Enone- and chalcone-chloroquinoline hybrid analogues: In silico guided design, synthesis, antiplasmodial activity, in vitro metabolism, and mechanistic studies

Article abstract of DOI:10.1021/jm200149e

Analogues of the previously reported antimalarial hybrid compounds 8b and 12 were proposed with the aim of identifying compounds with improved solubility and retained antimalarial potency. In silico characterization predicted improved solubilities of the analogues, particularly at low pH; they retained acceptable predicted permeability properties but were predicted to be susceptible to hepatic metabolism. These analogues were synthesized and found to exhibit notable in vitro antimalarial activity. Compounds 25 and 27 were the most active of the analogues. In vitro metabolism studies indicated susceptibility of the analogues to hepatic metabolism. There was also evidence of primary glucuronidation for analogues 24-27. Presumed cis-trans isomerism of 12, 22, and 23 under in vitro metabolism assay conditions was also observed, with differences in the nature and rates of metabolism observed between isomers. Biochemical studies strongly suggested that inhibition of hemozoin formation is the primary mechanism of action of these analogues.

Full text of DOI:10.1021/jm200149e

ARTICLE
pubs.acs.org/jmc
Enoneꢀ and ChalconeꢀChloroquinoline Hybrid Analogues:
In Silico Guided Design, Synthesis, Antiplasmodial Activity,
in Vitro Metabolism, and Mechanistic Studies
Eric M. Guantai,†,‡ Kanyile Ncokazi,† Timothy J. Egan,† Jiri Gut,§ Philip J. Rosenthal,§ Ravi Bhampidipati,||
Anitha Kopinathan,|| Peter J. Smith,‡ and Kelly Chibale*,†,^
†Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
‡Division of Pharmacology, University of Cape Town, Observatory 7925, South Africa
§Department of Medicine, San Francisco General Hospital, University of California at San Francisco, San Francisco,
California 94143, United States
Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus),
381 Royal Parade, Parkville, Victoria 3052, Australia
^Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
S Supporting Information
b
ABSTRACT: Analogues of the previously reported antimalarial
hybrid compounds 8b and 12 were proposed with the aim of
identifying compounds with improved solubility and retained
antimalarial potency. In silico characterization predicted im-
proved solubilities of the analogues, particularly at low pH; they
retained acceptable predicted permeability properties but were
predicted to be susceptible to hepatic metabolism. These
analogues were synthesized and found to exhibit notable in
vitro antimalarial activity. Compounds 25 and 27 were the most active of the analogues. In vitro metabolism studies indicated
susceptibility of the analogues to hepatic metabolism. There was also evidence of primary glucuronidation for analogues 24ꢀ27.
Presumed cisꢀtrans isomerism of 12, 22, and 23 under in vitro metabolism assay conditions was also observed, with differences in
the nature and rates of metabolism observed between isomers. Biochemical studies strongly suggested that inhibition of hemozoin
formation is the primary mechanism of action of these analogues.
’ INTRODUCTION
and relate these to easily measured or calculated physicochemical
and structural features of the compounds.11,12 In silico (compu-
tational) ADME prediction models and tools therefore comple-
ment in vitro and in vivo ADME experiments by facilitating the
characterization of compounds prior to their synthesis,9,11 there-
by focusing synthetic and experimental evaluation efforts on a
smaller number of particularly promising compounds. However,
the results from such predictions should, whenever possible, be
corroborated by in vitro and/or in vivo ADME assays.
For drugs to treat uncomplicated malaria, oral administration
is practically mandatory, as huge numbers of patients across the
developing world must be treated in facilities with limited
resources or infrastructure.10,13 The proportion of an orally
administered drug that is delivered into the systemic circulation
(i.e., its oral bioavailability) is heavily dependent on three primary
factors: solubility, permeability, and metabolic stability.14,15 The
in silico prediction and/or in vitro determination of these three
factors is therefore highly valuable in antimalarial drug design.
The World Health Organization (WHO) estimates that over
200 million episodes of malaria occur annually.1,2 These result in
0.6ꢀ1.2 million deaths a year, nearly all caused by Plasmodium
falciparum, with ∼85% of these deaths being of children below
the age of 5 years in sub-Saharan Africa.1 P. falciparum, the most
virulent species of the malaria parasite, has developed varying
degrees of resistance to many classes of drugs,3,4 including
possibly artemisinin-derived antimalarials.5,6 Thus, the develop-
ment of new, highly efficacious drugs to treat malaria remains a
key priority.7
Antimalarial drug discovery, and indeed drug discovery in
general, faces a myriad of challenges. For example, it is widely
acknowledged that challenging physicochemical and absorption,
distribution, metabolism, and excretion (ADME) properties
significantly hamper the development of many promising com-
pounds into leads and drug candidates.8,9 This has led to an
increased interest in the early determination (and/or prediction)
of ADME properties, with simultaneous filtering in vitro for
potency.8,10 The use of large compound and data sets has
dramatically increased the ability to model biological processes
Received: February 9, 2011
Published: April 18, 2011
r
2011 American Chemical Society
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Figure 1. Core structural features of 8b and 12.
Figure 2. Structures of analogues of 8b and 12 derived from replacement of the triazole linker.
In this study, in silico prediction tools were applied in the
characterization of a series of analogues proposed for synthesis.
These analogues were based on the promising antimalarial
chalconeꢀchloroquinoline hybrid compound 8b (and the lower
molecular weight intermediate 12) (Figure 1), both of which
were identified and reported previously.16 One of the main
concerns about compound 8b was its poor aqueous solubility,
which necessitated the use of up to 10% DMSO as co-solvent and
extended periods of sonication and agitation during the initial
dissolution of the compounds in the preparation of the stock
solutions for in vitro evaluation. The compound was thus
anticipated to have poor oral bioavailability.
The core structural features thought to be responsible for the
observed antimalarial activity of 8b and 12 and which were
therefore to be retained in the analogues were identified as the
chloroquinoline moiety and either the phenylbutenone moiety
(for 12) or the chalcone moiety (for 8b) (Figure 1): the
chloroquinoline moiety for its role in inhibition of hemozoin
formation17 and the chalcone and phenylbutenone moieties for
their possible dual roles in malarial cysteine protease inhibition18
and facilitating inhibition of hemozoin formation through πꢀπ
stacking interactions.
A small series of analogues of 8b and 12 was therefore proposed
based on the replacement of the triazole linker. These proposed
analogues 20ꢀ27 (Figure 2) contained aminoalkyl- or piperazi-
nyl-based linkers, thereby introducing the 4-amino substitution on
the chloroquinoline subunit and resulting in more polar com-
pounds with potentially enhanced (pH-dependent) solubility. In
addition, the piperazinyl-based linkers possess an additional pro-
tonatable nitrogen, which should further enhance the ionization of
the analogues bearing this type of linker. Analogous features are
present in chloroquine and amodiaquine, both of which are
established chloroquinoline antimalarials that possess a 4-amino
substitution and a second protonatable nitrogen and which have
been successfully formulated as water-soluble salts.
The in silico characterization of the proposed analogues and
their subsequent synthesis and biological evaluation are reported
and discussed herein.
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Figure 3. (A) Plots of predicted solubility (expressed as the logarithm, log Sol.) against the predicted n-octanolꢀwater partition coefficient (also
expressed as the logarithm, log D) at pH 5.0 and at pH 7.5. (B) Plots showing the proposed analogues projected onto the PLS models used to predict
Caco2 permeability and metabolic stability. The black dots represent the compounds comprising the models’ training data sets. The yellow dots
represent the test compounds. The blue regions indicate acceptable predicted permeability/metabolic stability, while the red zones indicate poor
predicted properties.
’ RESULTS AND DISCUSSION
attempt was made not to overinterpret the prediction results but
rather to use them as a guide in determining whether or not the
proposed compounds were likely to be improvements on the parent
compounds 8b and 12 and to highlight any potential liabilities.
Figure 3A shows the logarithm of the predicted aqueous
solubility (log Sol.) plotted against the predicted n-octanolꢀ
water partition coefficient (log D) at approximately physiological
pH (pH 7.5) and at pH 5.0 for the compounds of interest.
Chloroquine and amodiaquine showed significantly superior
predicted aqueous solubility at both pH values when compared
to 8b, 12, and the proposed analogues. At both pH 5.0 and pH
7.5, 8b showed the lowest predicted solubility; 12 was predicted
to be more soluble than 8b most likely because of its lower
molecular weight. As expected, a similar trend was seen for the
proposed compounds, whereby analogues of 12 were predicted
to be more soluble than the corresponding analogues of 8b.
In Silico Characterization of 8b, 12, and the Proposed
Analogues. Three software packages, with varying but comple-
mentary capabilities, were applied for the in silico prediction of
various parameters of interest. VolSurfþ was applied to predict
physicochemical and ADME parameters of the compounds, such
as aqueous solubility (at various pHs), Caco2 permeability, and
metabolic stability.19,20 MetaSite was applied to predict sites of
metabolism by hepatic cytochrome P450 enzymes and the
structures of possible metabolites.21,22 MoKa was used to predict
the pKa of ionizable compounds.23 These three software packages
are available from Molecular Discovery (http://www.moldiscovery.
com/software.php).
The chloroquinoline-based antimalarials chloroquine and amo-
diaquine were selected as reference compounds and were character-
ized with and compared to the study compounds. A deliberate
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Figure 4. Output from MetaSite showing the predicted sites of metabolism on the various analogues.
At pH 7.5, the predicted solubilities of the proposed analogues
were only slightly higher than those of the parent compounds.
However, as anticipated, there was a significant influence of pH
on the predicted solubilities, which were significantly higher at
pH 5.0 than at pH 7.5. This difference is attributable to the fact
that these basic compounds would exist predominantly in their
ionized state at lower pH levels, rendering them more soluble.
Analysis with MoKa predicted that compounds 20ꢀ27 were
>90% ionized at pH 5.0.
The number of protonatable centers was also predicted to
influence pH-dependent solubility. For example, among the
analogues of 12, the piperazinyl analogues 22, 24, and 26
(which have two protonatable centers) had higher predicted
solubilities at pH 5.0 than the aminoethoxy analogue 20, despite
the fact that these three piperazinyl derivatives have higher
molecular weights than 20. A similar trend was observed for
the analogues of 8b, and both observations can be attributed to
the positive influence on solubility of the higher charge-to-mass
ratio afforded by the diprotonation of the piperazinyl analogues.
Prediction of Caco2 permeability was qualitative. When
projected onto the two-dimensional PLS plot of the database
of compounds used to generate the Caco2 permeation model, all
the compounds (along with chloroquine and amodiaquine)
showed acceptable predicted Caco2 permeation, falling within
the blue (permeable) zone of the plot (Figure 3B).
compounds. 8b was predicted to be less stable to hepatic
metabolism than 12. The proposed analogues were also shown
to have relatively low predicted metabolic stabilities, particularly
compounds 22, 23, and 27. Site-of-metabolism analysis was
carried out on MetaSite in an attempt to shed light on these
findings (Figure 4).
The main route of metabolism for 8b was predicted to be
O-demethylation of the p-methoxy group on ring B, as indicated
by the blue ring highlighting this group; this group is absent in 12.
O-Demethylation on ring A and N-oxidation of the quinoline
ring were the main routes of metabolism predicted for 12, but
these may not be as efficient as O-demethylation on ring B, and
this is probably why this compound was predicted to be more
stable to metabolism than 8b.
N-Dealkylation of the nitrogen-containing side chain was the
principal route of metabolism for chloroquine and amodiaquine
predicted by MetaSite. This result is in agreement with reported
experimental findings, with their major metabolites identified as
N-desethylchloroquine and N-desethylamodiaquine, respecti-
vely.24,25 N-Oxidation of the quinoline nitrogen, although high-
lighted by MetaSite as a possible site of metabolism, is not a
favored route of metabolism for this class of compounds.
Unsurprisingly, N-dealkylation of the nitrogen-containing
linkers was predicted as a possible route of metabolism for the
proposed analogues. The piperazinyl-based linkers present in
22ꢀ27 were predicted as being particularly susceptible to
N-dealkylation, possibly explaining why these compounds were
predicted to be relatively unstable. This factor and the additional
possibility of O-demethylation on ring B most likely explain why
Amodiaquine and chloroquine were predicted to have inter-
mediate metabolic stability, falling somewhat between the blue
(stable) and red (unstable) zones on the PLS plot (Figure 3B);
they were predicted to be more stable to metabolism than the test
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compound 23 was predicted to be the least stable of the
analogues. The aminoethoxy linker present in 20 and 21 appears
to be somewhat less susceptible to N-dealkylation and was not
predicted as the primary target of metabolism for these com-
pounds, which were predicted to be the most stable of the studied
analogues.
the analogues were synthesized for in vitro antimalarial evalua-
tion to determine if they retained antimalarial activity.
The main alert for the analogues was predicted metabolic
instability, primarily due to N-dealkylation of the nitrogen-
containing linkers or O-demethylation of ring B of the chalcone
derivatives.
In summary, compounds 8b and 12 showed poor predicted
solubility profiles, suggesting limitations in oral bioavailability.
They had acceptable predicted Caco2 permeation when com-
pared to the reference compounds chloroquine and amodia-
quine, though their metabolic stability was predicted to be lower.
The proposed analogues had improved predicted solubilities
relative to their parent compounds, particularly at lower pH, and
this was a feature that could possibly be utilized during the
solubilization of these analogues for in vivo pharmacokinetic
(PK) studies. The analogues also maintained acceptably high
predicted Caco2 permeability. On the basis of these predictions,
Synthesis. The hydroxylated enones (15a and 15b) and
chalcones (16a and 16b) were intermediates in the synthesis of
these compounds; the synthesis of these intermediates is depicted
in Scheme 1 and involved the acid-catalyzed ClaisenꢀSchmidt
reaction.
Vanillin or 4-hydroxybenzaldehyde was stirred in acetone (as
reagent and solvent) in the presence of aqueous 5 M HCl at room
temperature for 24 h. The crude product of the reaction was
purified by silica column chromatography to furnish the pure 15a
and 15b in good yields. The synthesis of the chalcones 16a and
16b involved the reaction of vanillin or 4-hydroxybenzaldehyde
with 1.1 equiv of 20,40-dimethoxyacetophenone in methanol in
the presence of aqueous HCl as catalyst. The crude products
were then purified by silica gel column chromatography to
furnish the pure chalcone intermediates in average yields.
Scheme 2 shows the synthesis of the target analogues 20ꢀ23.
The required hydroxylated quinoline intermediates 17 and 18
were afforded by typical aromatic substitution reactions using
the (modified) method reported by Singh et al.26 Briefly, 4,7-
dichloroquinoline was heated with excess ethanolamine or with 1
equiv of 1-(2-hydroxyethyl)piperazine in the presence of potas-
sium carbonate to yield the target intermediates in moderate to
good yield after purification by crystallization. The intermediates
17 and 18 were then coupled to either 15a or 16a to yield the
target compounds 20ꢀ23. This coupling was achieved by apply-
ing the Mitsunobu reaction, a convenient, one-step SN2 reaction
using 1.2 equiv of triphenylphosphine (PPh3) and 1.2 equiv of
diisopropyl azodicarboxylate (DIAD) as described by Winssinger
et al.27
Scheme 1a
a Reagents andconditions: (i) acetone, 5 M HCl, room temp, 24 h; (ii) 20,
The synthesis of the proposed analogues 24ꢀ27 (Scheme 3)
40-dimethoxyacetophenone, 5 M HCl, MeOH, 50 ꢀC, 24 h.
began with the synthesis of the piperazinyl intermediate 19 via an
Scheme 2a
a Reagents and conditions: (i) ethanolamine, 130 ꢀC, 5 h; (ii) 1-(2-hydroxyethyl)piperazine, K2CO3, DMF, 80 ꢀC, 24 h; (iii) 15a, 15b, 16a or 16b, PPh3,
DIAD, DCM, 0 ꢀC to room temp, 6 h.
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Scheme 3a
a Reagents and conditions: (i) piperazine, K2CO3, NMP, 135 ꢀC, 4 h; (ii) 15a, 15b, 16a or 16b, HCHO, EtOH, reflux at 110 ꢀC, 12 h.
Mannich reaction was then applied to furnish the target analo-
gues using the (modified) method described by Chibale et al.28
This involved the refluxing of the appropriate phenolic inter-
mediate (15 or 16) and 1 equiv of 19 in ethanol in the presence of
10 equiv of aqueous formaldehyde for 12 h. The crude products
were then purified by silica gel column chromatography to afford
the pure target compounds in average yields, which could be
partly attributed to the fact that these reactions did not proceed
to completion even with the use of a large excess of formaldehyde
or after extended refluxing.
Table 1. In Vitro Antiplasmodial Activities against the D10,
Dd2, and W2 Strains of P. falciparum (IC50, μM)
IC50, μMa
compd
15a
D10
Dd2
W2
>20
5.3
2.6
5.2
0.7
1.0
0.8
1.2
1.2
1.0
0.5
1.7
0.6
0.8
0.04
0.017
>20
7.3
1.0
3.8
0.9
1.5
1.1
1.0
0.5
1.7
0.5
3.1
0.5
0.7
0.07
0.097
>10
>10
0.2
16a
17
18
1.0
1
All compounds were characterized by H NMR, 13C NMR,
19
0.3
low resolution mass spectrometry, IR spectroscopy, elemental
analysis, and melting point determination.
20
0.1
21
0.4
In Vitro Antiplasmodial Assay Results and Discussion. The
intermediates and target compounds were evaluated for their in
vitro antiplasmodial activity against the chloroquine sensitive
strain D10 and chloroquine resistant strains Dd2 and W2 of
P. falciparum (Table 1).
The hydroxylated enone intermediate 15a showed negligible
antiplasmodial activity in vitro across all three strains of P.
falciparum. However, the chalcone intermediate 16a showed
modest antiplasmodial activity against two of the three tested
strains. The chloroquinoline-based intermediates 17ꢀ19 exhib-
ited more potent antiplasmodial activity in vitro, with 19 showing
submicromolar IC50 values against all three strains.
22
0.5
23
0.6
24
0.5
25
0.4
26
0.7
27
0.3
12
ND
0.09
0.069
8b
chloroquine
a Average values from two independent determinations, each carried out
in duplicate.
All the target analogues exhibited notable antiplasmodial
activity, with IC50 values in the lower micromolar to mid-
nanomolar range. The related analogues 25 and 27 were the
most active of the analogues; both are chalcone derivatives with
piperazinyl linkers and had IC50 of 0.3ꢀ0.6 μM against all three
strains of P. falciparum. Compound 23 was also quite active, with
aromatic substitution reaction of 4,7-dichloroquinoline and 6
equiv of piperazine in the presence of potassium carbonate, using
the (modified) method reported by Singh et al.26 Purification by
silica gel column chromatography afforded 19 in good yield. The
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Table 2. Results from the in Vitro Metabolism Studies
degradation half-life (min)
microsome-predicted extraction ratio (EH)
compd
human
mouse
human
mouse
metabolites detecteda
8b
12
51.1
7.4
56.8
10.8
0.65
0.93
0.57
0.88
P þ 16
not detected
peak 1
peak 2
9.1
10.5
17.5
14.2
9.8
6.2
13.4
12.7
6.7
0.91
0.90
0.85
0.87
0.91
0.89
0.51
0.85
0.72
0.93
0.62
<0.28
0.92
0.85
0.86
0.92
0.93
0.89
0.34
0.84
0.87
0.84
0.63
0.31
20
21
22
P ꢀ 218
P þ 16 (ꢁ2), P þ 32 (ꢁ2), P ꢀ 14, P þ 16 þ 176
P ꢀ 14, P ꢀ 218
peak 1
peak 2
peak 1
peak 2
5.6
23
11.5
93.2
16.9
37.0
7.5
9.8
P þ 16 (ꢁ4), P ꢀ 14 (ꢁ2), P ꢀ 340
149.8
14.0
11.8
15.0
45.4
177.0
24
25
26
27
P þ 16, P ꢀ 204, P þ 176
P ꢀ 14 (ꢁ2), P ꢀ 326, P þ 176
P þ 16, P ꢀ 174, P þ 176
P þ 16 (ꢁ2), P ꢀ 14, P ꢀ 296, P þ 176
not undertaken
58.6
>250
chloroquine
a As represented by fragments detected after mass spectroscopy analysis and that had m/z corresponding to putative metabolites. P represents the
molecular mass of the parent compound. The interpretation of the results is contained in the section “Putative Metabolite Identification”.
mid-nanomolar IC50 against the Dd2 and W2 P. falciparum
strains.
chromatographic LCꢀMS analysis, most likely indicating cisꢀ
trans isomerism occurring under assay conditions. The rates of
degradation for each individual peak were therefore determined.
With respect to metabolic stability, the compounds exhibited
varying degradation half-lives in human and mouse microsomes;
however, the calculated hepatic extraction ratios (EH values)
were similar between the species. EH values take into account the
different hepatic blood flow rates between the species. The
similarities in these values suggest no significant interspecies
differences in the relative rates of metabolism.
Most of the compounds exhibited high EH values (>0.7),
suggesting notable susceptibility to hepatic metabolism. This is
particularly apparent when compared to the relatively low values
for chloroquine (e0.31) and is in agreement with the prediction
that this set of compounds would be less stable than chloroquine.
Notably, 8b and 27 had intermediate EH values, as did the isomer
of 23 corresponding to peak 2, indicating better metabolic
stability than that of the other tested compounds.
The majority of the test compounds showed no measurable
degradation in the microsomal matrix devoid of cofactors (i.e.,
controls), suggesting that there was no major non-cofactor-
dependent metabolism contributing to their rate of metabolism
in liver microsomes. The only exceptions were 12 (peak 2) and
22 (peak 2), both of which demonstrated apparent non-cofactor-
dependent degradation (which was not observed for the other
isomer), potentially indicating a stereospecific process.
As noted above, the two peaks observed for 12, 22, and 23
during chromatographic LCꢀMS analysis suggested that there
was possible cisꢀtrans isomerism of these compounds under
assay conditions. No obvious structural features specific to these
compounds that would selectively predispose them to isomerism
could be identified. The results suggest that one isomer of a
compound could be more susceptible than the other to meta-
bolism (e.g., compound 23) or to additional routes of metabo-
lism (e.g., peak 2 of compounds 12 and 22 to non-cofactor-
dependent hepatic metabolism). This nonuniform but notable
influence of isomerism on biological predisposition and the fact
Intermediate 19 was predicted by MetaSite as the main
metabolite of 24ꢀ27 (arising from N-dealkylation of the linker),
and the fact that it exhibited notable in vitro antiplasmodial
potency implies that this intermediate/metabolite may contri-
bute to in vivo antimalarial activity of these analogues and
thereby offset any potential liabilities of these compounds arising
from susceptibility to metabolism via this route.
In summary, the replacement of the triazole linker in 8b and 12
with the selected nitrogen-containing linkers was associated with
retention of antiplasmodial activity, though none were as active
as 8b.
In Vitro Metabolic Stability. The metabolic stability assay was
performed by separately incubating test compounds at 37 ꢀC (in
duplicate, substrate concentrations 1 μM) with human or mouse
liver microsomes (0.4 mg/mL protein concentration). The
reaction was initiated by the addition of an NADPH-regenerating
system (containing 1 mg/mL NADP, 1 mg/mL glucose 6-phos-
phate, 1 U/mL glucose 6-phosphate dehydrogenase) and
quenched at various time points over the incubation period by
the addition of acetonitrile. NADPH is the cofactor required for
cytochrome P450-mediated metabolism. Additional samples
with the dual cofactors NADPH and UDPGA (the latter the
cofactor for glucuronidation) were included in the incubations
for qualitative assessment of the potential for glucuronide for-
mation. Control samples (containing neither NADPH nor
UDPGA) were also included to monitor for potential degrada-
tion in the absence of cofactors. The concentrations of the
substrates after the various periods of incubation were quantified
against standard curves prepared in blank microsomal matrix by
LCꢀMS using a Waters/Micromass ZQ mass spectrometer
coupled to a Waters Alliance 2975 HPLC and an Ascentis
Express C18 column maintained at 40 ꢀC. Data processing was
performed using Quanlynx software.
The results of the in vitro metabolism studies are summarized
in Table 2. Two peaks were observed for 12, 22, and 23 during
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Table 3. Results from the Experimental Determination of
Solubility and Partition Coefficients
log D a
pH 7.4
solubility (μg/mL)
pH 2.0 pH 6.5
compd
pH 3.0
8b
12
20
21
22
23
24
25
26
27
4.3
3.5
1.8
2.8
1.2
2.2
0.9
2.1
0.8
2.0
0.4
4.3
3.6
3.0
4.2
4.2
5.3
3.9
5.0
4.2
5.3
2.0
<1.6
<1.6
Figure 5. Structures of the putative metabolites of 22ꢀ27 (A) and
20 (B).
3.1ꢀ6.3
50.0ꢀ100.0
>100
3.1ꢀ6.3
6.3ꢀ12.5
3.1ꢀ6.3
1.6ꢀ3.1
<1.6
>100
that such isomerism may be random and unpredictable even
among closely related compounds underscored the need for
close attention when studying compounds containing systems
potentially capable of isomerization, such as the R,β unsaturated
system present in enones and chalcones.
Putative Metabolite Identification. The MS scan data from
the metabolic stability assay of each compound were screened for
the presence of fragments corresponding to putative metabolites;
data acquisition was conducted in MS scan mode (spectra
acquired from 100 to 1000 Da). In the absence of authentic
metabolites, the chromatographic conditions in each case were
optimized for each parent compound and the identities of the
putative metabolites were not confirmed by dedicated MSꢀMS
experiments.
With respect to routes/sites of metabolism, the metabolite
profiles of all the examined compounds were found to be similar
across the two species tested. Oxygenation (P þ 16, P þ 32),
O-demethylation (P ꢀ 14) and N-dealkylation (P ꢀ 174, P ꢀ
218, P ꢀ 240, P ꢀ 296, P ꢀ 326, P ꢀ 326) appeared to be the
predominant pathways, where P is the molecular mass of the
parent compound.
This was consistent with the site-of-metabolism predictions
derived from MetaSite (Figure 4), where these three were the
major predicted routes of metabolism. However, there were
differences between the predicted routes of metabolism and
detected metabolites for some of the compounds; for example,
no predicted O-demethylation metabolites were detected for 8b
and 20 and no N-dealkylation metabolite was detected for 21.
For compounds with the piperazine linkers (22ꢀ27) a common
metabolite with m/z of 248 Da was found (i.e., P ꢀ 218 [22]; P ꢀ
340 [23]; P ꢀ 204 [24]; P ꢀ 326 [25]; P ꢀ 174 [26]; and P ꢀ
296 [27]). This was consistent with N-dealkylation of the side
chain from the piperazine and was confirmation of this route of
metabolism as predicted by MetaSite for all the piperazinyl-based
analogues. For 20, N-dealkylation of the aminoethoxy linker was
observed, resulting in the generation of a metabolite with m/z
179 Da (i.e. P ꢀ 218); this was not observed for 21, which also
bears an aminoethoxy linker. The putative metabolites from
these N-dealkylation reactions are shown in Figure 5.
The potential of the test compounds to undergo primary
glucuronidation was assessed in the microsomal incubation by
the addition of the cofactor UDPGA. There was no major increase
in the rate of degradation of 8b, 20, 21, 23, or chloroquine in
microsomal samples containing NADPH and UDPGA relative to
those containing NADPH alone, suggesting that these com-
pounds were not susceptible to primary glucuronidation. For 12
and 22, the impact of UDPGA on the overall rate of metabolism
could not be assessed, as the rate of NADPH-dependent degra-
dation alone was too rapid. However, for these two compounds,
there were no putative metabolites detected with molecular
weights consistent with glucuronidation.
>100
>100
1.6ꢀ3.1
<1.6
>100
>100
<1.6
>100
>100b
<1.6
>100b
chloroquine
a Value measured using the chromatographic gLogD technique. b Stock
solution of compound prepared in water at 10 mg/mL.
In contrast to the results discussed above, there was evidence
of primary glucuronidation for 24ꢀ27 in the microsomal assay,
where there was an increase in the relative rates of degradation of
test compounds in the presence of UDPGA and/or the detection
of a putative metabolite with a molecular weight consistent with
glucuronide conjugation [P þ 176]. Each of these compounds
bear a free phenolic OH group, and it is therefore not surprising
that they are substrates for glucuronidation. This route of
metabolism was not predicted by MetaSite, which only predicts
cytochrome P450-mediated phase I metabolism.
Physicochemical Properties: Solubility and Partition Coef-
ficients. The partition coefficients (log D) of the test compounds
were estimated using a modification of the method described by
Lombardo and co-workers29 (Table 3). The series exhibited
moderate to high partition coefficients, with measured log D7.4
values ranging from 3.0 to 5.3. Under acidic conditions (pH 3.0),
the majority of compounds showed a reduction in log D, con-
sistent with ionization of the weakly basic groups; however, the
log D values of the neutral 8b and 12 were unchanged across the
pH range tested. The above predictions were consistent with the
in silico predicted results. Compounds 24 and 26 had notably
lower log D3.0, reflecting the dual ionization of the piperazinyl
and quinoline nitrogens under acidic conditions and their lower
molecular weights (relative to 25 and 27, respectively). The
partition coefficients for chloroquine were lower than those
observed for the novel compounds.
The solubility of the test compounds was estimated using the
turbidimetric method described by Bevan and Lloyd,30 applying
standard test buffers (pH 2.0 and pH 6.5). As predicted, the
solubility characteristics of the series were relatively poor at pH
6.5, with only 20 showing moderate solubility under neutral
conditions (Table 3). Under acidic conditions, compounds
20ꢀ27 showed improved solubility consistent with ionization
of the basic centers within the structures. 8b and 12 showed no
increase in solubility under acidic conditions, consistent with the
neutral characteristics of these triazole substituted compounds.
Chloroquine showed good solubility under both neutral and
acidic conditions.
Mechanistic Studies. A limited investigation was carried out
on the synthesized analogues in an attempt to elucidate their mecha-
nisms of action. The mechanisms considered for investigation
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Table 4. Results from the Mechanistic Studies
15a, 15b, and 16a showed negligible β-hematin inhibition
activity.
β-hematin
falcipain 2, IC50 inhibition of sorbitol-
The analogues showed moderate to poor inhibition of falci-
pain 2 relative to E64, a known, potent inhibitor of the enzyme;
only compound 8b and the analogues 21, 23, 25, 26, and 27 had
lower micromolar IC50 values. The chalcone/enone intermedi-
ates 15 and 16 and the quinoline intermediates 17ꢀ19 did not
show appreciable falcipain 2 inhibition even at 50 μM. The
relatively low potencies of compounds 20, 22, and 24 as falcipain
2 inhibitors suggested that this was not the primary mechanism
of antimalarial action of these compounds. However, the most
potent analogues against falcipain 2, 23, 25, and 27, were among
the most potent against cultured parasites, suggesting that
protease inhibition may play a role in the mechanism of action
of these compounds.
a
compd
15a
formation, IC50
(μM)b
induced hemolysis (%)c
>5
>50
>50
>50
>50
>50
>50
>50
>50
22.9
>50
11.7
>50
9.1
21.1
ND
27.9
ND
0.9
15b
16a
16b
17
>5
>5
1.9
4.9
3.9
>5
18
ND
5.8
19
20
0.6
0.3
0.5
0.2
0.5
0.3
0.5
0.2
1.6
1.91
12.3
ND
ND
ND
16.8
3.9
21
22
All analogues tested were poor inhibitors of sorbitol-induced
lysis, with 24, the most active compound, showing only 17%
inhibition. This result discounts inhibition of NPPs as a mechan-
ism of action for these analogues.
23
24
25
26
29.9
10.0
10.8
11.6
ND
15.4
27
8b
’ SUMMARY AND CONCLUSIONS
chloroquine
E64
8b and 12 were selected for structural modification, with the
aim of identifying analogues with improved solubility and that
retained antimalarial potency. Eight analogues were proposed,
and these were characterized in silico for selected physicochem-
ical and ADME parameters. These analogues were predicted to
have improved solubilities relative to the parent compounds,
particularly at low pH; they retained acceptable predicted Caco2
permeability but were identified as possible substrates for
metabolism, principally N-dealkylation by hepatic cytochromes.
The proposed analogues were then synthesized and tested and
were found to exhibit notable in vitro antiplasmodial activity,
though none were as active as 8b. Compounds 25 and 27 were
the most active of the analogues. 19, an intermediate in the
synthesis (and a putative metabolite) of some of the analogues,
was also found to exhibit notable antiplasmodial activity.
There was no evidence of significant interspecies differences in
the relative rates of microsomal metabolism of these compounds
between mice and humans. However, most of the compounds
exhibited high EH values (>0.7), suggesting considerable sus-
ceptibility to hepatic metabolism. Oxygenation, O-demethyla-
tion, and N-dealkylation appeared to be the predominant
metabolic pathways, which was consistent with the site-of-
metabolism predictions derived from MetaSite. There was also
evidence of primary glucuronidation for 24ꢀ27. The majority of
the compounds showed no susceptibility to metabolism by
enzymes other than hepatic cytochromes and transferases.
Inhibition of hemozoin formation was found to be a likely
mechanism by which the tested analogues exert their antiplas-
modial activity. Analogues 20ꢀ27 were all more potent than
chloroquine in inhibiting β-hematin formation. Inhibition of
falcipain 2 could also contribute to the observed antiplasmodial
activities of some of these compounds, while inhibition of
parasite-induced transport channels did not appear to be a
notable mechanism of action.
0.055
a The IC50 values are averages of triplicate determinations and are
reported as equivalents of the compound (relative to hematin) that
inhibit the formation of β-hematin by 50%. b Average of duplicate
determinations. c Inhibition of sorbitol-induced lysis at 10 μM. ND:
not determined.
were based on literature accounts of the possible mechanisms by
which chalcones and quinoline-containing compounds exert
their antimalarial activities.
Inhibition of hemozoin formation is an attractive target for
antimalarial drugs, and quinoline-based antimalarials such as
chloroquine and amodiaquine are proposed to exert their anti-
malarial activity via this mechanism.17,31,32 The plasmodial
cysteine proteases involved in hemoglobin catabolism, such as
falcipain 2, have also been proposed as potential antimalarial
targets.33,34 By possessing R,β unsaturated carbonyl groups and
thereby able to act as Michael acceptors, chalcones could poten-
tially irreversibly inhibit the thiol-containing malarial cysteine
proteases;18 several quinolinyl chalcones that are moderate
inhibitors of falcipain 2 have been identified.35 Some chalcones
have also been shown to inhibit new permeability pathways
(NPPs), which are parasite-induced solute channels in erythro-
cyte membranes.36 Considering these potential targets, a selec-
tion of intermediates and analogues were evaluated for inhibition
of β-hematin formation, falcipain 2 activity, and NPP function
(sorbitol-induced hemolysis of infected erythrocytes) (Table 4).
Compound 8b, the most potent compound in vitro, was only
slightly more potent (IC50, 1.6 equiv) than chloroquine in
inhibiting β-hematin formation. Of the analogues tested, com-
pounds 20ꢀ27 were all very potent (more potent than
chloroquine) inhibitors of β-hematin formation. Compounds
23 and 27 (which were also some of the more active of these
analogues) had the lowest values of 0.2 equiv. Thus, inhibition of
hemozoin formation may be a primary mechanism of antimalarial
action of these compounds. The intermediates 17ꢀ19 showed
only moderate inhibition of β-hematin formation. Interestingly,
the hydroxylated chalcone 16b showed activity comparable to
that of chloroquine, while the chalcone and enone intermediates
The proposed analogues were by no means exhaustive but
represented compounds that did not represent significant devia-
tions from the molecules known to be active and were therefore
more likely to retain activity. They consisted of a small range of
compounds that that could be conveniently synthesized and
that offered possible improvements on certain physicochemical
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Journal of Medicinal Chemistry
ARTICLE
and/or ADME properties of the parent compounds, particularly
solubility. In addition, they opened up possibilities for further
derivatization in future.
The more promising compounds 25 and 27 were evaluated for
their cytotoxicity against the Chinese hamster ovarian (mammalian)
cell line. No cytotoxicity was observed at the highest concentra-
tion (100 μM) tested, an observation that was consistent with the
findings previously reported for compound 8b.16
132.6, 128.0, 125.1, 122.8, 122.5, 114.8, 110.2, 105.1, 98.8, 55.9, 55.8, 55.5;
LRMS (EI) m/z 314.0 (Mþ). Anal. (C18H18O5) C, H.
2.2. 1-(2,4-Dimethoxyphenyl)-3-(4-hydroxy-3-methoxyphenyl)pro-
penone 16b. Yellow solid (3.2 g, 59%); mp 92ꢀ94 ꢀC (from EtOAc/
Hex); Rf (EtOAc/Hex 4:6) 0.27; IR ν (KBr)/cmꢀ1 3204 (Ar OꢀH),
max
1635 (CdO), 1600 (Ar CdC); δH (400 MHz, CDCl3) 7.53 (2H, d,
J 8.7, H-1, 2), 7.51 (1H, d, J 8.7, H-30), 7.43 (1H, d, J 15.8, H-a), 7.28
(1H, d, J 15.7, H-b), 6.78 (2H, d, J 8.5, H-3, 4), 6.62 (1H, d, J 2.1, H-10),
6.58 (1H, dd, J 2.0 and 8.6, H-20), 3.83 (3H, s, H-40), 3.79 (3H, s, H-50);
δC (100 MHz, CDCl3) 189.9, 164.1, 160.4, 160.3, 142.4, 132.3, 130.8,
126.3, 124.3, 122.3, 116.4, 106.4, 99.2, 56.4, 56.0; LRMS (EI) m/z 284.0
(Mþ). Anal. (C17H16O4) C, H.
’ EXPERIMENTAL SECTION
To guide in the interpretation of the 1H NMR data presented below,
the complete (and numbered) structures of all the intermediates and
target compounds can be found in the Supporting Information. This
material is available free of charge via the Internet at http://pubs.acs.org.
Purity was determined by combustion analysis, and all compounds were
confirmed to have >95% purity.
3. 2-(7-Chloroquinolin-4-ylamino)ethanol 17. 4,7-Dichloro-
quinoline (2 g, 10 mmol) was heated with stirring in neat ethanolamine
(10 mL) at 130 ꢀC for 5 h. The mixture was then allowed to cool to room
temperature, during which the compound 17 precipitated. This crude
mixture was then suspended in water, filtered, and crystallized from hot
MeOH to yield pure 17 as a white solid (1.97 g, 89%). Mp 215ꢀ216 ꢀC
1. General Method A for the Preparation of Compounds
15a and 15b. Vanillin or 4-hydroxybenzaldehyde (20 mmol) was
dissolved in 30 mL of acetone, and 5 mL of 5 M HCl was then added
gradually to the stirring mixture. The reaction mixture was then stirred at
room temperature for 24 h. The reaction mixture was then neutralized
with 2.5 M NaOH, after which the acetone in the mixture was removed
at reduced pressure. The remaining compound mixture was then taken
up in 100 mL of EtOAc, washed three times with water, dried over
anhydrous Na2SO4, concentrated in vacuo, and purified by column
chromatography (EtOAc/hexane) to yield the pure target compound.
1.1. 4-(4-Hydroxy-3-methoxyphenyl)but-3-en-2-one 15a. Pale yellow
solid (3.2 g, 84%); mp 124ꢀ125 ꢀC (from EtOAc/Hex); Rf (EtOAc/
Hex 4:6) 0.30; IR νmax (KBr)/cmꢀ1 3261 (Ar OꢀH), 3000 (Ar CꢀH),
1668 (CdO), 1581 (Ar CdC); δH (400 MHz, CDCl3) 7.46 (1H, d,
J 16.2, H-a), 7.09 (1H, dd, J 1.9 and 8.2, H-2), 7.07 (1H, d, J 1.9, H-1),
6.94 (1H, d, J 8.1, H-3), 6.60 (1H, d, J 16.2, H-b), 5.98 (1H, s, H-5), 3.95
(3H, s, H-4), 2.37 (3H, s, H-b0); δC (100 MHz, CDCl3) 198.3, 150.4,
146.9, 143.5, 126.5, 125.0, 123.8, 114.9, 109.4, 55.8, 24.5; LRMS (EI)
m/z 193.1 (MHþ). Anal. (C11H12O3) C, H.
1.2. 4-(4-Hydroxyphenyl)but-3-en-2-one 15b. Dark yellow solid (2.4 g,
76%); mp 81ꢀ82 ꢀC (from EtOAc/Hex); Rf (EtOAc/Hex 4:6) 0.40; IR
νmax (KBr)/cmꢀ1 3150 (Ar CꢀH), 1666 (CdO), 1590 (Ar CdC); δH
(400 MHz, CDCl3) 7.50 (1H, d, J 16.2, H-a), 7.42 (2H, d, J 8.7, H-1, 2),
6.88 (2H, d, J 8.7, H-3, 4), 6.58 (1H, d, J 16.2, H-b), 2.35 (3H, s, H-b0); δC
(100 MHz, CDCl3) 200.0, 159.1, 144.8, 130.4 (2C), 126.5, 124.3 (2C),
116.2, 27.2; LRMS (EI) m/z 163.3 (MHþ). Anal. (C10H10O2) C, H.
2. General Method B for the Preparation of Compounds
16a and 16b. Vanillin or 4-hydroxybenzaldehyde (20 mmol) and 2,4-
dimethoxyacetophenone (4.0 g, 22 mmol) were dissolved in 20 mL of
MeOH. Then 5 mL of 5 M HCl was added gradually to the stirring
mixture. The reaction mixture was then stirred at 50 ꢀC for 24 h. The
reaction mixture was then allowed to cool to ambient temperature and
neutralized with 2.5 M NaOH, after which the MeOH in the mixture was
removed under reduced pressure. The remaining compound mixture
was then taken up in 100 mL of EtOAc, washed three times with water,
dried over anhydrous Na2SO4, concentrated in vacuo, and purified by
column chromatography (EtOAc/hexane) to yield the pure target
compound.
(fromMeOH) (lit. 214ꢀC)37 Rf (MeOH/EtOAc, 1:9) 0.20; δ (400MHz;
H
DMSO-d6) 8.41 (1H, d, J 5.4, H2), 8.30 (1H, d, J 9.0, H5), 7.81 (1H, d,
J 2.2, H3), 7.46 (1H, dd, J 2.2 and 8.9, H4), 7.26 (1H, t, J 5.2, H-6), 6.52
(1H, d, J 5.5, H1), 4.85 (1H, s, H-9), 3.70 (2H, t, J 5.9, H8), 3.38 (2H, q,
J 5.8, H7).
4. 2-[4-(7-Chloroquinolin-4-yl)piperazin-1-yl]ethanol 18.
4,7-Dichloroquinoline (2 g, 10 mmol) was dissolved in 10 mL of
anhydrous DMF, after which K2CO3 (2.1 g, 15 mmol) and 1-(2-
hydroxyethyl)piperazine (1.3 g, 10 mmol) were added. This reaction
mixture was then stirred at 80 ꢀC for 24 h, upon which TLC indicated
completion of the reaction. The mixture was then diluted in 100 mL of
EtOAc, washed with water (2 ꢁ 30 mL), dried over anhydrous Na2SO4,
and concentrated under reduced pressure. The resultant oily product
crystallized on standing, and these crystals were washed with hexane and
dried to yield 18 as pale yellow crystals (1.83 g, 63%). Mp 111ꢀ112 ꢀC
(from EtOAc); Rf (MeOH/EtOAc, 1:9) 0.24; IR ν (CHCl3)/cmꢀ1
max
3222 (OꢀH), 2903 (CꢀH), 1677 (CdO), 1569 (Ar CdC); δH (400
MHz, DMSO-d6) 8.66 (1H, d, J 5.0, H-2), 7.99 (1H, d, J 9.0, H-5), 7.95
(1H, d, J 2.2, H-3), 7.51 (1H, dd, J 2.2 and 9.0, H-4), 6.96 (1H, d, J 5.1,
H-1), 4.33 (1H, t, J 5.3, H-12), 3.55 (2H, q, J 6.0, H-11), 3.17 (4H, t,
J 4.8, H-6, 9), 2.70 (4H, t, J 4.8, H-7, 8), 2.51 (2H, t, J 6.2, H-10); δC (100
MHz, CDCl3) 156.2, 151.9, 149.6, 133.4, 127.9, 125.9, 125.5, 121.3,
109.1, 60.0, 58.5 (2C), 52.8 (2C), 51.7; LRMS (EI) m/z 290.5 (Mþ).
Anal. (C15H18ClN3O) C, H, N.
5. 7-Chloro-4-piperazin-1-ylquinoline 19. 4,7-Dichloroqui-
noline (2 g, 10 mmol) was dissolved in 15 mL of anhydrous NMP,
after which K2CO3 (2.8 g, 20 mmol) and piperazine (5.2 g, 60 mmol)
were added. This reaction mixture was then stirred at 135 ꢀC for 4 h,
upon which TLC indicated completion of the reaction. The mixture was
then diluted in 100 mL of EtOAc, washed with water (2 ꢁ 30 mL), dried
over anhydrous Na2SO4, concentrated under reduced pressure, and
purified by column chromatography (NH4OH/MeOH/EtOAC, 1:9:90)
to yield 19 as an off-white solid (1.9 g, 78%). Mp 111ꢀ112 ꢀC (lit.
113ꢀ115 ꢀC);26 δH (400 MHz, DMSO-d6) 8.68 (1H, d, J 5.0, H-2),
8.01 (1H, d, J 9.0, H-3), 7.89 (1H, d, J 2.0, H-5), 7.52 (1H, dd, J 2.1 and
9.0, H-4), 6.95 (1H, d, J 5.0, H-1), 3.05ꢀ3.11 (4H, m, H-6, 9),
2.92ꢀ2.97 (4H, m, H-7, 8), 3.19 (1H, br s, H-10); δC (100 MHz,
DMSO-d6) 157.4, 152.6, 150.2, 133.9, 128.5, 126.6, 121.9, 109.7, 53.7
(2C), 45.9 (2C).
2.1. 1-(2,4-Dimethoxyphenyl)-3-(4-hydroxy-3-methoxyphenyl)pro-
penone 16a. Yellow solid (4.3 g, 68%); mp 92ꢀ94 ꢀC (from EtOAc/
Hex); Rf (EtOAc/Hex 4:6) 0.30; IR ν (KBr)/cmꢀ1 3506 (Ar OꢀH),
6. General Method C for the Preparation of Compounds
20ꢀ23. Hydroxylated chloroquinoline intermediate 17 or 18 (1 equiv),
enone 15a or chalcone 16a (1 equiv), and triphenylphosphine (1.2
equiv) were dissolved in anhydrous DCM under a N2 atmosphere. The
solution was cooled to 0 ꢀC, and diisopropyl azodicarboxylate (DIAD,
1.2 equiv) was added slowly over 5 min. The stirred reaction mixture was
then allowed to warm to ambient temperature and was further stirred for
max
3113 (Ar CꢀH), 1640 (CdO), 1598 (Ar CdC); δH (400 MHz, CDCl3)
7.74 (1H, d, J 8.6, H-30), 7.61 (1H, d, J 15.7, H-a), 7.35 (1H, d, J 15.7,
H-b), 7.17 (1H, dd, J 1.9 and 8.3, H-2), 7.10 (1H, d, J 1.9, H-1), 6.94
(1H, d, J 8.3, H-3), 6.58 (1H, dd, J 2.3 and 8.6, H-20), 6.51 (1H, d, J 2.3,
H-10), 5.93 (1H, s, H-5), 3.94 (3H, s, H-40), 3.91 (3H, s, H-50), 3.88 (3H,
s, H-4); δC (100 MHz, CDCl3) 190.8, 163.9, 160.2, 147.8, 146.7, 142.6,
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6 h. The mixture was then concentrated under reduced pressure, and the
target compound was purified by silica column chromatography (5%
MeOH in EtOAc).
(1 equiv) and the piperazinylquinoline intermediate 19 (1 equiv) were
dissolved in EtOH. Formaldehyde (37% solution in water, 10 equiv) was
then added to the stirring solution, which was then refluxed at 110 ꢀC for
12 h. The reaction mixture was then allowed to cool to ambient
temperature, after which it was concentrated under reduced pressure
and purified by column chromatography (EtOAc) to yield the pure
target compounds.
6.1. 4-{4-[2-(7-Chloroquinolin-4-ylamino)ethoxy]-3-methoxyphe-
nyl}but-3-en-2-one 20. Yellow solid (289 mg, 73%); mp 143ꢀ144 ꢀC
max
(from MeOH/EtOAc); Rf (MeOH/EtOAc 1:9) 0.23; IR ν (KBr)/
cmꢀ1 3420 (NꢀH), 2972 (Ar CꢀH), 1686 (CdO), 1584 (Ar CdC);
δH (400 MHz, CDCl3) 8.57 (1H, d, J 5.3, H-9), 7.99 (1H, d, J 1.8, H-10),
7.76 (1H, d, J 8.9, H-12), 7.46 (1H, d, J 16.2, H-a), 7.39 (1H, dd, J 1.9 and
8.9, H-11), 7.13ꢀ7.11 (2H, m, H-1, 2), 6.98 (1H, d, J 8.0, H-3), 6.63
(1H, d, J 16.2, H-b), 6.49 (1H, d, J 5.3, H-8), 5.70 (1H, s, H-7), 4.39 (2H,
t, H-5.0, H-5), 3.92 (3H, s, H-4), 3.73 (2H, q, J 5.0, H-6), 2.37 (3H, s,
H-a0); δC (100 MHz, CDCl3) 198.2, 151.7, 150.1, 149.8, 148.9, 142.9,
135.1, 128.9, 125.9, 125.5, 122.6, 121.2, 117.4, 114.7, 110.5, 99.3, 67.7,
55.8, 42.3, 27.4, 21.9; LRMS (EI) m/z 395.3 (M ꢀ H)þ. Anal.
(C22H21ClN2O3) C, H, N.
7.1. 4-{3-[4-(7-Chloroquinolin-4-yl)piperazin-1-ylmethyl]-4-hydro-
xy-5-methoxyphenyl}but-3-en-2-one 24. Yellow solid (58%); mp
91ꢀ92 ꢀC (from EtOAc); Rf (EtOAc) 0.15; IR ν (KBr)/cmꢀ1 2943
max
(CꢀH), 1662 (CdO), 1575 (Ar CdC); δH (300 MHz, CDCl3) 8.73
(1H, d, J 5.0, H-11), 8.06 (1H, d, J 2.1, H-12), 7.89 (1H, d, J 9.0, H-14),
7.43 (1H, d, J 16.1, H-a), 7.40 (1H, dd, J 2.1 and 9.0, H-13), 7.03 (1H, d,
J 1.5, H-2), 6.91 (1H, d, J 1.5, H-1), 6.82 (1H, d, J 5.0, H-10), 6.58 (1H, d,
J 16.1, H-b), 3.91 (3H, s, H-3), 3.89 (2H, s, H-5), 3.28ꢀ3.34 (4H, m, H-7,
8), 2.90ꢀ2.96 (4H, m, H-6, 9), 2.35 (3H, s, H-a0); δC (75 MHz, CDCl3)
198.1, 156.4, 151.6, 149.9, 149.6, 148.4, 143.5, 135.2, 128.8, 126.5, 125.8,
124.9, 124.8, 122.5, 121.7, 120.9, 110.1, 109.1, 60.7, 55.9, 52.4 (2C), 51.9
6.2. 3-{4-[2-(7-Chloroquinolin-4-ylamino)ethoxy]-3-methoxyphenyl}-
1-(2,4-dimethoxyphenyl)propenone 21. Pale yellow solid (310 mg,
60%); mp 164ꢀ166 ꢀC (from MeOH/EtOAc); Rf (MeOH/EtOAc 1:9)
0.27; IR νmax (KBr)/cmꢀ1 3406 (NꢀH), 2942 (Ar CꢀH), 1646
(CdO), 1584 (Ar CdC); δH (400 MHz, CDCl3) 8.57 (1H, d, J 5.3,
H-9), 7.98 (1H, d, J 2.1, H-10), 7.78-7.74 (2H, m, H-30, 12), 7.61 (1H, d,
J 15.7, H-a), 7.39 (1H, d, J 15.7, H-b), 7.38 (1H, dd, J 2.1 and 8.9, H-11),
7.17 (1H, dd, J 1.7 and 8.2, H-2), 7.14 (1H, d, J 1.7, H-1), 6.96 (1H, d,
J 8.2, H-3), 6.57 (1H, dd, 2.2 and 8.6, H-20), 6.52 (1H, d, J 2.2, H-10), 6.48
(1H, d, J 5.3, H-8), 5.78 (1H, t, J 5.5, H-7), 4.39 (2H, t, J 5.2, H-5), 3.92
(3H, s, H-40), 3.90 (3H, s, H-50), 3.88 (3H, s, H-4), 3.72 (2H, q, J 5.2,
H-6); δC (100 MHz, CDCl3) 190.5, 164.1, 160.3, 152.0, 150.1, 149.6,
149.4, 149.2, 141.8, 134.9, 132.7, 130.2, 128.9, 126.1, 125.4, 122.3, 122.2,
121.2, 117.5, 115.0, 111.2, 105.2, 99.3, 98.8, 67.8, 55.9, 55.7, 55.5, 42.3;
LRMS (EI) m/z 517.1 (M ꢀ H)þ. Anal. (C29H27ClN2O5) C, H, N.
6.3. 4-(4-{2-[4-(7-Chloroquinolin-4-yl)piperazin-1-yl]ethoxy}-3-
methoxyphenyl)but-3-en-2-one 22. Pale yellow (316 mg, 68%); mp
(2C), 27.4; LRMS (EI) m/z 450.7 (M ꢀ H)þ. Anal. (C25H26ClN3O3
3
H2O) C, H, N.
7.2. 3-{3-[4-(7-Chloroquinolin-4-yl)piperazin-1-ylmethyl]-4-hydro-
xy-5-methoxyphenyl}-1-(2,4-dimethoxyphenyl)propenone 25. Deep
max
yellowsolid(66%);mp95ꢀ97 ꢀC(fromEtOAc);Rf (EtOAc) 0.20; IRν
(KBr)/cmꢀ1 2938 (CꢀH), 1648 (CdO), 1597 (Ar CdC); δH (400
MHz, CDCl3) 8.71 (1H, d, J 5.1, H-11), 8.10 (1H, d, J 1.9, H-12), 7.89
(1H, d, J 9.0, H-14), 7.72 (1H, d, J 8.6, H-30), 7.58 (1H, d, J 15.8, H-a),
7.44 (1H, dd, J 1.9 and 8.9, H-13), 7.33 (1H, d, J 15.7, H-b), 7.09 (1H,
d, J 1.8, H-2), 6.97 (1H, d, J 1.8, H-1), 6.84 (1H, d, J 5.1, H-10), 6.57
(1H, dd, J 1.7 and 8.6, H-20), 6.51 (1H, d, J 1.8, H-10), 3.92 (3H, s,
H-40), 3.91 (5H, s, H-5, 50), 3.87 (3H, s, H-3), 3.32ꢀ3.55 (4H, m,
H-7, 8), 2.90ꢀ2.96 (4H, m, H-6, 9); δC (100 MHz, CDCl3)
190.9, 163.9, 160.3, 156.7, 151.2, 149.4, 149.2, 148.3, 142.5, 135.5,
132.6, 128.4, 126.6, 124.9, 124.8, 122.7, 122.6, 121.6, 120.7, 110.8,
110.5, 109.0, 105.2, 98.8, 60.6, 56.0, 55.8, 55.5, 52.3 (2C), 51.8 (2C);
max
61ꢀ62 ꢀC (from MeOH/EtOAc); Rf (MeOH/EtOAc 1:9) 0.26; IR ν
LRMS (EI) m/z 572.7 (M ꢀ H)þ. Anal. (C32H32ClN3O5 H2O)
(KBr)/cmꢀ1 2977 (Ar CꢀH), 1714 (CdO), 1594 (Ar CdC); δH (300
MHz, CDCl3) 8.72 (1H, d, J 5.0, H-12), 8.04 (1H, d, J 2.0, H-13), 7.93
(1H, d, J 8.9, H-15), 7.46 (1H, d, J 16.1, H-a), 7.41 (1H, dd, J 2.2 and 9.0,
H-14), 7.12 (1H, dd, J 1.9 and 8.3, H-2), 7.09 (1H, d, J 1.9, H-1), 6.92
(1H, d, J 8.2, H-3), 6.83 (1H, d, J 5.0, H-11), 6.61 (1H, d, J 16.1, H-b),
4.25 (2H, t, J 5.9, H-5), 3.90 (3H, s, H-4), 3.28ꢀ3.25 (4H, m, H-8, 9),
2.99 (2H, t, J 5.9, H-6), 2.91ꢀ2.88 (4H, m, H-7, 10), 2.36 (3H, s, H-a0);
δC (75 MHz, CDCl3) 198.0, 156.9, 151.9, 150.5, 150.1, 149.7, 143.2,
134.8, 128.8, 127.8, 126.1, 125.4, 125.1, 122.7, 113.0, 110.3, 108.9, 67.0,
56.8, 55.9, 53.4, 52.9, 52.1, 27.3, 21.9; LRMS (EI) m/z 464.2 (M ꢀ H)þ.
Anal. (C26H28ClN3O3) C, H, N.
3
C, H, N.
7.3. 4-{3-[4-(7-Chloroquinolin-4-yl)piperazin-1-ylmethyl]-4-hydro-
xyphenyl}but-3-en-2-one 26. Pale yellow solid (45%); mp 83ꢀ85 ꢀC
(from EtOAc); Rf (EtOAc) 0.35; IR ν (KBr)/cmꢀ1 2823 (CꢀH),
max
1663 (CdO), 1578 (Ar CdC); δH (300 MHz, CDCl3) 8.71 (1H, d,
J 5.1, H-11), 8.07 (1H, d, J 2.1, H-12), 7.89 (1H, d, J 9.0, H-14), 7.44
(1H, d, J 16.4, H-a), 7.43 (1H, dd, J 2.1 and 9.0, H-13), 7.41 (1H, dd, J 2.3
and 8.4, H-2), 7.30 (1H, d, J 2.3, H-1), 6.86 (1H, d, J 8.4, H-3), 6.85 (1H,
d, J 5.1, H-10), 6.58 (1H, d, J 16.3, H-b), 3.88 (2H, s, H-5), 3.28ꢀ3.35
(4H, m, H-7, 8), 2.85ꢀ2.93 (4H, m, H-6, 9), 2.34 (3H, s, H-a0); δC
(75 MHz, CDCl3) 198.2, 160.1, 156.5, 151.5, 149.7, 143.2, 135.3, 129.8,
129.1, 128.7, 126.5, 126.0, 124.8, 124.6, 121.7, 121.1, 116.9, 109.1, 61.2,
52.4 (2C), 51.9 (2C), 27.4; LRMS (EI) m/z 420.7 (M ꢀ H)þ. Anal.
6.4. 3-(4-{2-[4-(7-Chloroquinolin-4-yl)piperazin-1-yl]ethoxy}-3-
methoxyphenyl)-1-(2,4-dimethoxyphenyl)propenone 23. Yellow solid
(435 mg, 74%); mp 62ꢀ63 ꢀC (from MeOH/EtOAc); Rf (MeOH/
EtOAc 1:9) 0.30; IR νmax (KBr)/cmꢀ1 2938 (CꢀH), 1648 (CdO),
1600 (Ar CdC); δH (300 MHz, CDCl3) 8.66 (1H, d, J 5.0, H-12), 8.01
(1H, d, J 9.0, H-15), 7.95 (1H, d, J 2.1, H-13), 7.56 (1H, d, J 8.5, H-30),
7.53 (1H, dd, J 2.3 and 9.0, H-14), 7.46 (1H, d, J 15.8, H-a), 7.37
(1H, d, J 15.8, H-b), 7.30 (1H, d, J 1.9, H-1), 7.23 (1H, dd, J 1.9 and
8.4, H-2), 7.05 (1H, d, J 8.4, H-3), 6.99 (1H, d, J 5.1, H-11), 6.67 (1H, d,
J 2.2, H-10), 6.62 (1H, dd, J 2.2 and 8.6, H-20), 4.19 (2H, t, J 5.8 H-5),
3.87 (3H, s, H-40), 3.84 (3H, s, H-50), 3.83 (3H, s, H-4), 3.19 (4H, t,
J 4.8, H-8, 9), 2.85 (2H, t, J 5.8, H-6), 2.79 (4H, t, J 4.8, H-7, 10); δC
(75 MHz, CDCl3) 189.7, 163.5, 159.9, 156.2, 151.9, 150.1, 149.6, 149.2,
141.6, 133.4, 131.5, 127.9, 125.8, 125.5, 125.1, 122.3, 121.7, 121.2,
113.3, 111.4, 109.2, 105.8, 98.7, 65.6, 56.3, 55.8 (2C), 52.8 (2C),
51.7 (2C); LRMS (EI) m/z 586.0 (M ꢀ H)þ. Anal. (C33H34ClN3O5)
C, H, N.
(C24H24ClN3O2 H2O) C, H, N.
3
7.4. 3-{3-[4-(7-Chloroquinolin-4-yl)piperazin-1-ylmethyl]-4-hydro-
xyphenyl}-1-(2,4-dimethoxyphenyl)propenone 27. Yellow solid
max
(49%); mp 85ꢀ87 ꢀC (from EtOAc); Rf (EtOAc) 0.40; IR ν
(KBr)/cmꢀ1 2935 (CꢀH), 1649 (CdO), 1598 (Ar CdC); δH (400
MHz, CDCl3) 8.68 (1H, d, J 5.0, H-11), 8.07 (1H, d, J 1.8, H-12), 7.89
(1H, d, J 9.0, H-14), 7.69 (1H, d, J 8.6, H-30), 7.57 (1H, d, J 15.7, H-a),
7.46 (1H, dd, J 1.9 and 8.5, H-2), 7.41 (1H, dd, J 1.8 and 8.9, H-13), 7.32
(1H, d, J 15.7, H-b), 7.24 (1H, d, J 1.9, H-1), 6.83 (1H, d, J 8.3, H-3), 6.82
(1H, d, J 4.9, H-10), 6.52 (1H, dd, J 2.0 and 8.6, H-20), 6.47 (1H, d, J 2.0,
H-10), 3.87 (3H, s, H-40), 3.85 (2H, s, H-5), 3.83 (3H, s, H-50),
3.28ꢀ3.34 (4H, m, H-7, 8), 2.84ꢀ2.90 (4H, m, H-6, 9); δC (100
MHz, CDCl3) 190.6, 163.9, 160.2, 159.6, 156.7, 151.1, 149.3, 142.1,
135.5, 132.6, 129.6, 129.3, 128.4, 127.1, 126.6, 124.8, 124.6, 122.5, 121.5,
120.8, 116.9, 108.9, 105.1, 98.7, 61.55, 55.7, 55.4, 52.4 (2C), 51.8 (2C);
7. General Method D for the Preparation of Compounds
24ꢀ27. The appropriate hydroxylated chalcone or enone (15 or 16)
LRMS (EI) m/z 542.8 (Mþ). Anal. (C31H30ClN3O4 H2O) C, H, N.
3
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Journal of Medicinal Chemistry
ARTICLE
8. Evaluation of in Vitro Antiplasmodial Activity. The in
vitro antiplasmodial activities of the compounds were evaluated against
D10, Dd2, and W2 strains of P. falciparum.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: þ27 21 650 2553. Fax: þ27 21 650 5195. E-mail: Kelly.
The parasites were maintained in continuous in vitro culture by
standard methods.38 The cultured parasites were, whenever necessary,
synchronized by treatment with 5% D-sorbitol (Sigma) at the ring stage.39
For the studies on the D10 and Dd2 strains, the assays were carried
out as previously reported by Guantai et al.16 The assays on the W2
strain were conducted as previously reported (Shenai et al.)40 with
parasites cultured in standard medium including 10% human serum
except that parasite development was assessed with a FACS-based assay,
as previously described (Sijwali and Rosenthal).41 The final concentra-
tion of DMSO that the parasites were exposed to did not exceed 0.5%,
which has no measurable effect on parasite viability.
9. log D Measurement. Partition coefficients (log D) of the test
compounds were estimated by correlation of their chromatographic
retention properties against the characteristics of a series of standard
compounds with known partition coefficient values. The method employed
was a gradient HPLC based derivation of the method developed by
Lombardo and co-workers.29
Chibale@uct.ac.za.
’ ACKNOWLEDGMENT
The authors thank Professor Susan A. Charman of the Centre
for Drug Candidate Optimization (CDCO), Monash University
(Australia), for kindly facilitating the in vitro physicochemical
and ADME analysis and for helpful discussions and assistance
with interpretation of the resultant data. Financial support from
the following sources is gratefully acknowledged: the South
African National Research Foundation (NRF) by way of the
Africa Scholarship (E.M.G.), the South African Research Chairs
Initiative (SARChI) of the Department of Science and Technol-
ogy (DST), the South African Medical Research Council
(MRC), the University of Cape Town and the European Union
through the AntiMal program (K.C.), the Doris Duke Charitable
Foundation, and the United States National Institutes of Health
(NIH) (P.J.R.).
10. Solubility Estimates. Compound in DMSO was spiked into
either pH 6.5 phosphate buffer or 0.01 M HCl (approximately pH 2.0)
with the final DMSO concentration being 1%. Samples were then
analysed via nephelometry to determine a solubility range, as described
by Bevan and Lloyd.30
’ ABBREVIATIONS USED
ADME, absorption, distribution, metabolism, and excretion; AQ,
amodiaquine; CQ, chloroquine; DIAD, diisopropyl azadicarboxylate;
NADP(H), nicotinamide adenine dinucleotide phosphate
(reduced); PK, pharmacokinetic; PPh3, triphenylphosphine;
UDPGA, uridine diphosphate glucuronic acid; WHO, World
Health Organization
11. Evaluation of β-Hematin Inhibition. β-Hematin inhibition
assays were conducted as previously reported.42
12. Evaluation of Falcipain 2 Inhibition. Falcipain 2 inhibition
assays were carried out as previously described except that the proteolytic
substrate was benzyloxycarbonyl-Phe-Leu-7-amino-4-methylcoumarin.43
13. Evaluation of Inhibition of Sorbitol-Induced Lysis. The
method described by Go et al.36 was applied for this assay, with modifications.
Briefly, chloroquine-sensitive P. falciparum D10 was cultured and trophozoite-
stage infected erythrocytes (10% parasitemia) were harvested by centrifuga-
tion (750 rpm, 3 min), washed twice with a solution of NaCl (150 mM)ꢀ
HEPES (20 mM) (pH 7.4, 304 mosM), and suspended in the same solution
to give a hematocrit of about 50%. Test compounds were dissolved in
dimethylsulfoxide (DMSO) to give 10 mM stock solutions, which were
diluted further with a solution comprising 300 mM sorbitolꢀ20 mM HEPES
(pH 7.4, 345 mosM) to give the test solutions of 100 μM.
In 10 mL screw-cap centrifuge tubes, 100 μL of the test solutions were
separately combined with an additional 700 μL of the sorbitolꢀHEPES
buffer and 200 μL of the parasitized cell suspension in NaClꢀHEPES
buffer. The final concentration of the test compounds was 10 μM. For
the negative control, 800 μL of the sorbitolꢀHEPES buffer and 200 μL
of the parasitized cell suspension in NaClꢀHEPES buffer were trans-
ferred into a 10 mL screw-cap centrifuge tube. For the blank, 800 μL of
the sorbitolꢀHEPES buffer and 200 μL of nonparasitized erythrocyte
suspension (50% hematocrit) in NaClꢀHEPES buffer were transferred
into a 10 mL screw-cap centrifuge tube.
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S
Supporting Information. Structures of intermediates
b
and target compounds. This material is available free of charge
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Journal of Medicinal Chemistry
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