Synthesis and antimalarial activity of new analogues of amodiaquine

Article abstract of DOI:10.1016/j.ejmech.2007.03.008

In order to determine the real significance of the 4′-phenolic group in the antimalarial activity and/or cytotoxicity of amodiaquine (AQ), analogues for which this functionality was shifted or modified were synthesized. Good in vitro antimalarial activity

Full text of DOI:10.1016/j.ejmech.2007.03.008

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 252e260
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antimalarial activity of new analogues
of amodiaquine
Sandrine Delarue-Cochin a,1, Emilia Paunescu b, Louis Maes c,2, Elisabeth Mouray d,
Christian Sergheraert a, Philippe Grellier d, Patricia Melnyk a,b,
*
a UMR CNRS 8525, Universite´ de Lille II, Institut Pasteur de Lille 1 rue du Professeur Calmette, B.P. 447, 59021 Lille cedex, France
b UMR CNRS 8161, Universite´s de Lille I & II, Institut Pasteur de Lille 1, rue du Professeur Calmette, B.P. 447, 59021 Lille cedex, France
c Tibotec, B-32800 Mechelen, Belgium
d USM 0504 De´partement ‘‘Re´gulations, De´veloppement, Diversite´ Chimique’’, Muse´um National d’Histoire Naturelle, 61 rue Buffon,
75005 Paris, France
Received 13 December 2006; received in revised form 2 March 2007; accepted 8 March 2007
Available online 3 April 2007
Abstract
In order to determine the real significance of the 40-phenolic group in the antimalarial activity and/or cytotoxicity of amodiaquine (AQ),
analogues for which this functionality was shifted or modified were synthesized. Good in vitro antimalarial activity was obtained for compounds
unable to form intramolecular hydrogen bond. Among the compounds synthesized, new amino derivative 5 displayed the greatest selectivity
index towards the most CQ-resistant strain tested and was active in mice infected by Plasmodium berghei.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Drug design; Plasmodium falciparum; Antimalarials; Amodiaquine; 4-Aminoquinolines
1. Introduction
parasites. The spread of CQ-resistance has prompted the re-ex-
amination of alternative antimalarials such as amodiaquine
(AQ, Chart 1), an other 4-aminoquinoline which proved to
be effective against CQ-resistant strains [2]. These early stud-
ies were confirmed by comparative trials of CQ and AQ for the
treatment of acute, uncomplicated infections in Gambia, West
and Central Africa and Nigeria. AQ was found to be superior
to CQ, displaying lower parasitological and clinical failure
rates [3e5]. However the use of AQ has been limited since
the mid-1980s because of the occurrence of numerous cases
of agranulocytosis and hepatotoxicity in adults taking the
drug prophylactically [6,7]. AQ toxicity has been explained
by the presence of its 4-hydroxyanilino moiety, which is be-
lieved to undergo extensive metabolization to its quinoneimine
variant [8,9]. Formation of this reactive species in vivo and
subsequent binding to cellular proteins and lipids could affect
cellular function either directly or by immunological response
[10,11]. This bio-activation was found to be accompanied by
the expression of a drug-related antigen on the cell surface,
Almost one-half of the world’s population is exposed to the
threat of malaria and the disease is responsible for two million
deaths each year [1]. Chloroquine (CQ, Chart 1) was a main-
stream drug in the fight against Plasmodium falciparum, but its
efficacy is being eroded by the emergence of resistant
Abbreviations: AQ, amodiaquine; CQ, chloroquine; DIEA, diisopropyle-
thylamine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide (thiazolyl blue).
* Corresponding author. Tel.: þ33 3 20 87 12 19; fax: þ33 3 20 87 12 33.
E-mail address: patricia.melnyk@ibl.fr (P. Melnyk).
Present address: Univ. Paris-Sud and CNRS, Equipe de Synthese Organi-
1
`
ˆ
que et Pharmacochimie, UMR CNRS 8076 BioCIS, Chatenay-Malabry,
F-92290.
2
Present address: Laboratory of Microbiology, Parasitology and Hygiene
(LMPH), Faculty of Pharmaceutical, Biomedical and Veterinary Sciences,
Antwerp University, campus Groenenborger-Groenenborgerlaan 171, B-2020
Antwerp, Wilrijk, Belgium.
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.03.008

S. Delarue-Cochin et al. / European Journal of Medicinal Chemistry 43 (2008) 252e260
OH
253
OH
NRR'
NEt2
NEt2
HN
HN
HN
N
N
Cl
Cl
Cl
N
Chloroquine (CQ)
Amodiaquine (AQ)
NRR' = NEt2 : Isoquine (IsQ)
NRR' = NHtBu : IsQ-tBu
Chart 1. Structure of chloroquine, amodiaquine and isoquine.
suggesting a type II hypersensitivity reaction [12] and causing
the myelotoxicity of AQ [13]. Besides, activity of AQ is
thought to be linked to an active conformation in which the in-
ternitrogen separation, i.e., between the quinoline nitrogen and
2. Chemistry
In the original synthesis of AQ, 4-hydroxyacetanilide was
subjected to a Mannich reaction in order to introduce the di-
ethylaminomethyl side chain ortho to the hydroxyl group
[23]. Following hydrolysis of the amide function, the amine
was condensed with 4,7-dichloroquinoline to give the desired
compound. Compound 1 was synthesized using a similar
method with inversion of the two steps (Scheme 1). Interme-
diate 7 was easily isolated by filtration in fairly good yield.
Lower yield observed for compound 1 was due to side-
reactions, such as formation of para-substituted product or
double-Mannich reaction, which caused difficulties during
purification. The same synthetic pathway could not be retained
for other compounds since the orientation of substituents is
different from that imposed by the Mannich reaction.
Compound 2 was prepared in four steps as described in
Scheme 2. Reductive amination of 4-hydroxy-benzaldehyde
with diethylamine gave intermediate 8, which was trans-
formed into the mono-nitro derivative 9 by using a solution
of nitric acid in diluted sulfuric acid. The last two steps con-
sisted of a reduction (iron powder in glacial acetic acid) of
the nitro group, then condensation of the resultant amine
with 4,7-dichloroquinoline which led, respectively, to com-
pounds 10 and 2. Low yields, which are not optimized, ob-
tained from this synthetic pathway were due to degradation
under reaction conditions.
˚
the diethylamino nitrogen, is approximately 8.30 A, like in CQ
[14]. This distance is similar to that measured by X-ray crys-
tallography between the central iron and the oxygens of the
carboxylate groups of haem [15], the putative ‘‘receptor’’ of
these 4-aminoquinolines. This active conformation is thought
to be the result of an intramolecular hydrogen bond between
the hydroxyl and the proton of the charged diethylamino func-
tion [16]. This hypothesis is at the basis of the design of fluoro
analogs [17] and isoquine IsQ (Chart 1). IsQ analog IsQetBu
is now planned for clinical trials [18].
Though resistance to AQ is developing, last WHO’s guide-
lines for treatment still recommend the use of AQ in combina-
tion with artemisinin derivatives or if not available with
sulfadoxine/pyrimethamine. As this antimalarial drug remains
of fundamental therapeutic interest [19], the search for even
more active analogs is of importance.
With the aim of studying the precise influence of the 40-
phenolic group of AQ for activity and toxicity, we report
here the synthesis and antimalarial activity of the 20-, 50-,
and 60-hydroxyl isomers with the diethylaminomethyl moiety
maintained in the 30-position (compounds 1, 2 and 4, Chart
2). As previous work in our group showed the interest on
amino substituent in 50-position [20], two derivatives (5 and
6, Chart 2) were also synthesized. As a comparison, deoxo-
AQ 3 [21] as well as the AQ isomer IsQ [22] (Charts 1 and
2), were also prepared and tested for their antimalarial activity
and cytotoxicity upon MRC-5 cells.
Compound 3 was prepared in three steps as outlined in
Scheme 3. Intermediate 11 was obtained by nucleophilic sub-
stitution of 3-nitrobenzylbromide with diethylamine. Nitro
function was reduced with tin chloride, then aromatic substitu-
tion of 4,7-dichloroquinoline with resulting amine 12 provided
compound 3.
X
X
Synthesis of compound 4 was carried out in four steps
(Scheme 4) with amino compound 5 as the intermediate. Ox-
idation of the previously described alcohol 13 [24], using the
conditions described previously, led to the corresponding alde-
hyde 14, which was directly transformed by reductive amina-
tion with diethylamine, to give compound 5. The global yield
of this one-pot synthesis was 30%. The last step comprised the
reaction of the primary aromatic amine with nitrous acid in the
presence of sulfuric acid as solvent, at 0 ꢀC, which led to the
diazonium salt. The latter gave the corresponding phenol 4 by
thermal decomposition at 50 ꢀC.
NEt2
NEt2
HN
HN
N
Y
Cl
Cl
N
3
4
5
6
X = H
X = OH
X = NH2
1
2
X = H Y = OH
X = OH Y = H
X = NH-(CH2)2-piperazine
Chart 2. General structures of compounds 1e6.

254
S. Delarue-Cochin et al. / European Journal of Medicinal Chemistry 43 (2008) 252e260
b
a
NEt2
HN
N
HN
N
CH3CONH
OH
OH
OH
Cl
Cl
7
1
Scheme 1. Synthesis of compound 1. Reagents: (a) HCl 20% then 4,7-dichloroquinoline, EtOH; (b) HCHO, HNEt2, EtOH.
Compound 6 was prepared in three steps as described in
around 100 nM upon Thai and FcB1R strains and superior
to 250 nM upon K1 strain).
Scheme 5. Substitution of N-(2-chloroethyl)piperidine with
previously described intermediate 13 [24] gave alcohol 15,
which was oxidized to aldehyde using MnO2 as reagent. Re-
ductive amination of the aldehyde 16 with diethylamine
gave compound 6.
The cytotoxicity of the different compounds was evaluated
upon human MRC-5 cells (Table 1). The MRC-5 cell is a dip-
loid human fibroblast which is frequently used for cytotoxicity
testing because of its higher sensitivity compared to some
other cell lines. On the other hand, isomers 1, 4 and the amino
analog 5 were found to be twice less toxic than AQ while com-
pound 3 has the same CC50 (concentration of drug causing
50% cytotoxicity) value as that of AQ. Secondary amino com-
pound 6 was the most toxic one. AQ-isomers 1 (20-isomer) and
2 (60-isomer) could be oxidized as AQ. For dehydroxy-AQ 3
the possibility of its 40-hydroxylation by metabolization in
vivo cannot be ruled out, generating problems of toxicity as
observed with AQ, agranulocytosis and hepatotoxicity. Com-
parison between 50-AQ isomer 4 and 50-amino analog 5, shows
that antimalarial activity is to the advantage of compound 5.
Four out of the compounds synthesized revealed a selectiv-
ity index (CC50/IC50 on K1 resistant strain) superior to that of
CQ. Hence it was encouraging to consider amino compound 5
for further evaluation. Compound 5 was tested for its ability to
inhibit haem polymerisation (Table 2). Results show that the
compounds inhibit haem polymerization three times less
than AQ and twice less than CQ.
3. Biological results and discussion
All the compounds were tested for their activity against
a CQ-sensitive strain Thai (IC50 (CQ) ¼ 14 nM), and two
CQ-resistant strains FcB1R (IC50 (CQ) ¼ 126 nM) and K1
(IC50 (CQ) ¼ 183 nM). IC50 values for AQ were found to be
4.6 nM, 4.8 nM and 9.4 nM, respectively, against the three
strains, therefore about a twice increase towards the most
CQ-resistant strain (Table 1).
By comparison of their activities with those for AQ, com-
pounds 1e6 can be ranged in four categories: (i) compound
3 (40-deoxo-AQ) shows a slight decrease of activity compared
with AQ or IsQ while IC50 remains stable upon all strains as
for IsQ; (ii) compounds 2, 5 and 6 show a slight decrease of
activity upon Thai and FcB1R strains (IC50s around 16 nM)
and an obvious variability upon the most resistant-CQ strain
(IC50s between 26 and 32 nM); (iii) compound 4 is less active
than the preceding ones and IC50s increase with the CQ-resis-
tance level of the strains; (iv) compound 1 (the 20-isomer of
AQ) is much less active than all the other derivatives (IC50s
Moreover as the basic phenol substituent of AQ is replaced
in compound 5 by a neutral aromatic amino group, compound
5 is expected to be accumulated less in the acidic food vacuole
of the parasite than AQ. Compound 5 was then tested in vivo
HO
HO
HO
HO
c
a
b
O
NEt2
NEt2
NEt2
O2N
H2N
H
8
9
10
d
HO
HN
NEt2
Cl
N
2
Scheme 2. Synthesis of compound 2. Reagents: (a) HNEt2, CH2Cl2 then NaHB(OAc)3; (b) HNO3, H2SO4, H2O; (c) Fe, HCl, EtOH; (d) 4,7-dichloroquinoline,
EtOH.

S. Delarue-Cochin et al. / European Journal of Medicinal Chemistry 43 (2008) 252e260
255
a
c
HN
N
O2N
O2N
N
Br
N
Cl
3
11 R = NO2
12 R = NH2
b
Scheme 3. Synthesis of compound 3. Reagents: (a) HNEt2, K2CO3, ACN, room temperature; (b) SnCl2, HCl 1 M, THF, reflux; (c) 4,7-dichloroquinoline, HCl 1 M,
ACN, reflux.
(Table 3) according to our typical procedure [25]. Compound
5 displayed a reasonable yet decreased activity when com-
pared with CQ and AQ.
towards the most CQ-resistant strain K1, constitutes a good
lead. Antimalarial activity of analog 6 is encouraging and
the synthesis of compounds bearing other amino groups in
50-position should be envisaged to decrease the cytotoxicity.
Moreover, as the replacement of OH group in compound 4
by amino group in compounds 5 and 6 provides an increase
in selectivity index, further work will comprise the synthesis
of analogues presenting a variety of substituents in 40-position.
High antimalarial activity against CQ-resistant strains is de-
scribed to be linked with the necessary formation of an intra-
molecular hydrogen bond between the hydroxyl function and
the diethylammmonium group. When the latter is roughly
well-positioned relative to the quinoline nitrogen [16] as in
the case for IsQ, the level of activity is retained. However, ac-
tivity is also maintained or slightly decreased, for compounds
2e5 where mobility of the diethylamino side chain is allowed.
This result was already observed for compound 3 by Hawley
[26]. It is only when a hydrogen-bonding interaction is likely
to engage the diethylamino nitrogen at a much smaller dis-
tance from the quinoline nitrogen, as for compound 1, that
a significant decrease in antimalarial activity is observed.
5. Materials and methods
5.1. Chemistry
All reactions were monitored by thin-layer chromatography
carried out on 0.2 mm E. Merck silica gel plates (60F-254) us-
ing UV light as a visualizing agent. Chromatography was un-
dertaken using silica gel 60 (230e400 mesh ASTM) from
Macherey-Nagel. Thick-layer chromatography (TLC) was per-
formed using silica gel from Merck, from which the com-
pounds were extracted by the following solvent system:
CH2Cl2/MeOH/NH4OH, 80:20:1. All melting points were
4. Conclusion
In conclusion, from among the AQ analogues studied,
amino compound 5 displaying both a notable in vitro and in
vivo antimalarial activity and the best selectivity index
determined on a Buchi melting point apparatus and were
¨
NH2
NH2
NH2
NH2
c
b
a
O
NEt2
OH
Cl
OH
HN
N
HN
N
HN
N
H2N
H
Cl
Cl
d
13
14
5
OH
NEt2
HN
N
Cl
4
Scheme 4. Synthesis of compounds 4 and 5. Reagents: (a) 4,7-dichloroquinoline, N-methylmorpholine, EtOH/CHCl3, 55:5 [23]; (b) MnO2, CH2Cl2; (c) HNEt2,
CH2Cl2 then NaHB(OAc)3; (d) NaNO2, H2SO4, 0 ꢀC then 50 ꢀC.

256
S. Delarue-Cochin et al. / European Journal of Medicinal Chemistry 43 (2008) 252e260
N
N
NH2
HN
HN
HN
N
HN
N
HN
N
R
a
b
OH
OH
Cl
Cl
Cl
13
15
16 R = CHO
c
6 R = CH2NEt2
Scheme 5. Synthesis of compound 6. Reagents: (a) N-(2-chloroethyl)piperidine, n-pentanol, reflux; (b) MnO2, CH2Cl2; (c) NaHB(OAc)3, CH2Cl2.
1
uncorrected. H NMR spectra were obtained using a Bruker
300 MHz spectrometer, chemical shifts (d) were expressed in
ppm relative to TMS used as an internal standard. Mass spec-
tra were recorded on a MALDI-TOF Voyager-DE STR
(Applied Biosystems, Palo Alto CA) with a trihydroxy-
acetophenone matrix. The purity of final compounds 1e6
was verified by high pressure liquid chromatography
(HPLC) using C18 Vydac (C18V) column. The purity of inter-
mediate compounds was verified using the same C18 V col-
umn (except compounds 11 and 12) or C18 TSK gel super
ODS (C18-TSK) column (intermediates 11 and 12) (PHPLC).
Analytical HPLC was performed on a Shimadzu system equip-
ped with a UV detector set at 254 nm. Compounds were dis-
solved in EtOH and injected through a 50 mL loop. The
following eluent systems were used: A (H2O/TFA, 100:0.05)
and B (CH3CN/H2O/TFA, 80:20:0.05). HPLC retention times
(tR) were obtained, at flow rates of 1 mL/min, using the fol-
lowing conditions. For C18 V column : a gradient run from
100% eluent A during 5 min, then to 100% eluent B over
the next 30 min, for C18-TSK column : a gradient run from
100% eluent A during 30 s, then to 100% eluent B over the
next 8 min. 4,7-dichloroquinoline was obtained from Acros
and other reagents from Acros, Aldrich, Avocado and
Lancaster.
5.1.1. Synthesis of compound 1
5.1.1.1. 2-(7-Chloroquinolin-4-ylamino)-phenol (7). A solu-
tion of 2-acetamidophenol (500 mg, 3.3 mmol) in 5 mL of
HCl 20% was heated at reflux for 2 h. A solution of 4,7-di-
chloroquinoline (655 mg, 3.3 mmol) in 8 mL of EtOH was
then added. After further stirring at reflux for 6 h, the mixture
was cooled to 0 ꢀC, filtered and the residue was washed by
ether and dried under vacuum to yield the desired compound
as a yellow solid (610 mg, 68% yield); Rf 0.60 (CH2Cl2/
MeOH, 9:1); mp ¼ 151 ꢀC; HPLC (C18V) PHPLC 100%, tR
1
14.25 min; H NMR (DMSO-d6) d 10.87 (s, 1H, OH), 10.24
(s, 1H, NH), 8.83 (d, J ¼ 9.2 Hz, 1H, Quin-H5), 8.48 (d,
J ¼ 7.0 Hz, 1H, Quin-H2), 8.16 (d, J ¼ 2.1 Hz, 1H, Quin-
H8), 7.85 (dd, J ¼ 9.1, 2.1 Hz, 1H, Quin-H6), 7.31 (m, 2H,
Ar-H6, Ar-H4), 7.15 (d, J ¼ 8.1 Hz, 1H, Ar-H3), 7.00 (t,
J ¼ 8.1 Hz, 1H, Ar-H3), 6.32 (d, J ¼ 7.0 Hz, 1H, Quin-H3);
m/z 271.5 (Mþ þ 1).
Table 1
In vitro antimalarial activity upon three P. falciparum strains, in vitro cytotox-
icity of compounds 1e6 and selectivity index against K1 strain
5.1.1.2.
2-(7-Chloroquinolin-4-ylamino)-6-diethylamino-
SIf
CC50
(mM) upon
MRC-5 cells
a
Compound IC50 (nM)
g
methyl-phenol (1). To a solution of compound 7 (610 mg,
2.3 mmol) in EtOH, were added formaldehyde 37% in water
(5 equiv.) and diethylamine (1 equiv.). After stirring at reflux
for 2 h, the mixture was concentrated and the residue was puri-
fied by TLC (CH2Cl2/MeOH/NH4OH, 95:5:1) to yield the de-
sired compound as a yellow solid (163 mg, 20% yield); Rf
0.75 (CH2Cl2/MeOH, 9:1); mp ¼ 118 ꢀC; HPLC (C18V) PHPLC
99%, tR 12.66 min; HPLC (C18N) PHPLC 98%, tR 13.60 min; 1H
NMR (DMSO-d6) d 9.65 (s, 1H, OH), 8.66 (s, 1H, NH), 8.44 (d,
J ¼ 9.1 Hz, 1H, Quin-H5), 8.33 (d, J ¼ 5.4 Hz, 1H, Quin-H2),
Thai
FcB1R
K1
CQ
14.3 ꢁ 2.4e
4.6 ꢁ 0.8e
4.3 ꢁ 0.5e
126 ꢁ 26e
4.8 ꢁ 0.9e
5.4 ꢁ 1.2b
183 ꢁ 35e
175 >32
AQ
IsQ
1
2
3
4
5
6
a
9.4 ꢁ 1.1e 1276
12
8
7.3 ꢁ 1.0e 1096
103.5 ꢁ 14.3e 95.2 ꢁ 21.8b 262 ꢁ 20.5d
15.2 ꢁ 1.9e 15.4 ꢁ 1.8b 32.6 ꢁ 6.1d
13.2 ꢁ 3.0e 15.8 ꢁ 4.6c 17.1 ꢁ 1.0d
25.3 ꢁ 1.1e 35.2 ꢁ 9.8c 42.8 ꢁ 6.7d
16.3 ꢁ 2.1e 18.1 ꢁ 0.8b 32.3 ꢁ 4.2d
15.6 ꢁ 0.1e 19.1 ꢁ 0.6b 26.5 ꢁ 5.9d
95 >25
583
731
19
12.5
584 >25
774 >25
152
4
Parasites were considered resistant to CQ for IC50 >100 nM.
n ¼ 3.
b
Table 2
Haem polymerisation inhibition
c
n ¼ 4.
d
n ¼ 5.
Compound
IC50 (mM)
e
n ¼ 6.
CQ
AQ
5
70
48
f
Selectivity index towards K1 strain.
CC50 is the concentration of drug causing 50% cytotoxicity, calculated on
the basis of two experiments.
g
153

S. Delarue-Cochin et al. / European Journal of Medicinal Chemistry 43 (2008) 252e260
257
Table 3
Antimalarial activity of compound 5 on P. berghei in mice
6.7 mmol). After stirring at reflux for 1 h, the mixture was fil-
tered on celite, the residue washed with 3 ꢂ 20 mL of MeOH
and the filtrate evaporated. The residue was then purified by
TLC (CH2Cl2/MeOH, 80:20) to yield compound 10 as a brown
oil (395 mg, 91% yield); Rf 0.35 (CH2Cl2/MeOH, 8.5:1.5);
Compound
Dose (mg/kg)
Reduction (%) of
parasitaemia on day 4
Excess MSTa (%)
CQ
AQ
5
10
10
20
40
100
100
96
Cb
C
1
HPLC (C18V) PHPLC 95%, tR 5.07 min; H NMR (DMSO-
63
C
d6) d 9.45 (broad s, 1H, OH), 6.71e6.68 (m, 2H, Ar-H3, Ar-
H6), 6.59 (dd, J ¼ 1.9, 8.0 Hz, 1H, Ar-H5), 4.68 (broad s,
2H, NH2), 3.98 (s, 2H, CH2), 2.88 (q, J ¼ 7.1 Hz, 4H,
CH2CH3), 1.21 (t, J ¼ 7.1 Hz, 6H, CH2CH3); m/z 195
(Mþ þ 1).
5
100
a
Excess MST is the change in the mean survival time of the treated mice,
calculated by comparing the mean survival time of the control mice with
the mean survival time of the treated mice.
b
C for ‘‘cured’’ indicates mice surviving the infection and that can be
termed cured definitively.
5.1.2.4. 3-(7-Chloroquinolin-4-ylamino)-4-diethylamino-methyl-
phenol (2). To a solution of compound 10 (240 mg, 1.2 mmol)
in 20 mL of EtOH, was added 4,7-dichloroquinoline (250 mg,
1.2 mmol) and the medium was alcanalized until pH 5.5. After
stirring at reflux for 8 h, the mixture was concentrated and the
residue purified by TLC (CH2Cl2/MeOH, 70:30) to yield com-
pound 2 as a yellow solid (30 mg, 7% yield); Rf 0.40 (CH2Cl2/
7.83 (d, J ¼ 2.2 Hz, 1H, Quin-H8), 7.49 (dd, J ¼ 9.0, 2.3 Hz, 1H,
Quin-H6), 7.10 (m, 2H, Ar-H3, Ar-H5), 6.81 (t, J ¼ 7.9 Hz, 1H,
Ar-H4), 6.22 (d, J ¼ 5.4 Hz, 1H, Quin-H3), 3.84 (s, 2H, CH2),
2.58 (q, J ¼ 7.2 Hz, 4H, CH2CH3), 1.02 (t, J ¼ 7.1 Hz, 6H,
CH2CH3); m/z 356.5 (Mþ þ 1).
1
MeOH, 7:3); HPLC (C18V) PHPLC 99%, tR 11.22 min; H
5.1.2. Synthesis of compound 2
NMR (DMSO-d6) d 9.47 (s, 1H, OH), 8.65 (s, 1H, NH), 8.42
(d, J ¼ 9.1 Hz, 1H, Quin-H5), 8.32 (d, J ¼ 5.4 Hz, 1H, Quin-
H2), 7.82 (d, J ¼ 2.2 Hz, 1H, Quin-H8), 7.48 (dd, J ¼ 9.0,
2.2 Hz, 1H, Quin-H6), 7.12 (d, J ¼ 1.9 Hz, 1H, Ar-H3), 7.03
(dd, J ¼ 8.2, 2.0 Hz, 1H, Ar-H5), 6.90 (d, J ¼ 8.2 Hz, 1H, Ar-
H6), 6.18 (d, J ¼ 5.4 Hz, 1H, Quin-H3), 3.42 (s, 2H, CH2),
2.39 (q, J ¼ 7.0 Hz, 4H, CH2CH3), 0.93 (t, J ¼ 7.1 Hz, 6H,
CH2CH3); m/z 356.5 (Mþ þ 1).
5.1.2.1. 4-Diethylaminomethyl-phenol (8). To a solution of 4-
hydroxybenzaldehyde (1 g, 8.2 mmol) in 90 mL of dry
CH2Cl2 were added diethylamine (2.54 mL, 24.6 mmol), after
2 h, NaHB(OAc)3 (5.22 g, 32.8 mmol). After stirring the mix-
ture at room temperature for 18 h, 10 mL of a solution of
NaHCO3 1 M was added. The solvent was then evaporated
and the residue was taken up in AcOEt. The reactive medium
was then filtrated, the filtrate was concentrated and the residue
purified by column chromatography (CH2Cl2/MeOH, 85:15)
to yield compound 8 as a yellow solid of the sodium phenolate
form (1.58 g, 96% yield); Rf 0.25 (CH2Cl2/MeOH, 8.5:1.5);
mp ¼ 79 ꢀC; HPLC (C18V) PHPLC 99%, tR 9.07 min; 1H
NMR (DMSO-d6) d 6.99 (d, J ¼ 8.5 Hz, 2H, Ar-H3), 6.60
(d, J ¼ 8.5 Hz, 2H, Ar-H2), 3.32 (s, 2H, CH2), 2.33 (q,
J ¼ 7.2 Hz, 4H, CH2CH3), 0.87 (t, J ¼ 7.1 Hz, 6H, CH2CH3).
5.1.3. Synthesis of compound 3
5.1.3.1. Diethyl-(3-nitro-benzyl)-amine (11). To a solution of
3-nitrobenzylbromide (200 mg, 0.93 mmol) in 25 mL of
ACN, were added K2CO3 (640 mg, 4.6 mmol) and after
20 min stirring diethylamine (115 mL, 1.1 mmol). After stir-
ring at room temperature for 8 h, the mixture was filtrated
and concentrated. 50 mL of saturated solution NaHCO3 was
added and the aqueous layer extracted by 2 ꢂ 50 mL of
CH2Cl2. The organic layers were then combined, dried over
MgSO4, the solvent was evaporated and the residue purified
by TLC (cyclohexane/EtOAc/NH4OH, 8:2:0.1) to yield com-
pound 11 as a yellow oil (187 mg, 97% yield); Rf 0.65 (cyclo-
hexane/EtOAc/NH4OH, 8:2:0.1); HPLC (C18V) PHPLC 99%,
5.1.2.2. 4-Diethylaminomethyl-2-nitrophenol (9). To a solution
of compound 8 (2.5 g, 12.5 mmol) in 5 mL of H2SO4, was
added dropwise, at 0 ꢀC, HNO3 (150 mL, 2.5 mmol). After
stirring at 0 ꢀC for 30 min, the mixture was neutralized, at
0 ꢀC, with Na2CO3, then concentrated. The residue was taken
up in AcOEt, filtered and purified by column chromatography
(CH2Cl2/MeOH, 90:10) to yield compound 9 as a yellow solid
(550 mg, 20% yield); Rf 0.85 (CH2Cl2/MeOH, 8.5:1.5);
1
tR 2.95 min; H NMR (CDCl3) d 8.22 (t, J ¼ 1.8 Hz, 1H, Ar-
H2), 8.06 (m, 1H, Ar-H4), 8.22 (dt, J ¼ 8.0, 1.8 Hz, 1H, Ar-
H6), 7.47 (t, J ¼ 8.0 Hz, 1H, Ar-H5), 3.66 (s, 2H, CH2), 2.54
(q, J ¼ 7.0 Hz, 2H, CH2CH3), 1.05 (t, J ¼ 7.0 Hz, 3H,
CH2CH3); m/z 209.2(Mþ þ 1).
1
mp ¼ 106 ꢀC; HPLC (C18V) PHPLC 98%, tR 10.03 min; H
NMR (DMSO-d6) d 7.79 (d, J ¼ 2.1 Hz, 1H, Ar-H3), 7.46
(dd, J ¼ 8.6, 2.2 Hz, 1H, Ar-H5), 7.06 (d, J ¼ 8.5 Hz, 1H,
Ar-H6), 3.50 (s, 2H, CH2), 2.47 (q, J ¼ 7.1 Hz, 4H,
CH2CH3), 0.97 (t, J ¼ 7.1 Hz, 6H, CH2CH3); m/z 225
(Mþ þ 1).
5.1.3.2. Diethyl-(3-amino-benzyl)-amine (12). To a solution of
nitro compound 11 (195 mg, 0.94 mmol) in 20 mL of THF,
was added a solution of tin chloride (711 mg, 3.7 mmol) in
5 mL of THF with HCl 1 M (2.8 mmol). After stirring at reflux
for 5 h, the mixture was concentrated, alkalinized with
NaHCO3 (pH 8) and the aqueous layer extracted by
5 ꢂ 50 mL of CH2Cl2. The organic layers were then com-
bined, dried over MgSO4, the solvent was evaporated and
5.1.2.3. 3-Amino-4-diethylaminomethyl-phenol (10). To a solu-
tion of compound 9 (500 mg, 2.2 mmol) in 25 mL of a EtOH/
H2O, 1:1 mixture, were added iron powder (375 mg,
6.7 mmol) and concentrated chlorhydric acid (560 mL,

258
S. Delarue-Cochin et al. / European Journal of Medicinal Chemistry 43 (2008) 252e260
the residue purified by TLC (CH2Cl2/MeOH/NH4OH,
9.5:0.5:0.2) to yield compound 12 as a yellow oil (111 mg,
67% yield); Rf 0.6 (CH2Cl2/MeOH/NH4OH, 9.5:0.5:0.2),
a solution of NaNO2 (23 mg, 0.33 mmol) in 2 mL of water.
After stirring the mixture at 0 ꢀC for 20 min, a urea crystal
is added and following further stirring at 30 ꢀC for an addi-
tional 15 min, a solution of NaHCO3 5% was added until neu-
tralisation and the aqueous layer extracted by 2 ꢂ 30 mL of
CH2Cl2. The organic layers were then combined, dried over
MgSO4, the solvent was evaporated and the residue purified
by TLC (CH2Cl2/MeOH/NH4OH, 90:10:1) to yield compound
4 as a yellow solid (40 mg, 40% yield); Rf 0.75 (CH2Cl2/
MeOH, 7.5:2.5); mp ¼ 77 ꢀC; HPLC (C18V) PHPLC 99%, tR
1
HPLC (C18V) PHPLC 95%, tR 2.17 min; H NMR (CDCl3)
d 7.06 (t, J ¼ 8.0 Hz, 1H, Ar-H5), 6.70 (m, 2H, Ar-H2, Ar-
H4), 6.53 (dt, J ¼ 8.0, 1.8 Hz, 1H, Ar-H6), 3.82 (broad s, 2H,
NH2), 3.49 (s, 2H, CH2), 2.54 (q, J ¼ 7.0 Hz, 2H, CH2CH3),
1.04 (t, J ¼ 7.0 Hz, 3H, CH2CH3); m/z 179.2(Mþ þ 1).
5.1.3.3. (7-Chloro-quinolin-4-yl)-(3-diethylaminomethyl-phe-
nyl)-amine (3). A solution of amine 12 (111 mg, 0.6 mmol)
in 10 mL of ACN was added a solution of 4,7-dichloroquino-
line (124 mg, 0.6 mmol) in 5 mL of ACN and 0.6 mL of HCl
1 M. After further stirring at reflux overnight, the mixture was
concentrated and purified by TLC (CH2Cl2/MeOH/NH4OH,
9.5:0.5:0.2) to yield compound 3 as a yellow solid (188 mg,
88% yield); Rf 0.8 (CH2Cl2/MeOH/NH4OH, 9.5:0.5:0.2);
1
11.60 min; H NMR (DMSO-d6) d 9.52 (s, 1H, OH), 9.01
(s, 1H, NH), 8.46 (d, J ¼ 5.3 Hz, 1H, Quin-H2), 8.41 (d,
J ¼ 9.2 Hz, 1H, Quin-H5), 7.88 (d, J ¼ 2.2 Hz, 1H, Quin-
H8), 7.55 (dd, J ¼ 9.0, 2.2 Hz, 1H, Quin-H6), 6.96 (d,
J ¼ 5.3 Hz, 1H, Quin-H3), 6.80e6.66e6.57 (3 s, 3H, Ar-H),
3.48 (s, 2H, CH2), 2.57 (q, J ¼ 7.0 Hz, 4H, CH2CH3), 1.02
(t, J ¼ 7.3 Hz, 6H, CH2CH3); m/z 355.5 (Mþ þ 1).
1
mp ¼ 142 ꢀC; HPLC (C18V) PHPLC 99%, tR 12.84 min; H
NMR (MeOD) d 8.26 (d, J ¼ 5.6 Hz, 1H, Quin-H2), 8.17 (d,
J ¼ 9.0 Hz, 1H, Quin-H5), 7.75 (d, J ¼ 2.1 Hz, 1H, Quin-
H8), 7.38 (dd, J ¼ 9.0, 2.1 Hz, 1H, Quin-H6), 7.30 (t,
J ¼ 7.8 Hz, 1H, Ar-H5), 7.25 (m,1H, Ar-H2), 7.18 (d,
J ¼ 7.8 Hz, 1H, Ar-H4), 7.08 (d, J ¼ 7.5 Hz, 1H, Ar-H6),
6.83 (d, J ¼ 5.6 Hz, 1H, Quin-H3), 3.57 (s, 2H, CH2), 2.52
(q, J ¼ 7.0 Hz, 2H, CH2CH3), 1.00 (t, J ¼ 7.0 Hz, 3H,
CH2CH3); m/z 340.2e342.2 (Mþ þ 1).
5.1.5. Synthesis of compound 6
5.1.5.1. [3-(7-Chloro-quinolin-4-ylamino)-5-(2-piperidin-1-yl-
ethylamino)-phenyl]-methanol (15). To a solution of com-
pound 13 (300 mg, 1 mmol) in 10 mL of n-pentanol, was
added N-(2-chloroethyl)piperidine hydrochloride (185 mg,
1 mmol) and N-ethylpiperidine (410 mL, 3 mmol). After stir-
ring at reflux for 24 h, the mixture was concentrated and the
residue purified by TLC (CH2Cl2/MeOH/NH4OH, 80:20:1)
to yield compound 14 as a yellow solid (240 mg, 57% yield);
Rf 0.25 (CH2Cl2/MeOH, 8:2); HPLC (C18V) PHPLC > 99%, tR
12.89 min; 1H NMR (DMSO-d6) d 8.9 (s, 1H, NH), 8.40e8.43
(m, 2H, Quin-H2, Quin-H5), 7.85 (d, J ¼ 2.1 Hz, 1H, Quin-
H8), 7.52 (dd, J ¼ 9.0, 2.2 Hz, 1H, Quin-H6), 6.92 (d,
J ¼ 5.4 Hz, 1H, Quin-H3), 6.51e6.42e6.37 (3 s, 3H, Ar-H),
5.53 (broad s, 1H, NH), 5.09 (broad s, 1H, OH), 4.49 (s,
2H, CH2OH), 3.12 (m, 2H, CH2NH), 2.46 (m, 2H, CH2e
CH2NH), 2.39 (m, 4H, piperaz-H), 1.49 (m, 4H, piperaz-H),
1.39 (m, 2H, piperaz-H), m/z 411.3 (Mþ þ 1).
5.1.4. Synthesis of compounds 4 and 5
5.1.4.1. N-(7-Chloroquinolin-4-yl)-5-diethylaminomethyl-1,3-
phenylenediamine (5). To a solution of compound 13 [24]
(300 mg, 1 mmol) in 15 mL of a CH2Cl2/DMF, 13:2 mixture,
were added DIEA (175 mL, 1 mmol) and MnO2 (1.3 g,
15 mmol). After stirring at room temperature for 18 h, the
mixture was filtered on celite and the residue washed with
20 mL of CH2Cl2. To the resulting mixture containing the al-
dehyde 14 were added diethylamine (155 mL, 1.5 mmol) and,
after 1 h, NaHB(OAc)3 (424 mg, 2 mmol). After stirring the
mixture at room temperature for 18 h, a solution of NaHCO3
1 M was added. Following further stirring of the mixture for
15 min, the layers were separated and the aqueous layer was
washed twice with CH2Cl2. The organic layers were then com-
bined, washed with brine, dried over MgSO4 then the solvent
was evaporated and the residue purified by TLC (CH2Cl2/
MeOH, 80:20) to yield compound 5 as a yellow solid
(110 mg, 31% yield); Rf 0.15 (CH2Cl2/MeOH, 8:2);
mp ¼ 61 ꢀC; HPLC (C18V) PHPLC 99%, tR 11.33 min; 1H
NMR (DMSO-d6) d 8.88 (s, 1H, NH), 8.49e8.43 (m, 2H,
Quin-H2, Quin-H5), 7.85 (d, J ¼ 2.2 Hz, 1H, Quin-H8), 7.51
(dd, J ¼ 9.0, 2.2 Hz, 1H, Quin-H6), 6.89 (d, J ¼ 5.4 Hz, 1H,
Quin-H3), 6.48e6.45e6.37 (3 s, 3H, Ar-H), 5.19 (s, 2H,
NH2), 3.46 (s, 2H, CH2), 2.53 (q, J ¼ 7.0 Hz, 4H, CH2CH3),
1.00 (t, J ¼ 6.9 Hz, 6H, CH2CH3); m/z 355.5 (Mþ þ 1).
5.1.5.2. [3-(7-Chloro-quinolin-4-ylamino)-5-(2-piperidin-1-yl-
ethylamino)-phenyl]-methanal (16). To a solution of com-
pound 15 (500 mg, 1.2 mmol) in 40 mL of CH2Cl2, was added
MnO2 (1.59 g, 18.3 mmol). After stirring at room temperature
for 18 h, the mixture was filtered on celite and the residue was
washed with MeOH. The filtrate was then concentrated and
the residue purified by TLC (CH2Cl2/MeOH, 85:15) to yield
compound 15 as a yellow solid (140 mg, 30% yield); Rf 0.50
(CH2Cl2/MeOH, 85:15); HPLC (C18V) PHPLC 99%, tR
1
13.73 min; H NMR (DMSO-d6) d 10.19 (s, 1H, CHO), 9.60
(broad s, 1H, NH), 8.76 (m, 2H, Quin-H2, Quin-H5), 8.18
(d, J ¼ 2.1 Hz, 1H, Quin-H8), 7.84 (dd, J ¼ 9.0, 2.2 Hz, 1H,
Quin-H6), 7.31 (d, J ¼ 5.4 Hz, 1H, Quin-H3), 7.41e7.25e
7.22 (3s, 3H, Ar-H), 6.85 (s, 1H, NH), 3.30 (m, 2H,
CH2NH), 2.78 (m, 6H, CH2eCH2NH, piperaz-H), 2.02 (m,
4H, piperaz-H), 1.49 (m, 2H, piperaz-H), m/z 409.2 (Mþ þ 1).
5.1.4.2. 3-(7-Chloroquinolin-4-ylamino)-5-diethylaminomethyl
phenol (4). To
a
solution of compound
5
(100 mg,
5.1.5.3. N-(7-Chloroquinolin-4-yl)-5-diethylaminomethyl-N0-
piperidin-1-ylethyl-1,3-phenylenediamine (6). To a solution
0.28 mmol) in 5 mL of H2SO4 5%, was added, at 0 ꢀC,

S. Delarue-Cochin et al. / European Journal of Medicinal Chemistry 43 (2008) 252e260
259
of aldehyde 16 (135 mg, 0.33 mmol) in 10 mL of CH2Cl2,
were added diethylamine (170 mL, 1.65 mmol.) and, after
1 h, NaHB(OAc)3 (140 mg, 0.66 mmol.). After stirring the
mixture at room temperature for 18 h, a solution of NaHCO3
1 M was added. Following further stirring of the mixture for
15 min, the layers were separated and the aqueous layer was
washed twice with CH2Cl2. The organic layers were then com-
bined, washed with brine, dried over MgSO4 then the solvent
was evaporated and the residue purified by (CH2Cl2/MeOH/
NH4OH, 90:10:1) to yield compound 6 as a yellow solid
(70 mg, 46% yield); Rf 0.2 (CH2Cl2/MeOH, 8:2); mp ¼ 51 ꢀC;
(RPMI þ 5% FCS). The final DMSO concentration in the cul-
ture remained below 0.5%. The mammalian cell line was incu-
bated with several concentrations of compounds (between 32
and 1.6 mM) at 37 ꢀC in 5% CO2e95% air for 7 days. Un-
treated cultures were included as controls. The cytotoxicity
was determined using the colorimetric MTT assay [29] and
scored as a % reduction of absorption at 540 nm of treated cul-
tures versus untreated control cultures.
5.2.3. Heam polymerization inhibition
The ability of a compound to inhibit haem polymerization
induced by lipids [30] was determined using the methods de-
veloped by Ayad [31]. Hemin and 1-monooleoylglycerol were
purchased from Sigma. Experiments were carried out in dupli-
cate, in 96-deep-wells. In each well, 250 mL of a solution of
700 mM of hemin in 25 mM NaOH were added to 250 mL of
a suspension of 1 mM 1-monooleoylglycerol in 90 mM so-
dium acetate at pH 5. Drugs were added from DMSO stock so-
lutions (5 mL). Microplates were incubated for 24 h at 37 ꢀC.
Controls contained an equal amount of DMSO. Following in-
cubation, the samples were centrifuged at 4000 tr/min at 4 ꢀC
for 30 min. The pellet of b-hematin was washed with 10 mM
sodium phosphate, pH 7.4, containing 2.5% SDS, and was vor-
texed for 10 min at 20 ꢀC before repelleting until the superna-
tant was colorless (5 times). Dissolution of b-hematin was
achieved by addition of 450 mL of 10 mM sodium phosphate,
pH 7.4, containing 2.5% SDS and 25 mL of NaOH 1 M. Con-
centration of haem was calculated from absorbance at 405 nm.
1
HPLC (C18V) PHPLC 98%, tR 12.76 min; H NMR (DMSO-
d6) d 8.88 (s, 1H, NH), 8.40 (m, 2H, Quin-H2, Quin-H5),
7.83 (d, J ¼ 2.1 Hz, 1H, Quin-H8), 7.50 (dd, J ¼ 9.0, 2.2 Hz,
1H, Quin-H6), 6.89 (d, J ¼ 5.4 Hz, 1H, Quin-H3), 6.49e
6.40e6.36 (3 s, 3H, Ar-H), 5.50 (broad s, 1H, NH2), 3.39 (s,
2H, CH2), 3.07 (m, 2H, CH2NH), 2.49 (m, 6H, CH2e
CH2NH), 2.44 (q, J ¼ 7.1 Hz, 4H, CH2CH3), 2.34 (m, 4H, pi-
peraz-H), 1.47 (m, 4H, piperaz-H), 1.35 (m, 2H, piperaz-H),
0.95 (t, J ¼ 7.1 Hz, 6H, CH2CH3), m/z 466.2 (Mþ þ 1).
5.2. Biological evaluation
5.2.1. In vitro P. falciparum culture and drug assays
P. falciparum strains were maintained continuously in cul-
ture on human erythrocytes as described by Trager and Jensen
[27]. In vitro antiplasmodial activity was determined using
a modification of the semi-automated microdilution technique
of Desjardins [28]. P. falciparum CQ-sensitive (Thai/Thai-
land) and CQ-resistant (FcB1R/Colombia and K1/Thailand)
strains were used in sensitivity testing. Stock solutions of chlo-
roquine diphosphate and test compounds were prepared in
sterile distilled water and DMSO, respectively. Drug solutions
were serially diluted with culture medium and introduced to
asynchronous parasite cultures (0.5% parasitaemia and 1% fi-
nal hematocrite) on plates comprising 96-wells for 24 h at
37 ꢀC prior to the addition of 0.5 mCi of [3H]hypoxanthine
(1e5 Ci/mmol; Amersham, Les Ulis, France) per well, for
24 h. After freezing and thawing, the cells were harvested
from each well onto glass fiber filters and the dried filters
were counted in a scintillation spectrometer. The growth inhi-
bition for each drug concentration was determined by compar-
ison of the radioactivity incorporated into the parasite nucleic
acids with that in the control culture (without drug) maintained
on the same plate. The concentration causing 50% inhibition
(IC50) was obtained from the drug concentrationeresponse
curve and the results were expressed as the mean ꢁ the stan-
dard deviations determined from several independent experi-
ments. The DMSO concentration never exceeded 0.1% and
did not inhibit the parasite growth.
5.2.4. In vivo drug assays on P. berghei
Antimalarial activity was determined in mice infected with
P. berghei (ANKA 65 strain). Four-week-old female Swiss
mice (CD-1, 20 ge25 g) were intraperitoneally infected with
about 107 parasitized erythrocytes, collected from the blood
of an acutely infected donor animal. At the same time, the an-
imals (three per group) were orally treated with the test com-
pound at 40 mg/kg (drug formulation in 100% DMSO). In
order not to interfere with the IP infection, this first treatment
dose is given orally. The treatment was continued during the
four following days by the intraperitoneal route because the
absorption is more extensive with less first-pass metabolism,
if any, again maximizing the treatment to show activity. Un-
treated control animals generally die between 7 and 10 days
following infection. Drug activity was evaluated by the reduc-
tion of parasites on day 4 and the prolongation of the mean
survival time compared to that of untreated controls. Three in-
fected DMSO-dosed mice were used as controls.
Acknowledgments
´
5.2.2. Cytotoxicity test upon MRC-5 cells
We express our thanks to Gerard Montagne for NMR
A human diploid embryonic lung cell line (MRC-5, Bio-
Whittaker 72211D) was used to assess the cytotoxic effects to-
wards host cells. MRC-5 cells were seeded at 5000 cells per
well. After 24 h, the cells were washed and two-fold dilutions
of the drug were added in 200 mL standard culture medium
experiments, Marie-Ange Debreu-Fontaine and Joseline
Akana-Momo for their contribution in organic synthesis and
´
Herve Drobecq for MS spectra. This work was supported by
CNRS, Universite de Lille II and MNHN. S.D. was a recipient
´
´
of fellowships from the Region Nord-Pas de Calais and EP

260
S. Delarue-Cochin et al. / European Journal of Medicinal Chemistry 43 (2008) 252e260
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S.A. Ward, B.K. Park, J. Med. Chem. 40 (1997) 437e448.
[16] H.L. Koh, M.L. Go, T.L. Ngiam, J.W. Mak, Eur. J. Med. Chem. 29
(1994) 107e113.
from the Gouvernement franc¸ais, the Gouvernement Roumain,
´
Erasmus/Socrates and Universite Lille II.
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