4-Thiophenoxy-2-trichloromethyquinazolines display in vitro selective antiplasmodial activity against the human malaria parasite Plasmodium falciparum

Article abstract of DOI:10.1016/j.bmcl.2011.06.113

A series of original quinazolines bearing a 4-thiophenoxy and a 2-trichloromethyl group was synthesized in a convenient and efficient way and was evaluated toward its in vitro antiplasmodial potential. The series revealed global good activity against the

Full text of DOI:10.1016/j.bmcl.2011.06.113

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6003–6006
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
4-Thiophenoxy-2-trichloromethyquinazolines display in vitro selective
antiplasmodial activity against the human malaria parasite
Plasmodium falciparum
Pierre Verhaeghe a, Aurélien Dumètre b, Caroline Castera-Ducros a, Sébastien Hutter b, Michèle Laget b,
Cyril Fersing a, Marion Prieri a, Julien Yzombard a, France Sifredi a, Sylvain Rault c, Pascal Rathelot a,
b,
Patrice Vanelle a, , Nadine Azas
⇑
⇑
a Laboratoire de Pharmacochimie Radicalaire, Faculté de Pharmacie, Universités d’Aix-Marseille I, II et III – 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, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05,
France
c Centre d’Etudes et de Recherche sur le Médicament de Normandie, 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:
A series of original quinazolines bearing a 4-thiophenoxy and a 2-trichloromethyl group was synthesized
in a convenient and efficient way and was evaluated toward its in vitro antiplasmodial potential. The ser-
ies revealed global good activity against the K1-multi-resistant Plasmodium falciparum strain, especially
Received 7 June 2011
Revised 25 June 2011
Accepted 26 June 2011
Available online 2 July 2011
with hit compound 5 (IC50 = 0.9 lM), in comparison with chloroquine and doxycycline chosen as refer-
ence-drugs. Both the in vitro cytotoxicity study which was conducted on the human HepG2 cell line
and the in vitro antitoxoplasmic screening against Toxoplasma gondii indicate that this series presents
an interesting selective antiplasmodial profile. Structure–activity- and toxicity relationships highlight
that the trichloromethyl group plays a key role in the antiplasmodial activity and also show that the mod-
ulation of the thiophenol moiety influences the toxicity/activity ratio.
Keywords:
Plasmodium falciparum
Quinazoline
Trichloromethyl group
HepG2 cell line
Ó 2011 Elsevier Ltd. All rights reserved.
Malaria is an infectious erythrocytopathy caused by protozoan
parasites belonging to the Plasmodium genus. In 2009, according
to the WHO, malaria affected about 250 million people worldwide
and was responsible for the death of almost 900.000 people,
mainly African children aged less than 5 years.1 Plasmodium
falciparum is the causative agent of the most severe type of
malaria: cerebral malaria. Moreover, many P. falciparum strains
have developed increasing resistance to most of marketed antima-
larial drugs leading to a major public health issue. Thus, the Roll
Back Malaria consortium calls for the development of a robust
pipeline of new antiplasmodial molecules displaying original
mechanisms of action.2
trichloromethylquinazolines8 display good in vitro antiplasmodial
activity, we decided to develop new close structural analogues
bearing a thiophenoxy moiety at position 4, for the sake of improv-
ing the antiplasmodial potency and decreasing the toxicity of the
2-trichloromethylquinazoline scaffold.
The 4-thiophenoxy-2-trichloromethylquinazoline series was
obtained from 4-chloro-2-trichloromethyl-quinazoline 1 which
we already prepared previously by using an efficient microwave
assisted method.12 Thiophenolates, obtained by reaction of thi-
ophenols with sodium hydride in DMSO, were reacted with
R1
1) NaH 95% (2 eq.)
In continuation of our research program centred on the design
and synthesis of original molecules with anti-infectious proper-
ties,3–5 we focused on the preparation of new antiplasmodial
compounds in quinazoline series. Effectively, various original
4-substituted-quinazoline derivatives have recently demonstrated
interesting potential as antiplasmodial agents.6–11 Thus, after
identifying that some 4-aryl-,6 4-anilino-,7 and 4-phenoxy-2-
N
DMSO, 20 min, N2, RT
Thiophenol + DMSO
N
CCl3
Cl
2)
2-18
1
N
29-88%
N
CCl3
1 eq., 1 h, RT
⇑
Corresponding authors.
E-mail address: patrice.vanelle@pharmacie.univ-mrs.fr (P. Vanelle).
Scheme 1. Simple preparation of compounds 2–18 by a SNAr reaction.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.113

6004
P. Verhaeghe et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6003–6006
did not present any antiplasmodial activity (IC50 = 55
4-thiophenoxy-2-trichloromethylquinazoline exerts
activity (IC50 = 1.9 M). The antiplasmodial activity was signifi-
cantly improved by introducing an electro-attracting group such
as a fluorine, chlorine or trifluoromethyl group on the thiophenoxy
moiety. More precisely, best activities are achieved with molecules
bearing electron-withdrawing groups at the para position of the
thiophenoxy moiety, which clearly appears by comparing para-
l
M) while
Cl
1) NaH 95% (2 eq.)
SH
Cl
2
a
good
l
DMSO, 20 min, N2, RT
S
N
DMSO
+
Cl
N
2)
R2
N
1 eq.
1 eq.
N
:
R2
chloro-substituted hit compound 5 (IC50 = 0.9
ortho-methyl-substituted compound 12 (IC50 = 7
l
M) with less active
M). Contrary to
R2 = -H
5h, 75 °C
19 50%
20 83%
R2 = -CF3 : 1h, 20 °C
l
the results observed previously in 4-anilino-2-trichloromethylqui-
nazoline series,7 there was no improvement of activity with com-
pound 18, bearing two chlorine substituents at positions ortho
and para, indicating that the 4-thiophenoxyquinazoline chemical
scaffold may differ from the 4-anilino one as regards of biological
profile.
Scheme 2. Preparation of compounds 19 and 20.
substrate 1 in DMSO, under nitrogen atmosphere at RT, to afford
the corresponding target compounds 2–18 (Scheme 1).13
Nontrichloromethylated compounds 1914 and 20, which were
synthesized for structure–activity relationships study purposes,
were prepared by a similar SNAr reaction approach, with slight
modifications, from commercial 4-chloro- or 4-chloro-2-trifluoro-
methyl-quinazoline, as described in Scheme 2.
Concerning cytotoxicity, the whole series was rather nontoxic
(12 < CC50 < 52 lM) on the metabolizing HepG2 cell line, in
comparison with both doxorubicin toxicity positive control and
commercial drug-compounds chloroquine and doxycycline
(Table 1). Thus, selectivity indexes of the series range from 7.3 to
>28, in between the ones of commercial doxycycline and chloro-
quine and are close to those obtained in the previously tested 2-tri-
chloromethylquinazoline series. Because of the presence of a
trichloromethyl group in the structure of hit compound 5, its
toxicological study was completed by performing Ames test.17 As
presented in Table 2, molecule 5 did not show any mutagenic
activity at the tested concentrations.
In order to demonstrate the key-role of the trichloromethyl
group in the activity of hit compound 5, this group was substituted
by a hydrogen atom (molecules 19) or a trifluoromethyl group
(molecule 20). As shown in Table 1, the absence of the trichloro-
methyl group leads to a 5- to >17-fold reduction of antiplasmodial
activity. Such an observation, added to the lack of activity of tri-
chloromethylated compound 1, indicates that the presence of a
CCl3 group at position 2 of the quinazoline ring is necessary for
providing antiplasmodial activity but that this group does not con-
fer activity alone to the quinazoline scaffold when this last does
not bear an appropriate substituent at position 4, such as a thio-
phenoxy group.
Compounds 1–20 were then assessed for their in vitro antiplas-
modial activity against the K1-multi-resistant P. falciparum strain
by using the SYBR Green I fluorescence-based method.15 Their
inhibitory concentrations 50% (IC50) were then determined and
compared with those of chloroquine and doxycycline, chosen as
reference-drugs (Table 1). In parallel, their in vitro cytotoxicity
(CC50) toward the human HepG2 cell line was evaluated,16 in order
to calculate, for each molecule, the corresponding selectivity index
(SI).
As already noted on the W2 P. falciparum strain,6 despite the
presence of a trichloromethyl group at position 2, compound 1
Table 1
Antiplasmodial activity and human cell toxicity in 4-thiophenoxyquinazoline series
Entry R1
R2
Activity
against
Cytotoxicity Selectivity
HepG2 CC50 indexb
K1 Plasmodium
falciparum
(l
M)a
(SI)
IC50 (l
M)a
e
1
2
3
4
5
6
7
8
Cl–
–CCl3 55.0
–CCl3 1.9
–CCl3 2.8
–CCl3 2.2
–CCl3 0.9
–CCl3 2.3
–CCl3 1.9
–CCl3 1.2
–CCl3 2.5
–CCl3 2.2
–CCl3 1.0
–CCl3 7.0
–CCl3 1.9
–CCl3 1.6
–CCl3 3.9
–CCl3 2.0
–CCl3 2.5
>125
35
26
>2.3
18.4
9.3
7.3
>28
12.2
8.2
>25
11.2
8
Finally, hit molecule 5 was tested on an other apicomplexan
parasite, Toxoplasma gondii, in order to assess whether or not such
Ph-S–
2-Cl-Ph-S–
3-Cl-Ph-S–
4-Cl-Ph-S–
2-F-Ph-S–
3-F-Ph-S–
4-F-Ph-S–
2-CF3-Ph-S–
3-CF3-Ph-S–
4-CF3-Ph-S–
2-CH3-Ph-S–
3-CH3-Ph-S–
4-CH3-Ph-S–
2-OCH3-Ph-S–
3-OCH3-Ph-S–
4-OCH3-Ph-S–
molecule displays
a selective action toward the Plasmodium
16
genus.18 As shown in Table 2, in comparison with pyrimethamine
used as an antitoxoplasmic drug of reference, compound 5 does
not affect the in vitro development of the Toxoplasma protozoa
>25e
28
15.5
>30e
28
17.5
13
(IC50 > 31 lM). Then, from this preliminary study, in vitro hit
9
10
11
12
13
14
15
16
17
18
19
20
Ref.
Ref.
Ref.
13
52
7.4
18.4
>19
8.2
6
>3.2
12.1
3.8
ND
60
Table 2
35
Complementary investigations about hit compound 5
>30e
32
Entry
Activity against Toxoplasma
Ames testb
gondii IC50 (l
M)a
12
>8e
18.2
16.7
>15e
30
Cl
2,4-di-Cl-Ph-S– –CCl3 1.5
4-Cl-Ph-S–
–H
–CF3
4.4
>15e
0.5
5.0
–
4-Cl-Ph-S–
S
N
Negative
( S9mix)
>31d
0.8
Chloroquinec
Doxycyclinec
Doxorubicind
5
N
20
0.2
4
–
CCl3
a
Pyrimethaminec
–
Mean of three independent experiments.
Selectivity index was calculated according to the following formula:
SIW2Plasmodium = HepG2 Human cell lineCC50/K1 Plasmodium falciparum IC50
b
a
Mean of three independent experiments.
A product is considered mutagenic when it induces, at least, a twofold increase
.
b
c
Chloroquine and doxycycline were used as antiplasmodial drug-compounds of
of the number of revertants in comparison with negative control.
reference.
c
d
e
Pyrimethamine was used as an antitoxoplasmic reference-drug.
Doxorubicin was used as a drug-compound of reference for human cell toxicity.
Molecule could not be tested at higher concentration because of lack of solu-
d
Molecule 5 could not be tested at higher concentrations because of insufficient
solubility.
bility in the culture medium.

P. Verhaeghe et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6003–6006
6005
dichloromethane-petroleum ether 1:1) as a white solid in 68% yield; mp
187 °C. 1H NMR (200 MHz, CDCl3) d: 7.42–7.46 (m, 2H), 7.58–7.62 (m, 2H),
7.70–7.78 (m, 1H), 7.92–8.01 (m, 1H), 8.12 (d, J = 8.5 Hz, 1H), 8.22 (d, J = 7.6 Hz,
1H). 13C NMR (50 MHz, CDCl3) d: 96.9 (C), 121.9 (C), 123.6 (CH), 124.9 (C),
129.2 (CH), 129.3 (CH Â 2), 129.8 (CH), 134.8 (CH), 136.2 (C), 136.9 (CH Â 2),
147.9 (C), 159.6 (C), 172.6 (C). Anal. Calcd for C15H8Cl4N2S: C, 46.18; H, 2.07; N,
7.18. Found: C, 46.58; H, 2.13; N, 7.15. Compound 6 was obtained, after
purification by column chromatography (eluent: petroleum ether-ethyl
acetate 9:1) as a white solid in 48% yield; mp 139 °C. 1H NMR (200 MHz,
CDCl3) d: 7.20–7.29 (m, 2H), 7.48–7.68 (m, 2H), 7.71–7.79 (m, 1H), 7.93–8.01
(m, 1H), 8.11–8.15 (m, 1H), 8.24–8.28 (m, 1H). 13C NMR (50 MHz, CDCl3) d:
96.8 (C), 113.9 (C, d, J = 18.3 Hz), 116.2 (CH, d, J = 22.7 Hz), 122.0 (C), 123.8
(CH), 124.7 (CH, d, J = 3.6 Hz), 129.1 (CH), 129.8 (CH), 132.6 (CH, d, J = 8.4 Hz),
134.7 (CH), 137.4 (CH), 148.0 (C), 159.6 (C), 163.3 (C, d, J = 250.7 Hz), 171.6 (C).
Anal. Calcd for C15H8Cl3FN2S: C, 48.22; H, 2.16; N, 7.50. Found: C, 48.35; H,
quinazoline 5 appears to be a new promising candidate for contrib-
uting to the identification and development of original antimalarial
pharmacophores.
Acknowledgments
This work was supported by the Centre National de la Recherche
Scientifique (CNRS) and the Université de la Méditerranée
(Aix-Marseille II). The authors thank Vincent Remusat for NMR
spectra recording.
References and notes
2.12; N, 7.48. Compound
7 was obtained, after purification by column
chromatography (eluent: petroleum ether-ethyl acetate 9:1) as a white solid
in 64% yield; mp 130 °C. 1H NMR (200 MHz, CDCl3) d: 7.16–7.25 (m, 1H), 7.42–
7.47 (m, 3H), 7.71–7.79 (m, 1H), 7.93–8.02 (m, 1H), 8.11–8.15 (m, 1H), 8.20–
8.25 (m, 1H). 13C NMR (50 MHz, CDCl3) d: 96.8 (C), 116.9 (CH, d, J = 20.8 Hz),
121.9 (C), 122.7 (CH, d, J = 23.1 Hz), 123.6 (CH), 128.3 (C, d, J = 8.4 Hz), 129.2
(CH), 129.8 (CH), 130.2 (CH, d, J = 8.1 Hz), 131.2 (CH, d, J = 3.3 Hz), 134.8 (CH),
148.0 (C), 159.7 (C), 162.6 (C, d, J = 248.8 Hz), 172.4 (C). Anal. Calcd for
1. World Malaria Report 2010, World Health Organisation. www.who.int/
malaria/world_malaria_report_2010/malaria2010_summary_keypoints_en.
pdf.
2. Global Malaria Action Plan 2008, Roll Back Malaria. www.rbm.who.int/gmap/
gmap.pdf.
3. Delmas, F.; Gasquet, M.; Timon-David, P.; Madadi, N.; Vanelle, P.; Vaille, A.;
Maldonado, J. Eur. J. Med. Chem. 1993, 28, 23.
C15H8Cl3FN2S: C, 48.22; H, 2.16; N, 7.50. Found: C, 48.60; H, 2.22; N, 7.37.
Compound was obtained, after purification by column chromatography
8
4. El-Kashef, H. S.; El-Emary, T. I.; Gasquet, M.; Timond-David, P.; Maldonado, J.;
Vanelle, P. Pharmazie 2000, 55, 572.
5. Upcroft, J. A.; Dunn, L. A.; Wright, J. M.; Benakli, K.; Upcroft, P.; Vanelle, P.
Antimicrob. Agents Chemother. 2006, 50, 344.
6. Verhaeghe, P.; Azas, N.; Gasquet, M.; Hutter, S.; Ducros, C.; Laget, M.; Rault, S.;
Rathelot, P.; Vanelle, P. Bioorg. Med. Chem. Lett. 2008, 18, 396.
7. Verhaeghe, P.; Azas, N.; Hutter, S.; Castera-Ducros, C.; Laget, M.; Dumètre, A.;
Gasquet, M.; Reboul, J.-P.; Rault, S.; Rathelot, P.; Vanelle, P. Bioorg. Med. Chem.
2009, 17, 4313.
(eluent: petroleum ether–ethyl acetate 9:1) as a pale yellow solid in 88% yield;
mp 155 °C. 1H NMR (200 MHz, CDCl3) d: 7.12–7.24 (m, 2H), 7.59–7.78 (m, 3H),
7.93–8.01 (m, 1H), 8.10–8.15 (m, 1H), 8.21–8.26 (m, 1H). 13C NMR (50 MHz,
CDCl3) d: 96.9 (C), 116.4 (CH Â 2, d, J = 22.0 Hz), 121.6 (C, d, J = 3.7 Hz), 121.9
(C), 123.6 (CH), 129.1 (CH), 129.8 (CH), 134.7 (CH), 138.0 (CH Â 2, d, J = 8.8 Hz),
147.9 (C), 159.7 (C), 163.8 (C, d, J = 250.0 Hz), 173.1 (C). Anal. Calcd for
C15H8Cl3FN2S: C, 48.22; H, 2.16; N, 7.50. Found: C, 48.34; H, 2.25; N, 7.23.
Compound was obtained, after purification by column chromatography
9
(eluent: petroleum ether–ethyl acetate 9:1) as a white solid in 31% yield; mp
152 °C. 1H NMR (200 MHz, CDCl3) d: 7.62–7.67 (m, 2H), 7.73–7.90 (m, 3H),
7.93–8.02 (m, 1H), 8.11–8.15 (m, 1H), 8.25–8.29 (m, 1H). 13C NMR (50 MHz,
CDCl3) d: 96.7 (C), 121.8 (C), 123.8 (CH), 125.3 (C), 127.3 (CH, q, J = 5.0 Hz),
129.2 (CH), 129.7 (C, q, J = 273.0 Hz), 129.8 (CH), 130.3 (CH), 132.2 (CH), 134.4
(C, q, J = 30.0 Hz), 134.8 (CH), 140.4 (CH), 147.9 (C), 159.5 (C), 172.8 (C). Anal.
Calcd for C16H8Cl3F3N2S: C, 45.36; H, 1.90; N, 6.61. Found: C, 45.31; H, 2.12; N,
6.65. Compound 10 was obtained, after purification by column
chromatography (eluent: petroleum ether–ethyl acetate 9:1) as a white solid
in 40% yield; mp 139 °C. 1H NMR (200 MHz, CDCl3) d: 7.57–7.65 (m, 1H), 7.73–
7.88 (m, 3H), 7.95–8.03 (m, 2H), 8.12–8.17 (m, 1H), 8.22–8.26 (m, 1H). 13C
NMR (50 MHz, CDCl3) d: 96.7 (C), 121.9 (C), 123.5 (CH), 126.5 (CH, q, J = 4.0 Hz),
127.9 (C), 129.1 (C, q, J = 272.0 Hz), 129.3 (CH), 129.5 (CH), 129.9 (CH), 131.6 (C,
q, J = 33.0 Hz), 132.8 (CH, q, J = 4.0 Hz), 134.9 (CH), 138.7 (CH), 148.0 (C), 159.6
(C), 172.0 (C). Anal. Calcd for C16H8Cl3F3N2S: C, 45.36; H, 1.90; N, 6.61. Found:
C, 45.52; H, 1.89; N, 6.66. Compound 11 was obtained, after purification by
column chromatography (eluent: petroleum ether–ethyl acetate 9:1) as a
white solid in 79% yield; mp 131 °C. 1H NMR (200 MHz, CDCl3) d: 7.71–7.86 (m,
5H), 7.95–8.03 (m, 1H), 8.13–8.17 (m, 1H), 8.22–8.27 (m, 1H). 13C NMR
(50 MHz, CDCl3) d: 96.8 (C), 121.9 (C), 123.6 (CH), 123.9 (C, q, J = 272.0 Hz),
125.8 (CH Â 2, q, J = 3.6 Hz), 129.3 (CH), 129.9 (CH), 131.4 (C), 131.7 (C, q,
J = 33.0 Hz), 134.9 (CH), 135.8 (CH Â 2), 148.0 (C), 159.6 (C), 171.9 (C). Anal.
Calcd for C16H8Cl3F3N2S: C, 45.36; H, 1.90; N, 6.61. Found: C, 45.48; H, 1.96; N,
6.75. Compound 12 was obtained, after purification by column
chromatography (eluent: petroleum ether–ethyl acetate 9:1) as a white solid
in 69% yield; mp 140 °C. 1H NMR (200 MHz, CDCl3) d:2.42 (s, 3H), 7.22–7.30 (m,
1H), 7.39–7.43 (m, 2H), 7.56–7.61 (m, 1H), 7.70–7.78 (m, 1H), 7.91–8.00 (m,
1H), 8.09–8.14 (m, 1H), 8.27–8.31 (m, 1H). 13C NMR (50 MHz, CDCl3) d: 20.7
(CH3), 96.9 (C), 122.1 (C), 123.9 (CH), 126.0 (C), 126.6 (CH), 128.9 (CH), 129.7
(CH), 130.5 (CH), 130.7 (CH), 134.5 (CH), 136.6 (CH), 144.0 (C), 147.9 (C), 159.7
(C), 173.1 (C). Anal. Calcd for C16H11Cl3N2S: C, 51.98; H, 3.00; N, 7.58. Found: C,
51.54; H, 2.97; N, 7.45. Compound 13 was obtained, after purification by
column chromatography (eluent: petroleum ether–ethyl acetate 9:1) as a pale
yellow solid in 31% yield; mp 139 °C. 1H NMR (200 MHz, CDCl3) d: 2.40 (s, 3H),
7.30–7.53 (m, 4H), 7.70–7.77 (m, 1H), 7.92–8.00 (m, 1H), 8.10–8.14 (m, 1H),
8.24–8.28 (m, 1H). 13C NMR (50 MHz, CDCl3) d: 21.3 (CH3), 97.0 (C), 122.0 (C),
123.7 (CH), 126.1 (C), 128.9 (CH), 129.0 (CH), 129.7 (CH), 130.5 (CH), 132.3
(CH), 134.6 (CH), 136.3 (CH), 138.9 (C), 147.9 (C), 159.7 (C), 173.3 (C). Anal.
Calcd for C16H11Cl3N2S: C, 51.98; H, 3.00; N, 7.58. Found: C, 51.46; H, 2.96; N,
7.43. Compound 14 was obtained, after purification by column
chromatography (eluent: petroleum ether–ethyl acetate 9:1) as a white solid
in 30% yield; mp 146 °C. 1H NMR (200 MHz, CDCl3) d: 2.43 (s, 3H), 7.16–7.36
(m, 2H), 7.52–7.57 (m, 2H), 7.73–7.76 (m, 1H), 7.91–7.99 (m, 1H), 8.09–8.14
(m, 1H), 8.23–8.28 (m, 1H). 13C NMR (50 MHz, CDCl3) d: 21.4 (CH3), 97.0 (C),
122.0 (C), 122.9 (C), 123.7 (CH), 128.9 (CH), 129.7 (CH), 129.9 (CH Â 2), 134.6
(CH), 135.5 (CH Â 2), 139.9 (C), 147.9 (C), 159.7 (C), 173.5 (C). Anal. Calcd for
8. Castera-Ducros, C.; Azas, N.; Verhaeghe, P.; Hutter, S.; Garrigue, P.; Dumètre, A.;
Mbatchi, L.; Laget, M.; Remusat, V.; Sifredi, F.; Rault, S.; Rathelot, P.; Vanelle, P.
Eur. J. Med. Chem. in press. doi:10.1016/j.ejmech.2011.06.021.
9. Mishra, A.; Srivastava, K.; Tripathi, R.; Puri, S. K.; Batra, S. Eur. J. Med. Chem.
2009, 44, 4404.
10. Madapa, S.; Tusi, Z.; Mishra, A.; Srivastava, K.; Pandey, S. K.; Tripathi, R.; Puri, S.
K.; Batra, S. Bioorg. Med. Chem. 2009, 17, 222.
11. Kabri, Y.; Azas, N.; Dumètre, A.; Hutter, S.; Laget, M.; Verhaeghe, P.; Gellis, A.;
Vanelle, P. Eur. J. Med. Chem. 2010, 45, 616.
12. Verhaeghe, P.; Rathelot, P.; Gellis, A.; Rault, S.; Vanelle, P. Tetrahedron 2006, 62,
8173.
13. Chemistry; materials, methods and products description: 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 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 chromatography using Kieselgel 60 F254 (Merck)
plates. General procedure for the preparation of compounds 2–18: 1 equiv of
the appropriate thiophenol was dissolved in DMSO, then added onto 2 equiv of
NaH 95%, and stirred under N2 for 20 min. 1 equiv of 4-chloro-2-
trichloromethylquinazoline was dissolved in DMSO, then added to the
mixture, and stirred at room temperature for 1 h. The reaction medium was
then extracted three times by dichloromethane. The organic layer was washed
with water five times, dried over anhydrous Na2SO4 and concentrated in vacuo.
The crude product was purified by column chromatography on silica gel with
appropriate solvent to afford the corresponding nucleophilic aromatic
substitution product. Compound
2 was obtained, after purification by
column chromatography (eluent: cyclohexane-ethyl acetate 8:2) as a white
solid in 65% yield; mp 123 °C. 1H NMR (200 MHz, CDCl3) d: 7.44–7.50 (m, 3H),
7.64–7.77 (m, 3H), 7.91–7.99 (m, 1H), 8.09–8.13 (m, 1H), 8.23–8.27 (m, 1H).
13C NMR (50 MHz, CDCl3) d: 97.0 (C), 121.9 (C), 123.7 (CH), 126.4 (C), 129.0
(CH), 129.1 (CH Â 2), 129.7 (CH), 129.8 (C), 134.6 (CH), 135.7 (CH Â 2), 147.9
(C), 159.7 (C), 173.2 (C). Anal. Calcd for C15H9Cl3N2S: C, 50.65; H, 2.55; N, 7.88.
Found: C, 50.59; H, 2.49; N, 7.69. Compound 3 was obtained, after purification
by column chromatography (eluent: dichloromethane-petroleum ether 1:1) as
a white solid in 75% yield; mp 135 °C. 1H NMR (200 MHz, CDCl3) d: 7.31–7.49
(m, 2H), 7.57–7.61 (m, 1H), 7.69–7.77 (m, 2H), 7.91–7.99 (m, 1H), 8.12 (dd,
J = 0.5 and 8.5 Hz, 1H), 8.25 (dd, J = 0.5 and 8.5 Hz, 1H). 13C NMR (50 MHz,
CDCl3) d: 96.8 (C), 121.9 (C), 123.7 (CH), 126.0 (C), 127.3 (CH), 129.0 (CH), 129.7
(CH), 130.2 (CH), 131.5 (CH), 134.7 (CH), 138.0 (CH), 140.1 (C), 147.9 (C), 159.5
(C), 171.8 (C). Anal. Calcd for C15H8Cl4N2S: C, 46.18; H, 2.07; N, 7.18. Found: C,
46.46; H, 2.05; N, 7.10. Compound 4 was obtained, after purification by column
chromatography (eluent: dichloromethane-petroleum ether 1:1) as a white
solid in 70% yield; mp 147 °C. 1H NMR (200 MHz, CDCl3) d: 7.36–7.48 (m, 2H),
7.56 (dd, J = 1.4 and 7.0 Hz, 1H), 7.71–7.78 (m, 2H), 7.93–8.01 (m, 1H), 8.11–
8.24 (m, 2H). 13C NMR (50 MHz, CDCl3) d: 96.9 (C), 121.9 (C), 123.6 (CH), 128.3
(C), 129.2 (CH), 129.8 (CH), 129.9 (CH), 130.0 (CH), 133.5 (CH), 134.6 (C), 134.9
(CH), 135.6 (CH), 148.0 (C), 159.7 (C), 172.3 (C). Anal. Calcd for C15H8Cl4N2S: C,
C16H11Cl3N2S: C, 51.98; H, 3.00; N, 7.58. Found: C, 51.78; H, 2.98; N, 7.52.
Compound 15 was obtained, after purification by column chromatography
(eluent: petroleum ether–ethyl acetate 9:1) as a white solid in 53% yield; mp
133 °C. 1H NMR (200 MHz, CDCl3) d: 3.78 (s, 3H), 7.00–7.08 (m, 2H), 7.46–7.62
(m, 2H), 7.68–7.76 (m, 1H), 7.90–7.98 (m, 1H), 8.08–8.12 (m, 1H), 8.28–8.32
(m, 1H). 13C NMR (50 MHz, CDCl3) d: 55.8 (OCH3), 97.1 (C), 111.3 (CH), 114.4
(C), 121.0 (CH), 122.2 (C), 124.1 (CH), 128.8 (CH), 129.6 (CH), 132.2 (CH), 134.4
46.18; H, 2.07; N, 7.18. Found: C, 46.52; H, 2.05; N, 7.24. Compound
5
was obtained, after purification by column chromatography (eluent:

6006
P. Verhaeghe et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6003–6006
(CH), 137.3 (CH), 148.0 (C), 159.7 (C), 160.4 (C), 172.8 (C). Anal. Calcd for
16H11Cl3N2OS: C, 49.82; H, 2.87; N, 7.26. Found: C, 49.90; H, 2.95; N, 7.14.
re-suspended cells were transferred to 96-well flat bottom nonsterile black
plates (Greiner Bio-one) already containing 15 L of the SYBR Green lysis
C
l
Compound 16 was obtained, after purification by column chromatography
(eluent: petroleum ether-dichloromethane 1:1) as a white solid in 29% yield;
mp 139 °C. 1H NMR (200 MHz, CDCl3) d: 3.84 (s, 3H), 7.01–7.06 (m, 1H), 7.24–
7.27 (m, 2H), 7.34–7.42 (m, 1H), 7.69–7.77 (m, 1H), 7.92–8.00 (m, 1H), 8.10–
8.14 (m, 1H), 8.23–8.27 (m, 1H). 13C NMR (50 MHz, CDCl3) d: 55.5 (OCH3), 97.0
(C), 116.4 (CH), 120.5 (CH), 122.0 (C), 123.7 (CH), 127.4 (C), 127.6 (CH), 129.0
(CH), 129.7 (CH), 129.8 (CH), 134.7 (CH), 147.9 (C), 159.7 (C), 159.9 (C), 173.1
(C). Anal. Calcd for C16H11Cl3N2OS: C, 49.82; H, 2.87; N, 7.26. Found: C, 50.09;
H, 2.99; N, 7.06. Compound 17 was obtained, after purification by column
chromatography (eluent: petroleum ether–ethyl acetate 9:1) as a white solid
in 69% yield; mp 182 °C. 1H NMR (200 MHz, CDCl3) d: 3.87 (s, 3H), 6.97–7.02
(m, 2H), 7.54–7.58 (m, 2H), 7.68–7.75 (m, 1H), 7.90–7.98 (m, 1H), 8.08–8.13
(m, 1H), 8.23–8.27 (m, 1H). 13C NMR (50 MHz, CDCl3) d: 55.4 (OCH3), 97.0 (C),
114.8 (CH Â 2), 116.8 (C), 121.9 (C), 123.7 (CH), 128.9 (CH), 129.7 (CH), 134.6
(CH), 137.3 (CH Â 2), 147.8 (C), 159.7 (C), 161.0 (C), 173.9 (C). Anal. Calcd for
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 (chloroquine, 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 (IC50 K1)
were calculated from three independent experiments.
16. The evaluation of the tested molecules cytotoxicity was conducted on the
HepG2 (purchased from ATCC, ref HB-8065) and HFF cell lines. Briefly, cells in
100
serum, 1%
(100 g/mL)] were inoculated into each well of 96-well plates and incubated
at 37 °C in humidified 6% CO2 with 95% air atmosphere. After 24 h
incubation, 100 L of medium with various product concentrations was added
l
L of complete medium, [RPMI supplemented with 10% foetal bovine
L-glutamine (200 mM) and penicillin (100 U/mL)/streptomycin
l
a
a
l
C16H11Cl3N2OS: C, 49.82; H, 2.87; N, 7.26. Found: C, 49.84; H, 2.82; N, 6.91.
and the plates were incubated for 72 h. At the end of the treatment and
incubation, each plate-well was microscope-examined for detecting possible
Compound 18 was obtained, after purification by column chromatography
(eluent: petroleum ether–ethyl acetate 9:1) as a beige solid in 35% yield; mp
117 °C. 1H NMR (200 MHz, CDCl3) d: 7.33–7.39 (m, 1H), 7.61–7.69 (m, 2H),
7.72–7.80 (m, 1H), 7.94–8.02 (m, 1H), 8.12–8.16 (m, 1H), 8.22–8.26 (m, 1H).
13C NMR (50 MHz, CDCl3) d: 96.7 (C), 121.9 (C), 123.6 (CH), 124.7 (C), 127.7
(CH), 129.2 (CH), 129.8 (CH), 130.2 (CH), 134.8 (CH), 137.1 (C), 138.6 (CH),
141.0 (C), 148.0 (C), 159.5 (C), 171.3 (C). Anal. Calcd for: C15H7Cl5N2S: C, 42.43;
H, 1.66; N, 6.60. Found: C, 42.70; H, 1.75; N, 6.37. Compound 19 was described
precipitate formation before the medium was aspirated from the wells. 10
of MTT solution (5 mg MTT/mL in PBS) were then added to each well with
100 L of medium without foetal calf serum. Cells were incubated for 2 h at
37 °C to allow MTT oxidation by mitochondrial dehydrogenase in the viable
cells. After this time, the MTT solution was removed and DMSO (100 L) was
lL
l
l
added to dissolve the resulting blue formazan crystals. Plates were shaken
vigorously (300 rpm) for 5 min. The absorbance was measured at 570 nm with
630 nm as reference wavelength with a microplate spectrophotometer. DMSO
was used as blank and doxorubicine as positive control. Cell viability was
calculated as percentage of control (cells incubated without compound). The
50% cytotoxic concentration was determined from the dose–response curve.
17. Compounds were assessed for mutagenicity by a modified version of the liquid
incubation assay of the classical Ames test at five concentrations (25–125 nM).
Salmonella tester strains (TA97a, TA98, TA100 and TA102 with and without
S9mix) were grown overnight in a Nutrient Broth n°2 (Oxoid, France). After this
period, products dissolved in DMSO (Sigma) were added to 0.1 mL of culture
and incubated for 1 h at 37 °C with shaking. Each sample was assayed in
duplicate. After incubation, 2 mL of molten top agar were mixed gently with
the pre-incubated solution and poured onto Vogel-Bonner minimal agar plates.
After 48 h at 37 °C, the number of spontaneous- and drug-induced revertants
per plate was determined for each dose with a bacterial colony counter. A
product was considered mutagenic when it induced a two-fold increase of the
number of revertants, compared with the spontaneous frequency (negative
control). For each Salmonella strain, a specific positive- and solvent-control
were performed with and without S9mix.
previously:13
1 equiv
of
4-chlorothiophenol
was
dissolved
in
dimethylsulfoxyde (DMSO), then added onto 2 equiv of NaH 95%, and stirred
under N2 for 20 minutes 1 equiv of 4-chloroquinazoline was dissolved in
DMSO, then added to the mixture, and stirred at 75 °C for 5 h. The mixture was
poured into an ice-water solution and the white solid which precipitated was
collected by filtration and then purified by column chromatography (silica gel,
eluent: petroleum ether–ethyl acetate 8:2) in 50% yield; mp 139 °C, Lit:13
139 °C. 1H NMR (200 MHz, CDCl3) d: 7.45–7.59 (m, 4H), 7.63–7.72 (m, 1H),
7.88–7.96 (m, 1H), 8.03–8.07 (m, 1H), 8.19–8.23 (m, 1H), 8.87 (s, 1H). 13C NMR
(50 MHz, CDCl3) d: 123.1 (C), 123.7 (CH), 125.3 (C), 127.9 (CH), 128.4 (CH),
129.8 (CH Â 2), 134.4 (CH), 136.4 (C), 137.0 (CH Â 2), 147.6 (C), 153.3 (CH),
171.7 (C). Anal. Calcd for: C14H9ClN2S: C, 61.65; H, 3.33; N, 10.27. Found: C,
61.68; H, 3.31; N, 10.14. Preparation of the compound 20: 1 equiv of 4-
chlorothiophenol was dissolved in DMSO, then added onto 2 equiv of NaH 95%,
and
stirred
under
N2
for
20 min.
1 equiv
of
4-chloro-2-
trifluoromethylquinazoline was dissolved in DMSO, then added to the
mixture, and stirred at 20 °C for 1 h. The mixture was poured into an ice-
water solution and the beige solid which precipitated was collected by
filtration and then purified by column chromatography (silica gel, eluent:
petroleum ether-dichloromethane 1:1) in 83% yield; mp 196 °C. 1H NMR
(200 MHz, CDCl3) d: 7.43–7.89 (m, 4H), 7.71–7.79 (m, 1H), 7.93–8.01 (m, 1H),
8.09–8.13 (m, 1H), 8.21–8.25 (m, 1H). 13C NMR (50 MHz, CDCl3) d: 119.5 (C, q,
J = 276.0 Hz), 123.1 (C), 123.7 (CH), 124.5 (C), 129.5 (CH Â 3), 129.6 (CH), 135.0
(CH), 136.4 (C), 136.7 (CH Â 2), 147.9 (C), 151.3 (C, q, J = 37.0 Hz), 173.0 (C).
Anal. Calcd for C15H8ClF3N2S: C, 52.87; H, 2.37; N, 8.22. Found: C, 53.13; H,
2.45; N, 8.17.
18. The effects of the tested compounds on the growth of Toxoplasma gondii
tachyzoites (PRU-b-Gal strain) were assessed by a colorimetric microtiter
assay. 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 3.104 HFF cells per well
and allowed to grow to confluence in RPMI 1640 (without phenol red)
supplemented with 10% FCS and 1% L-glutamine/penicillin-streptomycin mix
at 37 °C with 6% CO2. Cell monolayers were infected with 1.104 parasites per
well and incubated for 3 h at 37 °C with 6% CO2. Then, various
concentrations 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%
14. Badiger, I. V. V.; Nargund, K. S. J. Karnatak Univ. 1960, 5, 18.
15. In this study, a K1 culture-adapted P. falciparum strain (clone of W2), resistant
to chloroquine, pyrimethamine, and proguanil was used in an in vitro culture.
Cultures were maintained in fresh A+ human erythrocytes at 2.5% haematocrit
in complete medium (RPMI 1640 with 25 mM HEPES, 25 mM NaHCO3, 10% of
A+ human serum) at 37 °C under reduced O2 atmosphere (gas mixture 5% O2,
5% CO2, and 90% 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 a SYBR Green I fluorescence-based method using a
96-well fluorescence plate reader. Parasite culture was synchronised at ring
stage with 5% sorbitol. Compounds were incubated in a total assay volume of
CO2, cell medium was removed and 100 lL of a 1 mM chlorophenol red-b-D-
galactopyranoside (CPRG) solution were added to each well. The plates were
incubated at 37 °C with 6% CO2 for 6 h, at which time b-galactosidase
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
200
l
L (RPMI, 2% haematocrit and 1% parasitaemia) for 72 h in a humidified
parasite growth (IC50 was calculated by nonlinear regression analysis
)
atmosphere (5% O2 and 5% CO2) at 37 °C, in 96-well flat bottom plates.
processed on dose-response curves, using the Table Curve software 2D
v.5.0. IC50 values represent the mean value calculated from three
independent experiments.
Triplicate assays were performed for each sample. After incubation, 170
supernatant was discarded and cells were washed with 150
L 1Â PBS. 15
l
l
L
L
l