Novel amodiaquine congeners as potent antimalarial agents

Article abstract of DOI:10.1016/j.bmc.2008.05.068

To develop new classes of antimalarial agents, the possibility of replacing the phenolic ring of amodiaquine, tebuquine, and isoquine with other aromatic nuclei was investigated. Within a first set of pyrrole analogues, several compounds displayed high ac

Full text of DOI:10.1016/j.bmc.2008.05.068

Bioorganic & Medicinal Chemistry 16 (2008) 6813–6823
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel amodiaquine congeners as potent antimalarial agents
Manolo Casagrande a, Nicoletta Basilico b, Silvia Parapini b, Sergio Romeo a, Donatella Taramelli b,
Anna Sparatore a,
*
a Istituto di Chimica Farmaceutica e Tossicologica ‘Pietro Pratesi’, University of Milan, Via Mangiagalli 25, 20133 Milan, Italy
b Dipartimento di Sanità Pubblica–Microbiologia–Virologia, University of Milan, Via Pascal, 36 20133 Milan, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 February 2008
Revised 9 May 2008
Accepted 28 May 2008
Available online 16 June 2008
To develop new classes of antimalarial agents, the possibility of replacing the phenolic ring of amodia-
quine, tebuquine, and isoquine with other aromatic nuclei was investigated. Within a first set of pyrrole
analogues, several compounds displayed high activity against both D10 (CQ-S) and W-2 (CQ-R) strains of
Plasmodium falciparum. The isoquine structure was also modified by replacing the diethylamino group
with more metabolically stable bicyclic moieties and by replacing the aromatic hydroxyl function with
a chlorine atom. Among these compounds, two quinolizidinylmethylamino derivatives (6f and 7f) dis-
played high activity against both CQ-S and CQ-R strains.
Keywords:
4-Aminoquinoline derivatives
Amodiaquine analogues
Chloroquine
Ó 2008 Elsevier Ltd. All rights reserved.
Antimalarial agents
1. Introduction
AQ/SP combination in pregnant women has recently been
demonstrated.4
Novel, effective, safe, and inexpensive antimalarial agents are
required for the management of malaria in tropical and subtropical
regions, where this disease afflicts approximately 500 million peo-
ple annually.1,2
Presently, the most promising and so far successful strategy in
fighting malaria is a combination chemotherapy (ACT), in which an
artemisinin derivative is used together with a conventional antima-
larial drug to improve efficacy and delay onset of resistance.1
Despite the worldwide diffusion of resistance of Plasmodium fal-
ciparum to chloroquine (CQ), the 4-aminoquinoline derivatives
continue to attract interest because the resistance seems to be
compound specific and not related to changes in the structure of
the drug target.
From the amodiaquine scaffold, tebuquine (2, R = OH) was
developed,5 which resulted more active than CQ and AQ, but again
chronic toxicity was seen.6
Since toxicity of AQ and tebuquine is related with the possibility
to undergo in vivo oxidation to reactive quinoneimine derivatives,
the structures of these drugs were modified in order to prevent this
kind of metabolic activation. Thus, in a number of analogues the
hydroxyl group was either suppressed (2, R = H)7 or replaced with
a fluorine atom (1 and 2, R = F),7,8 or shielded with an aliphatic
chain (linear or branched) in ortho-position (3)6,9 to hinder or slow
down its oxidation. Moreover in most analogues the terminal
diethylamino group, that characterizes both CQ and AQ, was re-
placed with a cyclic basic head or with a tert-butylamino group
in order to prevent also the side chain metabolization, a process
that produces N-dealkyl metabolites that are less potent against
CQ-R strains. All these analogues resulted more resistant to meta-
bolic oxidation and maintained the antimalarial efficacy.
From the SAR studies, the aromatic hydroxyl function of these
compounds appears to be important for increasing the antimalarial
activity. Therefore, in another set of analogues, the oxidation to
toxic quinoneimine metabolites was prevented by the interchange
of the positions of the basic head and the hydroxyl group, leading
to isoquine (4).10 This compound displayed potent antimalarial
activity against the CQ-S and CQ-R strains of P. falciparum; in par-
ticular, against the CQ-R, K-1 strain isoquine was 10 or 30 times
more active than CQ, when tested as free base or diphosphate,
respectively. Isoquine exhibited also excellent oral activity in mice
infected with Plasmodium yoelii NS strain.
Indeed several CQ analogues, bearing different basic moieties,
retain potent activity against CQ-resistant (CQ-R) strains of P.
falciparum.
Also, amodiaquine (AQ) (1, R = H, Fig. 1) is active against many
CQ-R strains of P. falciparum, but its clinical use has been severely re-
stricted due to hepatotoxicity and agranulocytosis associated with
long-term prophylactic use. The situation changed in the recent
years with the use of AQ in association with sulfadoxine/pyrimeth-
amine (SP) or artesunate as first line treatment for uncomplicated
P. falciparum malaria in different African countries.3 The efficacy,
safety, and tolerability of a short-term treatment of AQ alone or
* Corresponding author. Tel.: +39 02 50319365; fax +39 02 50319359.
E-mail address: anna.sparatore@unimi.it (A. Sparatore).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.068

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M. Casagrande et al. / Bioorg. Med. Chem. 16 (2008) 6813–6823
Cl
R
OH
R
R
H
H
N
N
N
HN
HN
N
HN
N
3
1
2
Cl
N
Cl
Cl
R = OH: tebuquine
R = H, F
R= OH: amodiaquine
R= F
HC CH3
CH3
CH2CH2CH3;
R =
R
N
N
CF3
N
N
HN
OH
5
4
Cl
Cl
N
isoquine
CH3 ; R'-C6H4
R =
Figure 1.
Recently, we have synthesized new 4-aminoquinoline deriva-
tives endowed with a high activity against CQ-S and CQ-R strains
of P. falciparum and we have demonstrated that the presence of a
bulky, strongly basic, and lipophilic bicyclic moiety (such as the
quinolizidine or the pyrrolizidine ring), which is not supposed to
be easily metabolized, appears an interesting structural feature
to overcome resistance.11,12
roquinoline derivatives (5, Fig. 1), that were poorly active against
the W-2 CQ-resistant strain of P. falciparum, with IC50 = 13.8
l
M
for the best compound (5, R = CH3). In these compounds, that lack
any basic head, the pyrazole ring is linked to the quinoline nucleus
without the usual intercalating NH group. This supports the claim
that the resonance between the nitrogen atoms of the imino group
and of the quinoline is fundamental to explain the association of
CQ-like drugs with ferriprotoporphyrin IX, and hence their antima-
larial activity.14
Pursuing our research, we explored and here we report the ef-
fect of replacing the diethylamino group of isoquine with the bulky
(pyrrolizidin-7a-yl)alkylamino and (quinolizidin-1
a
-yl)methyl-
On this ground, we managed to replace the benzene ring of AQ-
like drugs with a pyrrole nucleus, still linked to the quinoline moi-
ety through the usual NH group. The pyrrole ring retains the high
reactivity of phenol, resulting well suitable for the Mannich reac-
tion used to introduce different types of basic heads (8–10, Fig. 2).
amino moieties (compounds 6d–f, Fig. 2) that were present in
our previous highly active CQ analogues. We reasoned that the
high lipophilicity of these substituents could improve the cellular
permeation, while the presence of an additional protonable nitro-
gen atom should promote the endocellular accumulation of the
drug.
2. Chemistry
To further analyze the importance of the aromatic hydroxyl
function, the hydroxyl group of compound 6f was replaced by a
chlorine atom (7f). Indeed, the 40-dehydroxy-40-fluoroamodiaqu-
ine,8 while resulting more resistant to oxidation, was shown to
maintain the same level of activity of AQ against CQ-S and CQ-R
strains of P. falciparum.
We decided also to investigate the possibility to replace the
benzene ring of AQ and of its analogues with other aromatic nuclei.
Recently, Cunico et al.13 described a set of 4-(pyrazol-1-yl)-7-chlo-
The isoquine analogues 6d–f (Fig. 2) were prepared in three
steps as indicated in Scheme 1. The proper amine (free base or
dihydrochloride) was reacted with formaldehyde and 3-acetami-
dophenol in absolute ethanol; the acetamido derivative was
hydrolized with 20% HCl and the resulting amine was heated with
4,7-dichloroquinoline in ethanol.
Alternatively, in order to spare the costly bicyclic amines, these
compounds could be prepared by reacting first 4,7-dichloroquino-
R
R'
H3C
R'
R'
N
N
N
R''
N
R''
N
R''
HN
HN
N
R
HN
N
CH3
CH3
Cl
N
Cl
Cl
8a-d, f
R = OH : 6d-f
R = Cl : 7f
R = H : 9a-c, f, g
R = Cl : 10a, b
H
N
H
N
N
H
R'
;
H
N
H
N
;
;
;
;
=
;
N
HN
N
H
N
N
N
N
R''
a
b
c
d
e
g
f
Figure 2. Structures of the investigated compounds.

M. Casagrande et al. / Bioorg. Med. Chem. 16 (2008) 6813–6823
6815
R'
R'
R'
N
N
Cl
N
N
R''
OH
OH
R''
OH
R''
a
b
c, d
HN
N
OH
+
Cl
N
H
HN
NH2
Cl
O
O
6d-f
11d-f
Scheme 1. Reagents and conditions: (a) CH2O, HNR0R00, EtOH, reflux, 24 h; (b) 20% HCl, reflux, 8 h; (c) EtOH, reflux, 8 h; (d) concd NH3, pH 8.
line with 3-aminophenol, in the presence of KI,15 and then con-
densing the 7-chloro-4-(3-hydroxyphenylamino)quinoline (12)
with formaldehyde and the proper amine (Scheme 2).
Compound 7f was obtained by coupling 2-chloro-4-nitroben-
zoic acid with lupinylamine in the presence of DCC; amido and
nitrogroup were reduced, respectively, with BH3 in THF and with
iron in acetic acid.16,17 Finally, the aromatic aminocompound was
reacted as usual with 4,7-dichloroquinoline (Scheme 3).
The 4-(pyrrol-1-yl)aminoquinoline derivatives 8 (Fig. 2) were
obtained in two steps. First, the 7-chloro-4-hydrazinoquinoline
was condensed with 2,5-hexanedione or 1-phenyl-1,4-pentandi-
one in acetic acid, giving rise through a Paal–Knorr reaction to a
pyrrole derivative, which was reacted with formaldehyde and the
proper amine in acetic acid (Scheme 4) to obtain compounds 8a–
d, f.
(20 and 21) by the action of the relevant amines in the presence
of trimethylaluminum in toluene.18,19 Amides were, finally, re-
duced to the expected aminocompounds 9a, b, f, g, and 10a, b
by means of either lithium aluminum hydride or diphenylsilane
in the presence of tris(triphenylphosphine)rhodium(I)carbonyl
hydride20,21 (Scheme 5), thus preventing the dehalogenation of
the phenyl ring.
The required 1,4-diketones of Scheme 4 were commercially
available, while the diketoesters of Scheme 5 were prepared
according to the literature22,23 by reacting phenacyl bromide or
4-chlorophenacyl bromide with sodium ethyl acetacetate.
The amido compound 20c, probably due to an overwhelming
steric hindrance, could not be obtained through the above de-
scribed method, thus an alternative synthetic pathway was set
up for compound 9c (Scheme 6). tert-Butylamine was reacted with
diketene to obtain N-(tert-butyl)acetacetamide,24 that with phen-
acylbromide gave the 1,4-diketone (22) required to form the pyr-
role derivative 20c and then the amido compound was reduced
to the amine 9c.
Starting from the pyrrole 17, bearing on positions 2 and 5 differ-
ent substituents, the Mannich reaction with diethylamine gave two
isomeric 3- and 4-(diethylamino)methyl derivatives (as indicated
by the NMR spectra) whose separation was very difficult and te-
dious. Thus, in this way only the 3-substituted isomer 9a was iso-
lated in a pure form.
Finally, the (hexahydro-1H-pyrrolizin-7a-yl)methyl- and ethyl-
amine and the (1S,9aR)-(octahydro-2H-quinolizin-1-yl)methyl-
and ethylamine (lupinylamine and homo-lupinylamine), required
to obtain compounds of Figure 2, were prepared, respectively, as
previous described by Oka et al.,25–27 Sparatore et al.,28 and Boido
et al.29
Therefore, in order to obtain compounds
7-chloro-4-hydrazinoquinoline was condensed with ethyl
-phenacyl- or -(4-chlorophenacyl)-acetoacetate, and the ob-
9 and 10, the
a
a
tained pyrrole derivatives (18 and 19) were converted to amides
R'
N
Cl
OH
HN
N
OH
R''
b
a
+
HN
N
OH
Cl
N
NH2
Cl
Cl
6d-f
12
Scheme 2. Reagents and conditions: (a) 2 N HCl, KI, EtOH, reflux; (b) CH2O, HNR0R00, EtOH, reflux.
H
H
N
N
H
O
N
N
N
NH2
H
COOH
Cl
N
H
H
Cl
H
Cl
Cl
e, f
a
c, d
b
7f
+
N
NO2
NO2
NO2
NH2
13
14
15
Scheme 3. Reagents and conditions: (a) DCC, DMAP, CH2Cl2, rt; (b) BH3–THF, THF, 0 °C to reflux; (c) Fe, AcOH, EtOH/H2O, reflux; (d) concd NH3; (e) 4,7-dichloroquinoline, 6 N
HCl, KI, EtOH, reflux; (f) aq NaOH.
R
R=CH3
8a-d, f
9a
NHNH2
N
O
HN
N
a
b
R'
+
+ HN
CH2O
+
R
R''
Cl
N
O
Cl
R= C6H5
R = CH3; 16
R = C6H5; 17
Scheme 4. Reagents and conditions: (a) AcOH, 65 °C, 3 h; (b) AcOH, rt, 2–3 h.

6816
M. Casagrande et al. / Bioorg. Med. Chem. 16 (2008) 6813–6823
R
R
R'
R''
COOEt
b
c; R=H
d; R=Cl
N
CO N
NHNH2
+
N
O
COOEt
O
9a, b, f, g
10a, b
HN
N
HN
N
a
Cl
N
R
Cl
Cl
R= H 20a, b, f, g
R= Cl 21a, b
R= H 18
R= Cl 19
Scheme 5. Reagents and conditions: (a) AcOH, reflux; (b) AlMe3, HNR0R00, PhMe, reflux; (c) LiAlH4, Et2O, reflux; (d) Ph2SiH2, (PPh3)3(CO)HRh, THF.
H
H
N
O N
O
N
O
O
O
a
b
c
HN
N
O
d
+ H2N
O
9c
N
H
O
Cl
22
20c
Scheme 6. Reagents and conditions: (a) Et3N, CH2Cl2, 0° to rt; (b) phenacyl bromide, EtONa, Et2O, reflux; (c) 7-chloro-4-hydrazinoquinoline, AcOH, reflux; (d) LiAlH4, Et2O,
reflux.
Structures of most final compounds and intermediates were
supported by 1H NMR and high-resolution mass spectra; final com-
pounds 8a and 8b were characterized by 1H NMR and elemental
analyses, while the intermediate 11e, which was used without a
complete purification, was characterized by 1H NMR and low reso-
lution mass spectrum.
compounds were not tested simultaneously). The ratios between
the IC50 of each compound against the two strains of P. falciparum
are also indicated. The last value is suggestive of the susceptibility
of the drug to the resistance mechanisms (resistance factor).
All tested compounds exhibited from moderate to high activity
on the CQ-S (D-10) strain, with IC50 ranging from 8.5 to 222.2 nM.
CQ IC50 was 27.2 nM (range 15–39 nM), thus the tested compounds
were from 0.1- to 2.8-fold as active as CQ.
3. Results and discussion
Five of the new compounds also exhibited a strong activity
against the CQ-R (W-2) strain, resulting from 10 to 28 times more
active than CQ, with IC50 values as low as 14–59 nM (9a, 10b, 10a,
6f, and 7f) compared to 481.2 nM of CQ (range 165–805 nM). Four
additional compounds (6e, 6d, 8f, and 9b) were in the order from
2- to 4.2-fold more active than the reference drug, while the
remaining seven compounds were from 0.5 to 1.2 times as active
as CQ on the W-2 strain.
The 16 compounds of Figure 2 were tested in vitro against D-10
(CQ-S) and W-2 (CQ-R) strains of P. falciparum. The antimalarial
activity was quantified as inhibition of parasite growth, measured
with the production of parasite lactate dehydrogenase (pLDH).30
Cytotoxicity on mammalian cell lines was assayed using the MTT
test.31
Table 1 shows the IC50 (nM) and the means of the ratios
between the IC50 of CQ and that of each compound against D-10 or
W-2 strains, calculated for each single experiment (since all the
Moreover, for the five most potent compounds the resistance
factor ranged from 1.2 to 1.7, resulting about ten times lower than
Table 1
In vitro antimalarial activity against D-10 and W-2 strains of P. falciparum and cytotoxicity against human (HMEC-1 and K562) and murine (WEHI) cell lines of compounds 6–10
Compound
D-10 (CQ-S)
IC50 (nM)
Ratio IC50
W-2 (CQ-R)
IC50 (nM)
Ratio IC50
Ratio IC50
HMEC-1
IC50 (nM)
K562
IC50 (nM)
WEHI 13
IC50 (nM)
CQ/Comp.b
CQ/Comp.b
CQ-R/CQ-Sc
a
a
d
d
d
6d
6e
6f
7f
8a
8b
8c
8d
8f
9a
9b
9c
9f
9g
10a
10b
Isoquine
CQ
81.9 3.4
90.3 48.5
8.5 1.9
0.4
0.3
2.8
1.8
0.2
0.2
0.3
0.1
0.4
0.7
0.7
0.4
0.8
0.2
0.9
0.8
1.3
—
76.96 14.6
153.1 24.7
14.3 7.8
2.2
2.1
22.0
28.0
1.2
0.7
0.9
0.5
3.1
9.9
4.2
0.7
1.1
0.9
1.7
1.7
1.2
2.8
4.2
5.3
1.6
0.8
1.4
3.3
18.5
14.1
5.0
n.t.
n.t.
7938 1242
5517 1193
n.t.
n.t.
n.t.
n.t.
5398 841
16,694 5919
12,874 2824
n.t.
13,451 7111
14,599 5031
5812 1511
7176 1383
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
12,211 986
4427 655
2769 1278
n.t.
n.t.
n.t.
23.6 5.1
69.0 25.3
80.7 27.1
48.6 10.5
222.2 15.6
70.7 5.3
42.7 8.9
42.9 13.0
63.5 18.2
36.5 12.6
154.1 37.5
26.1 8.3
30.7 4.3
23.7 3.0
27.2 3.5
28.8 4.5
192.9 4.0
336.3 64.3
255.2 53.7
358.3 20.6
53.4 16.4
59.2 27.4
139.5 56.0
1176.1 538.9
515.0 252.8
776.2 386.2
42.5 19.1
49.6 5.2
n.t.
n.t.
5508 3827
4380 2105
3878 638
n.t.
7441 1254
7219 1325
1446 670
2060 125
n.t.
3616 1268
2920 866
n.t.
1683 394
2614 629
n.t.
0.8
18.5
15.8
17.6
—
1.6
1.6
1.3
17.7
n.t.
n.t.
30.2e
481.2 183.9
>32,000
29,030 5701
>38,000
n.t., not tested.
a
The results are expressed as IC50 SD of at least three different experiments each performed in duplicate or triplicate.
b
c
Mean of ratios between the IC50 of chloroquine and that of each compound against D-10 or W-2 strains of P. falciparum calculated for each single experiment.
Ratios between the IC50 values of each compound against the two strains of P. falciparum.
The cytotoxic activity was assayed in vitro on different cell line using the MTT assay.
d
e
Mean of two experiments each performed in duplicate.

M. Casagrande et al. / Bioorg. Med. Chem. 16 (2008) 6813–6823
6817
that of CQ (17.7), suggesting that they were not (or only poorly) af-
fected by the resistance mechanisms.
5. Experimental
5.1. General
With regards to the structure–activity relationships among the
isoquine analogues, it has been observed that the quinolizidinylm-
ethylamino derivative (6f) exhibited a 10-fold higher activity than
the pyrrolizidinylalkylamino analogues 6d and 6e. Such a differ-
ence was unexpected, since in our previously described CQ ana-
logues,11,12 characterized also by the presence of these basic
moieties, the pyrrolizidinylalkylamino derivatives were somewhat
more active than the quinolizidinylalkylamino compounds.
Within the present series of compounds, the unsuitability of the
pyrrolizidinyl moiety was further observed in compound 8d, that
was the least active among all.
All commercially available solvents and reagents were used
without further purification, unless otherwise stated. CC = flash
column chromatography. Mps: Büchi apparatus, uncorrected. 1H
NMR spectra: Varian Mercury 300VX spectrometer; CDCl3 or
DMSO-d6 with Me4Si as internal standard; d in parts per million,
J in Hertz. Elemental analyses (of compounds 8a and 8b) were per-
formed on a Carlo Erba-EA-1110 CHNS-O instrument in the Micro-
analysis Laboratory of the Department of Pharmaceutical Sciences
of Genoa University. Mass spectra were recorded on a LCQ Advan-
tage (Thermofinnigan) mass spectrometer and high-resolution
mass spectra (HRMS) on a APEX II ICR-FTMS Bruker Daltonics mass
spectrometer in positive or negative electro spray ionization (ESI).
Interestingly, the replacement of the hydroxyl group of 6f with
a chlorine atom (7f) enhanced the activity against the CQ-R strain
of P. falciparum (ratio IC50CQ/compound), further supporting the
observation of O’Neill et al.8 that the hydroxyl is useful but not
absolutely necessary for activity.
5.2. N-[3-Hydroxy-4-[(N0-substituted-amino)methyl]
phenyl]acetamide (11d–f): general method
O’Neill et al.10 tested isoquine against a CQ-R strain of P. falcipa-
rum (K1) different from that used by us, thus not allowing a direct
comparison with our compounds. Such a comparison appears even
more difficult, since isoquine exhibited different degrees of activity
when assayed as free base or as diphosphate salt.
3-Acetamidophenol (756 mg, 5 mmol) was added to a stirred
solution of amine (free base, 5 mmol) and 37% aqueous formalde-
hyde (5 mmol, 0.37 ml) in 4 ml of absolute EtOH. The resulted solu-
tion was heated at reflux for 24 h under an atmosphere of N2. After
cooling, organic layer was evaporated and the obtained solid was
purified on silica gel column (CC) using CH2Cl2–MeOH (with or
without concd NH3) as eluent to afford the title compounds.
Therefore, we prepared isoquine according to O’Neill et al. pro-
cedure10 and tested it as free base against the same CQ-S and CQ-
R strains used for our compounds. Thus, we observed that isoquine
was 17.6 times more active than CQ on W-2 strain of P.falciparum,
while 6f and 7f were, respectively, 22 and 28 times more active.
The replacement of the phenolic ring, typical of AQ, tebuqu-
ine, and isoquine, with a pyrrolic ring is associated with a good
antimalarial activity. Therefore, these compounds represent a
new class of antimalarial agents worthy of further investigation.
Within this class of compounds the activity increased with the
increasing lipophilicity; thus 2-methyl-5-(4-chlorophenyl)- and
2-methyl-5-phenyl-derivatives (10a, 10b, and 9a–9f, respec-
tively), more resembling tebuquine, were more active than the
corresponding 2,5-dimethyl derivatives (8a–8f). With regards to
the effects of the basic heads on the activity, it is observed that
the smaller diethylamino and pyrrolidino moieties are more
profitable when a phenyl substituent is present on the pyrrole
ring, while the bulkier quinolizidinylmethylamino group (8f) is
advantageous within the 2,5-dimethylpyrrole subset of
compounds.
5.2.1. N-[4-[[(Hexahydro-1H-pyrrolizin-7a-yl)methyl
amino]methyl]-3-hydroxyphenyl]acetamide (11d)
CC (CH2Cl2/MeOH/concd NH3; from 93:6.3:0.7 to 90:9:1).
Amorphous solid rinsed with ethyl ether. Yield: 18%. 1H NMR
(DMSO-d6): d 9.72 (s, 1H); 7.02 (d, J = 1.92 Hz, 1H); 6.95 (d,
J = 8.20 Hz, 1H); 6.85 (dd, J = 8.20, 1.92 Hz, 1H); 3.72 (s, 2H);
2.90–2.75 (m, 2H); 2.33 (s, 2H); 1.98 (s, 3H); 1.80–1.35 (m, 10H).
HRMS (ESI) m/z calcd for C17H26N3O2 [M+H]+: 304.20195; found:
304.20222.
5.2.2. N-[4-[[2-(Hexahydro-1H-pyrrolizin-7a-yl)
ethylamino]methyl]-3-hydroxyphenyl]acetamide (11e)
CC (CH2Cl2/MeOH/concd NH3; up to 77:22:1). Amorphous solid
rinsed with a mixture of ethyl ether/CH2Cl2 (1:1). Yield: 14%. 1H
NMR (CDCl3):
d 6.87 (dd, J = 8.20, 1.92 Hz, 1H); 6.62 (d,
The most active compounds (6f, 7f, 8f, 9a, 10a, and 10b) exhibit
low toxicity against three different human or mouse cell lines with
a selectivity index ranging between 555 and 34.
J = 8.20 Hz, 1H); 6.50 (d, J = 1.92 Hz, 1H); 3.90 (s, 2H); 3.00–2.90
(m, 2H); 2.65–2.50 (m, 2H); 2.15 (s, 3H); 1.90–1.35 (m, 12H). MS
(ESI): m/z 318 [M+H]+.
5.2.3. N-[3-Hydroxy-4-[[((1S,9aR)-octahydro-2H-quinolizin-1-
yl)methylamino]methyl]phenyl]acetamide (11f)
4. Conclusions
CC (CH2Cl2/MeOH; 92:8). Solid rinsed with ethyl ether. Yield:
21%. Mp 117.8–122 °C. 1H NMR (DMSO-d6): d 7.20 (s, 1H); 7.05
(dd, J = 8.20, 1.92 Hz, 1H); 6.88 (d, J = 8.20 Hz, 1H); 6.80 (d,
J = 1.92 Hz, 1H); 3.90 (s, 2H); 2.90–2.70 (m, 4H); 2.10 (s, 3H);
In order to develop new classes of antimalarial agents, the pos-
sibility of replacing the phenolic ring of AQ, tebuquine, and isoqu-
ine with other kinds of aromatic nuclei was investigated.
Within a first set of pyrrole analogues, several compounds (8f,
9a, 10a, and 10b) displayed high activity against W-2 CQ-R strain
of P. falciparum with good selectivity indexes.
2.10–1.40 (m, 14H). HRMS (ESI) m/z calcd for
C19H30N3O2
[M+H]+: 332.23325; found: 332.23328.
The isoquine structure was also modified by replacing the
diethylamino group with some more metabolically stable bicyclic
moieties. Compound (6f), bearing a quinolizidinylmethylamino
residue, displayed high activity against both CQ-S and CQ-R strains.
By replacing the aromatic hydroxyl function with a chlorine atom
(7f), the activity ratio with CQ was further increased. On the basis
of these results, the exchange of phenol ring of amodiaquine-like
drugs with other aromatic moieties, as well as the replacement
of the usual amino groups with pyrrolizidine and quinolizidine-de-
rived basic heads will be further investigated.
5.3. 5-[(7-Chloroquinolin-4-yl)amino]-2-[(substituted-amino)
methyl]phenol (6d–f): general method
A stirred solution of the above acetamide (1 mmol) in 3 ml of
20% HCl was refluxed for 8 h under atmosphere of N2. The solution
was evaporated to dryness under vacuum, the residue was dis-
solved in 3 ml of absolute EtOH and 4,7-dichloroquinoline
(198 mg, 1 mmol) was added. The stirred mixture was heated for
8 h under N2. After cooling, the organic layer was evaporated, the

6818
M. Casagrande et al. / Bioorg. Med. Chem. 16 (2008) 6813–6823
residue was dissolved in water, alkalized with concd NH3 and ex-
tracted with CH2Cl2. The organic layer was dried on anhydrous
Na2SO4 and evaporated; the obtained solid was purified on silica
gel column (CC) using CH2Cl2–MeOH (with or without concd
NH3) as eluent to afford the title compounds.
The crude product was chromatographed on a column of silica
gel (CH2Cl2/MeOH, from 97:3 to 92:8). The resulted solid was
washed with ethyl ether. Yield: 52%. Mp 126.1–128.3. 1H NMR
(CDCl3): d 8.78 (s, 1H); 8.30 (s, 1H); 8.15 (d, J = 9.08 Hz, 1H); 7.75
(d, J = 9.08 Hz, 1H); 3.75–3.55 (m, 2H); 2.90–2.70 (m, 2H); 2.40–
1.15 (m, 14H). HRMS (ESI) m/z calcd for C17H23ClN3O3 [M+H]+:
352.14225; found: 352.14330.
5.3.1. 5-[(7-Chloroquinolin-4-yl)amino]-2-[[(hexahydro-1H-
pyrrolizin-7a-yl)methylamino]methyl]phenol (6d)
CC (CH2Cl2/MeOH; up to 83:17); followed by a preparative
TLC on silica gel (Kieselgel 60 F254, 1 mm, CH2Cl2/MeOH/concd
NH3; 10:2:0.2). Solid washed with ethyl ether. Yield: 6%. Mp
109–120.5 °C. 1H NMR (DMSO-d6): d 8.90 (s, 1H); 8.45–8.25
(m, 2H); 7.85 (s, 1H); 7.53 (d, J = 9.07 Hz, 1H); 7.07 (d,
J = 9.07 Hz, 1H); 6.90 (s, 1H); 6.60–6.75 (m, 2H); 3.80 (s, 2H);
2.90–2.80 (m, 2H); 2.36 (s, 2H); 1.80–1.00 (m, 10H). HRMS
5.6. N-(2-Chloro-4-nitrobenzyl)((1S,9aR)-octahydro-2H-quinolizin-
1-yl)methanamine (14)
A solution of 13 (380 mg, 1.08 mmol) in 3 ml of anhydrous THF
was added dropwise to a stirred, ice-cold solution of BH3 in THF
(1 N, 17 ml, 17 mmol). When addition was completed, the mixture
was heated at reflux for 6 h under N2. After, to the ice-cooled solu-
tion, 7 ml of 10 N HCl were carefully added and the solution was
heated at reflux for 10 min. The THF was removed under vacuum;
the resulting acid solution was saturated with NaOH pellets and
extracted three times with CH2Cl2. The solvent was removed and
the crude oil was purified by CC (silical gel, CH2Cl2/MeOH; from
98:2 to 94:6). The obtained oil was taken up in petroleum ether
and, after filtration, the solvent was removed to leave a sticky oil.
Yield: 87%. 1H NMR (CDCl3): d 8.22 (s, 1H); 8.10 (d, J = 9.08 Hz,
1H); 7.70 (d, J = 9.08 Hz, 1H); 3.95 (s, 2H); 2.95–2.65 (m, 4H);
2.25–1.00 (m, 14H). HRMS (ESI) m/z calcd for C17H25ClN3O2
[M+H]+: 338.16298; found: 338.16309.
(ESI) m/z calcd for
423.19524.
C
24H28ClN4O [M+H]+: 423.19462; found:
5.3.2. 5-[(7-Chloroquinolin-4-yl)amino]-2-[[2-(hexahydro-1H-
pyrrolizin-7a-yl)ethylamino]methyl]phenol (6e)
CC (CH2Cl2/MeOH; from 95:5 to 88:12). Solid rinsed with a mix-
ture of ethyl ether–CH2Cl2. Yield: 38%. Mp 204–206.7 °C. 1H NMR
(CDCl3): d 8.50 (d, J = 5.50 Hz, 1H); 8.00 (d, J = 1.93 Hz, 1H); 7.80
(d, J = 9.07 Hz, 1H); 7.40 (dd, J = 1.93, 9.08 Hz, 1H); 7.10–6.80 (m,
3H); 6.70 (dd, J = 1.92, 7.70 Hz, 1H); 4.00 (s, 2H); 3.00–2.80 (m,
2H); 2.80–2.40 (m, 4H); 1.70–1.40 (m,10H). HRMS (ESI) calcd for
C
25H30ClN4O [M+H]+: 437.21027; found: 437.21110. The free base
was converted to tri-hydrochloride (mp 247.2–256.7 °C) for biolog-
5.7. 3-Chloro-4-[[((1S,9aR)-octahydro-2H-quinolizin-1-yl)meth-
ical tests.
ylamino]methyl]benzenamine (15)
5.3.3. 5-[(7-Chloroquinolin-4-yl)amino]-2-[[((1S,9aR)-octahydro-
2H-quinolizin-1-yl)methylamino]methyl]phenol (6f)
Iron powder (270 mg, 4.8 mmol), freshly washed with 1 N HCl
and distilled water, was added to
a stirred solution of 14
CC (CH2Cl2/MeOH/concd NH3; from 96.5:3.2:0.3 to 96:3.6:0.4).
Solid rinsed with ethyl ether. Yield: 34%. Mp 136–137.5 °C. 1H
NMR (CDCl3): d 8.53 (d, J = 5.50 Hz, 1H); 8.00 (d, J = 1.93 Hz, 1H);
7.88 (d, J = 9.07 Hz, 1H); 7.45 (dd, J = 1.93, 9.08 Hz, 1H); 7.10–
6.90 (m, 2H); 6.75 (s, 1H); 6.70 (dd, J = 1.92, 7.70 Hz, 1H); 4.00 (s,
2H); 3.00–2.80 (m, 4H); 2.40–1.20 (m, 14H). HRMS (ESI) m/z calcd
for C26H32ClN4O [M+H]+: 451.22592; found: 451.22538.
(400 mg, 1.18 mmol) in 8.7 ml of ethanol/water (2:1), to which
0.3 ml of glacial acetic acid was then added. The suspension was
heated at reflux with vigorous stirring for 20 min under N2 atmo-
sphere. After cooling, the suspension was alkalized with concd
NH3 and filtered through a pad of CeliteÒ. The filtrate was concen-
trated under vacuum, diluted with water, and extracted with ethyl
acetate. The organic layer was dried and evaporated, and resulting
crude oil was purified by CC (Grade III alumina; cyclohexane/ethyl
acetate, 70:30) to afford compound 15 as an oil. Yield: 75%. 1H
NMR (CDCl3): d 7.10 (d, J = 9.05 Hz, 1H); 6.65 (s, 1H). 6.50 (d,
J = 9.05 Hz, 1H); 3.78–3.50 (m, 4H); 2.90–2.55 (m, 4H); 2.18–1.00
5.4. 5-[(7-Chloroquinolin-4-yl)amino]-2-[[((1S,9aR)-octahydro-
2H-quinolizin-1-yl)methylamino]methyl]phenol (6f): alternative
method
(m, 14H). HRMS (ESI) m/z calcd for
C
17H27ClN3 [M+H]+:
Aq. formaldehyde (37%, 0.14 ml, 1.5 mmol) was added to a stir-
red solution of aminolupinane (252 mg, 1.5 mmol) in 10 ml of
absolute ethanol; after a few minutes compound 1215 (406 mg,
1.5 mmol) and other 5 ml of ethanol were added and the mixture
was heated for 6 h under N2 atmosphere. After cooling, ethanol
was evaporated and the residue was chromatographed on a col-
umn of silica gel using CH2Cl2/MeOH/concd NH3 (96:3.6:0.4) as
eluent. Solid rinsed with a mixture of petroleum ether/ethyl ether
(90:10). Yield: 40%. Mp 139–142 °C.
308.18880; found: 308.18917.
5.8. 7-Chloro-4-[N-[3-chloro-4-[[((1S,9aR)-octahydro-2H-quinolizin-
1-yl)methylamino]methyl]phenyl]amino]quinoline (7f)
To
a stirred suspension of 4,7-dichloroquinoline (181 mg,
0.92 mmol) in 4 ml of EtOH 0.33 ml of 6 N HCl was added. When
the 4,7-dichloroquinoline was dissolved, compound 15 (270 mg,
0.88 mmol) and KI (2 mg) were added and the mixture was stirred
and heated at reflux for a night under N2 atmosphere.
5.5. 2-Chloro-4-nitro-N-[((1S,9aR)-octahydro-2H-quinolizin-1-
yl)methyl]benzamide (13)
After cooling, the solvent was removed, the residue was taken
up in 2 N NaOH and the mixture was extracted with ethyl ace-
tate. The crude product was purified by CC (silica gel; CH2Cl2/
MeOH/concd NH3; from 92:8:0 to 90:9.5:0.5). Solid rinsed with
ethyl ether. Yield: 79%. Mp 140.3–142.4 °C. 1H NMR (CDCl3): d
8.60 (d, J = 5.23 Hz, 1H); 8.04 (d, J = 1.93 Hz, 1H); 7.85 (d,
J = 9.08 Hz, 1H); 7.50–7.40 (m, 2H); 7.30 (d, J = 2.20 Hz, 1H);
7.15 (dd, J = 8.25, 2.20 Hz, 1H); 6.97 (d, J = 5.22 Hz, 1H); 6.65
(s, 1H); 3.85 (s, 2H); 2.90–2.75 (m, 4H); 2.15–1.35 (m, 14H).
HRMS (ESI) m/z calcd for C26H31Cl2N4 [M+H]+: 469.19203; found:
469.19200.
To
a solution of 2-chloro-4-nitrobenzoic acid (900 mg,
4.46 mmol) in 12 ml of anhydrous dichloromethane DCC (1.06 g,
5.13 mmol) and DMAP (16.5 mg, 0.134 mmol) were added. The
solution was stirred at rt for 15 min, a solution of aminolupinane28
(750 mg, 4.46 mmol) in 4 ml of anhydrous dichloromethane was
slowly added dropwise and the mixture was further stirred for
4 h at rt. The precipitate was filtered off and the organic layer
was washed with 2 N NaOH, then with brine, dried, and finally
evaporated in vacuo.

M. Casagrande et al. / Bioorg. Med. Chem. 16 (2008) 6813–6823
6819
5.9. 7-Chloro-4-[N-(2,5-dimethyl-1H-pyrrol-1-
yl)amino]quinoline (16)
5.11.2. 7-Chloro-4-[N-[2,5-dimethyl-3-((pyrrolidin-1-yl)methyl)-
1H-pyrrol-1-yl]amino]quinoline (8b)
CC (CH2Cl2/MeOH; from 95:5 to 88:12); solid rinsed with a mix-
2,5-Hexandione (767 mg, 6.72 mmol) was added to a stirred
solution of 7-chloro-4-hydrazinoquinoline (1.084 g, 5.6 mmol)
in glacial acetic acid (5.6 ml), and the mixture was heated at
65 °C under N2 for 3 h. After cooling, the solution was concen-
trated under vacuum, the residue dissolved in 50 ml of water
and alkalized with concd NH3; the precipitate was filtrated,
washed with cool water, and purified by CC (silica gel; CH2Cl2
containing 0.5–1% of MeOH). Then the resulting solid was
rinsed with ethyl ether. Yield: 90%. Mp 226–227.5 °C. 1H NMR
(DMSO-d6): d 10.33 (s, 1H); 8.44 (d, J = 4.95 Hz, 1H); 8.31 (d,
J = 9.07 Hz, 1H); 7.90 (d, J = 1.92 Hz, 1H); 7.57 (dd, J = 9.08,
1.93 Hz, 1H); 5.83 (s, 2H); 5.61 (d, J = 5.03 Hz, 1H); 1.95 (s,
6H). HRMS (ESI) m/z calcd for C15H15ClN3 [M+H]+: 272.09490;
found: 272.09469.
ture of EtOH–ethyl ether. Yield: 64%. Mp 160.5–162.5 °C. 1H NMR
(CDCl3):
d 8.50 (d, J = 4.67 Hz, 1H); 8.03 (s, 1H); 7.90 (d,
J = 8.25 Hz, 1H); 7.47 (dd, J = 9.08, 1.92 Hz, 1H); 5.95 (s, 1 H);
5.80 (d, J = 4.95 Hz, 1H); 3.45 (s, 2H); 2.55 (s, 4H); 2.15–1.95 (m,
6H); 1.80 (s, 4H). Anal. Calcd for C20H23Cl1N4 + 1/2H2O: C, 66.01;
H, 6.65; N, 15.40. Found: C, 65.84; H, 6.43; N, 15.42.
5.11.3. 7-Chloro-4-[N-[3-((tert-butylamino)methyl)-2,5-dimethyl-
1H-pyrrol-1-yl]amino]quinoline (8c)
CC (CH2Cl2/MeOH/concd NH3; from 96:3.6:0.4 to 94:5.4:0.6);
the resulting solid was dissolved into a mixture of cyclohexane/
dry ethyl ether (50:50), the solution was filtered and the sol-
vent was evaporated to leave pure compound. Yield: 27%. Mp
103.5–107.5 °C. 1H NMR (CDCl3): d 8.50 (d, J = 4.67 Hz, 1H);
8.03 (s, 1H); 7.90 (d, J = 8.25 Hz, 1H); 7.70 (br s, 1H); 7.47
(dd, J = 9.08, 1.92 Hz, 1H); 5.97 (s, 1H); 5.86 (d, J = 4.95 Hz,
1H); 3.60 (s, 2H); 2.10–1.90 (m, 6H); 1.22 (s, 9H). HRMS
5.10. 7-Chloro-4-[N-(2-methyl-5-phenyl-1H-pyrrol-1-yl)amino]-
quinoline (17)
(ESI) m/z calcd for
C
20H26ClN4 [M+H]+: 357.18405; found:
1-Phenyl-1,4-pentandione (846 mg, 4.8 mmol) was added to a
stirred solution of 7-chloro-4-hydrazinoquinoline (774 mg,
4 mmol) in glacial acetic acid (4 ml) and the mixture was re-
fluxed under N2 for 2 h. After cooling, the solution was concen-
trated under vacuum and the residue dissolved in CH2Cl2; the
organic phase was washed twice with 1 N NaOH, dried (Na2SO4),
the solvent removed and the crude solid was purified by CC (sil-
ica gel; CH2Cl2 up to 4% of MeOH). The violet resulting solid was
further purified by chromatography on grade I alumina (CH2Cl2/
cyclohexane from 90:10 to 95:5) to give a white solid. Yield:
44%. Mp 195.5–196.2 °C. 1H NMR (DMSO-d6): d 10.60 (s, 1H);
8.39 (d, J = 4.70 Hz, 1H); 8.30 (d, J = 9.08 Hz, 1H); 7.85 (s, 1H);
7.61 (d, J = 8.53 Hz, 1H); 7.50 (d, J = 7.70 Hz, 2H); 7.18 (t,
J = 7.43 Hz, 2H); 7.07 (d, J = 7.15 Hz, 1H); 6.40 (d, J = 3.3 Hz,
1H); 6.10 (s, 1H); 5.60 (d, J = 4.40 Hz, 1H); 2.02 (s, 3H). HRMS
357.18396.
5.11.4. 7-Chloro-4-[N-[2,5-dimethyl-3-[[(hexahydro-1H-pyrrolizin-
7a-yl)methylamino]methyl]-1H-pyrrol-1-yl]amino]quinoline (8d)
CC (CH2Cl2/MeOH/concd NH3; from 96:3.6:0.4 to 94:5.4:0.6);
solid rinsed with dry ethyl ether. Yield: 23%. Mp 92–97 °C (dec).
1H NMR (CDCl3): d 8.56 (s, 1H); 8.45 (d, J = 4.67 Hz, 1H); 8.00 (s,
1H); 7.90 (d, J = 8.25 Hz, 1H); 7.47 (dd, J = 9.08, 1.92 Hz, 1H); 5.90
(s, 1H); 5.86 (d, J = 4.95 Hz, 1H); 3.62 (s, 2H); 3.35–3.15 (m, 2H);
3.10 (br s, 1H); 2.75–2.60 (m, 2H); 2.20–1.50 (m, 16H). HRMS
(ESI) m/z calcd for
424.22695.
C
24H31Cl1N5 [M+H]+: 424.22625; found:
5.11.5. 7-Chloro-4-[N-[2,5-dimethyl-3-[[[((1S,9aR)-octahydro-2H-
quinolizin-1-yl)methyl]amino]methyl]-1H-pyrrol-1-yl]amino]-
quinoline (8f)
(ESI) m/z calcd for
C
20H17ClN3 [M+H]+: 334.11055; found:
334.11143.
CC (CH2Cl2/MeOH/concd NH3; from 96:3.6:0.4 to 95:4.5:0.5);
solid rinsed with dry ethyl ether. Yield: 35%. Mp 188–191 °C. 1H
NMR (CDCl3): d 8.40 (d, J = 4.59 Hz, 1H); 8.10–7.80 (m, 2H); 7.40
(d, J = 9.08, 1H); 5.97–5.65 (m, 2H); 3.65–3.30 (m, 2H); 3.30–2.95
(m, 2H); 2.95–2.65 (m, 2H); 2.65–0.60 (m, 20H). Anal. Calcd for
5.11. 7-Chloro-4-[N-[2,5-dimethyl-3-[(N-substituted)amino-
methyl]-1H-pyrrol-1-yl]amino]quinoline (8a–d and f): general
method
C26H34Cl1N5: C, 69.08; H, 7.58; N, 15.49. Found: C, 69, 07; H,
The proper amine (as free base or dihydrochloride; 1.5 mmol,
for secondary amines; 1.65 mmol, for primary amines), aq formal-
dehyde (37%, 1.5 mmol, for secondary amines; 1.65 mmol, for pri-
mary amines) were added to 0.5 ml of glacial acetic acid in an ice-
cooled flask. The solution was stirred for 2–3 min, and then poured
into a flask containing 16 (407.6 mg, 1.5 mmol). The mixture was
stirred at rt under N2 for 2 h, water (15 ml) was added and the
solution alkalized with 2 N NaOH. The resulting suspension was
extracted three times with CH2Cl2 and the collected organic phases
were dried (Na2SO4) and evaporated to give a crude derivative,
which was purified by CC (silica gel; CH2Cl2–MeOH, with or with-
out concd NH3).
7.22; N, 15.26.
5.12. Ethyl 2-acetyl-4-oxo-4-[(4-substituted)phenyl]butanoate:
general method
According to literature,22,23 ethyl acetoacetate (1.56 g,
12 mmol) was added dropwise to a stirred suspension of sodium
ethoxide (680 mg, 10 mmol) in 15 ml of anhydrous ethyl ether at
rt under N2. After 10 min a solution of phenacylbromide or 4-chlo-
rophenacylbromide (10 mmol) in 26 ml of anhydrous ethyl ether
was added dropwise, and the resulting mixture was heated at re-
flux for 2 h.
After cooling, the NaBr was filtered, washed twice with dry
ether, and the joined organic phases were evaporated to give a
product further purified by CC (silica gel; elution conditions as
indicated for each compound).
5.11.1. 7-Chloro-4-[N-[3-((diethylamino)methyl)-2,5-dimethyl-
1H-pyrrol-1-yl]amino]quinoline (8a)
CC (CH2Cl2/MeOH/concd NH3; from 97:2.7:0.3 to 95:4.5:0.5);
solid rinsed with petroleum ether/ethyl ether (30:70). Yield: 65%.
Mp 248–251 °C (dec.). 1H NMR (CDCl3): d 8.51 (d, J = 4.67 Hz,
1H); 8.04 (s, 1H); 7.92 (d, J = 8.25 Hz, 1H); 7.80 (s, 1H); 7.48 (dd,
J = 9.07, 1.92 Hz, 1H); 5.94 (s, 1H); 5.81 (d, J = 4.95 Hz, 1H); 3.45
(s, 2H); 2.55 (q, J = 6.88 Hz, 4H); 2.15–1.95 (m, 6H); 1.10 (t,
J = 6.82 Hz, 6H). Anal. Calcd for C20H25ClN4 + H2O: C, 64.07; H,
7.25; N, 14.94. Found: C, 63.68; H, 7.52; N, 14.71.
5.12.1. Ethyl 2-acetyl-4-oxo-4-phenylbutanoate22,23
CC (cyclohexane/CH2Cl2; from 60:40 to 40:60). Yield: 93%. Oil.
1H NMR (CDCl3): d 7.97 (d, J = 7.43 Hz, 2H); 7.58 (t, J = 7.43 Hz,
1H); 7.48 (t, J = 7.43 Hz, 2H); 4.25–4.18 (m, 3H); 3.67 (dd,
J = 18.43, 8.25 Hz, 1H); 3.43 (dd, J = 18.43, 8.25 Hz, 1H); 2.43 (s,
3H); 1.30 (t, J = 6.82 Hz, 3H).

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M. Casagrande et al. / Bioorg. Med. Chem. 16 (2008) 6813–6823
5.12.2. Ethyl 2-acetyl-4-(4-chlorophenyl)-4-oxobutanoate23
CC (cyclohexane/AcOEt; 60:40); resulting solid rinsed with
petroleum ether/dry ethyl ether (80:20). Yield: 81%. Mp 64.8–
66.6 °C. 1H NMR (CDCl3): d 7.92 (dd, J = 1.93, 6.88 Hz, 2H); 7.43
(dd, J = 1.93, 6.88 Hz, 2H); 4.25–4.18 (m, 3H); 3.67 (dd, J = 18.40,
8.25 Hz, 1H); 3.43 (dd, J = 18.40, 8.25 Hz, 1H); 2.43 (s, 3H); 1.30
5.14.2. [1-[(7-Chloroquinolin-4-yl)amino]-2-methyl-5-phenyl-
1H-pyrrol-3-yl](pyrrolidin-1-yl)methanone (20b)
During the reaction, a suspension was formed and a first portion
of 20b was collected and joined to the product obtained at the end
of the procedure. CC (CH2Cl2/MeOH; 98:2). Yield: 86%. Mp 306.1–
308.5 °C. 1H NMR (DMSO-d6): d 10.80 (br s, 1H); 8.30 (s, 1H);
7.90 (s, 1H); 7.70–7.00 (m, 7H); 6.70 (s, 1H); 5.66 (s, 1H); 3.65 (s,
2H); 3.41 (s, 2H); 2.18 (s, 3H); 1.85 (s, 4H). HRMS (ESI) m/z calcd
for C25H24ClN4O [M+H]+: 431.16332; found: 431.16457.
(t, J = 6.82 Hz, 3H). HRMS (ESI) m/z calcd for
C14H15ClO4Na
[M+Na]+: 305.05511; found: 305.05570.
5.13. Ethyl 1-[(7-chloroquinolin-4-yl)amino]-2-methyl-5-[(4-substi-
tuted)phenyl]-1H-pyrrole-3-carboxylate (18 and 19): general
method
5.14.3. 1-[(7-Chloroquinolin-4-yl)amino]-2-methyl-5-phenyl-1H-
pyrrole-3-N-[((1S,9aR)-octahydro-2H-quinolizin-1-yl)methyl]-
carboxamide (20f)
The above diketoester (6.2 mmol) was added to a stirred solu-
tion of 7-chloro-4-hydrazinoquinoline (1.2 g, 6.2 mmol) in 7 ml
of glacial acetic acid. The mixture was refluxed under N2 for
2 h to give a violet solution, which, after cooling, was alkalized
with cold 2 N NaOH and extracted three times with CH2Cl2.
The joined organic layers were dried (Na2SO4); the solvent was
removed and the crude solid was purified by CC (silica gel; dif-
ferent ratio of ethyl acetate and cyclohexane as indicated for
each compound).
During the reaction, a suspension was formed and compound
20f was filtered and washed twice with toluene. This product
was used directly in the next step without any purification. Yield:
88%. Mp 291.5–293.0 °C. 1H NMR (DMSO-d6): d 10.75 (br s, 1H);
8.35 (s 1H); 8.10 (s, 1H); 7.90–7.65 (m, 2H); 7.65–7.00 (m, 6H);
6.90 (s, 1H); 5.50 (s, 1H); 3.35 (s, 2H); 2.85–2.60 (m, 2H); 2.30 (s,
3H); 2.00–1.10 (m, 14H). HRMS (ESI) m/z calcd for C31H35ClN5O
[M+H]+: 528.25246; found: 528.25451.
5.14.4. 1-[(7-Chloroquinolin-4-yl)amino]-2-methyl-5-phenyl-1H-
pyrrole-3-N-[2-((1S,9aR)-octahydro-2H-quinolizin-1-yl)ethyl]-
carboxamide (20g)
5.13.1. Ethyl 1-[(7-chloroquinolin-4-yl)amino]-2-methyl-5-phenyl-
1H-pyrrole-3-carboxylate (18)
CC (AcOEt/cyclohexane; 40:60); solid rinsed with a mixture of
ethyl ether–petroleum ether. Yield: 79%. Mp 165–168 °C. 1H NMR
(DMSO-d6): d 11.55 (s, 1/2H); 10.83 (s, 1/2H); 8.41 (s, 1H); 8.30
(s, 1/2H); 7.90 (s, 1/2H); 7.75–7.00 (m, 7H); 6.75 (s, 1H); 5.65 (s,
1/2H); 5.20 (s, 1/2H); 4.20 (q, J = 6.87 Hz, 2H); 2.30 (s, 3H); 1.30
CC (CH2Cl2/MeOH; from 94:6 to 90:10); solid rinsed with
CH2Cl2/ethyl ether (30:70). Yield: 39%. Mp 197.6–200.5 °C. 1H
NMR (DMSO-d6): d 10.77 (br s, 1H); 8.40 (s, 1H); 8.30 (s, 1H);
7.90 (s, 2H); 7.70–7.00 (m, 6H); 6.90 (s, 1H); 5.66 (s, 1H); 3.30–
3.00 (m, 2H); 2.80–2.58 (m, 2H); 2.25 (s, 3H); 2.10–1.10 (m,
16H). HRMS (ESI) m/z calcd for C32H37ClN5O [M+H]+: 542.26811;
found: 542.26899.
(t, J = 6.87 Hz, 3H). HRMS (ESI) m/z calcd for
C23H21ClN3O2
[M+H]+: 406.13168; found: 406.13256.
5.13.2. Ethyl 5-(4-chlorophenyl)-1-[(7-chloroquinolin-4-yl)amino]-
2-methyl-1H-pyrrole-3-carboxylate (19)
5.14.5. 5-(4-Chlorophenyl)-1-[(7-chloroquinolin-4-yl)amino]-2-
methyl-1H-pyrrole-3-(N,N-diethyl)carboxamide (21a)
CC (AcOEt/cyclohexane; 30:70); solid rinsed with ethyl ether.
Yield 74%. Mp 210–212 °C. 1H NMR (DMSO-d6): d 11.55 (s, 1/2H);
10.83 (s, 1/2H); 8.41 (s, 1H); 8.30 (s, 1/2H); 7.90 (s, 1/2H); 7.70–
7.10 (m, 6H); 6.75 (s, 1H); 5.65 (s, 1/2H); 5.18 (s, 1/2H); 4.20 (q,
J = 6.82 Hz, 2H); 2.30 (s, 3H); 1.30 (t, J = 6.82 Hz, 3H). HRMS (ESI)
CC (CH2Cl2/MeOH; 99:1); solid rinsed with ethyl ether. Yield:
61%. Mp 261.5–263.8 °C. 1H NMR (DMSO-d6): d 10.75 (br s, 1H);
8.45-8.15 (m, 2H); 7.90 (s, 1H); 7.65-7.35 (m, 3H); 7.21 (d,
J = 8.25 Hz, 2H); 6.48 (s, 1H); 5.61 (s, 1H); 3.45 (q, J = 6.90 Hz,
4H); 2.05 (s, 3H); 1.12 (t, J = 6.90 Hz, 6H). HRMS (ESI) m/z calcd
for C25H25Cl2N4O [M+H]+: 467.13999; found: 467.14081.
m/z calcd for
C
23H20Cl2N3O2 [M+H]+: 440.09271; found:
440.09350.
5.14.6. [5-(4-Chlorophenyl)-1-[(7-chloroquinolin-4-yl)amino]-2-
methyl-1H-pyrrol-3-yl](pyrrolidin-1-yl)methanone (21b)
5.14. 1-[(7-Chloroquinolin-4-yl)amino]-2-methyl-5-[(4-substi-
tuted)phenyl]-1H-pyrrole-3-(N-substituted)carboxamide (20a,
b, f, g, 21a, and b): general method
During the reaction, a suspension was formed and a first portion
of 21b was collected and then joined to the product obtained at the
end of the general procedure. CC (CH2Cl2/MeOH; 98:2). Yield: 92%.
Mp 324.5–326.5 °C (dec.). 1H NMR (DMSO-d6): d 11.00 (br s, 1H);
8.45–8.15 (m, 2H); 7.75 (s, 1H); 7.65–7.40 (m, 3H); 7.30 (d,
J = 8.25 Hz, 2H); 6.75 (s, 1H); 5.50 (s, 1H); 3.70 (br s, 2H); 3.40
(br s, 2H); 2.15 (s, 3H); 1.81 (s, 4H). HRMS (ESI) m/z calcd for
A 2 M solution of Al(CH3)3 in toluene (0.625 ml, 1.25 mmol) was
added dropwise to an ice-cooled stirred solution of the proper
amine (1.25 mmol) in 10 ml of anhydrous toluene. The resulting
mixture was stirred under N2 at rt for 40 min, then 18 or 19
(1 mmol) was added. The solution was heated at reflux for 24 h,
cooled and then treated with 2 N NaOH solution. The precipitate
was filtered and washed four times with AcOEt, the joined organic
layers were dried (Na2SO4) and evaporated.
C
25H23Cl2N4O [M+H]+: 465.12434; found: 465.12561.
5.15. 7-Chloro-4-[N-[2-methyl-5-phenyl-3-[(N-substituted-amino)-
methyl]-1H-pyrrol-1-yl]-amino]quinoline (9a, b, f, and g): general
method
The solid was purified by CC (silica gel; different ratio of CH2Cl2
and MeOH as indicated for each compound).
Each amido compound 20 (1 mmol) was suspended in 43 ml
of anhydrous ethyl ether under N2 and LiAlH4 (296 mg,
7.8 mmol) was carefully added; the mixture was heated at reflux
and stirred for 4 h. After cooling, 1 N NaOH was added carefully
and the suspension filtrated; alumina was washed twice with
ethyl ether and the organic phase was dried (Na2SO4) and evap-
orated. The crude solid was purified by CC (silica gel; CH2Cl2–
MeOH with or without concd NH3 as indicated for each
compound).
5.14.1. 1-[(7-Chloroquinolin-4-yl)amino]-2-methyl-5-phenyl-
1H-pyrrole-3-(N,N-diethyl)carboxamide (20a)
CC (CH2Cl2/MeOH; 95:5); solid rinsed with a mixture of ethyl
ether/ethanol (95:5). Yield: 51%. Mp 252.7–254.3 °C. 1H NMR
(DMSO-d6): d 10.83 (br s, 1H); 8.41 (s, 1H); 7.90 (s, 1H); 7.70–
7.00 (m, 7H); 6.50 (s, 1H); 5.66 (s, 1H); 3.41 (q, J = 6.82 Hz,
4H); 2.03 (s, 3H); 1.10 (t, J = 6.82 Hz, 6H). HRMS (ESI) m/z calcd
for C25H26ClN4O [M+H]+: 433.17897; found: 433.18035.

M. Casagrande et al. / Bioorg. Med. Chem. 16 (2008) 6813–6823
6821
5.15.1. 7-Chloro-4-[N-[3-[(diethylamino)methyl]-2-methyl-5-
phenyl-1H-pyrrol-1-yl]amino]quinoline (9a)
dium(I)
carbonyl
hydride
(45.94 mg,
0.05 mmol)
and
diphenylsilane (3.3 ml, 18 mmol) were added in five portions dur-
ing 2 h and the mixture was further stirred for 3 h, until the amide
was completely reduced. After cooling, THF was evaporated, and
the residue was partitioned between CH2Cl2 and 0.5 N HCl. The
acid phase was alkalized with a 30% NaOH solution and extracted
with CH2Cl2. The organic solution was dried (Na2SO4) and evapo-
rated to yield the crude product that was purified by CC (silica
gel; CH2Cl2/MeOH, 94:6).
CC (CH2Cl2/MeOH; 95:5); solid rinsed with ethyl ether/petro-
leum ether (40:60). Yield: 66%. Mp 156.5–160 °C (dec.). 1H NMR
(DMSO-d6): d 10.63 (s, 1H); 8.41 (d, J = 5.22 Hz, 1H); 8.30 (d,
J = 9.08 Hz, 1H); 7.90 (s, 1H); 7.60 (d, J = 8.80 Hz, 1H); 7.50 (d,
J = 7.43 Hz, 2H); 7.19 (t, J = 7.43 Hz, 2H); 7.08 (d, J = 7.30 Hz, 1H);
6.40 (s, 1H); 5.60 (d, J = 5.23 Hz, 1H); 3.45 (s, 2H); 2.01 (s, 3H);
1.02 (t, J = 6.87 Hz, 6H); four protons are missing probably because
covered by the DMSO signal. HRMS (ESI) m/z calcd for C25H28Cl1N4
[M+H]+: 419.19970; found: 419.19963.
5.17.1. 7-Chloro-4-[N-[5-(4-chlorophenyl)-3-[(diethylamino)-
methyl]-2-methyl-1H-pyrrol-1-yl]amino]quinoline (10a)
Solid rinsed with ethyl ether. Yield: 61%. Mp 215.6–220 °C
(dec.). 1H NMR (DMSO-d6): d 10.60 (s, 1H); 8.39 (d, J = 5.22 Hz,
1H); 8.30 (d, J = 9.08 Hz, 1H); 7.90 (s, 1H); 7.61 (d, J = 8.80 Hz,
1H); 7.51 (d, J = 8.25 Hz, 2H); 7.21 (d, J = 8.25 Hz, 2H); 6.42 (s,
1H); 5.77 (d, J = 5.22 Hz, 1H); 3.55 (s, 2H); 2.10 (s, 3H); 1.15 (t,
J = 6.87 Hz, 6H); four protons are missing probably because cov-
ered by the DMSO signal. HRMS (ESI) m/z calcd for C25H27 Cl2N4
[M+H]+: 453.16073; found: 453.16154. The free base was con-
verted to dihydrochloride (mp 185.5–191.8 dec.) for biological
tests.
5.15.2. 7-Chloro-4-[N-[2-methyl-5-phenyl-3-[(pyrrolidin-1-yl)methyl]-
1H-pyrrol-1-yl]amino]quinoline (9b)
CC (CH2Cl2/MeOH; 96:4); solid washed with ethyl ether. Yield:
51%. Mp 172.5–176 °C. 1H NMR (DMSO-d6): d 10.65 (s, 1H); 8.40 (d,
J = 5.22 Hz, 1H); 8.30 (d, J = 9.08 Hz, 1H); 7.90 (s, 1H); 7.60 (d,
J = 8.80 Hz, 1H, CH); 7.50 (d, J = 7.43 Hz, 2H); 7.20 (t, J = 7.43 Hz,
2H); 7.09 (d, J = 7.30 Hz, 1H); 6.41 (s, 1H); 5.60 (d, J = 5.23 Hz,
1H); 3.55 (s, 2H); 2.65–2.50 (m, 4H); 2.02 (s, 3H); 1.85–1.55 (m,
4H). HRMS (ESI) m/z calcd for C25H26ClN4 [M+H]+: 417.18405;
found: 417.18410.
5.15.3. 7-Chloro-4-[N-[2-methyl-3-[[((1S,9aR)-octahydro-2H-
quinolizin-1-yl)methyl]aminomethyl]-5-phenyl-1H-pyrrol-1-
yl]amino]quinoline (9f)
5.17.2. 7-Chloro-4-[N-[5-(4-chlorophenyl)-2-methyl-3-[(pyrrolidin-
1-yl)methyl]-1H-pyrrol-1-yl]amino]quinoline (10b)
Solid rinsed with ethyl ether. Yield: 41%. Mp 179–182.3 °C. 1H
NMR (DMSO-d6): d 10.65 (s, 1H); 8.40 (d, J = 5.22 Hz, 1H); 8.30
(d, J = 9.08 Hz, 1H); 7.90 (s, 1H); 7.65 (d, J = 8.80 Hz, 1H); 7.52 (d,
J = 8.25 Hz, 2H); 7.25 (d, J = 8.25 Hz, 2H); 6.45 (s, 1H); 5.60 (d,
J = 5.22 Hz, 1H); 3.45 (s, 2H); 3.30 (s, 2H); 2.00 (s, 3H); 1.70 (s,
4H); four protons are missing probably because covered by the
DMSO signal. HRMS (ESI) m/z calcd for C25H25Cl2N4 [M+H]+:
451.14508; found: 451.14505. The free base was converted to
dihydrochloride (mp 207.5–211.7 °C dec.) for biological tests.
CC repeated twice (CH2Cl2/MeOH/concd NH3; from 95:4.5:05 to
94:5.4:0.6). Solid rinsed with petroleum ether. Yield: 32%. Mp
93.5 °C (dec.). 1H NMR (CDCl3): d 8.50 (s, 1H); 8.15–7.90 (m, 1H);
7.75–7.60 (m, 1H); 7.60–7.30 (m, 3H); 7.22–7.00 (m, 3H); 6.39 (s,
1H); 6.05 (s, 1H); 3.70 (s, 2H); 3.00–2.70 (m, 4H); 2.10 (s, 3H);
1.95–1.10 (m, 14H). HRMS (ESI) m/z calcd for C31H37Cl1N5 [M+H]+:
514.27320; found: 514.27553. The free base was converted to trihy-
drochloride (mp 220–230 °C dec.), for biological tests.
5.15.4. 7-Chloro-4-[N-[2-methyl-3-[[2-((1S,9aR)-octahydro-2H-
quinolizin-1-yl)ethyl]aminomethyl]-5-phenyl-1H-pyrrol-1-yl]amino]-
quinoline (9g)
5.18. N-tert-Butyl-2-acetyl-4-oxo-4-phenylbutanamide (22)
A
solution of N-tert-butyl-3-oxobutanamide (500 mg,
CC (CH2Cl2-MeOH; 93:7); solid washed with petroleum ether.
Yield: 23%. Mp 104–107 °C (dec). 1H NMR (CDCl3): d 8.60–8.10
(m, 2H); 7.90 (s, 1H); 7.70–7.30 (m, 3H); 7.20–6.90 (m, 3H); 6.55
(s, 1H); 5.75 (s, 1H); 3.90–3.50 (m, 2H); 3.05–2.50 (m, 4H); 2.50–
1.90 (m, 5H); 1.90–0.95 (m, 14H). HRMS (ESI) m/z calcd for
3.8 mmol)25 in 3 ml of anhydrous ethyl ether was added dropwise
to a stirred suspension of sodium ethoxide (216 mg, 3.18 mmol) in
14 ml of the same solvent at rt under N2. After 10 min a solution of
phenacylbromide (633 mg, 3.18 mmol) in 6 ml of dry ether was
added dropwise and the resulting mixture was refluxed for 2 h.
After cooling, NaBr was filtered, washed twice with dry ether,
and the organic phase was evaporated to give a crude product that
was purified by CC (silica gel; cyclohexane/AcOEt, 85:15). Yield:
74%. Mp 111.6–113.7 °C. 1H NMR (CDCl3): d 7.97 (d, J = 7.43 Hz,
2H); 7.59 (t, J = 7.43 Hz, 1H); 7.47 (t, J = 7.43 Hz, 2H); 6.00 (s,
1H); 3.85 (t, J = 6.50 Hz, 1H); 3.67 (dd, J = 18.42, 6.50 Hz, 1H);
3.50 (dd, J = 18.42, 6.50 Hz, 1H); 2.35 (s, 3H); 1.35 (s, 9H). HRMS
(ESI) m/z calcd for C16H21NO3Na [M+Na]+: 298.14136; found:
298.14112.
C
32H39ClN5 [M+H]+: 528.28885; found: 528.28896.
5.16. 7-Chloro-4-[N-[3-[(diethylamino)methyl]-2-methyl-5-phenyl-
1H-pyrrol-1-yl]amino]quinoline (9a): alternative method
Diethylamine (0.16 ml, 1.5 mmol), aq formaldehyde (37%,
0.13 ml, 1.5 mmol) were added at 0.5 ml of glacial acetic acid in
an ice-cooled flask. The solution was stirred for 2–3 min and then
poured into a flask containing 17 (501 mg, 1.5 mmol). The mixture
was stirred at rt under N2 for 1.5 h, water (15 ml) was added, and
the solution alkalized with 2 N NaOH. The suspension was ex-
tracted with CH2Cl2 and the collected organic phases were dried
(Na2SO4) and evaporated to give a crude product from which pure
9a was obtained by three successive CC (silica, CH2Cl2 containing
3–7% MeOH). The joint solids containing the expected compound
were washed with petroleum ether. Yield: 5%.
5.19. 1-[(7-Chloroquinolin-4-yl)amino]-2-methyl-5-phenyl-1H-
pyrrole-3-(N-tert-butyl)carboxamide (20c)
Compound 22 (500 mg, 1.82 mmol) was added to a stirred solu-
tion of 7-chloro-4-hydrazinoquinoline (352 mg, 1.82 mmol) in
3 ml of glacial acetic acid. The mixture was stirred and refluxed un-
der N2 for 2 h to give a violet solution, which, after cooling, was
alkalinized with cold 2 N NaOH and extracted three times with
CH2Cl2. The extract was dried (Na2SO4) and the solvent removed,
leaving a solid that was purified by CC (silica gel; ethyl acetate/
cyclohexane; 20:80). The product was rinsed with ethyl ether/eth-
anol (85:15). Yield: 80%. Mp 266.3–269.1 °C. 1H NMR (DMSO-d6): d
10.72 (s, 1H); 8.52–8.21 (m, 2H); 7.90 (s, 1H); 7.70–7.10 (m, 7H);
5.17. 7-Chloro-4-[N-[5-(4-chlorophenyl)-3-[(substitutedamino)-
methyl]-2-methyl-1H-pyrrol-1-yl]amino]quinoline (10a and b):
general method
To a stirred suspension of amide 21 (1 mmol) in 25 ml of anhy-
drous THF heated at reflux under N2, tris(triphenylphosphine)rho-

6822
M. Casagrande et al. / Bioorg. Med. Chem. 16 (2008) 6813–6823
7.02 (s, 1H); 5.60 (s, 1H); 2.30 (s, 3H); 1.37 (s, 9H). HRMS (ESI) m/z
razolium bromide (MTT) (M-2128 Sigma) in PBS was added for
an additional 3 h at 37 °C. The plates were then centrifuged, the
supernatants discarded and the dark blue formazan crystals dis-
calcd for C25H26ClN4O [M+H]+: 433.17897; found: 433.18007.
5.20. 4-[N-[3-[(tert-Butylamino)methyl]-2-methyl-5-phenyl-1H-
solved using 100 lL of lysing buffer consisting of 20% (w/v) of a
pyrrol-1-yl]amino]-7-chloroquinoline (9c)
solution of SDS (Sigma), 40% of N,N-dimethylformamide (Merck)
in H2O, at pH 4.7 adjusted with 80% acetic acid. The plates were
then read on a microplate reader (Molecular Devices Co., Menlo
Park, CA, USA) at a test wavelength of 550 nm and a reference
wavelength of 650 nm. The results are expressed as IC50, which is
the dose of compound necessary to inhibit cell growth by 50%.
All the tests were performed in triplicate at least three times.
N-tert-Butyl-1-(7-chloroquinolin-4-ylamino)-2-methyl-5-phe-
nyl-1H-pyrrole-3-carboxamide (8a, 550 mg, 1.27 mmol) was sus-
pended in 70 ml of anhydrous ethyl ether under N2 and LiAlH4
(550 mg, 14.5 mmol) was carefully added; then the mixture was
refluxed and stirred for 5 h. After cooling, 1 N NaOH was added;
the filtrated alumina was washed twice with ethyl ether and the
organic layers were dried (Na2SO4) and evaporated. The crude
was purified by CC (silica gel; CH2Cl2/MeOH, 95:5) to afford com-
pound 2c, which was rinsed with ethyl ether. Yield: 28%.
Mp > 245 °C (dec.). 1H NMR (DMSO-d6): d 10.50 (s, 1H); 8.37 (d,
J = 5.21, 1H); 8.30 (d, J = 9.08, 1H); 7.85 (s, 1H); 7.70 (s, 1H);
7.61–7.42 (m, 3H); 7.18 (t, J = 7.43, 1H); 7.05 (d, J = 7.30, 1H);
6.41 (s, 1H); 5.61 (d, J = 5.21, 1H); 3.55 (s, 2H); 2.01 (s, 3H); 1.10
(s, 9H). HRMS (ESI) m/z calcd for C25H28ClN4 [M+H]+: 419.19970;
found: 419.20173.
Acknowledgments
Part of this manuscript was generated in the context of the
AntiMal project, funded under the 6th Framework Programme of
the European Community (Contract No. IP-018834). The authors
are solely responsible for its content, it does not represent the
opinion of the European Community, and the Community is not
responsible for any use that might be made of the information con-
tained therein.
The financial support from the University of Milan (FIRST 2006)
is also acknowledged.
5.21. Parasite cultures and drug susceptibility assay
We thank Dr. F. Ravagnani from the Blood Unit, National Cancer
Institute, Milan, Italy, for providing fresh red blood cells for P. fal-
ciparum growth.
Plasmodium falciparum cultures were carried out according to
Trager and Jensen with slight modifications.32 The CQ-sensitive,
strain D10 and the CQ-resistant, strain W2 were maintained at
5% hematocrit (human type A-positive red blood cells) in RPMI
1640 (EuroClone, Celbio) medium with the addition of 10% heat
inactivated A-positive human plasma, 20 mM Hepes, and 2 mM
glutammine. All the cultures were maintained at 37 °C in a stan-
dard gas mixture consisting of 1% O2, 5% CO2, and 94% N2. Com-
pounds were dissolved in either water or DMSO and then diluted
with medium to achieve the required concentrations (final DMSO
concentration <1%, which is non-toxic to the parasite). Drugs were
placed in 96-well flat-bottomed microplates (COSTAR) and serial
dilutions made. Asynchronous cultures with parasitaemia of 1–
1.5% and 1% final hematocrit were aliquoted into the plates and
incubated for 72 h at 37 °C. Parasite growth was determined spec-
trophotometrically (OD650) by measuring the activity of the para-
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5.22. Cell cytotoxicity assays
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