Reversal agent and linker variants of reversed chloroquines: Activities against Plasmodium falciparum

Article abstract of DOI:10.1021/jm900972u

We have shown that "reversed chloroquine molecules" constructed from chloroquine-like and resistance "reversal-agent"-like cores can be powerful drugs against malaria (J. Med. Chem. 2006, 49, 5623-5625). Several reversed chloroquines are now presented that probe parameters governing the activities against chloroquine-resistant and chloroquine-sensitive malaria strains. The design is tolerant to linker and reversal agent changes, but a piperazinyl group adjacent to the quinoline, at least for the group of compounds studied here, may be detrimental. 2009 American Chemical Society.

Full text of DOI:10.1021/jm900972u

916 J. Med. Chem. 2010, 53, 916–919
DOI: 10.1021/jm900972u
Reversal Agent and Linker Variants of Reversed Chloroquines: Activities against
Plasmodium falciparum
Simeon Andrews,† Steven J. Burgess,†,‡ Deborah Skaalrud,† Jane Xu Kelly,§,†,‡ and David H. Peyton*,†
†Department of Chemistry, Portland State University, P.O. Box 751, Portland, Oregon 97207-0751, ‡DesignMedix, Inc.,
2828 SW Corbett Avenue, Portland, Oregon, 97201, and Portland Veterans Affairs Medical Center, Portland, Oregon, 97239
§
Received July 1, 2009
We have shown that “reversed chloroquine molecules” constructed from chloroquine-like and
resistance “reversal-agent”-like cores can be powerful drugs against malaria (J. Med. Chem. 2006, 49,
5623-5625). Several reversed chloroquines are now presented that probe parameters governing the
activities against chloroquine-resistant and chloroquine-sensitive malaria strains. The design is tolerant
to linker and reversal agent changes, but a piperazinyl group adjacent to the quinoline, at least for the
group of compounds studied here, may be detrimental.
Introduction
Various molecules, termed reversal agents (RA), have been
identified that inhibit this CQ export from the DV in CQR
parasites.10-13 One RA pharmacophore maybedescribed as a
pair of aromatic rings, often with an aliphatic nitrogen atom a
few angstroms removed from the aromatic rings.14
In terms of human suffering, malaria is clearly the most
important parasitic disease. Furthermore, the worldwide
burden of malaria is increasing in part because of the spread
of resistance to most of the drugs that were once effective,
inexpensive, and safe.1 Among these drugs, chloroquine
(CQa) had been the prime therapy for nearly half a century.
CQ was safe, effective, remarkably inexpensive, and could be
administered to pregnant women and infants. Unfortunately,
P. falciparum, the cause of the most deadly malaria, is now
CQ-resistant (CQR) in most endemic regions. The continuing
spread of CQR, as well as resistance to alternative drugs, has
helped fuel a strong increase in incidence and consequence of
malaria worldwide.1
In considering new antimalarial drug candidates, it seemed
that CQ’s safety and economic advantages are simply too
strong to abandon. Others have sought CQ modifications to
combat drug resistance.2,3 We began a program to use CQ’s
quinoline core but linked to entities that are known to over-
come CQR, postulating that the resulting hybrid molecules
might give enough physicochemical flexibility to allow such
hybrids to be tailored to produce compounds that retain the
beneficial qualities of CQ and that can be combined with a
wide range of other drugs, in current use or in development,
for combination therapies. This is an important point because
it has become generally accepted that combination therapy
should be used to delay the emergence of resistance to new
antimalarial agents.1,4,5
In an earlier publication,15 we showed that it is possible to
synthesize a molecule that conceptually is a 7-chloro-4-alkyl-
aminoquinoline linked to a reversal agent (RA) via an alkyl
group. The RA was envisioned as inhibiting the P. falciparum
chloroquine resistance transporter (PfCRT) associated CQ
export from the DV in CQR parasites.10-13 If such an effect
were “perfect” (i.e., no drug export and no other resistance
mechanism), then there should be equal efficacy against CQS
and CQR strains. Such a construct would deliver the RA in a
1:1 ratio with the quinoline, lowering the RA dose required if
the two were given separately. We termed this conjugate drug
a “reversed chloroquine” (RCQ) compound and showed that
our first prototype, 1, has low nanomolar IC50 against CQS
(e.g., D6) and CQR (e.g., Dd2) strains ofP. falciparum malaria
in red cell culture. Further, 1 was able to clear parasitemia
from a mouse model to <1% via oral dosing. Encouraging as
all this was, 1 is quite lipophilic (ClogP ≈ 8.9), and there had
been no intentional effort to optimize it against P. falciparum.
We therefore undertook to modify the RCQ structure in an
effort to delineate the factors that govern efficacy against
P. falciparum. Others have also taken up the RCQ approach.19
Herein we report initial variations in the RCQ structure,
beginning with the linker and aromatic groups of the RA
moiety. The RCQ features evaluated include bridging bet-
ween the two RA aromatic rings (conversion of the diphenyl
to dibenzyl) in addition to some evaluation of the linker length
and flexibility between the RA end and the quinoline (Chart 1).
CQ resistance in P. falciparum malaria is strongly asso-
ciated with mutations in a parasite digestive vacuole (DV)
membrane protein, PfCRT. These mutations have been found
to be correlated with enhanced CQ export from the DV.6-9
Results and Discussion
A set of molecules, shown in Table 1, was synthesized as
outlined in the Supporting Information. This set was chosen
to be a minimal representation of variants including the linker
between the chloroquinoline ring and the RA, as well as vary-
ing the RA aromatics from diphenylamine to dibenzylamine
*To whom correspondence should be addressed. Phone: 503 725-
3875. Fax: 503 725-9525. E-mail: peytond@pdx.edu.
a Abbreviations: CQ, chloroquine; CQR, chloroquine-resistant; CQS,
chloroquine-sensitive; DV, digestive vacuole; PfCRT, P. falciparum
chloroquine resistance transporter; RA, reversal agent; RCQ, reversed
chloroquine.
r
pubs.acs.org/jmc
Published on Web 12/01/2009
2009 American Chemical Society

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 917
Table 1. RCQ Compound Activities against P. falciparum
a Evaluated using ChemDraw software. b Averages of at least three runs ((15%). The uncertainties are estimated on the basis of weighing
uncertainties for the various compounds (which are free bases and often oils) and on variability between determinations that were performed on different
weeks. c Cytotoxicities are against mouse spleen lymphocytes. These values are estimated to be (50% on the basis of weighing uncertainties for the
various compounds (which are free bases and often oils) and of variability between determinations that were performed on different weeks. N.D.: not
determined.
or dibenzylamide, as well as the length of the aliphatic
appendage. Thus, the linker was shortened from the imipra-
mine propyl in 1 to an ethyl group and to a piperazine
(formally two ethyl linkers). In addition, the methyl attached
to the aliphatic N of the RA aliphatic appendage was deleted.
Other changes made to the RA moiety included shortening
and lengthening this appendage by one methylene and con-
verting the amine proximal to the aromatic groups into an
amide. In each case, the ethyl bridge between the aromatics in
1 was removed to give diphenylamino and dibenzylamino

918 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Chart 1. Variations in the RCQ Structurea
Andrews et al.
Figure 1. IC50 values for CQ and 1-13. The gray bars are for the
CQS D6 strain and the black bars are for the CQR Dd2 strain, of
P. falciparum malaria.
a RA portions are shown in bold bonds.
functionalities. Although we did not produce every possible
combination of all these changes, the set was sufficient to
examine effects of these structural changes on the activities
against CQS and CQR P. falciparum malaria.
All of the compounds have significant activity (IC50
e
Figure 2. Piperaquine (1,3-bis[4-(7-chloroquinolyl-4)piperazinyl-1]-
propane).
125 nM; see Table 1 and Figure 1) against D6 (CQS) and
Dd2 (CQR) P. falciparum malaria strains. The CQR strain
generally gives a higher IC50 than the CQS strain but with the
ratio of IC50(CQR)/IC50(CQS) ranging only from about 1 to 3.
In fact, of the 11 compounds presented here, 6 have lower IC50
than does CQ against CQS P. falciparum and all have a lower
ratio of IC50(CQR)/IC50(CQS) than does CQ, by at least a
factor of 5. Although the low IC50 values against CQS and
CQR strains are probably the most important factor, the low
strain sensitivities also contribute to the drug-development
process. We conclude that the RCQ design is more general
than the single molecule, 1, which was presented in our earlier
manuscript on RCQ molecules.15
moieties. The reduced activity of 13 is surprising; the only
change is the conversion of each RA nitrogen proximal to the
aromatic groups into an amide, and this is seen not to be
detrimental in the case of the superior activity of 10. Both 7
and 13 are fairly large and complex molecules and so likely
would not be preferred drugs in the context of the developing
world where they are most needed. Compound 12 has a single
RA headgroup, a much lower ClogP than 13 (6.9 vs 8.2,
respectively), and significantly better IC50, comparable to
those of its reduced analogue 6.
8 and 11 each have a piperazine ringR to the 7-chloroquino-
line ring, as does piperaquine (Figure 2), a drug that has been
However, there are significant differences among the com-
pounds’ effects on the CQS and CQR strains. From a compar-
ison of 4 to 3, it is seen that the dibenzylamino moiety can be
advantageous relative to the diphenylamino group, although
this does not infer that the diphenylamino group is inherently
bad. 10 is an interesting case, demonstrating that changing the
amino R to the RA phenyls to an amide is tolerable, giving
IC50 below those of CQ itself and reducing the ClogP to
almost the same value as CQ. This helps substantially with
water solubility, which is needed to develop orally effective
drug candidates. Also, the diphenyl to dibenzyl amine advan-
tage noted above (e.g., 3 to 4) is not found for the amides,
going from 10 to 12. Also, from a comparison of 2 to 9 or of 4
to 6, the IC50 values change by only a small amount when the
linker is shortened from three to two methylenes, at least if the
RA portion has a dibenzylamino moiety. With the linker fixed
at three methylenes, varying the RA aliphatic length also does
not make a large difference (IC50: 4 < 5 < 2) when the RA
aromatic portion is dibenyzlamino. 7 and 13 were obtained as
sideproducts during thesyntheses of 6 and 10. They constitute
an unusual pair in that they give the lowest and nearly the
highest IC50 values, respectively. It was unsurprising that 7
was so effective, insofar as the PfCRT is presumed to be
unable to export it to a significant extent, having two RA
in use for some time and has been reported to have an IC50
<
10 nM against the D6 strain.16 This appears to be at odds to
the higher IC50 against even D6 presented by 8 and 11. Others
have explored arylpiperazines as an antimalarial scaffold but
focused on CQS/CQR cross-reactivity rather than maximizing
potency.17 However, piperaquine is a “bisquinoline”, and the
presence of two 7-chloroquinoline moieties is perhaps the
major contributor to its stronger efficacy. This may be balan-
ced by the lack of proton on the nitrogen at the quinoline
4-position, as has been pointed out by others.18
Toxicity is an important consideration in any drug devel-
opment program, and so we provide cytotoxicity data in
Table 1. Given the strong potencies of the compounds against
malaria, the cytotoxicities are encouraging, especially for
10-12. In fact, 10 has the combination of high efficacy and
low cytotoxicity for a “therapeutic index” (cytotoxicity/
efficacy) of 12 000 for D6 and of 4800 for Dd2. For compar-
ison, these values are far superior to our calculated “thera-
peutic index” values for CQ: 1700 for D6 and only 120 for
Dd2. Such numbers and the low ClogP(approximating thatof
CQ and indicating relatively high water solubility) suggest
that 10 and 12 could prove to be possible starting points,
leading to further progress in the drug development process.

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 919
In conclusion, linking any of several reversal-agent-like
moieties to a 4-amino-7-chloroquinoline yields good activity
against CQS or CQR P. falciparum malarias so that there is
considerable flexibility available to the drug designer.
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Acknowledgment. The authors thank the following for
supporting this research: the Medical Research Foundation
of Oregon (Grant 0530), the National Institutes of Health
(Grants AI067837 and AI072923) to DHP, and a grant from
the Murdock Charitable Trust for the NMR instruments.
Supporting Information Available: General experimental
methods; 1H and 13C NMR data. This material is available free
of charge via the Internet at http://pubs.acs.org.
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