Original quinazoline derivatives displaying antiplasmodial properties

Article abstract of DOI:10.1016/j.ejmech.2009.11.005

The multistep synthesis of new quinazoline-derived molecules and their in vitro antiplasmodial evaluation on the W2 chloroquino-resistant Plasmodium falciparum strain is described herein. These molecules have also been studied concerning their in vitro cytotoxicity toward two human cell lines (K652 and HepG2) in order to calculate their respective selectivity indexes (S.I.). Among the fourteen tested molecules, two exhibited both significant antiplasmodial activity (IC₅₀ = 0.95 and 1.3 μM) and low toxicity (IC₅₀ > 100 or 125 μM), compared with two reference drugs: chloroquine and doxycycline. The structure activity relationships establish that the molecular scaffold which exerts the best profile is the 6-nitro-2-(tosylmethyl)-N-(3-substituted-phenyl)-quinazolin-4-amine. The hit molecules were finally investigated regarding their potential action toward two other protozoa, Leishmania donovani and Toxoplasma gondii, showing that these molecules display a selective antiplasmodial activity.

Full text of DOI:10.1016/j.ejmech.2009.11.005

European Journal of Medicinal Chemistry 45 (2010) 616–622
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Original quinazoline derivatives displaying antiplasmodial properties
a
b
b
b
b
´
`
´
`
Youssef Kabri , Nadine Azas , Aurelien Dumetre , Sebastien Hutter , Michele Laget ,
Pierre Verhaeghe a, Armand Gellis a, Patrice Vanelle a
,
*
a Laboratoire de Pharmacochimie Radicalaire, Faculte´ de Pharmacie, Universite´s d’Aix-Marseille I, II et III – UMR CNRS 6264, Laboratoire Chimie Provence, 27 Boulevard Jean Moulin,
13385 Marseille cedex 05, France
b Relations Hoˆte-Parasites, Pharmacologie et The´rapeutique, UMR MD3, Faculte´ de Pharmacie, Universite´ de la Me´diterrane´e (Aix-Marseille II), 27 Boulevard Jean Moulin, 13385
Marseille cedex 05, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The multistep synthesis of new quinazoline-derived molecules and their in vitro antiplasmodial evalu-
ation on the W2 chloroquino-resistant Plasmodium falciparum strain is described herein. These molecules
have also been studied concerning their in vitro cytotoxicity toward two human cell lines (K652 and
HepG2) in order to calculate their respective selectivity indexes (S.I.). Among the fourteen tested
Received 2 September 2009
Received in revised form
23 October 2009
Accepted 2 November 2009
Available online 6 November 2009
molecules, two exhibited both significant antiplasmodial activity (IC50 ¼ 0.95 and 1.3
mM) and low
toxicity (IC50 > 100 or 125 M), compared with two reference drugs: chloroquine and doxycycline. The
m
structure activity relationships establish that the molecular scaffold which exerts the best profile is the
6-nitro-2-(tosylmethyl)-N-(3-substituted-phenyl)-quinazolin-4-amine. The hit molecules were finally
investigated regarding their potential action toward two other protozoa, Leishmania donovani and
Toxoplasma gondii, showing that these molecules display a selective antiplasmodial activity.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
6-Nitroquinazoline ring
Tosylmethyl group
Antiparasitic activity
Plasmodium falciparum
Cytotoxicity
Selectivity index
1. Introduction
Among the different heterocyclic structures which have been
studied for their antiplasmodial properties, quinazoline has quite
Malaria causes 1 million deaths each year worldwide among
about 250 million people suffering from this major parasitic
infection, according to the W.H.O. [1]. Because of the emergence of
parasites which have developed resistances to the most frequently
used drug-compounds, in particular chloroquine, the fight against
malaria, regarding the treatment level of action, requires the
discovery of new efficient molecules with new mechanisms of
action, as recommended by the W.H.O. [2]. Plasmodium falciparum,
the protozoan agent responsible for cerebral malaria, is the most
worrying species, in particular with chloroquino- and multi-resis-
tant strains such as W2. Effectively, its chemosensitivity appears
nowadays as nearly limited to the single artemisin-based combi-
nation therapies. In such a context, medicinal chemists must
investigate new pharmacophores in order to put forward new drug
candidates presenting low costs of production which could signif-
icantly contribute to improve the sanitary situation of many
developing countries.
recently showed increasing interest [3,4]. During the last two years,
our research team has particularly explored the antiplasmodial
potential of the quinazoline ring [5,6], with promising results, in
particular with the 4-anilino-2-trichloromethylquinazoline scaf-
fold (Fig. 1). Taking into account these studies, we chose to search
for new 4-anilinoquinazoline derivatives displaying significant
antiplasmodial properties, considering the possible affinity of such
molecular scaffold for Plasmodium kinases which are parasitic
targets of prime strategic importance for the future development of
new antimalarial drug-compounds [7–9]. In parallel, the cytotox-
icity of these newly synthesized molecules was assessed on two
different complementary human cell lines, in order to exclude the
ones showing unfavourable toxicological profile from further
development.
2. Results and discussion
2.1. Chemistry
2.1.1. Preparation of precursors 2, 4 and 5
From the quinazoline ring, three distinct molecular series were
prepared for structure–activity relationships purposes. The main
* Corresponding author. Tel.: þ33 491 835 528; fax: þ33 491 794 677.
E-mail address: patrice.vanelle@pharmacie.univ-mrs.fr (P. Vanelle).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.11.005

Y. Kabri et al. / European Journal of Medicinal Chemistry 45 (2010) 616–622
617
Consequently, two complementary 4-anilinoquinazoline-
derived series were prepared and tested, aiming at obtaining more
active molecules. The results obtained with series B (4-anilino-2-
(2-methylprop-1-enyl)-6-nitroquinazoline series) indicate a signif-
icant improvement in antiplasmodial activity for compounds on
which the aniline moiety is substituted, in particular when meta
substituted by a trifluoromethyl group (compound 12). The bene-
ficial effect of the meta-trifluoromethylaniline moiety, as a substit-
uent of the quinazoline ring toward antiplasmodial activity, had
already been reported previously [6].
Interestingly, molecules from series C (4-anilino-6-nitro-2-
tosylmethylquinazoline series) are globally much more active than
the previous, especially when the aniline moiety is 3-substituted by
a CF3 group or Cl atom (molecules 16 and 19), permitting to reach
good antiplasmodial IC50 values (respectively 0.95 and 1.3 mM), in
comparison with chloroquine and doxycycline, used as reference
drug-compounds.
Cl
Cl
HN
N
CF3
HN
N
N
CCl3
N
CCl3
W2 IC50 = 1.8 µM
Select. Index (HepG2) = 83
W2 IC50 = 0.4 µM
Select. Index (HepG2) = 40
Fig. 1. 4-anilinoquinazoline derivatives presenting antiplasmodial properties.
preparation goals we wanted to achieve was to reach target
compounds through convenient, fast, and low cost synthesis steps,
taking into account the economical limitations of developing
countries.
Briefly, we started by preparing three different substrates (2, 4
and 5) according to multistep pathways which we quite recently
presented [10,11]. Substrate 2 was prepared in three microwave-
assisted steps from 2-aminobenzamide (Scheme 1).
Substrates 4 and 5 were prepared in good yields through 4
synthesis steps from the same 2-aminobenzamide, as depicted in
Scheme 2.
When comparing the antiplasmodial activities of molecules 11
versus 15, 12 versus 16, and 13 versus 17, it appears that 4-anilino-
quinazoline derivatives bearing a tosylmethyl group on the 2 posi-
tion of the quinazoline ring are about 3 times as active as the ones
which are substituted by a 2-methyl-prop-1-enyl group. More
interesting is the observation that 4-anilinoquinazolines which are
substituted by a nitro group at the 6 position are between 9 and 16
times as active as their non-nitrated analogs (molecules 7 versus 16
and 8 versus 17). Thus, the combination of a nitro group on the 6
position and a tosylmethyl group on the 2 position of a 4-(3-
substituted-anilino)quinazoline gives access to soluble and good
antiplasmodial molecules, such as 16 and 19.
2.1.2. Preparation of target compounds: condensation
of 2, 4 and 5 with anilines
Precursors 2, 4 and 5, belonging to the 4-chloroquinazoline
series, are ideal substrates for nucleophilic aromatic substitution
reactions involving amine reagents. In order to prepare compounds
aiming at inhibiting kinases, these substrates were reacted with
various anilines, following two slightly different operating methods
(Schemes 3 and 4), affording the corresponding 4-anilinoquinazo-
line target compounds in high yields and very short reaction times.
Thus three distinct 4-anilinoquinazoline series were obtained: the
2-tosylmethylquinazoline series (series A), the 2-(2-methylprop-1-
enyl)-6-nitroquinazoline series (series B) and the 6-nitro-2-tosyl-
methylquinazoline series (series C) which were evaluated for their
antiplasmodial properties.
2.3. Complementary investigations
The exploration of newanti-infectious pharmacophores requires
particularly the parallel evaluation of their cytotoxic profile toward
human cells so as to indicate whether or not such structures present
some real further pharmaceutical potential. With such objective, the
same molecules were tested in vitro on two complementary human
cell lines, so as to define their cytotoxic IC50 values and calculate the
corresponding selectivity indexes (Table 2). The first human cell line
was the chronic myeloid leukemia K562, used in suspension, while
the second was the liver carcinoma HepG2, used as a cell cloth, this
latter human cell line allowing the evaluation of the metabolic
activation effect toward cytotoxicity [12].
2.2. Antiplasmodial activity
Three different series were successively synthesized and tested
in vitro on the W2 P. falciparum chloroquino-resistant strain. For
each compound, the inhibitory concentration 50% (IC50) was
determined, as presented in Table 1. Two molecules (6 and 18)
could not be evaluated because of their lack of solubility in the
culture media.
Molecules belonging to the A series (4-anilino-2-tosylme-
thylquinazoline series) appear to be both weakly active (IC50 values
from 13 to 20 mM) and hardly solubilizable. There is almost no effect
Molecules from series A could not be examined properly because
of their low solubility in the culture media. In series B, molecules 11–
14 did not present any significant cellular toxicity, apart molecule 13
on the HepG2 cell line (IC50 ¼ 27
mM). Thetwohitcompounds 16 and
19, from series C, presented elevated IC50 values on the two different
human cell lines, allowing them to reach very good selectivity
indexes (from >77 to >131) in comparisonwith chloroquine (46 and
43) and doxycycline (2.3 and 3.1).
noted when varying the aniline moiety substitution positions, apart
that the para position seems to be less favourable (compound 8).
In order to evaluate the selectivity of action of the hit compounds
among protozoa, molecules 16, 17 and 19 were also tested in vitro
Cl
O
O
O
N
iv
iii
i, ii
N
NH
NH
R1
NH2
NH2
Cl
N
R1
N
1
2
86%
88%
91%
1 = -CH2SO2Ph(pCH3)
R
Scheme 1. Synthesis of substrate 2. (i) Chloroacetyl chloride, MW, 150 W, 50 ꢀC, 5 min; (ii) K2CO3, H2O, MW, 500 W, 80 ꢀC, 1.5 h; (iii) 4-Methylbenzene-sulfonyl chloride, NaHCO3,
Na2SO3, H2O, MW, 300 W, 100 ꢀC, 1 h; (iv) POCl3, diethylaniline, MW, 500 W, 110 ꢀC, 2 h.

618
Y. Kabri et al. / European Journal of Medicinal Chemistry 45 (2010) 616–622
O
N
O
O
N
Cl
N
O2N
O2N
O2N
i
iii
ii
NH
NH
NH
R1
N
Cl
Cl
R1
75%
N
4
3
1
80%
75%
R
1 = -CH=C(CH3)2
iv
O
N
Cl
O2N
O2N
v
NH
R1
N
N
R1
R1 = -CH2SO2Ph(pCH3)
R1 = -CH2SO2Ph(pCH3)
5
93%
74%
Scheme 2. Synthesis of substrates 4 and 5. (i) H2SO4, HNO3, r.t, 4 h; (ii) Lithium 2-nitropropane salt, methanol, MW, 500 W, 70 ꢀC, 2 h; (iii) POCl3, diethylaniline, MW, 500 W, 110 ꢀC,
2 h; (iv) 4-Methylbenzene-sulfonyl chloride, NaHCO3, Na2SO3, H2O, MW, 300 W, 100 ꢀC, 1 h; (v) POCl3, diethylaniline, MW, 500 W, 110 ꢀC, 2 h.
toward Leishmania donovani promastigotes and Toxoplasma gondii,
results being presented in Table 2. It did not appear surprising to us
that these antiplasmodial molecules do not display antileishmanial
properties, in comparison with amphotericin B (drug-compound of
Plasmodium kinases inhibition mechanism of action is under active
investigation.
4. Experimental
reference) as Leishmania,
a kinetoplastid-member protozoa,
diverges significantly from the apicomplexa-member Plasmodium.
Conversely, it was more curious to note that the same molecules
don’t present any antitoxoplasmic properties, in comparison with
pyrimethamine (drug-compound reference), considering that
Toxoplasma also belongs to the apicomplexa group and that several
antimalarial drug-compounds, such as atovaquone, pyrimethamine
and sulfonamides derivatives also display antitoxoplasmic activity
[13–15]. Thus, taking into account these three protozoa, the hit
molecules 16, 17 and 19 present selective antiplasmodial properties.
4.1. Instrumentation and chemicals
Melting points were determined in open capillary tubes with
a Bu¨chi apparatus and are uncorrected. 1H and 13C NMR spectra
were determined on a Bruker Avance 200 MHz instrument, at the
´
Faculte de Pharmacie de Marseille. Chemical shifts are given in
values referenced to the solvent. Elemental analyses were carried
d
ˆ
out with a Thermo Finnigan EA 1112 apparatus at the Spectropole
´
´ ˆ
department of the Faculte des Sciences et Techniques de St Jerome.
Silica gel 60 (Merck 70-230) was used for column chromatography.
The progress of the reactions was monitored by thin layer chro-
matography on using Kieselgel 60 F254 (Merck) plates.
3. Conclusion
Three different series of 4-anilinoquinazoline derivatives were
prepared in high yields and short reaction times through a cheap
and convenient multistep synthesis pathway. These compounds
were evaluated on their in vitro W2 chloroquino-resistant P. falci-
parum antiplasmodial activity, permitting to identify two mole-
cules (16 and 19) with high potential (respective W2 IC50
4.2. Syntheses
4.2.1. Synthesis precursors
4.2.1.1. 2-Chloromethylquinazolin-4(3H)-one (1). Yield 78%. White
solid, mp 249 ꢀC. Literature mp ¼ 247–248 ꢀC [16].
values ¼ 0.95 and 1.3
mM) in comparison with chloroquine and
doxycycline, used as reference drug-compounds. The structure–
activity relationships clearly show that the 6-nitro and the
2-tosylmethyl groups have a great influence on antiplasmodial
activity. In parallel, the same molecules were investigated con-
cerning their in vitro cytotoxicity toward two complementary
human cell lines (K562 and HepG2), in order to determine their
selectivity indexes. Thus, the hit molecules 16 and 19 display
promising selectivity indexes (77–131 range). Finally, to complete
the antiprotozoal evaluation of the best molecules, antileishmanial
and antitoxoplasmic activity determinations were realized and
indicate a selective antiplasmodial activity. Regarding the 4-anili-
noquinazoline scaffold of such molecules, the hypothesis of some
4.2.1.2. 4-Chloro-2-(tosylmethyl)quinazoline (2). Yield 91%. Beige
solid, mp 153 ꢀC [11]
4.2.1.3. 2-Chloromethyl-6-nitroquinazolin-4(3H)-one (3). Yield 80%.
Yellow solid, mp 235 ꢀC [10]
4.2.1.4. 4-Chloro-2-(2-methylprop-1-enyl)-6-nitroquinazoline
(4). Yield 75%. Brown solid, mp 140 ꢀC [11]
4.2.1.5. 4-Chloro-6-nitro-2-(tosylmethyl)quinazoline (5). Yield 74%.
Beige solid, mp 228 ꢀC [11]
R2
CH2Cl2
Cl
HN
CH3
CH3
R1
Isopropanol
R1
N
O
N
O
R2
+
S
S
H2N
N
N
50 °C, 30 min
O
O
2 : R1 = H
Series A : R1 = H, 6-10
Series C : R1 = NO2, 15-19
5 : R1 = NO2
Scheme 3. Synthesis of 4-anilinoquinazoline derivatives belonging to the A and C series.

Y. Kabri et al. / European Journal of Medicinal Chemistry 45 (2010) 616–622
619
R2
HN
N
Cl
N
CH2Cl2
O2N
O2N
Isopropanol
N
CH3
N
CH3
CH3
R2
+
CH3
H2N
R.t, 15 min
4
Series B : 11-14
Scheme 4. Synthesis of 4-anilinoquinazoline derivatives belonging to the B series.
4.2.2. General operating procedure for the synthesis of 2-
(tosylmethyl)quinazolin-4-amine derivatives (6–10): series A
7.26 (d, J ¼ 7.5 Hz, 2H, tosyl 3-H, 5-H), 7.45–7.47 (m, 2H, aromatic
H), 7.60 (d, J ¼ 7.5 Hz, 2H, tosyl 2-H, 6-H), 7.69–7.78 (m, 2H,
aromatic H), 7.87–8.02 (m, 2H, aromatic H), 8.17 (m, 1H, aromatic
H), 8.57 (d, J ¼ 7.9 Hz, 1H, aromatic H), 10.03 (s, 1H, NH). 13C NMR
A
mixture of 4-chloro-2-(tosylmethyl)quinazoline 2 (0.2 g,
0.60 mmol) and corresponding arylamine (1.8 mmol, 3 equiv) in
dichloromethane/propan-2-ol (2:1) was stirred for 30 min at 50 ꢀC.
Water was then added and the mixture was extracted with
dichloromethane. The combined organic layers were washed with
water, dried over anhydrous magnesium sulphate and filtered.
After removal of the dichloromethane under reduced pressure, the
residue was purified by silica gel column chromatography using
dichloromethane/ethyl acetate (9:1), and recrystallized from
propan-2-ol.
(50 MHz, DMSO-d6),
d (ppm): 21.5 (CH3), 65.8 (CH2), 114.0, 118.2,
120.1, 123.5, 125.5, 125.7, 127.5, 128.0, 128.4, 128.5, 129.9, 134.1,
136.9, 140.1, 144.7, 150.2, 155.4, 157.9 (aromatic C). Anal. Calcd. for
C23H18F3N3O2S (457.47): C ¼ 60.39, H ¼ 3.97, N ¼ 9.19; found:
C ¼ 60.12, H ¼ 4.09, N ¼ 8.94.
4.2.2.3. N-(4-Fluorophenyl)-2-(tosylmethyl)quinazolin-4-amine
(8). Yield, 94%. Yellow solid; mp ¼ 203 ꢀC; 1H NMR (200 MHz,
DMSO-d6),
d (ppm): 2.37 (s, 3H, CH3), 4.68 (s, 2H, CH2), 6.89–6.97
4.2.2.1. N-Phenyl-2-(tosylmethyl)quinazolin-4-amine
(6). Yield,
(m, 2H, aromatic H), 7.22 (d, J ¼ 7.9 Hz, 2H, tosyl 3-H, 5-H), 7.51–7.57
91%. Beige solid; mp ¼ 181 ꢀC; 1H NMR (200 MHz, DMSO-d6),
(m, 3H, aromatic H), 7.69–7.79 (m, 5H, aromatic H), 8.00 (bs, 1H,
d
(ppm): 2.31 (s, 3H, CH3), 4.73 (s, 2H, CH2), 7.05–7.13 (m, 1H,
NH); 13C NMR (50 MHz, DMSO-d6),
d (ppm): 21.6 (CH3), 66.3 (CH2),
aromatic H), 7.22–7.35 (m, 5H, aromatic H), 7.57–7.75 (m, 5H,
113.4, 115.1, 115.6, 120.5, 122.9, 123.1, 127.1, 128.5, 128.6, 129.5, 133.2,
133.9,136.7,144.7,150.0,155.1,156.9,157.2 (aromatic C). Anal. Calcd.
for C22H18FN3O2S (407.46): C ¼ 64.85, H ¼ 4.45, N ¼ 10.31; found:
C ¼ 64.71, H ¼ 4.57, N ¼ 10.23.
aromatic H), 7.82–7.90 (m, 1H, aromatic H), 8.55 (d, J ¼ 8.2 Hz, 1H,
aromatic H), 9.75 (s,1H, NH). 13C NMR (50 MHz, DMSO-d6),
d (ppm):
21.7 (CH3), 66.1 (CH2), 114.2, 122.2, 123.6, 124.0, 127.2, 128.2, 128.7,
128.8, 130.1, 133.9, 137.1, 139.4, 144.8, 150.4, 155.8, 158.1 (aromatic
C). Anal. Calcd. for C22H19N3O2S (389.47): C ¼ 67.84, H ¼ 4.92,
N ¼ 10.79; found: C ¼ 67.51, H ¼ 4.88, N ¼ 10.70.
4.2.2.4. N-(3-Bromophenyl)-2-(tosylmethyl)quinazolin-4-amine
(9). Yield, 99%. Yellow solid; mp ¼ 193 ꢀC; 1H NMR (200 MHz,
DMSO-d6), d (ppm): 2.28 (s, 3H, CH3), 4.74 (s, 2H, CH2), 7.16–7.30 (m,
4.2.2.2. 2-(Tosylmethyl)-N-(3-(trifluoromethyl)phenyl)quinazolin-4-
4H, aromatic H), 7.61 (d, J ¼ 8.2 Hz, 2H, tosyl 2-H, 6-H), 7.67–7.77
(m, 3H, aromatic H), 7.85–7.92 (m, 1H, aromatic H), 8.04 (m, 1H,
aromatic H), 8.54 (d, J ¼ 8.3 Hz, 1H, aromatic H), 9.82 (s, 1H, NH);
amine (7). Yield, 80%. Yellow solid; mp ¼ 226 ꢀC; 1H NMR
(200 MHz, DMSO-d6),
d (ppm): 2.26 (s, 3H, CH3), 4.75 (s, 2H, CH2),
Table 1
In vitro antiplasmodial and cytotoxic IC50 values of the tested molecules (6–19).
Molecule/series
R1
R2
R3
W2 P. falciparum IC50
(m
M)a
K562 IC50
(m
M)a
HepG2 IC50
(
m
M)a
Corresponding select. index.b
6/A
7/A
8/A
9/A
(pCH3)Ph-SO2–CH2–
(pCH3)Ph-SO2–CH2–
(pCH3)Ph-SO2–CH2–
(pCH3)Ph-SO2–CH2–
(pCH3)Ph-SO2–CH2–
(CH3)2C]CH–
(CH3)2C]CH–
(CH3)2C]CH–
(CH3)2C]CH–
H–
H–
H–
H–
H–
3-CF3–
4-F–
3-Br–
2-Cl–
H–
3-CF3–
4-F–
3-Br–
H–
3-CF3–
4-F–
3-Br–
3-Cl–
Insufficient solubilityc
15
20
13
14
17
3.2
5.6
7.5
5
>25e
>20e
>25e
>1.7/>1.7
>1/>0.75
>1.2/>1.2
1.7/>3.6
>7/2.9
>39/31.3
>11/4.8
>8/>2
>15e
>15e
>50e
50
>15e
10/A
11/B
12/B
13/B
14/B
15/C
16/C
17/C
18/C
19/C
H–
24
NO2–
NO2–
NO2–
NO2–
NO2–
NO2–
NO2–
NO2–
NO2–
>125d
>125d
>62e
>100d
27
>62e
>15e
>25e
>100d
>50e
(pCH3)Ph-SO2–CH2–
(pCH3)Ph-SO2–CH2–
(pCH3)Ph-SO2–CH2–
(pCH3)Ph-SO2–CH2–
(pCH3)Ph-SO2–CH2–
Chloroquinef
>125d
>125d
>125d
>25/>5
>131/>105
>57/>23
0.95
2.2
Insufficient solubilityc
1.3
0.7
6.5
–
>125d
32
>100d
>96/>77
46/43
2.3/3.1
–
30
20
0.2
Doxycyclinef
15
0.06
Doxorubicing
a
Mean of three independent experiments.
R3
b
c
d
e
f
Selectivity indexes correspond to the ratios: Human cell IC50/W2 Plasmodium IC50
Products 6 and 18 could not be tested because of their lack of solubility in the culture medium.
No significant toxicity at the highest tested concentration.
Highest tested concentration for which molecules did not precipitate in the culture medium.
Chloroquine and doxycycline were used as antimalarial drug-compound references.
Doxorubicin was used as a drug compound of reference for human cell toxicity.
.
HN
R2
N
N
R1
g

620
Y. Kabri et al. / European Journal of Medicinal Chemistry 45 (2010) 616–622
Table 2
Antiparasitic profile of the hit molecules (16, 17, 19) toward 3 protozoa.
Compounds
R3
Plasmodium
falciparum
Toxoplasma
gondii
Leishmania
donovani
IC50
IC50
(
m
M)a
IC50
(
m
M)a
(m
M)a
16
17
19
3-CF3–
4-F–
3-Cl–
0.95
2.2
1.3
0.7
6.5
–
>25b
>25b
>25b
–
–
1.5
–
24
>25b
11
–
Chloroquinec
Doxycyclinec
Pyrimethamined
Amphotericin Be
–
–
0.06
–
a
Mean of three independent experiments.
No significant activity noted at the highest tested concentration.
Chloroquine and doxycycline were used as antimalarial drug-compound references.
Pyrimethamine was used as antitoxoplasmic drug-compound reference.
Amphotericin B was used as antileishmanial drug-compound reference.
R3
b
c
HN
N
CH3
O2N
d
e
N
O
S
O
13C NMR (50 MHz, DMSO-d6),
d
(ppm): 21.2 (CH3), 65.7 (CH2), 113.7,
aromatic H), 7.61–7.68 (m,1H, aromatic H), 7.79 (d, J ¼ 9.2 Hz,1H, 8-
H), 8.15 (d, J ¼ 7.5 Hz, 1H, aromatic H), 8.23 (m, 1H, aromatic H), 8.47
(dd, J ¼ 9.2, 2.3 Hz, 1H, 7-H), 9.54 (d, J ¼ 2.3 Hz, 1H, 5-H), 10.42 (s,
120.4, 121.3, 123.1, 123.9, 126.1, 127.0, 127.9, 128.1, 129.7, 130.3, 133.6,
136.6, 140.7, 144.3, 150.1, 155.1, 157.4 (aromatic C). Anal. Calcd. for
C22H18BrN3O2S (468.37): C ¼ 56.42, H ¼ 3.87, N ¼ 8.97; found:
C ¼ 56.30, H ¼ 3.89, N ¼ 8.98.
1H, NH); 13C NMR (50 MHz, CDCl3),
d (ppm): 20.5 (CH3), 28.1 (CH3),
112.2, 119.1, 120.6, 120.9, 124.2, 125.3, 126.4, 126.8, 129.3, 129.4,
129.8, 139.7, 143.9, 149.0, 154.0, 158.6, 163.7 (aromatic C). Anal.
Calcd. for C19H15F3N4O2 (388.34): C ¼ 58.76, H ¼ 3.89, N ¼ 14.43,
found: C ¼ 58.68, H ¼ 3.83, N ¼ 14.52.
4.2.2.5. N-(2-Chlorophenyl)-2-(tosylmethyl)quinazolin-4-amine
(10). Yield, 63%. Yellow solid; mp ¼ 132 ꢀC; 1H NMR (200 MHz,
DMSO-d6),
d (ppm): 2.30 (s, 3H, CH3), 4.71 (s, 2H, CH2), 7.01–7.08
(m,1H, aromatic H), 7.16–7.19 (m, 3H, aromatic H), 7.38–7.42 (m,1H,
aromatic H), 7.64–7.68 (m, 3H, aromatic H), 7.82–7.96 (m, 3H,
aromatic H), 8.20–8.34 (m, 2H, aromatic H, NH). 13C NMR (50 MHz,
4.2.3.3. N-(4-Fluorophenyl)-2-(2-methylprop-1-enyl)-6-nitro-
quinazolin-4-amine (13). Yield, 86%. Yellow solid; mp ¼ 228 ꢀC; 1H
NMR (200 MHz, CDCl3), d (ppm): 1.93 (s, 3H, CH3), 2.19 (s, 3H, CH3),
DMSO-d6),
d
(ppm): 21.5 (CH3), 66.1 (CH2), 113.7, 120.2, 122.4, 123.4,
6.22 (s, 1H, vinylic H), 7.20–7.29 (m, 2H, 30-H, 50-H), 7.72–7.81 (m,
3H, 8-H, 20-H, 60-H), 8.44 (dd, J ¼ 9.2, 2.3 Hz, 1H, 7-H), 9.50 (d,
J ¼ 2.3 Hz, 1H, 5-H), 10.28 (s, 1H, NH); 13C NMR (50 MHz, CDCl3),
124.2, 127.6, 128.5, 128.7, 128.8, 129.4, 133.4, 134.4, 136.2, 144.5,
149.8, 154.9, 156.6 (aromatic C). Anal. Calcd. for C22H18ClN3O2S
(423.92): C ¼ 62.33, H ¼ 4.28, N ¼ 9.91; found: C ¼ 61.89, H ¼ 4.17,
N ¼ 9.75.
d
(ppm): 20.6 (CH3), 28.1 (CH3), 112.1, 115.0, 115.4, 120.9, 125.3,
125.5, 126.6, 129.1, 135.0, 143.7, 148.8, 154.0, 158.8, 159.0, 163.9
(aromatic C). Anal. Calcd. for C18H15FN4O2 (338.34): C ¼ 63.90,
H ¼ 4.47, N ¼ 16.56; found: C ¼ 63.53, H ¼ 4.57, N ¼ 16.34.
4.2.3. General operating procedure for the synthesis
of 2-(2-methylprop-1-enyl)-6-nitroquinazolin-4-amine
derivatives (11–14): series B
4.2.3.4. N-(3-Bromophenyl)-2-(2-methylprop-1-enyl)-6-nitro-
A
mixture of 4-chloro-2-(2-methylprop-1-enyl)-6-nitro-
quinazolin-4-amine (14). Yield, 83%. Yellow solid; mp ¼ 217 ꢀC; 1H
quinazoline (0.2 g, 0.76 mmol) and adequate arylamine
4
NMR (200 MHz, CDCl3), d (ppm): 1.94 (s, 3H, CH3), 2.25 (s, 3H, CH3),
(2.27 mmol, 3 equiv) in dichloromethane/propan-2-ol (2:1) was
stirred for 15 min at rt. Water was then added and the mixture was
extracted with dichloromethane. The combined organic layers
were washed with water, dried over anhydrous magnesium
sulphate and filtered. After removal of the dichloromethane under
reduced pressure, the residue was purified by silica gel column
chromatography, using dichloromethane/ethyl acetate (95:5), and
recrystallized from propan-2-ol.
6.22 (s, 1H, vinylic H), 7.29–7.39 (m, 2H, 30-H, 50-H), 7.72 (d,
J ¼ 9.2 Hz, 1H, 8-H), 7.77–7.82 (m, 1H, aromatic H), 8.13 (m, 1H,
aromatic H), 8.41 (dd, J ¼ 9.2, 2.4 Hz,1H, 7-H), 9.47 (d, J ¼ 2.4 Hz,1H,
5-H), 10.24 (s, 1H, NH); 13C NMR (50 MHz, CDCl3),
d (ppm): 20.7
(CH3), 28.2 (CH3), 112.1, 120.8, 121.3, 121.4, 125.2, 125.4, 126.6, 126.8,
129.2, 130.4, 140.4, 143.8, 148.9, 153.9, 158.4, 163.7 (aromatic C).
Anal. Calcd. for C18H15BrN4O2 (399.24): C ¼ 54.15, H ¼ 3.79,
N ¼ 14.03; found: C ¼ 54.36, H ¼ 3.96, N ¼ 13.79.
4.2.3.1. 2-(2-Methylprop-1-enyl)-6-nitro-N-phenylquinazolin-
4.2.4. General operating procedure for the synthesis of 6-nitro-2-
(tosylmethyl)quinazolin-4-amine derivatives (15–19): series C
4-amine (11). Yield, 81%. Yellow solid; mp ¼ 205 ꢀC; 1H NMR
(200 MHz, CDCl3),
d
(ppm): 1.99 (s, 3H, CH3), 2.27 (s, 3H, CH3), 6.42
A mixture of 4-chloro-6-nitro-2-(tosylmethyl)quinazoline 5
(s, 1H, vinylic H), 7.18–7.23 (m, 1H, NH), 7.39–7.47 (m, 2H, aromatic
H), 7.73–7.78 (m, 3H, aromatic H), 7.88 (d, J ¼ 9.2 Hz, 1H, 8-H), 8.48
(dd, J ¼ 9.2, 2.3 Hz, 1H, 7-H), 8.88 (d, J ¼ 2.3 Hz, 1H, 5-H); 13C NMR
(0.2 g, 0.53 mmol) and adequate arylamine (1.59 mmol, 3 equiv) in
dichloromethane/propan-2-ol (2:1) was stirred for 30 min at 50 ꢀC.
Water was then added and the mixture was extracted with
dichloromethane. The combined organic layers were washed with
water, dried over anhydrous magnesium sulphate and filtered. After
removal of the dichloromethane under reduced pressure, the residue
was purified by silica gel column chromatography, using dichloro-
methane/ethyl acetate (9:1), and recrystallized from propan-2-ol.
(50 MHz, CDCl3), d (ppm): 20.8 (CH3), 28.3 (CH3), 111.8, 118.1, 122.6,
125.2, 126.4, 129.0, 129.8, 137.6, 143.9, 150.4, 154.3, 158.2, 164.9
(aromatic C); Anal. Calcd. for C18H16N4O2 (320.35): C ¼ 67.49,
H ¼ 5.03, N ¼ 17.49; found: C ¼ 67.30, H ¼ 5.25, N ¼ 17.48.
4.2.3.2. 2-(2-Methylprop-1-enyl)-6-nitro-N-[3(tri-
fluoromethyl)phenyl]quinazolin-4-amine (12). Yield, 87%. Yellow
4.2.4.1. 6-Nitro-N-phenyl-2-(tosylmethyl)-quinazolin-4-amine
solid; mp ¼ 221 ꢀC; 1H NMR (200 MHz, CDCl3),
d
(ppm): 1.95 (s, 3H,
(15). Yield, 86%. Yellow solid; mp ¼ 267 ꢀC; 1H NMR (200 MHz,
CH3), 2.23 (s, 3H, CH3), 6.26 (s, 1H, vinylic H), 7.50 (d, J ¼ 7.5 Hz, 1H,
DMSO-d6), d (ppm): 2.32 (s, 3H, CH3), 4.77 (s, 2H, CH2), 7.11–7.35 (m,

Y. Kabri et al. / European Journal of Medicinal Chemistry 45 (2010) 616–622
621
5H, aromatic H), 7.55–7.66 (m, 4H, aromatic H), 7.86 (d, J ¼ 9.1 Hz,
1H, 8-H), 8.55 (dd, J ¼ 9.1, 2.2 Hz, 1H, 7-H), 9.66 (d, J ¼ 2.2 Hz, 1H, 5-
DMSO. Stock solutions of reference drugs (doxorubicin, pyrimeth-
amine, amphotericin B, chloroquine and doxycycline) were prepared
in ultrapure H2O or DMSO. Flow cytometry was performed at the UFR
de Pharmacie de Marseille, using a FACSort flow cytometer apparatus
(Beckton Dickinson, Paris, France), equipped with an argon laser
(power of 15 mW, and wavelength of 488 nm).
H), 10.38 (s, 1H, NH); 13C NMR (50 MHz, DMSO-d6),
d (ppm): 21.6
(CH3), 66.0 (CH2), 113.4, 121.3, 122.6, 124.8, 127.4, 128.6, 128.8, 129.8,
130.1, 136.9, 138.6, 144.8, 145.3, 153.8, 158.9, 159.3 (aromatic C).
Anal. Calcd. for C22H18N4O4S (434.47): C ¼ 60.82, H ¼ 4.18,
N ¼ 12.90; found: C ¼ 60.84, H ¼ 4.22, N ¼ 12.89.
4.3.2. In vitro antiplasmodial evaluation
4.2.4.2. 6-Nitro-2-(tosylmethyl)-N-(3-(trifluoromethyl)phenyl)quinazo-
In this study, the W2 culture-adapted P. falciparum reference
strain was used. It is resistant to chloroquine, pyrimethamine and
proguanil. Maintenance in continuous culture was done as
described previously by Trager and Jensen [17]. Parasites were
cultivated in 75 cm2-flasks containing RPMI 1640 (20 mL) supple-
mented with 25 mM HEPES, 25 mM NaHCO3, 10% of Aþ human
serum and 1 mL of washed erythrocytes (final haematocrit 2.5%).
Parasitaemia was maintained daily between 1 and 6%. Dilutions
used non-infected Aþ erythrocytes. Cultures were incubated at
37 ꢀC, 10% O2, 6% CO2, 84% N2, with 90% humidity. Cultures were
monitored daily by microscopic examination of blood smears fixed
with methanol and stained with 10% Giemsa stain. Parasite growth
was assessed by flow cytometry according to a methodology
previously described using hydroethidine (HE, Interchim, Mon-
tluçon, France) that is converted by metabolically active parasites
into ethidium [18]. After incubation with hydroethidine, parasitized
and uninfected erythrocytes were all identified on the basis of
fluorescence intensity and size. Triplicate assays were performed in
96-well plates (Nunc Brand products, Fisher, Paris, France) con-
lin-4-amine (16). Yield, 75%. Yellow solid; mp ¼ 273 ꢀC; 1H NMR
(200 MHz, DMSO-d6),
d (ppm): 2.26 (s, 3H, CH3), 4.79 (s, 2H, CH2),
7.25 (d, J ¼ 8.1 Hz, 2H, tosyl 3-H, 5-H), 7.48–7.51 (m, 2H, aromatic
H), 7.60 (d, J ¼ 8.1 Hz, 2H, tosyl 2-H, 6-H), 7.88 (d, J ¼ 9.2 Hz, 1H, 8-
H), 7.99–8.03 (m,1H, aromatic H), 8.12 (m,1H, aromatic H), 8.57 (dd,
J ¼ 9.2, 2.3 Hz, 1H, 7-H), 9.65 (d, J ¼ 2.3 Hz, 1H, 5-H), 10.56 (s, 1H,
NH); 13C NMR (50 MHz, DMSO-d6),
d (ppm): 21.2 (CH3), 65.6 (CH2),
113.1, 118.4, 120.5, 120.9, 123.8, 125.7, 127.3, 128.2, 129.0, 129.6,
129.7, 136.4, 139.2, 144.5, 145.1, 153.4, 158.5, 158.7 (aromatic C).
Anal. Calcd. for C23H17F3N4O4S (502.47): C ¼ 54.98, H ¼ 3.41,
N ¼ 11.15; found: C ¼ 54.98, H ¼ 3.61, N ¼ 10.97.
4.2.4.3. N-(4-Fluorophenyl)-6-nitro-2-(tosylmethyl)quinazolin-
4-amine (17). Yield, 81%. Yellow solid; mp ¼ 270 ꢀC; 1H NMR
(200 MHz, DMSO-d6),
d (ppm): 2.32 (s, 3H, CH3), 4.77 (s, 2H, CH2),
7.08–7.16 (m, 2H, aromatic H), 7.33 (d, J ¼ 8.1 Hz, 2H, aromatic H),
7.56–7.65 (m, 4H, aromatic H), 7.86 (d, J ¼ 9.1 Hz, 1H, 8-H), 8.56 (dd,
J ¼ 9.1, 2.3 Hz, 1H, 7-H), 9.63 (d, J ¼ 2.3 Hz, 1H, 5-H), 10.43 (s, 1H,
NH); 13C NMR (50 MHz, DMSO-d6),
d
(ppm): 21.2 (CH3), 65.6 (CH2),
taining 200
mL of asynchronous parasite cultures at 2% of para-
113.0,114.9,115.3,120.9,124.2,124.3,127.1,128.3,129.5,129.7,134.5,
136.5, 144.5, 144.9, 153.4, 158.5, 158.8, 158.9 (aromatic C). Anal.
Calcd. for C22H17FN4O4S (452.46): C ¼ 58.40, H ¼ 3.79, N ¼ 12.38;
found: C ¼ 58.36, H ¼ 4.07, N ¼ 12.24.
sitaemia and 2% haematocrit, and 5
m
L of the appropriate extract
dissolved in DMSO or ultrapure H2O. Negative control, treated by
solvents (DMSO or H2O) and positive controls (chloroquine) were
added to each set of experiments. After 48 h incubation without
medium change, plates were centrifuged and the upper liquids
4.2.4.4. N-(3-Bromophenyl)-6-nitro-2-(tosylmethyl)quinazolin-
were replaced with 200 mL hydroethidine solution [0.05 mg/mL in
4-amine (18). Yield, 73%. Yellow solid; mp ¼ 265 ꢀC; 1H NMR
phosphate buffered saline (PBS)]. Plates were incubated 20 min in
the dark at 37 ꢀC and washed three times with PBS. Finally, cells
were suspended in 1 mL of PBS to allow determination of the
number of cell events (around 300 per s) and parasitaemia by flow
cytometry using a FACSort flow cytometer. Settings were: Forward
Scatter (FSC-H), size: Voltage E-1, gain 1, mode Log, Side Scatter
(SSC-H), granulosity: Voltage 250, gain 1, mode Log, Fluorescence 2
(FL2), red fluorescence: Voltage 459, gain 1, mode Log. The
concentrations of compounds required to induce a 50% decrease of
infected erythrocytes (IC50W2) were calculated from three inde-
pendent experiments.
(200 MHz, DMSO-d6),
d (ppm): 2.29 (s, 3H, CH3), 4.80 (s, 2H, CH2),
7.20–7.37 (m, 4H, aromatic H), 7.59–7.71 (m, 3H, aromatic H), 7.88
(d, J ¼ 9.1 Hz, 1H, 8-H), 8.00 (m, 1H, aromatic H), 8.56 (dd, J ¼ 9.1,
2.2 Hz, 1H, 7-H), 9.65 (d, J ¼ 2.2 Hz, 1H, 5-H), 10.42 (s, 1H, NH). 13C
NMR (50 MHz, DMSO-d6),
d (ppm): 21.2 (CH3), 65.7 (CH2), 113.1,
120.9, 121.3, 124.4, 126.9, 127.3, 128.2, 129.6, 129.7, 130.4, 136.4,
139.9, 144.5, 145.1, 153.4, 158.4, 158.7 (aromatic C). Anal. Calcd. for
C22H17BrN4O4S (513.36): C ¼ 51.47, H ¼ 3.34, N ¼ 10.91; found:
C ¼ 51.23, H ¼ 3.31, N ¼ 10.79.
4.2.4.5. N-(3-Chlorophenyl)-6-nitro-2-(tosylmethyl)quinazolin-
4-amine (19). Yield, 80%. Yellow solid; mp ¼ 250 ꢀC; 1H NMR
4.3.3. In vitro antileishmanial evaluation
(200 MHz, DMSO-d6),
d
(ppm): 2.28 (s, 3H, CH3), 4.80 (s, 2H, CH2),
The effects of the tested compounds on the growth of L. dono-
vani promastigotes (GFP-transfected strain HOM/IN/01/2001,
kindly provided by Dr. N. Singh, Lucknow, India) were assessed by
flow cytometry as described previously by Singh and Dube [19].
Briefly, promastigotes in log-phase in M199 medium supplemented
7.18–7.35 (m, 4H, aromatic H), 7.59–7.63 (m, 3H, aromatic H), 7.85–
7.90 (m, 2H, aromatic H), 8.56 (dd, J ¼ 9.1, 2.1 Hz, 1H, 7-H), 9.64 (d,
J ¼ 2.1 Hz, 1H, 5-H), 10.43 (s, 1H, NH). 13C NMR (50 MHz, DMSO-d6),
d
(ppm): 21.6 (CH3), 66.0 (CH2), 113.4, 120.8, 121.2, 122.0, 124.4,
127.6, 128.5, 129.9, 130.0, 130.4, 133.2, 136.7, 140.1, 144.8, 145.4,
153.7, 158.8, 159.1 (aromatic C). Anal. Calcd. for C22H17ClN4O4S
(468.91): C ¼ 56.35, H ¼ 3.65, N ¼ 11.95; found: C ¼ 56.29, H ¼ 3.71,
N ¼ 11.78.
with 10% FCS and 500 mg/mL of G418 were incubated at an average
density of 105 parasites/mL in 24-well plates with various
concentrations of compounds dissolved in DMSO (final concen-
tration less than 0.5% v/v) incorporated in duplicate. Amphotericin
B was used as the reference drug. Appropriate controls, treated by
DMSO or amphotericin B, were added to each set of experiments.
After a 72-h incubation period at 27 ꢀC, parasite growth was
determined using a FACSort flow cytometer, equipped with an
argon laser (power of 15 mW, and wavelength of 488 nm). Settings
were: Forward Scatter (FSC-H), size: Voltage E-1, gain 1, mode Lin,
Side Scatter (SSC-H), granulosity: Voltage 489, gain 1, mode Lin,
Fluorescence 1 (FL1), green fluorescence: Voltage 505, gain 1, mode
Log. The concentrations of compounds required to induce a 50%
4.3. Biological evaluation
4.3.1. General
Cell culture medium (RPMI 1640), foetal calf serum (FCS), L-gluta-
mine, non-essential amino acids and other medium additives were
from Eurobio (Paris, France). All other chemicals were of highest
chemical purity and were purchased from Sigma except contrary
mention. Stock solutions of quinazoline derivatives were prepared in

622
Y. Kabri et al. / European Journal of Medicinal Chemistry 45 (2010) 616–622
decrease of parasite growth (IC50) were calculated from three
independent experiments.
After 24 h incubation, 100 ml of medium with various product
concentrations were added 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 precipitate formation
4.3.4. In vitro antitoxoplasmic evaluation
The effects of the tested compounds on the growth of T. gondii
before the medium was aspirated from the wells. 10
mL MTT solu-
tachyzoites (PRU-b-Gal strain, kindly provided by Pr. I. Villena,
tion (5 mg MTT/mL in PBS) were then added to each well with
Reims, France) were assessed by a colorimetric microtiter assay
according to the method of McFadden [20] Briefly, tachyzoites were
maintained by serial passage in confluent human foreskin fibro-
blast (HFF) monolayer (a gift of Pr. I. Dimier-Poisson, Tours, France).
For assay, 96-well microtiter plates were seeded with 104 HFF cells
and allowed to grow to confluence in RPMI 1640 (without phenol
100 ml of medium without foetal calf serum. Cells were incubated
for 2 h at 37 ꢀC to allow MTT oxidation by mitochondrial dehy-
drogenase in the viable cells. After this time, the MTT solution was
aspirated and DMSO (100 mL) was 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 deter-
mined from the dose–response curve.
red) supplemented with 10% FCS and 1% L-glutamine/penicillin–
streptomycin mix at 37 ꢀC with 6% CO2. Cell monolayers were
infected with 2 ꢁ103 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. 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
4.3.7. Selectivity indexes (S.I.)
The selectivity indexes that are presented correspond to the
ratios between, respectively, the toxicity on K562 or HepG2 human
cell lines and the W2 antiplasmodial activity. They are calculated as
follows: S.I. W2 ¼ IC50(K562) or (HepG2)/IC50(W2).
and drug. After a 96-h incubation period at 37 ꢀC with 6% CO2, 20
of chlorophenol red- -galactopyranoside (CPRG) were added to
each well to give a final concentration of 500 M. The plates were
incubated at 37 ꢀC with 6% CO2 for an additional 24 h, at which time
-galactosidase activity was measured by reading plates at 570 and
mL
b-D
m
Acknowledgments
b
630 nm on a Biotek microtiter plate reader. Blanking was made on
the negative-control wells. The concentrations of compounds
required to induce a 50% decrease of parasite growth (IC50) were
calculated from three independent experiments.
´
This work was supported by the CNRS and the Universite de la
´
´
Mediterranee (Aix-Marseille II). The authors would like to thank
Vincent Remusat for recording the NMR spectra, Gilles Lanzada and
Lucile Chiari for their technical assistance.
4.3.5. In vitro K562 cytotoxicity test
Cytotoxicity was assessed on chemosensitive subline K562
derived from a chronic myeloid leukemia and purchased from
Dr J. Boutonnat [21]. Late log-phase K562 cells were incubated in
RPMI 1640 (without phenol red) supplemented with 10% FSC, 2%
References
[1] M. Aregawi, R. Cibulskis, M. Otten, R. Williams, C. Dye, World Malaria Report
2008. World Health Organization, 2008.
[2] Global malaria action plan [online]: http://rbm.who.int/gmap/gmap.pdf,
pp. 85–7.
[3] S. Madapa, Z. Tusi, A. Mishra, K. Srivastava, S.K. Pandey, R. Tripathi, S.K. Puri,
S. Batra, Bioorg. Med. Chem. 17 (2009) 222–234.
[4] A. Mishra, K. Srivastava, R. Tripathi, S.K. Puri, S. Batra, Eur. J. Med. Chem. 11
(2009) 4404–4412.
[5] P. Verhaeghe, N. Azas, M. Gasquet, S. Hutter, C. Ducros, M. Laget, S. Rault,
P. Rathelot, P. Vanelle, Bioorg. Med. Chem. Lett. 18 (2008) 396–401.
[6] P. Verhaeghe, N. Azas, S. Hutter, C. Castera-Ducros, M. Laget, A. Dume`tre,
M. Gasquet, J.-P. Reboul, S. Rault, P. Rathelot, P. Vanelle, Bioorg. Med. Chem. 17
(2009) 4313–4322.
[7] M. Knockaert, P. Greengard, L. Meijer, Trends Pharmacol. Sci. 23 (2002) 417–425.
[8] C. Doerig, L. Meijer, Expert Opin. Ther. Targets 11 (2007) 279–290.
[9] C. Doerig, O. Billker, T. Haystead, P. Sharma, A.B. Tobin, N.C. Waters, Trends
Parasitol. 24 (2008) 570–577.
[10] Y. Kabri, A. Gellis, P. Vanelle, Green Chem. 11 (2009) 201–208.
[11] Y. Kabri, A. Gellis, P. Vanelle, Eur. J. Org. Chem. (2009) 4059–4066.
[12] C. Rodriguez-Antona, M.T. Donato, A. Boobis, R.J. Edwards, P.S. Watts,
J.V. Castell, M.J. Gomez-Lechon, Xenobiotica 32 (2002) 505–520.
L
-glutamine and 1% penicillin–streptomycin mix (complete RPMI
medium) and a range of compound concentrations incorporated
in duplicate (final DMSO concentration less than 0.5%). Appro-
priate controls treated with or without solvent (DMSO), and
various concentrations of doxorubicin (positive control), chloro-
quine, doxycycline, amphotericin B and pyrimethamine were
added to each set of experiments. After 72 h incubation at 37 ꢀC
and 6% CO2, each plate-well was microscope-examined for
detecting possible precipitate formation before cell growth was
measured by flow cytometry after staining cells with 5
propidium iodide. Antiproliferative activity was evaluated by
counting the number of live cells in a 100 L sample. Inhibitory
mL of
m
concentration 50% (IC50 K562) was defined as the concentration
of drug required to induce a 50% decrease in the K562 cell
proliferation compared to the control. IC50 was calculated by non-
linear regression analysis 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.
`
[13] H.R. Chang, R. Comte, J.-C. Pechere, Antimicrob. Agents Chemother. 34 (1990)
775–780.
[14] J.A. Kovacs, Lancet 340 (1992) 637.
[15] P. Meneceur, M.-A. Bouldouyre, D. Aubert, I. Villena, J. Menotti, V. Sauvage,
J.-F. Garin, F. Derouin, Antimicrob. Agents Chemother. 52 (2008) 1269–1277.
[16] V. Bavetsias, Synth. Commun. 28 (1998) 4547–4559.
[17] W. Trager, J.-B. Jensen, Science 193 (1976) 673–675.
[18] N. Azas, N. Laurencin, F. Delmas, C. Di Giorgio, M. Gasquet, M. Laget, P. Timon-
David, Parasitol. Res. 88 (2002) 165–171.
[19] N. Singh, A. Dube, Am. J. Trop. Med. Hyg. 71 (2004) 400–402.
[20] D.C. McFadden, F. Seeber, J.C. Boothroyd, Antimicrob. Agents Chemother. 41
(1997) 1849–1853.
[21] M. Hadjeri, M. Barbier, X. Ronot, A.-M. Mariotte, A. Boumendjel,
J. Boutonnat, J. Med. Chem. 46 (2003) 2125–2131.
[22] T.J. Mosmann, Rapid colorimetric assay for cellular growth and survival. J. Immunol.
Methods 65 (1983) 55–63.
4.3.6. In vitro HepG2 cytotoxic test
The evaluation of the tested molecules cytotoxicity on the
HepG2 cell line was done according to the method of Mosmann
[22] with slight modifications. Briefly, cells in 100
medium, [RPMI supplemented with 10% foetal bovine serum, 1%
glutamine (200 mM) and penicillin (100 U/mL)/streptomycin
(100 g/mL)] were inoculated into each well of 96-well plates and
incubated at 37 ꢀC in a humidified 6% CO2 with 95% air atmosphere.
mL of complete
L-
m