Synthesis and in vitro antiplasmodial evaluation of 4-anilino-2-trichloromethylquinazolines

Article abstract of DOI:10.1016/j.bmc.2009.05.022

To identify a new safe antiplasmodial molecular scaffold, an original series of 2-trichloromethylquinazolines, functionalized in position 4 by an alkyl- or arylamino substituent, was synthesized from 4-chloro-2-trichloromethylquinazoline 1, via a cheap, f

Full text of DOI:10.1016/j.bmc.2009.05.022

Bioorganic & Medicinal Chemistry 17 (2009) 4313–4322
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro antiplasmodial evaluation
of 4-anilino-2-trichloromethylquinazolines
Pierre Verhaeghe a, Nadine Azas b, Sébastien Hutter b, Caroline Castera-Ducros a, Michèle Laget b,
Aurélien Dumètre b, Monique Gasquet b, Jean-Pierre Reboul a, Sylvain Rault c,
Pascal Rathelot a, Patrice Vanelle a,
*
a Laboratoire de Pharmacochimie Radicalaire, Faculté de Pharmacie, Universités d’Aix Marseille I, II et III-CNRS, UMR 6264, Laboratoire Chimie Provence,
27 Boulevard Jean Moulin, 13385 Marseille cedex 05, France
b Relations Hôte-Parasites, Pharmacologie et Thérapeutique, UMR MD3, Faculté de Pharmacie, 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, Faculté des Sciences Pharmaceutiques, 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:
To identify a new safe antiplasmodial molecular scaffold, an original series of 2-trichloromethylquinaz-
olines, functionalized in position 4 by an alkyl- or arylamino substituent, was synthesized from 4-
chloro-2-trichloromethylquinazoline 1, via a cheap, fast and efficient solvent-free operating procedure.
Among the 40 molecules prepared, several exhibit a good profile with both a significant antiplasmodial
Received 1 April 2009
Revised 5 May 2009
Accepted 11 May 2009
Available online 15 May 2009
activity on the W2 Plasmodium falciparum strain (IC50 values: 0.4–2.2 lM) and a promising toxicological
behavior regarding human cells (HepG2/W2 selectivity indexes: 40–83), compared to the antimalarial
drug compounds chloroquine and doxycycline. The in vitro antitoxoplasmic and antileishmanial evalua-
tions were conducted in parallel on the most active molecules, showing that these ones specifically dis-
play antiplasmodial properties.
Keywords:
Quinazoline
Trichloromethyl group
Plasmodium falciparum
In vitro antiplasmodial activity
Selectivity index
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
azine or oxadiazole.3 Recently, some 4-anilino-substituted
quinazolines have also shown a quite interesting antiplasmodial
In the World Malaria Report 2008,1 the W.H.O. estimated that
247 million people suffered from this parasitic infectious disease
world-wide in 2006, with about a million deaths mainly among
African children under 5 years of age. This led to the drafting of a
series of international recommendations presented in the W.H.O.
Global Malaria Action Plan,2 where in ‘opportunities to improve
treatments’, the W.H.O. requests ‘active ingredients with lower
cost of goods for treatment of Plasmodium falciparum malaria’.
Moreover, anticipating the future emergence of parasites resis-
tant to the current lead treatment for P. falciparum malaria (arte-
misinin-based combination therapies), the W.H.O. calls for ‘a
robust pipeline of new medicines. . ., including non-artemisinin-
based combinations with novel mechanisms of action’. In such a
context it appears that university academic research teams have
a major role to play by offering new original therapeutic candidates
meeting all these requirements, in order to effectively assist pa-
tients in developing countries suffering from malaria.
activity against the 3D7 chloroquine-sensitive P. falciparum strain.4
Our team, which is particularly involved in the research of new po-
tential anti-infectious agents,5 showed in 2008 that the 4-aryl-2-
trichloromethylquinazoline series (Fig. 1) displays interesting
in vitro antiplasmodial properties on the W2 chloroquine-resistant
P. falciparum strain, combined with a safe toxicological profile on
two human cell lines: THP1 (monocyte) and HepG2 (hepatocyte).6
Starting from these preliminary encouraging results, we
decided to explore the antiplasmodial potential (W2 strain) of
the 4-aminosubstituted-2-trichloromethylquinazoline series. In
parallel, in order to determine the selectivity indexes of our mole-
cules, we studied their in vitro toxicity toward two human cell
F
IC50(W2)
IC50(THP1) = 125 µM
IC50(HepG2) > 125 µM
= 2.5 µM
The trichloromethyl group is already known to confer antiplas-
modial activity to various molecular series, such as benzene, tri-
N
N
CCl3
* Corresponding author. Tel.: +33 491 835 528; fax: +33 491 794 677.
E-mail address: patrice.vanelle@pharmacie.univ-mrs.fr (P. Vanelle).
Figure 1. Hit compound in the 4-aryl-2-trichloromethyl-quinazoline series.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.022

4314
P. Verhaeghe et al. / Bioorg. Med. Chem. 17 (2009) 4313–4322
lines: K526 and HepG2. This latter cell line allows a thorough eval-
uation of the cytotoxic behavior of the tested molecules, following
their metabolic activation.7
R
R
H
N
H
N
N
N
N
N
Fe (15 eq.)
mean yield = 98%
(95-99%)
2. Results and discussion
2.1. Chemistry
AcOH
Reflux / 1 h
CCl3
CH3
10, 22, 33
39-41
Scheme 2. Preparation of compounds 39–41 by reductive dehalogenation of the
corresponding CCl3 substrates.
In order to prepare these molecules, the most adapted chemical
starting material appeared to be the 4-chloro-2-trichloromethyl-
quinazoline 1 which was synthesized via a microwave-assisted tet-
rachlorination protocol that we previously reported.8
We decided to react 1 successively with 3 primary amines, 3
secondary amines and 3 aromatic amines, so as to compare their
antiplasmodial potential and select the most suitable amine cate-
gory for preparing a complete series of promising derivatives.
Focusing on setting up a simple, cheap, fast and efficient synthesis
access, we chose to work on the definition of a general operating
procedure for each amine category.
2.2. In vitro antiplasmodial activity
As explained above, the initial step of our study consisted in
choosing, through the biological results obtained with the first 9
preliminary synthesized compounds (2–10), the most relevant cat-
egory of amine reagent (I, II, or anilines), so as to prepare an
homogenous complete series, with optimal potential.
Thus, the reaction of 1 with primary amines, consisting in a fu-
sion reaction with an excess of amine, was conducted according to
the reaction presented in Scheme 1, reducing as much as possible
both reaction time and temperature, so as to preserve the CCl3
group from reacting. This rapid reaction protocol resulted in the
formation of secondary aminoquinazolines 2–4 in good yields.
Substrate 1 was reacted very easily with secondary amines, fol-
lowing the procedure as above type of and conducted to the prep-
aration of tertiary aminoquinazolines 5–7 in very good yields
(Scheme 1).
Logically, the reaction of 1 with anilines required stronger oper-
ating conditions, elevated temperature and longer reaction time to
give the expected products in good yields, except for compounds
31 and 36 for which reaction yields were, respectively 25% and
35%, and particularly compound 32, with a reaction yield of only
9%, probably due to the steric hindrance affecting the amino group
on the 2,6-dichloro-bisubstituted-aniline. This reaction was ini-
tially carried out for the preparation of compounds 8–10 and then
applied to prepare a series of 31 molecules (8–38), as presented in
Scheme 1.
The antiplasmodial inhibitory concentrations 50% (IC50) of these
molecules, toward the W2 P. falciparum strain ranged from 5.1 to
14.3, 5.6 to 41.5 and 1.7 to 11 lM, respectively for the compounds
derived from primary amines, secondary amines and anilines,
pointing out a relative lack of efficiency for molecules 5–7, ob-
tained via the reaction of 1 with secondary amines (Table 1).
Cytotoxicity experiments conducted on the K562 cell line were
not discriminating, with no significant antiproliferative effect mea-
sured for any of the tested molecules at the tested concentrations,
whereas tests on the HepG2 cell line showed that molecules (2–4)
derived from primary amines, display some toxicity (Table 1).
Thus, compounds 8–10 being overall both the most active and less
toxic molecules, we decided to focus our work on the study of the
4-anilino-2-trichloromethylquinazoline series and prepared 31
additional molecules (11–41) for structure–activity relationship
purposes.
If we consider the W2 antiplasmodial inhibitory concentrations
determined in 4-anilino-2-trichloromethylquinazoline series
(Table 1), we first note all IC50 were located in a narrow range of
values, from 0.4
of the antiplasmodial IC50 values of these compounds are compara-
ble with the ones measured for chloroquine (0.7 M) or doxycy-
cline (6.5 M), two antiplasmodial drug compound references
presenting separate mechanisms of action.
In comparison with compound 8 which presents an unsubsti-
tuted aniline moiety (IC50 = 11 M), it appears that the substitution
lM (molecule 33) to 22 lM (molecule 32). Most
Three 2-dehalogenomethylated-quinazolines (39–41) were also
prepared from the corresponding 2-trichloromethylated-quinazo-
lines (10, 22, 33), through a reduction reaction (Scheme 2), in order
to compare the biological results and to demonstrate the key role
played by the CCl3 group toward antiplasmodial activity.
l
l
l
R
H
N
of the aniline moiety increases effectivity, whatever the substitu-
ent or the substitution position, apart for compounds 31 and 32.
The observation of compounds for which the aniline ring is
monosubstituted indicates a global better activity for para or
meta-aniline-substituted compounds, except those with nitro or
methoxy groups for which no effect of the substitution position
was noted on the antiplasmodial activity (compounds 11, 16, 17,
24–26). Among these para or meta-aniline-substituted molecules,
it also appears that bromine, chlorine and trifluoromethyl groups
are the substituents which most contribute to increasing the anti-
R
N
N
N
H
H (3 eq.)
73-79%
Solvent-free
1 min / 50 °C
CCl3
2-4
R
R'
N
H
N
N
N
Cl
N
R'
R
(3 eq.)
N
89-99%
Solvent-free
5 min / 25 °C
CCl3
CCl3
plasmodial activity (1.5–3 lM).
5-7
1
Then, if we have a look at the molecules in which the aniline
moiety is bisubstituted (molecules 30–36), we surprisingly note
that this subgroup is very heterogeneous, including both the best
and the worst activity values (respectively compounds 33 and
32) and, except for compound 33 (the most potent in the series),
the double substitution of the aniline ring does not improve anti-
plasmodial activity, in comparison with monosubstituted deriva-
tives, even when the most promising groups (Cl or CF3) are
combined on the two meta-aniline positions (molecules 34 and
36).
R
R
H
N
N
N
mean yield
= 68%
NH2
(3 eq.)
(9-98%)
Solvent-free
15 min / 140 °C
CCl3
8-38
Scheme 1. Operating procedures for the reaction of 1 with each category of amines.

P. Verhaeghe et al. / Bioorg. Med. Chem. 17 (2009) 4313–4322
4315
Table 1
Antiplasmodial activity and human cell toxicity of the studied molecules
R1
N
N
R2
Molecule
R1-
R2-
W2 Antiplasmodial
IC50 activity (lM)
Human cell toxicitya
(l
M)
W2 strain selectivity
index toward K562b
W2 strain selectivity
index toward HepG2b
K562 IC50
HepG2 IC50
2
3
4
5
6
7
n-Butyl-NH–
Cycloheptyl-NH–
Benzyl-NH–
N-Morpholinyl–
N-Pyrrolidinyl–
4-CH3-piperazin-1-yl–
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
14.3
5.1
6.9
14.9
41.5
5.6
>50f
>50f
>50f
>125f
>125f
65
16
19
13
>3.5
>9.8
>7.2
>8.4
>3
1.1
3.7
1.9
>2
>6
13.4
63
>250f
75
11.6
8
9
Ph-NH–
4-F-Ph-NH–
3-Cl-Ph-NH–
2-NO2-Ph-NH–
2-Cl-Ph-NH–
4-Cl-Ph-NH–
2-F-Ph-NH–
3-F-Ph-NH–
3-NO2-Ph-NH–
4-NO2-Ph-NH–
2-Br-Ph-NH–
3-Br-Ph-NH–
4-Br-Ph-NH–
2-CF3-Ph-NH–
3-CF3-Ph-NH–
4-CF3-Ph-NH–
2-OCH3-Ph-NH–
3-OCH3-Ph-NH–
4-OCH3-Ph-NH–
2-CH3-Ph-NH–
3-CH3-Ph-NH–
4-CH3-Ph-NH–
3,4-Di-Cl-Ph-NH–
2,3-Di-Cl-Ph-NH–
2,6-Di-Cl-Ph-NH–
2,4-Di-Cl-Ph-NH–
3,5-Di-Cl-Ph-NH–
2,5-Di-Cl-Ph-NH–
3,5-Di-CF3-Ph-NH–
4-OCF3-Ph-NH–
1-Naphthyl-NH–
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
–CCl3
11
5.4
1.7
3
4.6
3
9
4
3
2.8
3
2.2
1.5
7.4
1.8
1.6
6
4
6
7
2.3
2.4
5
14
21.9
0.4
4
1.7
3
2.9
6
>50f
>125f
>50f
>60f
>50f
14
>50f
>50f
>100f
>100f
25
9
10
16
10
>4.5
>23.1
>29.4
>20
>10.9
4.7
3.3
3.8
1.7
1.8
>10
>22.7
10
4.1
>69.4
2.5
10
2.3
1.3
1.6
3.9
2.5
1.8
>4.3
0.5
25
2.2
>4.7
3.3
2.1
>4.5
>9.3
>58.8
>33.3
5.5
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
30
15
5
5
1.1
4
3.3
2.1
>16.7
>45.5
6.7
1.4
83.3
3.8
10
0.1
1.2
3.4
5.2
3.3
1.6
7.1
1.1
40
6
>30f
>50f
15
>50f
>100f
10
30
10
>125f
4
150f
6
60
9
8
11
9
6
60
0.5
7
24
12
8
8
>100
23
16
10
30
3
9
>60
f
f
10
10
9
2.5
17.6
1
2.1
2.5
>8f
10
6
6
15
5
0.8
39
40
41
3-Cl-Ph-NH–
3-CF3-Ph-NH–
2,4-Di-Cl-Ph-NH–
–CH3
–CH3
–CH3
55
42.5
85
27
10
15
40
20
15
0.5
0.2
0.2
0.7
0.5
0.2
Ref.
Ref.
Ref.
Chloroquinec
Doxycyclined
Doxorubicine
0.7
6.5
—
32
15
0.06
30
20
0.2
45.7
2.3
—
42.9
3.1
—
In bold: hit compounds in the series.
a
Mean of three independent experiments.
b
Selectivity index was calculated according to the following formula: SIW2Plasmodium = Human cell IC50/W2 Plasmodium IC50
Chloroquine and doxycycline were used as antiplasmodial drug compounds of reference.
Doxorubicin was used as a drug compound of reference for human cell toxicity.
No toxicity noted at the highest concentration tested.
.
c,d
e
f
In the 4-anilino-2-trichloromethylquinazoline series, the most
cell lines in 4-anilino-2-trichloromethylquinazoline series were
quite similar. This indicates that the metabolism of these deriv-
atives by HepG2 cells does not modify their toxicological
behavior.
important structural element for conferring activity remains the
essential influence of the trichloromethyl group, as demonstrated
with dehalogenated molecules 39–41 which have completely lost
antiplasmodial activity (IC50 = 42.5–85
l
M).
Thus, considering together HepG2 and K562 cell lines, the spec-
trum of IC50 values appears rather broad, in the 0.5–150 lM range,
2.3. In vitro human cell toxicity assessment
traducing a large diversity in the toxicological profile inside the 4-
anilino-2-trichloromethylquinazoline series. In the same series, 9
Contrary to the results obtained for the compounds derived
from primary amines (2–4), for which no toxicity was noted
on the K562 cell line while a light toxicity was detected on
the HepG2 cell line, the results reported on these two distinct
molecules presented IC50 values superior to 50 lM on both human
cell lines in comparison with the two antiplasmodial reference
drugs tested for which toxicity values are comprised in between
15 and 32 lM on the two same human cell lines.

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P. Verhaeghe et al. / Bioorg. Med. Chem. 17 (2009) 4313–4322
Among molecules for which the aniline ring is monosubstituted,
and 33 were evaluated for their mutagenicity via the Ames test,
at a concentration of 5 mM, on 4 distinct Salmonella typhimurium
strains, in two different conditions including metabolic activation
(S9 mix). None of these molecules presented mutagenic activity
(Table 2).
We then wanted to explore the antiparasitic spectrum of this
molecular scaffold toward other protozoa. Thus, molecules 10,
19, 22 and 33 were evaluated in vitro against the Leishmania dono-
vani promastigotes. None of them displayed any antileishmanial
toxicity seems to be essentially influenced by the substitution po-
sition on the aniline ring and little by the nature of substituents,
even if the methyl group seems to promote cytotoxicity. In this
way, apart for compound 9, the para position is involved in ele-
vated toxicity whereas the ortho position appears as related with
the less toxic molecules, apart for compounds 14 and 21.
In the bisubstituted aniline moiety subgroup, only one molecule
(31) does not display any toxicity toward these two human cell
lines.
activity, their IC50 values being superior to 50 lM (Table 2).
Then, the same molecules were tested on Toxoplasma gondii.
2.4. Hit molecules in the series
Once again, none of these 4 molecules exerted any activity against
this apicomplexan related to Plasmodium, (IC50 >50 lM, Table 2).
Taking into account the antiplasmodial activity and the toxicity
of the synthesized molecules, their selectivity indexes (ratio IC50
human cell line/IC50W2 P. falciparum.) were calculated to identify
compounds with therapeutic potential. In the target 4-anilino-2-
trichloromethylquinazoline series, 5 molecules reveal selectivity
indexes included in the >23–83 range, considering the two human
cell lines tested, in comparison with chloroquine and doxycycline
which, respectively display selectivity indexes of 43–46 and 2.3–
3.1.
This last information is more surprising than the lack of efficacy to-
ward Leishmania, as several antimalarial drug compounds such as
atovaquone9,10 or doxycycline11 are well-known for being used in
the treatment of toxoplasmosis, particularly in immunosuppressed
patients. Therefore, this new antiprotozoal molecular scaffold
shows its selective antiplasmodial activity.
3. Conclusion
As a logical consequence in matching both activity and toxicity
structural criterias, the molecules substituted by a bromine, chlo-
rine or CF3 group on the meta position of the aniline moiety are
the most promising (molecules 10, 19 and 22).
Despite a non negligible toxicity, compound 33, maintains good
selectivity indexes (25 and 40) because of its high antiplasmodial
We identified a new cheap antiplasmodial molecular scaffold,
quickly and easily prepared in two steps from commercially avail-
able reagents, in the 4-anilino-2-trichloromethylquinazoline series.
Depending on the aniline moietysubstitution, the structure–activity
relationships reveal that 4 molecules (10, 19, 22 and 33) in the series
present both a potent antiplasmodial activity on the W2 chloro-
activity (IC50W2 = 0.4 lM).
quine-resistant P. falciparum strain (IC50 = 0.4–2.2
son with chloroquine (IC50 = 0.7 M) and doxycycline (IC50
6.5 M) and a safe toxicological profile toward two human cell lines,
lM) in compari-
The conversion from trichloromethyl to methyl group, operated
with molecules 39–41 for comparison purposes, leads to the com-
plete inversion of the selectivity indexes (0.2–0.7), which clearly
vouches for its crucial role in the antiplasmodial scaffold.
l
=
l
affording high selectivity indexes (>23–83). In this scaffold, the
trichloromethyl group appears to be key to both potent activity
and low toxicity. Moreover, none of these molecules presents any
mutagenic activity via the Ames test, at a concentration of 5 mM.
Furthermore, these molecules present a specific action on P. falcipa-
rum. Considering the partial ATP-competitive structure of our scaf-
fold,12 Plasmodium kinases appear interesting targets to investigate
so as to determine the antiplasmodial mechanism of action of our
molecules. Such hypothesis is currently under active investigation.
2.5. Complementary investigations
After establishing the precise structure of our in vitro antiplas-
modial pharmacophore, designed as 2-trichloromethyl-N-(3-
substituted-phenyl)quinazolin-4-amine with Br, Cl or CF3 as
substituents, we wanted to further investigate its toxicological
profile by testing its mutagenic potential. Compounds 10, 19, 22
Table 2
Survey of the antiparasitic profile displayed by the most promising compounds
Ar
NH
N
N
CCl3
Molecule
Ar-
W2 Antiplasmodial
Antitoxoplasmic
Antileishmanial
Human cell toxicitya
(lM)
Mutagenicity (AmesTest)
activity IC50
(l
M)
activity IC50
(l
M)
activity Promastigotes
IC50 (lM)
K562 IC50
HepG2 IC50
10
19
22
33
3-Cl-Ph–
3-Br-Ph–
3-CF3-Ph–
2,4-Di-Cl-Ph–
1.7
2.2
1.8
0.4
>50g
>50g
>50g
>50g
>50g
>50g
>50g
>50g
>50h
>50h
>125h
10
>100h
>100h
150
Negative
Negative
Negative
Negative
16
Ref.
Ref.
Ref.
Ref.
Ref.
Chloroquineb
Doxycyclinec
0.7
6.5
—
—
—
—
—
—
2
—
—
0.08
—
—
32
15
17
5
30
20
10
30
0.2
—
—
—
—
—
AmphotericinBd
Pyrimethaminee
Doxorubicinf
—
0.06
a
Mean of three independent experiments.
b,c
Chloroquine and doxycycline were used as antiplasmodial drug compounds of reference.
Amphotericin B was used as the antileishmanial drug compound of reference.
Pyrimethamine was used as the antitoxoplasmic drug compound of reference.
Doxorubicin was used as the drug compound of reference for human cell toxicity.
No activity noted at the highest concentration tested.
d
e
f
g
h
No toxicity noted at the highest concentration tested.

P. Verhaeghe et al. / Bioorg. Med. Chem. 17 (2009) 4313–4322
4317
chloro-2-trichloromethylquinazoline 1, without any solvent, at
50 °C for 1 min. The reaction mixture was then extracted with
dichloromethane. The organic layer was washed with water three
times, dried over anhydrous Na2SO4 and concentrated in vacuo to af-
ford a crude residue with was purified by chromatography on a silica
gel, eluting with dichloromethane. 4 was obtained as a white solid in
79% yield; mp 102 °C. 1H NMR (200 MHz, CDCl3) d: 4.94–4.96 (m,
2H), 6.59 (br s, 1H), 7.30–7.41 (m, 3H), 7.47–7.52 (m, 3H), 7.71–
7.81 (m, 2H), 8.00–8.04 (m, 1H). 13C NMR (50 MHz, CDCl3) d: 160.9
(C), 160.1 (C), 148.5 (C), 137.8 (C), 133.4 (CH), 129.1 (CH), 128.8
4. Experimental
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 Fa-
culté de Pharmacie de Marseille. Chemical shifts are given in d val-
ues referenced to the solvent. High resolution mass spectra were
recorded on a QStar Elite or a JEOL JMS GCMate spectrometer at
the Spectropôle department of the Faculté des Sciences et Tech-
niques de St Jérôme or at the Centre d’Etudes et de Recherche
sur le Médicament de Normandie. 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 chromatogra-
phy. The progress of the reactions was monitored by thin layer
chromatography on using Kieselgel 60 F254 (Merck) plates.
*
*
(CH 2), 128.6 (CH 2), 127.9 (CH), 127.6 (CH), 120.8 (CH), 113.4 (C),
97.6 (C), 45.7 (CH2). Anal. Calcd for C16H12Cl3N3: C, 54.49; H, 3.43;
N, 11.92. Found: C, 54.31, H, 3.53; N, 11.72.
4.1.7. General procedure for the preparation of compounds (5),
(6) and (7)
Three equivalents of the appropriate secondary amine reagent
were added dropwise onto 1 equiv of 4-chloro-2-trichloromethyl-
quinazoline 1. The solvent-free reaction mixture was then stirred
at room temperature for 5 min. The crude residue was then ex-
tracted with dichloromethane. The organic layer was washed with
water three times, dried over anhydrous Na2SO4 and concentrated
in vacuo to afford the corresponding nucleophilic aromatic substi-
tution product.
4.1.2. 4-Chloro-2-trichloromethylquinazoline (1)
Compound 1 was synthesized according to a microwave-as-
sisted procedure that we previously reported,8 and obtained as a
white solid in 75% yield; mp 127 °C (Lit.13 mp 125 °C). 1H NMR
(200 MHz, CDCl3) d: 7.82–7.90 (m, 1H), 8.03–8.12 (m, 1H), 8.22
(dd, J = 8.5 and 0.5 Hz, 1H), 8.36 (dd, J = 8.5 and 0.5 Hz, 1H). 13C
NMR (50 MHz, CDCl3) d: 164.0 (C), 159.9 (C), 150.2 (C), 135.9
(CH), 130.6 (CH), 129.7 (CH), 126.0 (CH), 122.9 (C), 95.9 (C). Anal.
Calcd for C9H4Cl4N2: C, 38.34; H, 1.43; N, 9.94. Found: C, 38.25,
H, 1.41; N, 9.60.
4.1.8. 4-(2-Trichloromethylquinazolin-4-yl)morpholine (5)
Compound 5 was obtained as a yellow solid in 99% yield; mp
101 °C. 1H NMR (200 MHz, CDCl3) d: 3.86–3.98 (m, 8H), 7.50–
7.58 (m, 1H), 7.76–7.85 (m, 1H), 7.89–7.93 (m, 1H), 8.02–8.07
(m, 1H). 13C NMR (50 MHz, CDCl3) d: 164.6 (C), 160.0 (C), 151.6
(C), 133.1 (CH), 129.6 (CH), 126.7 (CH), 124.5 (CH), 114.8 (C),
*
*
4.1.3. General procedure for the preparation of compounds (2)
and (3)
97.6 (C), 66.5 (CH2 2), 50.0 (CH2 2). Anal. Calcd for C13H12Cl3N3O:
C, 46.94; H, 3.64; N, 12.63. Found: C, 46.62, H, 3.67; N, 12.71.
Three equivalents of the appropriate primary amine reagent
were added dropwise onto 1 equiv of 4-chloro-2-trichloromethyl-
quinazoline 1. The solvent-free reaction mixture was then stirred
at 50 °C for 1 min. The crude residue was then extracted with
dichloromethane. The organic layer was washed with water three
times, dried over anhydrous Na2SO4 and concentrated in vacuo to
afford the corresponding nucleophilic aromatic substitution
product.
4.1.9. 4-(Pyrrolidin-1-yl)-2-trichloromethylquinazoline (6)
Compound 6 was obtained as a white solid in 89% yield; mp
130 °C. 1H NMR (200 MHz, CDCl3) d: 2.05–2.11 (m, 4H), 3.98–
4.05 (m, 4H), 7.41–7.50 (m, 1H), 7.70–7.77 (m, 1H), 7.94–7.98
(m, 1H), 8.18–8.22 (m, 1H). 13C NMR (50 MHz, CDCl3) d: 160.7
(C), 160.1 (C), 151.5 (C), 132.4 (CH), 129.1 (CH), 125.8 (CH), 125.1
(CH), 115.2 (C), 98.2 (C), 51.0 (CH2 2), 25.6 (CH2 2). Anal. Calcd
for C13H12Cl3N3: C, 49.32; H, 3.82; N, 13.27. Found: C, 49.58, H,
3.92; N, 13.27.
*
*
4.1.4. N-Butyl-2-(trichloromethyl)quinazolin-4-amine (2)
Compound 2 was obtained as a white solid in 73% yield; mp
113 °C (Lit14 mp = 113–114 °C). 1H NMR (200 MHz, CDCl3) d: 0.91
(t, J = 7.2 Hz, 3H), 1.30–1.48 (m, 2H), 1.60–1.75 (m, 2H), 3.70 (q,
J = 6.7 Hz, 2H), 6.57 (br s, 1H), 7.39–7.47 (m, 1H), 7.63–7.71 (m,
1H), 7.85–7.89 (m, 2H). 13C NMR (50 MHz, CDCl3) d: 161.3 (C),
160.4 (C), 148.8 (C), 133.0 (CH), 128.7 (CH), 127.1 (CH), 121.0
(CH), 113.5 (C), 98.2 (C), 41.1 (CH2), 31.0 (CH2), 19.9 (CH2), 13.7
(CH3). Anal. Calcd for C13H14Cl3N3: C, 49.00; H, 4.43; N, 13.19.
Found: C, 49.50, H, 4.62; N, 13.32.
4.1.10. 4-(4-Methylpiperazin-1-yl)-2-trichloromethyl-
quinazoline (7)
Compound 7 was obtained as a brown solid in 99% yield; dec.
90 °C. 1H NMR (200 MHz, CDCl3) d: 2.32 (s, 3H), 2.55–2.60 (m,
4H), 3.90–3.95 (m, 4H), 7.41–7.50 (m, 1H), 7.68–7.75 (m, 1H),
7.83–7.87 (m, 1H), 7.92–7.97 (m, 1H). 13C NMR (50 MHz, CDCl3)
d: 164.3 (C), 159.9 (C), 151.6 (C), 132.9 (CH), 129.4 (CH), 126.5
*
*
(CH), 124.7 (CH), 114.8 (C), 97.7 (C), 54.6 (CH2 2), 49.2 (CH2 2),
45.9 (CH3). HR MS: m/z 344.0364. Calcd for C14H15Cl3N4:
344.0362.
4.1.5. N-Cycloheptyl-2-trichloromethylquinazolin-4-amine (3)
Compound 3 was obtained as a white solid in 78% yield; mp
151 °C. 1H NMR (200 MHz, CDCl3) d: 1.60–1.65 (m, 8H), 2.15–
2.19 (m, 4H), 4.43–4.44 (m, 1H), 5.82–5.85 (m, 1H), 7.48–7.56
(m, 1H), 7.70–7.80 (m, 2H), 7.94–7.98 (m, 1H). 13C NMR (50 MHz,
CDCl3) d: 161.4 (C), 159.2 (C), 149.1 (C), 133.0 (CH), 129.5 (CH),
127.2 (CH), 120.4 (CH), 113.6 (C), 98.2 (C), 52.6 (CH), 34.4
4.1.11. General procedure for the preparation of compounds
(8)–(38)
Three equivalents of the appropriate aromatic amine reagent
were added dropwise onto 1 equiv of 4-chloro-2-trichloromethyl-
quinazoline 1. The solvent-free reaction mixture was then stirred
at 140 °C for 15 min, cooled down and then extracted with dichlo-
romethane. The organic layer was washed with water, dried over
anhydrous Na2SO4 and concentrated in vacuo. The crude residue
was purified by chromatography on a silica gel, eluting with
dichloromethane to afford the corresponding nucleophilic aro-
matic substitution product.
*
*
*
(CH2 2), 28.1 (CH2 2), 24.4 (CH2 2). Anal. Calcd for C16H18Cl3N3:
C, 53.58; H, 5.06; N, 11.71. Found: C, 53.35, H, 5.13; N, 12.00.
4.1.6. N-Benzyl-2-trichloromethylquinazolin-4-amine (4)
Compound 4 was prepared by reacting 570 mg (5.34 mmol,
3 equiv) of benzylamine with 500 mg (1.78 mmol, 1 eq.) of 4-

4318
P. Verhaeghe et al. / Bioorg. Med. Chem. 17 (2009) 4313–4322
4.1.12. N-Phenyl-2-trichloromethylquinazolin-4-amine (8)
Compound 8 was obtained as a beige solid in 62% yield; mp
150 °C. 1H NMR (200 MHz, CDCl3) d: 7.15–7.23 (m, 1H), 7.40–
7.48 (m, 2H), 7.62–7.71 (m, 2H), 7.84–7.88 (m, 1H), 7.93–7.99
(m, 3H), 8.07–8.11 (m, 1H). 13C NMR (50 MHz, CDCl3) d: 161.0
(C), 157.7 (C), 149.6 (C), 138.0 (C), 133.6 (CH), 130.1 (CH), 129.1
(CH 2), 128.2 (CH), 124.6 (CH), 120.9 (CH 2), 120.2 (CH), 113.8
(C), 97.7 (C). Anal. Calcd for C15H10Cl3N3: C, 53.20; H, 2.98; N,
12.41. Found: C, 52.94, H, 2.97; N, 12.01.
4.1.18. N-(2-Fluorophenyl)-2-trichloromethyl-quinazolin-4-
amine (14)
Compound 14 was obtained as a pink solid in 45% yield; mp
155 °C. 1H NMR (200 MHz, CDCl3) d: 7.04–7.30 (m, 3H), 7.65–
7.73 (m, 1H), 7.85–7.96 (m, 3H), 8.09 (d, J = 8.1 Hz, 1H), 8.96–
9.05 (m, 1H). 13C NMR (50 MHz, CDCl3) d: 160.8 (C), 157.3 (C),
152.9 (d, J = 243.0 Hz, C), 149.5 (C), 133.8 (CH), 130.1 (CH), 128.5
(CH), 126.7 (d, J = 8.7 Hz, C), 124.8 (d, J = 4.0 Hz, CH), 124.1 (d,
J = 7.7 Hz, CH), 122.3 (CH), 120.1 (CH), 114.6 (d, J = 19.0 Hz, CH),
113.9 (C), 97.6 (C). Anal. Calcd for C15H9Cl3FN3: C, 50.52; H, 2.54;
N, 11.78. Found: C, 50.68, H, 2.79; N, 11.57.
*
*
4.1.13. N-(4-Fluorophenyl)-2-trichloromethyl-quinazolin-4-
amine (9)
4.1.19. N-(3-Fluorophenyl)-2-trichloromethyl-quinazolin-4-
amine (15)
Compound 9 was obtained as a beige solid in 88% yield; mp
165 °C. 1H NMR (200 MHz, CDCl3) d: 7.05–7.14 (m, 2H), 7.58–
7.67 (m, 2H), 7.80–7.92 (m, 4H), 8.02–8.06 (m, 1H). 13C NMR
(50 MHz, CDCl3) d: 160.9 (C), 159.4 (d, J = 244.5 Hz, C), 157.7 (C),
149.5 (C), 133.9 (d, J = 2.9 Hz, C), 133.7 (CH), 129.9 (CH), 128.2
Compound 15 was obtained as a white solid in 82% yield; mp
182 °C. 1H NMR (200 MHz, CDCl3) d: 6.82–6.92 (m, 1H), 7.29–
7.41 (m, 1H), 7.46–7.51 (m, 1H), 7.64–7.71 (m, 2H), 7.85–7.95
(m, 2H), 8.06–8.14 (m, 2H). 13C NMR (50 MHz, CDCl3) d: 165.5
(C), 160.7 (d, J = 2.3 Hz, C), 157.5 (C), 149.5 (C), 139.5 (d,
J = 11.3 Hz, C), 133.8 (CH), 130.1 (d, J = 9.6 Hz, CH), 130.0 (CH),
128.4 (CH), 120.1 (CH), 115.8 (d, J = 3.0 Hz, CH), 113.7 (C), 111.1
(d, J = 21.6 Hz, CH), 108.5 (d, J = 28.4 Hz, CH), 97.6 (C). Anal. Calcd
for C15H9Cl3FN3: C, 50.52; H, 2.54; N, 11.78. Found: C, 50.64, H,
2.65; N, 11.67.
*
(CH), 122.7 (d, J = 7.6 Hz, CH 2), 120.2 (CH), 115.7 (d, J = 22.3 Hz,
*
CH 2), 113.6 (C), 97.8 (C). Anal. Calcd for C15H9Cl3FN3: C, 50.52;
H, 2.54; N, 11.78. Found: C, 50.46, H, 2.56; N, 11.64.
4.1.14. N-(3-Chlorophenyl)-2-trichloromethyl-quinazolin-4-
amine (10)
Compound 10 was obtained as a beige solid in 90% yield; mp
174 °C. 1H NMR (200 MHz, CDCl3) d: 7.11–7.17 (m, 1H), 7.25–
7.37 (m, 1H), 7.63–7.75 (m, 3H), 7.85–7.95 (m, 2H), 8.06–8.11
(m, 1H), 8.21–8.23 (m, 1H). 13C NMR (50 MHz, CDCl3) d: 160.8
(C), 157.5 (C), 149.6 (C), 139.1 (C), 134.8 (C), 133.8 (CH), 130.0
(CH), 129.9 (CH), 128.4 (CH), 124.4 (CH), 121.0 (CH), 120.1 (CH),
118.6 (CH), 113.6 (C), 97.6 (C). Anal. Calcd for C15H9Cl4N3: C,
48.29; H, 2.43; N, 11.26. Found: C, 48.26, H, 2.46; N, 11.42.
4.1.20. N-(3-Nitrophenyl)-2-trichloromethylquinazolin-4-
amine (16)
Compound 16 was obtained as a yellow solid in 63% yield; mp
227 °C. 1H NMR (200 MHz, DMSO-d6) d: 7.66–7.74 (m, 1H), 7.78–
7.86 (m, 1H), 7.99–8.01 (m, 3H), 8.41 (dd, J = 1.1 and 8.3 Hz, 1H),
8.70 (d, J = 8.3 Hz, 1H), 9.21 (s, 1H), 10.55 (s, 1H). 13C NMR
(50 MHz, DMSO-d6) d: 159.9 (C), 158.5 (C), 149.1 (C), 148.1 (C),
140.2 (C), 134.7 (CH), 130.0 (CH), 128.9 (CH), 128.7 (CH), 127.6
(CH), 123.5 (CH), 118.4 (CH), 116.1 (CH), 114.1 (C), 97.8 (C). Anal.
Calcd for C15H9Cl3N4O2: C, 46.96; H, 2.36; N, 14.60. Found: C,
46.55, H, 2.32; N, 14.32.
4.1.15. N-(2-Nitrophenyl)-2-trichloromethylquinazolin-4-
amine (11)
Compound 11 was obtained as a yellow solid in 94% yield; mp
181 °C. 1H NMR (200 MHz, CDCl3) d: 7.20–7.29 (m, 1H), 7.76–7.83
(m, 2H), 7.93–8.00 (m, 1H), 8.16 (dd, J = 2.4 and 8.2 Hz, 2H), 8.35
(dd, J = 1.5 and 8.5 Hz, 1H), 9.58 (d, J = 8.7 Hz, 1H), 11.77 (br s,
1H). 13C NMR (50 MHz, CDCl3) d: 160.4 (C), 157.7 (C), 149.9 (C),
136.5 (C), 136.4 (CH), 135.9 (C), 134.2 (CH), 130.2 (CH), 129.3
(CH), 126.2 (CH), 122.9 (CH), 122.7 (CH), 120.9 (CH), 114.8 (C),
97.5 (C). Anal. Calcd for C15H9Cl3N4O2: C, 46.96; H, 2.36; N,
14.60. Found: C, 47.14, H, 2.36; N, 14.52.
4.1.21. N-(4-Nitrophenyl)-2-trichloromethylquinazolin-4-
amine (17)
Compound 17 was obtained as a yellow solid in 77% yield; mp
294 °C. 1H NMR (200 MHz, DMSO-d6) d: 7.81–7.89 (m, 1H), 8.00–
8.08 (m, 2H), 8.27–8.39 (m, 4H), 8.74 (d, J = 8.3 Hz, 1H), 10.65 (s,
1H). 13C NMR (50 MHz, DMSO-d6) d: 159.8 (C), 158.4 (C), 149.3
(C), 145.4 (C), 142.6 (C), 134.9 (CH), 129.0 (CH), 128.9 (CH), 124.8
*
*
(CH 2), 123.7 (CH), 121.4 (CH 2), 114.4 (C), 97.7 (C). Anal. Calcd
for C15H9Cl3N4O2: C, 46.96; H, 2.36; N, 14.60. Found: C, 46.56, H,
2.34; N, 14.38.
4.1.16. N-(2-Chlorophenyl)-2-trichloromethyl-quinazolin-4-
amine (12)
Compound 12 was obtained as a white solid in 63% yield; mp
164 °C. 1H NMR (200 MHz, CDCl3) d: 7.06–7.15 (m, 1H), 7.37–
7.49 (m, 2H), 7.68–7.76 (m, 1H), 7.87–7.99 (m, 2H), 8.11 (d,
J = 7.7 Hz, 1H), 8.45 (br s, 1H), 9.12 (dd, J = 1.5 and 8.3 Hz, 1H).
13C NMR (50 MHz, CDCl3) d: 160.7 (C), 157.3 (C), 149.6 (C), 134.7
(C), 133.9 (CH), 130.2 (CH), 129.0 (CH), 128.6 (CH), 128.1 (CH),
124.4 (CH), 123.2 (C), 122.0 (CH), 120.2 (CH), 114.2 (C), 97.6 (C).
Anal. Calcd for C15H9Cl4N3: C, 48.29; H, 2.43; N, 11.26. Found: C,
48.28, H, 2.48; N, 11.09.
4.1.22. N-(2-Bromophenyl)-2-trichloromethyl-quinazolin-4-
amine (18)
Compound 18 was obtained as a white solid in 60% yield; mp
191 °C. 1H NMR (200 MHz, CDCl3) d: 7.00–7.09 (m, 1H), 7.42–
7.50 (m, 1H), 7.62–7.76 (m, 2H), 7.89–7.96 (m, 1H), 8.01 (d,
J = 8.0 Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H), 8.47 (br s, 1H), 9.12 (d,
J = 8.3 Hz, 1H). 13C NMR (50 MHz, CDCl3) d: 160.8 (C), 157.4 (C),
149.6 (C), 135.7 (C), 133.8 (CH), 132.3 (CH), 130.2 (CH), 128.7
(CH), 128.6 (CH), 124.9 (CH), 122.2 (CH), 120.3 (CH), 114.3 (C),
114.1 (C), 97.6 (C). Anal. Calcd for C15H9BrCl3N4: C, 43.15; H,
2.17; N, 10.06. Found: C, 43.33, H, 2.20; N, 9.96.
4.1.17. N-(4-Chlorophenyl)-2-trichloromethyl-quinazolin-4-
amine (13)
Compound 13 was obtained as a yellow solid in 66% yield; mp
208 °C. 1H NMR (200 MHz, CDCl3) d: 7.36–7.43 (m, 2H), 7.64–7.71
(m, 2H), 7.85–7.95 (m, 4H), 8.07–8.11 (m, 1H). 13C NMR (50 MHz,
CDCl3) d: 160.8 (C), 157.5 (C), 149.5 (C), 136.5 (C), 133.8 (CH),
130.0 (CH), 129.5 (C), 129.1 (CH 2), 128.3 (CH), 122.1 (CH 2),
120.1 (CH), 113.7 (C), 97.6 (C). Anal. Calcd for C15H9Cl4N3: C,
48.29; H, 2.43; N, 11.26. Found: C, 48.10, H, 2.41; N, 11.06.
4.1.23. N-(3-Bromophenyl)-2-trichloromethyl-quinazolin-4-
amine (19)
Compound 19 was obtained as a beige solid in 93% yield; mp
181 °C. 1H NMR (200 MHz, CDCl3) d: 7.25–7.29 (m, 2H), 7.63–
7.80 (m, 3H), 7.84–7.95 (m, 2H), 8.06–8.10 (m, 1H), 8.38–8.39
(m, 1H). 13C NMR (50 MHz, CDCl3) d: 160.8 (C), 157.5 (C), 149.6
*
*

P. Verhaeghe et al. / Bioorg. Med. Chem. 17 (2009) 4313–4322
4319
(C), 139.2 (C), 133.9 (CH), 130.2 (CH), 130.1 (CH), 128.4 (CH), 127.3
(CH), 123.9 (CH), 122.8 (C), 120.1 (CH), 119.1 (CH), 113.7 (C), 97.6
(C). Anal. Calcd for C15H9BrCl3N4: C, 43.15; H, 2.17; N, 10.06.
Found: C, 43.53, H, 2.26; N, 10.36.
1H), 7.14–7.33 (m, 2H), 7.66–7.70 (m, 2H), 7.84–7.93 (m, 2H),
8.05–8.12 (m, 2H). 13C NMR (50 MHz, CDCl3) d: 160.8 (C), 160.3
(C), 157.6 (C), 149.4 (C), 139.3 (C), 133.6 (CH), 130.1 (CH), 129.6
(CH), 128.2 (CH), 120.1 (CH), 113.8 (C), 112.3 (CH), 111.0 (CH),
106.0 (CH), 97.7 (C), 55.6 (OCH3). Anal. Calcd for C16H12Cl3N3O:
C, 52.13; H, 3.28; N, 11.40. Found: C, 52.35, H, 3.36; N, 11.07.
4.1.24. N-(4-Bromophenyl)-2-trichloromethyl-quinazolin-4-
amine (20)
Compound 20 was obtained as green crystals in 59% yield; mp
228 °C. 1H NMR (200 MHz, CDCl3) d: 7.59 (d, J = 8.9 Hz, 2H),
7.74–7.81 (m, 1H), 7.91–8.02 (m, 4H), 8.66 (d, J = 8.2 Hz, 1H),
10.28 (br s, 1H). 13C NMR (50 MHz, CDCl3) d: 160.1 (C), 158.4 (C),
4.1.30. N-(4-Methoxyphenyl)-2-trichloromethyl-quinazolin-4-
amine (26)
Compound 26 was obtained as a beige solid in 98% yield; mp
149 °C. 1H NMR (200 MHz, CDCl3) d: 3.81 (s, 3H), 6.90–6.94 (m,
2H), 7.59–7.63 (m, 2H), 7.79–7.88 (m, 4H), 8.00–8.04 (m, 1H). 13C
NMR (50 MHz, CDCl3) d: 160.9 (C), 157.6 (C), 156.5 (C), 149.1 (C),
*
149.1 (C), 138.3 (C), 134.4 (CH), 131.5 (CH 2), 128.8 (CH), 128.4
*
(CH), 123.9 (CH 2), 123.4 (CH), 116.0 (C), 114.1 (C), 98.0 (C). HR
MS (+ESI): m/z 415.9117 (M+H+). Calcd for C15H9BrCl3N3:
133.5 (CH), 131.0 (C), 129.5 (CH), 128.0 (CH), 122.6 (CH 2), 120.5
*
*
414.9045.
(CH), 114.1 (CH 2), 113.7 (C), 97.7 (C), 55.5 (OCH3). HR MS
(+ESI): m/z 368.0112 (M+H+). Calcd for C16H12N3OCl3: 367.0046.
4.1.25. 2-Trichloromethyl-N-(2-trifluoromethylphenyl)-
quinazolin-4-amine (21)
4.1.31. N-o-Tolyl-2-trichloromethylquinazolin-4-amine (27)
Compound 27 was obtained as a white solid in 80% yield; mp
149 °C. 1H NMR (200 MHz, CDCl3) d: 2.42 (s, 3H), 7.10–7.18 (m,
1H), 7.27–7.35 (m, 2H), 7.54 (br s, 1H), 7.59–7.68 (m, 1H), 7.82–
7.90 (m, 2H), 8.03–8.08 (m, 1H), 8.30–8.34 (m, 1H). 13C NMR
(50 MHz, CDCl3) d: 161.1 (C), 158.1 (C), 149.6 (C), 136.0 (C),
133.5 (CH), 130.6 (CH), 130.0 (CH), 129.3 (C), 128.1 (CH), 126.9
(CH), 125.2 (CH), 123.1 (CH), 120.1 (CH), 113.9 (C), 97.7 (C), 18.0
(CH3). Anal. Calcd for C16H12Cl3N3: C, 54.49; H, 3.43; N, 11.92.
Found: C, 54.05, H, 3.55; N, 11.77.
Compound 21 was obtained as a white solid in 40% yield; mp
128 °C. 1H NMR (200 MHz, CDCl3) d: 7.24–7.32 (m, 1H), 7.63–
7.76 (m, 3H), 7.85 (d, J = 8.4 Hz, 1H), 7.91–7.96 (m, 1H), 8.08–
8.12 (m, 2H), 8.90 (d, J = 8.4 Hz, 1H). 13C NMR (50 MHz, CDCl3) d:
160.7 (C), 157.7 (C), 149.7 (C), 135.8 (C), 134.0 (CH), 133.0 (CH),
130.2 (CH), 128.8 (CH), 126.2 (q, J = 5.5 Hz, CH), 124.5 (q,
J = 273.0 Hz, C4), 124.4 (CH), 124.2 (CH), 119.9 (CH), 119.8 (q,
J = 29.0 Hz, C4), 114.0 (C), 97.5 (C). Anal. Calcd for C16H9Cl3F3N3:
C, 47.26; H, 2.23; N, 10.33. Found: C, 47.24, H, 2.18; N, 10.18.
4.1.26. 2-Trichloromethyl-N-(3-trifluoromethylphenyl)-
quinazolin-4-amine (22)
4.1.32. N-m-Tolyl-2-trichloromethylquinazolin-4-amine (28)
Compound 28 was obtained as a pale beige solid in 87% yield;
mp 150 °C. 1H NMR (200 MHz, CDCl3) d: 2.40 (s, 3H), 6.96–7.00
(m, 1H), 7.30–7.33 (m, 1H), 7.60–7.74 (m, 3H), 7.85–7.91 (m,
3H), 8.03–8.07 (m, 1H). 13C NMR (50 MHz, CDCl3) d: 161.1 (C),
157.7 (C), 149.6 (C), 139.0 (C), 137.9 (C), 133.6 (CH), 130.0 (CH),
128.9 (CH), 128.1 (CH), 125.3 (CH), 121.5 (CH), 120.2 (CH), 117.8
(CH), 113.8 (C), 97.9 (C), 21.6 (CH3). Anal. Calcd for C16H12Cl3N3:
C, 54.49; H, 3.43; N, 11.92. Found: C, 54.27, H, 3.46; N, 11.73.
Compound 22 was obtained as a beige solid in 86% yield; mp
139 °C. 1H NMR (200 MHz, CDCl3) d: 7.41–7.58 (m, 2H), 7.66–
7.74 (m, 1H), 7.82–7.96 (m, 1H), 8.00–8.13 (m, 4H), 8.53 (br s,
1H). 13C NMR (50 MHz, CDCl3) d: 160.7 (C), 157.6 (C), 149.3 (C),
138.5 (C), 134.0 (CH), 131.6 (q, J = 32.6 Hz, C4), 129.9 (CH), 129.5
(CH), 128.5 (CH), 123.9 (q, J = 272.0 Hz, C4), 123.7 (CH), 121.0 (q,
J = 3.7 Hz, CH), 120.3 (CH), 118.0 (q, J = 4.0 Hz, CH), 113.6 (C),
97.3 (C). Anal. Calcd for C16H9Cl3F3N3: C, 47.26; H, 2.23; N, 10.33.
Found: C, 47.08, H, 2.21; N, 10.36.
4.1.33. N-p-Tolyl-2-trichloromethylquinazolin-4-amine (29)
Compound 29 was obtained as a yellow solid in 71% yield; mp
146 °C. 1H NMR (200 MHz, CDCl3) d: 2.23 (s, 3H), 7.03–7.07 (m,
2H), 7.43–7.52 (m, 1H), 7.69–7.83 (m, 5H), 7.91–7.95 (m, 1H). 13C
NMR (50 MHz, CDCl3) d: 161.0 (C), 157.6 (C), 149.5 (C), 135.4 (C),
4.1.27. 2-Trichloromethyl-N-(4-trifluoromethylphenyl)-
quinazolin-4-amine (23)
Compound 23 was obtained as a white solid in 61% yield; mp
157 °C. 1H NMR (200 MHz, DMSO-d6) d: 7.76–7.81 (m, 3H), 7.98–
7.99 (m, 2H), 8.30 (d, J = 8.4 Hz, 2H), 8.72 (d, J = 7.9 Hz, 1H),
10.46 (br s, 1H). 13C NMR (50 MHz, DMSO-d6) d: 160.3 (C), 158.9
(C), 149.5 (C), 142.9 (C), 134.9 (CH), 129.2 (CH), 128.9 (CH), 126.3
*
134.1 (C), 133.5 (CH), 129.8 (CH), 129.5 (CH 2), 128.0 (CH), 120.8
*
(CH 2), 120.2 (CH), 113.7 (C), 97.8 (C), 20.9 (CH3). Anal. Calcd for
C16H12Cl3N3: C, 54.49; H, 3.43; N, 11.92. Found: C, 54.11, H, 3.45;
N, 11.74.
*
(q, J = 3.7 Hz, CH 2), 124.9 (q, J = 272.0 Hz, C4), 124.2 (q,
*
J = 32.0 Hz, C4), 123.9 (CH), 122.0 (CH 2), 114.5 (C), 98.2 (C). HR
4.1.34. N-(3,4-Dichlorophenyl)-2-trichloromethyl-quinazolin-
4-amine (30)
MS (+ESI): m/z 405.9881 (M+H+). Calcd for C16H9F3N3Cl3: 404.9814.
Compound 30 was obtained as a white solid in 80% yield; mp
223 °C. 1H NMR (200 MHz, CDCl3) d: 7.46 (d, J = 8.8 Hz, 1H),
7.62–7.74 (m, 3H), 7.87–7.95 (m, 2H), 8.07–8.13 (m, 1H), 8.35 (d,
J = 2.5 Hz, 1H). 13C NMR (50 MHz, CDCl3) d: 160.8 (C), 157.4 (C),
149.8 (C), 137.4 (C), 134.0 (CH), 133.0 (C), 130.5 (CH), 130.3 (CH),
128.5 (CH), 127.6 (C), 122.7 (CH), 119.9 (CH), 119.8 (CH), 113.6
(C), 97.5 (C). Anal. Calcd for C15H8Cl5N3: C, 44.21; H, 1.98; N,
10.31. Found: C, 44.35; H, 2.00; N, 10.19.
4.1.28. N-(2-Methoxyphenyl)-2-trichloromethyl-quinazolin-4-
amine (24)
Compound 24 was obtained as a yellow solid in 86% yield; mp
199 °C. 1H NMR (200 MHz, CDCl3) d: 4.01 (s, 3H), 6.98–7.12 (m,
3H), 7.61–8.06 (m, 4H), 8.62 (br s, 1H), 9.03–9.09 (m, 1H). 13C
NMR (50 MHz, CDCl3) d: 161.4 (C), 157.2 (C), 149.5 (C), 148.3 (C),
133.4 (CH), 129.9 (CH), 128.1 (CH), 127.8 (C), 123.6 (CH), 121.4
(CH), 120.5 (CH), 120.3 (CH), 114.4 (C), 109.9 (CH), 97.9 (C), 56.1
(OCH3). Anal. Calcd for C16H12Cl3N3O: C, 52.13; H, 3.28; N, 11.40.
Found: C, 52.23, H, 3.31; N, 11.33.
4.1.35. N-(2,3-Dichlorophenyl)-2-trichloromethyl-quinazolin-
4-amine (31)
Compound 31 was obtained as a white solid in 25% yield; mp
231 °C. 1H NMR (200 MHz, CDCl3) d: 7.25–7.41 (m, 2H), 7.71–7.79
(m, 1H), 7.90–8.01 (m, 2H), 8.13 (d, J = 7.8 Hz, 1H), 8.50 (br s,
1H), 9.08 (dd, J = 1.7 and 8.1 Hz, 1H). 13C NMR (50 MHz, CDCl3)
d: 160.7 (C), 157.3 (C), 149.7 (C), 136.3 (C), 134.0 (CH), 132.8
4.1.29. N-(3-Methoxyphenyl)-2-trichloromethyl-quinazolin-4-
amine (25)
Compound 25 was obtained as a green solid in 90% yield; mp
142 °C. 1H NMR (200 MHz, CDCl3) d: 3.89 (s, 3H), 6.71–6.76 (m,

4320
P. Verhaeghe et al. / Bioorg. Med. Chem. 17 (2009) 4313–4322
(C), 130.3 (CH), 128.9 (CH), 128.1 (CH), 125.0 (CH), 121.8 (C),
120.1 (CH), 119.9 (CH), 114.2 (C), 97.5 (C). Anal. Calcd for
C15H8Cl5N3: C, 44.21; H, 1.98; N, 10.31. Found: C, 44.31, H,
1.96; N, 10.26.
7.69 (m, 2H), 7.83–8.08 (m, 5H). 13C NMR (50 MHz, CDCl3) d:
160.8 (C), 157.5 (C), 149.5 (C), 145.3 (C), 136.6 (C), 133.8 (CH),
*
*
130.1 (CH), 128.4 (CH), 121.8 (CH 2), 121.7 (CH 2), 120.5 (q,
J = 257.0 Hz, C4), 120.1 (CH), 113.6 (C), 97.6 (C). Anal. Calcd for
C16H9Cl3N3O: C, 45.47; H, 2.15; N, 9.94. Found: C, 45.51; H, 2.37;
N, 9.86.
4.1.36. N-(2,6-Dichlorophenyl)-2-trichloromethyl-quinazolin-
4-amine (32)
Compound 32 was obtained as a white solid in 9% yield; mp
164 °C. 1H NMR (200 MHz, CDCl3) d: 7.22–7.33 (m, 2H), 7.44–
7.48 (m, 2H), 7.66–7.74 (m, 1H), 7.87–7.96 (m, 1H), 8.02 (d,
J = 8.2 Hz, 1H), 8.11 (d, J = 8.2 Hz, 1H). 13C NMR (50 MHz, CDCl3)
4.1.42. N-(Naphtalen-1-yl)-2-trichloromethylquinazolin-4-
amine (38)
Compound 38 was obtained as a pale pink solid in 33% yield;
mp 179 °C. 1H NMR (200 MHz, CDCl3) d: 7.54–7.80 (m, 5H), 7.86–
8.13 (m, 6H), 8.41 (d, J = 7.1 Hz, 1H). 13C NMR (50 MHz, CDCl3) d:
161.1 (C), 158.9 (C), 149.8 (C), 134.2 (C), 133.7 (CH), 132.6 (C),
130.1 (CH), 129.0 (CH), 128.2 (CH), 127.4 (C), 126.5 (CH), 126.1
(CH), 126.0 (CH), 125.8 (CH), 121.1 (CH), 120.5 (CH), 120.4 (CH),
114.0 (C), 97.6 (C). Anal. Calcd for C19H12Cl3N3: C, 58.71; H, 3.11;
N, 10.81. Found: C, 58.96; H, 3.15; N, 10.64.
*
d: 160.9 (C), 159.0 (C), 150.0 (C), 133.9 (CH), 133.8 (C4 2), 132.7
*
(C), 130.0 (CH), 128.6 (CH), 128.5 (CH 2), 128.2 (CH), 120.9 (CH),
113.7 (C), 97.3 (C). HR MS (+ESI): m/z 405.9237 (M+H+). Calcd for
C15H8Cl5N3: 404.9161.
4.1.37. N-(2,4-Dichlorophenyl)-2-trichloromethyl-quinazolin-
4-amine (33)
Compound 33 was obtained as a white solid in 75% yield; mp
204 °C. 1H NMR (200 MHz, CDCl3) d: 7.40 (dd, J = 2.4 and 9.0 Hz,
1H), 7.48 (d, J = 2.4 Hz, 1H), 7.69–7.77 (m, 1H), 7.89–7.98 (m,
2H), 8.07–8.14 (m, 1H), 8.35 (br s, 1H), 9.10 (d, J = 9.0 Hz, 1H).
13C NMR (50 MHz, CDCl3) d: 160.6 (C), 157.2 (C), 149.6 (C), 134.0
(CH), 133.4 (C), 130.3 (CH), 128.9 (C), 128.8 (CH), 128.7 (CH),
128.2 (CH), 123.6 (C), 122.7 (CH), 120.0 (CH), 114.1 (C), 97.5 (C).
Anal. Calcd for C15H8Cl5N3: C, 44.21; H, 1.98; N, 10.31. Found: C,
44.15, H, 1.96; N, 10.25.
4.1.43. General procedure for the preparation of compounds
(39)–(41)
To a refluxing mixture of acetic acid and iron (15 equiv), 1 equiv
of the corresponding trichloromethylated precursor reactant
(respectively compounds 10, 22 and 33) was added. The reaction
mixture was then stirred for 1 h and filtered over Celite. Acetic acid
was evaporated under reduced pressure, giving an orange residue
which was dissolved in dichloromethane. The organic layer was
washed with water, dried over anhydrous Na2SO4 and concen-
trated in vacuo. The crude residue was purified by chromatography
on a silica gel, eluting with ethyl acetate to afford the correspond-
ing reduction product.
4.1.38. N-(3,5-Dichlorophenyl)-2-trichloromethyl-quinazolin-
4-amine (34)
Compound 34 was obtained as a white solid in 76% yield; mp
216 °C. 1H NMR (200 MHz, CDCl3) d: 7.14–7.15 (m, 1H), 7.65–
7.72 (m, 2H), 7.86–7.95 (m, 2H), 8.00 (d, J = 1,4 Hz, 2H), 8.09 (d,
J = 8.1 Hz, 1H). 13C NMR (50 MHz, CDCl3) d: 160.7 (C), 157.4 (C),
4.1.44. N-(3-Chlorophenyl)-2-methylquinazolin-4-amine (39)
Compound 39 was obtained as a beige solid in 99% yield; mp
171 °C. 1H NMR (200 MHz, CDCl3) d: 2.72 (s, 3H), 7.11 (dd, J = 0.9
and 8.0 Hz, 1H), 7.32 (dd, J = 8.0 and 8.2 Hz, 1H), 7.50 (dd, J = 0.9
and 8.0 Hz, 1H), 7.64–7.88 (m, 5H), 8.01 (br s, 1H). 13C NMR
(50 MHz, CDCl3) d: 163.9 (C), 156.9 (C), 150.4 (C), 139.8 (C),
134.6 (C), 133.1 (CH), 129.9 (CH), 128.2 (CH), 126.0 (CH), 123.9
(CH), 121.0 (CH), 120.0 (CH), 118.9 (CH), 113.0 (C), 26.4 (CH3).
HR MS (+ESI): m/z 270.0793 (M+H+). Calcd for C15H12ClN3:
269.0720.
*
149.8 (C), 139.7 (C), 135.3 (C4 2), 134.0 (CH), 130.2 (CH), 128.6
*
(CH), 124.2 (CH), 120.0 (CH), 119.1 (CH 2), 113.6 (C), 97.5 (C). Anal.
Calcd for C15H8Cl5N3: C, 44.21; H, 1.98; N, 10.31. Found: C, 44.14, H,
2.00; N, 10.19.
4.1.39. N-(2,5-Dichlorophenyl)-2-trichloromethyl-quinazolin-
4-amine (35)
Compound 35 was obtained as a white solid in 47% yield; mp
176 °C. 1H NMR (200 MHz, CDCl3) d: 7.06–7.12 (m, 1H), 7.40 (d,
J = 8.6 Hz, 1H), 7.71–8.16 (m, 4H), 8.41 (br s, 1H), 9.35–9.36 (m,
1H). 13C NMR (50 MHz, CDCl3) d: 160.7 (C), 157.1 (C), 149.8 (C),
135.4 (C), 134.1 (CH), 133.9 (C), 130.3 (CH), 129.5 (CH), 128.8
(CH), 124.2 (CH), 122.1 (CH), 121.1 (C), 121.0 (CH), 114.1 (C),
97.5 (C). Anal. Calcd for C15H8Cl5N3: C, 44.21; H, 1.98; N, 10.31.
Found: C, 44.18, H, 1.92; N, 10.11.
4.1.45. 2-Methyl-N-(3-trifluoromethylphenyl)-quinazolin-4-
amine (40)
Compound 40 was obtained as a beige solid in 99% yield; mp
178 °C. 1H NMR (200 MHz, CDCl3) d: 2.72 (s, 3H), 7.40 (d,
J = 7.7 Hz, 1H), 7.52 (dd, J = 7.3 and 7.7 Hz, 2H), 7.74–8.06 (m,
5H), 8.23 (br s, 1H). 13C NMR (50 MHz, CDCl3) d: 163.7 (C), 157.1
(C), 149.9 (C), 139.1 (C), 133.3 (C), 131.4 (q, J = 32.6 Hz, C4),
129.5 (CH), 127.8 (CH), 126.2 (CH), 124.1 (CH), 123.9 (q,
J = 272.2 Hz, C4), 120.6 (q, J = 3.6 Hz, CH), 120.3 (CH), 118.0 (q,
J = 4.1 Hz, CH), 112.9 (C), 26.2 (CH3). HR MS (+ESI): m/z 304.1058
(M+H+). Calcd for C16H12F3N3: 303.0983.
4.1.40. N-[3,5-bis(Trifluoromethyl)phenyl]-2-trichloro-
methylquinazolin-4-amine (36)
Compound 36 was obtained as a white solid in 35% yield; mp
179 °C. 1H NMR (200 MHz, CDCl3) d: 7.67–7.77 (m, 2H), 7.90–
7.97 (m, 2H), 8.02 (d, J = 8.2 Hz, 1H), 8.12 (d, J = 8.2 Hz, 1H),
8.56–8.57 (m, 2H). 13C NMR (50 MHz, CDCl3) d: 160.6 (C), 157.4
4.1.46. N-(2,4-Dichlorophenyl)-2-methylquinazolin-4-amine
(41)
*
(C), 149.8 (C), 139.4 (C), 134.3 (CH), 132.4 (q, J = 33.6 Hz, C4 2),
*
*
Compound 41 was obtained as a beige solid in 95% yield; mp
154 °C. 1H NMR (200 MHz, CDCl3) d: 2.75 (s, 3H), 7.32–7.38 (m,
1H), 7.45–7.46 (m, 1H), 7.52–7.60 (m, 1H), 7.77–7.85 (m, 1H),
7.90–7.94 (m, 2H), 8.19 (br s, 1H), 8.88 (d, J = 8.3 Hz, 1H). 13C
NMR (50 MHz, CDCl3) d: 163.6 (C), 156.6 (C), 149.9 (C), 134.0
(C), 133.3 (CH), 128.8 (CH), 128.4 (C), 128.0 (CH), 127.8 (CH),
126.4 (CH), 124.0 (C), 122.9 (CH), 120.1 (CH), 113.3 (C), 26.3
(CH3). HR MS (+ESI): m/z 304.0398 (M+H+). Calcd for C15H11Cl2N3:
303.0330.
130.2 (CH), 128.9 (CH), 123.1 (q, J = 273.0 Hz, C4 2), 120.6 (q,
J = 3.3 Hz, CH), 120.0 (CH), 117.5 (q, J = 3.7 Hz, CH 2), 113.5 (C),
97.2 (C). Anal. Calcd for C17H8Cl3F6N3: C, 43.02; H, 1.70; N, 8.85.
Found: C, 43.36, H, 1.84; N, 8.93.
4.1.41. 2-Trichloromethyl-N-(4-trifluoromethoxy-
phenyl)quinazolin-4-amine (37)
Compound 37 was obtained as a white solid in 70% yield; mp
153 °C. 1H NMR (200 MHz, CDCl3) d: 7.25–7.29 (m, 2H), 7.62–

P. Verhaeghe et al. / Bioorg. Med. Chem. 17 (2009) 4313–4322
4321
4.2. Biology/material and methods
trols, 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 in-
duce a 50% decrease of parasite growth (IC50) were calculated from
three independent experiments.
4.2.1. General
Cell culture medium (RPMI 1640), foetal calf serum (FCS), L-glu-
tamine, 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 DMSO. Stock solutions of reference drugs (doxorubicin,
pyrimethamine, amphotericin B, chloroquine and doxycycline)
were prepared in ultrapure H2O or DMSO. Flow cytometry was per-
formed at the Faculté 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).
4.2.4. Antitoxoplasmic activity
The effects of the tested compounds on the growth of T. gondii
tachyzoites (PRU-b-Gal strain, kindly provided by Pr. I. Villena, Re-
ims, France) were assessed by a colorimetric microtiter assay
according to the method of Mc Fadden et al.18 Briefly, tachyzoites
were maintained by serial passage in confluent human foreskin
fibroblast (HFF) monolayer (a gift of Pr. I. Dimier-Poisson, Tours,
France). For assay, 96-well microtiter plates were seeded with
104 HFF cells and allowed to grow to confluence in RPMI 1640
4.2.2. Antiplasmodial activity
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 de-
scribed previously by Trager and Jensen.15 Parasites were culti-
vated in 75 cm2-flasks containing RPMI 1640 (20 mL)
supplemented with 25 mM HEPES, 25 mM NaHCO3, 10% of A+ hu-
man serum and 1 mL of washed erythrocytes (final haematocrit
2.5%). Parasitaemia was maintained daily between 1% and 6%. Dilu-
tions used non-infected A+ 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 previ-
ously described using hydroethidine (HE, Interchim, Montluçon,
France) that is converted by metabolically active parasites into
ethidium.16 After incubation with hydroethidine, parasitized and
uninfected erythrocytes were all identified on the basis of fluores-
cence intensity and size. Triplicate assays were performed in 96-
well plates (Nunc Brand products, Fisher, Paris, France) containing
(without phenol red) supplemented with 10% FCS and 1% L-gluta-
mine/penicillin-streptomycin mix at 37 °C with 6% CO2. Cell mono-
layers were infected with 2.103 parasites per well and incubated for
3 h at 37 °C with 6% CO2. Then, various concentrations of com-
pounds dissolved in DMSO (final concentration less than 0.5% v/v)
were incorporated in triplicate. Pyrimethamine was used as the ref-
erence drug compound. Appropriate controls treated by DMSO or
pyrimethamine were added to each set of experiments. Negative
control consisted in cell monolayers incubated without parasite
and drug. After a 96-h incubation period at 37 °C with 6% CO2,
20
l
L of chlorophenol red-b-
D-galactopyranoside (CPRG) were
added to each well to give a final concentration of 500
lM. The
plates were incubated at 37 °C with 6% CO2 for an additional 24 h,
at which time b-galactosidase activity was measured by reading
plates at 570 and 630 nm on a Biotek microtiter plate reader. Blank-
ing 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.
200
l
L of asynchronous parasite cultures at 2% of parasitaemia and
L of the appropriate extract dissolved in
2% haematocrit, and 5
l
DMSO or ultrapure H2O. Negative control, treated by solvents
(DMSO or H2O) and positive controls (chloroquine and doxycy-
cline) were added to each set of experiments. After 48 h incubation
without medium change, plates were centrifuged and the upper
4.2.5. Cytotoxic assays on human myeloid leukemic K562
Cytotoxicity was assessed on chemosensitive subline K562 de-
rived from a chronic myeloid leukemia and purchased from Dr J.
Boutonnat (UMR-CNRS 5525, Université Joseph Fourier, La Tron-
che, France).19 Late log-phase K562 cells were incubated in RPMI
liquids were replaced with 200 lL hydroethidine solution
[0.05 mg/ml in phosphate buffered saline (PBS)]. Plates were incu-
bated 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; Fluo-
rescence 2 (FL2), red fluorescence: Voltage 459, gain 1, mode Log.
The concentrations of compounds required to induce a 50% de-
crease of infected erythrocytes (IC50W2) were calculated from
three independent experiments.
1640 (without phenol red) supplemented with 10% FSC, 2% L-gluta-
mine and 1% penicillin-streptomycin mix (complete RPMI med-
ium) and a range of compound concentrations incorporated in
duplicate (final DMSO concentration less than 0.5%). Appropriate
controls treated with or without solvent (DMSO), and various con-
centrations of doxorubicin (positive control), chloroquine, doxycy-
cline, amphotericin B and pyrimethamine were added to each set
of experiments. After 72 h incubation at 37 °C and 6% CO2, cell
growth was measured by flow cytometry after staining monocytes
with 5
lL of propidium iodide. Antiproliferative activity was eval-
uated by counting the number of live cells in a 100
lL sample.
4.2.3. Antileishmanial activity
Inhibitory concentration 50% (IC50 K562) was defined as the con-
centration of drug required to induce a 50% in the K562 cell prolif-
eration 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 va-
lue calculated from three independent experiments.
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.17
Briefly, promastigotes in log-phase in M199 medium supple-
mented with 10% FCS and 500 lg/mL of G418 were incubated at
an average density of 105 parasites/mL in 24-well plates with var-
ious concentrations of compounds dissolved in DMSO (final con-
centration less than 0.5% v/v) incorporated in duplicate.
Amphotericin B was used as the reference drug. Appropriate con-
4.2.6. MTT assays
The evaluation of the tested molecules’ cytotoxicity on the
HepG2 cell line was done according to the method of Mosmann20
with slight modifications. Briefly, cells in 100 lL of complete med-

4322
P. Verhaeghe et al. / Bioorg. Med. Chem. 17 (2009) 4313–4322
ium, [RPMI supplemented with 10% foetal bovine serum, 1%
tamine (200 mM) and penicillin (100 U/mL)/streptomycin (100
L
-glu-
g/
Acknowledgments
l
mL)] were inoculated into each well of 96-well plates and incu-
bated at 37 °C in a humidified 6% CO2 with 95% air atmosphere.
After 24 h incubation, 100 ll of medium with various product con-
centrations was added and the plates were incubated for 72 h. At
This work was supported by the CNRS and the Université de la
Méditerranée. The authors would like to thank the Assistance Pub-
lique-Hôpitaux de Marseille for hospital appointment, Vincent
Remusat for recording all the NMR spectra, France Sifredi for her
technical assistance, Rémi Legay (CERMN) for recording some of
the HR mass spectra and also Virginie Poncelet (J&J Pharmaceutical
Research Center, Val de Reuil) for her final contribution to this
work.
the end of the treatment and incubation, the medium was aspi-
rated from the wells and 10
lL MTT solution (5 mg MTT/mL in
PBS) was added to each well with 100
ll of medium without foetal
calf serum. Cells were incubated for 2 h at 37 °C to allow MTT oxi-
dation by mitochondrial dehydrogenase in the viable cells. After
this time, the MTT solution was aspirated and DMSO (100 lL)
References and notes
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 determined from the dose–re-
sponse curve.
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