Identification and Mechanistic Evaluation of Hemozoin-Inhibiting Triarylimidazoles Active against Plasmodium falciparum

Article abstract of DOI:10.1021/acsmedchemlett.6b00416

In a previous study, target based screening was carried out for inhibitors of β-hematin (synthetic hemozoin) formation, and a series of triarylimidazoles were identified as active against Plasmodium falciparum. Here, we report the subsequent synthesis and testing of derivatives with varying substituents on the three phenyl rings for this series. The results indicated that a 2-hydroxy-1,3-dimethoxy substitution pattern on ring A is required for submicromolar parasite activity. In addition, cell-fractionation studies revealed uncommonly large, dose-dependent increases of P. falciparum intracellular exchangeable (free) heme, correlating with decreased parasite survival for β-hematin inhibiting derivatives.

Full text of DOI:10.1021/acsmedchemlett.6b00416

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Letter
Identification and Mechanistic Evaluation of Hemozoin-Inhibiting
Triarylimidazoles Active against Plasmodium falciparum
Kathryn J. Wicht, Jill Michelle Combrinck, Peter J Smith, Roger Hunter, and Timothy J Egan
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00416 • Publication Date (Web): 24 Jan 2017
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Identification and Mechanistic Evaluation of HemozoinꢀInhibiting
Triarylimidazoles Active against Plasmodium falciparum
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Kathryn J. Wichta, Jill M. Combrinckb, Peter J. Smithb, Roger Huntera, Timothy J. Egana,*
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aDepartment of Chemistry, University of Cape Town, Rondebosch 7701, South Africa.
bDivision of Pharmacology, Department of Medicine, Faculty of Health Sciences, University of Cape Town, Observatory
7925, South Africa.
KEYWORDS: Antimalarial, Plasmodium falciparum, triarylimidazole, hemozoin
ABSTRACT: In a previous study, target based screening was
carried out for inhibitors of βꢀhematin (synthetic hemozoin)
formation and a series of triarylimidazoles were identified as active
against Plasmodium falciparum. Here we report the subsequent
synthesis and testing of derivatives with varying substituents on the
three phenyl rings for this series. The results indicated that a 2ꢀ
hydroxyꢀ1,3ꢀdimethoxy substitution pattern on ring A is required
for submicromolar parasite activity. In addition, cellꢀfractionation
studies revealed uncommonly large, doseꢀdependent increases of P.
falciparum intracellular exchangeable (free) heme, correlating with
decreased parasite survival for βꢀhematin inhibiting derivatives.
No new chemical classes of antimalarials have been
introduced into clinical practice since 1996 and the shortage of
novel, publicly accessible antimalarial scaffolds has had a
negative impact on drug discovery efforts.1,2 Past and present
therapies are based on a limited number of chemotypes (e.g.
quinolines, cycloguanils, tetracylines and sesquiterpene
lactones) which often leads to crossꢀresistance.3 Targetꢀbased
high throughput screening (HTS) of diverse libraries is a
popular approach to identifying novel drug scaffolds and
requires assays for inhibition of specific P. falciparum
pathways. Hemozoin (Hz) inhibition is one such valid
pathway, since this process is not directly affected by the
structureꢀspecific efflux mechanism which causes chloroquine
resistance in the parasite.4 Synthetic Hz, βꢀhematin (βH), can
be efficiently and reliably synthesized in the laboratory with
growth by ≥90% at 23 ꢁM, (32.3% of the 530 βH inhibitors)
and 25 compounds exhibited favorable IC50 values below 1
ꢁM. Furthermore, cell fractionation studies successfully
validated Hz formation as the mechanism of action in parasites
for a subset of tested compounds, which represented twelve of
the fourteen novel scaffolds identified. Doseꢀdependent
measurements of intracellular free heme and Hz revealed that
different levels of free heme were attained in the parasite,
depending on compound type. Three compounds caused
extraordinarily high levels of nonꢀHz free heme (30ꢀ50% of
total heme) at 2.5 times their respective IC50 when compared
to those seen with CQ and other quinolineꢀcontaining
scaffolds (8ꢀ14%).17,18 Two of these compounds were
benzamides, reported elsewhere,19 and the other,
a
triarylimidazole, which attained a free heme content of 43%.
The triarylimidazole class also displayed a high hit rate for
parasite activity, as six of seven βH inhibitors were active
against P. falciparum (Table 1).
minimal equipment using
a
variety of methods.5ꢀ8
Furthermore, detecting the formation of βH is easily achieved
through a number of spectroscopic techniques including IR,9
UVꢀvis of the dissolved crystals,10 or UVꢀvis of the
ferrihemochrome complex formed between “free” Fe(III)PPIX
and pyridine (free heme refers to nonꢀHz/βH, nonꢀhemoglobin
heme that coordinates to pyridine at pH ≈7).11,12 Several
Recently, in phenotypic screens carried out by GSK and
Novartis several triarylimidazoles were discovered to have
activity against the 3D7 strain of P. falciparum1, 20 and three
triarylimidazoles in the MMV Malaria Box were reported as
inhibitors of βH formation.21 Consequently, this scaffold was
selected as interesting for investigating Hz inhibition of this
class owing to the novelty of the chemotype. Furthermore,
additional analysis of the HTS hits and their derivatives with
potent βH inhibition activity, but with a range of parasite
activities and intracellular free heme levels was required for an
groups have endeavored to find novel βH inhibitors via targetꢀ
16
based HTS projects.13,14,15,
In the most recent, a total of
144,330 compounds from the Vanderbilt University (VU)
chemical compound library were screened for their ability to
inhibit βH formation using an NPꢀ40 based assay described
previously and the ferrihemochrome method of detection.8,15,16
Of the 530 confirmed βH inhibitors, 171 inhibited parasite
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ongoing study into the mechanistic aspects of nonꢀquinoline inhibition activity (Table 2). Ring B and C in the HTS hits
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Hz inhibitors. Therefore, the aim of this study was to
investigate a range of triarylimidazoles as probe compounds to
gain a better understanding of the relationships between
activity and levels of nonꢀHz free heme in the parasite. This
involved identifying the pharmacophore and preliminary
SARs for this series as a step towards exploring their
mechanism of action and antimalarial potential.
appeared to have a less important influence on βH activity
than ring A. As a result, rings B and C were removed to
evaluate whether they are required. The monoarylimidazoles
2ꢀphenylimidazole (8a) and 4ꢀ(1Hꢀimidazolꢀ2ꢀyl)phenol (8b)
were obtained commercially and tested in the NPꢀ40 assay.
Compound 8a was found to be entirely inactive against βH
formation, while 8b showed only very weak activity, more
than tenꢀfold less active than those possessing rings B and C,
confirming that they are essential for potent activity.
Additional data on the triarylimidazole chemotype, which was
made available via a collaboration between UCT and
Vanderbilt University (VU), indicated that the unsubstituted
2,4,5ꢀtriphenylimidazole (9) also did not inhibit βH formation.
Seven triarylimidazoles were found to inhibit parasite growth
in the HTS, with three showing IC50 values below 1 µM.16
Interestingly all seven were very good βH inhibitors, at least
twice as active as CQ, but parasite activity decreased
significantly upon variation of the 3,5ꢀdimethoxyꢀ4ꢀhydroxy
substituents on ring A. The identified compounds showed D6
IC50s from 0.3 to 11.7 µM (Table 1), with small structural
changes having significant influence on activity as evident
when comparing 1 (0.3 µM), 3 (0.7 µM), 5 (3.4 µM) and 6
(9.0 µM).
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The next evaluated derivative was the purchased compound 4ꢀ
(4,5ꢀdiphenylꢀ1Hꢀimidazolꢀ2ꢀyl)phenol (10), which was
identified as the simplest triphenylimidazole to strongly inhibit
βH formation (IC50 of 17.7 µM). In order to further investigate
the SARs for this scaffold, derivatives 14aꢀd were synthesized
using the standard method available in the literature by
refluxing the appropriate aldehyde, benzil and sodium acetate
in MeOH, according to Scheme 1. Target compounds 14e and
14f containing substituents on the B and C rings required 4ꢀ
chlorobenzil (13e) and 4,4’ꢀdimethoxybenzil (13f) as starting
materials respectively. Since these compounds were not
commercially available at the time of synthesis, they were
synthesized from the corresponding benzoins, 11e and 11f via
oxidation of the hydroxyl to the carbonyl with sodium hydride
and O2. Attempts to synthesize target compound 15 containing
a paraꢀamino group on the Aꢀring from pꢀaminobenzaldehyde
were unsuccessful. It is likely that the aldehyde preferentially
selfꢀcondenses under these conditions to give oligomeric
products, however this was not further investigated. An
alternative route was followed whereby the nitro derivative
(14g) was formed and then reduced to the amine using iron
powder and a catalytic amount of HCl. The conditions
reported by Wan et al.22 were followed for the formation of
both 14g and 15, which involved a 4 h glacial acetic acid
reflux at 118 °C for 14g and then a 6 h reflux at 80 °C with Fe
powder in an EtOH/water (2:1) mixture to afford 15.
Table 1. The seven βH inhibiting triarylimidazoles from the
HTS active against P. falciparum.16
Structure
βH IC50 (µM)
D6 IC50 (µM)a
1
12.6
0.31
2
3
6.2
2.4
0.61
0.70
4
12.7
2.24
2.40
5
6
7
18.7
5.6
9.06
(43% ∆ free
heme)
10.0
11.7
Scheme 1. Preparation of the triarylimidazole derivatives 14aꢀ
g and 15.
a Compounds are listed in order of decreasing D6 activity. The intracellular % free heme
increase is shown in bold for
6
.
The triarylimidazole scaffold was initially deconstructed in
order to determine the minimum structural features for βH
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Table 2. βH and D6 or NF54 and K1 parasite IC50 values. Derivatives 8b and 9, which were not βH inhibitors below 100
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4
5
6
7
8
ꢀM, also showed no parasite activity up to 20 µM.
Surprisingly, the other nonꢀinhibitor, 14d, showed a moderate
activity of 4.2 ꢀM. Derivative 16, which was a potent βH
inhibitor, showed weak activity of 10.8 µM in the CQꢀ
sensitive D6 strain. The remaining purchased and synthesized
triarylimidazoles were tested in the LDH parasite growth
inhibition assay using the CQ sensitive NF54 strain (Table 2).
Compound 10 was found to be inactive at concentrations up to
βH
inhibition
IC50 µM
D6d or
NF54
IC50 µM
K1 IC50
µM
(RI)
Code
CQa
Structure
0.140
(14)
0.010
31.5 ± 0.5
>1000
ND
>20
ND
ND
R2 = H
32 µM, despite displaying potent βH activity.
Other
8a
8b
9
derivatives which performed poorly in the assay were the 4ꢀ
nitro (14g) and 4ꢀamino (15), analogues of 10. This showed
that having substituents only in the para position of ring A
was not sufficient for parasite activity. For the βH inhibiting
compounds containing 3,5ꢀmethoxy groups on ring A, 14aꢀc,
the parasite activity improved from 7.0 to 1.3 µM with
variation of the para R2 substituent H < OMe < OH,
illustrating that the ring A substitution pattern of the parent
compound (1, Table 1) was superior for activity. The ability of
βH inhibiting derivatives with a greater number of hydroxyl
and methoxy substituents to inhibit parasite growth can be
seen graphically in Figure 1. This had a much greater impact
on parasite activity than did βH inhibition IC50. Indeed, the
relationship with βH activity was not statistically significant.
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347 ± 12
R2 = OH
X
R1
H
H
R2
R3
H
H
R4/R5
H
9
Ha
OHa
>1000
>20d
>32
ND
ND
10
H
17.7 ± 0.5
1.9 ± 0.8
(1.5)
14a
14b
OMe
OMe
OHa
OMe
OMe
H
H
10.2 ± ND
10.9 ± 0.3
1.3 ± 0.5
3 ± 2
OMea
ND
14cc
14dc
OMe
H
Ha
OMe
H
H
H
14.6 ± 0.2
>1000
7.0 ± 0.1
4.7 ± 0.1
(0.7)
a
OCF3
4.2 ± 0.1
1.7 ± 0.5
ND
14e
OMe
OMe
Ha
Ha
OMe
OMe
Cl/H
16.6 ± 0.6
13.8 ± 0.4
2.7 ± 0.4
(1.6)
14fc
OMe/
OMe
1.8 ± 0.5
(1.1)
1.7 ± 0.4
a
14gc
15
H
H
H
NO2
H
H
H
H
H
H
19 ± 1
15 ± 4
42 ± 16
10.8d
ND
ND
ND
a
NH2
ꢀb
16.1 ± 0.5
25.4 ± ND
16
a X = C, see Scheme 1
b X = N, see Scheme 1
c Cytotoxicity against CHO cells. 14c: >269 ꢀM (SI > 206); 14d: 29 ꢀM (SI =
6.9); 14f: > 240 ꢀM (SI > 141); 14g: 149 ꢀM (SI = 7.8).
d CQꢀsensitive D6 parasite strain from VU for 9 and 16
Compound 14a resembles the parent compound, 1, but lacks
the bromo substituent on ring B. This derivative maintained
potent activity against βH formation, confirming that the
substituents on ring B and C are not essential. The substituents
on ring A were varied by replacing the 4ꢀhydroxy group with a
methoxy group (14b) or a hydrogen atom (14c). These
analogues also maintained activity against βH formation,
demonstrating that with R1 = R3 = OMe, substituents at R2 are
not required. By contrast, βH inhibition IC50 data for
derivatives with R1 = R3 =H suggested that a substituent at R2
is necessary but not sufficient for activity. Specifically,
analogues with R2 = OH (10), NO2 (14g) and NH2 (15) were
βH inhibitors while those with R2 = H (9) or OCF3 (14d)
showed no inhibition up to 1000 ꢀM. This indicated that
compounds with both electron withdrawing and releasing
groups as well as both planar and nonꢀplanar substituents on
ring A are tolerated. Finally, analogues of 14c, which
contained either a 4ꢀchloro atom on ring B (14e) or 4ꢀmethoxy
groups on both rings B and C (14f) were evaluated as an
indication of the influence of substituents on these rings.
These compounds displayed similar activity to 14c, with IC50
values of 16.6 µM and 13.8 µM for the chloro and dimethoxy
analogues respectively. This also demonstrated that both
electron withdrawing and releasing as well as planar and nonꢀ
planar substituents are tolerated on rings B and C. Replacing
phenyl ring A with pyridyl, which corresponds to the active
compound 16 from VU, gave a βH inhibition IC50 of 25.4 µM.
The βH activity data for the triarylimidazoles are shown in
Table 2.
Figure 1. A linear correlation between the log of the parasite
activity with the number of hydroxyl or methoxy substituents
on the phenyl rings in a triarylimidazole given by log(NF54
IC50) = ꢀ0.30(# of OH or OMe) + 1.23; (r2 = 0.76, P = 0.0048).
The most active triarylimidazoles in the HTS, with
submicromolar activity, contained a para Br or OH substituent
on ring B (Table 1), suggesting that the effect of these groups,
while not a significant factor for βH inhibition, is to increase
parasite activity. The preparation of 14e and 14f was carried
out in order to determine whether the activity of
triarylimidazoles with other ring A substituents could also be
improved by attaching ring B and ring C substituents. The 4ꢀ
chloro and 4,4’ꢀdimethoxy analogues were tested and found to
improve activity from 7.0 µM for 14c to ≈1.7 µM for both 14e
and 14f. This >4ꢀfold activity improvement upon the
introduction of ring B and/or C substituents was almost
identical to that of 14c vs 1 (Table 1). This trend was also
observed when comparing 10 with poor activity (>32 µM) to
its 4ꢀbromo derivative 6 from the HTS, with IC50 of 9.1 µM.
Compounds 14a, 14c, 14e and 14f, were selected for testing in
the CQꢀresistant K1 strain of P. falciparum and were found to
have low resistance indices (RIs), indicating no significant CQ
cross resistance for this series. Furthermore, compounds 14c,
14f and 14g showed low cytotoxicity against Chinese hamster
ovarian (CHO) cells (>100 ꢀM), demonstrating specific
activity against the malaria parasite (Table 2 footnote). By
contrast, 14d was an order of magnitude more cytotoxic, with
a selectivity index of only 7, suggesting that its activity against
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Plasmodium is likely based on its cytotoxicity. The SAR is in view of the unusually high levels achieved. Investigations of
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3
4
5
6
7
summarized in Figure 2.
the relationship between cellular accumulation, free heme
levels and activity for this and other series are currently
ongoing.19 Such data are crucial for gaining a detailed
understanding of the mechanism of action of Hz inhibitors.
R3
R1 = OMe; R2 = OH; R3 = OMe
R1 = H; R2 = OH; R3 = H
R1 = H; R2 = NO2; R3 = H
R1 = H; R2 = NH2; R3 = H
R2
A
R1
N
HN
C
R5
B
R4 = Cl, Br or F; R5 = H or F
R4 = OMe or OH; R5 = OMe or H
8
R4
9
Figure 2. Favorable (blue) and unfavorable (red) substituents
for parasite activity within the triarylimidazole HTS hits and
synthesized analogues.
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The triarylimidazole 6 (Table 1) from the HTS was shown to
cause an increase in nonꢀHz, nonꢀhemoglobin “free” heme to
43% of total heme.16 Nevertheless, data for additional
derivatives with a range of potencies, were deemed necessary
in order to support the biological mechanism of action for this
series. Compounds 14c, 14f and 14g, which had different ring
A substituents from the HTS hits and showed a marked
difference in activity, were subjected to cell fractionation
studies. 14c and 14f were shown to cause a very large increase
in free heme in the cell (>40 fg/cell which corresponds to
>50% of total cellular heme) at 2.5 times the corresponding
IC50 of the compound (Figure 3a and S4.2a). 14g also caused
a large increase in free heme (>20 fg/cell, see S4.2b), smaller
than 14c and 14f, but larger than CQ.18 This study confirmed
that these triarylimidazoles inhibit Hz formation, causing an
increase in free heme in the malaria parasite in a dose
dependent manner that coincides with decreased parasite
survival. This strongly suggests that inhibition of Hz
formation is the mechanism of action of these compounds. By
contrast, 14d, which exhibited parasite activity despite
showing no βH inhibition activity, brought about no increase
in free heme with decreasing parasite survival (Figure 3b).
Thus, 14d appears to exhibit an entirely different mechanism
of action. This is reinforced by flow cytometry data, which
showed that parasite cells treated with 14d contained less
DNA and were considerably smaller and less complex than
those treated with 14c, 14f and 14g (see S4.2c-f). They also
appeared to be pyknotic by light microscopy (see S4.2g). It is
noteworthy that this compound is much more cytotoxic than
the others are and this may be the basis of its parasite activity.
Figure 3. Dose response curves for amount of free nonꢀHz
heme (fg/cell) and the percent parasite survival for a) 14f,
showing a cross over near the IC50 and b) 14d, showing no
increase in free heme with parasite growth inhibition.
ASSOCIATED CONTENT
Supporting Information
Supporting Information is available free of charge on the ACS
Publications website at DOI: XXX
Experimental methods including chemistry, detergent
mediated assay for βꢀhematin inhibition, LDH malaria
parasite survival assay, cell fractionation and
cytotoxicity (PDF). Additional cell fractionation and
flow cytometry data.
In this study, purchased or synthesized triarylimidazoles were
evaluated for their ability to inhibit βH formation and prevent
parasite growth. Furthermore, the RIs and SIs were assessed
for selected analogues, which showed low CQ crossꢀresistance
and high selectivity for P. falciparum over mammalian cells,
supporting the Hzꢀinhibition pathway as an effective and
unique drug target. Parasite activity relied on very specific
substituents, showing that the scaffold was not amenable to
structural changes on ring A. There did not appear to be a
direct correlation between βH inhibition and parasite activity.
This is not surprising given that access to the digestive
vacuole, including pH trapping, is likely to play a crucial role
in the relative potency of a compounds ability to inhibit
cellular hemozoin formation. Indeed, for this series there is
clear evidence of hemozoin inhibition in the cell. For the three
βH inhibitors selected for testing, intracellular free heme
levels correlated to parasite growth inhibition. These
derivatives are extraordinary probe molecules for investigating
the properties and effects of free heme in the malaria parasite
AUTHOR INFORMATION
Corresponding Author
* Email: timothy.egan@uct.ac.za
Author Contributions
The manuscript was written by K.J.W with guidance and editing
from T.J.E. and R.H. High throughput screening, scaffold
analysis, synthesis and SAR analysis was carried out by K.J.W
under the supervision of T.J.E and R.H. Parasite assays and cell
fractionation studies were conducted and analyzed by J.M.C
under the supervision of P.J.S and T.J.E. All authors have given
approval to the final version of the manuscript.
Funding Sources
The research reported in this publication was
supported by the National Institute of Allergy and
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M. A.; Taylor, D.; Smith, P. J.; Hoppe, H. C.; Egan, T. J.
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Wright, D. W.; Egan, T. J. Optimization of a MultiꢀWell
Colorimetric Assay to Determine Haem Species in
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Egan, T. J. Identification and SAR Evaluation of Hemozoinꢀ
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20. Plouffe, D.; Brinker, A.; McNamara, C.; Henson, K.; Kato,
N.; Kuhen, K.; Nagle, A.; Adrian, F.; Matzen, J. T.;
Anderson, P.; Nam, T.ꢀg.; Gray, N. S.; Chatterjee, A.; Janes,
J.; Yan, S. F.; Trager, R.; Caldwell, J. S.; Schultz, P. G.;
Zhou, Y.; Winzeler, E. A. In Silico Activity Profiling Reveals
the Mechanism of Action of Antimalarials Discovered in a
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Infectious Diseases of the National Institutes of
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Health under Award Number R01AI110329. The
content is solely the responsibility of the authors and
does not necessarily represent the official views of the
National Institutes of Health.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The authors wish to acknowledge Rebecca D. Sandlin and David
W. Wright from the Department of Chemistry, Vanderbilt
University, Nashville, TN, USA for collaboration on the HTS as
well as Carmen de Kock from the Division of Pharmacology,
Department of Medicine, University of Cape Town for providing
cytotoxicity data.
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ABBREVIATIONS
βH, βꢀhematin; Hz, hemozoin; LDH, lactate dehydrogenase; CQ,
chloroquine; RI, resistance index; SI, selectivity index; CHO,
Chinese hamster ovarian; VU, Vanderbilt University; UCT,
University of Cape Town.
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