N, 7.89.
4.1.4.7. 4-(2-Chlorophenoxy)-2-trichloromethylquinazoline (9). Com-
pound 9 was obtained, after purification by column chromatography
(eluent: dichloromethane-petroleum ether 1:1) as a white solid in
4.1.4.3. 4-(Naphthalen-2-yloxy)-2-trichloromethylquinazoline (5).
Compound 5 was obtained, after purification by column chroma-
tography (eluent: petroleum ether-ethyl acetate 9:1) as a pale beige
53% yield; mp 133 ꢀC 1H NMR (200 MHz, CDCl3)
d: 7.23e7.54 (m,
solid in 80% yield; mp 155 ꢀC 1H NMR (200 MHz, CDCl3)
d:
4H), 7.74e7.81 (m, 1H), 7.96e8.04 (m,1H), 8.14e8.18 (m,1H), 8.47 (d,
7.48e7.57 (m, 3H), 7.74e8.05 (m, 6H), 8.16e8.20 (m, 1H), 8.46e8.50
J ¼ 8.1 Hz, 1H). 13C NMR (50 MHz, CDCl3)
d: 96.5 (C), 114.9 (C), 123.7
(m, 1H). 13C NMR (50 MHz, CDCl3)
d
: 96.7 (C), 115.4 (C), 118.2 (CH),
(CH), 123.8 (CH), 126.9 (C), 127.1 (CH), 127.8 (CH), 128.9 (CH), 129.2
(CH), 130.4 (CH), 135.0 (CH), 148.3 (C), 151.1 (C), 159.9 (C), 166.8 (C).
Anal. Calcd for C15H8Cl4N2O: C, 48.16; H, 2.16; N, 7.49. Found: C,
47.83; H, 2.38; N, 8.06.
121.1 (CH), 123.7 (CH), 125.8 (CH), 126.6 (CH), 127.7 (CH), 127.8 (CH),
128.9 (CH), 129.1 (CH), 129.2 (CH), 131.4 (C), 133.8 (C), 134.9 (CH),
149.9 (C), 151.1 (C), 160.1 (C), 167.3 (C). Anal. Calcd for C19H11Cl3N2O:
C, 58.56; H, 2.85; N, 7.19. Found: C, 58.43; H, 3.06; N, 7.11.
4.1.4.8. 4-(4-Fluorophenoxy)-2-trichloromethylquinazoline (10). Com-
pound 10 was obtained, after purification by column chromatog-
raphy (eluent: petroleum ether-dichloromethane 1:1) as a white
4.1.4.4. 4-(3,4-Dimethylphenoxy)-2-trichloromethylquinazoline (6).
Compound 6 was obtained, after purification by column chroma-
tography (eluent: cyclohexane-ethyl acetate 8:2) as a white solid in
solid in 45% yield; mp 128 ꢀC 1H NMR (200 MHz, CDCl3)
d
: 7.54e7.59
(m, 1H), 7.74e7.85 (m, 3H), 7.99e8.08 (m, 1H), 8.17e8.22 (m, 1H),
8.41e8.46 (m, 1H). 13C NMR (50 MHz, CDCl3)
: 96.7 (C), 115.2 (C),
64% yield; mp 104 ꢀC 1H NMR (200 MHz, CDCl3)
d: 2.30 (s, 6H),
7.15e7.17 (m, 3H), 7.70e7.78 (m, 1H), 7.93e8.01 (m, 1H), 8.11e8.15
d
(m, 1H), 8.40 (dd, J ¼ 0.9 Hz and 8.2 Hz, 1H). 13C NMR (50 MHz,
115.8 (CH), 116.3 (CH), 123.0 (d, J ¼ 8.4 Hz, CH*2), 123.6 (CH), 129.0 (d,
J ¼ 8.8 Hz, CH*2), 134.9 (CH), 147.9 (d, J ¼ 3 Hz, C), 151.1 (C), 157.8 (C),
160.1 (C), 164.9 (d, J ¼ 232 Hz, C). Anal. Calcd for C15H8Cl3FN2O: C,
50.38; H, 2.25; N, 7.83. Found: C, 50.74; H, 2.22; N, 8.08.
CDCl3) d: 19.3 (C), 20.0 (C), 96.9 (C), 115.5 (C), 118.4 (CH), 122.2 (CH),
123.8 (CH), 128.9 (CH), 129.0 (CH), 130.2 (CH), 134.0 (C), 134.8 (CH),
137.8 (C), 150.2 (C), 151.0 (C), 160.3 (C), 167.4 (C). Anal. Calcd for
C17H13Cl3N2O: C, 55.54; H, 3.56; N, 7.62. Found: C, 56.00; H, 3.72; N,
7.66.
4.1.4.9. 4-(3-Fluorophenoxy)-2-trichloromethylquinazoline (11). Com-
pound 11 was obtained, after purification by column chromatography
(eluent: petroleum ether-ethyl acetate 8:2) as a white solid in 63%
4.1.4.5. 4-(4-Chlorophenoxy)-2-trichloromethylquinazoline (7). Com-
pound 7 was obtained, after purification by column chromatography
(eluent: dichloromethane-petroleum ether 1:1) as a white solid in
yield; mp 107 ꢀC 1H NMR (200 MHz, CDCl3)
d: 6.99e7.09 (m, 1H),
7.18e7.23 (m, 2H), 7.38e7.49 (m, 1H), 7.74e7.81 (m, 1H), 7.97e8.05
74% yield; mp 167 ꢀC 1H NMR (200 MHz, CDCl3)
d
: 7.32e7.46 (m, 4H),
7.73e7.81 (m, 1H), 7.95e8.04 (m, 1H), 8.13e8.17 (m, 1H), 8.38e8.42
(m, 1H). 13C NMR (50 MHz, CDCl3)
: 96.6 (C), 115.2 (C), 122.9 (CH*2),
(m, 1H), 8.14e8.19 (m, 1H), 8.41 (dd, J ¼ 0.6 and 8.2 Hz, 1H). 13C NMR
(50 MHz, CDCl3)
d
: 96.6 (C),109.7 (d, J ¼ 25 Hz, CH),112.9 (d, J ¼ 21 Hz,
d
CH), 115.2 (C), 117.3 (d, J ¼ 3 Hz, CH), 123.5 (CH), 129.0 (CH), 129.2
(CH), 130.2 (d, J ¼ 10 Hz, CH), 135.0 (CH), 151.1 (C), 152.9 (d, J ¼ 11 Hz,
C), 160.0 (C), 162.9 (d, J ¼ 247 Hz, C), 166.8 (C). Anal. Calcd for
C15H8Cl3FN2O: C, 50.38; H, 2.25; N, 7.83. Found: C, 50.73; H, 2.33; N,
7.74.
123.6 (CH), 129.0 (CH), 129.2 (CH), 129.5 (CH*2),131.2 (C),135.0 (CH),
150.6 (C), 151.1 (C), 160.0 (C), 167.0 (C). Anal. Calcd for C15H8Cl4N2O:
C, 48.16; H, 2.16; N, 7.49. Found: C, 48.46; H, 2.23; N, 7.49.
4.1.4.6. 4-(3-Chlorophenoxy)-2-trichloromethylquinazoline
(8)
[18]. Compound 8 was obtained, after purification by column
4.1.4.10. 4-(2-Fluorophenoxy)-2-trichloromethylquinazoline (12).
Compound 12 was obtained, after purification by column chro-
matography (eluent: dichloromethane-petroleum ether 1:1) as
chromatography (eluent: petroleum ether-ethyl acetate 8:2) as
a white solid in 80% yield; mp 109 ꢀC 1H NMR (200 MHz, CDCl3)
d:
a white solid in 53% yield; mp 116 ꢀC 1H NMR (200 MHz, CDCl3)
d:
7.27e7.48 (m, 4H), 7.74e7.82 (m, 1H), 7.97e8.05 (m, 1H),
8.14e8.19 (m, 1H), 8.38e8.42 (m, 1H). 13C NMR (50 MHz, CDCl3)
d
:
7.19e7.46 (m, 4H), 7.74e7.82 (m, 1H), 7.97e8.05 (m, 1H),
8.15e8.19 (m, 1H), 8.43e8.47 (m, 1H). 13C NMR (50 MHz, CDCl3)
96.5 (C), 114.8 (C), 116.8 (d, J ¼ 18 Hz, CH), 123.6 (d, J ¼ 4 Hz, CH),
96.6 (C), 115.1 (C), 119.9 (CH), 122.2 (CH), 123.5 (CH), 126.1 (CH),
129.0 (CH), 129.3 (CH), 130.1 (CH), 134.7 (C), 135.1 (CH), 151.1 (C),
d
:

C. Castera-Ducros et al. / European Journal of Medicinal Chemistry 46 (2011) 4184e4191
4189
123.7 (CH), 124.5 (d, J ¼ 4 Hz, CH), 127.2 (d, J ¼ 7 Hz, CH), 128.9
(CH), 129.2 (CH), 135.0 (CH), 139.5 (d, J ¼ 12 Hz, C), 151.1 (C), 154.3
chromatography (eluent: petroleum ether-ethyl acetate 8:2) as
a white solid in 70% yield; mp 174 ꢀC 1H NMR (200 MHz, CDCl3)
7.47e7.56 (m, 2H), 7.75e7.84 (m, 2H), 7.99e8.06 (m, 1H), 8.15e8.25
(m, 2H), 8.43e8.47 (m, 1H). 13C NMR (50 MHz, CDCl3)
: 96.2 (C),
114.7 (C), 123.7 (CH), 125.2 (CH), 126.0 (CH), 126.9 (CH), 129.0 (CH),
129.5 (CH), 134.9 (CH), 135.3 (CH), 141.9 (C), 145.3 (C), 151.2 (C), 159.5
(C), 166.8 (C). Anal. Calcd for C15H8Cl3N3O3: C, 46.84; H, 2.10; N,10.93.
Found: C, 47.24; H, 2.30; N, 10.58.
d:
(d,
J
¼
250 Hz, C), 160.0 (C), 166.7 (C). Anal. Calcd for
C15H8Cl3FN2O: C, 50.38; H, 2.25; N, 7.83. Found: C, 50.88; H, 2.45;
N, 7.77.
d
4.1.4.11. 2-Trichloromethyl-4-[4-(trifluoromethyl)phenoxy]quinazoline
(13). Compound 13 was obtained, after purification by column
chromatography (eluent: petroleum ether-ethyl acetate 8:2) as
a white solid in 31% yield; mp 120 ꢀC 1H NMR (200 MHz, CDCl3)
d:
4.1.4.17. 4-(4-Methylphenoxy)-2-trichloromethylquinazoline (19).
Compound 19 was obtained, after purification by column chro-
matography (eluent: petroleum ether-ethyl acetate 8:2) as a white
7.55e7.61 (m, 2H), 7.74e7.85 (m, 3H), 8.00e8.08 (m, 1H), 8.17e8.21
(m,1H), 8.41e8.46 (m,1H).13C NMR (50 MHz, CDCl3)
d: 96.5 (C),115.1
(C),121.9 (CH*2),123.5 (CH),124.0 (q, J ¼ 272 Hz, C),126.8 (q, J ¼ 4 Hz,
CH*2), 128.1 (q, J ¼ 33 Hz, C),129.1 (CH),129.4 (CH),135.2 (CH), 151.2
(C), 154.6 (C), 159.9 (C), 166.7 (C). Anal. Calcd for C16H8Cl3F3N2O: C,
47.15; H, 1.98; N, 6.87. Found: C, 47.70; H, 2.20; N, 6.55.
solid in 79% yield; mp 138 ꢀC 1H NMR (200 MHz, CDCl3)
d
: 2.42 (s,
3H), 7.22e7.32 (m, 4H), 7.72e7.80 (m, 1H), 7.95e8.03 (m, 1H),
8.12e8.17 (m, 1H), 8.40e8.45 (m, 1H). 13C NMR (50 MHz, CDCl3)
d
:
21.0 (CH3), 96.8 (C), 115.4 (C), 121.1 (CH*2), 123.7 (CH), 128.9 (CH),
129.0 (CH), 129.9 (CH*2), 134.7 (CH), 135.4 (C), 150.0 (C), 151.0 (C),
160.2 (C), 167.4 (C). Anal. Calcd for C16H11Cl3N2O: C, 54.34; H, 3.14;
N, 7.92. Found: C, 54.87; H, 3.36; N, 7.88.
4.1.4.12. 2-Trichloromethyl-4-[3-(trifluoromethyl)phenoxy]quinazoline
(14). Compound 14 was obtained, after purification by column
chromatography (eluent: petroleum ether-ethyl acetate 9:1) as
a white solid in 55% yield; mp 84 ꢀC 1H NMR (200 MHz, CDCl3)
d:
4.1.4.18. 4-(3-Methylphenoxy)-2-trichloromethylquinazoline (20).
Compound 20 was obtained, after purification by column chro-
matography (eluent: petroleum ether-ethyl acetate 8:2) as a white
7.58e7.66 (m, 3H), 7.76e7.85 (m, 2H), 7.99e8.08 (m, 1H), 8.16e8.21
(m, 1H), 8.41e8.46 (m, 1H). 13C NMR (50 MHz, CDCl3)
d: 96.5 (C),115.1
(C), 119.1 (q, J ¼ 4 Hz, CH), 122.6 (q, J ¼ 4 Hz, CH), 123.5 (CH), 124.9
(CH), 123.6 (q, J ¼ 272 Hz, C), 129.1 (CH), 129.4 (CH), 130.0 (CH), 131.9
(q, J ¼ 33 Hz, C), 135.2 (CH), 151.2 (C), 152.1 (C), 159.9 (C), 166.7 (C).
Anal. Calcd for C16H8Cl3F3N2O: C, 47.15; H, 1.98; N, 6.87. Found: C,
47.40; H, 2.01; N, 7.10.
solid in 70% yield; mp 116 ꢀC 1H NMR (200 MHz, CDCl3)
d
: 2.42 (s,
3H), 7.10e7.39 (m, 4H), 7.71e7.80 (m, 1H), 7.94e8.03 (m, 1H),
8.13e8.17 (m, 1H), 8.39e8.44 (m, 1H). 13C NMR (50 MHz, CDCl3)
d
:
21.4 (CH3), 96.8 (C), 115.4 (C), 118.3 (CH), 122.0 (CH), 123.7 (CH),
126.6 (CH), 128.9 (CH), 129.0 (CH), 129.1 (CH), 134.8 (CH), 139.5 (C),
151.0 (C), 152.1 (C), 160.2 (C),167.3 (C). Anal. Calcd for C16H11Cl3N2O:
C, 54.34; H, 3.14; N, 7.92. Found: C, 54.63; H, 3.28; N, 7.68.
4.1.4.13. 2-Trichloromethyl-4-[2-(trifluoromethyl)phenoxy]quinazo-
line (15). Compound 15 was obtained, after purification by column
chromatography (eluent: petroleum ether-ethyl acetate 7:3) as
4.1.4.19. 4-(2-Methylphenoxy)-2-trichloromethylquinazoline (21).
Compound 21 was obtained, after purification by column chro-
matography (eluent: petroleum ether-ethyl acetate 8:2) as a white
a beige solid in 68% yield; mp 116 ꢀC 1H NMR (200 MHz, CDCl3)
d:
7.42e7.46 (m, 1H), 7.67e7.84 (m, 4H), 7.98e8.06 (m, 1H), 8.15e8.20
(m, 1H), 8.42e8.46 (m, 1H). 13C NMR (50 MHz, CDCl3)
d: 96.5 (C),
solid in 65% yield; mp 91 ꢀC 1H NMR (200 MHz, CDCl3)
d
: 2.22 (s,
3H), 7.21e7.34 (m, 4H), 7.73e7.81 (m, 1H), 7.95e8.04 (m, 1H),
8.14e8.18 (m, 1H), 8.43e8.45 (m, 1H). 13C NMR (50 MHz, CDCl3)
115.0 (C), 122.8 (q, J ¼ 31 Hz, C), 123.1 (q, J ¼ 273 Hz, C), 123.7 (CH),
124.3 (CH), 125.9 (CH), 127.0 (q, J ¼ 5 Hz, CH), 128.9 (CH), 129.5 (CH),
132.9 (CH), 135.1 (CH), 149.5 (C), 149.6 (C), 151.2 (C), 159.8 (C), 166.9
(C). Anal. Calcd for C16H8Cl3F3N2O: C, 47.15; H, 1.98; N, 6.87. Found:
C, 47.64; H, 1.94; N, 6.98.
d
:
16.4 (CH3), 96.7 (C), 115.1 (C), 121.7 (CH), 123.7 (CH), 126.1 (CH),
126.9 (CH), 128.9 (CH), 129.0 (CH), 130.4 (C), 131.2 (CH), 134.8 (CH),
150.8 (C),151.0 (C),160.3 (C),167.2 (C). Anal. Calcd for C16H11Cl3N2O:
C, 54.34; H, 3.14; N, 7.92. Found: C, 54.21; H, 3.05; N, 7.70.
4.1.4.14. 4-(4-Nitrophenoxy)-2-trichloromethylquinazoline (16). Com-
pound 16 was obtained, after purification by column chromatog-
raphy (eluent: petroleum ether-dichloromethane 6:4) as a white
4.1.4.20. 4-(4-Methoxyphenoxy)-2-trichloromethylquinazoline (22).
Compound 22 was obtained, after purification by column chro-
matography (eluent: dichloromethane-petroleum ether 1:1) as
solid in 75% yield; mp 158 ꢀC 1H NMR (200 MHz, CDCl3)
d
: 7.59e7.66
(m, 2H), 7.78e7.86 (m, 1H), 8.01e8.09 (m, 1H), 8.18e8.22 (m, 1H),
8.36e8.45 (m, 3H). 13C NMR (50 MHz, CDCl3)
: 97.9 (C), 115.0 (C),
a white solid in 64% yield; mp 129 ꢀC 1H NMR (200 MHz, CDCl3)
d:
d
3.86 (s, 3H), 6.93e7.01 (m, 2H), 7.26e7.34 (m, 2H), 7.70e7.79 (m,
122.4 (CH*2), 123.4 (CH), 125.3 (CH*2), 129.2 (CH), 129.6 (CH), 135.4
(CH), 145.4 (C), 151.3 (C), 156.8 (C), 159.7 (C), 166.4 (C). Anal. Calcd for
C15H8Cl3N3O3: C, 46.84; H, 2.10; N, 10.93. Found: C, 47.35; H, 2.11; N,
11.07.
1H), 7.93e8.02 (m, 1H), 8.12e8.16 (m, 1H), 8.39e8.43 (m, 1H). 13C
NMR (50 MHz, CDCl3) d: 55.6 (CH3), 96.8 (C), 114.3 (CH*2), 115.4 (C),
122.2 (CH*2), 123.7 (CH), 128.9 (CH), 129.0 (CH), 134.7 (CH), 145.7
(C), 151.0 (C), 157.2 (C), 160.2 (C), 167.5 (C). Anal. Calcd for
C16H11Cl3N2O2: C, 51.99; H, 3.00; N, 7.58. Found: C, 52.01; H, 3.09; N,
7.46.
4.1.4.15. 4-(3-Nitrophenoxy)-2-trichloromethylquinazoline (17). Com-
pound 17 was obtained, after purification by column chromatog-
raphy (eluent: petroleum ether-ethyl acetate 8:2) as a white solid in
4.1.4.21. 4-(3-Methoxyphenoxy)-2-trichloromethylquinazoline (23).
Compound 23 was obtained, after purification by column chro-
matography (eluent: dichloromethane-petroleum ether 1:1) as
73% yield; mp 116 ꢀC 1H NMR (200 MHz, CDCl3)
d
: 7.64e7.74 (m, 1H),
7.79e7.87 (m, 2H), 8.01e8.10 (m, 1H), 8.19e8.25 (m, 2H), 8.38e8.47
(m, 2H). 13C NMR (50 MHz, CDCl3)
: 96.4 (C), 114.9 (C), 117.4 (CH),
d
a yellow solid in 25% yield; mp 101 ꢀC 1H NMR (200 MHz, CDCl3)
d:
120.9 (CH), 123.4 (CH), 128.0 (CH), 129.2 (CH), 129.5 (CH), 130.0 (CH),
135.4 (CH), 148.8 (C), 151.3 (C), 152.3 (C), 159.7 (C), 166.5 (C). Anal.
Calcd for C15H8Cl3N3O3: C, 46.84; H, 2.10; N, 10.93. Found: C, 47.23; H,
2.27; N, 10.59.
3.83 (s, 3H), 6.83e6.89 (m, 1H), 6.96e7.05 (m, 2H), 7.32e7.40 (m,
1H), 7.72e7.80 (m, 1H), 7.95e8.03 (m, 1H), 8.13e8.17 (m, 1H),
8.39e8.43 (m, 1H). 13C NMR (50 MHz, CDCl3)
d: 55.5 (CH3), 96.8 (C),
107.2 (CH), 112.0 (CH), 113.5 (CH), 115.4 (C), 123.7 (CH), 128.9 (CH),
129.1 (C), 129.7 (CH), 134.8 (CH), 151.0 (C), 153.1 (C), 160.1 (C), 160.4
(C), 167.4 (C). Anal. Calcd for C16H11Cl3N2O2: C, 51.99; H, 3.00; N,
7.58. Found: C, 52.16; H, 3.07; N, 7.67.
4.1.4.16. 4-(2-Nitrophenoxy)-2-trichloromethylquinazoline (18). Com-
pound 18 was obtained, after purification by column

4190
C. Castera-Ducros et al. / European Journal of Medicinal Chemistry 46 (2011) 4184e4191
4.1.4.22. 4-(2-Methoxyphenoxy)-2-trichloromethylquinazoline (24).
Compound 24 was obtained, after purification by column chro-
matography (eluent: dichloromethane-petroleum ether 1:1) as
parasite in human erythrocytes according to an SYBR Green I
fluorescence-based method [20] using a 96-well fluorescence
plate reader. Parasite culture was synchronized at ring stage with
5% sorbitol. Compounds were incubated in a total assay volume of
a white solid in 71% yield; mp 85 ꢀC 1H NMR (200 MHz, CDCl3)
d:
3.72 (s, 3H), 7.01e7.07 (m, 2H), 7.27e7.32 (m, 2H), 7.73e7.78 (m,
200 ml (RPMI, 2% hematocrit and 1% parasitaemia) for 72 h in
1H), 7.93e7.98 (m, 1H), 8.12e8.16 (m, 1H), 8.43e8.48 (m, 1H). 13C
a humidified atmosphere (5% O2 and 5% CO2) at 37 ꢀC, in 96-well
flat bottom plates. Triplicate assays were performed for each
NMR (50 MHz, CDCl3) d: 55.7 (CH3), 96.8 (C), 112.4 (CH), 115.0 (C),
120.7 (CH), 122.4 (CH), 123.9 (CH), 127.0 (CH), 128.7 (CH), 128.8
(CH), 134.6 (CH), 141.1 (C), 150.9 (C), 151.3 (C), 160.2 (C), 167.3 (C).
Anal. Calcd for C16H11Cl3N2O2: C, 51.99; H, 3.00; N, 7.58. Found: C,
52.54; H, 3.26; N, 7.28.
sample. After incubation, 170
cells were washed with 150
were transferred to 96-well flat bottom non-sterile black plates
(Greiner Bio-one) already containing 15 l of the SYBR Green lysis
m
l supernatant was discarded and
m
l 1ꢁ PBS. 15 l re-suspended cells
m
m
buffer (2XSYBR Green, 20 mM Tris base pH 7.5, 20 mM EDTA,
0.008% w/v saponin, 0.08% w/v Triton X-100). Negative control,
treated by solvents (DMSO or H2O) and positive controls (chlo-
roquine, doxycycline) were added to each set of experiments.
Plates were incubated for 15 min at 37 ꢀC and then read on
a TECAN Infinite F-200 spectrophotometer with excitation and
emission wavelengths at 497 and 520 nm, respectively. The
concentrations of compounds required to induce a 50% decrease
of parasite growth (IC50W2) were calculated from three indepen-
dent experiments.
4.1.5. Preparation of 4-(4-chlorophenoxy)-2-methylquinazoline
(25)
To a refluxing mixture of acetic acid and iron (15 eq.), 1 eq. of 4-
(4-chlorophenoxy)-2-trichloromethylquinazoline (7) was added.
The reaction mixture was then stirred for 1 h and filtered over
celite. Acetic acid was evaporated under reduced pressure, afford-
ing a yellow residue which was dissolved in dichloromethane. The
organic layer was washed with water, dried over anhydrous Na2SO4
and concentrated in vacuo. Compound 25 was obtained, after
purification by silica column chromatography, eluting with
dichloromethane-ethyl acetate (8:2), as a white solid in 90% yield;
4.2.2. Antitoxoplasmic evaluation
The effects of the tested compounds on the growth of T. gondii
tachyzoites (PRU-b-Gal strain) [21] were assessed by a colorimetric
microtiter assay adapted from Mc Fadden et al. [22]. Briefly,
tachyzoites were maintained by serial passage in confluent
monolayer of human foreskin fibroblasts HFF (ATCC, Manassas,
USA). For assay, 96-well microtiter plates were seeded with 3ꢁ104
HFF cells per well and allowed to grow to confluence in RPMI 1640
mp 95 ꢀC 1H NMR (200 MHz, CDCl3)
2H), 7.38e7.46 (m, 2H), 7.54e7.62 (m, 1H), 7.82e7.94 (m, 2H),
8.27e8.31 (m, 1H). 13C NMR (50 MHz, CDCl3)
: 26.2 (CH3), 114.2 (C),
d
: 2.63 (s, 3H), 7.19e7.25 (m,
d
123.3 (CH*2), 123.4 (CH), 126.7 (CH), 127.0 (CH), 129.5 (CH*2), 130.9
(C), 134.1 (CH), 150.9 (C), 151.9 (C), 163.7 (C), 166.2 (C). Anal. Calcd
for C15H11ClN2O: C, 66.55; H, 4.10; N,10.35. Found: C, 66.93; H, 4.34;
N, 10.13.
(without phenol red) supplemented with 10% FCS and 1% L-gluta-
4.1.6. Preparation of 4-(4-chlorophenoxy)-2-trifluoromethylquina-
zoline (26)
mine/penicillin-streptomycin mix at 37 ꢀC with 6% CO2. Cell
monolayers were infected with 1ꢁ104 parasites per well and
incubated for 3 h at 37 ꢀC with 6% CO2. Then, various concentra-
tions of compounds dissolved in DMSO (final concentration less
than 0.5% v/v) were incorporated in triplicate. Appropriate controls
treated by DMSO or the reference-drug pyrimethamine were
added to each set of experiments. Negative control consisted in cell
monolayers incubated without parasite and drug. After a 72 h
incubation period at 37 ꢀC with 6% CO2, cell medium was removed
One eq. of 4-chlorophenol was dissolved in dimethylsulfoxyde
(DMSO), then added onto 2 eq. of NaH 95%, and stirred under N2 for
20 min. 1 eq. of 4-chloro-2-trifluoromethylquinazoline was dis-
solved in DMSO, then added to the mixture, and stirred at RT for
24 h. The organic layer was then extracted three times by
dichloromethane. The organic layer was washed with water five
times, dried over anhydrous Na2SO4 and concentrated in vacuo.
Compound 26 was obtained after purification by silica column
chromatography, eluting with dichloromethane/petroleum ether
(1:1) as a white solid in 78% yield; mp 143 ꢀC 1H NMR (200 MHz,
and 100
(CPRG) solution were added to each well. The plates were incu-
bated at 37 ꢀC with 6% CO2 for 6 h, at which time
-galactosidase
ml of a 1 mM chlorophenol red-b-D-galactopyranoside
b
CDCl3)
(m, 1H), 7.99e8.07 (m, 1H), 8.14e8.18 (m, 1H), 8.41e8.45 (m, 1H).
13C NMR (50 MHz, CDCl3)
122.9 (CH*2); 123.7 (CH); 128.8 (CH); 129.6 (CH); 129.7 (CH*2);
131.5 (C); 135.2 (CH); 150.4 (C); 151.1 (C); 151.6 (q, J ¼ 36.9 Hz, C);
167.5 (C). Anal. Calcd for C15H8ClF3N2O: C, 55.49; H, 2.48; N, 8.63.
Found: C, 55.71; H, 2.50; N, 8.62.
d
: 7.27 (d, J ¼ 8.8 Hz, 2H), 7.45 (d, J ¼ 8.8 Hz, 2H), 7.77e7.85
activity was measured by reading plates at 570 and 630 nm on
a Biotek microtiter plate reader. Blanking was made on the
negative-control wells. The concentration of compounds required
to induce a 50% decrease of parasite growth (IC50) was calculated
by non-linear regression analysis processed on doseeresponse
curves, using the Table Curve software 2D v.5.0. IC50 values
represent the mean value calculated from three independent
experiments.
d
: 116.4 (C); 119.4 (q, J ¼ 275.5 Hz, C);
4.2. Biology
4.2.3. Cytotoxicity evaluation
4.2.1. Antiplasmodial evaluation
The evaluation of the tested molecules cytotoxicity on the
HepG2 (purchased from ATCC, ref HB-8065) and HFF cell lines
was done according to the method of Mosmann [23] with
In this study, a W2 culture-adapted P. falciparum strain resis-
tant to chloroquine, pyrimethamine and proguanil was used in an
in vitro culture. Maintenance in continuous culture was done as
described previously by Trager and Jensen [19]. Cultures were
maintained in fresh Aþ human erythrocytes at 2.5% hematocrit 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 5% O2, 5% CO2, and 90% N2). Para-
sitaemia 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
slight modifications. Briefly, cells in 100
[RPMI supplemented with 10% foetal bovine serum, 1%
mine (200 mM) and penicillin (100 U/mL)/streptomycin (100
m
L of complete medium,
-gluta-
g/
L
m
mL)] were inoculated into each well of 96-well plates and
incubated at 37 ꢀC in a humidified 6% CO2 with 95% air atmo-
sphere. After a 24 h incubation, 100 ml of medium with various
product concentrations was added and the plates were incu-
bated for 72 h. At the end of the treatment and incubation, each
plate-well was microscope-examined for detecting possible

C. Castera-Ducros et al. / European Journal of Medicinal Chemistry 46 (2011) 4184e4191
4191
precipitate formation before the medium was aspirated from the
wells. 10 L of MTT solution (5 mg MTT/mL in PBS) were then
added to each well with 100
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This work was supported by the Centre National de la Recherche
Scientifique (CNRS), the Université de la Méditerranée (Aix-Mar-
seille II) and the Assistance Publique-Hôpitaux de Marseille
(APHM).