Identification of a Potential Antimalarial Drug Candidate from a Series of 2-Aminopyrazines by Optimization of Aqueous Solubility and Potency across the Parasite Life Cycle

Article abstract of DOI:10.1021/acs.jmedchem.6b01265

Introduction of water-solubilizing groups on the 5-phenyl ring of a 2-aminopyrazine series led to the identification of highly potent compounds against the blood life-cycle stage of the human malaria parasite Plasmodium falciparum. Several compounds displayed high in vivo efficacy in two different mouse models for malaria, P. berghei-infected mice and P. falciparum-infected NOD-scid IL-2R^null mice. One of the frontrunners, compound 3, was identified to also have good pharmacokinetics and additionally very potent activity against the liver and gametocyte parasite life-cycle stages.

Full text of DOI:10.1021/acs.jmedchem.6b01265

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Article
Identification of a potential anti-malarial drug candidate from
a series of 2-aminopyrazines by optimization of aqueous
solubility and potency across the parasite life-cycle.
Claire Le Manach, Aloysius T Nchinda, Tanya Paquet, Diego Gonzalez Cabrera, Yassir Younis
Adam, Ze Han, Sridevi Bashyam, Mohammed Zabiulla, Dale Taylor, Nina Lawrence, Karen
L. White, Susan A. Charman, David Waterson, Michael J. Witty, Sergio Wittlin, Mariette E.
Botha, Sindisiswe H. Nondaba, Janette Reader, Lyn-Marie Birkholtz, Maria Belen Jimenez-
Diaz, Maria S. Martínez-Martínez, Santiago Ferrer-Bazaga, Iñigo Angulo-Barturen, Stephan
Meister, Yevgeniya Antonova-Koch, Elizabeth A Winzeler, Leslie J. Street, and Kelly Chibale
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01265 • Publication Date (Web): 17 Oct 2016
Downloaded from http://pubs.acs.org on October 18, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Identification of a potential antiꢀmalarial drug
candidate from a series of 2ꢀaminopyrazines by
optimization of aqueous solubility and potency
across the parasite lifeꢀcycle.
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Claire Le Manach†, Aloysius T. Nchinda†, Tanya Paquet†, Diego Gonzàlez Cabrera†,
Yassir Younis†, Ze Han†, Sridevi Bashyamζ, Mohammed Zabiullaζ, Dale Taylor∞, Nina
Lawrence∞, Karen L. WhiteΨ, Susan A. CharmanΨ, David Waterson‡, Michael J. Witty‡,
Sergio Wittlin◊,∫, Mariëtte E. BothaÞ, Sindisiswe H. NondabaÞ, Janette ReaderÞ, Lynꢀ
Marie BirkholtzÞ, María Belén JiménezꢀDíaz¥, María Santos Martínez¥, Santiago Ferrer¥,
Iñigo AnguloꢀBarturen¥, Stephan Meisterº, Yevgeniya AntonovaꢀKochº, Elizabeth A.
Winzelerº, Leslie J. Street† and Kelly Chibale*,† ,#
†Drug Discovery and Development Center (H3D), University of Cape Town, Rondebosch
ζ
7701, South Africa; Syngene International Ltd., Biocon Park, Plot No. 2 & 3,
Bommasandra IV Phase, Jigani Link Road, Bangalore 560099, India; ∞Division of
Clinical Pharmacology, Department of Medicine, University of Cape Town, Observatory,
7925, South Africa; Ψ Centre for Drug Candidate Optimisation, Monash University, 381
‡
Royal Parade, Parkville, Victoria 3052 Australia; Medicines for Malaria Venture, ICC,
◊
Route de PréꢀBois 20, PO Box 1826, 1215 Geneva, Switzerland; Swiss Tropical and
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Public Health Institute, Socinstrasse 57, 4002 Basel, Switzerland; University of Basel,
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4003 Basel, Switzerland; Department of Biochemistry, Centre for Sustainable Malaria
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Control, University of Pretoria, Private bag X20, Hatfield 0028, South Africa;
¥GlaxoSmithKline, Tres Cantos Medicines Development Campus, Severo Ochoa, 2,
28760 Tres Cantos, Madrid, Spain; º School of Medicine, Department of Pediatrics,
Pharmacology & Drug Discovery, University of California, San Diego (UCSD), 9500
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#
Gilman Drive, La Jolla, California 92093, United States ; South African Research
Council, Drug Discovery and Development Research Unit, Department of Chemistry and
Institute of Infectious Disease and Molecular Medicine, University of Cape Town,
Rondebosch 7701, South Africa.
ABSTRACT: Introduction of waterꢀsolubilizing groups on the 5ꢀphenyl ring of a 2ꢀ
aminopyrazine series led to the identification of highly potent compounds against the
blood lifeꢀcycle stage of the human malaria parasite Plasmodium falciparum. Several
compounds displayed high in vivo efficacy in two different mouse models for malaria, P.
bergheiꢀinfected mice and P. falciparumꢀinfected NODꢀscid ILꢀ2Rγnull mice. One of the
frontrunners, compound 3, was identified to also have good pharmacokinetics, and
additionally, very potent activity against the liver and gametocyte parasite lifeꢀcycle
stages.
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The recent rise of resistant mutants against frontline malaria treatments in SouthꢀEast
Asia and South America1 is a reminder of the constant need for novel antimalarial drugs.
In order to counter new resistance mechanisms as quickly as possible when they emerge,
molecules with different mechanisms of action that are not prone to resistanceꢀ
development need to be continually fed into the drug pipeline. In previous papers we
reported the antimalarial efficacy of aminopyridine2,3 and aminopyrazine4 derivatives,
including the potency of 2 particular compounds, 12 and 24 (Figure 1). In the latter study,4
the in vivo potency of 2 was particularly highlighted, but its low aqueous solubility at
physiological pH is likely to pose challenges during development. Further optimization
was therefore initiated in order to identify aminopyrazine derivatives with improved
aqueous solubility at physiological pH, while retaining the good in vivo efficacy of
previous analogues. Herein we describe the synthesis and lead optimization of the 2ꢀ
aminopyrazine series, focusing on structureꢀactivity relationship (SAR) studies around
the 3ꢀ and 5ꢀpositions along with the introduction of water solubilizing groups. The in
vitro and in vivo biological activities are described, as well as pharmacokinetic
characteristics and the hERG profiles. This work led to the identification of highly potent
compounds in vitro and in vivo, with significantly improved aqueous solubility, of which
the piperazine amide 3 (Figure 2) proved to be particularly promising.
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Chemistry: The cyclic amide derivatives (3, 9ai-li (i= 1,2), 10, 11 and 12) were prepared
following a 6ꢀ to 7ꢀstep synthetic route (Scheme 1) from the commercially available 2ꢀ
aminopyrazine. Briefly, 2ꢀaminopyrazine was brominated at the 5ꢀposition using Nꢀ
bromosuccinimide (NBS) to give bromopyrazine 4. Subsequently, a Suzuki crossꢀ
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coupling reaction5 with 4ꢀmethoxycarbonylphenyl boronic acid was performed at the 5ꢀ
position, where after the resulting intermediate 5 was brominated at the 3ꢀposition with
NBS to give intermediate 6. After lithium hydroxide hydrolysis of the methylester, the
resulting carboxylic acid 7 was subjected to amidation with a cyclic amine, mediated by
EDCI, Et3N, and HOBt, to give the amides 8a-l. A second Suzuki crossꢀcoupling reaction
was then carried out with the desired boronic ester, either 2ꢀ(trifluoromethyl)pyridineꢀ5ꢀ
boronic pinacol ester or 4ꢀ((trifluoromethyl)phenyl)boronic acid pinacol ester, to afford
compounds 9ai-li (i= 1,2). Finally, when applicable, the amide moiety was deprotected to
give target compounds 3, 10, 11, and 12. All intermediates and target compounds were
purified using column chromatography and characterized by various spectroscopic and
analytical methods.
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Carboxylic acids 13 and 14, and methylester 15 were prepared using similar chemistry
as shown in Scheme 2.
Likewise, for amides 16 and 17 and compounds 18-33 a similar 4ꢀstep route was
followed, using the same reaction conditions. The commercially available 2ꢀ
aminopyrazine was brominated at the 5ꢀposition, before the first Suzuki crossꢀcoupling
reaction was performed with the relevant boronic acid. Bromination at the 3ꢀposition was
then carried out, followed by a second Suzuki coupling reaction with the appropriate
boronic acid delivered the desired compounds 16-33 (scheme 3).
In vitro antiplasmodial activity: All compounds were evaluated for in vitro
antiplasmodial activity against a drug sensitive (NF54) and a multiꢀdrug resistant (K1)
strain of P. falciparum. Chloroquine and artesunate were used as references in all
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experiments. The in vitro antiplasmodial activities of the target compounds, as indicated
by their IC50 values, are summarized in Tables 1 and 2. All compounds were equipotent
against both strains, indicating that crossꢀresistance against chloroquineꢀresistant
parasites is unlikely. In addition, selected compounds have been test against field
resistant isolates. As shown in Table C1 of the supporting information, compounds were
generally equipotent against all the strains tested, suggesting the absence of any crossꢀ
resistance.
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Selected compounds were tested for cytotoxicity (Table D1 in the Supporting
Information). All tested compounds displayed a selectivity index greater than 1800.
Therefore, cytotoxicity was not considered to be a concern for this series.
Previous studies on related 2ꢀaminopyridines have shown that substitution at the 4ꢀ
position of the 5ꢀphenyl ring was optimal for in vitro blood stage activity3. In addition,
former SAR studies around the 2ꢀaminopyrazine scaffold suggested that the SAR was
conserved across the two series2,4. Based on these results, lead optimization in the 2ꢀ
aminopyrazine series focused on exploring substitution of the 5ꢀphenyl group with
hydrophilic moieties at the paraꢀposition in order to improve solubility.
When comparing the 4ꢀtrifluoromethylpyridine derivatives with their direct phenyl
analogues at the 3ꢀposition (Table 1), it appears that, in most cases, the 4ꢀtrifluorophenyl
derivatives are slightly more potent against blood stage parasites than the pyridine
derivatives. Incorporation of amides derived from cyclic amines on the 5ꢀphenyl
(compounds 3, 9ai, 9di-li, 10ꢀ12) led to highly active compounds with NF54 IC50s ≤10
nM. These amides were more active than the related carboxamides (16 and 17). The size
of the cycloamine ring had little effect on activity as compounds with 4ꢀ, 5ꢀ, 6ꢀ, and 7ꢀ
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membered rings showed comparable IC50s. The Nꢀmethylated analogues of the
cycloamines also maintained comparable activity to their NH counterparts. For the
azetidine, pyrrolidine, and piperidine examples, substitution of the ring with waterꢀ
solubilizing groups were well tolerated based on in vitro blood stage activity. Piperidines
seemed to be slightly more active than pyrrolidines, while a hydroxy substituent led to
more active compounds than those with an amino group. The enantiomers of 9g2, 9h and
9i, were separated and evaluated. The Rꢀenantiomer 9i was found to be the more active
form with a NF54 IC50 of 6 nM compared to 22 nM for 9h. Hydroxyazetidine derivative
9f also showed potent activity (K1/NF54 IC50 = 7.0/9.2 nM) and was more amenable to
progressing due to the lack of chirality. Carboxylic acids 14 and 13 also displayed good
potency (K1/NF54 IC50 = 33/32 nM and 26/19 nM respectively), while methylester 15
was not active in comparison (NF54 IC50 = 2630 nM). The antiplasmodial activity data
for the carboxylic acids generated excitement as, to date, there are no known antiꢀ
malarials in development, which contain a carboxylic acid functionality6,7. In addition,
the introduction of a carboxylic acid moiety could potentially improve aqueous solubility
at physiological pH.
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The introduction of substituted pyridines at the 5ꢀposition was generally detrimental to
activity, e.g. 25 NF54 IC50 = 48 nM compared with its phenyl analogue 2 NF54 IC50 = 10
nM, or 20 NF54 IC50 = 2708 nM compared to 9g2 NF54 IC50 = 94. However, pyridine
carboxamide 22 (NF54 IC50 = 5.2 nM) exhibited improved activity over the analogous
phenyl compound 16 (NF54 IC50 = 20 nM).
An alternative strategy to improve solubility was to replace the CF3ꢀpyridine at the 3ꢀ
position with aminopyridine or phenylcarboxamide moieties (Table 2). Although
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relatively good activities were maintained, this approach generally led to a 2ꢀ6 fold drop
in activity compared to the CF3ꢀpyridine analogues (e.g. 9l1 K1/NF54 IC50 = 27/38 nM
compared to 2 K1/NF54 IC50 = 8.4/10 nM).
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Assessment for hERG activity: The activity against the hERG potassium channel was
determined using in vitro IonWorks patchꢀclamp electrophysiology8. Selected
compounds were evaluated for hERG activity with the goal of identifying compounds
with activity greater than 10 µM or with a selectivity index (SI) > 1000 in order to
minimize cardiotoxicity risks. Nꢀmethyl 1,4ꢀdiazepane derivative 9d2 and Nꢀtertꢀbutyl
piperazine 9e2 had low micromolar activity against the potassium channel with IC50s of 5
µM and 2 µM respectively. Piperazine amide 3 and aminoꢀpyrrolidine amide 9j2 had
IC50s around 10 µM and selectivity indexes of 1900 and 526 respectively. Based on their
relatively low hERG activity and high selectivity indexes, cardiotoxicity is not likely a
major risk for these two compounds. 4ꢀHydroxypiperidine amides 9l2 and 9l1,
hydroxypyrrolidine amide 9k1, and carboxylic acid 14 were all considered inactive
against the hERG potassium channel since they displayed IC50’s >33 µM and SI greater
than 1000 (>868 for 9l1).
Aqueous solubility: As previously stated, the aim of this work was to identify
compounds with potent activity against blood stage malaria parasites whilst improving
aqueous solubility over the phenylsulfoneꢀbased analogues, exemplified by 2. The
strategy employed was to lower the logD (based on calculated values) by introducing
hydrophilic water solubilizing groups. Several approaches were accordingly investigated.
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The first strategy involved replacement of the hydrophobic trifluoromethylpyridine
group of 2 with an aminopyridine group as in compound 29 improved solubility at pH 2
and at the more relevant physiological pH 6.5 (200/149 µM, respectively). However, this
trend was not consistent across the analogues made with an aminopyridine group at the 3ꢀ
position (Table 2), as illustrated by compounds 27 and 30, which remained highly
insoluble at pH 6.5. In addition, such a change negatively impacted in vivo efficacy (see
below).
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A second strategy to reduce logD and improve aqueous solubility was to use a pyridine
instead of a phenyl substituent at the 5ꢀposition. As with the aminopyridine analogues at
the 3ꢀposition, some enhancement of solubility was observed at lower pH (24: 199 µM at
pH 2), but a lack of solubility at pH 6.5 (24: 34 µM) proved the phenylpyridine change
not to be effective.
A further strategy was to substitute the 5ꢀphenyl ring with carboxamides and cyclic
amides at the 4ꢀposition. A range of cyclic amides, including amides derived from
substituted and unsubstituted azetidines, pyrrolidines, piperazines, piperidines, and 1,4ꢀ
diazepanes, was assessed. Carboxylic acids were also investigated at the 4ꢀposition. As
shown in Table 1, the acids and most amides had lower logD than 2.
The carboxamides remained relatively insoluble (pH 2/pH 6.5: 17: 78/<5 µM; 16:
<5/<5 µM). Generally, pendent primary amines resulted in good solubility at pH 2 and
moderate to good solubility at pH 6.5. Replacing the amine with a hydroxy group resulted
in a similar improvement in aqueous solubility. Indeed, hydroxypyrrolidines 9i (pH 2/pH
6.5: 103/90 µM) and 9h (pH 2/pH 6.5: 93/68 µM) displayed moderate to good solubility
at both acidic and physiological pH’s, as did the hydroxyazetidine derivative 9f (pH 2/pH
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6.5: 110/55 µM), and the hydroxypiperazine 9-l1 (pH 2/pH 6.5: 200/184 µM). The
methylated or NH 1,4ꢀdiazepane and piperazine amides showed good solubility, with the
NHꢀpiperazine amide derivatives 3, and 10 standing out with > 150 µM solubility at both
pH’s (pH 2/pH 6.5: 192/158 µM; 200/198 µM, respectively).
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Finally, the two carboxylic acids 14 and 13 had good solubility at physiological pH (pH
6.5: 95 and 178 µM, respectively).
Metabolic stability in hepatic liver microsomes: Metabolic stability of the lead
compounds was assessed in vitro in human, rat, and mouse liver microsomal
preparations. All active compounds were submitted for a turnover assay that determines
the percentage compound remaining after a 30ꢀminute incubation in the presence of liver
microsomes. Subsequently, microsomeꢀpredicted hepatic extraction ratios (EH) were
determined for selected compounds using a 5ꢀpoint assay (60 minutes). All the results are
summarized in the Supporting Information (Table F1). All of the tested compounds
proved to be stable across the 3 species with >90% of compound remaining after 30
minutes, except for the Nꢀtertꢀbutyl piperazine derivatives 9e2 and 9e1, which was
expected, as one can anticipate metabolism on the tertꢀbutyl group.
In vivo efficacy and pharmacokinetic studies: A selection of compounds with suitable
in vitro properties were tested in vivo in the P. bergheiꢀinfected and P. falciparumꢀ
infected NODꢀscid ILꢀ2Rγnull (SCID)9,10 mouse models for malaria. Efficacy in the P.
berghei assay was determined following oral administration (p.o.) of 10 mg/kg/day
and/or 3 mg/kg/day for 4 consecutive days (4ꢀday Peters’ test11). The parasitemia was
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measured at day 7 and the mice were monitored for symptoms for up to 30 days. The
efficacy results are summarized in Table 3. The Nꢀmethylpiperazines 9a1 and 9a2 were
efficacious at 4x10 mg/kg with >99.8% reduction in parasitemia. For 9a2 average mouse
survival days (MSD) was 26 days, and 2 mice out of 3 were cured (defined as having no
detectible parasites at day 30). 9a2 was also active at 4x3 mg/kg, but the MSD was only
12 days. Four compounds afforded a complete cure (3/3 mice cured and MSD >30 days)
with ≥99.9% activity at 4x10 mg/kg, namely NHꢀpiperazine 3, carboxamide 17, 2ꢀ
hydroxyꢀazetidine 9f, and carboxylic acid 14. These four compounds were also very
potent at 4x3 mg/kg with >99% reduction in parasitemia at day 7. However, the MSD
remained lower than 20 days and no complete cure was achieved at this dose. Other
compounds of interest include the 3ꢀhydroxypyrrolidines 9i and 9h, 4ꢀhydroxypiperidine
9l2, and carboxylic acid 13 that cleared >99% of parasites at 4x10 mg/kg and >90% at
4x3 mg/kg as measured on day 7.
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Interestingly, neither of the 3ꢀaminopyrrolidines (9j2 and 9j1) or the 4ꢀaminopiperidines
(9h and 9i) were active at 4x3 mg/kg, which suggests that the amino substituent is subꢀ
optimal for in vivo efficacy in these instances at this dosing regimen. The pyridine
analogue of 3, namely 10, was not efficacious at 4x10 mg/kg (<40% parasite reduction),
and neither were the NH 1.4ꢀdiazepane derivatives 12 and 11. Lastly, it is interesting to
note that the pyridine derivatives were generally less active than the related phenyl
analogues. In an attempt to rationalize the varying in vivo efficacies, drug plasma levels
from the in vivo efficacy studies were measured for 3, 9a2, 9e1, 9g2, 9i, 13, 14, 16, and
17. As shown in Figure 3, all compounds with >90% reduction in parasitemia at 4x3
mg/kg had a good plasma exposure, especially compounds 13, 14, 16 and 17. Although
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the plasma drug levels of 3 were not as high as for that of primary amides 16 and 17 or
carboxylic acids 13 and 14, the compound remained very active in vivo. The intrinsic
potency and plasma protein binding may play a role in the in vivo efficacy. On the other
hand, for compound 9e1, the reduced in vivo efficacy could be due to a higher clearance
rate as the drug levels dropped drastically after 4h.
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In addition, the ED90s (the dose that reduces parasitemia at day 7 after infection by 90%
with respect to vehicleꢀtreated mice) for compounds 3, 17, 9i, and 14 were determined in
the P. berghei model. All four compounds were highly potent with ED90’s ≤ 1mg/kg (1.0,
0.3ꢀ1.0, 1.1, and 0.56 mg/kg respectively).
Further to the P. berghei efficacy studies, promising compounds (3, 9i, 9f, 9l2, 13, 14,
and 17) were also evaluated in the P. falciparum SCID mouse model. Efficacy was
assessed following administration of one oral dose per day for four consecutive days. The
parameter estimated in this model were the ED90 in mg/kg and the estimated average
daily exposure in whole blood necessary to achieve the ED90, denoted as AUCED90. The
results are summarized in Table 3. All tested compounds were efficacious in the P.
falciparum SCID mouse model with ED90’s <0.25 mg/kg and related AUCED90’s between
0.02 (9f) and 0.20 (14). ED90’s from the in vivo P.b. and P.f. SCID studies are
comparable, showing good correlation between the two different in vivo efficacy models
of malaria.
Blood concentrations were measured following the first oral administration of compound
to all P. falciparumꢀinfected SCID mice in the efficacy studies. PK parameters are
summarized in Table I1 of the Supporting Information. All compounds showed
increasing exposure with increasing dose. Remarkably, for all compounds, the dose level
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of 1 mg·kgꢀ1 gave higher parasite clearance than that at 10 mg·kgꢀ1 even though mice
treated at 10 mg·kgꢀ1 showed the highest exposure in blood. Further detailed PK/PD
studies should be performed to elucidate whether this phenomenon is caused by
decreased compound exposure upon multiple dosing at the highest dose levels
(pharmacokinetics) or unexpected reduction of parasite clearance (pharmacodynamics).
In addition to the efficacy studies, the pharmacokinetics of the aforementioned seven
compounds in rats was determined. The data is shown in Table 4. 3 and 14 clearly stood
out with a bioavailability of 98% and 94% respectively. They also proved to have a good
plasma halfꢀlife (5.2 h and 4.8 h respectively) and low clearance and volume of
distribution. 9i also had a good bioavailability of 71% in rat and long halfꢀlife (7.2h).
However its plasma clearance rate and volume of distribution appeared to be higher than
for 3 and 14. The other compounds in the study only achieved 20 to 38 % oral
biovailability and were generally cleared from plasma at a higher rate.
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In vitro evaluation in the P. berghei (P.b.) liver and P. falciparum gametocyte
malaria parasite lifecycle stages: The lead compounds 3, 9i, 9f, 9l2, 13, 14 , and 17
were profiled against the liver and gametocyte parasite lifeꢀcycle stages to evaluate their
potential further. Activities are reported in Table 5. IC50’s were obtained for the P.b. liver
stage12 and P. falciparum early and late stage gametocytes13 (EG and LG respectively).
The piperazine amide 3 was highlighted as the most active compound in the series against
the P.b. liver assay and in the gametocyte assays. Excitingly, it demonstrated highly
potent activity against the liver stage with a subnanomolar IC50 (0.92 nM), and displayed
good activity against the early and late stage gametocytes (IC50 = 134/66 nM
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respectively). 9f, 9l2, and 9i also had activities below 15 nM (2.7, 4.1, and 14 nM
respectively) against the liver stage. However, they were less active against early stage
gametocytes (IC50 >200 nM). They remained active against late stage gametocytes,
especially compound 9f, which had a 45 nM IC50. The two carboxylic acids 13, and 14,
and carboxamide 19 were less active in these assays.
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Conclusion: Several strategies were adopted to improve aqueous solubility and
developability of compounds in the 2ꢀaminopyrazine series. The most successful
approach was to incorporate amides containing a water solubilizing group at the 4ꢀ
position of the 5ꢀphenyl ring. Cyclic amides proved to be highly efficacious in vivo in
both the P. falciparum SCID and P. berghei mouse models for malaria, with ED90s ≤ 1
mg/kg.
The piperazine amide 3 in particular was shown to be one of the most potent compounds
in vitro against asexual blood, liver, and gametocyte stages of the malaria parasite. With
the aid of its good pharmacokinetics properties, including a long halfꢀlife and
bioavailability of 98%, in vitro activity translated into potent in vivo efficacy. Good
selectivity over inhibition of the hERG channel was also achieved. The overall profiles of
compound 3 demonstrates its potential to become a drug candidate for the treatment of
malaria and further profiling is in progress. It is an attractive potential followꢀon
compound to the previously described 2ꢀaminopyridine 12 a novel Plasmodium PI4K
inhibitor that is currently in clinical trials for the treatment of malaria.
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Experimental section:
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All commercially available chemicals were purchased from either SigmaꢀAldrich or
CombiꢀBlocks. All solvents were dried by appropriate techniques. Unless otherwise
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stated, all solvents used were anhydrous. H NMR spectra were recorded on a Varian
Mercury Spectrometer at 300 MHz or a Varian Unity Spectrometer at 400 MHz. 13C
NMR spectra were recorded at 75 MHz on a Varian Mercury Spectrometer or at 100
MHz on Varian Unity Spectrometer. Highꢀresolution mass spectra were recorded on a
VG70 SEQ micromass spectrometer. Melting points (M.P.) were determined by
Differential Scanning Calorimetry (DSC) using TA Q200/Q2000 DSCfrom TA
Instruments. Analytical thinꢀlayer chromatography (TLC) was performed on aluminiumꢀ
backed silicaꢀgel 60 F254 (70ꢀ230 mesh) plates. Column chromatography was performed
with Merck silicaꢀgel 60 (70ꢀ230 mesh). Chemical shifts (δ) are given in ppm downfield
from TMS as the internal standard. Coupling constants, J, are recorded in Hertz (Hz).
Purity was determined by HPLC and all compounds were confirmed to have > 95%
purity.
The data that is not shown below is supplied in the Supporting Information.
General procedure for the bromination at the 3-position: To a suspension of 2ꢀ
aminopyrazine (1 eq) in dry DCM (1 M) was added Nꢀbromosuccinimide (1 eq) in
portion at 0º C under nitrogen. The reaction mixture was stirred at RT for 1.5 h, before
being concentrated under vacuum. Water (30 mL) was added and the resulting solid was
filtered off. The filtrate was extracted with DCM:MeOH (9:1, x 4), and the combined
organic layers were washed with brine, dried over anhydrous Na2SO4 and concentrated
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under vacuum. The crude solid was purified by column chromatography over silica gel
(230ꢀ400 mesh) by using 25ꢀ28% of ethyl acetate in petroleum ether as an eluent to
afford product 4 (52%) as a yellow solid.
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1H NMR (400 MHz, DMSOꢀd6): δ 8.03 (d, J = 1.3 Hz, 1H), 7.68 (d, J = 1.3 Hz, 1H), 6.66
(s, 2H). LCꢀMS APCI: found m/z = 176.0 [M+H]+, (calculated for C4H4BrN3: 172.95,
174.95); Purity by LCꢀMS: 99.8%.
General procedure for the first Suzuki cross-coupling reaction: To a solution of 4 (1
eq) in 1,4ꢀdioxane (resulting solution at 0.6 M) of was added 4ꢀ
methoxycarbonylphenylboronic acid (1.1 eq) followed by potassium phosphate tribasic
(1.2 eq) and water (0.068 Voldioxane mL) at RT. The reaction mixture was purged with
nitrogen for 1 h. Bis(triphenylphosphine)palladium(II)chloride (0.07 eq) was then added
to the reaction mixture. The reaction mixture was heated to reflux for 16h, cooled to RT
and concentrated under vacuum to remove dioxane. 50 mL of water was added and the
resulting solid was filtered off, washed with water (20 mL x 3), dried and again washed
with MeOH (10 mL x 4), and dried to afford compound 5.
Methyl 4ꢀ(5ꢀaminopyrazinꢀ2ꢀyl)benzoate 5: 91%, pale yellow solid.
1H NMR (400 MHz, DMSOꢀd6): δ 8.62 (d, J = 1.20 Hz, 1H), 8.07 (d, J = 8.56 Hz, 2H),
7.98ꢀ8.00 (m, 3H), 6.78 (s, 2H), 3.86 (s, 3H). LCꢀMS APCI: found m/z = 230.2 [M+H]+,
(calculated for C12H11N3O2: 229.23), purity by LCꢀMS: 84.5%.
General procedure for the second bromination: To a suspension of compound 5 at 0ºC
(1 eq) in dry DCM (0.5 M) was added Nꢀbromosuccinimide (1.1 eq) in portions under
nitrogen. The resulting mixture was stirred at RT for 1 h. To the reaction mixture was
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added 20 mL of water and the resulting mixture was extracted with DCM:MeOH.
Combined organic layers were washed with brine, dried over anhydrous Na2SO4 and
concentrated under vacuum. The crude material was purified by column chromatography
over silica gel 230ꢀ400 mesh using 24% of ethyl acetate and 4% of DCM in petroleum
ether as an eluent to afford compound 6.
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Methyl 4ꢀ(5ꢀaminoꢀ6ꢀbromopyrazinꢀ2ꢀyl)benzoate 6: 47%, pale yellow solid
1H NMR (300 MHz, DMSOꢀd6): δ 8.70 (s, 1H), 7.98ꢀ8.05 (m, 4H), 7.08 (s, 2H), 3.85 (s,
3H). LCꢀMS APCI: found m/z = 309.8 [M+H]+, (calculated for C12H10BrN3O2: 306.99,
308.99), purity by LCꢀMS: 95.2%.
General procedure for the ester hydrolysis: To a solution of compound 6 (4.76 g,
15.44 mmol) in THF (50 mL) was added lithium hydroxide monohydrate (1.5 eq) at RT
and the resulting mixture was stirred for 16 h. It was then concentrated under vacuum,
before 10 mL of water were added. Concentrated HCl was added dropwise until the pH
of the solution was acidic. The solid was filtered off, washed with water, dried and again
washed with DCM to give compound 7.
4ꢀ(5ꢀaminoꢀ6ꢀbromopyrazinꢀ2ꢀyl)benzoic acid 7: 90%, pale yellow solid.
1H NMR (300 MHz, DMSOꢀd6): δ 13.00 (bs, 1H), 8.68 (s, 1H), 7.95ꢀ8.02 (m, 4H), 7.04
(s, 2H). LCꢀMS APCI: found m/z = 295.6 [M+H]+, (calculated for C11H8BrN3O2: 292.98,
294.98), purity by LCꢀMS: 94.7%.
General procedure for the amide coupling: To a suspension of compound 7 (1 eq) in
dry THF (0.2M) was added EDCI.HCl (1.2 eq), HOBt (0.1 eq) and triethylamine (2.5 eq)
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at RT under nitrogen. The resulting mixture was stirred for 1 h. Nꢀtertꢀbutyl piperazine
was then added (532 mg, 3.74 mmol) at RT and stirring was allowed for 16 h. Water was
added and the solution was extracted with ethyl acetate. The combined organic layers
were washed with brine, dried over anhydrous Na2SO4 and concentrated under vacuum.
The crude material was purified by column chromatography over neutral alumina using
3% MeOH in DCM as an eluent to yield the desired compounds 8a-l.
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Second Suzuki coupling: To a solution of compound 8 (1 eq) in 1, 4ꢀdioxane (0.2 M)
was added the appropriate boronic acid pinacol ester (1.1 eq) at RT and the solution was
purged with nitrogen for 30 minutes. Bis(triphenylphosphine)palladium(II)chloride (0.07
eq) and 1 M aqueous solution of potassium carbonate (1.2 eq, preꢀpurged with nitrogen)
were added to the reaction mixture. The reaction mixture was heated to reflux for 16 h
and cooled to RT. Brine solution was added to the reaction mixture and extraction with
ethyl acetate was performed. Combined organic layers were dried over anhydrous
Na2SO4 and concentrated under vacuum. The crude material was purified by column
chromatography over silica gel 230ꢀ400 mesh by using 3.5% of MeOH in DCM as an
eluent to afford the desired compounds 9ai-li,(i = 1,2)
Boc-deprotection: The protected amides were stirred in neat TFA for 12 hours, at which
time TFA was removed under pressure. The residue was dissolved in DCM/MeOH (9:1)
and stirred with Amberlyst A21 for 1 h. The resin was filtered off, the solvents were
removed under pressure and the residue was purified by column chromatography.
(4ꢀ(5ꢀaminoꢀ6ꢀ(4ꢀ(trifluoromethyl)phenyl)pyrazinꢀ2ꢀyl)phenyl)(piperazinꢀ1ꢀ
yl)methanone 3:
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1H NMR (300 MHz, DMSOꢀd6): δ 8.69 (s, 1H), 8.09 (d, J = 8.20 Hz, 2H), 8.03 (d, J =
8.04 Hz, 2H), 7.88 (d, J = 8.16 Hz, 2H), 7.53 (d, J = 8.24 Hz, 2H), 6.63 (s, 2H), 3.60ꢀ3.40
(m, 5H), 2.81 (brs, 4H). 13C NMR (100 MHz, DMSOꢀd6) δ: 45.3 (2C), 65.4, 123.4, 125.3
(2C), 126.0 (2C), 128.0 (2C), 129.4 (q), 129.6 (2C), 135.3, 136.7, 138.2, 139.3, 139.7,
142.0, 153.0, 169.5. HPLC/MS: (ESI)+: found m/z = 428.2 [M+H]+, (calculated for
C22H20F3N5O: 427.16), purity by HPLC: 98.8% ; M.P.: 195.4 oC (DSC)
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(4ꢀ(5ꢀaminoꢀ6ꢀ(4ꢀ(trifluoromethyl)phenyl)pyrazinꢀ2ꢀyl)phenyl)(3ꢀhydroxyazetidinꢀ1ꢀ
yl)methanone 9f:
1H NMR (400 MHz, DMSOꢀd6): δ 8.70 (s, 1H), 8.08 (d, J = 8.8 Hz, 2H), 8.03 (d, J = 8.7
Hz, 2H), 7.88 (d, J = 8.7 Hz, 2H), 7.70 (d, J = 8.8 Hz, 2H), 6.60 (s, 2H), 5.74 (d, J = 5.6
Hz,1H), 4.57ꢀ4.45 (m, 2H), 4.27 (brs, 1H), 4.08 (brs, 1H), 3.81 (brs, 1H). 13C NMR (100
MHz, DMSOꢀd6) δ: 58.9, 63.3, 123.4, 125.1 (2C), 126.0, 126.0, 128.7 (2C), 129.3 (q),
129.6 (2C), 132.7, 136.9, 139.2, 139.4, 139.8, 142.0, 153.0, 169.3. LCꢀMS APCI: found
m/z = 415.2 [M+H]+, (calculated for C22H19F3N4O2: 414.13), purity by HPLC: >99.0%.
(R)ꢀ(4ꢀ(5ꢀaminoꢀ6ꢀ(4ꢀ(trifluoromethyl)phenyl)pyrazinꢀ2ꢀyl)phenyl)(3ꢀ
hydroxypyrrolidinꢀ1ꢀyl)methanone 9i:
1H NMR (400 MHz, DMSOꢀd6): δ 8.69 (s, 1H), 8.03ꢀ8.07 (m, 4H), 7.88 (d, J = 8.20 Hz,
2H), 7.58ꢀ7.61 (m, 2H), 6.62 (s, 2H), 4.95ꢀ5.04 (m, 1H), 4.24ꢀ4.33 (m, 1H), 3.40ꢀ3.63
(m, 4H), 1.81ꢀ1.97 (m, 2H). 13C NMR (100 MHz, DMSOꢀd6) δ: 32.6, 34.9, 44.6, 47.4,
54.9, 57.6, 68.5, 69.9, 123.4, 125.0 (2C), 126.1, 126.2, 128.1, 128.2, 129.1 (q), 129.6
(2C), 136.4, 136.8, 138.4, 139.4, 139.7, 142.0, 152.9, 168.7, 168.7. LCꢀMS APCI: found
m/z = 429.2 [M+H]+, (calculated for C22H19F3N4O2: 428.15), purity by HPLC: 98.0%.
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(4ꢀ(5ꢀaminoꢀ6ꢀ(4ꢀ(trifluoromethyl)phenyl)pyrazinꢀ2ꢀyl)phenyl)(4ꢀhydroxypiperidinꢀ1ꢀ
yl)methanone 9l2:
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1H NMR (400 MHz, DMSOꢀd6): δ 8.68 (s, 1H), 8.03ꢀ8.07 (m, 4H), 7.88 (d, J = 8.28 Hz,
2H), 7.45 (d, J = 8.20 Hz, 2H), 6.61 (s, 2H), 4.81 (d, J = 3.92 Hz, 1H), 4.01 (bs, 1H),
3.72ꢀ3.76 (m, 1H), 3.54 (bs, 1H), 3.18 (bs, 1H), 3.16 (bs, 1H), 1.73ꢀ1.78 (m, 2H), 1.36
(bs, 2H). 13C NMR (100 MHz, DMSOꢀd6) δ: 33.9 (2C), 44.2(2C), 65.6, 123.5, 124.8
(2C), 125.6, 125.6, 127.3 (2C), 128.9 (q), 129.1(2C), 135.4, 136.3, 137.6, 139.0, 139.2,
141.6, 152.5, 168.8. LCꢀMS APCI: found m/z = 443.2 [M+H]+, (calculated for
C23H21F3N4O2: 442.16), purity by HPLC: 98.1%.
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4ꢀ(5ꢀaminoꢀ6ꢀ(6ꢀ(trifluoromethyl)pyridinꢀ3ꢀyl)pyrazinꢀ2ꢀyl)benzoic acid 13:
1H NMR (400 MHz, DMSOꢀd6) δ 9.16 (d, J = 2.1 Hz, 1H), 8.74 (s, 1H), 8.47 (dd, J =
8.1, 2.1 Hz, 1H), 8.13 (d, J = 8.5 Hz, 2H), 8.04ꢀ8.00 (m, 3H), 6.64 (s, 2H). 13C NMR (101
MHz, DMSOꢀd6) δ 167.5, 153.5, 150.1, 141.0, 140.6, 139.6, 138.3, 137.1, 134.6, 130.5,
130.3, 125.4 (2C), 121.1(2C). HPLC/MS: (ESI)+: found m/z = 361.1 [M+H]+, (calculated
for C17H11F3N4O2: 360.08), purity = 99%
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4ꢀ(5ꢀaminoꢀ6ꢀ(4ꢀ(trifluoromethyl)phenyl)pyrazinꢀ2ꢀyl)benzoic acid 14: H NMR (400
MHz, DMSOꢀd6): δ 12.91 (1H, broad s), 8.68 (1H, s), 8.10 (2H, d, J = 8.2 Hz), 7.99 (4H,
m), 7.86 (2H, d, J = 8.2Hz), 6.69 (2H, s). 13C NMR (100 MHz, DMSOꢀd6): δ 167.5,
153.1, 141.9, 141.3, 140.1, 139.0, 137.1, 130.3 (2C), 130.1, 129.6 (2C), 129.3 (q), 126.1,
126.0, 125.3 (2C), 123.4. LCꢀMS: (ESI)+: found m/z = 360.1 [M+H]+, (calculated for
C18H12F3N3O2: 359.08), purity = 99.9% ; M.P.: 267.6 oC (DSC)
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4ꢀ(5ꢀaminoꢀ6ꢀ(4ꢀ(trifluoromethyl)phenyl)pyrazinꢀ2ꢀyl)benzamide) 17:
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1H NMR (400 MHz, DMSOꢀd6): δ 8.71 (s, 1H), 8.01ꢀ8.08 (m, 5H), 7.94 (d, J = 8.40 Hz,
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2H), 7.87 (d, J = 8.24 Hz, 2H), 7.37 (s, 1H), 6.62 (s, 2H). C NMR (100 MHz, DMSOꢀ
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d6) δ: 123.4, 125.0 (2C), 126.0, 126.0, 128.5 (2C), 129.1 (q), 129.6 (2C), 133.6, 136.9,
139.3, 139.8, 139.9 142.0, 153.0, 168.0. LCꢀMS APCI: found m/z = 359.2 [M+H]+,
(calculated for C18H13F3N4O: 358.10), purity by HPLC: 99.6%.
In vitro P. falciparum assay and in vivo P. berghei antimalarial efficacy studies.
Compounds were screened against multidrug resistant (K1) and sensitive (NF54) strains
of P. falciparum in vitro using the modified [3H]ꢀhypoxanthine incorporation assay14. In
vivo P. berghei efficacy was conducted as previously described3, with the modification
that mice (n = 3) were infected with a GFPꢀtransfected P. berghei ANKA strain (donated
by A. P. Waters and C. J. Janse, Leiden University, The Netherlands) and parasitemia
was determined using standard flow cytometry techniques. The detection limit was 1
parasite in 1,000 erythrocytes (that is, 0.1%). Activity was calculated as the difference
between the mean per cent parasitemia for the control and treated groups expressed as a
per cent relative to the control group. Compounds were dissolved or suspended in a nonꢀ
solubilizing, standard suspension vehicle called HPMC (0.5% [wt/vol]
hydroxypropylmethylcellulose, 0.5% [vol/vol] benzyl alcohol, 0.4% [vol/vol] Tween 80,
and 0.9% [wt/vol] sodium chloride in water), and administered orally as four consecutive
daily doses (4, 24, 48 and 72 h after infection). Blood samples for the quadrupleꢀdose
regimens were collected on day 4 (96 h after infection) on 2 of the 3 mice.
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In vitro gametocytocidal activity:
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In vitro gametocytocidal activity was determined using luciferase reporter lines
specifically enabling screening against early stage gametocytes (>90% stage II/III) and
late stage gametocytes (>95% stage IV/V) as previously reported13. More details are
given in section J of the Supporting Information.
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In vitro P.b. liver assays:
This assay is based on the murine malaria parasite Plasmodium berghei transformed with
Luciferase. Hepatic human transformed cells (HepG2), pretreated for 18 hours with the
compound to investigate, are infected with freshly dissected P. berghei Luciferase sporozoites.
After another 48 hours of incubation with the compound to investigate, the viability of P. berghei
exoerythrocytic forms (EEF) and the HepG2 host cells is measured by bioluminescence.
This assay allows us to identify compounds with an eventual activity against sporozoite infection
of liver cells as well the viability of liver schizonts. More details are provided in section K of the
Supporting Information.
ASSOCIATED CONTENT:
Supporting Information Available: Additional details of the characterization of selected
compounds and the procedures used for the in vitro and in vivo antimalarial studies as
well as PK and metabolism studies. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION:
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Corresponding author
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* Phone: +27ꢀ21ꢀ6502557. Fax: +27ꢀ21ꢀ6505195. Eꢀmail: Kelly.Chibale@uct.ac.za
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ACKNOWLEDGEMENTS: We thank Medicines for Malaria Ventures (MMV) and
the South African Technology Innovation Agency (TIA) for financial support of this
research (Project MMV09/0002). The University of Cape Town, South African Medical
Research Council, and South African Research Chairs Initiative of the Department of
Science and Technology, administered through the South African National Research
Foundation are gratefully acknowledged for support (K.C). We thank Christoph Fischli,
Jolanda Kamber, Sibylle Sax, Christian Scheurer and Ursula Lehmann for assistance in
performing antimalarial assays. At UCT, we thank Carmen de Kock, Virgil Verhoog and
Sumaya Salie for running the antimalarial assays, Nesia Barnes and Warren Olifant for
running the ADME assays and Trevor Finch for assistance with the animal work. We
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thank L. D. Shultz and The Jackson Laboratory for providing access to nonobese diabetic
null
scid IL2Rγ
mice (NSG mice) through their collaboration with GSK Tres Cantos
Medicines Development Campus.
ABBREVIATIONS USED: SAR, structureꢀactivity relationships; ADME, absorption,
distribution, metabolism and excretion; CQ, chloroquine; p.o., oral administration; i.v.,
intraveneous administration; MSD, mean survival days; PK, pharmacokinetics; NMR,
nuclear magnetic resonance; M.P.,melting point; DSC, differential scanning calorimetry;
RT, room temperature; MMV, Medicines for Malaria Venture.
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Figure 1. Structures of compounds 1 and 2:
Figure 2.
Figure 3. Exposure and PK parameters after oral administration (4x3 mg/kg) for
representative compounds (snapshot mouse PK).
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MMV #
Dose
App.
AUC
App.
Cmax
(µM)
4
App.
Cav24
(µM)
1.8
(mg/kg)
(µM.h)
42.3
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69.3
5
2.9
5.4
0.4
0.2
9a2
14
13
9e1
9g2
9i
c.n.c.*
79.7
1.6
c.n.c.*
3.5
5
30.81
4.34
2.29
1.52
3.61
3.29
1.48
0.57
2.06
1.93
34.7
33.19
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Scheme 1: Preparation of compounds 3, 9ai-li (i= 1,2), 10,11 and12.
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Reagents and conditions: (i) NBS (1.1 eq), DCM, RT, 1.5h, 52% yield; (ii) 4ꢀCOOMeꢀ
PhꢀB(OH)2 (1.1 eq), Pd(PPh3)2Cl2 (0.07 eq), aq. K2CO3 (1M) (1.2 eq), dioxane,
reflux,16h, 91% yield; (iii) NBS (1.1 eq), DCM, RT, 1h, 47% yield; (iv) LiOH ( 1.5 eq),
THF, 16h, 90% yield; (v) NHR’R (eq), EDCI.HCl (1.2 eq), Et3N (eq), HOBt (0.1eq),
THF, RT, 16h, 63ꢀ88% yield; (vi) 4ꢀCF3ꢀPhꢀB(OH)2,pinacol ester or 4ꢀCF3ꢀPyridineꢀ
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