Synthesis and antiplasmodial activity of new 4-aryl-2-trichloromethylquinazolines

Article abstract of DOI:10.1016/j.bmcl.2007.10.027

A series of original 4-aryl-substituted 2-trichloromethylquinazoline derivatives was synthesized using a microwave-assisted Suzuki-Miyaura cross-coupling approach. Antiplasmodial activity was evaluated on both chloroquino-resistant and -sensitive Plasmodi

Full text of DOI:10.1016/j.bmcl.2007.10.027

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 396–401
Synthesis and antiplasmodial activity of new
4-aryl-2-trichloromethylquinazolines
a
b
b
b
´
Pierre Verhaeghe, Nadine Azas, Monique Gasquet, Sebastien Hutter,
b
c
d
`
Christophe Ducros, Michele Laget, Sylvain Rault,
Pascal Rathelota and Patrice Vanellea,*
aLaboratoire de Chimie Organique Pharmaceutique, UMR CNRS 6517, Faculte´ de Pharmacie, Universite´ de la Me´diterrane´e,
27 Bd. Jean Moulin, 13385 Marseille Cedex 05, France
bLaboratoire de Parasitologie, EA 864, Faculte´ de Pharmacie, Universite´ de la Me´diterrane´e,
27 Bd. Jean Moulin, 13385 Marseille Cedex 05, France
cLaboratoire de Microbiologie, EA 864, Faculte´ de Pharmacie, Universite´ de la Me´diterrane´e,
27 Bd. Jean Moulin, 13385 Marseille Cedex 05, France
dCentre d’Etudes et de Recherche sur le Me´dicament de Normandie, UPRES EA 3915, UFR des Sciences Pharmaceutiques,
Universite´ de Caen, 5 rue Vaube´nard, 14032 Caen Cedex, France
Received 17 September 2007; revised 5 October 2007; accepted 6 October 2007
Available online 17 October 2007
Abstract—A series of original 4-aryl-substituted 2-trichloromethylquinazoline derivatives was synthesized using a microwave-
assisted Suzuki-Miyaura cross-coupling approach. Antiplasmodial activity was evaluated on both chloroquino-resistant and -sen-
sitive Plasmodium falciparum strains, and the selectivity indexes for THP1 and HepG2 human cells were also calculated, revealing
their antiplasmodial potential.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Malaria remains one of the most lethal diseases, killing
more than 1 million people each year according to the
WHO. The therapeutic management of this infectious
disease has been considerably hindered because of the
emergence of chloroquino-resistant strains of Plasmo-
dium falciparum. Moreover, malaria mainly affects
developing countries in which treatment cost is a major
constraint on medical support. In such a context, it
appears essential to identify new molecules with anti-
plasmodial potential, with a view to developing new
pharmacological agents which could contribute to solv-
ing this public health challenge.
in position 2, via a microwave-assisted Suzuki-Miyaura
cross-coupling reaction.
Certain molecules including a 2-trichloromethyl- substi-
tuted quinazoline ring present pharmacological properties
valuable for oncology5,6 and infectiology.7 Furthermore,
other quinazoline-derived molecules appear to be anti-
malarial agents.8 In parallel, trichloromethylated struc-
tures such as triazine derivatives show the same anti-
infectious activity.9
We recently developed a microwave-assisted chlorina-
tion reaction protocol suitable for a-methylazahetero-
cycles, and one of the molecules prepared during this
In continuation of previous studies1–4 resulting from our
research program directed toward the preparation of
new azaheterocyclic compounds and their anti-infec-
tious evaluation, we synthesized a series of new tricyclic
quinazoline derivatives bearing a trichloromethyl group
work was 4-chloro-2-trichloromethyl-quinazoline
1
(Fig. 1).10 This molecule, obtained in good yields with
O
N
Cl
N
H
POCl3
N
N
PCl5
Keywords: Malaria; Quinazoline; Microwave-assisted chemistry; Suzu-
ki-Miyaura cross-coupling reaction.
800 W / 15 min
CH3
CCl3
75%
1
*
Corresponding author. Tel.: +33 4 91 83 55 80; fax: +33 4 91 79 46
77; e-mail: patrice.vanelle@pharmacie.univ-mrs.fr
Figure 1. Synthesis of 4-chloro-2-trichloromethylquinazoline 1.
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.027

P. Verhaeghe et al. / Bioorg. Med. Chem. Lett. 18 (2008) 396–401
397
a short reaction time, also presented the advantage of
being functionalized in position 4. It was initially evalu-
ated for its antiplasmodial potential, which appeared
limited.
Eleven reaction parameters were tested through 50 trials
and the best conditions were finally defined (Table 1),
permitting the synthesis of 4-phenyl-2-trichloromethyl-
quinazoline 213 with 65% yield in only 2 h, using the sim-
ple Pd(OAc)2 catalyst.
We therefore investigated the possibility of using 4-
chloro-2-trichloromethylquinazoline 1 as a substrate
for a Suzuki-Miyaura cross-coupling reaction, hoping
that the substitution of an aryl group for the chlorine
atom in position 4 would improve biological activity.
Using microwave-assisted general operating procedure,
we prepared a series of 4-aryl-2-trichloromethylquina-
zolines14 by varying the boronic acid reagents (Table
2). The reaction yields obtained were little influenced
by the nature of the boronic acid (from 50% to 65%).
In a recent publication, Connolly and co-workers stud-
ied Suzuki-Miyaura cross-coupling reaction from 2-
substituted-4-chloroquinazolines, using Pd(PPh3)4 as a
catalyst. This reaction seems to be greatly influenced
by the nature of the quinazoline substituent in position
2, varying the reaction yields from 53% to 93%.11
In order to demonstrate that the trichloromethyl
group was mainly responsible for the biological activ-
ity of our molecules, we evaluated a dehalogenated
analog, 2-methyl-4-phenylquinazoline 13,13 and we
also included 4-phenyl-2-trichloromethylquinazoline 2
in a nucleophilic substitution with methylamine, lead-
ing to the amidine N,N0-dimethyl-4-phenylquinazoline-
2-carboximidamide hydrochloride 1415 (Fig. 3).
A trichloromethyl group was not automatically compat-
ible with such a reaction mainly because of the sensitivity
of the trichloromethyl group to alkaline mediums and
because of the formation of Csp3/Csp3 homocoupling
by-products that we observed in nitrotrichloromethyl-
quinoline and trichloromethylbenzoxazole series. These
side reactions were also noted in trichloromethylquino-
line and trichloromethylbenzene series by Folli and co-
workers,12 in reactions using other metals such as iron.
All molecules were assessed for their in vitro antiplas-
modial activity against two different strains of P. falcipa-
rum: W2 chloroquino-resistant and 3D7 chloroquino-
sensitive. In order to identify our molecules’ potential,
a drug compound of reference, chloroquine, was tested
in the same conditions. The IC50 values were then calcu-
lated (Table 2).2,16,17
This cross-coupling reaction was therefore studied with
the assistance of a liquid chromatography/mass spec-
trometry apparatus, affording the possibility of studying
the influence of each reaction parameter in an efficient
way. All reaction trials were conducted with 4-chloro-
2-trichloromethylquinazoline 1 as a substrate and phen-
ylboronic acid as a reagent, in a synthesis microwave
oven, inside miniaturized sealed reactors, so as to realize
each trial on limited amounts of reagents (Fig. 2).
Usually, gem-trichloromethylated compounds exert an
anti-infectious activity which is directly linked to their
toxicity. To synthesize molecules of therapeutic interest,
it was necessary for us to determine whether our mole-
cules were presenting a toxic profile on human cells, or
if they could offer in vitro selective antiparasitic proper-
ties. Thus, the IC50 were determined for two different
human cell lines (THP1 and HepG2), using doxorubicin
as reference of cellular toxicity.17–19
Then the selectivity indexes of all compounds and refer-
ence drug compounds were calculated (Table 2).20 Some
of the tested molecules emerge as interesting antiplasmo-
dial agents because of their safe and active profile.
Ar
N
Cl
N
Solvent
N
N
Ar B(OH)2
Base "Pd"
Microwave
CCl3
CCl3
1
Figure 2. General Suzuki-Miyaura cross-coupling reaction for the
preparation of 4-aryl-2-trichloromethylquinazolines from 1.
Apart from molecule 12, these quinazoline derivatives
do not display any in vitro toxic properties neither
Table 1. Parameters studied for the Suzuki-Miyaura reaction optimization
Type of parameter studied
Trials
Best reaction conditions
DMF
Solvent
Base
DME, dioxane, DMF
CsF, K3PO4, Na2CO3, NaHCO3, Cs2CO3
PdP(Ph3)4, Pd(OAc)2
None or CuI
Cs2CO3
Pd(OAc)2
None
1.3
Catalyst
Co-catalyst
Nb equiv boronic acid (equiv)
Nb equiv base (equiv )
Nb equiv catalyst (%)
Temperature (ꢁC)
M.W. power (W)
Pressure (bar)
Time (h)
1, 1.3, 1.5
2, 2.5, 3
2.5
2.5
2.5, 5
100, 150, 175
150
150
100, 150, 200, 250
+0, 0.5, 1, 4
0.5, 1, 1.5, 2, 2.5, 3
+0.5
2
Reactions were performed in a Biotage Initiatorꢂ monomode microwave reactor.
The analytical LC/MS system used was a Waters Allianceꢂ 2695.

Table 2. Antiplasmodial activity and human cell toxicity of quinazoline derivatives
R2
N
N
R1
Drugs
Quinazoline substituents
R2
Antiplasmodial
activitya (lM)
Human cell toxicitya (lM)
W2 strain selectivity
index toward HepG2b
W2 strain selectivity
index toward THP1b
R1
W2 IC50
3D7 IC50
MTT (HEP G2) IC50
THP1 IC50
THP1 LC50
1
CCl3
CCl3
Cl
Ph
54.5
8.0
73.2
20.4
>125e
>125e
>500e
125
>500e
>500e
>2.3
>15.6
>9.2
15.6
2
3
CCl3
CCl3
2-CH3-Ph
4-F-Ph
Insufficient solubilityf
4
2.5
3.0
>125e
>125e
>125e
>125e
125
125
500
>50.0
>4.3
50.0
>8.7
15.6
1.9
5
CCl3
CCl3
3,4,5-tri-OCH3-Ph
4-Cl-Ph
28.9
4.7
23.3
2.2
>250e
73.2
78.6
250
>250e
>500e
>500e
>250e
>500e
6
>26.6
>3.1
11.5
7
CCl3
CCl3
3-CF3-Ph
4-OCH3-Ph
2-Naphthyl
1-Naphthyl
2-Furyl
40.4
10.9
8.1
36.6
25.0
23.3
8
22.9
>61.7
9
CCl3
CCl3
>125e
>500e
>15.4
10
11
12
13
14
Insufficient solubilityf
CCl3
CCl3
20.2
8.7
30.0
24.0
>125e
25
>125e
>125e
61.6
50
>500e
125
>500e
>500e
>6.2
2.9
3.1
5.7
2.0
ꢀ5
5-CH3-2-thienyl
Ph
Ph
CH3
N,N0-Dimethyl-amidinyl
62.4
>100
>125
>100
125
>500e
>2
ꢀ1.25
42.9
Ref.
Ref.
Chloroquinec
Doxorubicind
0.7
0.08
30
0.2
40
0.05
250
2.5
57.1
a Mean of three independent experiments.
b Selectivity index was calculated according to the following formula: SIW2Plasmodium = human cell IC50/W2 Plasmodium IC50
c Chloroquine was used as the antiplasmodial drug compound of reference.
.
d Doxorubicin was used as the drug compound of reference for human cell toxicity.
e No toxicity at the highest tested concentration.
f Products 3 and 10 could not be evaluated because of their lack of solubility in the culture medium.

P. Verhaeghe et al. / Bioorg. Med. Chem. Lett. 18 (2008) 396–401
399
times higher. However, if we consider the 9 lM W2
IC50 value of doxycycline, another common commer-
cial antimalarial agent,26 we note that molecules 2,
4, 6, 8, 9, and 12 work in the same concentration
range.
HCl
DMF
N
N
H3C NH2
N
150˚C / 10 h
N
CH3
N
CCl3
14
2
HN
30%
CH3
Finally, molecules 2, 4, 6, 8, and 9 show a favorable anti-
plasmodial activity profile toward THP1 and HepG2
human cell lines. Molecule 4, hit compound in this series
(W2 IC50 = 2.5 lM), reaches quite good selectivity
indexes (50), similar to those of chloroquine, and so, de-
spite superior IC50 values because of its safer profile to-
ward human cells.
Figure 3. Preparation of amidine hydrochloride 14.
toward THP1 cell line nor toward HepG2. HepG2 is a
commonly used human-derived hepatoma cell line that
has shown characteristics similar to those of primary
hepatocytes. The goal of cytotoxic assay using HepG2,
in addition to classic THP1 cells, was to evaluate the
impact of metabolic activation of the tested
compounds on the viability of a human hepatic cell
line.21,22
The next step of our research program will consist in
studying the mechanism by which these molecules pro-
duce their antiplasmodial effect.
For most of these molecules, the comparison of the
two IC50 values calculated for each Plasmodium strain
seems to indicate that their mode of action is not af-
fected by the resistance to chloroquine, as both chloro-
quino-resistant and -sensitive strains require roughly
the same IC50.
Acknowledgments
´
This work was supported by the CNRS and l’Universite
de la Mediterranee. The authors thank the Assistance
´
´
ˆ
´
Publique-Hopitaux de Marseille, for hospital appoint-
1
ment, V. Remusat for the H and 13C NMR spectra
recording and are thankful to F. Sifredi and L. Hadjadj
for their technical support.
Moreover, an antileshmanial evaluation was performed
on Leishmania donovani promastigotes and gave IC50
values >100 lM for all molecules (reference drug com-
pound being amphotericin B with a IC50 = 0.2 lM),
indicating that these structures do not display any
antileishmanial activity.23–25 This difference observed
between antiplasmodial and antileishmanial activities
suggests that these molecules may exert specific antipar-
asitic effects on coccidies, with no effect on kinetoplast-
ids. We are planning to check this hypothesis by
testing the same molecules on other plasmatic coccidies
such as Toxoplasma.
References and notes
1. Rathelot, P.; Vanelle, P.; Gasquet, M.; Delmas, F.;
Crozet, M. P.; Timon-David, P.; Maldonado, J. Eur. J.
Med. Chem. 1995, 30, 5031.
2. Hout, S.; Azas, N.; Darque, A.; Robin, M.; Di Giorgio,
C.; Gasquet, M.; Galy, G.; Timon-David, P. Parasitology
2004, 129, 525.
3. Verhaeghe, P.; Rathelot, P.; Rault, S.; Vanelle, P. Lett.
Org. Chem. 2006, 3, 891.
´
4. Crozet, M. D.; Remusat, V.; Curti, C.; Vanelle, P. Synth.
Commun. 2006, 36, 3639.
Having a look at results observed with test compounds
13 and 14 (without a CCl3 group), it appears that the
molecular skeleton responsible for the antiplasmodial
activity is the 2-trichloromethylquinazoline. However,
this skeleton, chlorinated in position 4 (test compound
1), does not present satisfying IC50 values on Plasmo-
dium strains.
5. Liu, X. P.; Narla, K.; Uckun, F. M. Bioorg. Med. Chem.
Lett. 2003, 13, 581.
6. Sielecki, T. M.; Johnson, T. L.; Liu, J.; Muckelbauer, J.
K.; Grafstrom, R. H.; Cox, S.; Boylan, J.; Burton, C. R.;
Chen, H.; Smallwood, A.; Chang, C. H.; Boisclair, M.;
Benfield, P. A.; Trainor, G. L.; Seitz, S. P. Bioorg. Med.
Chem. Lett. 2001, 11, 1157.
7. Elslager, E. F.; Colbry, N. L.; Davoll, J.; Hutt, M. P.;
Johnson, J. L.; Werbel, L. M. J. Med. Chem. 1984, 27,
1740.
8. Genther, C. S.; Smith, C. C. J. Med. Chem. 1977, 20, 237.
9. Werbel, L. M.; Elslager, E. F.; Hess, C.; Hutt, M. P.
J. Med. Chem. 1987, 30, 1943.
10. Verhaeghe, P.; Rathelot, P.; Gellis, A.; Rault, S.; Vanelle,
P. Tetrahedron 2006, 62, 8173.
11. Connolly, D. J.; Lacey, P. M.; McCarthy, M.; Saunders,
C. P.; Carroll, A. M.; Goddard, R.; Guiry, P. J. J. Org.
Chem. 2004, 69, 6572.
From the results obtained, it appears that antiplasmo-
dial activity can be significantly improved by adding
an aryl group in position 4 of the quinazoline ring.
Among 4-aryl-2-trichloromethylquinazolines, the best
activity values are noted with substituted phenyl groups.
Note that the nature and the position of substituents on
the phenyl group play an important role in antiplasmo-
dial activity. Thus, favorable influence is noted with hal-
ogen atoms in the para position, especially with fluorine
(test compound 4).
12. Folli, U.; Goldoni, F.; Larossi, D.; Sbardellati, S.; taddei,
F. J. Chem. Soc., Perkin Trans. 2 1995, 5, 1017.
13. Bergman, J.; Brynolf, A.; Elman, B.; Vuorinen, E.
Tetrahedron 1986, 42, 3697.
In comparison with the commercial drug compound
of reference, chloroquine, the best IC50 values ob-
tained on the chloroquino-resistant W2 strain with
4-aryl-2-trichloromethylquinazolines are about 3–6
14. Compound 3: Yield 60%; white powder, mp 142 ꢁC; 1H
NMR (200 MHz; CDCl3) d 2.25 (3H, s), 7.37–7.47 (4H,
m), 7.66–7.71 (1H, m), 7.80–7.84 (1H, m), 7.96–8.03 (1H,

400
P. Verhaeghe et al. / Bioorg. Med. Chem. Lett. 18 (2008) 396–401
m), 8.24–8.28 (1H, m). 13C NMR (50 MHz; CDCl3) d 20.0
Calcd for C19H11Cl3N2 (373.66) C, 61.07; H, 2.97; N, 7.50;
found C, 60.72; H, 3.06; N, 7.57.
Compound 11: Yield 64%; yellow powder, mp 120 ꢁC; H
(CH3), 97.4 (C), 122.9 (C), 125.6 (CH), 127.2 (CH), 129.2
(CH), 129.6 (CH), 129.8 (CH), 129.9 (CH), 131.1 (CH),
134.7 (CH), 135.5 (C), 136.9 (C), 150.2 (C), 160.5 (C),
171.4 (C). Anal. Calcd for C16H11Cl3N2 (337.63) C, 56.92;
H, 3.28; N, 8.30; found C, 56.85; H, 3.36; N, 8.76.
Compound 4: Yield 60%; white powder, mp 130 ꢁC; 1H
NMR (200 MHz; CDCl3) d 7.25–7.34 (2H, m), 7.70–7.78
(1H, m), 7.87–7.94 (2H, m), 7.97–8.05 (1H, m), 8.18–8.27
(2H, m). 13C NMR (50 MHz; CDCl3) d 97.3 (C), 115.9
(2CH, d, J = 21.9 Hz), 121.7 (C), 126.7 (CH), 129.4 (CH),
129.8 (CH), 132.50 (2CH, d, J = 8.8 Hz), 132.54 (C, d,
J = 3.6 Hz), 134.58 (CH), 150.75 (C), 160.7 (C), 164.3 (C,
d, J = 251.7 Hz), 168.6 (C). Anal. Calcd for C15H8Cl3FN2
(341.59) C, 52.74; H, 2.36; N, 8.20; found C, 52.33; H,
2.38; N, 8.63.
1
NMR (200 MHz; CDCl3) d 6.70–7.72 (1H, m), 7.72–7.83
(3H, m), 7.92–8.00 (1H, m), 8.14–8.18 (1H, m), 9.01–9.06
(1H, m). 13C NMR (50 MHz; CDCl3) d 97.4 (C), 112.8
(CH), 117.9 (CH), 119.6 (C), 126.9 (CH), 129.4 (CH),
129.7 (CH), 134.4 (CH), 146.7 (CH), 151.6 (C), 153.4 (C),
156.1 (C), 160.8 (C). Anal. Calcd for C13H7Cl3N2O
(313.57) C, 49.79; H, 2.25; N, 8.93; found C, 49.75; H,
2.26; N, 8.80.
1
Compound 12: Yield 65%; yellow powder, mp 123 ꢁC; H
NMR (200 MHz; CDCl3) d 2.62 (3H,s), 6.95 (1H, dd,
J = 3.7 Hz, J = 1Hz), 7.71–7.79 (1H, m), 7.83 (1H, d,
J = 3.7 Hz), 7.92–8.01 (1H, m), 8.17–8.20 (1H, m), 8.56–
8.60 (1H, m). 13C NMR (50 MHz; CDCl3) d 15.7 (CH3),
97.3 (C), 120.5 (C), 126,1 (CH), 127.2 (CH), 129.3 (CH),
129.8 (CH), 132.8 (CH), 134.3 (CH), 138.3 (C), 147.7 (C),
151.2 (C), 160.4 (C), 161.5 (C). Anal. Calcd for
C14H9Cl3N2S (343.66) C, 48.93; H, 2.64; N, 8.15; found
C, 48.90; H, 2.65; N, 8.04.
Compound 5: Yield 55%; yellow crystals, mp 151 ꢁC; 1H
NMR (200 MHz; CDCl3) d 3.94 (6H, s), 3.96 (3H, s), 7.08
(2H, s), 7.70–7.78 (1H, m), 7.97–8.05 (1H, m), 8.23–8.31
(2H, m). 13C NMR (50 MHz; CDCl3) d 56.4 (2CH3), 60.97
(CH3), 97.3 (C), 108.0 (2CH), 121.9 (C), 127.1 (CH), 129.2
(CH), 129.8 (CH), 131.7 (C), 134.5 (CH), 140.4 (C), 150.8
(C), 153.5 (2C), 160.7 (C), 169.6 (C). Anal. Calcd for
C18H15Cl3N2O3 (413.68) C, 52.26; H, 3.65; N, 6.77; found
C, 52.40; H, 3.66; N, 6.86.
1
15. Compound 14: Yield 30%; yellow powder, mp 149 ꢁC, H
NMR (200 MHz; CDCl3) d 3.05 (3H, s), 3.20 (3H, s),
7.54–7.61 (2H, m), 7.62–7.70 (2H, m), 7.78–7.83 (2H, m),
7.92–8.00 (1H, m), 8.14–8.20 (2H, m). 13C NMR (50 MHz;
CDCl3) d 35.0 (CH3), 38.4 (CH3), 122.4 (C), 127.2 (CH),
128,4 (CH), 128.6 (2CH), 128.9 (CH), 130.2 (2CH), 130.3
(CH), 134.3 (CH), 136.6 (C), 150.7 (C), 157.9 (C), 167.1
(C), 169.4 (C).
Compound 6: Yield 57%; white powder, mp 127 ꢁC; 1H
NMR (200 MHz; CDCl3) d 7.54–7.60 (2H, m), 7.69–7.77
(1H, m), 7.80–7.87 (2H, m), 7.96–8.04 (1H, m), 8.15–8.26
(2H, m). 13C NMR (50 MHz; CDCl3) d 97.2 (C), 121.6
(C), 126.6 (CH), 129.0 (2CH), 129.5 (CH), 129.8 (CH),
131.7 (2CH), 13.6 (CH), 134.8 (C), 137.1 (C), 150.7 (C),
160.7 (C), 168.5 (C). Anal. Calcd for C15H8Cl4N2 (358.05)
C, 50.32; H, 2.25; N, 7.82; found C, 50.00; H, 2.18; N,
7.73.
16. Van der Heyde, H. C.; Elloso, M. M.; vande Waa, J.;
Schell, K.; Weidanz, W. P. Clin. Diagn. Lab. Immunol.
1995, 2, 417.
17. Azas, N.; Laurencin, N.; Delmas, F.; DiGiorgio, C.;
Gasquet, M.; Laget, M.; Timon David, P. Parasitol. Res
2002, 88, 165.
Compound 7: Yield 50%; white powder, mp 108 ꢁC; 1H
NMR (200 MHz; CDCl3) d 7.72–7.82 (2H, m), 7.86–7.90
(1H, m), 8.00–8.16 (4H, m), 8.27–8.31 (1H, m). 13C NMR
(50 MHz; CDCl3)d 97.1 (C), 121.7 (C), 123.8 (C, q,
J = 272.6 Hz), 126.4 (CH), 127.1 (CH, q, J = 4 Hz), 127.2
(CH, q, J = 3.5 Hz), 129.4 (CH), 129.8 (CH), 130 (CH),
131.3 (C, q, J = 32.9 Hz), 133.6 (CH), 134.9 (CH), 137.2
(C), 150.8 (C), 160.8 (C), 168.3 (C). Anal. Calcd for
C16H8Cl3F3N2 (391.6) C, 49.07; H, 2.06; N, 7.15; found C,
48.91; H, 1.97; N, 7.24.
Compound 8: Yield 55%; pale yellow powder, mp 138 ꢁC;
1H NMR (200 MHz; CDCl3) d 3.83–4.02 (3H, m), 7.09–
7.15 (2H, m), 7.66–7.75 (1H, m), 7.87–8.01 (3H, m), 8.20–
8.29 (2H, m). 13C NMR (50 MHz; CDCl3) d 55.5 (CH3),
97.5 (C), 114.3 (2CH), 121.8 (C), 127.2 (CH), 128.9 (C),
129.0 (CH), 129.7 (CH), 132.5 (2CH), 134.3 (CH), 150.8
(C), 160.8 (C), 161.9 (C), 169.2 (C). Anal. Calcd for
C16H11Cl3N2O (353.63) C, 54.34; H, 3.14; N, 7.92; found
C, 54.14; H, 3.13; N, 7.87.
18. Mosmann, T. J. Immununol. Methods 1983, 65, 55.
19. MTT assay was done according to the method of
Mosmann with slight modifications: Briefly, 5 · 103 cells
in 100 ll of culture medium (RPMI + 10% CO2) were
inoculated into each well of 96-well plates. After 24-h
incubation, 100 ll of medium with various product
concentrations was added and the plates were incu-
bated for 72 h. At the end of the treatment and
incubation, the medium was aspirated 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 fetal
calf serum. Cells were incubated for 2 h at 37 ꢁC in a
humidified 6% CO2 with 95% air atmosphere to allow
MTT oxidation by mitochondrial dehydrogenase in the
viable cells. After 2 h, the MTT solution was aspirated
and DMSO (100 ll) 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. DMSO
was used as blank. Cell viability was calculated as
percentage of control (cells incubated without com-
pound). The 50% cytotoxic concentration was deter-
mined from the dose–response curve.
Compound 9: Yield 56%; white powder, mp 95 ꢁC; 1H
NMR (200 MHz; CDCl3) d 7.55–7.67 (2H, m), 7.70–7.78
(1H, m), 7.94–8.09 (5H, m), 8.26–8.36 (3H, m). 13C NMR
(50 MHz; CDCl3) d 97.4 (C), 122.1 (C), 126.9 (CH), 127.1
(CH), 127.2 (CH), 127.7 (CH), 127.8 (CH), 128.6 (CH),
128.9 (CH), 129.3 (CH),129.8 (CH), 130.9 (CH), 132.9 (C),
133.8 (C), 134.2 (C), 134.5 (CH), 150.79 (C), 160.8 (C),
169.9 (C). Anal. Calcd for C19H11Cl3N2 (373.66) C, 61.07;
H, 2.97; N, 7.50; found C, 60.68; H, 2.99; N, 7.29.
20. Rathelot, P.; Azas, N.; El-Kashef, H.; Delmas, F.;
DiGiorgio, C.; Timon David, P.; Vanelle, P. Eur. J.
Med. Chem. 2002, 37, 671.
´
21. Rodrıguez-Antona, C.; Donato, M. T.; Boobis, A.;
1
Compound 10: Yield 50%; white powder, mp 209 ꢁC; H
Edwards, R. J.; Watts, P. S.; Castell, J. V. Xenobiotica
2002, 32, 505.
22. Ogunkolade, B. W.; Colomb-Valet, I.; Monjour, L.;
Rhodes-Feuillette, A.; Abita, J. P.; Frommel, D. Acta
Trop. 1990, 47, 171.
NMR (200 MHz; CDCl3) d 7.38–7.80 (7H, m), 7.96–8.10
(3H, m), 8.28–8.32 (1H, m). 13C NMR (50 MHz; CDCl3) d
97.4 (C), 123.62 (C), 124.9 (CH), 125.4 (CH), 126.4 (CH),
127.0 (CH), 127.4 (CH), 128.5 (CH), 128.6 (CH), 129.3
(CH),129.6 (CH), 130.4 (CH), 131.5 (C), 133.5 (C), 133.8
(C), 134.8 (CH), 150.22 (C), 160.8 (C), 170.7 (C). Anal.
23. Azas, N.; DiGiorgio, C.; Delmas, F.; Gasquet, M.; Timon
David, P. Exp. Parasitol. 1997, 87, 1.

P. Verhaeghe et al. / Bioorg. Med. Chem. Lett. 18 (2008) 396–401
401
24. Delmas, F.; DiGiorgio, C.; Robin, M.; Azas, N.; Gasquet,
M.; Detang, C.; Costa, M.; Timon David, P.; Galy, J. P.
Antimicrob. Agents Chemother. 2002, 46, 2588.
25. Leishmania donovani promastigotes (strain MHOM/IN/
80/DD8) in log-phase were incubated at an average
density of 105 cells/ml. Various concentrations of active
compounds were aseptically dissolved in DMSO (final
concentration less than 0.5% v/v) and incorporated in
triplicate cell cultures. After a 48-h incubation period at
25 ꢁC, parasite growth and viability were determined by
flow cytometry after staining DNA by 5 ll propidium
iodide (1 mg/ml, Sigma). Amphotericin B was used as
reference drug.
26. Pradines, B.; Rogier, C.; Fusai, T.; Mosnier, J.; Daries,
W.; Barret, E.; Parzy, D. Antimicrob. Agents Chemother.
2001, 45, 1746.