Quinoline antimalarials containing a dibemethin group are active against chloroquinone-resistant plasmodium falciparum and inhibit chloroquine transport via the P. falciparum chloroquine-resistance transporter (PfCRT)

Article abstract of DOI:10.1021/jm2009698

A series of 4-amino-7-chloroquinolines with dibenzylmethylamine (dibemethin) side chains were shown to inhibit synthetic hemozoin formation. These compounds were equally active against cultures of chloroquine-sensitive (D10) and chloroquine-resistant (K1) Plasmodium falciparum. The most active compound had an IC₅₀ value comparable to that of chloroquine, and its potency was undiminished when tested in three additional chloroquine-resistant strains. The three most active compounds exhibited little or no cytotoxicity in a mammalian cell line. When tested in vivo against mouse malaria via oral administration, two of the dibemethin derivatives reduced parasitemia by over 99%, with mice treated at 100 mg/kg surviving the full length of the experiment. Three of the compounds were also shown to inhibit chloroquine transport via the parasite's chloroquine-resistance transporter (PfCRT) in a Xenopus oocyte expression system. This constitutes the first example of a dual-function antimalarial for which the ability to inhibit both hemozoin formation and PfCRT has been demonstrated directly.

Full text of DOI:10.1021/jm2009698

ARTICLE
pubs.acs.org/jmc
Quinoline Antimalarials Containing a Dibemethin Group Are Active
against Chloroquinone-Resistant Plasmodium falciparum and Inhibit
Chloroquine Transport via the P. falciparum Chloroquine-Resistance
Transporter (PfCRT)
Vincent K. Zishiri,† Mukesh C. Joshi,† Roger Hunter,† Kelly Chibale,†,‡ Peter J. Smith,§ Robert L. Summers,||
Rowena E. Martin,|| and Timothy J. Egan†,*
†Department of Chemistry, University of Cape Town, Private Bag, Rondebosch 7701, South Africa
‡Institute for Infectious Diseases and Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
§Division of Pharmacology, Department of Medicine, University of Cape Town, Observatory 7925, South Africa
Research School of Biology, The Australian National University, Canberra, ACT 0200, Australia
S Supporting Information
b
ABSTRACT: A series of 4-amino-7-chloroquinolines with di-
benzylmethylamine (dibemethin) side chains were shown to
inhibit synthetic hemozoin formation. These compounds were
equally active against cultures of chloroquine-sensitive (D10)
and chloroquine-resistant (K1) Plasmodium falciparum. The
most active compound had an IC50 value comparable to that of
chloroquine, and its potency was undiminished when tested in
three additional chloroquine-resistant strains. The three most
active compounds exhibited little or no cytotoxicity in a mamma-
lian cell line. When tested in vivo against mouse malaria via oral
administration, two of the dibemethin derivatives reduced
parasitemia by over 99%, with mice treated at 100 mg/kg surviving the full length of the experiment. Three of the compounds
were also shown to inhibit chloroquine transport via the parasite's chloroquine-resistance transporter (PfCRT) in a Xenopus oocyte
expression system. This constitutes the first example of a dual-function antimalarial for which the ability to inhibit both hemozoin
formation and PfCRT has been demonstrated directly.
’ INTRODUCTION
identified from a wide range of drug families and include the
calcium channel blocker verapamil, the antidepressant imipra-
mine (1) (Chart 1), and the antihistamine azatadine, as well as
antipsychotics such as chlorpromazine.7 Using the oocyte ex-
pression system, it was shown that CQ transport via PfCRTCQR
is inhibited by the resistance-reversers verapamil and primaquine,
thus providing a mechanistic explanation for the ability of these
compounds to reverse CQ-resistance.3 With the exception of
chlorpheniramine,8,9 however, these compounds have not been
tested clinically because most require unacceptably high con-
centrations to exert significant CQ-resistance reversing activity.
This leaves the development of new drugs as the only viable
alternative. One approach has been to modify a quinoline so as to
avoid cross-resistance. Examples of such re-engineered 4-amino-
7-chloroquinolines include ferroquine and isoquine, the first of
which is currently in development. These do not exhibit cross-
resistance with CQ.10,11
Chloroquine (CQ)-resistance has been a major setback to
malaria control worldwide1 and is primarily due to mutations in
the P. falciparum CQ-resistance transporter (PfCRT). PfCRT is
located in the membrane of the parasite’s digestive vacuole, the
organelle in which CQ exerts its antimalarial effects by interfering
with the formation of hemozoin.2 Expression of PfCRT at the
surface of Xenopus laevis oocytes has allowed the function of the
protein to be studied directly, leading to the demonstration that
the resistance-conferring form of PfCRT (PfCRTCQR) has the
ability to transport CQ out of the digestive vacuole, whereas the
CQ-sensitive form (PfCRTCQS) does not.3
Although parasites have been reported to recover CQ-sensi-
tivity after an extended period in the absence of CQ pressure,4 it
has been shown that this arises from expansion of a CQ-sensitive
subpopulation of parasites rather than back mutation of PfCRT.5
Thus, drug rotation is not a viable option as CQ pressure is likely
to result in rapid re-expansion of CQ-resistant strains.6 Com-
pounds that are able to partially reverse CQ-resistance (known as
chemosensitizing agents or CQ-resistance-reversers) have been
Received: July 20, 2011
Published: August 29, 2011
r
2011 American Chemical Society
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Chart 1. Imipramine (1), a Reversed-CQ (2) Containing a Side-Chain Related to 1, and (3) a Dibemethin Type Side Chaina
aDibemethin resistance-reversing agents were used in this work as side chains for attachment through the primary amine to 4,7-dichloroquinoline (4a ꢀ 15a).
A second strategy has been to generate hybrid compounds that
combine the antiplasmodial activity with CQ-resistance-reversing
ability.12,13 Burgess et al. have described such a reversed-CQ
compound that is active against both CQ-sensitive and CQ-
resistant parasites.12 It couples the hemozoin-inhibiting 4-amino-
7-chloroquinoline pharmacophore of CQ to a resistance-rever-
sing agent, such as 1, via a linker (2) (Chart 1). Similarly, Kelly
et al. have shown that an acridone derivative designed to combine
both heme-binding and resistance-reversing abilities exhibits ad-
ditive activity in combination with CQ against a CQ-sensitive
strain of P. falciparum but synergistic activity against a CQ-resistant
strain.14 Although the reversed-CQs and acridones have been
designed to inhibit PfCRT, this activity has not been demon-
strated directly.
as well as the ability to inhibit PfCRTCQR. Structureꢀactivity
relationships were determined for the series, and in vivo activity
in a mouse model of malaria was assayed. The findings reported
herein further validate the viability of the reversed-CQ strategy.
The compounds reported here are the subject of a recent
patent.17
’ CHEMISTRY
The target molecules (4ꢀ15) (Chart 2) were synthesized in a
single step by reaction of the appropriate dibemethin (4aꢀ15a,
described previously by Zishiri et al.)16 with excess commercially
available 4,7-dichloroquinoline in anhydrous N-methyl-2-pyrro-
lidone and in the presence of potassium carbonate and triethy-
lamine as bases. The reaction was conducted in sealed cycloaddition
tubes under N2 at 90ꢀ130 °C over periods ranging from 16ꢀ48 h.
The products were then extracted from an alkaline aqueous solu-
tion into ethyl acetate and purified by column chromatography to
afford modest yields (between 18 and 37%). All products were
characterized by infrared, 1H and 13C NMR, and mass spectro-
metry. Well-behaved solids were further characterized by melting
point determination and elemental combustion analysis, while
oils and hygroscopic solids were subjected to high-resolution
mass spectrometry to confirm identity and HPLC to confirm purity.
The prototype of this series of compounds (4) was crystallized and
the structure determined by single crystal X-ray diffraction analysis.
The structure is shown in Figure 1, and crystallographic data are
presented in Table 1 (see Supporting Information for further
details).
A recent patent13 has claimed a wide range of reversed-CQs
that incorporate the resistance-reversing pharmacophore features
identified by Bhattacharjee et al.15 This pharmacophore consists
of two suitably arranged aromatic rings, a three or four carbon
chain linked between the rings, and a terminal amino group. The
claim includes a dibemethin compound (3) with this arrange-
ment (Chart 1). In a recent study,16 we have shown that resistance-
reversing agents need not possess this precise structural motif
since a series of aminomethyldibemethins (4aꢀ15a) also show
strong resistance-reversing activity (Chart 1). These compounds
were also shown to interact directly with PfCRTCQR to inhibit
CQ transport in the Xenopus oocyte system.16
Here, we demonstrate that linking these molecules to a 7-chlor-
oquinoline nucleus results in compounds that possess antimalar-
ial activity against both CQ-sensitive and CQ-resistant parasites
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Chart 2. Compounds Synthesized in This Study
Determination of pKa Values and β-Hematin Inhibition
Activity. The pKa values of compounds 4ꢀ15 were determined
by pH titration and are reported in Table 2. As expected, all of the
compounds exhibit two pKa values. The lower one, correspond-
ing to the quinoline heteroaromatic N atom was found to lie
within a narrow range from 7.33 (15) to 7.63 (5). The higher
pKa, corresponding to the basic tertiary amino group in the
dibemethin side chain ranged from 9.6 (7) to 9.9 (6).
The IC50 values for β-hematin (synthetic hemozoin) inhibi-
tion are also presented in Table 2 and were determined using a
pyridine based 96-well plate method that we have previously
reported in full.18 The method relies on the fact that a 5% solution
(v/v) of aqueous pyridine dissolves hematin but not β-hematin at
pH 7.5. The extent of inhibition is then characterized by measuring
the intensity of the monomeric pyridineꢀhematin complex at
405 nm. The IC50 values for β-hematin inhibition varied signifi-
cantly, with the most potent inhibitor (6) producing an IC50 of 0.32
equivalents and the least active (12) a value of 1.44 equivalents.
are comparable between the CQ-sensitive D10 and CQ-resistant
K1 parasites.
The antimalarial properties of the most active of these com-
pounds (6) was tested against a further three strains of CQ-resistant
parasites Dd2, W2, and RSA11. As shown in Table 3, no significant
cross-resistance was observed. The three most potent in vitro
antimalarials (5, 6, and 15) were also tested for mammalian cyto-
toxicity in Chinese hamster ovarian (CHO) cells (Table 4). The
lowest selectivity index (SI = IC50 CHO/IC50 D10) was exhibited
by 5, but this exceeded 1000. No cytotoxicity was observed in 6
and 15 up to the maximum concentration tested (100 μg/mL; SI
exceeds 9000 and 4700, respectively).
The potent in vitro antimalarial properties of 4, 5, and 6, together
with the lack of toxicity of dibemethin derivatives in mammalian
cells and the availability of these materials as well behaved solids,
encouraged the in vivo testing of this initial subseries of com-
pounds in the P. berghei mouse model of malaria (Table 5). The
compounds were administered orally at 100 mg/kg and
30 mg/kg. Compound 4 was administered in four treatments
(on days 0, 1, 2, and 3 postinfection), whereas the activities of com-
pounds 5 and 6 were investigated using abbreviated regimes owing
to the reduced availability of material; compound 5 was adminis-
tered three times (on days 0, 1, and 2), and compound 6 was given
as a single treatment on day 0. All three compounds showed sig-
nificant activity, reducing parasitemia by over 99% after three or
four doses were administered (4 and 5) or by over 97% after one
dose was delivered (6). Compounds 4 and 5 caused all of the
mice treated at 100 mg/kg to survive the full 30 days of the
experiment, and the mice exhibited no evidence of parasitemia on
day 30. Further in vivo studies are being conducted.
To determine whether members of this series of compounds
interact with PfCRTCQR, we tested compounds 4ꢀ6 for the
ability to inhibit the flux of CQ via this protein. These experi-
ments made use of the recently described system for expressing
PfCRT in Xenopus oocytes3 with which direct measurements of
CQ transport via PfCRTCQR and its inhibition by potential
resistance-reversers or reversed-CQ compounds can be made.
The direction of CQ transport in the PfCRT expression system is
from the mildly acidic extracellular medium (pH 6.0) into the
oocyte cytosol (pH 7.2),3 which corresponds to the efflux of CQ
from the acidic digestive vacuole (pH ∼5) into the parasite cytosol
’ BIOLOGICAL TESTING
Compounds 4ꢀ15 were tested in human red blood cells
infected with either CQ-sensitive (D10) or CQ-resistant (K1)
parasites. The in vitro antimalarial activities of the compounds
were measured up to a concentration of 100 ng/mL in the D10-
infected cells and to 10 μg/mL in the K1-infected cells. With these
cutoff concentrations, three compounds (7, 9, and 14) were
inactive against the D10 strain, and one (9) was inactive in the K1
strain. The IC50 values for the dibemethin derivatives ranged
from 27 nM (6) to 178 nM (13) in the D10 strain and from
24 nM (6) to 1178 nM (7) in the K1 strain (Table 2). By contrast,
the IC50 values determined for CQ against these two strains were
23 nM and 144 nM, respectively. Thus, the most active dibe-
methin compound has an IC50 in CQ-resistant K1 parasites,
which is comparable to that of CQ in the CQ-sensitive D10 strain.
Compounds 5 and 15 also exhibited potent in vitro antimalarial
activities against the D10 and K1 strains, with IC50 values
between 37 and 48 nM. The resistance index (RI = IC50 in the
K1 divided by the IC50 in the D10 strain) determined for each
compound ranged from 0.6 (8) to 1.4 (4). This variation is small
and indicates that the antimalarial activities of these compounds
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ARTICLE
Figure 1. ORTEP drawing of the molecular structure of compound 4 with an ellipsoidal model at 50% probability level, showing the atomic
numbering scheme. The molecule exhibits a folded conformation in which the terminal dibemethin phenyl ring wraps back around toward the
quinoline ring. The position of the quinoline N relative to the two aromatic rings of the dibemethin side chain resembles the arrangement of the N
atom relative to the two aromatic rings in the resistance-reversing analogues of 1.15 Pairs of molecules interact via π-stacking of the dibemethin
phenyl rings closest to the quinoline group. An interesting feature of the packing (see Supporting Information) is large solvent accessible voids in
which no electron density peaks could be located. We suspect that these voids contain highly disordered solvent molecules (hexane and/or ethyl
acetate).
Table 1. Crystal Structure Data for Compound 4
Table 2. Properties of Compounds 4ꢀ15a
empirical formula
formula weight
crystal system, space group
a (Å)
C25 H24ClN3
401.92
IC50 D10 IC50 K1
(nM)c
code
R
pKa1 pKa2 BHIA50
(nM)c
RIf
a,b
monoclinic, P21/c
13.1065(2)
15.7570(3)
21.7792(5)
90
4
5
6
7
8
9
VZ1
VZ2
VZ3
0
0
0
7.57 9.85 1.1(2)
140(2)
41(5)b
22(3)b
NDd
122(24)
43(2)b
26(3)b
0.9
1.0
1.2
7.63 9.77 0.69(5)
7.56 9.90 0.32(4)
b (Å)
c (Å)
2VZ1 ꢀ0.15 7.44 9.60 1.4(2)
2VZ2 ꢀ0.15 7.55 9.89 0.66(5)
2VZ3 ꢀ0.15 7.44 9.74 0.34(4)
1128(117)
85(15)
NDe
α (deg)
138(7)
NDd
0.6
β (deg)
90.5650(10)
90
γ (deg)
10 3VZ1 ꢀ0.51 7.47 9.85 0.46(4)
11 3VZ2 ꢀ0.51 7.38 9.67 0.52(7)
12 3VZ3 ꢀ0.51 7.44 9.70 1.44(9)
13 4VZ1 ꢀ0.92 7.40 9.71 0.48(2)
14 4VZ2 ꢀ0.92 7.36 9.67 0.5(3)
15 4VZ3 ꢀ0.92 7.33 9.66 0.4(2)
175
134(13)
120(12)
0.8
1.3
0.7
0.8
V (Å3)
4497.60(15)
8
91
Z
130(14) 88(10)
λ (MoꢀKα) (Å)
F (000)
0.71073
178
NDd
149(3)
281(29)
37(3)
1696
crystal size (mm)
range scanned θ (deg)
range of indices
0.16 ꢁ 0.11 ꢁ 0.09
3.02ꢀ25.37
h: ( 15
48(2)
0.9
CQ
8.4 10.8 1.9(3)18 23(4)b
144(19)b 6.3
a Resonance constant (R)(see Chart 2) of each compound is listed along
with measured acid dissociation constant (pKa) values, BHIA50 value,
and in vitro antimalarial activities (IC50 values) against the D10 and K1
strains of P. falciparum, and RI. b Equivalents relative to hematin with
β-hematin formation mediated by 4.5 M acetate, pH 4.5. c Mean (SEM,
n = 3). d Errors are for duplicate determination except where stated
k: ( 18
l: ( 26
no. reflections collected
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no. unique reflections
8209
S
1.104
otherwise. e Not determined, IC50 > 100 ng/mL. Not determined,
f
IC50 > 10 μg/mL. g RI = IC50 (K1)/IC50 (D10).
R1
wR2
0.0476
0.1061
ΔF excursions/e (Åꢀ3
)
ꢀ0.379, 0.329
the results are presented in Figure 2 and Table 6. All three com-
pounds blocked the PfCRTCQR-mediated transport of CQ.
The ability of the most active dibemethin derivative (6) to
enhance the activity of CQ in drug-resistant parasites was also
(pH 7.3).19 The concentration dependence of inhibition of the
CQ transport was determined for each of these compounds, and
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investigated. Isobolograms were constructed for drug-resistant (Dd2)
and sensitive (D10) strains over a concentration range that was
comparable to the in vitro antimalarial IC50 value for compound 6.
As shown in Figure 3, compound 6 and CQ were additive in CQ-
sensitive parasites but exhibited synergism in the CQ-resistant
strain (see Supporting Information for further details).
from this group by six bonds. This means that the influence of
the terminal functional group on pKa must be a through-space
interaction. The crystal structure of 4 illustrates that these
molecules can adopt a folded structure in which the quinoline
comes into relatively close contact with the terminal phenyl ring
of the side chain. It is possible that electron-withdrawing groups
favor intramolecular interactions between the aromatic rings,
promoting protonation because of the resulting cationꢀπ
interactions. However, without a detailed structural investiga-
tion of these intramolecular interactions, this remains spec-
ulative, and the observed trend in pKa values is difficult to
rationalize. Given the narrow range of values, we have not
attempted to explore this further.
’ DISCUSSION
Dependence of pKa on the Identity of the Terminal Group
on the Side Chain. Although the pKa values for compounds
4ꢀ15 vary over only a small range of about 0.3 log units, a sta-
tistically significant correlation with the physical characteristics of
the functional group attached to the terminal phenyl ring of the
side chain was observed (Figure 4). Unexpectedly, both pKa1 and
pKa2 increase as the functional group becomes less resonance-
releasing (i.e., as the parameter R becomes more positive). One
would expect that an electron-releasing group would strengthen
the NꢀH+ bonds, making deprotonation more difficult and
raising rather than decreasing the pKa. Furthermore, the quino-
line N atom is separated from the functional group on the terminal
phenyl ring by 15 bonds, and the dibemethin N atom is separated
Using eq 1, the predicted accumulation ratio of 4ꢀ15 in the
digestive vacuole of the parasite (VAR) can be calculated.20
ꢀ pHv
ꢀ pHe
þ pKa2 ꢀ 2pHv
þ pKa2 ꢀ 2pHe
a1
a1
a1
½Qꢂv
½Qꢂe
1 þ 10pK
1 þ 10pK
þ 10pK
þ 10pK
VAR ¼
¼
a1
ð1Þ
Here, [Q]v is the concentration of the compound in the digestive
vacuole of the parasite, [Q]e is its concentration in the extra-
cellular medium, pHv is the digestive vacuole pH (taken as 5, the
midpoint between two recent estimates of 4.8 and 5.2),19,21 and
pHe is the pH of the external medium (7.4). Values are given in
Table 7. An accumulation-normalized IC50 for antimalarial activity
can be calculated by multiplying the observed IC50 by the VAR
value for each compound. Since 4-aminoquinolines are believed
to act by inhibiting hemozoin formation in the digestive vacuole,
these numbers may be more relevant than the observed IC50.
They are, therefore, reported in Table 7.
Table 3. In Vitro Antimalarial Activities of Compound 6 and
CQ against the CQ-sensitive D10 Strain and Three CQ-
Resistant Strains of P. falciparuma
IC50 Dd2 (nM) RI IC50 W2 (nM) RI IC50 RSA11(nM) RI
CQ
150 ( 5b
26 ( 1
6.5
1.2
125 ( 33
23 ( 2
5.4
1.0
170 ( 17
38 ( 4
7.4
1.7
6
a Mean ( SEM, n = 4. b Mean ( SEM, n = 3.
Correlations among Dibemethin Structure, Biological
Activity, and β-Hematin Inhibition. In the case of compounds
4ꢀ9, the β-hematin inhibition activity increases markedly in the
order of ortho- to meta- to para- derivatives. However, this pat-
tern is reversed in the series 10ꢀ12, and no significant differ-
ences are seen between compounds 13ꢀ15. Thus, the abilities of
these compounds to inhibit hemozoin formation appears to
depend on both the site of attachment of the aminoquinoline
to the dibemethin side chain and the identity of the group on the
terminal phenyl ring of the dibemethin, and in a manner that is too
complex to determine from the limited number of derivatives used
in this study.
Table 4. In Vitro Cytotoxicity of Selected Compounds in
CHO Cellsa
IC50 D10 (μM) IC50 K1 (μM) RI IC50 CHO (μM) SIb
5
0.055 ( 0.004 0.065 ( 0.013 1.2
0.027 ( 0.002 0.025 ( 0.002 0.9
0.047 ( 0.001 0.036 ( 0.002 0.8
70c
1273
6
>249
>9222
>4787
15
>225
emetine
0.08 ( 0.02
a mean ( SEM, n = 3. b IC50 (CHO)/IC50 (D10). c Close to the
maximum concentration tested, estimated by interpolation of data
points.
Table 5. In Vivo Antimalarial Activity of Selected Compounds in P. berghei-Infected Micea
% parasitized red blood cellsa
mouse survival/days
dose (mg/kg)
av
% of control
% activity
av
4
4 ꢁ 100
4 ꢁ 30
3 ꢁ 100
3 ꢁ 30
1 ꢁ 100
1 ꢁ 30
0.10
0.11
0.09
0.08
0.21
1.72
0.08
0.71
0.10
0.10
0.10
1.89
0.10
0.07
0.08
0.12
0.06
2.88
0.09b
0.30b
0.09b
0.10b
0.12b
2.16c
78c,d
0.12
0.38
0.11
0.13
0.16
2.76
99.9
99.6
99.9
99.9
99.8
97
30e
30e
30e
16
10
7
30e
16
30e
16
10
6
30e
16
30e
18
10
7
30.0e
20.7
30.0e
16.7
10.0
6.7
5
6
control
4f
a The compounds were administered orally. b At day 4, each column represents one of three mice used in the experiment. Parasites could not be
detected by microscopy. d Parasites observed by microscopy. e Average of 66.56, 80.24, 70.68, 89.02, and 85.86%. f Survived for the full length of the study
with no detectable parasitemia at day 30. g The parasitemia of all five control mice was measured at day 4, and they were then sacrificed. Control mice in
this model die between day 6 and day 7.
c
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Figure 2. Concentration-dependent effects of 4, 5, and 6 on the uptake of [3H]CQ into oocytes expressing Dd2 PfCRTCQR (b) or D10 PfCRTCQS
(O). IC50 values derived from these data are shown in Table 3. In all panels, uptake is shown as the mean ( SEM from 4ꢀ5 separate experiments, within
which measurements were made from 10 oocytes per treatment.
Table 6. IC50 Values for the Inhibition of PfCRTCQR
Mediated CQ Transport by 4, 5, and 6a
-
compd
IC50 (μM)
4
5
6
58 ( 6 (n = 4)
36 ( 4 (n = 4)
69 ( 5 (n = 3)
a PfCRTCQR-mediated CQ transport was calculated by subtracting the
uptake measured in oocytes expressing PfCRTCQS from that in oocytes
expressing PfCRTCQR. The data are shown in Figure 2. IC50 values were
derived by least-squares fit of the equation Y = Ymin + [(Ymax ꢀ Ymin)/
(1 + ([inhibitor]/IC50)C] where Y is PfCRTCQR-mediated CQ trans-
port, Ymin and Ymax are the minimum and maximum values of Y, and C is
a constant. All values are the mean ( SEM from 4ꢀ5 separate experi-
ments, within which measurements were made from 10 oocytes per
treatment.
Figure 3. Isobolograms for compound 6 and CQ in the CQ-sensitive
D10 (square symbols) and CQ-resistant Dd2 (circular symbols) strains
of P. falciparum. The straight line represents an additive relationship.
Curves for combinations falling below this line are synergistic, whereas
those located above the line are antagonistic.
IC50 values were not determined for three of the compounds
against the D10 strain of parasite, whereas values were obtained
for 11 of the 12 compounds against the K1 strain. Hence, we used
the K1 IC50 data for analyses of structureꢀactivity relationships.
Plots of log IC50 for in vitro antimalarial activity versus log IC50
for inhibition of β-hematin formation (log BHIA50) indicate
that there was a general decrease in biological activity as the
β-hematin inhibition activity decreases (Figure 5a). However,
the trend was not statistically significant unless compound 12
was omitted. The correlation was only marginally improved if
the antimalarial activity was normalized for accumulation in the
digestive vacuole (Figure 5b) and was again only statistically
significant if compound 12 was omitted. While this may suggest that
either the BHIA50 or the IC50 value for 12 is an outlier, it is more
likely an indication that factors additional to vacuolar accumulation
and hemozoin inhibition play a role in biological activity.
In a previous study of short-chain analogues of CQ, vacuolar
accumulation and β-hematin inhibition alone correlated with
activity.2 In this study, the structure of the side chain was not kept
constant, necessitating the inclusion of a structural descriptor
(the position number two, three, and four for ortho-, meta-, and
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Figure 4. Correlations between pKa values and resonance constants (R) of the group attached to the terminal phenyl ring (see Table 2). (a) Statistically
significant correlation for the quinoline N, pKa1 (r2 = 0.69, P = 0.0008). (b) Correlation for the dibemethin tertiary amino group, pKa2. In this case, the
correlation is weaker but is statistically significant if all of the data points are included (r2 = 0.35, P = 0.042). Omission of a single data point (O) for
compound 7 improves the fit considerably (r2 = 0.65, P = 0.0028).
wild-type protein,24 and this is thought to be because of the ability of
Table 7. Calculated Vacuolar Accumulation Ratios (VARs)
and Accumulation-Normalized IC50 Values (VAR IC50) in
the K1 Strain of P. falciparum
the resistance-reverser to inhibit an essential physiological func-
3
tion fulfilled by PfCRTCQR
.
Cross-Resistance, Cytotoxicity, and Oral Activity. There
was no indication of significant cross-resistance of the most active
compound in this series (6) with CQ in four different strains of
the CQ-resistant parasite (K1, Dd2, W2, and RSA11). The lack
of cross-resistance with CQ could be the result of the resistance-
reversing moiety binding to, and inhibiting, PfCRTCQR. Alter-
natively, the modified quinoline may bind to PfCRTCQR but then
fail to undergo translocation because of interference by the altered
side chain.
VARa
VAR IC50 (mM)
3
4
15029
15843
14892
13173
14753
13620
12318
13181
12608
12031
11601
2.82
1.03
0.37
14.85
1.25
1.83
1.44
1.16
1.93
3.38
0.43
5
6
7
8
10
11
12
13
14
15
The three most active compounds (5, 6, and 15) show little or
no cytotoxicity in a mammalian cell line (CHO cells), whereas com-
pounds 4, 5, and 6 all exhibit oral antimalarial activity in the
P. berghei mouse model. Although none of the three compounds
completely eliminated parasites from the mice after four days,
four doses of compound 4and three doses of 5(both at100 mg/kg)
appear to have cured the mice since all six mice survived the 30-
day duration of the experiment, and no parasites were detected at
the end of the experiment. Lower concentrations (30 mg/kg) or
a single treatment with 6 either at 100 or 30 mg/kg were much
less effective. Lower doses of 4 and 5 reduced the parasite load at
day 4 to an extent similar to that of high doses, but mouse
survival was reduced from 30 days to between 16ꢀ30 days. In this
experiment, compound 4 was comparable to CQ , which resulted
in an average mouse survival of 29.8 days when administered
at 100 mg/kg and 20 days when delivered at 30 mg/kg. These
studies demonstrate that the parent compounds are orally
active despite not being optimized for oral delivery. Preparation
of salts of these compounds and improved formulation would
probably increase in vivo activity considerably. Further pharmaco-
logical and whole organism toxicity studies will be required to
investigate the potential of these compounds for development as
antimalarials.
a According to eq 1, assuming a vacuolar pH of 5.0.
para- positions, respectively) in the correlation analyses. The log
IC50 is significantly correlated with position in the order ortho- >
meta- > para- (Figure 5c). This correlation improved slightly if
the accumulation-normalized IC50 values were used (r2 = 0.55
and P = 0.0087 versus r2 = 0.52 and P = 0.012 without normal-
ization), and it improved further when the analysis was expanded
to include the log BHIA50 values (Figure 5d). However, the
strongest correlation was achieved when vacuolar accumulation
was considered as a third variable in multiple correlation analysis
with raw log IC50 values (Figure 5e).
The factors affecting in vitro antimalarial activities of this series
of compounds are in agreement with our previous study on short
chain CQ analogues2 and with recent studies of reversed-CQ
compounds,22,23 with the additional observation of an indepen-
dent influence of side-chain structure. The origin of this effect is
unknown. It could indicate enhanced uptake of the para-substituted
compounds and/or a decrease in the digestive vacuole concen-
tration of ortho-compounds (perhaps as a result of preferential
binding to constituents of the cell or culture medium). An alter-
native explanation is that the ortho-, meta-, or para-substitution
pattern of the dibemethin derivatives affects their interaction
with PfCRTCQR. CQ resistance-reversers are known to exhibit
greater intrinsic antiplasmodial activity in parasite strains posses-
sing resistant haplotypes of PfCRT than in those containing the
Inhibition of PfCRTCQR. Compounds 4, 5, and 6 are the first
examples of dual action antimalarials, which have been directly
demonstrated to inhibit CQ transport via PfCRTCQR. It should
be noted that the micromolar concentrations of the compounds
used here to inhibit PfCRTCQR are physiologically relevant; the
dibemethin derivatives are expected to exceed these concentra-
tions within the acidic environment of the parasite’s digestive
vacuole as a result of weak-base trapping (Table 7). At present, it
is not clear whether these and other CQ resistance-reversers inhibit
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Figure 5. Correlations of biological activity (IC50) with vacuolar accumulation ratio (VAR), IC50 for BHIA50, and a molecular structure descriptor pos
(ortho-, meta- or para- indicated by 2, 3, or 4, respectively, and referring to compounds 4, 7, 10, and 13; 5, 8, 11, and 14; and 6, 9, 12, and 15,
respectively). (a) Linear correlation between log IC50 and log BHIA50 is not statistically significant (r2 = 0.30, P = 0.083) unless the point for compound
12 is omitted (open circle), which considerably improves the correlation (r2 = 0.50, P = 0.021). (b) Linear correlation between the vacuolar
accumulation-normalized log IC50 (log VAR IC50) and log BHIA50 is also not statistically significant (r2 = 0.33, P = 0.064) unless the point for
3
compound 12 is omitted (open circle), which again considerably improves the correlation (r2 = 0.51, P = 0.012). (c) Statistically significant linear
correlation between log IC50 and a structural descriptor for the ortho-, meta-, and para- substituent pattern on the side-chain was observed (r2 = 0.52, P =
0.012). (d) Multiple linear correlation between the log of the vacuolar accumulation-normalized IC50 (log VAR IC50) and both log BHIA50 and the
3
structural descriptor (pos) conform to the equation log VAR IC50 = 0.84 ꢁ log BHIA50 ꢀ 0.34 ꢁ pos ꢀ 1.65. F = 10.3 > Fcrit = 8.65 at the 99%
3
confidence level. (e) Multiple linear correlation of log IC50 with VAR, log BHIA50, and pos. Here, the data conform to the equation log IC50 = 0.95 ꢁ log
BHIA50 ꢀ 0.35 ꢁ pos ꢀ 3.87 ꢁ log VAR + 10.24. F = 9.70 > 8.45 at the 95% confidence level. Open circles represent the available IC50 values for the D10
strain, which were not used in the correlation.
CQ transport by competing with CQ for translocation via
PfCRTCQR or whether the resistance-reversers bind but fail to
complete the translocation step. Given the lack of CQ cross-
resistance displayed by this series of compounds, if the former
applies, the efficiency of transport of compounds 4, 5, and 6
via PfCRTCQR must be far lower than that of CQ. Regardless
of the mode of inhibition, the fact that these dibemethin
derivatives possess the ability to block PfCRTCQR supports
the hypothesis that reversed-CQ compounds could combat
CQ resistance by inhibiting mutant PfCRT. The observed ability
of compound 6 to enhance CQ activity in a CQ-resistant strain,
and at physiologically relevant concentrations, further supports
this idea.
’ CONCLUSIONS
The series of novel dibemethin-coupled 4-aminoquinolines
described here are structurally simple and achiral. They can be
synthesized in a short four-step sequence from relatively cheap
starting materials. The final step, which couples the dibemethin
side chain to 4-amino-7-chloroquinoline, exhibited relatively low
yields and would need to be optimized if these compounds are to
be developed further. Several of these compounds (5, 6, and 15)
displayed potent in vitro antimalarial activities against both CQ-
sensitive (D10) and CQ-resistant (K1) strains of parasite, with
IC50 values below 100 nM. None of the tested compounds
showed a significant cross-resistance with CQ. The activity of the
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most potent compound (6) in four CQ-resistant strains was
comparable to that of CQ in the CQ-sensitive strain. The three
most active compounds (5, 6, and 15) exhibited little or no
evidence of cytotoxicity, and the three prototype compounds (4,
5, and 6) all possessed significant oral in vivo activity against
mouse malaria.
The activity of this class of compound results from their ability
to accumulate in the parasite digestive vacuole and inhibit hemozoin
formation, a finding which is consistent with previous structureꢀ
activity studies of other CQ analogues. However, a hitherto un-
known structural influence of the side chain on activity was un-
covered and warrants future investigation. These compounds
represent the first examples of reversed-CQ antimalarials that have
been directly demonstrated to inhibit both hemozoin formation and
the CQ-resistance mechanism. Given this dual-functionality, and
their likely increased cost of synthesis, it is conceivable that reversed-
CQ compounds like these could be administered in combination
with CQ. Such a combination would be expected to have an additive
effect in CQS parasites, and a synergistic effect in CQR parasites,
providing a cost-effective treatment for drug-resistant malaria.
using mixtures of ethyl acetate/hexane (30:70) to (80:20) as eluent to
give 4 as a white crystalline solid (0.26 g, 37%): mp (EtOAc/hexane)
101ꢀ103 °C; IR (DMSO) νmax 3583, 3384ꢀ3256, 2595, 2140, 2050,
1975, 1659, 1580; 1H NMR (CDCl3, 300 MHz) δ 8.53 (1H, d, J = 5.1
Hz, H-1), 7.89 (1H, d, J = 2.0 Hz, H-8), 7.49ꢀ7.19 (10H, m, Ar H and
H-5), 6.85 (1H, dd, J = 2.0, 9.0 Hz, H-6), 6.52 (1H, d, J = 5.1 Hz, H-2),
4.43 (2H, d, J = 5.1 Hz, H-10), 3.61 (2H, s, H-17), 3.58 (2H, s, H-19),
2.14 (3H, s, H-18), 1.27 (1H, s, NH); 13C NMR (CDCl3, 75 MHz) δ
152.0 (C-1), 150.2 (Arqu), 149.3 (Arqu), 137.4 (Arqu), 137.2 (Arqu),
137.1 (Arqu), 134.5 (Arqu), 132.0 (C-15), 130.5 (ArCꢀH), 129.9 (C-12),
129.9 (ArCꢀH),128.4 (C-21/25), 128.4 (C-22/24), 127.9 (C-8), 127.6
(ArCꢀH), 124.7 (C-6), 122.2 (C-5), 117.8 (Arqu), 99.0 (C-2), 61.9 (C-17),
61.0 (C-19), 46.6 (C-10), 41.9 (C-18); Anal. (C25H24N3Cl) C, H, N.
N-[{3-(N-Benzyl-N-methylaminomethyl)phenyl}methyl]-
7-chloro-4-quinolinamine (5). To a stirred solution of 5a (0.50 g,
2.08 mmol) in anhydrous N-methyl-2-pyrrolidone (5 mL) under N2
were added triethylamine (1.45 mL, 10.4 mmol), K2CO3 (0.57 g, 4.16
mmol), and 4,7-dichloroquinoline (2.06 g, 10.4 mmol). The mixture was
heated under pressure in a cyclo-addition tube at 130 °C overnight. After
the mixture was allowed to cool to room temperature, it was poured into
saturated brine (20 mL) and extracted with ethyl acetate (3 ꢁ 50 mL).
The organic layer was further washed with saturated brine (5 ꢁ 50 mL)
to ensure the removal of any traces of pyrrolidone. The organic layer was
dried (Na2SO4) and concentrated in vacuo to afford a crude product,
which was purified by column chromatography using mixtures of ethyl
acetate/hexane (50:50) to (90:10) as eluent to give 5 as a white solid
(0.19 g, 23%): mp (EtOAc/hexane) 103ꢀ104 °C; IR (DMSO) νmax
3582, 3395ꢀ3279, 2596, 2143, 2060, 1972, 1660, 1579; 1H NMR
(CDCl3, 300 MHz) δ 8.51 (1H, d, J = 5.1 Hz, H-1), 7.98 (1H, d, J = 2.6
Hz, H-8), 7.69 (1H, d, J = 9.0 Hz, H-5), 7.37 (1H, dd, J = 2.6, 9.0 Hz,
H-6), 7.34ꢀ7.20 (9H, m, ArH), 6.45 (1H, d, J = 5.1 Hz, H-2), 5.43 (1H,
br t, J = 5.1 Hz, NH), 4.51 (2H, d, J = 5.1 Hz, H-10), 3.53 (2H, s, H-17),
3.51 (2H, s, H-19), 2.19 (3H, s, NꢀCH3); 13C NMR (CDCl3, 75 MHz)
δ 152.1 (C-1), 149.5 (Arqu), 149.2 (Arqu), 140.4 (Arqu), 139.1 (Arqu),
137.2 (Arqu), 134.9 (Arqu), 128.9 (ArCꢀH), 128.8 (ArCꢀH), 128.8 (C-21/
25), 128.5 (ArCꢀH), 128.2 (C-22/24), 127.9 (C-8), 126.9 (ArCꢀH),
126.1 (ArCꢀH), 125.4 (C-6), 120.9 (C-5), 117.2 (Arqu), 99.7 (C-2), 61.9
(C-17), 61.6 (C-19), 47.6 (C-10), 42.3 (NꢀCH3). Anal. (C25H24N3Cl)
C, H. N: calcd, 10.45%; found, 9.87%.
N-[{4-(N-Benzyl-N-methylaminomethyl)phenyl}methyl]-
7-chloro-4-quinolinamine (6). To a stirred solution of 6a (0.35 g,
1.46 mmol) in anhydrous N-methyl-2-pyrrolidone (3.5 mL) under N2
were added triethylamine (1.02 mL, 7.25 mmol), K2CO3 (0.57 g, 4.16
mmol), and 4,7-dichloroquinoline (2.06 g, 10.4 mmol). The mixture was
heated under pressure in a cyclo-addition tube at 90 °C for 48 h. After
the mixture was allowed to cool to room temperature, it was poured into
saturated brine (20 mL) and extracted with ethyl acetate (3 ꢁ 50 mL).
The organic layer was further washed with saturated brine (5 ꢁ 50 mL)
to ensure the removal of any traces of pyrrolidone. The organic layer was
dried (Na2SO4) and concentrated in vacuo, and the resulting crude
product was purified by column chromatography using mixtures of ethyl
acetate/hexane (50:50) to (90:10) as eluent to give 6 as a colorless solid
(0.19 g, 32%): mp (EtOAc/hexane) 120ꢀ122 °C; IR (DMSO) νmax
3603, 3394ꢀ3225, 2598, 2349, 2140, 2056, 1969, 1903, 1657; 1H NMR
(CDCl3, 300 MHz) δ 8.53 (1H, d, J = 5.1 Hz, H-1), 7.98 (1H, d, J = 1.9
Hz, H-8), 7.68 (1H, d, J = 8.3 Hz, H-5), 7.41ꢀ7.22 (9H, m, ArH), 7.28
(1H, dd, J = 1.9, 8.3 Hz, H-6), 6.46 (1H, d, J = 5.1 Hz, H-2), 5.29 (1H, br s,
NH), 4.49 (2H, d, J = 5.1 Hz, H-10), 3.53 (2H, s, H-17), 3.51 (2H, s,
H-19), 2.19 (3H, s, H-18); 13C NMR (CDCl3, 75 MHz) δ 152.1 (C-1),
149.5 (Arqu), 149.2 (Arqu), 139.5 (Arqu), 139.3 (Arqu), 135.8 (Arqu),
134.9 (Arqu), 129.5 (C-21/25), 129.0 (C-23), 128.8 (C-22/24), 128.2
(C-13/15), 127.6 (C-12/16), 127.0 (C-8), 125.5 (C-6), 120.9 (C-5),
117.1 (Arqu), 99.6 (C-2), 61.9 (C-17), 61.4 (C-19), 47.4 (C-10), 42.3
(C-18). Anal. (C25H24N3Cl) C, H. N: calcd, 10.45%; found, 10.00%.
’ EXPERIMENTAL SECTION
Chemistry. Solvents, acids, and common salts were obtained from
Sarchem, Krugersdorp, South Africa, and 4,7-dichloroquinoline was
obtained from Sigma-Aldrich, Vorna Valley, South Africa. The synthesis
of the dibemethin side chains has been described elsewhere.16 Precoated
silica gel plates as well as silica and alumina for column chromatography
were obtained from Merck, South Africa. 1H and 13C NMR spectra were
recorded on a Varian Mercury spectrometer at 300 MHz and a Varian
Unity spectrometer at 400 MHz. All spectra were recorded in d3-chloro-
form or d4-methanol. Infrared spectra were recorded on a Perkin-Elmer
Paragon 1000 FT-IR spectrophotometer in the range 3600ꢀ800 cmꢀ1
.
Mass spectra were recorded on a VG Micromass 16F spectrometer
operating at 70 eV with an accelerating voltage of 4 kV and a variable
temperature source. Accurate mass determinations were performed on a
Kratos Limited MS9/50 spectrometer. All mass spectra were obtained
using electron-impact techniques. All compounds for which elemental
analyses could not be obtained as well as compounds 5 and 6 were
subjected to analytical HPLC to confirm purity using a Spectra Series
HPLC with Phenomenex-Luna, 3 m C18 column, and a run time of 13
min (see Supporting Information). Purity exceeded 95% except where
otherwise stated. Extensive attempts were made to purify 7; however,
neither crystallization nor chromatography, including preparative thin layer
chromatography, could improve the purity of this compound above 86%.
Details for the crystal structure determination can be found in the
Supporting Information. CCDC 835831 contains the supplementary
crystallographic data for this paper. These data can be obtained free of
charge from the Cambridge Crystallographic Data Centre via www.ccdc.
cam.ac.uk/data_request/cif.
N-[{2-(N-Benzyl-N-methylaminomethyl)phenyl}methyl]-
7-chloro-4-quinolinamine (4). To a stirred solution of 4a (0.42 g,
1.73 mmol) in anhydrous N-methyl-2-pyrrolidone (2 mL) under N2
were added triethylamine (1.21 mL, 8.67 mmol), K2CO3 (0.48 g, 3.47
mmol), and 4,7-dichloroquinoline (1.72 g, 8.67 mmol), and the mixture
was heated at 90 °C for 48 h. After the mixture was allowed to cool to
room temperature, it was poured into saturated brine (20 mL) and
extracted with ethyl acetate (3 ꢁ 50 mL). The combined organic extracts
were further washed 5 times with saturated brine to ensure the removal
of any traces of pyrrolidone before being dried over anhydrous Na2SO4
and filtered. Following solvent removal, the resulting crude product was
dried under reduced pressure and purified by silica-gel chromatography
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N-[{2-(N-p-Chlorobenzyl-N-methylaminomethyl)phenyl}-
methyl]-7-chloro-4-quinolinamine (7). To a stirred solution of
7a (0.20 g, 0.73 mmol) in anhydrous N-methyl-2-pyrrolidone (2 mL)
under N2 were added triethylamine (0.58 mL, 4.17 mmol), K2CO3 (0.23 g,
1.46 mmol), and 4,7-dichloroquinoline (0.82 g, 4.14 mmol). The mixture
was heated under pressure in a cyclo-addition tube at 120 °C overnight.
After the mixture was allowed to cool to room temperature, it was
poured into saturated brine (20 mL) and extracted with ethyl acetate
(3 ꢁ 50 mL). The organic layer was further washed with saturated brine
(5 ꢁ 50 mL) to ensure the removal of any traces of pyrrolidone. The
organic layer was dried (Na2SO4) and concentrated in vacuo to afford a
crude product, which was purified by column chromatography using
mixtures of ethyl acetate/hexane (50:50) to (80:10) as eluent to give 7 as
a yellow oil (98 mg, 31%): IR (DMSO) νmax (cmꢀ1) 3595, 3378ꢀ3231,
2142, 2067, 1975, 1657; 1H NMR (CDCl3, 300 MHz) δ 8.53 (1H, d,
J = 5.4 Hz, H-1), 7.91 (1H, d, J = 2.4 Hz, H-8), 7.41ꢀ7.12 (8H, m, ArH),
7.26 (1H, d, J = 9.0 Hz, H-5), 6.98 (1H, dd, J = 2.4, 9.0 Hz, H-6), 6.52
(1H, d, J = 5.4 Hz, H-2), 5.29 (1H, s, NH), 4.44 (2H, d, J = 3.0 Hz,
using mixtures of ethyl acetate/hexane (50:50) to (90:10) as eluent to
give 9 as a colorless crystalline solid (0.19 g, 21%): mp (DCM/hexane)
101ꢀ104 °C; IR (DMSO) νmax (cmꢀ1) 3607, 3362ꢀ3195, 2344, 2146,
2059, 1947, 1905, 1677, 1627; 1H NMR (CDCl3, 300 MHz) δ 8.47 (1H,
d, J = 5.4 Hz, H-1), 7.95 (1H, d, J = 1.8 Hz, H-8), 7.37ꢀ7.25 (1H, d,
J = 9.0 Hz, H-5), 7.32 (9H, m, H-6 and ArH), 6.40 (1H, d, J = 5.4 Hz,
H-2), 5.73 (1H, br s, NH), 4.48 (2H, d, J = 3.9 Hz, H-10), 3.50 (2H, s,
H-17), 3.47 (2H, s, H-19), 2.17 (3H, s, H-18); 13C NMR (CDCl3, 75
MHz) δ 151.6 (C-1), 149.8 (Arqu), 149.8 (Arqu), 139.0 (Arqu), 137.8
(Arqu), 135.8 (Arqu), 135.0 (Arqu), 130.1 (C-21/25), 129.4 (C-22/24),
128.4 (Arqu), 128.3 (C-13/15), 127.5 (C-8), 127.5 (C-12/16), 125.4
(C-6), 121.2 (C-5), 117.1 (Arqu), 99.5 (C-2), 61.4 (C-17), 61.0 (C-19),
47.3 (C-10), 42.2 (C-18). Found, 436.1335; C25H23N3Cl2 (M + H)+
requires 436.1342.
7-Chloro-N-[{2-(N-p-methoxybenzyl-N-methylamino-
methyl)phenyl}methyl]-4-quinolinamine (10). To a stirred
solution of 10a (0.35 g, 1.30 mmol) in anhydrous N-methyl-2-pyrroli-
done (3.50 mL) under N2 were added triethylamine (0.91 mL, 6.50
mmol), K2CO3 (0.54 g, 3.90 mmol), and 4,7-dichloroquinoline (2.06 g,
10.4 mmol). The mixture was heated under pressure in a cyclo-addition
tube at 120 °C overnight. After the mixture was allowed to cool to room
temperature, it was poured into saturated brine (20 mL) and extracted
with ethyl acetate (3 ꢁ 50 mL). The organic layer was further washed
with saturated brine (5 ꢁ 50 mL) to ensure the removal of any traces of
pyrrolidone. The organic layer was dried (Na2SO4) and concentrated in
vacuo, and the resulting crude product was purified by column chro-
matography using mixtures of ethyl acetate/hexane (50:50) to (100:0)
as eluent to give 10 as a dark-yellow oil (0.12 g, 21%): IR (DMSO) νmax
(cmꢀ1) 3618, 3376-3177, 2633, 2595, 2548, 2149, 2059, 1972, 1903,
1681; 1H NMR (CDCl3, 400 MHz) δ 8.53 (1H, d, J = 5.4 Hz, H-1), 7.91
(1H, d, J = 2.3 Hz, H-8), 7.62 (1H, s, NH), 7.34 ꢀ 7.31 (4H, m, ArH),
7.27 (1H, d, J = 9.0 Hz, H-5), 7.09 (2H, d, J = 8.9 Hz, H-21/25), 6.92
(1H, dd, J = 2.3, 9.0 Hz, H-6), 6.75 (2H, d, J = 8.9 Hz, H-22/24), 6.53
(1H, d, J = 5.4 Hz, H-2), 4.42 (2H, s, H-10), 3.77 (3H, s, OMe), 3.61
(2H, s, H-17), 3.53 (2H, s, H-19), 2.16 (3H, s, H-18); 13C NMR
(CDCl3, 75 MHz) δ 159.1 (Arqu), 151.7 (C-1), 150.3 (Arqu), 149.0
(Arqu), 137.2 (Arqu), 137.1 (ArCꢀH), 134.6 (Arqu), 132.0 (ArCꢀH), 131.0
(C-21/25), 130.5 (Arqu), 129.3 (Arqu), 128.3 (ArCꢀH), 128.1 (ArCꢀH),
127.9 (C-8), 124.7 (C-6), 122.3 (C-5), 117.8 (Arqu), 113.8 (C-22/24), 99.0
(C-2), 61.5 (C-17), 60.9 (C-19), 55.2 (OMe), 46.5 (C-10), 41.7 (C-18).
HRMS (ESI): found, 432.1860; C26H26N3O Cl (M + H)+ requires
432.1877.
7-Chloro-N-[{3-(N-p-methoxybenzyl-N-methylaminome-
thyl)phenyl}methyl]-4-quinolinamine (11). To a stirred solu-
tion of 11a (0.40 g, 1.48 mmol) in anhydrous N-methyl-2-pyrrolidone
(4.00 mL) under N2 were added triethylamine (1.04 mL, 7.40 mmol),
K2CO3 (0.61 g, 4.44 mmol), and 4,7-dichloroquinoline (1.47 g, 7.40
mmol). The mixture was heated under pressure in a cyclo-addition tube
at 120 °C overnight. After the mixture was allowed to cool to room
temperature, it was poured into saturated brine (20 mL) and extracted
with ethyl acetate (3 ꢁ 50 mL). The organic layer was further washed
with saturated brine (5 ꢁ 50 mL) to ensure the removal of any traces of
pyrrolidone. The organic layer was dried (Na2SO4) and concentrated in
vacuo, and the resulting crude product was purified by column chro-
matography using mixtures of ethyl acetate/hexane (50:50) to (90:10)
as eluent to give 11 as a light-brown oil (0.13 g, 20%): IR (DMSO) νmax
(cmꢀ1) 3600, 3363ꢀ3169, 2599, 2350, 2148, 2059, 1973, 1907, 1679,
1618; 1H NMR (CDCl3, 300 MHz) δ 8.51 (1H, d, J = 5.4 Hz, H-1), 7.98
(1H, d, J = 1.8 Hz, H-8), 7.70 (1H, d, J = 9.0 Hz, H-5), 7.40ꢀ7.24 (4H,
m, ArH), 7.36 (1H, dd, J = 2.1, 9.0 Hz, H-6), 7.21 (2H, d, J = 8.7 Hz,
H-21/25), 6.80 (2H, d, J = 8.7 Hz, H-22/24), 6.45 (1H, d, J = 5.4 Hz,
H-2), 5.44 (1H, br s, NH), 4.51 (2H, d, J = 5.4 Hz, H-10), 3.78 (3H, s,
OMe), 3.50 (2H, s, H-17), 3.45 (2H, s, H-19), 2.17 (3H, s, H-18); 13C
NMR (CDCl3, 75 MHz) δ 158.6 (Arqu), 152.0 (C-1), 149.6 (Arqu),
H-10), 3.60 (2H, s, H-17), 3.54 (2H, s, H-19), 2.16 (3H, s, H-18); 13
C
NMR (CDCl3, 75 MHz) δ 151.9 (C-1), 150.1 (Arqu), 149.1 (Arqu),
137.1 (Arqu), 136.9 (Arqu), 135.9 (Arqu), 134.8 (Arqu), 133.5 (Arqu),
132.0 (ArCꢀH), 131.1 (C-21/25), 130.5 (ArCꢀH), 128.6 (C-22/24),
128.5 (ArCꢀH), 128.4 (ArCꢀH), 128.0 (C-8), 124.9 (C-6), 121.8 (C-5),
118.0 (Arqu), 99.1 (C-2), 61.4 (C-19), 60.8 (C-17), 46.5 (C-10), 42.0
(C-18). HRMS (ESI): found, 436.1344; C25H23N3Cl2 (M + H)+ requires
436.1342. HPLC, 86.1%.
N-[{3-(N-p-Chlorobenzyl-N-methylaminomethyl)phenyl}-
methyl]-7-chloro-4-quinolinamine (8). To a stirred solution of
8a (0.45 g, 1.64 mmol) in anhydrous N-methyl-2-pyrrolidone (4 mL)
under N2 were added triethylamine (1.16 mL, 8.32 mmol), K2CO3 (0.46 g,
3.33 mmol), and 4,7-dichloroquinoline (1.65 g, 8.32 mmol). The mixture
was heated under pressure in a cyclo-addition tube at 120 °C overnight.
After the mixture was allowed to cool to room temperature, it was
poured into saturated brine (20 mL) and extracted with ethyl acetate
(3 ꢁ 50 mL). The organic layer was further washed with saturated brine
(5 ꢁ 50 mL) to ensure the removal of any traces of pyrrolidone. The
organic layer was dried (Na2SO4) and concentrated in vacuo to afford a
crude product, which was purified by column chromatography using
mixtures of ethyl acetate/hexane (50:50) to (90:10) as eluent to give 8 as
a white solid (0.19 g, 27%): mp (DCM/hexane) 103ꢀ104 °C; IR
(DMSO) νmax (cmꢀ1) 3620, 3372ꢀ3193, 2594, 2345, 2150, 2054,
1971, 1911, 1676, 1626; 1H NMR (CDCl3, 300 MHz) δ 8.48 (1H, d,
J = 5.3 Hz, H-1), 7.97 (1H, d, J = 2.4 Hz, H-8), 7.16 (1H, d, J = 8.7 Hz,
H-5), 7.37ꢀ7.23 (9H, m, H-6 and ArH), 6.42 (1H, d, J = 5.3 Hz, H-2),
5.62 (1H, br s, NH), 4.51 (2H, d, J = 4.8 Hz, H-10), 3.50 (2H, s, H-17),
3.45 (2H, s, H-19), 2.15 (3H, s, H-18); 13C NMR (CDCl3, 75 MHz) δ
151.5 (C-1), 149.8 (Arqu), 149.8 (Arqu), 140.1 (Arqu), 137.6 (Arqu),
137.2 (Arqu), 135.1 (Arqu), 132.6 (Arqu), 130.0 (C-21/25), 128.9 (C-8),
128.4 (ArCꢀH), 128.4 (ArCꢀH), 128.3 (C-22/24), 127.8 (ArCꢀH), 126.2
(ArCꢀH), 125.5 (C-6), 121.2 (C-5), 117.1 (Arqu), 99.6 (C-2), 61.6 (C-17),
61.0 (C-19), 47.5 (C-10), 42.2 (C-18). Found, 436.1335; C25H23N3Cl2
(M + H)+ requires 436.1342.
N-[{4-(N-p-Chlorobenzyl-N-methylaminomethyl)phenyl}-
methyl]-7-chloro-4-quinolinamine (9). To a stirred solution of
9a (0.57 g, 2.08 mmol) in anhydrous N-methyl-2-pyrrolidone (6 mL))
under N2 were added triethylamine (1.45 mL, 10.4 mmol), K2CO3 (0.57 g,
4.16 mmol), and 4,7-dichloroquinoline (2.06 g, 10.40 mmol). The mixture
was heated under pressure in a cyclo-addition tube at 120 °C overnight.
After the mixture was allowed to cool to room temperature, it was
poured into saturated brine (20 mL) and extracted with ethyl acetate
(3 ꢁ 50 mL). The organic layer was further washed with saturated
brine (5 ꢁ 50 mL) to ensure the removal of any traces of pyrrolidone.
The organic layer was dried (Na2SO4) and concentrated in vacuo, and
the resulting crude product was purified by column chromatography
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Journal of Medicinal Chemistry
ARTICLE
149.1 (Arqu), 140.4 (Arqu), 137.2 (Arqu), 134.9 (Arqu), 131.0 (Arqu),
130.0 (C-22/24), 128.8 (C-8), 128.7 (ArCꢀH), 128.5 (ArCꢀH), 128.0
(ArCꢀH), 126.1 (ArCꢀH), 125.4 (C-6), 121.1 (C-5), 117.2 (Arqu), 113.6
(C-21/25), 99.6 (C-2), 61.4 (C-17), 61.2 (C-19), 55.2 (OMe), 47.5
(C-10), 42.1 (C-18). HRMS (ESI): found 432.1823; C26H26N3OCl
(M + H)+ requires 432.1877.
(4.00 mL) under N2 were added triethylamine (1.00 mL, 7.19 mmol),
K2CO3 (0.58 g, 4.23 mmol), and 4,7-dichloroquinoline (1.39 g, 7.04
mmol). The mixture was heated under pressure in a cyclo-addition tube
at 120 °C overnight. After the mixture was allowed to cool to room
temperature, it was poured into saturated brine (20 mL) and extracted
with ethyl acetate (3 ꢁ 50 mL). The organic layer was further washed
with saturated brine (5 ꢁ 50 mL) to ensure the removal of any traces of
pyrrolidone. The organic layer was dried (Na2SO4) and concentrated in
vacuo, and the resulting crude product was purified by column chro-
matography using mixtures of hexane/ethyl acetate/methanol (50:50:0)
to (0:90:10) as eluent to give 14 as a dark-yellow oil (0.14 g, 22%): IR
(DMSO) νmax (cmꢀ1) 3363ꢀ3169, 2599, 2350, 2148, 2059, 1973, 1907,
1679, 1618; 1H NMR (CDCl3, 400 MHz) δ 8.47 (1H, d, J = 5.2 Hz, H-1),
7.95 (1H, d, J = 2.0 Hz, H-8), 7.70 (1H, d, J = 8.8 Hz, H-5), 7.38ꢀ7.21
(4H, m, ArH), 7.31 (1H, dd, J = 2.0, 8.8 Hz, H-6), 7.13 (2H, d, J = 8.6 Hz,
H-21/25), 6.61 (2H, d, J = 8.6 Hz, H-22/24), 6.41 (1H, d, J = 5.2 Hz,
H-2), 5.58 (1H, br s, NH), 4.47 (2H, d, J = 4.8 Hz, H-10), 3.47 (2H, s,
7-Chloro-N-[{4-(N-p-Methoxybenzyl-N-methylaminome-
thyl)phenyl}methyl]-4-quinolinamine (12). To a stirred solu-
tion of 12a (0.50 g, 1.85 mmol) in anhydrous N-methyl-2-pyrrolidone
(5.00 mL) under N2 were added triethylamine (1.30 mL, 9.25 mmol),
K2CO3 (0.77 g, 5.55 mmol), and 4,7-dichloroquinoline (1.84 g, 9.25
mmol). The mixture was heated under pressure in a cyclo-addition tube
at 120 °C overnight. After the mixture was allowed to cool to room tem-
perature, it was poured into saturated brine (20 mL) and extracted with
ethyl acetate (3 ꢁ 50 mL). The organic layer was further washed with
saturated brine (5 ꢁ 50 mL) to ensure the removal of any traces of
pyrrolidone. The organic layer was dried (Na2SO4) and concentrated in
vacuo, and the resulting crude product was purified by column chro-
matography using mixtures of ethyl acetate/hexane (50:50) to (100:0)
as eluent to give 12 as a dark-yellow oil (0.19 g, 24%): IR (DMSO) νmax
H-17), 3.40 (2H, s, H-19), 2.88 (6H, s, NMe2), 2.15 (3H, s, H-18); 13
C
NMR (CDCl3, 75 MHz) δ 152.4 (Arqu), 152.0 (C-1), 149.9 (Arqu),
149.6 (Arqu), 140.7 (Arqu), 137.2 (Arqu), 135.0 (Arqu), 130.1 (Arqu),
129.8 (C-21/25), 128.8 (C-8), 128.8 (ArCꢀH), 128.6 (ArCꢀH), 128.1
(ArCꢀH), 126.1 (ArCꢀH), 125.5 (C-6), 121.0 (C-5), 117.2 (Arqu), 112.5
(C-22/24), 99.7 (C-2), 61.3 (C-17), 61.3 (C-19), 47.7 (C-10), 42.2
(C-19), 40.7 (NMe2). HRMS (ESI): found, 445.2138; C27H29N4Cl
(M + H)+ requires 445.2159.
1
(cmꢀ1) 3589, 3365ꢀ3255, 2148, 2059, 1977, 1910, 1661; H NMR
(CDCl3, 400 MHz) δ 8.46 (1H, d, J = 5.7 Hz, H-1), 7.94 (1H, d, J = 2.1 Hz,
H-8), 7.69 (1H, d, J = 9.0 Hz, H-5), 7.38ꢀ7.25 (7H, m, H-6 and ArH),
6.86 (2H, d, J = 8.7 Hz, H-22/24), 6.40 (1H, d, J = 5.7 Hz, H-2), 5.57
(1H, br s, NH), 4.49 (2H, d, J = 4.8 Hz, H-10), 3.76 (3H, s, OMe), 3.47
(2H, s, H-17), 3.44 (2H, s, H-19), 2.15 (3H, s, H-18); 13C NMR
(CDCl3, 75 MHz) δ 158.7 (Arqu), 151.5 (C-1), 149.9 (Arqu), 149.9 or
148.4 (Arqu), 139.4 (Arqu), 135.7 (Arqu), 135.2 (Arqu), 131.1 (Arqu),
130.1 (C-22/24), 129.6 (C-13/15), 128.3 (C-8), 127.6 (C-12/16),
125.6 (C-6), 121.2 (C-5), 117.1 (Arqu), 113.7 (C-21/25), 99.5 (C-2),
61.2 (C-17), 61.2 (C-19), 55.3 (OMe), 47.4 (C-10), 42.1 (C-18).
HRMS (ESI): found, 432.1835; C26H26N3OCl (M + H)+ requires
432.1877.
7-Chloro-N-[{4-(N-p-dimethylaminobenzyl-N-methylamino-
methyl)phenyl}methyl]-4-quinolinamine (15). To a stirred
solution of 15a (0.40 g, 1.41 mmol) in anhydrous N-methyl-2-pyrroli-
done (4.00 mL) under N2 were added triethylamine (1.00 mL, 7.19
mmol), K2CO3 (0.58 g, 4.23 mmol), and 4,7-dichloroquinoline (1.39 g,
7.04 mmol). The mixture was heated under pressure in a cyclo-addition
tube at 120 °C overnight. After the mixture was allowed to cool to room
temperature, it was poured into saturated brine (20 mL) and extracted
with ethyl acetate (3 ꢁ 50 mL). The organic layer was further washed
with saturated brine (5 ꢁ 50 mL) to ensure the removal of any traces of
pyrrolidone. The organic layer was dried (Na2SO4) and concentrated in
vacuo, and the resulting crude product was purified by column chro-
matography using mixtures of hexane/ethyl acetate/methanol (50:50:0)
to (0:90:10) as eluent to give 15 as a dark-yellow oil (0.16 g, 26%): IR
(DMSO) νmax (cmꢀ1) 3616, 3378ꢀ3212, 2596, 2150, 2062, 1977,
1904, 1674, 1635; 1H NMR (CDCl3, 400 MHz) δ 8.43 (1H, d, J = 5.4
Hz, H-1), 7.92 (1H, d, J = 2.0 Hz, H-8), 7.70 (1H, d, J = 9.2 Hz, H-5),
7.34ꢀ7.28 (4H, m, ArH), 7.26 (1H, dd, J = 2.4, 6.4 Hz, H-6), 7.19 (2H,
d, J = 8.4 Hz, H-21/25), 6.68 (2H, d, J = 8.4 Hz, H-22/24), 6.37 (1H, d,
J = 5.4 Hz, H-2), 5.82 (1H, br s, NH), 4.43 (2H, d, J = 4.8 Hz, H-10), 3.46
(2H, s, H-17), 3.42 (2H, s, H-19), 2.89 (6H, s, NMe2), 2.15 (3H, s,
NꢀCH3); 13C NMR (CDCl3, 75 MHz) δ 151.7 (C-1), 149.7 (Arqu),
149.7 (Arqu), 148.8 (Arqu), 139.2 (Arqu), 135.6 (Arqu), 134.8 (Arqu),
129.7 (C-13/15), 129.3 (C-21/25), 128.3 (C-8), 127.3 (C-12/16),
126.7 (C-20), 125.2 (C-6), 121.3 (C-5), 117.1 (Arqu), 112.4 (C-22/24),
99.5 (C-2), 61.2 (C-17 or 19), 61.0 (C-19 or 17), 47.1 (C-10), 42.0
(NꢀCH3), 40.6 (NMe2). HRMS (ESI): found, 445.2171; C27H29N4Cl
(M + H)+ requires 445.2159.
Biological Testing. In Vitro Antimalarial Testing. Continuous in
vitro cultures of asexual erythrocyte stages of P. falciparum were maintained
using a modified method of Trager and Jensen.25 A quantitative assessment
of antimalarial activity in vitro was determined via the parasite lactate
dehydrogenase assay using a modified method described by Makler.26
The samples were prepared to a 2 mg/mL stock solution in 100%
DMSO or 100% methanol and sonicated to enhance solubility. Samples
were tested as a suspension if not completely dissolved. Stock solutions
were stored at ꢀ20 °C. Further dilutions were prepared on the day of the
experiment. CQ was used as the reference drug in all experiments. A full
doseꢀresponse was performed for all compounds to determine the
7-Chloro-N-[{2-(N-p-dimethylaminobenzyl-N-methylamino-
methyl)phenyl}methyl]-4-quinolinamine (13). To a stirred
solution of 13a (0.40 g, 1.41 mmol) in anhydrous N-methyl-2-pyrroli-
done (4.00 mL) under N2 were added triethylamine (1.00 mL, 7.19
mmol), K2CO3 (0.58 g, 4.23 mmol), and 4,7-dichloroquinoline (1.39 g,
7.04 mmol). The mixture was heated under pressure in a cyclo-addition
tube at 120 °C overnight. After the mixture was allowed to cool to room
temperature, it was poured into saturated brine (20 mL) and extracted
with ethyl acetate (3 ꢁ 50 mL). The organic layer was further washed
with saturated brine (5 ꢁ 50 mL) to ensure the removal of any traces of
pyrrolidone. The organic layer was dried (Na2SO4) and concentrated in
vacuo, and the resulting crude product was purified by column chro-
matography using mixtures of hexane/ethyl acetate/methanol (50:50:0)
to (0:90:10) as eluent to give 13 as a dark-yellow oil (112 mg, 18%): IR
(DMSO) νmax (cmꢀ1) 3633, 3360ꢀ3104, 2706, 2641, 2598, 2342,
2147, 2056, 1970, 1901; 1H NMR (CDCl3, 300 MHz) δ 8.54 (1H, d,
J = 5.4 Hz, H-1), 7.89 (1H, d, J = 2.1 Hz, H-8), 7.79 (1H, br s, NH),
7.40ꢀ7.10 (4H, m, ArH), 7.04 (2H, d, J = 8.6 Hz, H-21/25), 6.89 (1H, d,
J = 2.4 Hz, H-5), 6.86 (1H, d, J = 2.1 Hz, H-6), 6.57 (2H, d, J = 8.6 Hz,
H-22/24), 6.54 (1H, d, J = 5.4 Hz, H-2), 4.41 (2H, s, H-10), 3.61 (2H, s,
H-17), 3.50 (2H, s, H-19), 2.92 (6H, s, NMe2), 2.14 (3H, s, H-18); 13
C
NMR (CDCl3, 75 MHz) δ 152.0 (C-1), 150.3 (Arqu), 150.0 (Arqu),
149.2 (Arqu), 137.4 (Arqu), 137.4 (Arqu), 134.4 (Arqu), 132.0 (ArCꢀH),
130.8 (C-21/25), 130.5 (ArCꢀH), 128.2 (C-8), 128.0 (ArCꢀH), 127.8
(ArCꢀH), 124.8 (Arqu), 124.7 (C-6), 122.6 (C-5), 117.9 (Arqu), 112.3 (C-
22/24), 98.8 (C-2), 61.6 (C-19), 61.0 (C-17), 46.5 (C-10), 41.5 (C-18),
40.4 (NMe2). HRMS (ESI): found, 445.2172; C27H29N4Cl (M + H)+
requires 445.2159. HPLC, 93.6%.
7-Chloro-N-[{3-(N-p-dimethylaminobenzyl-N-methylamino-
methyl)phenyl}methyl]-4-quinolinamine (14). To a stirred
solution of 14a (0.40 g, 1.41 mmol) in anhydrous N-methyl-2-pyrrolidone
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Journal of Medicinal Chemistry
ARTICLE
concentration inhibiting 50% of parasite growth (IC50 value). Test
samples were tested at a starting concentration of 100 ng/mL or 10 μg/mL,
which was then serially diluted 2-fold in complete medium to give 10
concentrations. The same dilution technique was used for all samples.
CQ was tested at a starting concentration of 1.15 μg/mL. The highest
concentration of solvent to which the parasites were exposed had no
measurable effect on the parasite viability (data not shown). The IC50
values were obtained using a nonlinear doseꢀresponse curve fitting
analysis via Graph Pad Prism v.3.0 software.27
For the construction of the isobolograms, mixtures consisting of 0, 20,
40, 60, 80, and 100% by mass CQ were prepared with compound 6. The
IC50 values were determined in the D10 and Dd2 strains by the same
method used for pure substances described above. Values were con-
verted from units of ng/mL to nanomolar based on the masses and
corresponding molecular masses of each component in the mixture.
Fractional IC50 values (FICs) were then calculated for CQ and 6 for each
mixture (data are presented in Supporting Information) and used to plot
the isobolograms.
Cytotoxicity Testing. The MTT assay was used as a colorimetric assay
for cellular growth and survival and compares well with other available
assays.28 The tetrazolium salt MTT was used to measure all growth and
chemosensitivity. Compound 4 was tested in triplicate on three separate
occasions.
The compound was dissolved in 10% DMSO. The initial concentra-
tions of stock solutions were 2 mg/mL. The compound was tested as a
suspension and stored at ꢀ20 °C until use. The highest concentration of
solvent to which the cells were exposed had no measurable effect on the
cell viability (data not shown). Emetine was used as the reference drug in
all experiments. The initial concentration of emetine was 100 μg/mL,
which was serially diluted in complete medium with 10-fold dilutions to
give 6 concentrations, the lowest being 0.001 μg/mL. The same dilution
technique was applied to the test sample with an initial concentration of
100 μg/mL to give 5 concentrations, with the lowest concentration
being 0.01 μg/mL.
oocytes was performed as described elsewhere.3 Briefly, oocytes were
injected with cRNA encoding PfCRT (30 ng per oocyte), and the uptake
of [3H]CQ (0.15 μM; 15 Ci/mmol) was measured 4ꢀ6 days postinjec-
tion using a method described previously.30 The influx measurements
were made over 1ꢀ2 h at 27.5 °C and in medium that contained 96 mM
NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 10 mM MES, 10 mM
Tris-base (pH 6.0), and 15 μM unlabeled CQ. Statistical comparisons
were made with the Student’s t-test for paired or unpaired samples or
with ANOVA in conjunction with Tukey’s multiple comparisons test.
Physico-Chemical Measurements. β-Hematin inhibition was mea-
sured using a method described in detail elsewhere without modification.18
’ ASSOCIATED CONTENT
S
Supporting Information. Crystal structure data includ-
b
ing a packing diagram, tables of bond lengths, angles, and torsion
angles for compound 4; details of data used to construct isobolo-
grams; HPLC traces and details. This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: +27-21-650-2528. Fax: +27-21-650-5195. E-mail:
Timothy.Egan@uct.ac.za.
’ DISCLOSURE
Any opinion, findings and conclusions or recommendations ex-
pressed in this material are those of the authors and do not
necessarily reflect the views of the funding agencies involved.
’ ACKNOWLEDGMENT
This work was primarily supported by the South African
Malaria Initiative (SAMI). We also acknowledge the National
Research Foundation (Grant No. 2061833), the Australian National
Health and Medical Research Council (NHMRC) (grant 471472),
the Medical Research Council of South Africa, and the University of
Cape Town for financial support. R.E.M. was supported by an
NHMRC Australian Biomedical Fellowship (fellowship 520320).
The 50% inhibitory concentration (IC50) values for these samples
were obtained from doseꢀresponse curves, using a nonlinear doseꢀ
response curve fitting analysis via GraphPad Prism v.3.0 software.27
In Vivo Antimalarial Testing. Compounds 4, 5, and 6 were sent to the
Parasite Chemotherapy Unit of the Swiss Tropical Institute for testing on
P. berghei infected female NMRI mice using a modified Peters four-day
test.29 Heparinized blood was taken from a donor mouse with approxi-
mately 30% parasitemia and diluted in physiological saline to 108
parasitized erythrocytes per milliliter. Of this suspension, 0.2 mL was
injected intravenously (i.v.) into experimental groups of 3 mice and a
control group of 5 mice. Four hours postinfection the experimental
groups were treated with a single oral dose; the experimental groups
were treated with a further single daily dose of 4 and 5 at 24 and 48 h
postinfection and with a dose of 4 at 72 h postinfection (owing to
problems handling 5 and 6 resulting from the hygroscopic nature of
these compounds, insufficient material was available for a fourth treatment
with 5 and for any additional tests with 6). One microliter of tail blood
was taken 24 h after the last drug treatment, and the parasitemia was
determined with a FACScan. The difference between the mean value of
the control group and those of the experimental groups was calculated
and expressed as a percent relative to the control group (=activity). The
survival of the animals was monitored up to 30 days. Mice surviving for
30 days were checked for parasitemia. A compound was considered
curative if the animal survived to day 30 postinfection with no detectable
parasites. The results were expressed as (1) the reduction of parasitemia
on day 4 in % as compared to the untreated control group and (2) mean
survival time.
’ ABBREVIATIONS USED:
BHIA50, 50% β-hematin inhibitory activity; CHO, Chinese hamster
ovarian; CQ, chloroquine; dibemethin, N,N-dibenzylmethylamine;
PfCRT, Plasmodium falciparum chloroquine-resistance transporter;
PfCRTCQR, chloroquine-resistant form of PfCRT; PfCRTCQS
,
chloroquine-sensitive form of PfCRT; RI, resistance index; SI,
selectivity index; VAR, vacuolar accumulation ratio
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