Synthesis, antimalarial activity, and preclinical pharmacology of a novel series of 4'-fluoro and 4'-chloro analogues of amodiaquine. identification of a suitable "back-up" compound for N-tert-butyl isoquine

Article abstract of DOI:10.1021/jm8012757

On the basis of a mechanistic understanding of the toxicity of the 4-aminoquinoline amodiaquine (1b), three series of amodiaquine analogues have been prepared where the 4-aminophenol "metabolic alert" has been modified by replacement of the 4'-hydroxy gro

Full text of DOI:10.1021/jm8012757

1828
J. Med. Chem. 2009, 52, 1828–1844
Synthesis, Antimalarial Activity, and Preclinical Pharmacology of a Novel Series of 4′-Fluoro
and 4′-Chloro Analogues of Amodiaquine. Identification of a Suitable “Back-Up” Compound
for N-tert-Butyl Isoquine
Paul M. O’Neill,*,†,‡ Alison E. Shone,‡ Deborah Stanford,† Gemma Nixon,† Eghbaleh Asadollahy,† B. Kevin Park,‡
James L. Maggs,‡ Phil Roberts,‡ Paul A. Stocks,† Giancarlo Biagini,§ Patrick G. Bray,§ Jill Davies,§ Neil Berry,†
Charlotte Hall,§ Karen Rimmer,§ Peter A. Winstanley,‡ Stephen Hindley,† Ramesh B. Bambal,| Charles B. Davis,|
Martin Bates, Stephanie L. Gresham,# Richard A. Brigandi, Federico M. Gomez-de-las-Heras,O Domingo V. Gargallo,O
Silvia Parapini,¶ Livia Vivas,[ Hollie Lander,[ Donatella Taramelli,¶ and Stephen A. Ward§
Department of Chemistry, UniVersity of LiVerpool, LiVerpool L69 7ZD, U.K., MRC Centre for Drug Safety Science, Department of Pharmacology and
Therapeutics, School of Biomedical Sciences, UniVersity of LiVerpool, LiVerpool L69 3GE, U.K., LiVerpool School of Tropical Medicine, Pembroke
Place, LiVerpool L3 5QA, U.K., Drug Metabolism and Pharmacokinetics, GlaxoSmithKline Drug DiscoVery, 1250 South CollegeVille Road,
CollegeVille, PennsylVania 19426, Medicines Research Centre, GlaxoSmithKline, Gunnels Wood Road, SteVenage, Hertfordshire SG1 2NY, U.K.,
Safety Assessment, GlaxoSmithKline, The Frythe, Welwyn, Hertfordshire AL6 9AR, U.K., DiscoVery Medicine Infectious Diseases CEDD, DDW DPU,
GlaxoSmithKline, Upper ProVidence, PennsylVania, GlaxoSmithKline, S.A., Parque Tecnolo´gico de Madrid, SeVero Ochoa, 2, 28760 Tres Cantos,
Madrid, Spain, Dipartimento di Sanita` Pubblica, Microbiologia, Virologia, UniVersita` degli Studi di Milano, Via Pascal, 36, 20133 Milan, Italy, and
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, U.K.
ReceiVed October 8, 2008
On the basis of a mechanistic understanding of the toxicity of the 4-aminoquinoline amodiaquine (1b),
three series of amodiaquine analogues have been prepared where the 4-aminophenol “metabolic alert” has
been modified by replacement of the 4′-hydroxy group with a hydrogen, fluorine, or chlorine atom. Following
antimalarial assessment and studies on mechanism of action, two candidates were selected for detailed ADME
studies and in vitro and in vivo toxicological assessment. 4′-Fluoro-N-tert-butylamodiaquine (2k) was
subsequently identified as a candidate for further development studies based on potent activity versus
chloroquine-sensitive and resistant parasites, moderate to excellent oral bioavailability, low toxicity in in
vitro studies, and an acceptable safety profile.
Introduction
Malaria is endemic in many of the poorest countries in the
world, causing between 1-3 million deaths each year.1,2 More
than 90% of the deaths occur in sub-Saharan Africa, and nearly
all the deaths are in children under the age of 5 years. The
morbidity and mortality from malaria have been increasing over
recent decades because of deterioration in health systems,
growing drug3 and insecticide resistance,4 climate change, civil
and social instability, and population displacement.2
The hemoglobin degradation pathway in Plasmodium falci-
parum has been successfully exploited as the therapeutic target
of chloroquine (1a, Figure 1) and other 4-aminoquinolines.5-7
As the parasite catabolizes human hemoglogin, the hematin
biproduct (1c) is detoxified by crystallization to hemozoin.
Chloroquine interacts with the porphyrin ring of the hematin
by a π-π stacking interaction of the quinoline ring and
accumulation of the heme/drug complex within the erythrocyte
ultimately poisons the parasite.8-10 Resistance to this mechanism
Figure 1. Structures of chloroquine (1a), amodiaquine (1b), and drug
target hematin (1c).
* To whom correspondence should be addressed. Address: Department
of Chemistry, University of Liverpool, Liverpool, L69 7ZD, UK. Phone:
of action has proven difficult to induce. Chloroquine resistance,
which developed relatively slowly and only after considerable
human exposure, is the result of point mutations in a parasite
protein known as Plasmodium falciparum chloroquine resistance
transporter (PfCRTa).11
0151-794-3553. Fax: XXXX. E-mail: p.m.oneill01@liv.ac.uk.
†
Department of Chemistry, University of Liverpool.
Department of Pharmacology and Therapeutics, University of Liverpool.
Liverpool School of Tropical Medicine.
GlaxoSmithKline Drug Discovery.
‡
§
|
Medicines Research Centre, GlaxoSmithKline.
Safety Assessment, GlaxoSmithKline.
#
a Abbreviations: ADME, absorption, distribution, metabolism, and excre-
tion; PfCRT, Plasmodium falciparum chloroquine resistance transporter;
GSK, GlaxoSmithKline; NOAEL, no observable adverse effect level;
MNLD, maximum nonlethal dose; LAR, lipid accumulation ratio; VAR,
vacuolar accumulation ratio.
DiscoveryMedicineInfectiousDiseasesCEDD,DDWDPU,GlaxoSmith-
Kline.
O
GlaxoSmithKline, S.A., Spain.
Universita` degli Studi di Milano.
London School of Hygiene and Tropical Medicine.
¶
[
10.1021/jm8012757 CCC: $40.75
2009 American Chemical Society
Published on Web 03/13/2009

Analogues of Amodiaquine
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1829
Scheme 1. Oxidation of Amodiaquine to a Reactive
Quinoneimine Metabolite
amodiaquine is replaced by either fluorine or chlorine; the
intention, as in previous work, was to produce analogues
incapable of producing quinoneimine metabolites by P450
oxidation. The array of target molecules is highlighted in Chart
1. For comparison purposes, we have also examined the
antimalarial profile of two 4′-dehydroxy analogues of amodi-
aquine. Following completion of the syntheses and appropriate
antimalarial assessment and preclinical pharmacological assess-
ment, we aimed to select suitable back-up compounds to 1e
with appropriate properties suitable for drug development.
Chemistry
Although the original reported route to 4-fluoroamodiaquine
was relatively efficient, we reasoned that shortening the number
of synthetic steps and developing a route where a point of
diversity appears in the penultimate step of the synthesis could
achieve a significant improvement to the synthesis of target
molecules in Chart 1. The alternative route adopted for the
synthesis of 4′-chloro and 4′-fluoro analogues is shown in
Scheme 2.
Amodiaquine (1b) is active against chloroquine-resistant
strains of P. falciparum.12 Until recently the use of this drug
has somewhat been restricted by adverse effects in humans
including hepatotoxicity and agranulocytosis.13,14 This appears
to be the result of bioactivation to form a reactive quinoneimine
metabolite that binds irreversibly to cellular macromolecules
and may lead to direct toxicity as well as immune-mediated
hypersensitivity reactions (Scheme 1).15-18 IgG antibodies have
been detected in patients with adverse reactions to 1b.19,20
In order to enhance the metabolic stability of amodiaquine,
we demonstrated that introduction of fluorine into the 4-hy-
droxyanilino nucleus increases the oxidation potential of the
molecule and reduces the in vivo oxidation of the molecule to
a cytotoxic quinoneimine.21 From this earlier work, we dem-
onstrated that the 4′-hydroxyl group could be replaced with a
4′-fluorine atom to produce an amodiaquine analogue, fluoroa-
modiaquine (2d), with antimalarial activity in the low nanomolar
range.21
An alternative approach to circumvent the facile oxidation
of 1b involves isomerization of the 3′ and 4′ groups. It was
proposed that interchange of the 3′-hydroxyl and the 4′ Mannich
side chain function of 1b would provide a new series of
analogues that cannot form toxic quinoneimine metabolites via
cytochrome P450 mediated metabolism.22 Isomeric amodiaquine
analogues were prepared using a simple two-step procedure and
then tested against K1 (chloroquine-resistant) and HB3 (chlo-
roquine-sensitive) strains of P. falciparum in vitro.22,23 While
several analogues displayed potent antimalarial activity against
both strains, isoquine (1d), the direct isomer of amodiaquine,
was selected for in vivo antimalarial assessment. The potent in
vitro antimalarial activity of 1d was translated into excellent
oral in vivo ED50 activity of 1.6 and 3.7 mg/kg against the P.
yoelii NS strain compared to 7.9 and 7.4 mg/kg for amodiaquine.
Subsequent metabolism studies in the rat model demonstrated
that isoquine does not undergo in vivo bioactivation, as
evidenced by the complete lack of glutathione metabolites in
bile. A drawback with 1d is the low oral bioavailability in animal
models, and this was demonstrated to be due to extensive
metabolism of the N-diethylamino Mannich side chain. Further
studies in this area have revealed that 1e (Figure 2) has superior
pharmacokinetic and pharmacodynamic profiles compared to
isoquine in four animal species including primates. This
compound has completed preclinical evaluation with Glaxo-
SmithKline pharmaceuticals (GSK), and the phase I clinical trial
commenced in April 2008.
For the synthesis of 4′-chloro analogues, nitro reduction of
3a was easily achieved using iron powder and HCl in refluxing
aqueous ethanol. The resulting amine 3b was then allowed to
react with 4,7-dichloroquinoline to provide 3c in excellent yield
as a yellow powder following standard workup. Quinoline 3c
proved to be quite insoluble in dichloromethane, so a combina-
tion of dichloromethane, DMF, and a small amount of diiso-
propylethylamine was used as solvent to dissolve the alcohol
for MnO2 mediated oxidation. After 18 h of reflux, precipitates
derived from the oxidant MnO2 were filtered off. In many runs,
significant losses occurred at this stage and a more satisfactory
workup procedure involved the use of a precolumn with ethyl
acetate prior to flash column chromatography using MeOH/
dichloromethane as eluent. The key aldehyde was obtained in
moderate to good yields following silica gel chromatography.
The final step involved reductive amination of the aldehyde 13
with a variety of different amines to produce a small array of
target molecules depicted in Chart 1. In all cases the reductive
amination procedure worked well and column chromatography
gave the products 3d-m in good yields (Table 1).
Scheme 2 also depicts the synthesis of a fluorinated series of
analogues, and the approach used is essentially the same as that
discussed with the exception that alternative conditions were
explored for the reduction of the nitro group of 2a and the
oxidation of the benzylic alcohol 2c to the corresponding
aldehyde. Disappointingly, the use of powdered iron/HCl gave
the desired amine 3b in low yields following basification and
workup. Alternative reducing conditions using methanol, am-
monium chloride, and zinc powder or granulated tin and
concentrated hydrochloric acid provided the product in good
yield following addition of sodium hydroxide solution to the
amine in the workup phase of the reaction. The synthesis of 2b
from 2c proceeded in high yields without event. In contrast to
the 4′-chloro series, oxidation of 2c with MnO2 gave the desired
aldehyde in poor yields, possibly because of the insolubility of
the quinoline aldehyde. An alternative method based on chro-
mium(VI) was explored that provided the aldehyde in moderate
yield. Reductive amination and workup provided the desired
compounds as a dark-brown solid. NMR analysis of the products
prior to chromatography indicated complex formation (very
broad peaks in the aromatic and aliphatic regions), the nature
of which remains undefined. Acidification (HCl) and basification
(sodium bicarbonate) provided the free bases which were pure
by NMR and CHN analysis following silica gel column
The aim of this research was to investigate two series of
amodiaquine analogues where the 4′-hydroxyl function of

1830 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
O’Neill et al.
Figure 2. Rational drug design of amodiaquine analogues based on metabolism/toxicity considerations. Clinical candidate 1e has an exceptional
pharmacodynamic and pharmacokinetic profile.
Chart 1. Structures of 4′-Chloro and 4′-Fluoro Analogues of Amodiaquine
Table 1. Yields for Reductive Amination of Aldehydes 2c and 3c
Scheme 2. Synthesis of 4′-Fluoro and 4′-Chloro Analogues by
Reductive Amination of Aldehydes 2c and 3ca
aldehyde
amine
Fluoro Series
diethylamine
product
yield (%)
2c
2c
2c
2c
2c
2c
2c
2d
2e
2f
24
20
31
31
27
42
23
pyrrolidine
piperidine
N-methylpiperazine
dipropylamine
dibutylamine
2g
2h
2i
morpholine
2j
Chloro Series
3c
3c
3c
3c
3c
3c
3c
3c
3c
3c
diethylamine
pyrrolidine
piperidine
N-methylpiperazine
dipropylamine
3d
3e
3f
3g
3h
3i
3j
3k
3l
74
61
92
89
85
24
63
59
98
56
dibutylamine
N-ethylpiperazine
tert-butylamine
1-(2-pyridyl)-piperazine
N-isopropylpiperazine
a Reagents and conditions (i) Fe/HCl/H2O, reflux; (ii) 4,7-dichloroquino-
line, EtOH, reflux; (iii) DIEA, CH2Cl2, DMF, MnO2; (iv) amine, then
NaHB(OAc)3.
3m
chromatography. Although the new route examined provided
the final reductive amination step in the 4′-fluoro series requires
target molecules in acceptable yields for the 4′-chloro series,
optimization (Table 1).

Analogues of Amodiaquine
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1831
Scheme 3. Synthesis of 4-Dehydroxy, 4′- Chloro, and 4′-Fluoro Analogues of Amodiaquinea
a Reagents and conditions: (i) NBS, AIBN, reflux; (ii) amine, K2CO3, CH3CN, reflux; (iii) MeOH, room temp, then 0 °C, NaHB(OAc)3; (iv) Sn/HCl,
H2O, reflux; (v) 4,7-dichloroquinoline, EtOH, HCl, reflux.
Table 2. In Vitro Antimalarial Activities24 versus Chloroquine Sensitive
3D7 Strain of Plasmodium falciparuma
Table 3. In Vitro Antimalarial Activities24 of 4-Chloro Series versus
Chloroquine-Sensitive HB3 Strain and TM6 Chloroquine-Resistant
Plasmodium falciparuma
compdb
IC50 (nM) 3D7
compdb
IC50 (nM) 3D7
IC50 (nM)
2d
2e
2f
2g
2h
2i
2j
2k
2l
27.3 (3) ( 11.5 3d
29.7 (3) ( 11.1 3f
33.2 (3) ( 15.2 3g
30.4 (3) ( 15.2
25.7 (3) ( 10.6
86.8 (3) ( 43.1
277.3 (3) ( 34.6
345.2 (3) ( 9.3
39.7 (3) ( 9.3
7.8 (6) ( 3.2
27.2 (3) ( 18.1
4.7 (3) ( 2.9
6.0 (6) ( 4.0
compd
TM6
HB3
3d
3e
3f
3g
3i
3j
3k
3l
10.2 ( 5.3
4.2 ( 1.3
9.4 ( 6.7
24.4 ( 4.8
96.3 ( 11.3
10.3 ( 4.3
4.5 ( 3.2
500 ( 22.4
6.4 ( 2.3
5.0 ( 2.2
95.1 ( 6.5
7.2 ( 3.3
12.3 ( 5.1
15.2 ( 3.7
12.3 ( 3.2
54.5 ( 8.3
144.3 ( 12.8
9.6 ( 3.4
8.1 ( 3.2
485 ( 14.1
9.1 ( 2.1
35.1 (3) ( 8.7
3h
85.2 (3) ( 18.1 3i
95.2 (3) ( 30.2 3j
550.7 (3) ( 90.4 3k
12.1 (3) ( 5.3
27.2 (3) ( 3.4
9.1 (3) ( 4.9
7.8 (6) ( 2.2
3l
amodiaquine (1b)
1e
chloroquine (1a)
isoquine (1d)
a 3D7 is a chloroquine sensitive strain of P. falciparum. b Amodiaquine
and 1e were tested as the hydrochloride salt. Chloroquine was tested as the
diphosphate. All other compounds were tested as free bases.
3m
4d
10.1 ( 3.2
20.2 ( 10.1
5.5 ( 3.2
chloroquine (1a)
amodiaquine (1b)
a TM6 is a chloroquine resistant strain of P. falciparum, and HB3 is a
chloroquine sensitive strain of P. falciparum.
For this reason, the previously used linear approach was
employed for selected analogues as shown Scheme 3. The first
step in the sequence involved free radical bromination of
2-fluoro-5-nitrotoluene (or 2-chloro-5-nitrotoluene) using NBS/
AIBN to give the benzylic bromide 4b as a crystalline solid in
good yield. The resulting benzyl bromide 4b was then allowed
to react with the appropriate amine to provide intermediates
(4c, 4d, 5c-f, 6b, and 6c) that were reduced and coupled with
4,7-dichloroquinoline as shown. Benzyl bromides 4b, 5b, and
6a were also allowed to react with imidazole, and this enabled
the synthesis of novel weak base imidazole derivatives 2l, 3p,
and 6g. For intermediates 5c-e, 2-chloro-5-nitrobenzaldehyde
was also used as starting material in a reductive amination
approach as shown.
and 2i of >5.89). The N-tert-butyl analogue 2k (log P ) 4.74)
was the most potent in this series with activity equivalent to
that of chloroquine and isoquine (1d). The 4′-chloro analogues
tested were all active around 30 nM with the exception of
dipropyl and dibutyl analogues 3g and 3h, and in line with the
4′-fluoro series, the 4′-chloro-N-tert-butyl analogue 3k expressed
the highest antimalarial activity. Interestingly, within these two
templates, the side chain alkylamine or cyclic amine groups can
be replaced with imidazole to produce analogues 2l and 3p that
had antimalarial activity in the low nanomolar range.27
Table 3 lists activity for the 4′-chloro series versus a
chloroquine-resistant TM6 and sensitive HB3 strain. The most
potent analogue was the N-tert-butyl analogue 3k. Analogues
3e, 3f, 3j, and 3m also expressed excellent activities against
these two strains. For the series as a whole there is a clear lack
of cross-resistance for the 4′-chloro series. The replacement of
the 4′-F or 4′-Cl with hydrogen coupled with N-tert-butyl
substitution provides (6f) an analogue with equipotent activity
against the HB3 and TM6 strains of Plasmodium falciparum.
Despite the good activity of this molecule, it was not selected
for in vivo evaluation because of the concerns that metabolically
4-hydroxyanilino ring system may be susceptible to P450
Biology
In Vitro Antimalarial Activity versus Plasmodium
falciparum.24,25 Initial testing24 was carried out on a chloro-
quine-resistant strain of Plasmodium falciparum (3D7) (Table
2). In the 4′-fluoro series the weak base morpholine analogue
2j and analogues 2h and 2i expressed relatively poor antimalarial
activity versus the 3D7 strain. The former observation has been
seen previously with short chain analogues of chloroquine;26
for 2h and 2i it would appear that increasing the lipophilicity
has a detrimental effect on antimalarial activity (ClogP for 2h

1832 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
O’Neill et al.
Table 4. Expanded in Vitro Antimalarial Activities24 of 2e and 2k
versus a Panel of Plasmodium falciparum Isolatesa
Table 6. Therapeutic Efficacy of 2k and Isoquine Derivatives against
Plasmodium yoelii 17X Infection in Micea
IC50 (nM)
mean
(mg/kg)
IC 95%b
(mg/kg)
compd
test
parameter
strain chloroquine (1a) amodiaquine (1b)
2e
2k
c
chloroquine (1a)
4-day
ED50
3.3
2.8-3.7
4.0-4.9
NAf
2.1-3.2
3.3-4.1
NAf
4.6-5.8
6.9-8.3
NAf
5.1-5.9
7.6-8.5
NAf
TM6
K1
112.0 ( 15.3
170.6 ( 22.3
16.0 ( 3.4
30.8 ( 12.3
112.8 ( 22.2
59.3 ( 11.1
154.0 ( 17.3
133.5 ( 14.5
6.91 ( 3.2
20.0 ( 2.3
8.5 ( 3.4
15.21 ( 4.4
15.1 ( 3.3
9.5 ( 1.4
60.8 ( 8.9
45.9 ( 12.2 22.0 ( 3.4
63.7 ( 11.2 14.6 ( 5.4
25.0 ( 8.2
46.9 ( 18.1 17.3 ( 4.9
36.9 ( 4.9
52.5 ( 9.5
34.7 ( 8.3 29.24 ( 4.5
21.9 ( 5.4
ED90
NRLd
ED50
ED90
NRL
ED50
ED90
NRL
ED50
ED90
NRL
ED50
ED90
NRL
4.4
>40e
2.6
HB3
PH3
TM4
DD2
V1S
J164
amodiaquine (1b)
isoquine (1d)
desethylisoquine (1f)
2k
4-day
4-day
4-day
4-day
25.8 ( 6.7
3.7
>40e
5.2
22.4 ( 1.6
26.1 ( 7.6
8.9 ( 4.6
18.5 ( 4.2
7.6
>40e
5.5
a TM6, K1, TM4, V1S, J164 are all chloroquine-resistant strains of P.
falciparum. The rest of the strains are chloroquine-sensitive.
8.0
>40e
7.7
Table 5. Therapeutic Efficacy of 2k, 2m, Isoquine, and
Desethylisoquine against Plasmodium berghei ANKA Infection in Mice
Using the Standard 4-Day Test Assaya
6.7-8.9
9.4-11.8
NAf
10.6
>40e
a Standard 4-day test assay. Chloroquine and amodiaquine were included
as control compounds.28 b Interval of confidence of the mean. c Dose at
which a 50% of reduction of parasitemia in peripheral blood is achieved at
day 4 after infection. d Nonrecrudescence level (NRL) is defined as the
minimum dose at which no recrudescence is observed. Highest dose
deployed in this study was 40 mg/kg. e NRL level is only defined for
treatments that make parasitemia undetectable by flow cytometry at day 4
in the “4-day test”. At the highest dose deployed chloroquine, amodiaquine,
isoquine, desethylisoquine, and 2k (40 mg/kg) were unable to render
parasitemia undetectable; therefore, it is not possible to evaluate NRL level
for these compounds accurately. It is higher than 40 mg/kg. f NA ) not
applicable.
ED50 95% confidence ED90 95% confidence
compd
(mg/kg)
interval
(mg/kg)
interval
amodiaquine (1b)
isoquine (1d)
desethyl isoquine (1f)
2.1
3.7
5.3
5.3
13.4
2.0-2.2
3.4-3.9
4.1-6.8
4.1-6.8
11.2-15.6
3.2
5.3
7.3
12.4
27.3
2.9-3.4
4.3-6.6
6.4-8.4
2k
3k
10.7-14.4
14.3-30.2
a Groups of five mice were inoculated iv with 107 infected erythrocytes/
mice. The compound was administered orally once a day for 4 days starting
1 h after infection. Seven drug doses were used ranging from 40 to 0.31
mg/kg to determine ED50 and ED90 values. Parasitemia after treatment was
measured in peripheral blood by flow cytometry 24 h after the administration
of the fourth dose.
Scheme 4. Potential Binding Interactions for Drug (L) with
Heme (P)a
mediated hydroxylation that would produce 4-aminophenol or
2-aminophenol quinoneimine precursors.
As this project progressed, the excellent profiles displayed
by the isoquine analogue 1d and in particular the metabolic
advantages provided by the N-tert-butyl side chain seen in the
development of 1e led to the selection of 2k for further expanded
biological assessment against eight additional strains of Plas-
modium falciparum. In the expanded testing contained in Table
4, data are also included for the pyrollidine analogue 2e.
The data indicate that 2k is superior to the additional control
analogue 2e against all of the strains examined. It is also clear
that N-tert-butyl analogue 2k is potent against chloroquine
resistant strains, though it is not quite as active as amodiaquine
against both chloroquine-sensitive and resistant parasites. On
the basis of the activity presented in Table 4, 2k was selected
alongside its direct 4-chloro analogue for further studies in the
mouse model of malaria.
In Vivo Antimalarial Activity against Plasmodium
berghei ANKA.28 The therapeutic efficacy of 2k, 3k, and
selected 4-aminoquinolines was evaluated in a standard “4-day
test” in CD1 mice infected intravenously with the murine
pathogen P berghei ANKA at GSK, Tres Cantos, Spain (Table
5).
Isoquine (1d) showed in vivo therapeutic efficacy against
Plasmodium berghei ANKA with ED50 and ED90 in the same
range as that of amodiaquine. Analogue 2k was also potent in
this in vivo assay with an ED50 value of 5.3 mg/kg, whereas
chloro analogue 3k was less potent with an ED50 of 13.4 mg/
kg. The therapeutic efficacy of 2k, isoquine (1d), 1f, and the
two control compounds amodiaquine and chloroquine were also
evaluated in the standard “4-day test” against the murine
pathogen P. yoelii 17X. Mice were infected with 6.4 × 106
parasitized erythrocytes/mouse iv. Treatments started 1 h after
infection, and each drug was administered once a day po in
saline. Parasitemias in peripheral blood were measured 96 h
after infection (day 4) and then every 2-3 days until day 23
a After the first association of porphyrin with drug, there are two distinct
binding events in which either another drug or porphyrin molecule
coordinate to the intermediate adduct.
after infection in order to assess recrudescence after treatment.
The results (ED50, ED90, and nonrecrudescence level) are shown
in Table 6.
Mechanism of Action. Hematin Binding Studies. Recent
efforts have centered on developing titration methods to
determine accurate equilibrium constants and binding stoichi-
ometries for the most widely used quinoline antimalarials.29-36
We have applied a UV-visible spectroscopic method for
determining accurate binding equilibrium constants for several
clinically used antimalarials and compared the results with fluoro
analogue 2k. Initially, the titrations were carried out in a mixed
aqueous/organic solvent to minimize porphyrin aggregation
effects and µ-oxo-dimer formation. Buffered 40% DMSO was
used to provide a strictly monomeric heme species in solution.35
The mode of action for antimalarial binding was investigated.
The potential binding processes 1 and 2 shown in Scheme 4
were mathematically modeled and examined as possible best
fits for the titrations. In model 1 there is a stepwise bonding of
2 equiv of drug to 1 molecule of heme. In model 2 there is a
stepwise addition of 2 additions of heme to 1 equiv of drug.
The UV-visible spectra obtained after each titrated addition
were analyzed and stacked against the corresponding absor-
bances. The data were transferred for analysis using the Pro-
Fit nonlinear curve fitting program licensed from Quansoft. The
data were fitted to achieve ꢀ2 at a minima to produce K1 and K2
fitted parameters for both models. This was initially done at

Analogues of Amodiaquine
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1833
after administration of a single dose of [3H]2k to cannulated
rats 55.94 ( 5.82% of the dose could be accounted for in the
organs, bile, and urine (Figure 3). The pattern of distribution
followed that seen previously following metabolism of other
4-aminoquinolines such as amodiaquine and chloroquine, with
the majority of the dose being retained in the liver (17.49 (
0.82%) and skin (12.44 ( 1.76%). Elimination of the drug
seemed to be favored by neither biliary nor urinary excretion
systems, with 9.16 ( 1.29% of the dose being excreted in bile
and 8.06 ( 1.12% being excreted in the urine.
Table 7. Equilibrium Constants for the Binding of Antimalarials with
Heme in DMSO Solutions
best fit
compound
solvent
log K1
log K2
model
chloroquine (1a)
amodiaquine (1b) 40% DMSO 4.40 ( 0.1 6.20 ( 0.1
quinine
mefloquine
2k
40% DMSO 4.60 ( 0.1 6.70 ( 0.1
2
2
1
1
2
40% DMSO 4.40 ( 0.1 4.10 ( 0.1
40% DMSO 3.84 ( 0.1 3.17 ( 0.1
40% DMSO 5.45 ( 0.1 5.68 ( 0.1
Table 8. Results of the ꢁ Hematin Inhibition Assay (BHIA) Assay37
As might be expected, the pattern of distribution of [3H]3k
in the tissues of male Wistar rats followed the same pattern as
the other 4-aminoquinoline compounds tested. Slightly less
radioactivity than 2k was recovered in the tissues, bile, and
urine, 47.35 ( 4.77%, the majority of which could be found in
the liver (12.41 ( 0.29%). Significant differences in areas of
accumulation were seen with the kidneys, liver, biliary, and
urinary output. Five hours after dosing with [3H]3k there was
significantly more radioactivity found in the kidneys than
observed with [3H]2k, 5.43 ( 0.28% versus 3.97 ( 0.13% (p
) 0.0054). In the liver, there was significantly less [3H]3k than
[3H]2k (p ) 0.0209). This was followed by a significant increase
in biliary excretion after dosing with [3H]3k (14.88 ( 0.83%,
p ) 0.0052). The increased biliary excretion of 3k compared
to 2k could be attributed to increased lipophilicity (log P )
5.59 ( 0.51 versus log P ) 5.15 ( 0.56).
On comparison of the tissue distribution of the two novel
compounds with amodiaquine (1b), 5 h after administration it
can be observed that at an equivalent dose, the halogenated
analogues appear to accumulate in the major organs to a much
lesser extent than amodiaquine (Figure 4). In the brain, kidneys,
liver, and skin it can be seen that there was significantly less
retention with the halogenated analogues of 1b.
Animals were dosed with either [3H]2k or [3H]3k and kept
in metabolism cages for a period of 24 h in order to see if the
drugs were effectively eliminated or if they would accumulate
in the tissues over time (Figure 5). Results showed that both
drugs were effectively eliminated after 24 h, with the total
amount of radioactivity recovered with [3H]2k and [3H]3k being
51.36 ( 8.68% and 61.00 ( 5.93%, respectively. In the kidneys,
almost 2-fold more drug was found in rats dosed with [3H]2k
than those dosed with [3H]3k, 1.48 ( 0.11% versus 0.31 (
0.07%; this was found to be highly significant (p < 0.0001).
The same was observed on comparison of the radioactivity
retained in the liver 24 h after dosing. After dosing with [3H]2k,
6.57 ( 0.27% of the dose remained in the liver, while after
dosing with the chloro analogue only 1.87 ( 0.21% remained
(3.5-fold less). This showed mathematical significance with p
< 0.0001. The lungs were another example where the choice of
halogen substitute appeared to make a great difference to the
retention of the drug. After dosing with [3H]2k, 1.17 ( 0.06%
of the dose remained. In comparison, after dosing with [3H]3k,
only 0.25 ( 0.08% remained, a decrease of almost 5-fold (p <
0.0001).
Although the amount of [3H]2k remaining in the rat after
24 h was relatively low (14.76 ( 2.47) (compared to 38.54 (
3.41% after 5 h) and significantly lower in the liver and skin (p
) 0.0286 and p ) 0.0011, respectively), it was deemed
appropriate to investigate to further time points (Figure 6). After
48 h 72.79 ( 2.56% of the dose could be accounted for in
tissues, feces, and urine. Of this, less than 8% remained in the
tissues; the remainder had been excreted, mainly by biliary
excretion. After 1 week (168 h) very little radioactivity remained
inside the rat (1.22 ( 0.24%). After 10 days (240 h) radioactivity
a
compd
IC50 molar ratio for drug/hemin
2k
2l
3k
3pb
3m
6f
1.66
1.78
1.66
1.58
1.66
1.65
2.65
1.78
0.85
6g
chloroquine (1a)
amodiaquine (1b)
a The IC50 represents the molar equivalents of test compounds, relative
to hematin, required to inhibit ꢁ-hematin formation. b Compound not
completely dissolved.
one wavelength and then simultaneously at 10-15 wavelengths
to give a more unbiased approach to fitting and more accurate
values in comparison to fitting at just a single wavelength. Table
7 above shows the K1 and K2 values obtained from this method
for chloroquine, amodiaquine, quinine, 2k, and mefloquine, with
the best fitted model shown.
An important observation indicated from the resulting fits is
that the binding stoichiometries for the quinoline methanols
quinine and mefloquine are different from the 4-aminoquinolines
included in this work; the second binding event in the chloro-
quine titrations involves the addition of another molecule of
heme, whereas the second binding to the quinine-heme adduct
involves a second molecule of ligand. This is in line with earlier
studies derived from NMR experiments suggesting that the
binding modes of 4-aminoquinoline antimalarials, such as
chloroquine and amodiaquine, and quinolinemethanol antima-
larials, such as quinine and mefloquine, are different.35,36
The data measured are in good agreement with the literature
and demonstrate that the fluoro analogue 2k can form a 2:1
complex in solution with clearly defined association constants.
Selected analogues from the new series of 4-aminoquinolines
were also examined for their ability to inhibit ꢁ-hematin
formation using the method of Parapini et al.37
The drug concentration required to inhibit ꢁ-hematin forma-
tion by 50% (IC50) was determined for each compound, and
the results listed in Table 8 are the mean IC50 of two different
experiments performed in duplicate. All of the compounds tested
had excellent activity in this assay including the imidazole
containing analogues 2l, 3p, and 6g. These molecules are
interesting in the sense that in addition to π-π-stacking over
the porphyrin ring system of hematin these agents may have
the capacity to interfere with hematin dimerization or crystal-
lization by binding to iron by axial ligation.27 Both 2k and chloro
variant 3k had superimposible activity in the assay.
Disposition and Metabolism Studies. In order to determine
the effect of replacing the 4′-hydroxy group in the amodiaquine-
like framework with fluorine and chlorine atoms, distribution
and metabolism studies were carried out in the rat. The requisite
[3H] labeled 4′-fluoro derivative 2k and 4′-chloro analogue 3k
were prepared by the route depicted in Scheme 2, and further
details are available in the Supporting Information. Five hours

1834 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
O’Neill et al.
Figure 3. Percentage of radioactive dose recovered in tissues 5 h after administration of [3H]2k and [3H]3k (54 µmol/kg, 20 µCi/rat) to male
Wistar rats (n ) 4). Results are expressed as mean percentage of dose ( SEM. Statistics were performed using a one-way ANOVA test for
parametric data and Kruskal-Wallis test for nonparametric data: (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.
Figure 4. Percentage of radioactive dose recovered in tissues 5 h after administration of [3H]amodiaquine, [3H]2k, and [3H]3k (54 µmol/kg, 20
µCi/rat) to male Wistar rats (n ) 4). Results are expressed as mean percentage of dose ( SEM. Statistics were performed using a one-way ANOVA
test for parametric data and Kruskal-Wallis test for nonparametric data: (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.
was negligible in the tissues of the rat, indicating a lack of
accumulation and the effective clearance of [3H]2k after a single
dose of 54 µmol/kg.
major biliary metabolite of [3H]2k was shown to yield m/z
469; however, a corresponding structure could not be
assigned. In urine [3H]2k was excreted mainly as the parent
compound. There was no evidence for P450 hydroxylation
of the 4′-fluoro aromatic ring to produce aminophenol
metabolites capable of reactive metabolite generation. This
was further backed up by the observation that no glutathione
conjugates were identified indicating that the incorporation
of fluorine into the amodiaquine backbone has eliminated
the possibility of the formation of toxic quinoneimine
metabolites.
Metabolism studies on [3H]3k were carried out in male
Wistar rats (54 µmol/kg, 20 µCi, iv). All major metabolites
were structurally assigned (Scheme 6, Table 10). The major
route of metabolism appeared to be oxidation to the side chain
carboxylic acid IV (Scheme 6). Oxygenation of the nitrogen
of the quinoline ring to form the N-oxide III was also
observed. No evidence of P450 hydroxylation of the 4′-chloro
ring system was obtained and no bioactivation to reactive
From these dispositional in vivo studies it is clear that both
2k and 3k show a similar pattern of distribution to other
4-aminoquinolines of the same class, with the majority of the
dose being found in the liver and skin. Incorporation of a
halogen appears to reduce the retention within the tissues
compared to 1b; however, the choice of halogen is important
because the chloro analogue appears to have significantly less
retention and was cleared much more effectively by biliary
excretion. After studies have been carried out for up to 10 days,
it has been shown that [3H]2k was effectively cleared from the
rat and there were no issues of irreversible retention.
Metabolism Studies on 2k and 3k. Following administra-
tion of [3H]2k (54 µmol/kg, 20 µCi) to male Wistar rats, a
total of nine metabolites were found in bile and urine in rats
in vivo (Table 9). Of these, four have been identified and
the structures elucidated (Scheme 5). (For full methods of
analysis and protocols, see Supporting Information.) The

Analogues of Amodiaquine
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1835
Figure 5. Percentage of radioactive dose recovered in tissues 24 h after administration of [3H]2k and [3H]3k (54 µmol/kg, 20 µCi/rat) to male
Wistar rats (n ) 4). Results are expressed as mean percentage of dose ( SEM. Statistics were performed using a one-way ANOVA test for
parametric data and Kruskal-Wallis test for nonparametric data: (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.
Figure 6. Percentage of radioactive dose recovered in tissues 5, 24, 48, 168, and 240 h after administration of [3H]2k (54 µmol/kg, 20 µCi/rat) to
male Wistar rats (n ) 4). Results are expressed as mean percentage of dose ( SEM. Statistics were performed using a one-way ANOVA test for
parametric data and Kruskal-Wallis test for nonparametric data: (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.
Table 9. Summary of the Metabolites Produced in the Bile and Urine of Male Wistar Rats after Dosing with [3H]2k (54 µmol/kg, 20 µCi)a
metabolite proportion
retention
time (min)
metabolite
identity
(% of radiolabeled
metabolites, 0-5 h)
excreted in
bile/urine
metabolite
m/z
I-IIIb
3.60, 3.80, 6.26
8.36
12.36
16.26
10.91
NDc
469
358
NDc
388
374
564
NDc
NDc
2k
2.43 ( 0.21, 0.47 ( 0.03, 3.57 ( 2.31
62.5 ( 9.37
bile and urine
bile
bile and urine
bile
urine
urine
IV
V
VI
VII
VIII
IX
5.35 ( 2.22, 67.53 ( 9.22
19.0 ( 3.49
NDc
carboxylic acid in side chain of 2k
2k-N-oxide
glucuronide of carboxylic acid side chain derivative
14.22 ( 2.21
13.8
15.75
3.55 ( 0.21
9.95 ( 3.11
urine
a Metabolites were characterised by LC-MS. b Metabolites I-III were minor metabolites. c ND ) not determined.
metabolites was observed, as indicated by the lack of
glutathione conjugate formation.
following iv and po administration to the mouse and rat. For
the purposes of comparison we included two other lead
4-aminoquinolines 1d and 1f as comparators. Table 11 compares
the blood clearance and oral bioavailability in the mouse and
the rat. Blood clearance of comparator molecule 1d was very
high, exceeding the rate of blood flow to the liver in both species
Pharmacokinetics
Given the higher potency of 2k in the in vivo studies, this
molecule was selected for comparative pharmacokinetic studies

1836 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
O’Neill et al.
Scheme 5. Structures of the Metabolites Identified in the Bile
and Urine of Male Wistar Rats Dosed iv with [3H]2k (54
µmol/kg, 20 µCi)a
related 4-aminoquinoline derivatives, using sensitive screening
assays employing purified recombinant enzymes and fluorescent
probe substrates. The results of these studies are summarized
in Table 13. Inhibition of 2D6-mediated 4-methylamino-7-
methoxycoumarin O-dealkylation was observed for isoquine,
amodiaquine, desethyl amodiaquine, and 2k. For this isozyme,
IC50 values ranged from 0.3 to 7 µM across this series.
In Vitro Cytotoxicity Studies in Rat Hepatocytes. Rat
hepatocytes, isolated using a two-step collagen digestion method
(Supporting Information), were incubated with amodiaquine, 2k,
or 3k at concentrations of 0, 20, 50, 100, 200, and 500 µM
(Figure 8). Results are expressed as the percentage of viable
cells, measured microscopically, using a trypan blue exclusion
test (as a percentage of the control). The results from this study
show that the most profound difference in toxicity for rat
hepatocytes was observed with 2k (Figure 8). By replacement
of the hydroxyl group of amodiaquine with fluorine and
incorporation of a tert-butyl group into the amine side chain,
the TD50 decreases from 78 to 385 µM, a 5-fold difference. 3k
also showed less toxicity than amodiaquine after incubation with
a Gluc ) glucuronide.
Scheme 6. Structures of the Metabolites Identified in the Bile
and Urine of Male Wistar Rats Dosed iv with [3H]3k (54
µmol/kg, 20 µCi)
rat hepatocytes, although to a lesser extent than 2k (TD50
)
120 µM). The in vitro therapeutic indexes of 2k and 3k were
observed to be very high at 29 167 and 11 881, respectively,
showing that the amount of compound required to kill the
parasite is much lower than that which causes toxicity to
hepatocytes (Table 14).
Genotoxicity. The potential genotoxicity of 2k and 3k has
been determined by the Salmonella typhimurium SOS/umu assay
in two strains: TA1535/pSK1002 and NM2009.38 This assay is
based on the ability of DNA damaging agents to induce the
expression of the umu operon. The Salmonella strains have a
plasmid pSK1002 that carries a umuC-lacZ fused gene that
produces a hybrid protein with ꢁ-galactosidase activity and
whose expression is controlled by the umu regulatory region.
Since many compounds do not exert their mutagenicity effect
until they have been metabolized, the assay was also performed
in the presence of rat liver S9-mix. Positive control agents (4-
nitroquinoline 1-oxide and 2-aminoanthracene) were used to test
the response of the tester strains. Both 2k and 3k were negative
at the highest concentration tested (140 µM).38
studied. Mean oral bioavailability of this molecule was less than
20%. Both 1f and 2k had substantially lower blood clearance
compared to 1d. The oral bioavailability of 2k was 100% in
the rat and 37% in the mouse in contrast to 1d which had low
clearance values of 21% and 17%, respectively.
Preliminary in Vivo Toxicity in Uninfected (Healthy)
Mice. The final part of this study was to determine the no
observed adverse effects level (NOAEL) of 2k in the mouse
after oral administration of either a single dose or four doses
(single daily dose for 4 days according to the same administra-
tion schedule used in therapeutic efficacy studies). Chloroquine
and amodiaquine were included as control compounds.
The dose framework for NOAEL definition was established
from oral MNLD (maximal nonlethal dose) determinations
shown in Table 15. The parameters evaluated for oral NOAEL
included major apparent signs of toxicity after dosing, body
weights evolution, biochemical analysis of serum (general,
hepatic and renal toxicity biomarkers), and hematology evalu-
ation. A summary of results can be found in Table 16.
The three 4-aminoquinoline derivatives appeared to have
similar toxicities with NOAELs of 100 mg/kg for the four oral
doses, with 300 mg/kg being the first dose with detected toxic
effects. The four oral dose NOAELs for amodiaquine and
chloroquine were 100 and 300 mg/kg, respectively; for 2k the
four oral dose NOAEL was 100 mg/kg.
In terms of the pharmacokinetics of 2k in the mouse, exposure
was comparable to chloroquine after administration of a single
10 mg/kg oral dose of drug with equivalent AUC over 24 h
and similar elimination half-lives (∼7 h in noninfected mice)
(Figure 7). Whole blood concentrations were significantly higher
in infected animals compared to noninfected animals (data not
shown). This presumably represents selective accumulation into
parasitized red cells as seen in in vitro studies. The poor
exposure to amodiaquine in studies conducted in infected
animals reflects its efficient first pass metabolism to desethyl
amodiaquine in vivo.
Intrinsic Clearance. Intrinsic clearance of 2k and related
4-aminoquinoline derivatives was compared in animal and
human hepatocytes in vitro. The results are summarized in Table
12. As expected, amodiaquine had high intrinsic clearance in
mouse, rat, dog, monkey, and human hepatocytes. Isoquine had
high intrinsic clearance in mouse, rat, dog, and monkey
hepatocytes but moderate intrinsic clearance in human hepato-
cytes. The major metabolite of amodiaquine, the result of
N-dealkylation, had lower intrinsic clearance compared to the
parent drug. Compared with amodiaquine and isoquine, 2k had
lower intrinsic clearance across all species.
Discussion
Human P450 Inhibition. The potential for inhibition of the
major human cytochrome P450 isozymes was compared for
The necessity for a new 4-aminoquinoline antimalarial to
supersede chloroquine and amodiaquine is apparent. The

Analogues of Amodiaquine
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1837
Table 10. Summary of the Metabolites Produced in the Bile and Urine of Male Wistar Rats after Dosing with [3H]3k (54 µmol/kg, 20 µCi)a
metabolite proportion
retention
time (min)
metabolite
identity
(% of radiolabeled
metabolites, 0-5 h)
excreted in
bile/urine
metabolite
m/z
I,b II,b V, and VI
7.30, 8.02, 16.09, 18.09
NDc
NDc
1.08 ( 0.21, 1.63 ( 0.26,
12.48 ( 2.94, 19.53 ( 6.82
4.52 ( 0.03, 34.43 ( 1.67
60.76 ( 2.34 28.48 ( 3.33
bile
III
IV
13.29, 14.99
14.73, 11.93
390
404
3k-N-oxide
bile and urine
bile and urine
carboxylic acid side
chain derivative of 3k
parent compound 3k
benzoic acid derivative of 3k
VII
VIII
13.46
16.52
374
333
55.70 ( 6.31
11.39 ( 2.99
urine
urine
a Metabolites were characterised by LC-MS. b Metabolites I and II were minor metabolites. c ND ) not determined.
Table 11. Blood Clearance and Oral Bioavailability of Desethylisoquine
(1f), Isoquine (1d), and 2k in Animalsa
equal concern is that the long half-life of piperaquine may lead
to toxicity through accumulation, as has been observed for
chloroquine. Piperaquine does not, however, contain any obvious
structural alert for metabolic activation.
parameter
species
mouse
1f
1d
2k
40.4
blood clearance
((mL/min)/kg)
blood clearance
((mL/min)/kg)
oral F (%)
43.5
219
Given the half-life issues with 9, it is important to identify
and design a drug from the 4-aminoquinoline class that has a
half-life shorter than the half-lives of chloroquine and amodi-
aquine. As noted earlier, our approach to addressing the toxicity
concerns with amodiaquine has involved two different strategies
to modify the 4-aminophenol unit. The first approach involved
the interchange of the hydroxyl group of amodiaquine with the
amino side chain leading to the isoquine series of amodiaquine
analogues. Following extensive antimalarial and preclinical
evaluation N-tert-butylisoquine (1e) was selected from this group
as a drug for clinical trials in humans and the phase 1 study
was initiated in April 2008. On the basis of the concept of
removing the 4-aminophenol “metabolic alert”, a number of
different groups have prepared novel amodiaquine derivatives.
For example, Sergheraert and co-workers have synthesized a
series of novel 4-aminoquinolines that retain the aromatic ring
of amodiaquine but lack the 4′-hydroxyl group responsible for
the transformation to the toxic quinoneimine in vivo (Chart 2,
e.g., 10).44 The strategy of the Sergheraert group involved the
incorporation of a basic side chain in both the 3′ and 4′ positions
of the aromatic nucleus to prevent nucleophilic addition of
proteins during metabolism. The results of this work revealed
several compounds with high antimalarial activity, with no cross-
resistance in vitro and low cytotoxicity in tests using a human
lung cell line.44
More recently, similar structural modifications to tebuquine
(3-((tert-butylamino)methyl)-4′-chloro-5-(7-chloroquinolin-4-
ylamino)biphenyl-2-ol) have been reported. Lin described a
series of isotebuquine analogues (11) with a hydroxy group meta
rather than para to the amino group of the aniline moiety (Chart
2).45 In vitro testing of these novel isotebuquine analogues
against chloroquine-sensitive and chloroquine-resistant parasites
showed them to exhibit activity similar to that of tebuquine.45
However, during in vivo analysis the new isotebuquine ana-
logues showed only marginal antimalarial activity, possibly due
to poor solubility in vehicle and water.45 Sparatore and
co-workers also have designed isoquine and tebuquine ana-
logues, and some of these compounds (for example, pyrrole
derivative 12) have good activity versus chloroquine resistant
strains of P. falciparum.46 Other 4-aminoquinolines under
development include the chloroquine analogue N1-(7-chloro-
quinolin-4-yl)-N4,N4-diethylbutane-1,4-diamine (AQ13)47 and
the organometallic analogue ferroquine (7-chloro-4-[(2-N,N-
dimethylaminomethyl)ferrocenylmethylamino]quinoline),48 both
of which have entered clinical trials in man.
rat
61.5 ( 21.6
89.2 ( 4.3
35 ( 4.2
mouse
rat
100
21
37
100
oral F (%)
60 ( 26
17.0 ( 4.0
a For studies of the rat, data are expressed as mean ( SD (n ) 3). For
composite mouse studies variability of parameters has not been estimated.
Figure 7. Concentration-time profile for 2k, amodiaquine (1b),
chloroquine (1a), in the uninfected mouse after a single oral dose of
10 mg/kg.
PfCRT-mediated resistance to chloroquine11 and the toxicity
of amodiaquine have prompted a re-examination of the phar-
macology of new and existing 4-aminoquinoline antimalarials.22
Piperaquine (9), a bisquinoline compound (Chart 2), is currently
undergoing phase III trials in combination with dihydroarte-
misinin (DHA).39,40 Piperaquine was introduced as a mono-
therapy for the treatment of malaria in the 1960s, and it was
considered at least as effective as chloroquine against P.
falciparum and P. ViVax malaria and was considered to be better
tolerated. Its use declined in the 1980s because of the emergence
of piperaquine-resistant strains of P. falciparum and the avail-
ability of the artemisinin derivatives.41 In the 1990s, a group
of Chinese scientists rediscovered piperaquine and used it as
one of the components of short-course artemisinin-based
combination therapies to achieve a high cure rate without
significant adverse effects. A number of clinical trials carried
out by White et al. describe dihydroartemisinin (DHA)/
piperaquine as a highly efficacious and well-tolerated combina-
tion therapy for the treatment of malaria.40 Concerns with this
combination lie in the fact that the calculated terminal half-life
for piperaquine is around 16.5 days.42 Compared to that of DHA
(approximately 0.5 h),43 the development of resistance could
be a possibility due to prolonged exposure of piperaquine at
subtherapeutic levels and the prior emergence of extensive
parasites’ resistant to monotherapy with this drug in China. Of
In this publication we have presented extensive metabolic
and dispositional studies on candidates 2k and 3k, making direct
comparisons to chloroquine, amodiaquine, and isoquine (1d).
Each compound was synthesized via highly efficient and

1838 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
O’Neill et al.
Table 12. Comparison of the Intrinsic Clearance of Isoquine, Amodiaquine, Desethylamodiaquine, and 2k in Animal and Human Hepatocytes
intrinsic clearance ((mL/min)/g liver)
compd
mouse
>50
41 ( 7
11 ( 2
16 ( 1.2
rat
dog
monkey
human
amodiaquine (1b)
isoquine (1d)
desethylamodiaquine
2k
9.9 ( 5.3
11 ( 4
1.9 ( 0.4
3.6 ( 0.7
12.0 ( 0.5
23 ( 1
4.4 ( 0.3
4.5 ( 1.0
29 ( 1
21 ( 1
1.6 ( 0.2
12.3 ( 1.1
9.8 ( 0.2
3.3 ( 0.1
<0.5
2.96 ( 0.3
Table 13. Comparative P450 Inhibition Potential of Isoquine, Amodiaquine, Desethylamodiaquine, and 2ka
inhibition potential IC50 (µM)
compd
3A4 DEF
3A4 PPR
1A2
2D6
2C8
2C9
2C19
amodiaquine (1b)
isoquine (1d)
desethyl amodiaquine
2k
28 ( 6
>100
>100
>100
>100
15 ( 5
23 ( 8
28 ( 11
8.8 ( 1.9
6.1 ( 0.5
7.0 ( 0.2
6.4 ( 0.1
0.3 ( 0.1
2.7 ( 1.4
13 ( 2
62 ( 9
75 ( 8
58 ( 2
52, >100
g85
Ab
39 ( 11
54 ( 11
64, >100
24 ( 3
Ab
20 ( 4
9.8 ( 3.9
a Mean and standard deviation of three independent IC50 (µM) determinations reported. b Activation observed.
Figure 8. Viability of male Wistar rat hepatocytes incubated for 6 h with various concentrations of 2k or 3k and stained with trypan blue dye as
an indicator for cell death. Results are expressed as mean percentage of dose ( SEM. Statistics were performed using a one-way ANOVA test for
parametric data and Kruskal-Wallis test for nonparametric data: (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.
Table 14. In Vitro Therapeutic Index of Amodiaquine, 2k, and 3k as a
Measure of the Cytotoxicity in Male Wistar Rat Hepatocytes
gesting a common mechanism of action. Table 17 contains
measured pKa values for amodiaquine, chloroquine, 2k, and 3k.
On the basis of these data, we have calculated the ClogP and
log D at medium pH (7.4). From these values and the drug pKa
values we have estimated the vacuolar accumulation ratio (VAR)
and lipid accumulation ratio (LAR) of each drug, using the
model of Warhurst et al.49 and assuming a digestive vacuole
pH of 5.2.50 There is a good range of in vitro antimalarial
activity (4-fold) among these compounds, but their interaction
with hematin is very similar (Table 8). Since the differences in
activity cannot be attributed to their hematin binding affinities,
it is interesting to compare the VAR and LAR accumulation
parameters to gain some insights into the antimalarial mode of
action of these drugs (Table 17). Considering the very high
predicted accumulation of diprotic weak bases in the acid
vacuole water by a proton trapping mechanism, it is tempting
to speculate that the important drug-heme interaction is taking
place in the aqueous environment. If so, then we would expect
the VAR to be well correlated with antimalarial activity. On
the contrary, the drugs with the highest VAR (e.g., chloroquine)
have the lowest antimalarial activity and vice versa. In fact,
antimalarial activity appears to be well correlated with the LAR
among this series of 4-aminoquinolines (R2 ) 0.97). These
findings suggest that the interaction of hematin with uncharged
drug species may be more important than previously suspected.
These data should be considered in light of recent findings from
IC50 HB3
Tox50 rat
in vitro
compd
strain (nM) hepatocytes (µM) therapeutic index
amodiaquine (1b)
2k
3k
8.5 ( 3.4
14.6 ( 5.4
8.1 ( 3.2
78 ( 2.3
385 ( 25.6
120 ( 14.5
9176
26369
14814
Table 15. Single Oral Dose Maximal Nonlethal Dose (MNLD)
compd
route
MNLD (mg/kg)
chloroquine (1a)
amodiaquine (1b)
2k
po
po
po
840
>500
610
inexpensive routes, allowing for in depth in vitro and in vivo
pharmacological testing to be performed. More importantly, the
chemistry should enable the delivery of 4-aminoquinolines with
acceptable cost of goods based on the commercial costs of
starting materials. It is clear from the studies that fluoro and
chloro analogues of amodiquine have excellent in vitro anti-
malarial activity against a range of different strains of Plasmo-
dium falciparum including chloroquine-resistant parasites such
as TM6, K1, TM4, V1S, and J164.
In terms of mechanism of action we have definitively shown
that both 2k and 3k have identical activity in the BHIA assay;37
in terms of equilibrium binding 2k had similar equilibrium
binding constants to both chloroquine and amodiaquine, sug-

Analogues of Amodiaquine
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1839
Table 16. NOAEL (No Observed Adverse Effect Levels)a
NOAEL, 1
dose (mg/kg)
NOAEL, 4
doses (mg/kg)
toxicity observed
above NOAELs
compd
chloroquine (1a)
amodiaquine (1b)
2k
300
100
300
300
100
100
hepatic toxicity (ALP, ALT, AST, LAP), steatosis
hepatic (LAP, CHOL, AST) and hematological (neutrophils) toxicity
hepatic (ALT, AST, TBIL, TG), renal (BUN), and hematological
(neutrophils) toxicity
a Single and four oral doses.
Chart 2. 4-Aminoquinoline Derivatives
4-day test, 2k was selected for further profiling. In comparison
to isoquine (1d), 2k had moderate to excellent oral bioavail-
ability in the two animal studies performed. Overall, the
exposure pharmacology of 2k appears to be similar to chloro-
quine whereas total radioactivity distribution of this fluorinated
analogue reflects that of amodiaquine. Fluoro analogue 2k was
shown to inhibit CYP 2D6 which was predicted since many
drugs in this class have this potential.54 Both 2k and 3k
demonstrate excellent in vitro safety profiles with large thera-
peutic indices when antimalarial activity is compared to
mammalian cytotoxicity. In preliminary screens we have
demonstrated a lack of mutagenicity, and in vivo MNLD and
NOEL were similar for 2k, chloroquine, and amodiaquine.
In conclusion, fluoro analogue 2k represents an excellent
candidate for further studies with potent activity against
chloroquine-resistant parasites, an excellent safety profile, and
acceptable pharmacokinetic and exposure profiles. These results
establish N-tert-butylfluoro amodiaquine (2k) as a suitable back-
up compound for clinical candidate N-tert-butylisoquine (1e).
Experimental Section
All reactions that employed moisture sensitive reagents were
performed in dry solvent under an atmosphere of nitrogen in oven-
dried glassware. All reagents were purchased from Sigma Aldrich
or Alfa Aesar chemical companies and were used without purifica-
tion. Thin layer chromatography (TLC) was carried out on Merck
silica gel 60 F-254 plates, and UV inactive compounds were
visualized using iodine or anisaldehyde solution. Flash column
chromatography was performed on ICN Ecochrom 60 (32-63
mesh) silica gel eluting with various solvent mixtures and using
an air line to apply pressure. NMR spectra were recorded on a
Brucker AMX 400 (1H, 400 MHz; 13C, 100 MHz) spectrometer.
Chemical shifts are described on parts per million (δ) downfield
from an internal standard of trimethylsilane. Microanalyses were
performed in the University of Liverpool Microanalysis Laboratory.
Mass spectra were recorded on a VG analytical 7070E machine
and Fisons TRIO spectrometers using electron ionization (EI) and
chemical ionization (CI).
the Sullivan laboratory, that hemozoin microcrystals appear to
be surrounded by neutral lipid “nanospheres” inside the digestive
vacuole.51,52
Following the in vitro analysis of both sets of molecules, 2k
and 3k were examined in the Peter’s 4-day test.28 The fluoro
analogue 2k was slightly less potent than amodiaquine and
isoquine; chloro analogue 3k, although more potent than
amodiaquine in vitro, was significantly less potent in the mouse
model with an ED50 of 13 mg/kg.
The disposition studies reveal a similar pattern to other
4-aminoquinolines in the class, and metabolism studies confirm
that both 2k and 3k do not produce aminophenol metabolites
capable of undergoing oxidation to chemically reactive quino-
neimines or other electrophilic metabolites. This demonstrates
that halogenation of the arylamino ring system imparts metabolic
stability for these compounds. Since 2k outperformed 3k in the
Chemistry. General Procedure 1. To a solution of fluoroal-
dehyde (250 mg) in dry CH2Cl2 (10 mL) and dry DMF (0.25 mL)
was added the appropriate amine (5 equiv). The mixture was
Table 17. Measured pKa Values, ClogP, log D, Vacuolar Accumulation Ratio (VAR), and Lipid Accumulation Ratio (LAR) for Selected
4-Aminoquinolinesa
drug (IC50 HB3 strain)
amodiaquine (5.5 nM)
equation
measured pKa
ClogP
log D (7.4)
VAR (pHvac ) 5.2)
LAR
X- + H+ h XH
11.73 ( 0.31
8.40 ( 0.12
7.31 ( 0.18
10.31 ( 0.46
8.22 ( 0.27
9.45 ( 0.26
7.37 ( 0.39
9.33 ( 0.42
7.18 ( 0.38
4.24
4.24
4.24
4.79
4.79
4.87
4.87
5.32
5.32
3.08
3.08
3.08
0.06
0.06
2.40
2.40
3.00
3.00
10756.8
10756.8
10756.8
21834.0
21834.0
12151.7
12151.7
9474.0
908.4
908.4
908.4
9.97
9.97
340.1
340.1
1520.6
1520.6
XH + H+ h XH2
+
XH2+ + H+ h XH3
B + H+ h BH+
2+
chloroquine (20.2 nM)
2k (14.6 nM)
BH+ + H+ h BH2
B + H+ h BH+
BH+ + H+ h BH2
B + H+ h BH+
BH+ + H+ h BH2
2+
2+
3k (8.1 nM)
2+
9474.0
a The sample pKa values were determined using the fast-DPAS ((D-PAS ) dip probe absorption spectroscopy) method. All samples were titrated at a
concentration of ∼50 µM between pH 2 and pH 12. All pKa values were obtained by extrapolating the data to aqueous conditions by the Yasuda-Shedlovsky
method (see http://www.sirius analytical.com). From measured pKa values, the vacuolar accumulation ratio (VAR) and lipid accumulation ratio (LAR) of
each drug were determined using the model of Warhurst et al.49 and assuming a digestive vacuole pH of 5.2.50 ClogP values were calculated using the
AlogPS 2.1 program.53

1840 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
O’Neill et al.
allowed to stir at room temperature for 2 h. After this time,
NaHB(OAc)3 (0.50 g, 3 equiv) was added to the solution and the
mixture was stirred overnight at room temperature. The solvent
was removed in vacuo and the residue was purified via column
chromatography (10% MeOH in CH2Cl2) to give the desired
product.
(1H, d, J ) 5.32 Hz, Ar-H), 6.65 (1H, s, NH), 3.60 (2H, s, CH2N),
2.39 (4H, m, 2 × NCH2CH2), 1.49 (4H, m, 2 × CH2CH2-CH3),
0.83 (6H, t, J ) 7.04 Hz, 2 × CH3-CH2); 13C NMR (100 MHz,
CDCl3) δ 160.2, 152.4, 152.3, 150.1, 148.7, 135.7, 129.5, 126.7,
126.6, 123.9, 123.8, 121.5, 116.7, 116.5, 102.3, 62.8, 56.6, 51.3,
20.7, 12.3; MS (CI) m/z 386 ([M + H]+, 100), 388 (30), 390 (1),
352 (100); HRMS m/z 386.179 55 (C22H2635ClFN3 requires
386.179 93). Anal. (C, H, N).
N-(3-((Dibutylamino)methyl)-4-fluorophenyl)-7-chloroquinolin-
4-amine (2i). The sample was prepared according to general
procedure 1 using dibutylamine to give 2i as a yellow solid: 0.29 g,
42%; mp ) 120-128 °C; 1H NMR (CDCl3, 400 MHz) δ 8.51 (1H,
d, J ) 5.49 Hz, Ar-H), 8.02 (1H, d, J ) 2.19 Hz, Ar-H), 7.85
(1H, d, J ) 9.07 Hz, Ar-H), 7.43 (2H, m, Ar-H), 7.15 (2H, m,
Ar-H), 6.80 (1H, d, J ) 5.49 Hz, Ar-H), 6.60 (1H, s, NH), 3.67
(2H, s, CH2N), 2.50 (4H, t, J ) 6.87 Hz, 2 × NCH2), 1.20-1.55
(8H, m, 2 × CH2CH2), 0.89 (6H, t, J ) 6.88 Hz, 2 × CH3); 13C
NMR (CDCl3, 100 MHz) δ 160.1, 157.7, 152.1, 150.1, 148.9, 135.7,
129.1, 128.9, 126.7, 126.4, 123.6, 121.6, 118.1, 116.5, 102.2, 53.7,
53.5, 32.2, 20.0, 13.7; MS (EI) m/z 414 [M + H]+ (100), 416 (23);
HRMS m/z 414.2097 (C24H3035ClFN3 requires 414.2112). Anal. (C,
H, N).
N-(3-((Diethylamino)methyl)-4-fluorophenyl)-7-chloroquinolin-
4-amine (2d). The sample was prepared according to general
procedure 1 using diethylamine to give 2d as a yellow solid: 0.14 g,
24%; mp ) 130-133 °C; 1H NMR (CDCl3, 400 MHz) δ 8.50 (1H,
d, J ) 5.32 Hz, Ar-H), 8.02 (1H, d, J ) 2.04 Hz, Ar-H), 7.85
(1H, d, J ) 8.96 Hz, Ar-H), 7.45 (2H, m, Ar-H), 7.17 (1H, m,
Ar-H), 7.10 (1H, dd, J ) 8.96, 2.04 Hz, Ar-H), 6.80 (1H, d, J )
5.32 Hz, Ar-H), 6.61 (1H, s, NH), 3.65 (2H, s, CH2N), 2.60 (4H,
q, J ) 7.12 Hz, 2 × CH2), 1.06 (6H, t, J ) 7.10 Hz, 2 × CH3); 13
C
NMR (CDCl3, 100 MHz) δ 160.2, 157.8, 152.3, 150.0, 148.8, 135.5,
129.3, 128.9, 126.7, 126.4, 123.9, 121.6, 118.2, 116.5, 102.3, 50.2,
47.5, 12.2; MS (CI) m/z 358 [M + H]+ (100), 360 (32), 253 (44),
74 (72); HRMS m/z 358.148 19 (C20H2235ClFN3 requires 358.148 62).
Anal. (C, H, N).
7-Chloro-N-(4-fluoro-3-((pyrrolidin-1-yl)methyl)phenyl)quinolin-
4-amine (2e). The sample was prepared according to general
procedure 1 to give 12e as a yellow solid: 0.06 g, 20%; mp )
156-158 °C; 1H NMR (CDCl3, 400 MHz) δ 8.55 (1H, d, J ) 9.10
Hz, Ar-H), 8.45 (1H, d, J ) 6.54 Hz, Ar-H), 7.95 (1H, d, J )
2.09 Hz, Ar-H), 7.82 (1H, dd, J ) 6.33, 2.68 Hz Ar-H), 7.70
(1H, dd, J ) 9.10, 2.09 Hz Ar-H), 7.62 (1H, m, Ar-H), 7.42
(1H, t, J ) 9.18 Hz, Ar-H), 7.02 (1H, d, J ) 6.54 Hz, Ar-H),
4.90 (1H, s, NH), 3.70 (2H, s, CH2N), 3.45 (4H, m, 2 × NCH2),
2.10 (4H, m, 2 × CH2); 13C NMR (CDCl3, 100 MHz) δ 162.6,
160.1, 155.4, 147.9, 144.3, 140.4, 136.5, 130.9, 130.6, 129.0, 126.3,
123.5, 121.6, 119.3, 102.9, 55.6, 52.4, 24.4; MS (CI) m/z 356 ([M
+ H]+, 100), 358 (32), 253 (54), 70 (72); HRMS m/z 356.132 77
(C20H2035ClFN3 requires 356.132 97). Anal. (C, H, N).
7-Chloro-N-(4-fluoro-3-((piperidin-1-yl)methyl)phenyl)quinolin-
4-amine (2f). The sample was prepared according to general
procedure 1 using piperidine to give 2f as a yellow solid: 0.19 g,
31%; mp ) 195-201 °C; 1H NMR (CDCl3, 400 MHz) δ 8.54 (1H,
d, J ) 5.25 Hz, Ar-H), 8.03 (1H, d, J ) 2.02 Hz, Ar-H), 7.85
(1H, d, J ) 8.99 Hz, Ar-H), 7.45 (1H, dd, J ) 8.99, 2.02 Hz,
Ar-H), 7.35 (1H, dd, J ) 6.53, 2.95 Hz, Ar-H), 7.20 (1H, m,
Ar-H), 7.12 (1H, t, J ) 8.81 Hz, Ar-H), 6.80 (1H, d, J ) 5.25
Hz, Ar-H), 6.50 (1H, bs, NH), 3.56 (2H, s, CH2N), 2.51 (4H, m,
2 × CH2N), 1.52-1.47 (6H, m, 3 × CH2); 13C NMR (CDCl3, 100
MHz) δ 157.7, 1521, 150.0, 148.6, 135.6, 129.6, 128.9, 126.8,
126.5, 123.5, 121.3, 118.1, 116.5, 102.4, 100.1, 55.8, 46.4, 27.5,
25.4; MS (CI) m/z 370 ([M + H]+, 100), 372 (30), 253 (42), 86
(85); HRMS m/z 370.147 68 (C21H2235ClFN3 requires 370.148 62).
Anal. (C, H, N).
7-Chloro-N-(4-fluoro-3-(morpholinomethyl)phenyl)quinolin-4-
amine (2j). The sample was prepared according to general procedure
1 using dibutylamine to give 2j as a yellow solid: 0.14 g, 23%; mp
1
) 225-230 °C; H NMR (CDCl3, 400 MHz) δ 8.53 (1H, d, J )
5.26 Hz, Ar-H), 8.04 (1H, d, J ) 2.06 Hz, Ar-H), 7.85 (1H, d,
J ) 8.94 Hz, Ar-H), 7.45 (1H, dd, J ) 8.94, 2.06 Hz, Ar-H),
7.35 (1H, dd, J ) 6.16, 2.57 Hz, Ar-H), 7.19 (1H, m, Ar-H),
7.11 (1H, t, J ) 8.97 Hz, Ar-H), 6.80 (1H, d, J ) 5.26 Hz, Ar-H),
6.60 (1H, bs, NH), 3.71 (4H, t, J ) 4.48 Hz, 2 × CH2O), 3.65
(2H, s, CH2N), 2.55 (4H, t, J ) 4.38 Hz, 2 × CH2N); 13C NMR
(CDCl3, 100 MHz) δ 157.8, 152.3, 150.1, 148.8, 135.8, 129.6,
128.9, 126.9, 126.6, 124.5, 121.4, 118.2, 116.5, 102.4, 100.0, 67.4,
55.8, 53.9; MS (CI) m/z 372 ([M + H]+, 100), 374 (30), 253 (32),
88(92);HRMSm/z372.127 47(C20H2035ClFN3Orequires372.127 87).
Anal. (C, H, N).
[3-(tert-Butylaminomethyl)-4-fluorophenyl](7-chloro-quinolin-
4-yl)amine (2k). The sample was prepared according to the method
in Supporting Information from 4e (by method in Scheme 3) to
give 2k as a pale-yellow solid: 0.37 g, 29%; 1H (CDCl3, 400 MHz)
δ 8.52 (1H, d, J ) 5.32 Hz, Ar-H), 8.02 (1H, d, J ) 2.10 Hz,
Ar-H), 7.83 (1H, d, J ) 8.92 Hz, Ar-H), 7.43 (1H, dd, J ) 2.10
Hz, 8.92 Hz, Ar-H), 7.35 (1H, dd, J ) 2.68 Hz, 6.48 Hz, Ar-H),
7.18-7.13 (1H, m, Ar-H), 7.07 (1H, t, J ) 8.92 Hz), 6.76 (1H, d,
J ) 5.32, Ar-H), 6.63 (1H, bs, NH), 3.80 (2H, s, CH2), 1.18 (9H,
s, (CH3)3); 13C (100 Mz, CDCl3) δ 160.0, 157.6, 152.4, 150.1, 148.6,
135.7, 129.5, 126.2, 126.2, 124.0, 121.4, 118.2, 116.8, 116.6, 102.4,
51.3, 40.8, 29.5; MS (CI) m/z 358 ([M + H]+, 94), HRMS m/z
358.1488 (C20H2235ClFN3 requires 358.1486). Anal. (C, H, N).
General Procedure 2. HCl (5 drops) was added to a solution
of the appropriate amine (0.52 mmol) and 4,7-dichloroquinoline
(1 equiv) in EtOH (20 mL). The mixture was stirred at reflux for
24 h. The reaction mixture was concentrated, and 1 M NaOH was
added to the residue. The aqueous mixture was washed with CH2Cl2
(×3). The organic layers were combined, dried over MgSO4,
filtered, and concentrated to give the crude product. Purification
by column chromatography (silica gel, 10% MeOH in CH2Cl2) gave
the desired product.
7-Chloro-N-(4-fluoro-3-((4-methylpiperazin-1-yl)methyl)phe-
nyl)quinolin-4-amine (2g). The sample was prepared according to
general procedure 1 using piperidine to give 2f as a yellow solid:
1
0.20 g, 31%; mp ) 165-169 °C; H NMR (400 MHz, CDCl3) δ
8.54 (1H, d, J ) 5.33 Hz, Ar-H); 8.01 (1H, d, J ) 2.10 Hz, Ar-H),
7.90 (1H, d, J ) 8.96 Hz, Ar-H), 7.41 (1H, dd, J ) 2.10 Hz, 8.96
Hz, Ar-H), 7.34 (1H, dd, J ) 2.72 Hz, 6.22 Hz, Ar-H), 7.18
(1H, m, Ar-H), 7.08 (1H, t, J ) 8.95 Hz, Ar-H), 6.80 (1H, d, J
) 5.33 Hz, Ar-H), 3.60 (2H, s, CH2N), 2.60 (4H, s, 2 × CH2N),
2.54 (4H, s, 2 × CH2N), 2.25 (3H, s, CH3-N); 13C NMR (400
MHz, CDCl3) δ 160.3, 157.9, 152.2, 150.0, 148.8, 135.8, 129.2,
126.9, 126.4, 124.4, 121.8, 118.2, 116.9, 116.7, 102.3, 55.4, 55.3,
53.1, 46.0; MS (CI) m/z 385 ([M + H]+, 100), 387 (27), 389 (2);
HRMS m/z 385.158 06 (C21H2235ClFN4 requires 385.159 52). Anal.
(C, H, N).
(7-Chloroquinolin-4-yl)(4-fluoro-3-imidazol-1-ylmethylphenyl)-
amine (2l). General procedure 2 provided 2l as an off-white solid:
1
0.91 g, 46%; H (400 MHz, MeOD) δ 8.37 (1H, d, J ) 5.56 Hz,
Ar-H), 8.23 (1H, d, J ) 9.03 Hz, Ar-H), 7.86 (1H, d, J ) 2.28
Hz, Ar-H), 7.78 (1H, s, Ar-H), 7.49 (1H, dd, J ) 2.09 Hz, 9.11
Hz, Ar-H), 7.37 (1H, m, Ar-H), 7.24 (2H, m, Ar-H), 7.17 (1H,
s, Ar-H), 7.02 (1H, s, Ar-H), 6.78 (1H, d, J ) 5.56 Hz, Ar-H),
4.82 (2H, s, CH2); 13C (100 MHz, MeOD) δ 160.7, 158.2, 152.7,
151.7, 150.4, 139.2, 138.3, 137.4, 130.0, 128.2, 127.4, 127.1, 125.0,
121.3, 119.8, 118.4, 118.1, 102.9, 45.8; MS (CI) m/z 353 ([M +
H]+, 100), 355 (32), 357 (1), 319 (34); HRMS m/z 353.095 93
(C19H15ClFN4 requires 353.096 92). Anal. (C, H, N).
N-(3-((Dipropylamino)methyl)-4-fluorophenyl)-7-chloroquinolin-
4-amine (2h). The sample was prepared according to general
procedure 1 using dipropylamine to give 2h as a yellow solid:
1
0.17 g, 27%; mp ) 130-132 °C; H NMR (400 MHz, CDCl3) δ
8.45 (1H, d, J ) 5.32 Hz, Ar-H), 8.01 (1H, d, J ) 1.94 Hz, Ar-H),
7.86 (1H, d, J ) 8.92 Hz, Ar-H), 7.85 (1H, dd, J ) 8.92, 1.94
Hz, Ar-H), 7.44 (1H, m, Ar-H), 7.15-7.04 (2H, m, Ar-H), 6.79

Analogues of Amodiaquine
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1841
Compounds 3d-m were prepared according to the general
procedure 3 in Supporting Information.
7.86 (1H, d, J ) 8.98 Hz, Ar-H), 7.56 (1H, d, J ) 2.43 Hz, Ar-H),
7.47 (1H, dd, J ) 2.11, 8.98 Hz, Ar-H), 7.36 (1H, d, J ) 8.46
Hz, Ar-H), 7.09 (1H, dd, J ) 2.65, 8.53 Hz, Ar-H), 6.92 (1H, d,
J ) 5.27 Hz, Ar-H), 6.55 (1H, bs, NH), 3.69 (2H, s, CH2), 2.48
(4H, t, J ) 7.92 Hz, 2 × NCH2), 1.45 (4H, m, CH2CH2NCH2CH2),
1.29 (4H, m, 2 × CH2CH3), 0.87 (6H, t, J ) 7.32, 2 × CH2CH3);
13C (100 MHz, MeOD) δ 150.4, 148.9, 146.5, 138.7, 136.9, 136.6,
130.6, 126.6, 125.9, 125.7, 123.8, 123.3, 118.1, 101.8, 54.9, 53.4,
50.4, 32.8, 32.5, 22.6, 20.8, 20.5, 14.4, 14.2; MS (ES+) m/z 430
([M + H]+, 100); HRMS m/z 430.1804 (C24H3035Cl2N3 requires
430.1817). Anal. (C, H, N).
N-(3-((Diethylamino)methyl)-4-chlorophenyl)-7-chloroquinolin-
1
4-amine (3d). Yellow solid; 0.22 g, 74%; H NMR (CDCl3, 400
MHz) δ 8.55 (1H, d, J ) 5.28 Hz, Ar-H), 8.05 (1H, d, J ) 2.09
Hz, Ar-H), 7.84 (1H, d, J ) 8.96 Hz, Ar-H), 7.54 (1H, d, J )
2.71 Hz, Ar-H), 7.45 (1H, dd, J ) 8.96, 2.09 Hz, Ar-H), 7.36
(1H, d, J ) 8.46 Hz, Ar-H), 7.12 (1H, dd, J ) 8.46, 2.77 Hz,
Ar-H), 6.92 (1H, d, J ) 5.28 Hz, Ar-H), 6.61 (1H, s, NH), 3.68
(2H, s, CH2N), 2.60 (4H, q, J ) 7.14 Hz, 2 × CH2), 1.06 (6H, t,
J ) 7.10 Hz, 2 × CH3); 13C NMR (CDCl3, 100 MHz) δ 152.3,
150.0, 147.9, 140.2, 138.9, 135.8, 130.6, 129.57, 129.21, 126.61,
124.6, 122.0, 121.5, 118.5, 103.0, 54.8, 47.8, 12.4; MS (CI) m/z
374 [M + H]+ (100), 376 (73), 340 (28), 72 (24); HRMS m/z
374.118 83 (C20H2235Cl2N3 requires 374.119 08) Anal. (C, H, N).
(4-Chloro-3-pyrrolidin-1-ylmethyl-phenyl)(7-chloroquinolin-4-
7-Chloro-N-(4-chloro-3-((4-ethylpiperazin-1-yl)methyl)phenyl)quin-
olin-4-amine (3j). Yellow solid; 0.207 g, 63%; 1H NMR (400 MHz,
CDCl3) δ 8.56 (1H, d, J ) 5.32 Hz, Ar-H), 8.04 (1H, d, J ) 2.08
Hz, Ar-H), 7.87 (1H, d, J ) 8.92 Hz, Ar-H), 7.46 (1H, dd, J )
2.08 Hz, 8.92 Hz, Ar-H), 7.44 (1H, d, J ) 2.64 Hz, Ar-H), 7.37
(1H, d, J ) 8.52 Hz, Ar-H), 7.14 (1H, dd, J ) 2.64 Hz, 8.52 Hz,
Ar-H), 6.92 (1H, d, J ) 5.32 Hz, Ar-H), 6.70 (1H, s, NH), 3.61
(2H, s, CH2N), 2.47 (4H, bs, 2(-NCH2N-)), 2.57 (4H, bs,
2(-NCH2N-)), 2.40 (2H, q, J ) 7.28 Hz, CH2CH3), 1.07 (3H, t,
J ) 7.20 Hz, CH3CH2); 13C NMR (100 MHz, CDCl3) δ 152.3,
150.2, 147.7, 138.7, 138.2, 135.8, 130.8, 130.1, 129.5, 126.6, 124.6,
122.4, 121.6, 118.6, 103.2, 59.4, 53.8, 53.5, 53.2, 52.7, 42.3, 12.1;
MS (ES-) m/z 413 ([M - H]-, 100), 415 (48), 417 (6); HRMS
m/z 413.1297 (C22H2335Cl2N4 requires 413.1300). Anal. (C, H, N).
[3-(tert-Butylaminomethyl)-4-chlorophenyl](7-chloroquinolin-4-
1
yl)amine (3e). Yellow solid; 0.18 g, 61%; H NMR (400 MHz,
MeOD) δ 8.41 (1H, d, J ) 5.52 Hz, Ar-H), 8.27 (1H, d, J ) 8.99
Hz, Ar-H), 7.87 (1H, d, J ) 2.04 Hz, Ar-H), 7.59 (1H, d, J )
2.67 Hz, Ar-H), 7.52-7.41 (2H, m, Ar-H), 7.28 (1H, dd, J )
2.04, 8.99 Hz, Ar-H), 6.97 (1H, d, J ) 5.52 Hz, Ar-H), 3.81
(2H, s, CH2), 2.66 (4H, s, CH2-N-CH2), 1.83 (4H, s, CH2CH2);
13C NMR (100 MHz, MeOD) δ 152.9, 151.1, 150.7, 140.7, 139.0,
137.3, 131.9, 131.0, 128.3, 127.3, 126.9, 125.1, 124.7, 120.1, 103.5,
58.1, 55.5, 55.2, 24.8, 24.7; MS (CI) m/z 272 ([M + H]+, 41), 269
(60); HRMS m/z 372.102 59 (C20H20Cl2N3 requires 372.103 42).
Anal. (C, H, N).
1
yl)amine (3k). Yellow solid; (0.25 g, 59%); H NMR (400 MHz,
MeOD) δ 8.47 (1H, d, J ) 5.72 Hz, Ar-H), 8.32 (1H, d, J ) 8.92
Hz, Ar-H), 7.90 (1H, d, J ) 2.12 Hz, Ar-H), 7.65 (1H, d, J )
2.64 Hz, Ar-H), 7.53 (2H, m, Ar-H), 7.39 (1H, dd, J ) 2.64 Hz,
8.52 Hz, Ar-H), 7.08 (2H, d, J ) 5.72 Hz, Ar-H), 4.81 (1H, bs,
NH), 3.79 (2H, s, CH2N), 1.21 (9H, s, (CH3)3); 13C NMR (100
MHz, MeOD) δ 152.9, 150.9, 150.7, 140.9, 140.6, 137.2, 131.8,
130.6, 128.4, 127.3, 126.8, 125.0, 124.7, 120.1, 103.6, 52.7, 45.9,
29.3; MS (ES+) m/z 374 ([M + H]+, 100), 376 (38), 378 (2);
HRMS m/z 374.1187 (C20H2235Cl2N3 requires 374.1191). Anal. (C,
H, N).
7-Chloro-N-(4-chloro-3-((piperidin-1-yl)methyl)phenyl)quinolin-
4-amine (3f). Yellow solid; 0.30 g, 92%; H NMR (400 MHz,
1
CDCl3) δ 8.55 (1H, d, J ) 5.25 Hz, Ar-H), 7.90 (1H, d, J ) 2.06
Hz, Ar-H), 7.49 (1H, d, J ) 2.57 Hz, Ar-H), 7.40 (1H, dd, J )
2.06, 8.90 Hz, Ar-H), 7.34 (1H, d, J ) 8.90 Hz, Ar-H), 7.14
(1H, d, J ) 2.7 Hz, Ar-H), 7.12 (1H, d, J ) 2.7 Hz, Ar-H), 6.90
(1H, d, J ) 5.25 Hz, Ar-H), 6.80 (1H, s, NH), 3.56 (2H, s, CH2N),
2.46 (4H, t, J ) 4.45 Hz, 2 × CH2), 1.56 (4H, m, 2 × CH2), 1.25
(2H, t, J ) 7.15 Hz, CH2); 13C NMR (100 MHz, CDCl3) δ 152.3,
150.2, 147.8, 138.7, 138.7, 135.8, 130.7, 130.0, 129.4, 126.6, 124.6,
122.2, 121.7, 118.6, 103.1, 60.1, 55.1, 53.8, 26.4, 24.6, 21.3; MS
(CI) m/z 386 ([M + H]+, 100), 388 (54); HRMS m/z 386.1191
(C21H2235Cl2N3 requires 386.1167). Anal. (C, H, N).
7-Chloro-N-(4-chloro-3-((4-(pyridin-2-yl)piperazin-1-yl)meth-
1
yl)phenyl)quinolin-4-amine (3l). Yellow solid: 0.36 g, 98%; H
NMR (400 MHz, CDCl3) δ 8.57 (1H, d, J ) 5.32 Hz, Ar-H),
8.17 (1H, m, -HCdN-), 8.04 (1H, d, J ) 2.12 Hz, Ar-H), 7.87
(1H, d, J ) 8.92 Hz, Ar-H), 7.51 (1H, d, J ) 2.84 Hz, Ar-H),
7.48-7.45 (1H, m, Ar-H), 7.40 (1H, d, J ) 8.56 Hz, Ar-H), 7.33
(1H, m, Ar-H), 7.17 (1H, dd, J ) 2.64 Hz, 8.52 Hz, Ar-H), 6.94
(1H, d, J ) 5.32 Hz, Ar-H), 6.65-6.60 (2H, m, Ar-H), 3.67 (2H,
s, CH2N), 3.56 (4H, t, J ) 4.96 Hz, 2 × CH2N), 2.67 (4H, t, J )
5.12 Hz, 2 × CH2); 13C NMR (100 MHz, CDCl3) δ 159.8, 150.1,
149.5, 148.2, 147.7, 138.4, 137.9, 136.6, 130.9, 130.6, 127.1, 126.7,
125.2, 122.9, 122.8, 118.2, 113.7, 107.5, 102.3, 59.3, 51.0, 45.6;
MS (ES-) m/z 462 ([M - H]-, 100), 464 (50), 466 (4); HRMS
m/z 462.1258 (C25H2235Cl2N5 requires 462.1252). Anal. (C, H, N).
[4-Chloro-3-(4-isopropylpiperazin-1-ylmethyl)phenyl](7-chloro-
7-Chloro-N-(4-chloro-3-((4-methylpiperazin-1-yl)methyl)phe-
1
nyl)quinolin-4-amine (3g). Yellow solid; 0.28 g, 89%; H NMR
(400 MHz, CDCl3) δ 8.54 (1H, d, J ) 5.25 Hz, Ar-H), 8.01(1H,
d, J ) 2.10 Hz, Ar-H), 7.91 (1H, d, J ) 9.06 Hz, Ar-H), 7.43
(1H, dd, J ) 2.10 Hz, 9.06 Hz, Ar-H), 7.40 (1H, d, J ) 2.07 Hz,
Ar-H), 7.36 (1H, d, J ) 8.42 Hz, Ar-H), 7.12 (1H, dd, J ) 5.72,
8.42 Hz, Ar-H), 6.90 (1H, d, J ) 5.25 Hz, Ar-H), 6.59 (1H, s,
NH), 3.62 (2H, s, CH2), 2.56 (4H, s, 2 × CH2N), 2.43 (4H, s, 2 ×
CH2N), 2.26 (3H, s, CH3N); 13C NMR (100 MHz, CDCl3) δ 151.8,
149.7, 147.6, 138.4, 137.8, 135.4, 130.4, 129.7, 128.9, 126.2, 124.4,
122.1, 121.5, 118.3, 102.7, 59.0, 55.2, 55.1, 53.2, 53.5, 46.0; MS
(ES-) m/z 399 ([M - H]-, 100), 401 (50), 403 (5); HRMS m/z
399.1154 (C21H2135Cl2N4 requires 399.1143), 401.1118
(C21H2135Cl37ClN4 requires 401.1114). Anal. (C, H, N).
N-(3-((Dipropylamino)methyl)-4-chlorophenyl)-7-chloroquino-
lin-4-amine (3h). Yellow solid; 0.27 g, 85%; 1H NMR (400 MHz,
CDCl3) δ 8.45 (1H, d, J ) 5.32 Hz, Ar-H), 8.04 (1H, d, J ) 2.08
Hz, Ar-H), 7.86 (1H, d, J ) 8.92 Hz, Ar-H), 7.57 (1H, d, J )
2.63 Hz, Ar-H), 7.47 (1H, dd, J ) 2.08 Hz, 8.92 Hz, Ar-H),
7.35 (1H, d, J ) 8.36 Hz, Ar-H), 7.09 (1H, dd, J ) 2.84 Hz, 8.36
Hz, Ar-H), 6.94 (1H, d, J ) 5.32 Hz, Ar-H), 6.56 (1H, s, NH),
3.69 (2H, s, CH2N), 2.46 (4H, t, J ) 1.59 Hz, 2 × CH2), 1.46 (4H,
m, 2 × CH2), 0.84 (6H, t, J ) 7.31 Hz, 2 × CH3); 13C NMR (100
MHz, CDCl3) δ 151.2, 149.1, 148.7, 139.7, 138.6, 136.2, 130.6,
129.9, 128.4, 128.2, 125.6, 124.9, 122.5, 122.4, 118.4, 102.7, 56.7,
55.8, 50.1, 20.5, 12.2; MS (ES-) m/z 400 ([M - H]-, 100), 402
(45), 404 (3); HRMS m/z 400.1358 (C22H24Cl2N3 requires 400.1347).
Anal. (C22H25Cl2N3) C, H, N.
1
quinolin-4-yl)amine (3m). Yellow solid (0.31 g, 61%); H (400
MHz, MeOD) δ 8.43 (1H, d, J ) 5.53 Hz, Ar-H), 8.29 (1H, d, J
) 9.03, Ar-H), 7.89 (1H, d, J ) 2.05 Hz, Ar-H), 7.53 (1H, dd,
J ) 2.05, 9.03 Hz, Ar-H), 7.46 (1H, d, J ) 8.53 Hz, Ar-H), 7.30
(1H, dd, J ) 2.68, 8.53 Hz, Ar-H), 6.98 (2H, d, J ) 5.55 Hz,
Ar-H), 3.72 (2H, s, CH2), 3.33 (1H, s, CH), 2.82 (4H, bs, 2 ×
CH2), 2.71 (4H, bs, 2 × CH2), 1.19 (6H, s, (CH3)2); 13C (100 MHz,
MeOD) δ 152.9, 151.1, 150.6, 140.8, 138.1, 137.3, 132.0, 131.3,
128.3, 127.4, 126.8, 125.2, 124.8, 120.2, 103.6, 60.1, 57.4, 55.2,
53.5, 18.7; MS (ES-) m/z 427/429/431 ([M - H]-); HRMS m/z
427.1454 (C23H2535Cl2N4 requires 427.1456). Anal. (C, H, N).
(7-Chloroquinolin-4-yl)(4-chloro-3-imidazol-1-ylmethylphenyl)-
amine (3n). Yellow solid; 71.9 mg, 33%; 1H (400 MHz, MeOD) δ
8.43 (1H, d, J ) 5.83, Ar-H), 8.30 (1H, d, J ) 9.06, Ar-H), 7.91
(1H, d, J ) 2.09 Hz, Ar-H), 7.85 (1H, s, Ar-H), 7.58 (1H, dd, J
) 2.09 Hz, 9.06 Hz, Ar-H), 7.56 (1H, d, J ) 8.54 Hz), 7.40 (1H,
dd, J ) 2.66 Hz, 8.54 Hz, Ar-H), 7.22 (1H, s, Ar-H), 7.12 (1H,
d, J ) 2.47 Hz, Ar-H), 7.06 (1H, s, Ar-H), 6.94 (1H, d, J ) 5.83
Hz, Ar-H), 4.81 (2H, s, CH2); 13C (100 MHz, MeOD) δ 150.3,
(4-Chloro-3-dibutylaminomethylphenyl)(7-chloroquinolin-4-
yl)amine (3i). Yellow solid; 0.08 g, 24%; 1H (400 MHz, MeOD) δ
8.56 (1H, d, J ) 5.27 Hz, Ar-H), 8.05 (1H, d, J ) 2.11 Hz, Ar-H),

1842 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
O’Neill et al.
147.7, 144.5, 138.4, 138.3, 135.8, 130.8, 130.2, 129.5, 126.7, 125.4,
124.8, 124.6, 122.4, 121.5, 118.7, 113.5, 103.2, 46.0; MS (CI) m/z
369 ([M + H]+, 100), 371 (67), 373 (15); HRMS m/z 369.067 94
(C19H15Cl2N4 requires 369.067 38). Anal. (C, H, N).
appropriate. Chloroquine or amodiaquine was included as assay
quality control for each in vivo assay of P. yoelii or P. berghei,
respectively.
Disposition and Metabolism Studies on 2k and 3k.
Materials. [3H]2k (24.08 Ci/mmol; radiochemical purity by HPLC
greater than 97%) and [3H]3k (46.50 Ci/mmol; radiochemical purity
by HPLC greater than 98%) were prepared from [3H]4,7-dichlo-
roquinoline (general method 1a). Tissue solubilizer-450 (0.5 N
quaternary ammonium hydroxide in toluene) was purchased from
Beckmann Chemicals (Bremen, Germany). Ultima Flo and Ultima
Gold scintillant were from Packard Bioscience BV (Groningen, The
Netherlands). HPLC grade solvents were products of Fisher
Scientific (Loughborough, Leicestershire, U.K.).
Adult male Wistar rats (200-400 g) were obtained from Charles
River (Margate, U.K.). The protocols described were undertaken
in accordance with criteria outlined in a license granted under the
Animals (Scientific Procedures) Act of 1986 and approved by the
University of Liverpool Animal Ethics Committee.
Methods. In Vivo Metabolism of [3H]2k and [3H]3k in the
Rat (5 h). Adult male Wistar rats (250-400 g, n ) 4) were
anesthetized with urethane (1.4 g/mL in isotonic saline; 1 mL/kg,
ip). Polyethylene cannulae were inserted into the trachea, femoral
vein, and common bile duct, and the penis was ligated. Drug-blank
bile was collected for approximately 20 min before treatment.
[3H]2k or [3H]3k (54 µmol/kg, 20 µCi/rat) was dissolved in saline
(0.5-1 mL depending on animal weight) and was injected over 10
min (iv). Bile was collected hourly for a time period of 5 h into
preweighed Eppendorf tubes. Samples were weighed after collection
to determine the extent of biliary secretion and stored at -30 °C
until analysis by LC-MS.
After 5 h, urine was aspirated from the bladder and transferred
to a preweighed Eppendorf tube and a cardiac puncture was
performed. The blood was centrifuged (1000g, 6 min) and the
plasma transferred to a separate tube. The plasma from each
experiment was pooled together, and then blood proteins were
removed by the exhaustive addition of MeOH (5 equiv) followed
by centrifugation (1200g, 10 min). The supernatant was removed
and concentrated by evaporation before redissolving it in MeOH
(200 µL). The plasma and urine samples were stored at -30 °C
until analysis by LC-MS. Aliquots of bile and urine (30 µL) were
mixed with scintillant (4 mL) for determination of radioactivity.
LC-MS Analysis of Bile, Urine, and Plasma Samples. Aliquots
of bile, urine, and plasma (100 µL) were analyzed at room
temperature on a Hypersil 5-µm HyPurity Elite C-18 column (150
mm × 4.6 mm; Thermo Hypersil-Keystone, Runcorn, Cheshire,
U.K.) with a gradient of acetonitrile (10-50% over 30 min (2k),
20-50% over 30 min (3k)) in trifluoroacetic acid (0.1%, v/v). The
LC system consisted of two Jasco PU980 pumps (Jasco UK, Great
Dunmow, Essex, U.K.) and a Jasco HG-980-30 mixing module.
The flow rate was 0.9 mL/min. Eluate split-flow to the LC-MS
interface was ∼40 µL/min. A Quattro II mass spectrometer
(Micromass MS Technologies, Manchester, U.K.) fitted with the
standard coaxial electrospray source was used in the positive-ion
mode. Nitrogen was used as the nebulizing and drying gas. The
interface temperature was 80 °C; the capillary voltage was 3.9 kV.
Spectra were acquired between m/z 100 and 1050 over a scan
duration of 5 s. Fragmentation of analyte ions was achieved at a
cone voltage of 50-70 V. Data were processed with MassLynx
3.5 software (Micromass). Radiolabeled analytes in the remainder
of the eluate were detected with a Radiomatic A250 flow detector
(Packard, Pangbourne, Berkshire, U.K.). Eluate was mixed with
Ultima-Flo AP scintillant (Packard Bioscience BV, Groningen, The
Netherlands) at 1 mL/min.
[3-(tert-Butylaminomethyl)phenyl](7-chloroquinolin-4-yl)amine
(6f). Preparation according to general procedure 3, using 6d, gave
6f as a yellow solid after purification by column chromatography
1
(20% MeOH in CH2Cl2): 0.89 g, 39%; H (400 MHz, MeOD) δ
8.64 (1H, d, J ) 9.12 Hz, Ar-H), 8.45 (1H, d, J ) 7.06 Hz, Ar-H),
8.01 (1H, d, J ) 1.96 Hz, Ar-H), 7.73 (1H, dd, J ) 1.96, 9.12
Hz, Ar-H), 7.75-7.73 (2H, m, Ar-H), 7.65-7.57 (2H, m, Ar-H),
7.03 (1H, d, J ) 7.04 Hz, Ar-H), 3.33 (2H, s, CH2), 1.53 (9H, s,
(CH3)3); 13C (100 MHz, CDCl3) δ 150.1, 148.0, 145.5, 138.1, 135.8,
130.3, 129.8, 129.5, 126.7, 121.9, 118.8, 115.2, 114.9, 113.5, 103.6,
51.2, 46.9, 29.8, 29.5; MS (CI) m/z 340 ([M + H]+, 100); HRMS
m/z 340.157 29 (C20H23ClN3requires 340.158 02). Anal. (C, H, N).
(7-Chloroquinolin-4-yl)(3-imidazol-1-ylmethylphenyl)amine (6g).
Preparation according to general procedure 3, using 6e, gave 6g as
a yellow solid after purification by column chromatography (5%
1
MeOH in CH2Cl2): 162 mg, 25%; H (400 MHz, MeOD) δ 8.35
(1H, d, J ) 5.50 Hz, Ar-H), 8.22 (1H, d, J ) 9.06), 7.84 (1H, d,
J ) 2.09 Hz, Ar-H), 7.77 (1H, s, Ar-H), 7.44 (1H, dd, J ) 2.09
Hz, 9.06 Hz, Ar-H), 7.42 (1H, t, J ) 7.79 Hz, Ar-H), 7.32 (1H,
d, J ) 7.97 Hz, Ar-H), 7.22 (1H, t, J ) 1.71 Hz, Ar-H), 7.15
(1H, t, J ) 1.33, Ar-H), 7.09 (1H, dd, J ) 0.57 Hz, 7.59 Hz,
Ar-H), 7.02 (1H, t, J ) 1.14 Hz, Ar-H), 6.90 (1H, d, J ) 5.50
Hz, Ar-H), 4.86 (2H, s, CH2); 13C (100 MHz, MeOD) δ 152.8,
151.2, 150.9, 142.4, 140.5, 139.1, 137.2, 131.6, 129.9, 128.2, 127.2,
125.3, 125.1, 124.2, 123.5, 121.4, 120.3, 103.4, 51.8; MS (CI) m/z
335 ([M + H]+, 70), 337 (30), 339 (3), 301 (56); HRMS m/z
335.105 68 (C19H16ClN4requires 335.106 32). Anal. (C, H, N).
Antimalarial Activity. Parasites were maintained in culture
according to previously published work.22 In vitro antimalarial
activity was assessed using the methods described by Smilkstein.24
For this assay, after 48 h of growth, 100 µL of SYBR Green I in
lysis buffer (0.2 µL of SYBR Green I/mL of lysis buffer) was added
to each well, and the contents were mixed until no visible
erythrocyte sediment remained. After 1 h of incubation in the dark
at room temperature, fluorescence was measured using a Thermo
Electron Corporation VARIOSKAN fluorescent plate reader, with
excitation and emission wavelength bands centered at 485 and 530
nm, respectively, and processed using the Thermo SkanIt RE
proprietry software package. The background reading for an empty
well was subtracted to yield fluorescence counts for analysis.
Analysis of the counts obtained by both assay methods was
performed with the Grafit data analysis and graphics program
(Erithacus software, version 3.01). The counts were plotted against
the logarithm of the drug concentration and curve fitting was done
by nonlinear regression (sigmoidal dose-response/variable slope
equation) to yield the drug concentration that produced 50% of the
observed decline from the maximum counts in the drug-free control
wells (IC50).
In Vivo Antimalarial Activity. The efficacy of selected 4-ami-
noquinolines was measured against P. yoelii or P. berghei in a “4-
day test” (Peters et al.).28 Cohorts of age-matched female CD1 mice
were infected iv with 6.4 × 106 or 10.0 × 106 parasites obtained
from infected donors, and the mice were randomly distributed in
groups of n ) 5 mice/group (day 0). Treatments were administered
from day 0 (1 h after infection) until day 3. The percentage of
YOYO-1+ murine erythrocytes in peripheral blood at day 4 after
infection was measured (Cytometry, Part A, 2005, 67, 27-36) and
recrudescence up to days 22-25 if the parasitemias were below
our detection limit (0.01%) at day 4 after infection. The therapeutic
efficacy of compounds was expressed as the effective dose (mg ·kg/
(body weight)) that reduces parasitemia by 50% (ED50) and 90%
(ED90) with respect to vehicle treated groups (ED90) and the dose
that achieved eradication of parasitemia until day 23 after infection
(NRL). All compounds and corresponding vehicles were admin-
istered orally at 20 mg ·kg-1 or subcutaneously at 10 mg ·kg-1, as
In Vivo Retention Studies of [3H]2k and [3H]3k in the Rat. Adult
male Wistar rats (200-400 g, n ) 4) were administered a solution
of [3H]2k or [3H]3k (54 µmol/kg, 20 µCi/rat) dissolved in saline
(0.5-1 mL, ip). The animals were then transferred to individual
metabolism cages equipped with a well for collecting urine and
faeces. The rats were allowed food and drink ad libitum for a 24 h
period before administration of a lethal dose of pentabarbitone.

Analogues of Amodiaquine
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1843
Aliquots of urine (30 µL) were collected and mixed with
scintillant for determination of radioactivity. The collected feces
were weighed and dissolved in distilled water for a period of 16 h
before determination of radioactivity. The major organs were then
removed and assessed for radioactivity.
Acknowledgment. This work was supported by grants from
the EU (Antimal, FP6 Malaria Drugs Initiative), GlaxoSmith-
kline, and the Medicines for Malaria Venture (MMV, Geneva).
Supporting Information Available: Synthetic procedures and
characterization data for intermediates, procedures for equilibrium
binding studies, metabolism and disposition studies, and protocols
for pharmacokinetics/safety evaluation. This material is available
free of charge via the Internet at http://pubs.acs.org.
In Vivo Retention Studies of [3H]2k and [3H]3k in the
Rat (48 h). Adult male Wistar rats (n ) 4) were administered a
solution of either [3H]2k or [3H]3k (54 µmol/kg, 20 µCi/rat)
dissolved in saline (0.5-1 mL, ip). The animals were kept in a
normal animal cage for a period of 24 h before they were transferred
to individual metabolism cages equipped with a well for collecting
urine and faeces. The rats remained in the metabolism cages for a
further 24 h, with full access to food and drink, before overdose
using pentabarbitone.
References
(1) White, N. J.; Nosten, F.; Looareesuwan, S.; Watkins, W. M.; Marsh,
K.; Snow, R. W.; Kokwaro, G.; Ouma, J.; Hien, T. T.; Molyneux,
M. E.; Taylor, T. E.; Newbold, C. I.; Ruebush, T. K.; Danis, M.;
Greenwood, B. M.; Anderson, R. M.; Olliaro, P. Averting a malaria
disaster. Lancet 1999, 353, 1965–1967.
(2) Greenwood, B. M.; Fidock, D. A.; Kyle, D. E.; Kappe, S. H. I.; Alonso,
P. L.; Collins, F. H.; Duffy, P. E. Malaria: progress, perils, and
prospects for eradication. J. Clin. InVest. 2008, 118, 1266–1276.
(3) Tilley, L.; Davis, T. M. E.; Bray, P. G. Prospects for the treatment of
drug-resistant malaria parasites. Future Microbiol. 2006, 1, 127–141.
(4) Kelly-Hope, L.; Ranson, H.; Hemingway, J. Lessons from the past:
managing insecticide resistance in malaria control and eradication
programmes. Lancet Infect. Dis. 2008, 8, 387–389.
(5) Goldberg, D. E. Plasmodial hemoglobin degradation: an ordered
pathway in a specialized organelle. Infect. Agents Dis. 1992, 1, 207–
211.
(6) Francis, S. E.; Sullivan, D. J.; Goldberg, D. E. Hemoglobin metabolism
in the malaria parasite Plasmodium falciparum. Annu. ReV. Microbiol.
1997, 51, 97–123.
(7) Mungthin, M.; Bray, P. G.; Ridley, R. G.; Ward, S. A. Central role of
hemoglobin degradation in mechanisms of action of 4-aminoquinolines,
quinoline methanols, and phenanthrene methanols. Antimicrob. Agents
Chemother. 1998, 42, 2973–2977.
(8) Bray, P. G.; Mungthin, M.; Ridley, R. G.; Ward, S. A. Access to
hematin: the basis of chloroquine resistance. Mol. Pharmacol. 1998,
54, 170–179.
(9) O’Neill, P. M.; Bray, P. G.; Hawley, S. R.; Ward, S. A.; Park, B. K.
4-Aminoquinolines. Past, present, and future: a chemical perspective.
Pharmacol. Ther. 1998, 77, 29–58.
(10) Hawley, S. R.; Bray, P. G.; Mungthin, M.; Atkinson, J. D.; O’Neill,
P. M.; Ward, S. A. Relationship between antimalarial drug activity,
accumulation, and inhibition of heme polymerization in Plasmodium
falciparum in vitro. Antimicrob. Agents Chemother. 1998, 42, 682–
686.
(11) Bray, P. G.; Martin, R. E.; Tilley, L.; Ward, S. A.; Kirk, K.; Fidock,
D. A. Defining the role of PfCRT in Plasmodium falciparum
chloroquine resistance. Mol. Microbiol. 2005, 56, 323–333.
(12) Hawley, S. R.; Bray, P. G.; Park, B. K.; Ward, S. A. Amodiaquine
accumulation in Plasmodium falciparum as a possible explanation for
its superior antimalarial activity over chloroquine. Mol. Biochem.
Parasitol. 1996, 80, 15–25.
(13) (a) Neftel, K. A.; Woodtly, W.; Schmid, M.; Frick, P. G.; Fehr, J.
Amodiaquine induced agranulocytosis and liver-damage. Br. Med. J.
1986, 292, 721–723. (b) Although there are toxicity concerns with
amodiaquine, a new combination of artesunate and amodiaquine
(ASAQ) has been launched and amodiaquine continues to be used
for uncomplicated malaria in many African countried in combination
with sulfadoxine/pyrimethamine.
(14) Schulthess, H. K.; von Felten, A.; Gmur, J.; Neftel, K. Amodiaquine-
induced agranulocytosis during suppressive treatment of malarias
demonstration of an amodiaquine-dependent granulocytotoxic anti-
body. Schweiz. Med. Wochenschr. 1983, 113, 1912–1913.
(15) Maggs, J. L.; Tingle, M. D.; Kitteringham, N. R.; Park, B. K. Drug-
protein conjugates. 14. Mechanisms of formation of protein-arylating
intermediates from amodiaquine, a myelotoxin and hepatotoxin in man.
Biochem. Pharmacol. 1988, 37, 303–311.
Aliquots of urine (30 µL) were collected and mixed with
scintillant for determination of radioactivity. The collected feces
were weighed and dissolved in distilled water for a period of 16 h
before determination of radioactivity). The major organs were then
removed and assessed for radioactivity.
In Vivo Retention Studies of [3H]2k and [3H]3k in the Rat
(168-240 h). Adult male Wistar rats (n ) 4) were administered a
solution of [3H]2k or [3H]3k (54 µmol/kg, 20 µCi/rat) dissolved in
saline (0.5-1 mL, ip). The animals were kept in a normal animal
cage for a period of 168 or 240 h with full access to food and
drink before administration of a lethal dose of pentabarbitone. The
major organs were then removed and assessed for radioactivity.
Tissue Distribution of [3H]2k and [3H]3k in the Rat. At the end
of the relevant time point (5, 24, 48, 168, or 240 h) the animals
were killed by a lethal dose of pentabarbitone and the major organs
were excised (brain, eyes, heart, lungs, liver, kidneys, spleen, testes,
and skin) and stored at -30 °C. Duplicate portions (50-100 mg)
of the tissues and feces were weighed and solubilized in tissue
solubilizer (0.75 mL) at 50 °C for 16 h. The solutions were
decolorized with hydrogen peroxide (200 µL) over 1 h and then
neutralized using glacial acetic acid (30 µL) before leaving in the
darkness for 16 h to prevent chemoluminescence. Ultima Gold
scintillant (10 mL) was added before the radioactivity was
determined by scintillation counting.
Cytotoxicity of 2k and 3f in Isolated Rat Hepatocytes. Male
Wistar rats (200-250 g) (n ) 3) were terminally anesthetised with
pentobarbitone sodium (90 mg/kg in isotonic saline ip). Hepatocytes
were isolated using a two-step collagenase perfusion method
adapted from that of Guguen-Guillouzo.55 The abdomen was opened
and the liver cannulated through the portal vein using a gauge
catheter. Before perfusion, the heart was removed to allow free
loss of perfusion buffers. The liver was washed with calcium-free
HEPES buffer for 10 min at a flow rate of 40 mL/min. The liver
was then perfused with digestion buffer, a mixture of wash buffer
with 5% CaCl2 solution, for 6-10 min. The digested tissue was
then excised and placed in a Petri dish containing wash buffer and
DNAase (0.1% w/v). The tissue was anchored, and the Glison’s
capsule was disrupted and disturbed to release the cells. The cell
suspension was filtered through gauze to eliminate cell debris, blood,
and sinusoidal cells. The cells were washed three times in DNAase
wash buffer before resuspending in incubation buffer (HEPES buffer
and MgSO4 ·7H20 (0.25% w/v)). Cell viability was then assessed
microscopically using the trypan blue exclusion test. The cells were
then incubated with various concentrations of the relevant com-
pound for a period of 6 h before determining cell viability in the
presence of test compound using the trypan blue exclusion test and
MTS assay.
(16) Maggs, J. L.; Park, B. K. Cyto-toxicity of the quinoneimine of
Statistical Analysis. All results are expressed as the mean (
standard error of the mean (SEM). Values to be compared were
analyzed for non-normality using the Shapiro-Wilk test. Student
t tests were used when normality was indicated. A Mann-Whitney
U test was used for nonparametric data. For analysis of sets of
data with variance, a one-way analysis of variance (ANOVA) test
was used for parametric data and a Kruskell-Wallis test was used
for nonparametric data. All calculations were performed using
StatsDirect statistical software, and results were considered to be
significant when P < 0.05.
amodiaquine. Br. J. Clin. Pharmacol. 1988, 25, P143.
(17) Maggs, J. L.; Colbert, J.; Winstanley, P. A.; Orme, M. L.; Park, B. K.
Irreversible binding of amodiaquine to human-liver microsomes:
chemical and metabolic factors. Br. J. Clin. Pharmacol. 1987, 23,
P649.
(18) Harrison, A. C.; Kitteringham, N. R.; Clarke, J. B.; Park, B. K. The
mechanism of bioactivation and antigen formation of amodiaquine in
the rat. Biochem. Pharmacol. 1992, 43, 1421–1430.
(19) Rouveix, B.; Coulombel, L.; Aymard, J. P.; Chau, F.; Abel, L.
Amodiaquine-induced immune agranulocytosis. Br. J. Clin. Haematol.
1989, 71, 7–11.

1844 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
O’Neill et al.
(20) Clarke, J. B.; Neftel, K.; Kitteringham, N. R.; Park, B. K. Detection
of antidrug IgG antibodies in patients with adverse drug-reactions to
amodiaquine. Int. Arch. Allergy Appl. Immunol. 1991, 95, 369–375.
(21) O’Neill, P. M.; Harrison, A. C.; Storr, R. C.; Hawley, S. R.; Ward,
S. A.; Park, B. K. The effect of fluorine substitution on the metabolism
and antimalarial activity of amodiaquine. J. Med. Chem. 1994, 37,
1362–1370.
(22) O’Neill, P. M.; Mukhtar, A.; Stocks, P. A.; Randle, L. E.; Hindley,
S.; Ward, S. A.; Storr, R. C.; Bickley, J. F.; O’Neil, I. A.; Maggs,
J. L.; Hughes, R. H.; Winstanley, P. A.; Bray, P. G.; Park, B. K.
Isoquine and related amodiaquine analogues: a new generation of
improved 4-aminoquinoline antimalarials. J. Med. Chem. 2003, 46,
4933–4945.
(23) Lawrence, R. M.; Dennis, K. C.; O’Neill, P. M.; Hahn, D. U.; Roeder,
M.; Struppe, C. Development of a scalable synthetic route to
GSK369796 (N-tert-butyl isoquine), a novel 4-aminoquinoline anti-
malarial drug. Org. Process Res. DeV. 2008, 12, 294–297.
(24) Smilkstein, M.; Sriwilaijaroen, N.; Kelly, J. X.; Wilairat, P.; Riscoe,
M. Simple and inexpensive fluorescence-based technique for high-
throughput antimalarial drug screening. Antimicrob. Agents Chemother.
2004, 48, 1803–1806.
(25) Trager, W.; Jenson, J. B. Cultivation of malarial parasites. Nature 1978,
273, 621–622.
(26) Stocks, P. A.; Raynes, K. J.; Bray, P. G.; Park, B. K.; O’Neill, P. M.;
Ward, S. A. Novel short chain chloroquine analogues retain activity
against chloroquine resistant K1 Plasmodium falciparum. J. Med.
Chem. 2002, 45, 4975–4983.
(27) Gemma, S.; Campiani, G.; Butini, S.; Kukreja, G.; Coccone, S. S.;
Joshi, B. P.; Persico, M.; Nacci, V.; Fiorini, I.; Novellino, E.;
Fattorusso, E.; Taglialatela-Scafati, O.; Savini, L.; Taramelli, D.;
Basilico, N.; Parapini, S.; Morace, G.; Yardley, V.; Croft, S.; Coletta,
M.; Marini, S.; Fattorusso, C. Clotrimazole scaffold as an innovative
pharmacophore towards potent antimalarial agents: design, synthesis,
and biological and structure-activity relationship studies. J. Med.
Chem. 2008, 51, 1278–1294.
(38) Oda, Y.; Funasaka, K.; Kitano, M.; Nakama, A.; Yoshikura, T. Use
of a high-throughput umu-microplate test system for rapid detection
of genotoxicity produced by mutagenic carcinogens and airborne
particulate matter. EnViron. Mol. Mutagen. 2004, 43, 10–19.
(39) Davis, T. M. E.; Hung, T. Y.; Sim, I. K.; Karunajeewa, H. A.; Ilett,
K. F. Piperaquine: a resurgent antimalarial drug. Drugs 2005, 65, 75–
87.
(40) Hien, T. T.; Dolecek, C.; Mai, P. P.; Dung, N. T.; Truong, N. T.;
Thai, L. H.; An, D. T. H.; Thanh, T. T.; Stepniewska, K.; White, N. J.;
Farrar, J. Dihydroartemisinin-piperaquine against multidrug-resistant
Plasmodium falciparum malaria in Vietnam: randomised clinical trial.
Lancet 2004, 363, 18–22.
(41) Chen, L.; Qu, F.; Zhou, Y. Field observations on the antimalarial
piperaquine. Chin. Med. J. 1982, 95, 281–286.
(42) Price, R. N.; Hasugian, A. R.; Ratcliff, A.; Siswantoro, H.; Purba,
H. L. E.; Kenangalem, E.; Lindegardh, N.; Penttinen, P.; Laihad, F.;
Ebsworth, E. P.; Anstey, N. M.; Tjitra, E. Clinical and pharmacological
determinants of the therapeutic response to dihydroartemisinin-
piperaquine for drug-resistant malaria. Antimicrob. Agents Chemother.
2007, 51, 4090–4097.
(43) Khanh, N. X.; de Vries, P. J.; Ha, L. D.; van Boxtel, C. J.; Koopmans,
R.; Kager, P. A. Declining concentrations of dihydroartemisinin in
plasma during 5-day oral treatment with artesunate for falciparum
malaria. Antimicrob. Agents Chemother. 1999, 43, 690–692.
(44) Delarue, S.; Girault, S.; Maes, L.; Debreu-Fontaine, M. A.; Labaeid,
M.; Grellier, P.; Sergheraert, C. Synthesis and in vitro and in vivo
antimalarial activity of new 4-anilinoquinolines. J. Med. Chem. 2001,
44, 2827–2833.
(45) Miroshnikova, O. V.; Hudson, T. H.; Gerena, L.; Kyle, D. E.; Lin,
A. J. Synthesis and antimalarial activity of new isotebuquine analogues.
J. Med. Chem. 2007, 50, 889–896.
(46) Casagrande, M.; Basilico, N.; Parapini, S.; Romeo, S.; Taramelli, D.;
Sparatore, A. Novel amodiaquine congeners as potent antimalarial
agents. Bioorg. Med. Chem. 2008, 16, 6813–6823.
(47) Mzayek, F.; Deng, H. Y.; Mather, F. J.; Wasilevich, E. C.; Liu, H. Y.;
Hadi, C. M.; Chansolme, D. H.; Murphy, H. A.; Melek, B. H.;
Tenaglia, A. N.; Mushatt, D. M.; Dreisbach, A. W.; Lertora, J. J. L.;
Krogstad, D. J. Randomized dose-ranging controlled trial of AQ-13,
a candidate antimalarial, and chloroquine in healthy volunteers. PloS
Clin. Trials 2007, 2, e6.
(48) Dive, D. Ferrocene conjugates of chloroquine and other antimalarials:
the development of ferroquine, a new antimalarial. ChemMedChem
2008, 3, 383–391.
(28) Peters, W.; Robinson, B. L. Handbook of Animal Models of Infection;
Academic Press: San Diego, CA, 1999; pp 757-773.
(29) Vippagunta, S. R.; Dorn, A.; Ridley, R. G.; Vennerstrom, J. L.
Characterization of chloroquine-hematin mu-oxo dimer binding by
isothermal titration calorimetry. Biochim. Biophys. Acta 2000, 1475,
133–140.
(49) Warhurst, D. C.; Craig, J. C.; Adagu, P. S.; Guy, R. K.; Madrid, P. B.;
Fivelman, Q. L. Activity of piperaquine and other 4-aminoquinoline
antiplasmodial drugs against chloroquine-sensitive and resistant blood-
stages of Plasmodium falciparum: role of beta-haematin inhibition and
drug concentration in vacuolar water- and lipid-phases. Biochem.
Pharmacol. 2007, 73, 1910–1926.
(50) Kuhn, Y.; Rohrbach, P.; Lanzer, M. Quantitative pH measurements
in Plasmodium falciparum-infected erythrocytes using pHluorin. Cell.
Microbiol. 2007, 9, 1004–1013.
(51) Pisciotta, J. Neutral lipid microspheres in Plasmodium falciparum
digestive vacuoles mediate heme crystallization. Am. J. Trop. Med.
Hyg. 2005, 73, 188.
(52) Pisciotta, J. M.; Coppens, I.; Tripathi, A. K.; Scholl, P. F.; Shuman,
J.; Bajad, S.; Shulaev, V.; Sullivan, D. J., Jr. The role of neutral lipid
nanospheres in Plasmodium falciparum haem crystallization. Biochem.
J. 2007, 402, 197–204.
(53) Tetko, I. V.; Gasteiger, J.; Todeschini, R.; Mauri, A.; Livingstone,
D.; Ertl, P.; Palyulin, V. A.; Radchenko, E. V.; Zefirov, N. S.;
Makarenko, A. S.; Tanchuk, V. Y.; Prokopenko, V. V. Virtual
computational chemistry laboratory: design and description. J. Com-
put.-Aided Mol. Des. 2005, 19, 453–463.
(54) Wennerholm, A.; Nordmark, A.; Pihlsgard, M.; Mahindi, M.; Bertils-
son, L.; Gustafsson, L. L. Amodiaquine, its desethylated metabolite,
or both, inhibit the metabolism of debrisoquine (CYP2D6) and losartan
(CYP2C9) in vivo. Eur. J. Clin. Pharmacol. 2006, 62, 539–546.
(55) Guguen-Guillouzo, C.; Guillouso, A. Methods for Preparation of Adult
and Fetal Hepatocytes. In Research in Isolated and Cultured Hepa-
tocytes; Guillouzo, A., Guguen-Guillouzo, C., Eds.; John Libbey
Eurotext/INSERM: London/Paris, 1986; pp 1-12.
(30) Dorn, A.; Vippagunta, S. R.; Matile, H.; Jaquet, C.; Vennerstrom, J. L.;
Ridley, R. G. An assessment of drug-haematin binding as a
mechanism for inhibition of haematin polymerisation by quinoline
antimalarials. Biochem. Pharmacol. 1998, 55, 727–736.
(31) Vippagunta, S. R.; Dorn, A.; Matile, H.; Bhattacharjee, A. K.; Karle,
J. M.; Ellis, W. Y.; Ridley, R. G.; Vennerstrom, J. L. Structural
specificity of chloroquine-hematin binding related to inhibition of
hematin polymerization and parasite growth. J. Med. Chem. 1999, 42,
4630–4639.
(32) Egan, T. J. Haemozoin formation. Mol. Biochem. Parasitol. 2008, 157,
127–136.
(33) Egan, T. J. Recent advances in understanding the mechanism of
hemozoin (malaria pigment) formation. J. Inorg. Biochem. 2008, 102,
1288–1299.
(34) Egan, T. J.; Ncokazi, K. K. Effects of solvent composition and ionic
strength on the interaction of quinoline antimalarials with ferriproto-
porphyrin IX. J. Inorg. Biochem. 2004, 98, 144–152.
(35) Egan, T. J.; Mavuso, W. W.; Ross, D. C.; Marques, H. M.
Thermodynamic factors controlling the interaction of quinoline anti-
malarial drugs with ferriprotoporphyrin IX. J. Inorg. Biochem. 1997,
68, 137–145.
(36) Casabianca, L. B.; An, D.; Natarajan, J. K.; Alumasa, J. N.; Roepe,
P. D.; Wolf, C.; de Dios, A. C. Quinine and chloroquine differentially
perturb heme monomer-dimer equilibrium. Inorg. Chem. 2008, 47,
6077–6081.
(37) Parapini, S.; Basilico, N.; Pasini, E.; Egan, T. J.; Olliaro, P.; Taramelli,
D.; Monti, D. Standardization of the physicochemical parameters to
assess in vitro the beta-hematin inhibitory activity of antimalarial drugs.
Exp. Parasitol. 2000, 96, 249–256.
JM8012757