Synthesis, Structure-activity relationship, and mode-of-action studies of antimalarial reversed chloroquine compounds

Article abstract of DOI:10.1021/jm1006484

We have previously shown that a "reversed chloroquine (RCQ)" molecule, composed of a chloroquine-like moiety and a resistance reversal-like moiety, can overcome chloroquine resistance in P. falciparum (Burgess, S. J.; Selzer, A.; Kelly, J. X.; Smilkstein, M. J.; Riscoe, M. K.; Peyton, D. H. J. Med. Chem. 2006, 49, 5623. Andrews, S.; Burgess, S. J.; Skaalrud, D.; Kelly, J. X.; Peyton, D. H. J. Med. Chem. 2010, 53, 916). Here, we present an investigation into the Structure-activity relationship of the RCQ structures, resulting in an orally active molecule with good in vitro and in vivo antimalarial activity. We also present evidence of the mode of action, indicating that the RCQ molecules inhibit hemozoin formation in the parasite's digestive vacuole in a manner similar to that of chloroquine.

Full text of DOI:10.1021/jm1006484

J. Med. Chem. 2010, 53, 6477–6489 6477
DOI: 10.1021/jm1006484
Synthesis, Structure-Activity Relationship, and Mode-of-Action Studies of Antimalarial Reversed
Chloroquine Compounds
Steven J. Burgess,†,‡ Jane X. Kelly,†,§ Shawheen Shomloo,†,‡ Sergio Wittlin, ,^ Reto Brun, ,^ Katherine Liebmann,† 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, §Portland Veterans Affairs Medical Center, Portland, Oregon 97239, Swiss Tropical and Public Health
Institute, Basel, Switzerland, and ^University of Basel, Basel, Switzerland
Received May 28, 2010
We have previously shown that a “reversed chloroquine (RCQ)” molecule, composed of a chloroquine-
like moiety and a resistance reversal-like moiety, can overcome chloroquine resistance in P. falciparum
(Burgess, S. J.; Selzer, A.; Kelly, J. X.; Smilkstein, M. J.; Riscoe, M. K.; Peyton, D. H. J. Med. Chem.
2006, 49, 5623. Andrews, S.; Burgess, S. J.; Skaalrud, D.; Kelly, J. X.; Peyton, D. H. J. Med. Chem. 2010,
53, 916). Here, we present an investigation into the structure-activity relationship of the RCQ struc-
tures, resulting in an orally active molecule with good in vitro and in vivo antimalarial activity. We also
present evidence of the mode of action, indicating that the RCQ molecules inhibit hemozoin formation
in the parasite’s digestive vacuole in a manner similar to that of chloroquine.
Introduction
(P. falciparum chloroquine resistance transporter), located in
the parasite’s digestive vacuole (DV) membrane.7-10 In chlor-
oquine resistant (CQR) P. falciparum there is reduced accu-
mulation of CQ in the DV due to increased efflux.9,11 PfCRT
is a putative member of the drug/metabolite transporter super-
family, and recent evidence suggests that the mutated forms
are in fact the transporters directly responsible for exporting
CQ from the DV of P. falciparum.12
Malaria remains among the most important diseases, with
nearly half of the world’s population at risk of infection and
∼250 million cases reported annually.1 Malaria kills nearly a
million people each year, 90% of whom are African children.
Of the different Plasmodium parasites known to give human
malaria infections, P. falciparum is the most deadly, although
the others still result in serious illness that can turn fatal.
There are a number of drugs available to treat malaria, yet
P. falciparum has developed resistance to almost all of them.
Even the artemisinin class of drugs is now showing worrying
signs of reduced efficacy,1-6 and so there is a continuing need
for a pipeline of new antimalarial treatments to combat the
disease.
There are molecules such as verapamil and desipramine
that have been found to reverse the effects of CQ resist-
ance.11,13,14 These are known as reversal agents (RAs) or che-
mosensitizers. Many of these compounds are existing drugs,
such as antidepressants or antihistamines, and at the high
doses often required to achieve optimal reversal activity there
can be problems with unpleasant side effects.15 A pharmaco-
phore consisting of two aromatic rings and an aliphatic nitrogen
a few angstroms away has been derived from a set of such RAs.16
Our previous work demonstrated that the hybrid molecule
1 can be synthesized, containing elements from both CQ and
the RA pharmacophore (Figure 1).17 This prototype “re-
versed chloroquine” (RCQ) molecule gave IC50 values low-
er than CQ for both CQR and chloroquine sensitive (CQS)
P. falciparum strains and demonstrated oral efficacy against
P. chabaudiin mice. A subsequent structure-activity relation-
ship (SAR) study demonstrated that the linkage between the
7-chloro-4-aminoquinoline moiety from CQ and the aromatic
rings of the RA headgroup could be varied in length without
serious loss of activity and that the RA portion itself could
be substantially varied without serious loss of activity
against CQR or CQS P. falciparum malaria strains.18
The work presented here is the result of a more extensive
SAR study, with further variations to both the linkage and the
aromatic headgroup of the RA moiety. The result is an orally
efficacious molecule with good in vitro and in vivo antimalar-
ial activity.
Chloroquine (CQa) was first introduced in the 1940s and
quickly became the drug of choice for the treatment of
malaria. CQ has several advantages over other antimalarial
drugs: its low cost made it available to everyone; its low
toxicity meant it was safe for children and pregnant women,
themostvulnerablevictimsof malaria;its good efficacy meant
the treatment regime was simple and easy to administer. How-
ever, resistance developed within about a decade and spread
to such an extent that today the World Health Organization
(WHO) recommends CQ not be used for the treatment of
P. falciparum malaria except in specific areas.1
CQ resistance in P. falciparum is strongly linked to muta-
tions in the gene pfcrt that gives rise to the protein, PfCRT
*To whom correspondence should be addressed. Phone: 503-725-
3875. Fax: 503-725-9525. E-mail: peytond@pdx.edu.
a Abbreviations: CQ, chloroquine; WHO, World Health Organiza-
tion; PfCRT, Plasmodium falciparum chloroquine resistance transpor-
ter; DV, digestive vacuole; CQR, chloroquine resistant; CQS, chloro-
quine sensitive; RA, reversal agent; RCQ, reversed chloroquine com-
pound; SAR, structure-activity relationship; RBC, red blood cell;
PRBC, parasitized red blood cell.
r
2010 American Chemical Society
Published on Web 08/04/2010
pubs.acs.org/jmc

6478 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Burgess et al.
Chemistry
linker analogue of 1, was synthesized by treating 3a with
desipramine hydrochloride. The intermediate compound 14
was made by first treating 2b with methanesulfonyl chloride,
then adding an excess of piperazine (Scheme 1). This was
subsequently treated with trityl chloride to give 15. 16 and 17
were synthesized from diphenylacetaldehyde and 2-adaman-
tanone, respectively, by reductive amination onto 14.19 The
dipyridyl analogues were synthesized by first treating 4-ami-
nopiperidine with 2,20-dipyridyl ketone to give 18 (Scheme 3).
The synthesis of 3b has been previously described,17 and 3a
was similarly synthesized (Scheme 1). These were treated with
1-(diphenylmethyl)piperazine or27-29 (Scheme 2) to give the
RCQ compounds 4-12. RCQ compound 13, the two-carbon
Scheme 2. Synthesis of RA Headgroups 27-29a
Figure 1. Prototype reversed chloroquine molecule 1. The dashed
boxes show the chloroquine and reversal agent (imipramine) por-
tions of the molecule.
a Reagents and conditions: R-Br or R-Cl, homopiperazine, chloro-
form, reflux.
Scheme 1. Synthesis of the Intermediate Molecules 2a,b, 3a,b, and 14 and the Reversed Chloroquine Molecules 4-13 and 15-17a
a Reagents and conditions: (i) 2-aminoethanol or 3-aminopropanol, 140 ꢀC; (ii) methanesulfonyl chloride, Et3N, dichloromethane, 5 ꢀC; (iii) app-
ropriate piperazine or homopiperazine compound, Et3N, 3a or 3b, THF, 70 ꢀC; (iv) (1) desipramine HCl, NaHCO3, dichloromethane; (2) 3a, Et3N,
THF, 60 ꢀC; (v) 2b, Et3N, methanesulfonyl chloride, THF, 0 ꢀC, piperazine, reflux; (vi) trityl chloride, K2CO3, acetonitrile, reflux; (vii) diphenyl-
acetaldehyde, Na(AcO)3BH, acetic acid, THF, room temperature; (viii) 2-adamantanone, Na(AcO)3BH, acetic acid, THF, room temperature.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6479
Scheme 3. Synthesis of the Dipyridyl Compounds 21 and 22a
a Reagents and conditions: (i) 2,20-dipyridylketone, pTSA, toluene, reflux; (ii) 3a or 3b, K2CO3, acetonitrile, 70 ꢀC; (iii) NABH4, methanol, room
temperature.
Scheme 4. Synthesis of the Four-Carbon Analogue 25a
a Reagents and conditions: bromobutylphthalimide, K2CO3, acetonitrile, reflux; (ii) hydrazine hydrate, ethanol, reflux; (iii) 4,7-dichloroquinoline,
Et3N, ethanol, reflux.
keeping with thepublished pharmacophore,16 the1-(diphenyl-
methyl)piperazine retained the two aromatic rings and the
Scheme 5. Synthesis of 26, the RA Headgroup without a
Quinoline Ring Attacheda
protonatable nitrogen. However, the connector was now a
piperazinyl ring which, it was hoped, would make the com-
pounds more stable to metabolic cleavage. A secondary
advantage was that it was easy to design a SAR study around
this structure from inexpensive and readily available starting
materials. Thus compounds 6-12 and 15-17 were also
synthesized, varying the length of the chain from the quinoline
to the piperazine ring, changing the piperazine to a homo-
piperazine, or varying the RA aromatic headgroup. Com-
a Reagents and conditions: K2CO3, KI, DMF, 70 ꢀC.
pound 13 gave the two-carbon linker analogue of 1. As can be
seen in Table 1, the in vitro results from these first compounds
against P. falciparum malaria strains were favorable. While
This was then treated with methane sulfonate esters 3a and 3b
some did not quite match CQ against CQS D6, they all were
to give 19 and 20, respectively, which were reduced in the pre-
more active than CQ against the two tested CQR strains.
sence of sodium borohydride to give 21 and 22. Initial attem-
The general cytotoxic effect was assessed by functional
pts to make 25, the four-carbon linker analogue of 4 and 5, by
assay using mitogen-stimulated murine spleenic lymphocytes.
an analogous route failed because of intramolecular cycliza-
The therapeutic indices for the RCQ compounds were calcu-
tion of the activated alcohol to form a pyrrolidine ring at the
lated as the ratio of cytotoxicity IC50 to antimalarial IC50
4-position of the quinoline. Therefore, a different route was em-
against Dd2 (Table 1).
ployed (Scheme 4). 1-(Diphenylmethyl)piperazine was treated
Although the in vitro data for 1, 4, 5-13, and 15-17 were
with N-(4-bromobutyl)phthalamide to give 23, then depro-
promising, all of these compounds suffer from high ClogP
tected with hydrazine to give 24. This was treated with 4,7-
values. This suggested that they could have limited water solu-
dichloroquinoline to give 25. To make the unattached RA
bility, and so oral availability might be impaired, although
headgroup 26, chlorodiphenylmethane was treated with 1-(2-
formulating as salts and/or cocrystals or other strategies may
hydroxyethyl)piperazine in the presence of potassium carbo-
mitigate this concern. A modification to the aromatic head-
nate (Scheme 5).
group was designed using pyridines in place of the phenyl
groups. Compounds 21 and 22 had ClogP values of 3.3 and
Results and Discussion
3.6, respectively, and IC50 values comparable to the best of the
After the success of the prototype molecule 1 in overcoming
CQ resistance, compounds 4 and 5 were designed to modify
thestructureoftheRAheadgroup slightly, moving awayfrom
the initial, tricyclic antidepressant, imipramine structure. In
previous compounds (Table 1). For comparison, CQ has a
ClogP value of 5.1. The therapeutic index values for these
compounds are substantially greater than 10-fold above that
of CQ. The result of the SAR is a collection of compounds

6480 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Burgess et al.
Table 1. IC50 of RCQ Compounds against Three P. falciparum Strains CQS D6, CQR Dd2, and 7G8, ClogP, Cytotoxicity Data against Mouse Spleen
Lymphocytes, and a Therapeutic Index Calculated from the Cytotoxicity and Dd2 IC50
IC50 (nM) for P. falciparuma
compd
D6
Dd2
102
7G8
106
cytotoxicity (nM)b
therapeutic index (Dd2 strain)c
ClogP d
CQ
1
6.9
2.9
1.0
1.9
4.8
2.6
3.2
6.6
2.0
9.2
21
12000
700
122
132
5.1
8.8
7.2
7.4
7.4
7.8
8.0
8.0
7.6
7.0
7.3
8.5
3.4
8.9
6.4
6.5
3.3
3.6
7.5
3.2
5.3
3.6
2.6
4.1
2.0
3.5
5.7
2.4
9.6
15
4.0
4.3
11
4
1900
1100
700
528
423
5
6
15
10
171
700
7
1400
800
8
12
22
229
158
9
900
10
11
12
13
14
15
16
17
21
22
25
26
34
41
4100
2500
2500
1400
23000
57000
23000
130000
29000
6500
1100
130000
1710
260
56
167
269
3.6
4.3
2.0
0.5
0.8
1.1
0.9
1.3
5.2
6.8
4.2
3.3
1.8
3.9
1.6
1.3
>2500
21
nte
10
3380
13600
6970
69400
7310
4060
846
3.4
10
7.4
1.8
7.2
>2500
>2500
>50
a Averages of at least three runs ((15%). The uncertainties are estimated based on weighing uncertainties for the various compounds (free bases and
often oils) and on variability between determinations that were performed in different weeks. In order to compare results run on different days and with
different batches of each stain, CQ was run as a positive control. All results obtained were “normalized” to the CQ values of 6.9 nM for D6, 102 nM for
Dd2, and 108 nM for 7G8. For example, the normalized IC50 for an RCQ compound tested against a D6 strain is [6.9/IC50(CQ (D6))] ꢀ IC50(RCQ
compound (D6)). b Cytotoxicity is tested against mouse spleen lymphocytes. c Therapeutic index for the Dd2 strain is given by the ratio of the cyctoxicity
divided by the IC50 for Dd2. d ClogP values calculated for the un-ionized forms of the compounds, using ChemDrawUltra 11.0. e nt: not tested.
Table 2. In Vivo Data for Mice Infected with P. berghei, Tested at the
Swiss Tropical and Public Health Institutea
with high potencies against CQS and CQR malaria strains, low
cytotoxicities, and with ClogP values bracketing that of CQ.
Ithaspreviouslybeenshownthatsimply changingthe chain
length of CQ can circumvent the PfCRT-associated CQR
mechanism.20-22 All of the RCQ molecules thus far synthe-
sized had either a two- or three-carbon chain between the
tertiary nitrogen and the aminoquinoline, so 25, which has the
same length linker between the quinoline ring and the alipha-
tic nitrogen as CQ, was made and evaluated by the in vitro
methods applied above. As can be seen in Table 1, the activity
of 25 against both CQS and CQR P. falciparum is better than
that of CQ and comparable to both 4 and 5, the two- and
three-carbon analogues. These results, combined with other
work from our group,18 demonstrate that the ability to over-
come CQR by the addition of a RA headgroup to the
4-aminoquinoline ring is, in fact, independent of the chain
length between them, at least if the chain length is between two
and four carbons.
In Vivo Efficacy against Plasmodium berghei. 1, 4, 15, and
22 were tested in vivo in a P. berghei rodent model. Three sets
of conditions were used: 1 dose of 30 mg/kg administered
orally, 1 dose of 30 mg/kg administered subcutaneously, and
4 ꢀ 30 mg/kg dose delivered orally. The subcutaneous series
provided a convenient alternative to the oral route, in case
absorption across the intestine was problematic. The percent
activity was calculated as the difference between the mean
percent parasitemia for the control and treated groups
expressed as a percent relative to the control group:
drug dosage (mg/kg)
route
% activity mouse survival (days)
99.7
99.5 10
CQ
1
1 ꢀ 30
1 ꢀ 30
4 ꢀ 30
1 ꢀ 30
1 ꢀ 30
4 ꢀ 30
1 ꢀ 30
1 ꢀ 30
4 ꢀ 30
1 ꢀ 30
1 ꢀ 30
4 ꢀ 30
1 ꢀ 30
1 ꢀ 30
4 ꢀ 30
oral
9
subcutaneous
oral
oral
99.9 23 (3/10 mice cured)
mice euthanized day 3
<40
subcutaneous
oral
oral
92
96
7
8
4
>99.9
8
mice euthanized day 3
subcutaneous <40
oral
oral
>99.9 16
99.7
15
22
7
mice euthanized day 3
>99.9 20 (1/3 mice cured)
subcutaneous <40
oral
oral
94
99.3
7
9
subcutaneous
oral
>99.9 27 (2/3 mice cured)
a Historical data for CQ is included for comparison. The no-drug
controls were euthanized on day 4.
neously. 4 and 15 were opposite to this, and 22 was fairly
good both orally and subcutaneously. However, the average
survival time of the mice in all cases was under 10 days, indi-
cating that even if the drug dramatically reduced the para-
sitemia initially, there was recrudescence. In this protocol,
CQ gave similar results. Improvements were seen in some
cases of the 4 ꢀ 30 mg/kg dosage experiment: 1 showed little
difference, 4 showed high activity and about a 10 day in-
crease in survival days compared to control animals, and
both 15 and 22 were encouraging, with 22 resulting in two of
the three treated mice being parasite-free on day 30 post-
infection. The change from phenyls in 4 to pyridyls in 22
seemed to result in a drug that was orally active and showed
no obvious signs of toxicity when administered to mice. 15
was a surprise in that it showed good oral activity but was
% activity ¼ 100 ꢀ ½ðparasitemia control group
- parasitemia treated groupÞ=parasitemia control groupꢁ
The results for 1 ꢀ 30 mg/kg dose experiments were varied
(Table 2). In terms of the activity of the drug, 1 did poorly
when administered orally but better when given subcuta-

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6481
Table 3. Second in Vivo Trial against P. berghei, with Dosage Adjusted
To Be Equimolar to 30 mg/kg CQa
drug dosage (mg/kg) route % activity mouse survival (days)
CQ
21
4 ꢀ 30
4 ꢀ 44
4 ꢀ 46
oral
oral
oral
99.9
>99.9
>99.9
23 (3/10 mice cured)
>30 (3/3 mice cured)
30 (9/10 mice cured)
22
a Historical data for CQ are included for comparison.
poor when administered subcutaneously, even though it had
a ClogP value of 8.9. It is noted that ClogP is a calculated
value that gives only an indication of aqueous solubility but
should not be used on its own as an equivalent to bioavail-
ability.23 Another possible reason for the good in vivo acti-
vity of 15 is that the trityl group was being cleaved from the
rest of the molecule after oral administration. This would
result in the starting material, 14, which has a ClogP value of
3.4 (consistent with good oral activity) and low IC50 values
against P. falciparum strains (Table 1).
Figure 2. Time dependent decline of drug concentration in culture
medium for each drug/P. falciparum strain combination. The graph
shows the first 60 min. The uncertainties are estimated to be (5%
based on variations measured in multiple trials of similar experi-
ments performed under identical experimental conditions.
Because of the difference in molecular weights, a dose of
30 mg/kg of CQ actually resulted in many more molecules
being administered to the mouse than in a dose of 30 mg/kg
of any of the RCQ compounds. Accordingly, a second in
vivo experiment was carried out for 21 and 22, wherein the
dosage was adjusted such that each compound was adminis-
tered in an equimolar amount to 30 mg/kg of CQ. In this test
22 was administered to 10 mice to obtain a more reliable eva-
luation of oral activity in this mammalian model (Table 3).
21 had 3 out of 3 mice cured, and 22 had 9 out of 10, with the
10th mouse reaching 29 days. Parasitemia determination on
day 30 showed that the surviving mice were parasite-free.
Accumulation of 1 in the DV of P. falciparum. When re-
versal agents such as verapamil or imipramine are coadmi-
nistered with CQ, the enhanced CQ activity is limited to CQR
parasites; no effect is seen against CQS P. falciparum.11-13
However, several of the RCQ compounds show IC50 values
lower than CQ against even CQS parasites. One hypothesis
to explain this enhanced potency against CQS parasites is
that the RA headgroup also had antimalarial potency and
that its intrinsic antimalarial effect was enhancing activity of
the total RCQ molecule. To investigate this possibility, com-
pound 26, which lacks the quinoline moiety, was tested
against both CQS and CQR P. falciparum. As can be seen
in Table 1, 26 showed no effect up to 2500 nM, and so the RA
headgroup has no strong intrinsic antimalarial activity.
A second hypothesis to explain the enhanced activity of
the RCQ molecules is that there was increased accumulation
in the DV of the parasite. To test this, an accumulation ex-
periment was devised, similar to one used by Kelly et al. to
show accumulation of xanthones in the DV of Plasmodium
parasites.24 The experimental design provides two results:
first, it allows the amount of drug accumulated in the DV to
be measured; second, by demonstrating that the retention
time is unchanged for the released drug, it shows that the vast
majority of the drug is not modified by the parasite. As
shown in Figure 2, 1 accumulated in the parasitized red
blood cells (PRBCs) in a manner similar to that of CQ
(Figure 2) in that there was a rapid initial uptake over the
first minute followed by a slow uptake until an equilibrium
state was reached by about 60 min. After this time the
accumulated amount remained nearly constant.
Table 4. Uptake, DV Concentration, and Accumulation Ratio for
Drug/P. falciparum Strain Combinationsa
drug/Pf
strain
uptakeb
(mol)
concentration in DV
(mM)
Aaccumulation
ratioc
1/D6
1/Dd2
CQ/D6
1.6 ꢀ 10-8
1.8 ꢀ 10-8
0.7 ꢀ 10-8
65
70
29
20
32000
38000
9000
CQ/Dd2 0.5 ꢀ 10-8
6000
a The concentration in DV is calculated assuming 6 ꢀ 107 PRBCs
present and an average DV volume of 4 fL. The uncertainties are
estimated to be (5%, based on variations measured in multiple trials
of similar experiments performed under identical experimental condi-
tions. b Uptake is the number of moles of drug taken up by the 6 ꢀ 107
PRBCs after 1 h of incubation, calculated by the ratio [(concentration in
medium after NH4Cl addition - concentration in medium after 1 h of
incubation)]/5 mL (the volume of medium used). c Accumulation ratio is
[concentration in DV/concentration in medium].
in a rapid return of each drug into the culture medium (data
not shown). Both 1 and CQ returned to very close to their
respective starting levels, and the HPLC retention times were
not changed, indicating that the chemical composition of
each of the drugs was, in fact, not altered while in the PRBCs
over the course of the experiment.
By taking the difference between the amount of each drug
in the medium after 60 min of incubation and the amount
after the addition of ammonium chloride to the medium, it is
possible to deduce how much of each drug was taken up by
the parasites. To estimate the concentration of the drug in the
DV, it was first considered that each flask contained a 50 μL
pellet of red blood cells (RBCs) and that each RBC has a
volume of 80 fL.25 Therefore, each flask contained 6 ꢀ 108
cells. Given the parasitemia level was about 10%, there were
6 ꢀ 107 PRBCs present. Assuming the DV has an average
volume of 4 fL,25 the total DV volume for the population
of parasites is (6 ꢀ 107) ꢀ (4 ꢀ 10-15) = 2.5 ꢀ 10-7 L. The
concentration of each drug in the DV can then be calculated
(Table 4). This calculation is based on the assumption that
the drug is completely released by the ammonium chloride.
However, a small portion of the drug may still be associated
with heme and thus not be released to the medium.
The accumulation ratio for each drug can be estimated
from these data, with the assumption that the drugs accu-
mulate within the parasite DV; we provide evidence for this,
below. From an initial medium concentration of ∼5 μM, 1
accumulates in the DV of D6 P. falciparum to an equilibrium
concentration of about 65 mM, a 13000-fold increase, yielding
The initial concentration of 1 was slightly higher than CQ
(5.5-4.9 μM), but after 1 h the concentration of 1 in the med-
ium was lower than CQ in both the D6 and Dd2 experiments.
Addition of the ammonium chloride to the PRBCs resulted

6482 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Burgess et al.
Table 5. Apparent Dissociation Equilibrium Constants Obtained from
Optical Titration of Heme with Antimalarial Agents, Assuming a 2:1
Heme to Drug Stoichiometry, and IC50 for the Inhibition of β-Hematin
by RCQ Compounds
drug
Kd [μM (app)]
β-hematin inhibition (μM)
CQ
1
4.0
8.6
3.3
3.0
7.3
5.4
2.5
1.0
1.7
24
9.0
4
2.5
3.5
5
13
15
17
21
22
10
insoluble
14
2.0
Figure 3. Microscope images from showing D6 and Dd2 parasites
in RBCs: (top) D6 parasites clearly showing hemozoin in the control
and CQ samples and showing the enlarged DV with little or no
hemozoin with 1 and 22; (bottom) Dd2 parasites with 22, showing
little or no hemozoin formation.
1.6
an accumulation ratio ([drug in the DV]/[drug in medium at
equilibrium]) of 32 000. Most significantly, the accumulation
ratio of 1 is more than triple that of CQ. This is in agreement
with the IC50 values, in that the values for CQ are well over
twice those of 1, even for CQS D6 P. falciparum (Table 1).
However, the DV is known to swell as it accumulates quino-
line-based antimalarial drugs,26,27 and so the accumulation
ratio presented here may be too large because the calculation
is based on a DV volume of 4 fL. Yet whether the drug accu-
mulation causes or is enabled by the swelling of the DV does
not detract from the fact that the molar uptake of 1 is more
than double that of CQ, irrespective of DV volume. In any
event, these experiments were carried out at significantly
higher drug concentrations than the IC50 determinations
presented in Table 1 and may reflect mechanisms in addition
to simple differences in accumulation and inhibition of hemo-
zoin formation.28,29 In fact, the higher concentrations may re-
present at least a significant portion of the CQ blood concen-
tration time-course during malaria chemotherapy.30,31
In Vitro Heme Binding and β-Hematin Inhibition. It is
known that CQ binds to heme dimers in vitro and also can
inhibit the formation of β-hematin, the in vitro analogue of
hemozoin.32-35 A selection of RCQ compounds was tested
for whether addition of the RA headgroup affected the com-
pounds’ abilities to bind to heme and to inhibit the formation
of β-hematin. As can be seen from Table 5, there was no
significant difference between CQ and the RCQ compounds’
ability to bind heme in solution (Table 5, Kd values). Alth-
ough the numbers range from 1 to 8.3 μM, they are all in the
micromolar range, with CQ about in the middle. A weak,
positive correlation was noted between the RCQ in vitro pot-
ency (Table 1) and its Kd value (R2 ≈ 0.17). 26 was tested and
showed no activity, suggesting that the quinoline portion of
the molecules physically interacted with the heme.
Regarding β-hematin inhibition, the IC50 values ranged
from 24 μM for CQ to about 2 μM for 4, 21, and 22 (Table 5).
While these are all in the micromolar range, there is a trend
toward (R2 ≈ 0.66) enhanced potency, coinciding with lower
IC50 values for the RCQ compounds against P. falciparum
strains (Table 1).
Hemozoin Inhibition in Vivo. The hemozoin inhibition
properties of 1 and 22 in cell culture were examined in a series
of experiments, monitoring in parallel with microscopy (to
characterize morphological change of the parasites) and a
colorimetric assay (to provide a semiquantitative assessment
of hemozoin suppression). The images obtained by micro-
scopy show hemozoin in the control samples, and even in the
100 nM CQ samples, but compound 22, at as low as 10 nM,
appeared to preclude hemozoin formation in both D6 and
Dd2 strains. 1 at 100 nM showed some hemozoin present in
the D6 sample, so inhibition was not complete even at this
concentration in a CQS strain. This was also the case with the
CQR Dd2 stain (not shown).
The images (Figure 3) also show an enlarged DV in the
parasites incubated in the presence of drugs. In the drug-free
control, the DV is fairly small relative to the parasite and is
almost entirely filled by several large crystals of hemozoin. It
is known that the DV swells in the presence of quinoline-
based antimalarial drugs,26,27 and this can clearly be seen
with both 1 and 22 (and to a lesser extent with CQ), where
the almost clear DV is easy to distinguish from the darker
parasite.
For the colorimetric assay, synchronized D6 and Dd2 P.
falciparum parasites were incubated with various concentra-
tions of each drug for 24 h, then lysed by treatment with a
saponin-containing lysis buffer. The hemozoin was extracted
by centrifugation, then washed with acetone and PBS buffer
to remove residual protein. The pellet was then dissolved in
0.2 N sodium hydroxide solution, and the absorbance at 400
nm was measured. By use of an extinction coefficient of 91000
cm-1 M-1, the amount of heme was calculated.36-38 The
amount of heme per parasitized erythrocyte was calculated on
the basis of the number of erythrocytes in the culture and the
percent parasitemia obtained after growing synchronized
culture for 24h. Baseline hemozoin production was calculated
by processing the cell culture at 0 h in an identical way.
The results from the colorimetric assays indicate that the
RCQ molecules did indeed decrease the hemozoin produc-
tion of P. falciparum, in a manner analogous to but more
potently than CQ (Figure 4). At 10 nM against D6, 22 nearly
completely inhibited hemozoin production. Dd2 required
100 nM 22 for the same percentage decrease, but this was
much lower than the 1000 nM required by CQ.
The results for 1 against Dd2 are between those for 22 and
CQ, a result consistent with the in vitro IC50 values (Table 1).
The relatively low activity of 1 against D6 seems inconsistent
with its low IC50 value against CQS P. falciparum. However,
these results are for very high concentrations. While it is
possible that hemozoin inhibition is not the only significant
mode of action for the RCQ molecules and that the low IC50
value is due, in part, to another antimalarial mechanism,
further investigation would be needed before such a conclu-
sion is reached.
These in vivo hemozoin inhibition experiments indicate
that the RCQ molecules act in a manner similar to that of
CQ against CQS P. falciparum. The enlarged DV caused by
accumulation of the drugs can be seen clearly, as can the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6483
determined by HPLC, measuring by UV detection at 254 and
325 nm, using a Varian ProStar 325 UV/vis dual wavelength
detector. HPLC method A was done with a Microsorb-MV 100
CN 5 μm, 4.6 mm ꢀ 250 mm column, eluting with 100% met-
hanol for 30 min unless otherwise stated. HPLC method B
was performed using a SUPELCO Ascentis RP-Amide 5 μm, 4.6
mm ꢀ 150 mm column, eluting with 100% methanol for 30 min
unless otherwise stated. HPLC method C was performed using a
SUPELCO Ascentis C18 5 μm, 4.6 mm ꢀ 150 mm column,
eluting with a 30 min gradient, from 95:5 to 5:95 water with
0.1% formic acid (v/v)/acetonitrile. High resolution mass spec-
trometry was performed on a Bruker micrOTOF-Q instrument.
Results were obtained using electospray ionization (ESI) in the
positive mode, at a flow rate of 0.4 mL/min with 1:1 methanol-
water. 1H, 13C, and 2D NMR experiments were run on a Bruker
400 MHz AMX or AVANCE-IIþ instrument, using the stan-
dard pulse sequences provided, including zg30, zgpg30 co-
sygpqf, hsqcetgpsi2, hmbcgplpndqf, and noesyph, at 25 ꢀC.
The syntheses of 1, 2b, and 3b have been previously described.17
2-(7-Chloroquinolin-4-ylamino)ethan-1-ol (2a). A mixture of
4,7-dichloroquinoline (4.95 g, 0.025 mol) and ethanolamine
(15.27 g, 15.0 mL, 0.25 mol) was heated with stirring at 130-
140 ꢀC for 24 h. After cooling, the mixture was poured into water
(150 mL) and filtered. After air drying, the solid was boiled in
methanol (100 mL), allowed to cool to room temperature, then
cooled in ice. The solid was filtered, then washed with a small
amount of ice cold methanol to give 2a (3 g, 54%) as an off-white
solid. HPLC (method A) tR = 6.99 min (99% pure). 1H NMR
δ (ppm) (400 MHz, CH3OH-d4): 3.48-3.55 (2 H, m), 3.85 (2 H,
q, J = 5.80 Hz), 6.60 (1 H, d, J = 5.67 Hz), 7.43 (1 H, dd, J =
9.02, 2.20 Hz), 7.80 (1 H, d, J = 2.19 Hz), 8.11 (1 H, t, J = 9.02
Hz), 8.38 (1 H, d, J = 5.64 Hz). 13C NMR δ (ppm) (100 MHz,
CH3OH-d4): 46.2, 60.7, 99.7, 118.8, 124.3, 126.1, 127.6, 136.4,
149.7, 152.5, 152.9.
2-(7-Chloroquinolin-4-ylamino)ethyl Methanesulfonate (3a).
To a suspension of 2a (1.5 g, 6.7 mmol) in anhydrous dichlor-
omethane (25 mL) under a nitrogen atmosphere was added
triethylamine (2 mL, 14.3 mmol). The mixture was cooled to
below 0 ꢀC. Methanesulfonyl chloride (0.57 mL, 7.41 mmol) was
added slowly, keeping the temperature below 5 ꢀC, and the
mixture was stirred in an ice bath for 1 h. The mixture was added
to a saturated NaHCO3 solution (100 mL), and the organic layer
was separated and washed with saturated NaHCO3 solution (25
mL). The combined aqueous layers were extracted with dichlor-
omethane (2 ꢀ 20 mL). The combined organic extracts evapo-
rated to leave 3a (1.19 g, 59%) as an off-white solid.
General Procedure for the Preparation of Compounds 4-13. A
mixture of the respective piperazine or homopiperazine com-
pound (1.2 equiv), triethylamine (2.0 equiv), and appropriate
methylsulfonate ester (1.0 equiv) was heated to 70 ꢀC in THF for
3 days with stirring. After the mixture was cooled to room
temperature, 50% K2CO3 solution was added. The mixture was
shaken, and the THF layer was separated. The aqueous layer
was extracted with ethyl acetate. The extracts were combined
with the THF layer and washed with water. After the mixture
was dried and evaporated, the residue was purified.
Figure 4. Graph of hemozoin production in synchronized D6 and
Dd2 P. falciparum when incubated with various concentrations of
CQ, 1, and 22. After the parasites were lysed, the hemozoin was
collected by centrifugation, washed, and dissolved in 0.2 N sodium
hydroxide solution. The absorbance at 400 nm was measured, and
the amount of heme was calculated using an extinction coefficient of
91 000 cm-1 M-1 The amount of heme per parasitized erythrocyte
was calculated based on the number of erythrocytes in the culture
and the percent parasitemia obtained after growing synchronized
culture for 24 h. Blank regions represent concentration of drug not
tested for a particular cell line. Uncertainties are estimated to be at
least (15%, based on estimated uncertainties due to the extraction
of hemozoin from the cells.
reduction in hemozoin production. These effects are most pro-
nounced for 22, requiring substantially lower concentrations
than CQ to effect almost complete inhibition of hemozoin.
Summary and Conclusion
This work strongly supports the hypothesis that an im-
proved drug can be made by combining elements of CQ and a
reversal agent. The in vitro results clearly show that the RCQ
compounds have great efficacy against P. falciparum, and the
invivoresultsdemonstrate thatthe compoundsare efficacious
in a mouse model. In the in vivo test, 22 stands out as an
excellent lead compound for full preclinical testing, with good
activity via the oral route of administration, a low ClogP, and
no obvious signs of toxicity. Further testing will also be car-
ried out using a wider range of drug-resistant parasite strains
to demonstrate thatthese compounds truly are promising lead
compounds for all CQR Plasmodia.
While the mode of action of these compounds has not been
fully elucidated, the experiments described above show that
the RCQ compounds appear to act in a manner similar to that
of CQ. Compound 22 showed the highest level of hemozoin
inhibition, both in vitro and in vivo. Taken together, the
results suggest that the addition of the RA moiety to the
4-aminoquinoline can enhance the mode of antimalarial
activity, at least in part by acting to increase the accumulation
in the parasite’s DV.
N-(2-(4-Benzhydrylpiperazin-1-yl)ethyl)-7-chloroquinolin-4-
amine (4). The title compound was prepared from 1-(diphenyl-
methyl)piperazine (0.4 g, 0.0016 mol), triethylamine (0.27 g,
0.0027 mol), and 3a (0.4 g, 0.00133 mol) in THF (12 mL) ac-
cording to the general procedure. The crude compound was
purified by recrystallization in ethyl acetate to give an off-white
solid (0.13 g, 21%). HPLC (method B) tR = 2.74 min (97%
pure). 1H NMR δ (ppm) (400 MHz, CHCl3-d) 2.51 (8 H, d, J =
40.09 Hz), 2.78 (2 H, t, J = 5.91 Hz), 3.29 (2 H, q, J = 5.32 Hz),
4.26 (1 H, s), 5.96 (1 H, s), 6.35 (1 H, d, J = 5.36 Hz), 7.21-7.14
(2 H, m), 7.31-7.25 (4 H, m), 7.39 (1 H, dd, J = 8.90, 2.20 Hz),
7.46-7.40 (4 H, m), 7.65 (1 H, d, J = 8.92 Hz), 7.95 (1 H, d, J =
2.16 Hz), 8.52 (1 H, d, J = 5.31 Hz). 13C NMR δ (ppm) (100
MHz, CHCl3-d) 38.9, 52.1, 52.9, 55.4, 76.2, 99.3, 117.4, 121.1,
Experimental Section
Chemistry. All chemicals were obtained from Sigma-Aldrich
Chemical Co. Purities of all final products were g95% as

6484 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Burgess et al.
125.3, 127.0, 127.9, 128.5, 128.8, 134.8, 142.6, 149.1, 149.8,
152.2. MS (ESI): m/z 457.2149 M þ H (calculated 457.2154).
N-(3-(4-Benzhydrylpiperazin-1-yl)propyl)-7-chloroquinolin-4-
amine (5). The title compound was prepared from 1-(diphenyl-
methyl)piperazine (0.56 g, 0.0022 mol), triethylamine (0.43 g,
0.0042 mol), and 3b (0.65 g, 0.0021 mol) in THF (15 mL)
according to the general procedure. The crude compound was
purified by recrystallization in ethyl acetate to give an off-white
solid (0.32 g, 31%). HPLC (method B) tR = 3.22 min (97%
pure). 1H NMR δ (ppm) (400 MHz, CHCl3-d) 1.97-1.89 (2 H,
m), 2.87-2.32 (10 H, m), 3.35 (2 H, q, J = 5.12 Hz), 4.42 (1 H,
s), 6.29 (1 H, d, J = 5.40 Hz), 7.13 (1 H, dd, J = 8.92, 2.18 Hz),
7.28-7.20 (2 H, m), 7.36-7.29 (4 H, m), 7.46-7.41 (4 H, m),
7.61 (1 H, s), 7.78 (1 H, d, J = 8.95 Hz), 7.92 (1 H, d, J = 2.16
Hz), 8.48 (1 H, d, J = 5.35 Hz). 13C NMR δ (ppm) (100 MHz,
CHCl3-d) 23.3, 44.5, 51.6, 54.0, 58.9, 75.9, 98.4, 117.5, 122.4,
124.6, 127.2, 128.2, 128.5, 128.6, 134.6, 141.9, 149.1, 150.6,
152.2. MS (ESI): m/z 471.2293 M þ H (calculated 471.2310).
N-(3-(4-Benzhydryl-1,4-diazepan-1-yl)propyl)-7-chloroquino-
lin-4-amine (6). The title compound was prepared from 27 (0.22
g, 0.001 mol), triethylamine (0.18 g, 0.0018 mol), and 3b (0.20 g,
0.0006 mol) in THF (15 mL) according to the general procedure.
The crude compound was purified by chromatography on
alumina, eluting with ethyl acetate/hexanes 40:60) to give a solid
(0.07 g, 24%). HPLC (method C) tR = 10.76 min (95% pure).
1H NMR δ (ppm) (400 MHz, CHCl3-d): 1.85-1.86 (4 H, m),
2.72-2.73 (6 H, m), 2.78 (2 H, d, J = 6.40 Hz), 2.87 (2 H, t, J =
5.56 Hz), 3.34 (2 H, q, J = 5.22 Hz), 4.63 (1 H, s), 6.29 (1 H, d,
J = 5.39 Hz), 7.18-7.20 (2 H, m), 7.27-7.27 (5 H, m), 7.43 (4 H,
d, J = 7.75 Hz), 7.62 (1 H, s), 7.72 (1 H, d, J = 8.92 Hz), 7.92
(1 H, d, J = 2.18 Hz), 8.48 (1 H, d, J = 5.34 Hz). 13C NMR δ
(ppm) (100 MHz, CHCl3-d): 24.5, 27.8, 44.5, 52.9, 53.0, 54.3,
57.0, 57.9, 75.7, 98.5, 117.6, 122.1, 124.8, 127.0, 128.0, 128.5,
128.6, 134.6, 143.2, 149.2, 150.6, 152.2. MS (ESI): m/z 485.2454
M þ H (calculated 485.2467).
150.5, 152.2. MS (ESI): m/z 505.1911 M þ H (calculated
505.1920).
7-Chloro-N-(3-(4-((4-chlorophenyl)(phenyl)methyl)-1,4-diaze-
pan-1-yl)propyl)quinolin-4-amine (9). The title compound was
prepared from 28 (0.17 g, 0.000 57 mol), triethylamine (0.18 g,
0.0018 mol), and 3b (0.20 g, 0.000 64 mol) in THF (15 mL)
according to the general procedure. The crude compound was
purified by chromatography on alumina, eluting with ethyl
acetate/hexanes (40:60) to give a solid (0.21 g, 71%). HPLC
(method B) tR = 4.66 min (97% pure). 1H NMR δ (ppm) (400
MHz, CHCl3-d) 1.81-1.92 (4 H, m), 2.74-2.75 (8 H, m), 2.88 (2
H, t, J = 5.53 Hz), 3.37 (2 H, q, J = 5.13 Hz), 4.61 (1 H, s), 6.31
(1 H, d, J = 5.41 Hz), 7.26-7.28 (5 H, m), 7.35-7.40 (4 H, m),
7.53 (1 H, s), 7.72 (1 H, d, J = 8.93 Hz), 7.93 (1 H, d, J = 2.16
Hz), 8.50 (1 H, d, J = 5.36 Hz). MS (ESI): m/z 519.2079 M þ H
(calculated 519.2077).
N-(3-(4-(Bis(4-fluorophenyl)methyl)piperazin-1-yl)propyl)-7-
chloroquinolin-4-amine (10). The title compound was prepared
from 1-(bis(4-fluorophenyl)methyl)piperazine (0.52 g, 0.0018
mol), triethylamine (0.20 g, 0.0019 mol), and 3b (0.41 g, 0.0013
mol) in THF (15 mL) according to the general procedure. The
crude compound was purified by chromatography on alumina,
eluting with ethyl acetate/hexanes (40:60) to give a solid (0.12 g,
18%). HPLC (method B) tR = 3.3 min (98% pure). 1H NMR δ
(ppm) (400 MHz, CHCl3-d) 1.97 (3 H, m), 2.57 (8 H, bs), 2.68 (3
H, t, J = 5.60 Hz), 3.40 (3 H, m), 4.38 (1 H, s), 6.34 (1 H, d, J =
5.47 Hz), 7.04 (4 H, t, J = 8.55 Hz), 7.22 (1 H, dd, J = 8.90, 2.05
Hz), 7.40 (4 H, dd, J = 8.47, 5.45 Hz), 7.67 (1 H, bs), 7.84 (1 H, d,
J = 8.89 Hz), 8.00 (1 H, s), 8.51 (1 H, d, J = 5.44 Hz).
N-(2-(4-(9H-Fluoren-9-yl)-1,4-diazepan-1-yl)ethyl)-7-chloro-
quinolin-4-amine (11). The title compound was prepared from 29
(0.24 g, 0.0010 mol), triethylamine (0.69 g, 0.0068 mol), and 3a
(0.30 g, 0.0010 mol) in THF (15 mL) according to the general
procedure. The crude compound was purified by chromatogra-
phy on alumina, eluting with ethyl acetate/hexanes (40:60) to
give a solid (0.05 g, 11%). HPLC (method C) tR = 10.88 min
(95% pure). 1H NMR δ (ppm) (400 MHz, CHCl3-d): 1.79-1.80
(2 H, m), 2.67-2.70 (2 H, m), 2.74-2.78 (2 H, m), 2.84 (2 H, t,
J = 5.93 Hz), 2.91 (4 H, dt, J = 12.45, 5.98 Hz), 3.23 (2 H, q, J =
5.25 Hz), 4.90 (1 H, s), 6.17 (1 H, s), 6.34 (1 H, d, J = 5.34 Hz),
7.26 (2 H, s), 7.37 (3 H, d, J = 7.82 Hz), 7.63 (1 H, s), 7.69 (3 H, d,
J = 7.26 Hz), 7.96 (1 H, d, J = 2.18 Hz), 8.52 (1 H, d, J = 5.28
Hz). 13C NMR δ (ppm) (100 MHz, CHCl3-d): 29.4, 39.7, 53.4,
54.9, 56.5, 71.6, 99.3, 119.8, 121.2, 125.3, 125.5, 127.1, 128.1,
128.8, 134.8, 140.8, 144.9, 149.1, 149.9, 152.1. MS (ESI): m/z
469.2143 M þ H (calculated 469.2154).
7-Chloro-N-(2-(4-((4-chlorophenyl)(phenyl)methyl)piperazin-
1-yl)ethyl)quinolin-4-amine (7). The title compound was pre-
pared from 1-[(4-chlorophenyl)(phenyl)methyl]piperazine
(0.46 g, 0.0016 mol), triethylamine (0.27 g, 0.0027 mol), and 3a
(0.4 g, 0.00133 mol) in THF (12 mL) according to the general
procedure. The crude compound was purified by recrystalliza-
tion in ethyl acetate to give an off-white solid (0.32 g, 31%). The
crude compound was purified by recrystallization in ethyl
acetate/hexanes (70/30) to give an off-white solid (0.20 g,
30%). HPLC (method A) tR = 7.30 min (96% pure). 1H
NMR δ (ppm) (400 MHz, CHCl3-d) 2.50 (8 H, d, J = 44.37
Hz, 2.78 (2 H, t, J = 5.91 Hz), 3.29 (2 H, q, J = 5.32 Hz), 4.24
(1 H, s), 5.94 (1 H, s), 6.35 (1 H, d, J = 5.36 Hz), 7.31-7.19 (5 H,
m), 7.41-7.35 (5 H, m), 7.64 (1 H, d, J = 8.93 Hz), 7.95 (1 H, d,
J = 2.16 Hz), 8.52 (1 H, d, J = 5.31 Hz). 13C NMR δ (ppm) (100
MHz, CHCl3-d) 38.9, 52.0, 52.8, 55.4, 75.4, 99.3, 117.3, 121.1,
125.3, 127.2, 127.8, 128.6, 128.7, 128.8, 129.1, 132.6, 134.8,
141.2, 142.1, 149.1, 149.7, 152.1. MS (ESI): m/z 491.1745 M þ
H (calculated 491.1764).
N-(3-(4-(9H-Fluoren-9-yl)-1,4-diazepan-1-yl)propyl)-7-chloro-
quinolin-4-amine (12). The title compound was prepared from 29
(0.31 g, 0.0012 mol), triethylamine (0.25 g, 0.0025 mol), and 3b
(0.37 g, 0.0012 mol) in THF (15 mL) according to the general
procedure. The crude compound was purified by chromatogra-
phy on alumina, eluting with ethyl acetate/hexanes (40:60) to
give a solid (0.29 g, 50%). HPLC (method C) tR = 11.24 min
(97% pure). 1H NMR δ (ppm) (400 MHz, CHCl3-d): 1.88-1.93
(4 H, m), 2.08 (1 H, s), 2.77 (3 H, s), 2.83 (2 H, t, J = 5.89 Hz),
2.94 (2 H, t, J = 5.29 Hz), 3.00 (1 H, s), 3.34 (2 H, t, J = 5.75 Hz),
4.92 (1 H, s), 6.27 (1 H, d, J = 5.53 Hz), 7.38 (2 H, t, J = 7.47
Hz), 7.64 (2 H, d, J = 7.49 Hz), 7.69 (2 H, d, J = 7.56 Hz),
7.96-7.96 (2 H, m), 8.48 (1 H, s). 13C NMR δ (ppm) (100 MHz,
CHCl3-d): 23.3, 24.1, 27.6, 43.5, 50.2, 51.7, 53.5, 56.8, 57.4, 71.3,
98.3, 117.5, 119.9, 122.7, 125.1, 125.5, 127.2, 127.7, 128.3, 135.0,
140.9, 144.5, 148.3, 151.0, 151.3, 177.3. MS (ESI): m/z 483.2297
M þ H (calculated 483.2310).
7-Chloro-N-(3-(4-((4-chlorophenyl)(phenyl)methyl)piperazin-
1-yl)propyl)quinolin-4-amine (8). The title compound was pre-
pared from 1-[(4-chlorophenyl)(phenyl)methyl]piperazine (0.63
g, 0.0022 mol), triethylamine (0.43 g, 0.0042 mol), and 3b (0.65 g,
0.0021 mol) in THF (15 mL) according to the general procedure.
The crude compound was purified by recrystallization from
ethyl acetate to give an off-white solid (0.38 g, 34%). HPLC
1
(method A, 40 min) tR = 9.67 min (98% pure). H NMR δ
(ppm) (400 MHz, CHCl3-d) 1.92 (2 H, p, J = 5.38 Hz), 2.70-
2.52 (10 H, m), 3.34 (2 H, q, J = 5.11 Hz), 4.37 (1 H, s), 6.29 (1 H,
d, J = 5.40 Hz), 7.15 (1 H, dd, J = 8.90, 2.17 Hz), 7.32-7.22 (3
H, m), 7.38-7.30 (2 H, m), 7.42-7.35 (4 H, m), 7.52 (1 H, s), 7.76
(1 H, d, J = 8.95 Hz), 7.92 (1 H, d, J = 2.15 Hz), 8.48 (1 H, d,
J = 5.35 Hz). 13C NMR δ (ppm) (100 MHz, CHCl3-d) 23.3,
44.4, 51.6, 53.9, 58.8, 75.3, 98.4, 117.4, 122.3, 124.6, 127.5,
128.1, 128.6, 128.7, 129.3, 132.7, 134.6, 140.8, 141.2, 149.1,
N1-(7-chloroquinolin-4-yl)-N2-(3-(10,11-dihydro-5H-dibenzo-
[b,f]azepin-5-yl)propyl)-N2-methylethane-1,2-diamine (13). Desi-
pramine hydrochloride (0.44 g, 0.00145 mol) was dissolved in
water (7 mL), and solid NaHCO3 (0.24 g, 0.0029 mol) was added
with stirring. After addition of dichloromethane (8 mL), two
clear layers resulted. The aqueous layer was removed and
extracted with dichloromethane (2 ꢀ 7 mL). The combined

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6485
organic layers were evaporated to leave desipramine free base as
a yellow oil. To this oil were added anhydrous THF (12 mL) and
3a (0.35 g, 0.001 16 mol) followed by triethylamine (0.32 mL,
0.002 32 mol). After being stirred at 50-60 ꢀC for 72 h, the
mixture was allowed to cool to room temperature. The mixture
was diluted with 50% K2CO3 solution (30 mL), and the THF
layer was separated. The aqueous layer was extracted with ethyl
acetate (2 ꢀ 10 mL). The extracts were combined with the THF
layer and washed with water (10 mL). After the mixture was
dried and evaporated, the residue was chromatographed on
alumina (MCB type F20, 80-200 mesh), eluting with ethyl
acetate:/hexanes (70:30) to give a yellow oil (0.42 g, 77%). HPLC
(method A) tR = 9.12 min (96% pure). 1H NMR δ (ppm) (400
MHz, CHCl3-d): 1.78 (2 H, p, J = 6.79 Hz), 2.23 (3 H, s), 2.46
(2 H, t, J = 7.00 Hz), 2.68 (2 H, t, J = 5.81 Hz), 3.03 (4 H, s),
3.26-3.16 (2 H, m), 3.79 (2 H, t, J = 6.54 Hz), 5.82 (1 H, s), 6.32
(1 H, d, J = 5.35 Hz), 6.84 (2 H, td, J = 7.14, 1.61 Hz),
7.09-6.95 (6 H, m), 7.23 (1 H, dd, J = 8.90, 2.19 Hz), 7.44 (1 H,
d, J = 8.93 Hz), 7.95 (1 H, d, J = 2.16 Hz), 8.52 (1 H, d, J = 5.31
Hz). 13C NMR δ (ppm) (100 MHz, CHCl3-d): 24.7, 31.0, 38.5,
40.9, 47.0, 53.3, 54.3, 98.2, 116.3, 118.6, 120.0, 121.5, 124.3,
125.3, 127.7, 128.8, 133.1, 133.7, 147.0, 148.1, 148.6, 151.0. MS
(ESI): m/z 471.2316 M þ H (calculated 471.2310).
sodium triacetoxyborohydride (0.52 g, 0.0025 mol) followed by
acetic acid (0.1 g, 0.001 75 mol).19 The mixture was stirred under
nitrogen and at room temperature for 7 days. The reaction was
quenched with saturated NaHCO3 solution (50 mL) and ex-
tracted into dichloromethane (3 ꢀ 10 mL). The extracts were
washed with brine (10 mL), then dried and evaporated. The
residue was chromatographed to give pure product.
7-Chloro-N-(3-(4-(2,2-diphenylethyl)piperazin-1-yl)propyl)-
quinolin-4-amine (16). The title compound was prepared from
diphenylacetaldehyde (0.32 g, 0.001 64 mol) according to the
general procedure. The crude compound was purified by chro-
matography on silica, eluting with ethyl acetate/ammonium
hydroxide (99:1) to give an off-white solid (0.43 g, 54%). HPLC
(method A) tR = 7.87 min (98% pure). 1H NMR δ (ppm) (400
MHz, CHCl3-d): 1.87-1.94 (2 H, m), 2.48-2.62 (6 H, m), 2.64 (4
H, s), 3.07 (2 H, d, J = 7.54 Hz), 3.34 (2 H, q, J = 5.12 Hz), 4.24
(1 H, t, J = 7.52 Hz), 6.30 (1 H, d, J = 5.41 Hz), 7.17-7.23 (2 H,
m), 7.24-7.33 (9 H, m), 7.58 (1 H, s), 7.87 (1 H, d, J = 8.94 Hz),
7.93 (1 H, d, J = 2.15 Hz), 8.49 (1 H, d, J = 5.36 Hz). 13C NMR
δ (ppm) (100 MHz, CHCl3-d): 23.3, 44.5, 48.9, 53.5, 53.6, 58.8,
63.8, 98.5, 117.5, 122.5, 124.6, 126.3, 128.2, 128.4, 128.7, 134.6,
143.7, 149.1, 150.6, 152.2. MS (ESI): m/z 485.2479 M þ H
(calculated 485.2467).
7-Chloro-N-(3-(piperazin-1-yl)propyl)quinolin-4-amine (14). 2b
(2.7 g, 0.0116 mol) was finely ground and suspended in dry THF
(50 mL). Triethylamine (2.9 g, 0.29 mol) was added, and the
mixture was cooled to below 0 ꢀC. Methanesulfonyl chloride
(1.46 g, 0.0128 mol) was added slowly, keeping the temperature
below 5 ꢀC. After the mixture was stirred in an ice bath for 1 h,
TLC (alumina plate, run in ethyl acetate) indicated no 2b left in
the mixture. Piperazine (10 g, 0.116 mol) was added, and the mix-
ture was heated to reflux. After 2 h TLC indicated the reaction
was complete, and the mixture was allowed to cool to room tem-
perature. Then 150 mL of saturated NaHCO3 solution was added
and the solutionwas extractedwithethylacteate(3 ꢀ 50mL). The
combined organic extracts were washed with water (5 ꢀ 25 mL),
dried, and evaporated to give a cream solid (1.2 g, 34%). HPLC
(method C) tR = 2.81 min (93% pure). 1H NMR δ (ppm) (400
MHz, CH3OH-d4): 1.79-1.89 (2 H, m), 2.38-2.49 (6 H, m),
2.76-2.83 (4 H, m), 3.33 (2 H, t, J = 6.79 Hz), 6.44 (1 H, d, J =
5.69 Hz), 7.27-7.33 (1 H, m), 7.68 (1 H, d, J = 2.18 Hz), 7.98 (1
H, d, J = 9.01 Hz), 8.25 (1 H, d, J = 5.65 Hz). 13C NMR δ (ppm)
(100 MHz, CH3OH-d4): 25.8, 42.6, 46.1, 54.6, 58.0, 99.6, 118.7,
124.3, 126.0, 127.6, 136.4, 149.6, 152.4, 152.8. MS (ESI): m/z
305.1538 M þ H (calculated 305.1528).
N-(3-(4-(Adamant-2-yl)piperazin-1-yl)propyl)-7-chloroquinolin-
4-amine (17). The title compound was prepared from 2-ada-
mantanone (0.5 g, 0.003 28 mol) according to the general pro-
cedure. The crude compound was purified by chromatography
on silica, eluting with ethyl acetate/ammonium hydroxide
(99.3:0.7) to give an off white solid (0.33 g, 46). HPLC
(method A) tR = 11.75 min (96% pure). 1H NMR δ (ppm) (400
MHz, CHCl3-d): 1.42 (2 H, d, J = 11.72 Hz), 1.79 (5 H, m),
1.86-2.02 (5 H, m), 2.10 (5 H, m,), 2.60-2.66 (10 H, m), 3.38 (2
H, q, J = 5.12 Hz), 6.31 (1 H, d, J = 5.41 Hz), 7.32 (1 H, dd, J =
8.95, 2.14 Hz), 7.81 (1 H, s), 7.91-7.95 (2 H, m), 8.50 (1 H, d, J =
5.37 Hz). 13C NMR δ (ppm) (100 MHz, CHCl3-d): 23.2, 27.3,
27.6, 29.0, 31.4, 37.3, 37.8, 44.7, 49.7, 54.3, 59.0, 68.3, 98.4,
117.5, 122.7, 124.7, 128.6, 134.6, 149.2, 150.7, 152.2. MS (ESI):
m/z 439.2631 M þ H (calculated 439.2623).
N-(Dipyridin-2-ylmethylene)piperidin-4-amine (18). 2,20-Di-
pyridylketone (25.95 g, 0.141 mol) was dissolved in toluene
(500 mL), and 4-aminopiperidine (16.2 g, 0.162 mol) was added
followed by p-toluenesulfonic acid (∼0.5 g). The mixture was
heated to reflux for 3 days, with a Dean and Stark trap to
remove water. After the mixture was cooled to room tempera-
ture, the toluene was removed to leave a crude oil (37.5 g, 99%),
which was used without further purification.
7-Chloro-N-(3-(4-tritylpiperazin-1-yl)propyl)quinolin-4-amine
(15). 14 (0.3 g, 0.000 98 mol) was dissolved in acetonitrile (15 mL),
and potassium carbonate (0.2 g, 0.0015 mol) was added, followed
by trityl chloride (0.25 g, 0.000 89 mol). The mixture was heated to
reflux with stirring for 3 h, after which TLC indicated completion.
After cooling to room temperature, the mixture was poured into
water (50 mL) and ethyl acetate (20 mL) was added. The insoluble
precipitate was filtered, washed with water and ethyl acetate, and
dried. The ethyl acetate layer was separated and the aqueous layer
extracted with ethyl acetate (2 ꢀ 20 mL). The combined organic
phases were dried, filtered through an alumina plug, and evapo-
rated. The residue was combined with the insoluble solid from
above to give a white solid (0.15 g, 31%). HPLC (method A) tR =
7-Chloro-N-(2-(4-(dipyridin-2-ylmethyleneamino)piperidin-1-yl)-
ethyl)quinolin-4-amine (19). 18 (1.74 g, 0.0065 mol crude) was
dissolved in acetonitrile (20 mL), and 3a (1.31 g, 0.004 36 mol)
and K2CO3 (1.2 g, 0.008 71 mol) were added. The mixture was
stirred at 70 ꢀC for 2 days. After the mixture was cooled to room
temperature, water (100 mL) was added and the mixture was
stirred for 30 min. The solid was filtered, washed with water, and
recrystallized from toluene/hexanes twice to give a solid (0.68 g,
33%). HPLC (method A) tR = 7.80 min (99% pure). MS (ESI):
m/z 471.2054 M þ H (calculated 471.2058).
7-Chloro-N-(3-(4-(dipyridin-2-ylmethyleneamino)piperidin-1-yl)-
propyl)quinolin-4-amine (20). 18 (1.02 g, 0.003 82 mol crude) was
dissolved in acetonitrile (12 mL), and 3b (1 g, 0.003 18 mol) and
K2CO3 (0.88 g, 0.006 36 mol) were added. The mixture was
stirred at 70 ꢀC overnight. TLC (alumina plate, run in ethyl
acetate/methanol 9:1) indicated some 3b was still present, so a
further solution of 18 (0.25 g, 0.000 954 mol) in acetonitrile
(5 mL) was added, and heating continued overnight. After the
mixture was cooled to room temperature, the solvent was
evaporated and the residue was slurried in water (30 mL). The
solid was filtered, washed with water, and recrystallized from
toluene/hexanes. The solid was dissolved in ethyl acetate/metha-
nol 50:50 and stirred with alumina and charcoal for 30 min.
After filtration through Celite, the solvents were removed to give
1
8.63 min (97% pure). H NMR δ (ppm) (400 MHz, CHCl3-d):
1.90-1.97 (4 H, m), 2.53 (2 H, bs), 2.67-2.72 (2 H, m), 3.06 (2 H,
bs), 3.16 (2 H, bs), 3.31 (2 H, q, J= 4.99 Hz), 6.22 (1 H, d, J = 5.41
Hz), 6.62(1H, dd, J=8.95, 2.19 Hz), 7.26-7.34 (10 H, m), 7.52 (6
H, bs), 7.67 (1 H, s), 7.84 (1 H, d, J = 2.16 Hz), 8.43 (1 H, d, J =
5.35 Hz). 13C NMR δ (ppm) (100 MHz, CH3OH-d4): 22.8, 44.7,
47.8, 54.6, 59.6, 98.1, 117.2, 122.1, 125.0, 126.4, 127.2, 127.7, 127.8,
127.9, 129.4, 134.8, 146.9, 150.8, 151.5. MS (ESI): m/z 547.2637
M þ H (calculated 547.2623).
General Method for the Preparation of Compounds 16 and 17.
14 (0.5 g, 0.001 64 mol) and the respective carbonyl compound
were mixed together in dry THF (5 mL) and then treated with

6486 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Burgess et al.
a solid (0.78 g, 50%). HPLC (method B) tR = 2.41 min (96%
pure). MS (ESI): m/z 485.2230 M þ H (calculated 485.2215).
7-Chloro-N-(2-(4-(dipyridin-2-ylmethylamino)piperidin-1-yl)-
ethyl)quinolin-4-amine (21). 19 (0.53 g, 0.001 13 mol) was dis-
solved in methanol (40 mL) and cooled in an ice/water bath.
Sodium borohydride (0.13 g, 0.003 38 mol) was added in por-
tions, and the mixture was stirred overnight at room tempera-
ture. After evaporation of the methanol, water (40 mL) was
added to the residue, and the resulting suspension was stirred for
30 min. The mixture was extracted with dichloromethane (3 ꢀ
20 mL), and the combined extracts were washed with water (10
mL), then dried and evaporated. Chromatography on alumina,
eluting with dichloromethane/methanol (95:5), gave an oil (0.17
g, 34%). HPLC (method B) tR = 2.30 min (84% pure). 1H NMR
δ (ppm) (CHCl3-d): 1.50-1.62 (2 H, m), 1.83 (1 H, s), 1.95 (2 H,
d, J = 13.24 Hz), 2.04 (2 H, t, J = 11.64 Hz), 2.44-2.53 (1 H, m),
2.71 (2 H, t, J = 5.88 Hz), 2.89 (2 H, d, J = 11.30 Hz), 3.26 (2 H,
q, J = 5.27 Hz), 5.24 (1 H, s), 6.09 (1 H, s), 6.34 (1 H, d, J = 5.37
Hz), 7.14 (2 H, ddd, J = 7.48, 4.87, 1.18 Hz), 7.36 (1 H, dd, J =
8.89, 2.19 Hz), 7.42 (2 H, dt, J = 7.88, 1.05 Hz), 7.62 (2 H, td,
J = 7.67, 1.83 Hz), 7.64 (1 H, d, J = 8.94 Hz), 7.94 (1 H, d, J =
2.17 Hz), 8.51 (1 H, d, J = 5.32 Hz), 8.56 (2 H, ddd, J = 4.89,
1.81, 0.92 Hz). 13C NMR δ (ppm) (CHCl3-d): 33.0, 39.0, 51.9,
52.7, 55.3, 66.2, 99.2, 117.3, 121.2, 122.2, 122.4, 125.4, 128.7,
134.8, 136.7, 149.1, 149.2, 149.8, 152.1, 161.6. MS (ESI): m/z
473.2227 M þ H (calculated 473.2215).
cool to room temperature. The ethanol was evaporated, and the
residue was partitioned between saturated NaHCO3 solution
(20 mL) and dichloromethane (20 mL). The organic layer was
separated, and the aqueous layer was extracted with dichlor-
omethane (2 ꢀ 20 mL). The combined dichloromethane layers
were washed with saturated NaHCO3 solution (20 mL), then
dried and evaporated. The resulting oil was chromatographed
on silica, eluting with ethyl acetate/triethylamine (99:1), to give
an off-white solid (0.10 g, 17%). HPLC (method B) tR = 2.97
min (95% pure). 1H NMR δ (ppm) (400 MHz, CHCl3-d): 1.67
(2 H, t, J = 6.86 Hz), 1.80 (2 H, p, J = 6.95 Hz), 2.37-2.54 (10
H, m), 3.24-3.33 (2 H, m), 4.19-4.24 (1 H, m), 5.38 (1 H, s), 6.38
(1 H, t, J = 6.53 Hz), 7.14-7.22 (2 H, m), 7.25-7.31 (4 H, m),
7.32 (1 H, dd, J = 8.92, 2.20 Hz), 7.41 (4 H, d, J = 7.61 Hz), 7.66
(1 H, d, J = 8.94 Hz), 7.95 (1 H, d, J = 2.18 Hz), 8.52 (1 H, d,
J = 5.36 Hz). 13C NMR δ (ppm) (100 MHz, CHCl3-d): 24.7,
26.4, 43.2, 51.8, 53.5, 57.7, 76.2, 99.0, 117.2, 121.1, 125.1, 127.0,
127.9, 128.5, 128.9, 134.8, 142.6, 149.2, 149.7, 152.1. MS (ESI):
m/z 485.2456 M þ H (calculated 485.2467).
2-(4-Benzhydrylpiperazin-1-yl)ethanol (26). 1-(2-Hydroxyethyl)-
piperazine (2 g, 0.0154 mol) was dissolved in DMF (20 mL), and
potassium carbonate (4.27 g, 0.0308 mol) was added, followed by a
catalytic amount of potassium iodide. The mixture was stirred at
room temperature, and chlorodiphenylmethane (3.12 g, 0.0154
mol) was added dropwise. After the addition, the mixture was stir-
red for a further 2 h at room temperature, then warmed to 70 ꢀC,
and held there overnight. After the mixture was cooled to room
temperature, water (100 mL) was added, and the mixture was
extracted with diethyl ether (3 ꢀ 20 mL). The combined organic
extracts were washed with brine solution (30 mL), then dried and
7-Chloro-N-(3-(4-(dipyridin-2-ylmethylamino)piperidin-1-yl)-
propyl)quinolin-4-amine (22). 20 (0.60 g, 0.001 24 mol) was dis-
solved in methanol (40 mL) and cooled in ice. Sodium boro-
hydride (0.14 g, 0.0037 mol) was added in portions, and the
mixture was stirred at room temperature overnight. After eva-
poration of the methanol, the residue was stirred with water (50
mL) for 30 min and then extracted with dichloromethane (3 ꢀ 20
mL). The extracts were washed with water (20 mL), dried, and
evaporated to give a solid (0.59 g, 99%). HPLC (method B) tR =
evaporated to give an oil (1.7 g, 37%). HPLC (method B) tR
=
2.25 min (95% pure). 1HNMRδ(ppm) (400 MHz, CHCl3-d): 2.43
(5 H, bs, J = 8.59 Hz), 2.54 (5 H, m, J = 5.45 Hz), 3.58 (2 H, t, J =
5.41 Hz), 4.22 (1 H, s), 7.14-7.19 (2 H, m), 7.24-7.30 (4 H, m),
7.38-7.43 (4 H, m). 13C NMR δ(ppm) (100 MHz, CHCl3-d): 52.0,
53.1, 57.7, 59.1, 76.2, 126.9, 127.9, 128.5, 142.7.
1
2.49 min (97% pure). H NMR δ (ppm) (400 MHz, CHCl3-d):
1.57-1.70 (2 H, m), 1.87-1.95 (2 H, m), 1.99 (5 H, d, J = 11.77
Hz), 2.56 (2 H, t, J = 5.17 Hz), 2.59-2.68 (1 H, m), 3.00 (2 H, d,
J= 11.29 Hz), 3.35 (2 H, q, J=5.10Hz),5.25(1H, s), 6.29(1H, d,
J = 5.42 Hz), 7.16 (2 H, ddd, J = 7.47, 4.88, 1.19 Hz), 7.44 (1 H,
dd, J = 8.91, 2.18 Hz), 7.47 (2 H, d, J = 7.89 Hz), 7.65 (2 H, td,
J = 7.67, 1.83 Hz), 7.74 (1 H, s), 7.84 (1 H, d, J = 8.96 Hz),
7.91-7.94 (1 H, m), 8.49 (1 H, d, J = 5.37 Hz), 8.59 (2 H, ddd,
J = 4.89, 1.80, 0.92 Hz). 13C NMRδ (ppm) (100 MHz, CHCl3-d):
23.7, 33.1, 44.5, 52.8, 53.1, 58.7, 66.5, 98.3, 117.6, 122.2, 122.4,
122.4, 125.0, 128.5, 134.6, 136.7, 149.1, 149.1, 150.7, 152.1, 162.1.
MS (ESI): m/z 487.2355 M þ H (calculated 487.2371).
General Method for the Preparation of Compounds 27-29.
The halo compound was placed in chloroform, and homopiper-
azine was added. The mixture was stirred and heated at reflux
for 3 days. After the mixture was cooled to room temperature, a
saturated NaHCO3 solution was added and the mixture was
shaken. The organic layer was separated and washed with water.
The aqueous layers were combined with the NaHCO3 layer and
extracted with dichloromethane. The combined organic layers
were dried and evaporated.
1-Benzhydryl-1,4-diazepane (27). The title compound was
prepared from chlorodiphenylmethane (0.61 g, 0.003 mol) and
homopiperazine (1.5 g, 0.015 mol) according to the general
procedure. The crude compound was purified by chromatogra-
phy on alumina, eluting with chloroform/methanol (95:5) to
give an oil (0.35 g, 44%).
1-((4-Chlorophenyl)(phenyl)methyl)-1,4-diazepane (28). The
title compound was prepared from chloro(4-chlorophenyl)phe-
nylmethane (0.71 g, 0.003 mol) and homopiperazine (1.5 g, 0.015
mol) according to the general procedure. The crude compound
was purified by chromatography on alumina, eluting with
chloroform/methanol (95:5) to give an oil (0.39 g, 43%).
1-(9H-Fluoren-9-yl)-1,4-diazepane (29). The title compound
was prepared from 9-bromofluorene (1.47 g, 0.006 mol) and
homopiperazine (3 g, 0.03 mol) according to the general proce-
dure. The crude compound was purified by chromatography on
alumina, eluting with chloroform/methanol (95:5) to give an oil
(0.55 g, 37%).
Inhibition of P. falciparum Growth. CQS (D6) and CQR (Dd2
and 7G8) P. falciparum maintained continuously in culture were
used.40 Asynchronous cultures were diluted with uninfected
RBCs and complete medium (RPMI-1640 with 0.5% Albumax
II) to achieve 0.2% parasitemia and 2% hematocrit. In 96-well
microplates, CQ (positive control) or RCQ diluted in complete
medium from 10 mM stock in DMSO was added to the cell
2-(4-(4-Benzhydrylpiperazin-1-yl)butyl)isoindoline-1,3-dione
(23). A mixture of N-(4-bromobutyl)phthalimide (0.5 g, 0.001 77
mol), 1-(diphenylmethyl)piperazine (0.47 g, 0.001 86 mol), and
K2CO3 (0.61 g, 0.004 43 mol) was stirred and heated in acetoni-
trile (25 mL) to reflux for 3 h.39 After the mixture was cooled, the
acetonitrile was evaporated and the residue partitioned between
water (20 mL) and ethyl acetate (20 mL). The aqueous layer was
extracted with ethyl acetate (2 ꢀ 10 mL) and the combined
organic layers were dried and evaporated to give a solid (0.74 g,
92%), which was used without further purification.
4-(4-Benzhydrylpiperazin-1-yl)butan-1-amine (24). 23 (0.74 g,
0.001 63 mol crude) was dissolved in ethanol (5 mL), and hydra-
zine hydrate (0.25 g, 0.0049 mol) was added.39 The mixture was
stirred and heated to reflux for 3 h, then allowed to cool to room
temperature. The solid was filtered off, and the filter cake was
washed with cold ethanol. The filtrate was evaporated to give an
oil (0.53 g, ∼100%), which solidified on contact with air. This
was used without further purification.
N-(4-(4-Benzhydrylpiperazin-1-yl)butyl)-7-chloroquinolin-4-
amine (25). 4,7-Dichloroquinoline (0.24 g, 0.0012 mol) was
dissolved in ethanol (10 mL), and 24 (0.53 g, 0.001 63 mol crude)
was added, followed by triethylamine (0.33 g, 0.003 26 mol). The
mixture was stirred and refluxed for 10 days, then allowed to

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6487
mixture to yield triplicate wells with drug concentrations ran-
ging from 0 to 10-4 M in a final well volume of 100 μL. After 72 h
of incubation under standard culture conditions, plates were
harvested and read by the SYBR Green I fluorescence-based
method40 using a 96-well fluorescence plate reader (Gemini-
EM, Molecular Devices), with excitation and emission wave-
lengths at 497 and 520 nm, respectively. The fluorescence read-
ings were plotted against log [drug], and the IC50 values were
obtained from curve fitting performed by nonlinear regression
using either Prism (GraphPad) or Excel (Microsoft) software. In
order to better compare results run on different days and with
different batches of each stain, CQ was run as a positive control.
All results obtained were “normalized” to the CQ values of 6.9
nM for D6, 102 nM for Dd2, and 108 nM for 7G8. The
normalization for D6 strains, using the CQ value of 6.9 nM as
the control value is shown below:
1 had a retention time of 14 min and CQ had 5 min. Each drug
sample was monitored at 325 nm and was compared to a
standard curve for quantification.
In Vitro Heme Binding and β-Hematin Inhibition. For heme-
drug binding studies, a 1 mM stock solution of chloroquine or
test compound was prepared in distilled water, methanol, or
dimethyl sulfoxide (DMSO), depending on solubility, and soni-
cated to ensure complete dissolution. A 5 mM stock solution of
heme was prepared by dissolving heme chloride in 0.1 mM
NaOH by incubating at 37 ꢀC for 30 min. The solution was
stored at 4 ꢀC for up to 1 month. At the beginning of each experi-
ment, the stock heme solution was diluted to 5 μM in phosphate
buffer (100 mM, pH 5.7) and allowed to equilibrate for 4 h. The
4 h equilibration allowed for the initial heme absorbance to
stabilize prior to beginning the titration. Optical titrations with
each compound were performed by successive addition of
aliquots of its stock solution to the 5 μM heme solution. The
pH was monitored throughout the procedure with only negli-
gible ((0.05 pH units) changes. Equilibrium binding constants
were determined by nonlinear least-squares analysis.41
Hemin chloride (16.3 mg) was dissolved in 1 mL of DMSO.
The solution was passed through a 0.2 μm pore membrane filter
to remove insoluble particles and kept at 4 ꢀC for up to 1 month
as a stock solution.42 In order to determine heme concentration
of the stock solution, a sample was diluted in 2.5% sodium
dodecyl sulfate in 0.1 M NaOH and an absorbance reading
taken at 400 nm. The heme concentration was calculated using
Beer’s law with a molar absorptivity ε = 105 mol L-1 cm-1. The
optimal heme and Tween-20 concentrations for promoting
heme crystallization were calculated by the procedure described
by Huy.43 The RCQ compounds were screened for their inhibi-
tory capacity, and IC50 values were determined. Assays were run
in duplicate twice. Incubations were conducted in the dark to
ensure that light did not interfere. A series of solutions were
made consisting of 300 μL of varying concentrations of the
compound under study in distilled water, 700 μL of 1 M acetate
buffer, 300 μL of a 200 μM heme solution freshly buffered by
1 M sodium acetate (pH 4.8), and 200 μL of 0.0375 g/L Tween-20
solution. This provided a final 40 μM heme solution buffered by
0.67 M sodium acetate at pH 4.8 and 0.0005 g/L Tween20, with
the test compound ranging in concentration from 0 to 1000 μM.
The mixtures were incubated for 24 h at 37 ꢀC,35 then mixed and
transferred to a cuvette for a 415/630 nm absorbance reading.
IC50 values were calculated by (Dmax - Dinitial)/2 where Dmax
represents the lowest concentration of compound under study to
provide maximal absorbance readings indicating maximal free
heme, and Dinitial represents the lowest concentration of drug to
provide any increase in absorbance over a solution with no drug.
In Vivo Hemozoin Inhibition Experiment. P. falciparum strains
D6 and Dd2 were synchronized to the ring stage (early
trophozoites) with 5% sobitol solution.24 After the synchroni-
zation of the parasites, the erythrocytes were suspended in
culture medium at 1% hematocrit and aliquots of 1 mM stock
solution of drug were added to the culture flasks. Drug treated
cultures, along with the no-drug control culture, were incubated
for 24 h at 37 ꢀC under a gas mixture of 5% O2, 5% CO2, and
90% N2 and then transferred to 15 mL centrifuge tubes. One
2 μL aliquot was used from each tube to obtain Giemsa-stained
smear for determination of the parasitemia and morphology
examination by microscopy.
normalized IC50ðRCQ compound ðD6ÞÞ
¼ ½6:9=IC50ðCQ ðD6ÞÞꢁ ꢀ IC50ðRCQ compound ðD6ÞÞ
Mouse Efficacy against P. berghei. Compounds were formu-
lated in a solution consisting of 70% Tween-80 (d = 1.08 g/mL)
and 30% ethanol (d = 0.81 g/mL), followed by a 10-fold dilu-
tion in water. On day 0, heparinized blood (containing 100 μL of
200 u/mL heparin) was taken from a donor NMRI mouse with
approximately 30% parasitemia. The blood was diluted in
physiological saline to 108 parasitized erythrocytes per milliliter.
From this suspension 0.2 mL was injected intravenously (iv) into
experimental groups of three female NMRI mice and a control
group of five mice. Compounds were administered in a volume
of 10 mL/kg either as single dose 24 h after infection (day 1)
either by oral gavage (po) or subcutaneous injection or as four
consecutive daily po doses 4, 24, 48, and 72 h after infection
(days 0-3).
On day 3 (with the single-dose regimen) or on day 4 (with the
quadruple-dose regimen), 1 μL of tail blood was taken and dis-
solved in 1 mL of PBS buffer. Parasitemia was determined with a
FACScan (Becton Dickinson) by counting 100 000 RBCs. 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). Animals
receiving no compound would die typically 5-6 days postinfec-
tion and were therefore euthanized right after determination of
parasitemia. The survival of the animals was monitored up to 30
days. Mice surviving for 30 days were checked for parasitemia
and subsequently euthanized. A compound was considered
curative if the animal survived to 30 days postinfection with
no detectable parasites by microscopy, with a detection limit of
1 parasite in 10 000 erythrocytes (that is, 0.01%).
Accumulation Experiment. Synchronized PRBCs were ob-
tained following two cycles of sorbitol-induced lysis of an
asynchronous stock culture. Incubation for an additional
20-24 h provided a population of mature trophozoites that
were added to the culture medium at 2% v/v (about/10%
parasitemia).
An aliquot of a 10 mM solution of 1 was added to a culture
flask containing 5 mL of PRBCs suspended in complete medium
(10% parasitemia) such that the initial medium concentration of
1 was ∼5 μM. Samples were removed from the flask at various
intervals and centrifuged; the supernatant fluid was then re-
moved and refrigerated before analysis. 1 was added to flasks
containing both CQS D6- and CQR Dd2-infected RBCs and, as
a control, to a flask containing uninfected RBCs. For the
purpose of estimating the amount of 1 accumulated within the
DV, NH4Cl (10 mM) was added 10 min prior to sampling in
order to basify the acidic subcellular compartments and cause
the release of accumulated 1. The samples were analyzed by
reverse-phase HPLC, using a C18 column, eluting with an
isocratic mixture of 75% acetonitrile/25% 5 mM phosphate
buffer (pH 11). A parallel experiment was performed with CQ,
also at ∼5 μM, for comparative purposes. With these conditions
Parasites were isolated by one freeze-thaw cycle at -20 ꢀC
and treated with a saponin-containing lysis buffer (Tris (20 mM,
pH 7.5), EDTA (5 mM), saponin (0.008%, w/v), and Triton
X-100 (0.08%, v/v)40 at 37 ꢀC for 30 min. The hemozoin was
pelleted by centrifugation at 215,000 g for 30 min at 25 ꢀC. The
supernatant was removed, and the pellet consisting primarily of
hemozoin was washed two times with acetone to remove re-
sidual proteins. Insoluble material was then washed three times
with PBS (pH 7.4) buffer and collected by centrifugation at
215000g for 30 min at 25 ꢀC. The pellet was dissolved with 0.2 N

6488 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Burgess et al.
sodium hydroxide for 2 h at 37 ꢀC and periodic mixing of the
sample. The absorbance at 400 nm was measured, and the
amount of heme was calculated using an extinction coefficient
(15) Kelly, J. X.; Smilkstein, M. J.; Cooper, R. A.; Lane, K. D.;
Johnson, R. A.; Janowsky, A.; Dodean, R. A.; Hinrichs, D. J.;
Winter, R.; Riscoe, M. Design, synthesis, and evaluation of 10-
N-substituted acridones as novel chemosensitizers in Plasmo-
dium falciparum. Antimicrob. Agents Chemother. 2007, 51, 4133–
4140.
(16) Bhattacharjee, A. K.; Kyle, D. E.; Vennerstrom, J. L.; Milhous,
W. K. A 3D QSAR pharmacophore model and quantum chemical
structure-activity analysis of chloroquine (CQ)-resistance rever-
sal. J. Chem. Inf. Model. 2002, 42, 1212–1220.
(17) Burgess, S. J.; Selzer, A.; Kelly, J. X.; Smilkstein, M. J.; Riscoe,
M. K.; Peyton, D. H. A chloroquine-like molecule designed to
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(18) Andrews, S.; Burgess, S. J.; Skaalrud, D.; Kelly, J. X.; Peyton,
D. H. Reversal agent and linker variants of reversed chloroquines:
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(19) Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff,
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of 91 000 cm-1 M-1 36-38
The amount of heme per parasitized
.
erythrocyte was calculated on the basis of the number of
erythrocytes in the culture and the percent parasitemia obtained
after growing synchronized culture for 24 h. Cultures at 0 h
processed with the identical method were used for determination
of the baseline hemozoin production.
Acknowledgment. The authors thank the following for
supporting this research: The Medical Research Foundation
of Oregon (Grant 0530) and the National Institutes of Health
(Grants AI067837 and AI072923) to D.H.P., as well as the
Murdock Charitable Trust for a grant for the NMR instru-
ments. The authors also thank Westin Morrill for his help in
preparing this manuscript.
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