Structure-activity-relationship studies around the 2-amino group and pyridine core of antimalarial 3,5-diarylaminopyridines lead to a novel series of pyrazine analogues with oral in vivo activity

Article abstract of DOI:10.1021/jm401278d

Replacement of the pyridine core of antimalarial 3,5-diaryl-2- aminopyridines led to the identification of a novel series of pyrazine analogues with potent oral antimalarial activity. However, other changes to the pyridine core and replacement or substitution of the 2-amino group led to loss of antimalarial activity. The 3,5-diaryl-2-aminopyrazine series showed impressive in vitro antiplasmodial activity against the K1 (multidrug resistant) and NF54 (sensitive) strains of Plasmodium falciparum in the nanomolar IC₅₀ range of 6-94 nM while also demonstrating good in vitro metabolic stability in human liver microsomes. In the Plasmodium berghei mouse model, this series generally exhibited good efficacy at low oral doses. One of the frontrunner compounds, 4, displayed potent in vitro antiplasmodial activity with IC ₅₀ values of 8.4 and 10 nM against the K1 and NF54 strains, respectively. When evaluated in P. berghei-infected mice, compound 4 was completely curative at an oral dose of 4 × 10 mg/kg.

Full text of DOI:10.1021/jm401278d

Article
pubs.acs.org/jmc
Structure−Activity-Relationship Studies around the 2‑Amino Group
and Pyridine Core of Antimalarial 3,5-Diarylaminopyridines Lead to a
Novel Series of Pyrazine Analogues with Oral in Vivo Activity
Yassir Younis,† Frederic Douelle,† Diego Gonzalez Cabrera,† Claire Le Manach,† Aloysius T. Nchinda,†
́
Tanya Paquet,† Leslie J. Street,† Karen L. White,‡ K. Mohammed Zabiulla,§ Jayan T. Joseph,§
Sridevi Bashyam,§ David Waterson,∥ Michael J. Witty,∥ Sergio Wittlin,⊥,# Susan A. Charman,‡
and Kelly Chibale*,†,∇
†Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
‡Centre for Drug Candidate Optimisation, Monash University (Parkville Campus), 381 Royal Parade, Parkville, Victoria 3052,
Australia
§Syngene International Ltd., Biocon Park, Plot No. 2 & 3. Bommasandra IV Phase, Jigani Link Road, Bangalore 560099, India
∥Medicines for Malaria Venture, ICC Building, Route de Pre-
́
Bois 20, P.O. Box 1826, 1215 Geneva, Switzerland
⊥Swiss Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel, Switzerland
#University of Basel, Socinstrasse 57, 4002 Basel, Switzerland
∇Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
S
* Supporting Information
ABSTRACT: Replacement of the pyridine core of antimalarial 3,5-diaryl-2-aminopyridines led to the identification of a novel
series of pyrazine analogues with potent oral antimalarial activity. However, other changes to the pyridine core and replacement
or substitution of the 2-amino group led to loss of antimalarial activity. The 3,5-diaryl-2-aminopyrazine series showed impressive
in vitro antiplasmodial activity against the K1 (multidrug resistant) and NF54 (sensitive) strains of Plasmodium falciparum in the
nanomolar IC50 range of 6−94 nM while also demonstrating good in vitro metabolic stability in human liver microsomes. In the
Plasmodium berghei mouse model, this series generally exhibited good efficacy at low oral doses. One of the frontrunner
compounds, 4, displayed potent in vitro antiplasmodial activity with IC50 values of 8.4 and 10 nM against the K1 and NF54
strains, respectively. When evaluated in P. berghei-infected mice, compound 4 was completely curative at an oral dose of 4 × 10
mg/kg.
affordable drugs against multidrug-resistant Plasmodium strains
are long-term and vital tasks for researchers.3−10
In our previous work, we disclosed the aminopyridine series
of compounds as novel orally active antimalarial agents.11 SAR
exploration around positions 3 and 5 of the 2-aminopyridine
core led to the identification of 1 and 2 as frontrunner
INTRODUCTION
■
Malaria is one of the most widespread and deadly infectious
diseases in the world and is responsible for an estimated 500
000−900 000 deaths each year, especially among children and
pregnant women.1 Plasmodium falciparum and Plasmodium
vivax are the two most prevalent species responsible for causing
disease in humans.2 Because of the continuing development of
the resistance of malaria parasites to conventional antimalarial
drugs, efforts to search for novel, structurally diverse, and
Received: August 19, 2013
Published: October 7, 2013
© 2013 American Chemical Society
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candidates (Figure 1).11,12 Inspired by this initial success and in
our quest to identify more potent analogs with improved
5-bromo-3-iodopyrazin-2-amine 24, which underwent Suzuki
cross-coupling reactions using 6-(trifluoromethyl)pyridin-3-
ylboronic acid or 4-(trifluoromethyl)phenylboronic acid
resulting in intermediates 25 and 25a (Scheme 5). The aryl
groups were introduced at position 5 of the 2-aminopyrazine
core via Suzuki cross-coupling reactions with appropriate
boronic acids to deliver the desired target compounds 4−10
and 26−36.11−13All compounds were purified using column
chromatography and were fully characterized by a range of
analytical and spectroscopic techniques.
In Vitro Antiplasmodial Activity. Initial studies involved
exploration around the 2-amino group and pyridyl core to
establish SAR, as shown in Tables 1 and 2, respectively. The in
vitro antiplasmodial activities of all synthesized compounds
were determined against a sensitive (NF54) and multidrug
resistant (K1) strain of P. falciparum.6 Chloroquine and
artesunate were used as the reference drugs in all of the
experiments. Among the 7 compounds evaluated with varying
2-amino group substituents, the unsubstituted 2-amino group
retained the best activity (1; IC50 = 51 nM (K1) and (NF54)).
Complete removal of the 2-amino substituent (13; IC50 = 538
nM (K1) and 421 nM (NF54)) led to a decrease in
antiplasmodial potency. Introducing electron-withdrawing or
-donating substituents in place of the 2-amino group resulted in
a significant loss in antiplasmodial activity as exemplified by 17
(IC50 = >24 505 nM (K1) and (NF54)) and 15 (IC50 > 28 083
nM (K1) and (NF54)). In general, the unsubstituted 2-amino
group of the pyridyl core is critical to potent in vitro
antiplasmodial activity, suggesting that this group is essential
for specific binding to an as yet unknown target.
The next phase of SAR exploration was directed at
optimizing the pyridyl core while fixing the optimal
unsubstituted 2-amino group. Compounds 18−21 (Table 2)
were synthesized for this purpose. It was observed that the 2-
aminopyridine core remained critical for antiplasmodial activity
(1; IC50 = 51 nM (K1) and (NF54)), whereas its replacement
with either phenyl, pyridazine, and/or thienopyridine groups
was detrimental to activity as was the case for 19 (IC50 >2821
nM (K1) and (NF54)), 20 (IC50 > 281 nM (K1) and (NF54)),
and 21 (IC50 = 1173 nM (K1) and >2433 nM (NF54)).
Moving the position of the pyridine nitrogen, as exemplified by
compound 18 (IC50 >28 136 nM (K1) and (NF54)), led to a
significant loss in antiplasmodial potency. However, introduc-
ing the pyrazine core (3; IC50 = 45 nM (K1) and 48 nM
(NF54)) resulted in significant potency, which was comparable
Figure 1. Structures of 2-aminopyridine frontrunners compounds, 1
and 2.9,10
ADME properties, our attention was diverted to the
investigation of SAR around the 2-amino group and pyridine
core.
In this article, we report the synthesis and SAR exploration
around the 2-amino group and the pyridine core of 3,5-
diarylaminopyridines, leading to the identification of the
corresponding 2-aminopyrazines with potent antimalarial
activity (Figure 2).
RESULTS AND DISCUSSION
■
Chemistry. Target compounds 1, 11, 13, and 18−20 were
synthesized using a previously reported general route, which
involved iodination followed by two Suzuki cross-coupling
reactions, as depicted in Scheme 1.11−13 Compounds 14−16
were obtained from compound 1 by converting the 2-amino
group of the pyridyl core to the corresponding 2-iodo-, 2-
ethoxy-, and 2-hydroxyl-substituted pyridines using previously
described procedures to give the desired products, as depicted
in Scheme 2.14−16 The 2-trifluoromethyl analogue 17 was
prepared from 2-iodo analogue 14 using methyl
(fluorosulfonyl)difluoroacetate and copper iodide in DMF, as
shown in Scheme 2.17 Compounds 12 and 21 were accessed as
outlined in Schemes 3 and 4, respectively. The aminopyrazine
analogues 4−10 and 26−36 shown in Table 3 were synthesized
from 2-aminopyrazine 22, which was brominated at position 5
using NBS in DCM at 25 °C for 4 h to afford intermediate 5-
bromopyrazin-2-amine 23. This intermediate was iodinated at
position 3 using NIS in DCM to give the second intermediate
Figure 2. SAR around 2-aminopyrdines led to 2-aminopyrazine series.
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a
Scheme 1. General Synthetic Approach for Synthesized Compounds 1, 3, 11, 13, and 18−20
a
Conditions: (a) I2, DMSO, 100 °C, 4 h, (75%); (b) 1,4-dioxane, K2CO3 (aq, 1 M), Pd(PPh3)2Cl2 (5 mol %), 110 °C, 16 h, (55%); (c) 1,4-dioxane,
K2CO3 (aq, 1 M), Pd(PPh3)2Cl2 (7 mol %), 110 °C, 16 h, (40−60%).
a
Scheme 2. General Synthetic Approach for Synthesized Compounds 14−17
a
Conditions: (a) CH2I2, t-BuONO, I2, 25 °C, 24 h, (49%); (b) DMF, CuI, FSO2CF2CO2Me, microW, 125 °C, 30 min (45%); (c) NaNO2, TFA, °C,
15 min, rt, 2 h, (88%); (d) CH3CH2I, K2CO3, THF/DMSO, 50 °C, 16 h, (52%).
to that of the corresponding 2-aminopyridine compound 1
(IC50 = 51 nM (K1) and (NF54)). 3 was profiled for its in vitro
ADME properties and found to have moderate metabolic
stability in human liver microsomes, with a predicted human
hepatic extraction ratio (EH) of 0.39. On the basis of our initial
SAR knowledge of the metabolic vulnerability of the methoxy
group of the 3-pyridyl substituent, a trifluoromethyl group was
introduced, leading to the identification of compound 4 with
impressive in vitro antiplasmodial activity (IC50 = 8.4 nM (K1)
and 10 nM (NF54)) and significantly improved metabolic
stability in human and rat liver microsomes (EH < 0.28 and <
0.19, respectively) (Figure 2). In addition, no cytotoxicity was
observed for compound 4 when tested against a Chinese
hamster ovary (CHO) cell line (IC50 >254 μM; selectivity
index, SI, >20 000). The 4-trifluoromethylphenyl substituent at
position 3 also led to impressive antiplasmodial activity in 5
(IC50 = 11 nM (K1) and (NF54)).
Aminopyrazines and their derivatives have been reported to
possess a variety of pharmacological activities, including
antioxidant and kinase inhibition activities.18−20 Prior to our
work, some examples of this class were reported as having in
vitro antimalarial activity among the many thousands of
compounds in the published GlaxoSmithKline (GSK) Tres
Cantos Antimalarial Set (TCAMS) and Novartis (GNF)
Malaria Screening.21−23 However, this activity has not been
followed up on in the literature. As demonstrated in previous
studies for the 5-aryl position,12 introducing polar, electron-
withdrawing substituents such as morpholinylcarbonyl, iso-
propylsufonyl, and methylsulfonamido resulted in superior
antiplasmodial activity, as displayed by compound 6 (IC50 = 10
nM (K1) and 9.0 nM (NF54)), 7 (IC50 = 7.0 nM (K1) and 6.0
nM (NF54)), 8 (IC50 = 17 nM (K1) and 20 nM (NF54)), 9
(IC50 = 11 nM (K1) and 12 nM (NF54)), and 10 (IC50 = 21
nM (K1) and 23 nM (NF54)). Adding cyclopropylmethylsul-
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a
Scheme 3. General Synthetic Approach for Synthesized Compound 12
a
Conditions: (a) (i) 1,4-diaminobutane, 115 °C, 16 h; (ii) (Boc)2O, DCM/MeOH, Et3N, rt, 16 h; (b) NBS, PEG, rt, 30 min; (c) 1,4-dioxane,
K2CO3 (aq, 1 M), Pd(PPh3)2Cl2 (5 mol %), 110 °C, 16 h, (55%); (d) NBS, PEG, rt, 30 min; (e) 1,4-dioxane, K2CO3 (aq, 1 M), Pd(PPh3)2Cl2 (7
mol %), 110 °C, 16 h, (60%); (f) (i) TFA, DCM, rt, 3h, (ii) Amberlyst, DCM/MeOH, rt, 3 min (89.8%).
a
Scheme 4. General Synthetic Approach for Synthesized Compounds 21
a
Conditions: (a) 1,4-dioxane, K2CO3 (aq, 1 M), Pd(PPh3)2Cl2 (5 mol %), 110 °C, 16 h, (55%); (b) NBS, DMF, rt, 15 min, (50%); (c) 1,4-dioxane,
K2CO3 (aq, 1 M), Pd(PPh3)2Cl2 (7 mol %), 110 °C, 16 h, (42%).
a
Scheme 5. General Synthetic Approach for Synthesized Compounds 4−10 and 26−36
a
Conditions: (a) NBS, DCM, rt, 4 h, (50.4%); (b) NIS, 1,4-dioxane, 80 °C, 4 h, (58%); (c) 1,4-dioxane, K2CO3 (aq, 1 M), Pd(PPh3)2Cl2 (5 mol %),
110 °C, 16 h, (55%); (d) 1,4-dioxane, K2CO3 (aq, 1 M), Pd(PPh3)2Cl2 (7 mol %), 110 °C, 16 h, (40−60%).
fonyl and cyclopropylsulfonyl aryl substituents at position 4
also resulted in good potency, as achieved with compounds 27
(IC50 = 13 nM (K1) and 19 nM (NF54)) and 26 (IC50 = 10
nM (K1) and 11 nM (NF54)), respectively. Introducing 4-
methylsulfoxide aryl substituent maintained good potency (36;
IC50 = 16 nM (K1) and 21 nM (NF54)) in comparison with
corresponding methylsulfonyl analogue 4. Moreover, we
investigated aryl substitution at position 5 of the 2-amino-
pyrazine core by introducing heteroaromatic substituents.
Interestingly, compound 28 (IC50 = 20 nM (K1) and 26 nM
(NF54)) with a 4-pyridyl substituent displayed good activity,
whereas 3-pyridyl-containing 29 (IC50 = 316 nM (NF54)) and
pyrimidine-based 30 (IC50 = 735 nM (K1) and 1043 nM
(NF54)) substituents showed significantly decreased in vitro
antiplasmodial activity. Furthermore, compounds containing
the 2-aminopyridyl 33 (IC50 = 28 nM (K1) and 33 nM
(NF54)) and 2-methylsulfonylpyridyl (35) (IC50 = 41 nM
(K1) and 48 nM (NF54)) substituents also demonstrated good
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Table 1. In Vitro Antiplasmodial Activity of Compounds 1 and 11−17
a
b
Mean from n values of ≥2 independent experiments. LogD pH 7.4 values were determined using a chromatographic estimation method; nd = not
determined. Data from Younis et al.11
c
activity. However, compounds with 2-fluoropyridyl 31 (IC50
=
speaking, an improvement in the hERG profile was observed
for some aminopyrazine analogues.
189 nM (K1) and 282 nM (NF54)) and 2-trifluoromethylpyr-
idyl 32 (IC50 = 680 nM (NF54)) substituents displayed inferior
activity. Generally speaking, 2-aminopyrazine analogues dis-
played potent antiplasmodial activity of slightly greater potency
than their previously reported corresponding 2-aminopyridines.
Toward hERG derisking, some analogues from the amino-
pyrazine series were tested for their activity against the hERG
potassium channel using in vitro IonWorks patch-clamp
electrophysiology. The data is shown in Table 3. Lead
compound 4, displayed a hERG IC50 value of 8.1 μM.
However, the high antiplasmodial (low IC50) potency of this
compound should ensure a good safety margin. Generally
In Vitro ADME Profiling. The physicochemical properties
of all of the compounds were evaluated through a combination
of in silico and experimental techniques,5,11,12 and the
parameters were generally found to be within the ranges
typically considered acceptable for “drug-like” compounds, as
shown in Tables 1, 2, and 3. Generally, most of the compounds
displayed poor to moderate kinetic solubility at pH 6.5 but
increased solubility under acidic conditions, consistent with
expected ionization behavior.
Partition coefficient values of the compounds were generally
moderate, with LogD values at pH 7.4 ranging from 1.7 to 3.2.
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Table 2. In Vitro Antiplasmodial Activity of Compounds 1, 3, and 18−21
a
b
Mean from n values of ≥2 independent experiments. LogD pH 7.4 values were determined using a chromatographic estimation method; nd = not
determined. Data from Younis et al.11
c
In vitro metabolic stability of all the compounds was evaluated
using human liver microsomes as a preliminary indication of the
likely in vivo metabolic clearance (Table 4). No measurable
degradation was observed for many of the compounds,
suggesting that they would have low hepatic clearance in
vivo. Only one compound showed instability in human liver
microsomes, 13 with (EH = 0.7), likely due to pyridine N-oxide
formation, whereas five compounds, 16, 3, 5, 9, and 10,
exhibited a moderate rate of degradation. Generally speaking,
the pyrazine analogues displayed good metabolic stability in
human liver microsomal preparation. Notably, compound 4
demonstrated stability in both rat and human liver microsomes.
Although some compounds in the aminopyrazine series
displayed superior mouse efficacy compared to the previously
disclosed aminopyridines, presumably because of superior in
vitro potency, generally, there was no improvement in the
ADME properties.
In Vivo Efficacy. The in vivo antimalarial activity of the
most promising compounds was evaluated using a P. berghei
mouse model.6 Parasitemia reduction and mean survival days
(MSD) for single- or multidose regimens were determined.
Table 5 summarizes the in vivo efficacy test results of
compounds 4−7 following oral (p.o.) administration compared
with the standard antimalarial chloroquine (CQ). As shown in
Table 5, compounds 6 and 7 at low daily oral doses of 4 × 10
and 4 × 3 mg/kg exhibited good in vivo activity with a >80%
reduction in parasitemia. Three compounds, 8−10, were
evaluated for their efficacy at a lower (4 × 3 mg/kg) oral
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Table 3. In Vitro Antiplasmodial Activity and hERG Data of Compounds 4−10 and 26−36
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Table 3. continued
a
b
Mean from n values of ≥2 independent experiments. LogD pH 7.4 values were determined using a chromatographic estimation method; nd = not
determined. Data from Younis et al.11 Average of two independent experiments. No hERG inhibition at the highest measured concentration (11
c
d
e
μM).
dose. Compound 8 was less potent at this dose, with a 45%
reduction in parasitemia (MSD 6 days). However, compounds
9 and 10 exhibited significant activity at 4 × 3 mg/kg with a
99.7 (MSD 8 days) and 99.6% (MSD 8 days) reduction in
parasitemia respectively. In general, 4 showed potent in vivo
activity at all low multiple and single doses, with a >99.0%
suppression of parasitemia. Interestingly, compound 4
remained active at a single oral dose of 3 mg/kg and multiple
oral doses of 4 × 1 mg/kg with 99.4 (MSD 9 days) and 99.9%,
(MSD 10 days) reduction in parasitemia, respectively. No
toxicity symptoms were observed during in vivo studies with
these compounds. Notably, an oral dose of 4 × 10 mg/kg of 4,
resulting in 99.9% reduction in parasitemia, was completely
curative (MSD >30 days).
Mouse Exposure Studies. To interpret the in vivo efficacy
results obtained, five representative analogues, 4, 5, 7, 9, and
10, were selected for mouse exposure studies on the basis of
their varying degrees of in vivo efficacy. The plasma
concentrations in P. berghei-infected mice were measured
after an oral dose of 3 mg/kg and followed for 24 h using a
limited sampling schedule (i.e; sampling at 1, 4, and 24 h), as
depicted in Figure 3 and Table 6.24 As shown in Table 6,
compound 5 exhibited a relatively lower Cmax but a more
extended duration of exposure. However, it was shown that
analogues 7, 9, and 10 exhibited a generally higher Cmax but a
short duration of exposure, as evidenced by plasma
concentrations being undetectable at 24 h after administration.
It is noteworthy that even the relatively low systemic exposure
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Table 4. In Vitro Microsomal Stability for Selected
Analogues
metabolism in human liver microsome
a
compound
t1/2 (min)
CLint (μL/min/mg)
EH
(1)
281
6.2
0.26
<0.2
<0.28
0.7
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(3)
c.n.c.
>250
41
c.n.c.
<7
11.3
7.3
238.4
c.n.c.
121.7
nd
0.29
<0.2
0.44
nd
b
c.n.c.
14.2
c
nd
c.n.c.
>250
154
c.n.c.
<7
11
<7
<7
<7
11
<7
<7
8
<0.2
<0.28
0.39
<0.28
<0.28
<0.28
0.39
<0.28
<0.28
0.31
0.46
Figure 3. Graphical representation for mouse exposure after p.o.
dosing (3 mg/kg) of selected analogues 4, 5, 7, 9, and 10.
(20)
(21)
(4)
>250
>250
>250
155
purification. Column chromatography was carried out using silica gel
60 (Fluka), particle size 0.063−0.2 mm (70−230 mesh ASTM), as the
stationary phase. Analytical TLC was performed on silica on TLC
aluminum foils, H × W = 20 cm × 20 cm, with fluorescent indicator
(200 μm thick, Fluka) and visualized under UV light. Melting points
were determined on a Reichert-Jung Thermovar hotstage microscope
and are uncorrected. Routine 1H and 13C NMR spectra were recorded
on either a Varian Mercury 300 (1H 300.13, 13C 75.5 MHz) or 400
MHz on a Bruker AV 400 (1H 300.13, 13C 75.5 MHz) instrument.
Spectra were recorded at ambient temperature unless otherwise stated.
Chemical shifts (δ) are reported in parts per million from low to high
field and are referenced to residual solvent. Standard abbreviations
indicating multiplicity are used as follows: br = broad, d = doublet, m =
multiplet, q = quartet, quint = quintet, s = singlet, and t = triplet. In
many cases DMSO-d6 was used as a solvent, and the 1H was
referenced to 2.500 ppm for the quintuplet downfield methyl signal.
13C was referenced to the methyl carbon septuplet at 39.52 ppm.
Atmospheric pressure chemical ionization (APCI) mass spectrometry
was carried out by the services at the Centre for Drug Candidate
Optimisation and Syngene. All final compounds were purified to
≥95% purity as determined by LC using a Phenomenox-Luna C18
(250 × 4.6) mm 5u column, 2.0 μL injection volume, flow 0.7 mL/
min, gradient: 30−100% B in 15 min (hold 2 min) (mobile phase A:
10 nM NH4OAc in H2O and mobile phase B: acetonitrile) with a
Thermo Separation Products (TSP) Spectra SERIES P200 pump
UV100 detector set at 254 nm.
(5)
(6)
>250
>250
217
(7)
(9)
(10)
111
12
a
b
Predicted EH based on in vitro intrinsic clearance. Value was not
c
measured, nd = not determined.
of 7, 9, and 10 was sufficient to significantly clear parasites in
the blood given the higher in vitro potency of 7 (IC50 = 7.0 nM
(K1) and 6.0 nM (NF54)), 9 (IC50 = 11 nM (K1) and 12 nM
(NF54)), and 10 (IC50 = 21 nM (K1) and 23 nM (NF54));
although, as expected, these compounds resulted in shorter
mouse survival periods.
CONCLUSIONS
■
A novel series of 3,5-diaryl-2-aminopyrazines has been
identified as orally active antimalarial agents. This series of
compounds demonstrated impressive in vitro antiplasmodial
activity, good in vivo efficacy, and metabolic stability in human
microsomes. Compound 4 combines good in vitro activity
against P. falciparum with oral efficacy in a P. berghei mouse
model. Furthermore, 4 shows good stability in human and rat
microsomes and is undergoing further profiling as a potential
antimalarial drug candidate.
5-Bromopyrazin-2-amine (23). To a suspension of 2-amino-
pyrazine 22 (5.0 g, 52.50 mmol) in dry DCM (50 mL) was added
N-bromosuccinimide (9.35 g, 52.50 mmol) portionwise under cold
conditions, and the mixture was allowed to stir at rt for 1.5 h. To the
reaction mixture was added water (50 mL), and the layers separated.
The aqueous layer was extracted with DCM (3 × 50 mL), and the
combined organic layers washed with brine solution (20 mL), dried
EXPERIMENTAL SECTION
■
General Comments on Experimental Data. All other reagents
were purchased from commercial sources and used without further
Table 5. Oral Antimalarial Efficacy Using a Single- or Multidose of Compounds 4−10 in P. berghei-Infected Mice
a
percent reduction in parasitemia (MSD)
compound
1 × 30 mg/kg
1 × 10 mg/kg
1 × 3 mg/kg
4 × 10 mg/kg
4 × 3 mg/kg
4 × 1 mg/kg
c
b
(4)
(5)
99.3(13)
99.4(9)
nd
99.3(14)
99.4(9)
61(7)
nd
99.9(>30)
99.9(14)
94(11)
81(9)
99.8(10)
66(10)
41(6)
85 (7)
nd
94(6)
nd
99.9(14)
99.4(9)
99.9(27)
nd
(6)
(7)
nd
nd
nd
99.9(10)
45(6)
(8)
nd
nd
nd
(9)
nd
nd
nd
nd
99.7(8)
99.6(8)
99.1(10)
nd
(10)
nd
nd
nd
nd
nd
c
c
c
Chloroquine
99.7(9)
99.5(7)
83(7)
99.9(16)
nd
a
b
c
MSD = mean survival time (in days). No detectable parasites at day 30 postinfection. Data from Younis et al.11
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Table 6. Mouse Parameters after Oral Dosing (3 mg/kg) of Selected Compounds 4, 5, 7, 9, and 10
*
[24 h] <5 ng/mL (approximately 0.01 μM).
over anhydrous Na2SO4, and concentrated under vacuum. The crude
material was purified by column chromatography over silica gel by
using 16−18% of ethyl acetate in hexane as an eluent to yield
chromatography on silica gel hexane/EtOAc to give the product as a
solid.
The following compounds were prepared according to the general
1
procedure given.
compound 23 (4.61 g, 50.4%) as a white solid. mp 88−91 °C. H
3-(6-Methoxypyridin-3-yl)-5-(4-(methylsulfonyl)phenyl)pyrazin-
2-amine (3). Yield 56%. mp 190−193 °C. 1H NMR (400 MHz,
DMSO-d6) δ 8.70 (s, 1H), 8.60 (s, 1H), 8.26 (d, J = 8.2 Hz, 2H), 8.11
(d, J = 7.5 Hz, 1H), 7.97 (d, J = 8.2 Hz, 2H), 6.98 (d, J = 8.5 Hz, 1H),
6.71 (s, 2H), 3.94 (s, 3H), 3.24 (s, 3H). 13C NMR (100 MHz, DMSO-
d6) δ 164.1, 153.6, 147.1, 142.2, 139.8, 139.6 (2C), 138.1, 136.7, 127.9
(2C), 127.2, 125.9 (2C), 111.0, 53.9, 44.2. Anal. RP-HPLC tR = 10.2
min (purity 98.5%). Calcd m/z [M + H]+ for C17H16N4O3S, 356.40;
found, 357.0.
5-(4-(Methylsulfonyl)phenyl)-3-(6-(trifluoromethyl)pyridin-3-yl)-
pyrazin-2-amine (4). According to GP, yield = 58%. mp 209−211 °C.
1H NMR (400 MHz, DMSO-d6) δ 9.15 (d, J = 1.7 Hz, 1H), 8.83 (d, J
= 9.5 Hz, 1H), 8.47 (dd, J = 1.8, 8.2 Hz, 1H), 8.28 (d, J = 8.5 Hz, 2H),
8.05 (d, J = 8.2 Hz, 1H), 7.97−8.00 (m, 2H), 6.94 (s, 2H), 3.25 (s,
3H). 13C NMR (100 MHz, DMSO-d6) δ 153.8, 150.2, 146.5, 146.1,
141.8, 141.1, 140.0, 138.5, 137.0, 134.7, 128.0, 126.0 (2C), 123.6,
121.2, 120.8, 44.1. Anal. RP-HPLC tR = 9.2 min (purity 96.1%). Calcd
m/z [M + H]+ for C17H13F3N4O2S, 394.37; found, 395.0.
NMR (400 MHz, DMSO-d6) δ 8.01 (s, 1H), 7.66 (s, 1H), 6.59 (br s,
2H). 13C NMR (100 MHz, DMSO-d6) δ 155.8, 144.0, 132.6, 124.1.
Calcd m/z [M + H]+ for C4H4BrN3, 174.0; found, 174.3.
5-Bromo-3-iodopyrazin-2-amine (24). NIS (3.10 g, 13.79 mmol)
was added to a solution of 5-bromopyrazin-2-amine 23 (2.0 g, 11.49
mmol) in 1,4-dioxane (20 mL), and the resulting mixture was stirred at
80 °C for 4 h. The reaction mixture was poured onto a saturated
Na2S2O5 aqueous solution (20 mL) and extracted with EtOAc (3 × 50
mL). The combined organic layers were washed with brine (25 mL),
dried over anhydrous MgSO4, and concentrated in vacuo. The residue
was purified by column chromatography on silica gel (20% hexane/
EtOAc), providing compound 24 (3.80 g, 58%) as a white solid. mp
125−127 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.04 (s, 1H), 6.76 (br
s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 156.3, 143.2, 121.8, 103.2.
Calcd m/z [M + H]+ for C4H3BrIN3, 299.9; found, 300.2.
General Procedure for the Cross-Coupling Reactions. To a
solution of the aryl halide (1.0 equiv) in 1,4-dioxane, was added the
appropriate boronic acid (1.1 equiv). The mixture was thoroughly
degassed with nitrogen for 15 min, and then Pd(PPh)3Cl2 (5−7 mol
%) was added under a nitrogen atmosphere followed by aqueous
K2CO3 (1 M, 3.0 equiv). The reaction mixture was stirred at 110 °C
for 16 h, poured into H2O, and extracted with EtOAc (3×). The
combined organic layers were washed with brine, dried over MgSO4,
and concentrated in vacuo. The residue was purified by column
5-(4-(Methylsulfonyl)phenyl)-3-(4-(trifluoromethyl)phenyl)-
1
pyrazin-2-amine (5). Yield 60%. mp 230−232 °C. H NMR (400
MHz, DMSO-d6) δ 8.76 (s, 1H), 8.25 (d, J = 8.5 Hz, 2H), 8.02 (d, J =
8.2 Hz, 2H), 7.97 (d, J = 8.5 Hz, 2H), 7.88 (d, J = 8.2 Hz, 2H), 6.76 (s,
2H), 3.24 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 153.0, 141.6,
141.4, 140.0, 139.5, 137.7, 136.7, 129.2 (2C), 129.0, 128.7, 128.4,
127.6, 125.7, 125.6, 125.4, 123.0, 43.7. Anal. RP-HPLC tR = 11.8 min
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(purity 99.4%). Calcd m/z [M + H]+ for C18H14F3N3O2S, 393.38;
found, 394.0.
Research Foundation; TCAMS, Tres Cantos Antimalarial Set;
HPLC, high-pressure liquid chromatography; HPMC, hydrox-
ypropyl methyl cellulose; i.p., intraperitoneal injection; MMV,
Medicines for Malaria Venture; MSD, mean survival time; NBS,
N-bromosuccinimide; NIS, N-iodosuccinimide; p.o., oral
administration; SAR, structure−activity relationship; s.c.,
subcutaneous; PK, pharmacokinetics; TLC, thin-layer chroma-
tography; TMS, tetramethylsilane
(4-(5-Amino-6-(4-(trifluoromethyl)phenyl)pyrazin-2yl)phenyl)-
1
(morpholino)methanone (7). Yield 48%. mp 205−207 °C. H NMR
(400 MHz, DMSO-d6) δ 8.67 (s, 1H), 8.02−8.08 (m, 4H), 7.87 (d, J =
8.2 Hz, 2H), 7.48 (d, J = 8.2 Hz, 2H), 6.61 (s, 2H), 3.63 (br, 8H). 13C
NMR (100 MHz, DMSO-d6) δ 169.4, 153.4, 150.2, 146.3, 146.0,
140.4, 139.6, 138.4, 138.1, 137.2, 135.1, 134.3, 128.1 (2C), 125.3,
123.6 (2C), 121.2 (2C), 120.9, 66.6, 40.6. Anal. RP-HPLC tR = 13.3
min (purity 96.3%). Calcd m/z [M + H]+ for C22H19F3N4O2, 428.41;
found, 429.1.
REFERENCES
■
5-(4-(Ethylsulfonyl)phenyl)-3-(6-(trifluoromethyl)pyridin-3-yl)-
1
pyrazin-2-amine (9). Yield 49%. mp 178−180 °C. H NMR (300
(1) World Health Organization. World Malaria Report 2012; World
Health Organization: Geneva, Switzerland, 2012; http://www.who.
int/malaria/publications/world_malaria_report_2012/report/en/.
(2) Arora, N.; Banerjee, A. K. New targets, new hope: Novel drug
targets for curbing malaria. Mini-Rev. Med. Chem. 2012, 12, 210−226.
(3) World Health Organization. Global Report on Antimalarial
Efficacy and Drug Resistance: 2000-2010; World Health Organization:
Geneva, Switzerland, 2010; http://www.who.int/malaria/
publications/atoz/9789241500470/en/.
MHz, DMSO-d6) δ 9.15 (d, J = 1.8 Hz, 1H), 8.81 (s, 1H), 8.45 (dd, J
= 1.8, 8.0 Hz, 1H), 8.28 (d, J = 8.6 Hz, 2H), 8.04 (d, J = 8.0 Hz, 1H),
7.93 (d, J = 8.6 Hz, 2H), 6.70 (b rs, 2H), 2.50 (m, 2H), 1.11(t, J = 7.3
Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 153.3, 150.1, 146.4,
146.0, 142.2, 140.5, 138.8, 138.2, 137.0, 135.8, 134.5, 129.0 (2C),
126.2 (2C), 120.0, 51.8, 6.9. Anal. RP-HPLC tR = 11.2 min (purity
94.5%). Calcd m/z [M + H]+ for C18H15F3N4O2S, 408.4; found, 408.1.
4-(5-Amino-6-(6-(trifluoromethyl)pyridin-3-yl)pyrazin-2-yl)-N-
1
methylbenzenesulfonamide (10). Yield 51%. mp 185−188 °C. H
(4) Burrows, N. J.; Chibale, K.; Wells, T. N. C. The state of the art in
anti-malarial drug discovery and development. Curr. Top. Med. Chem.
2011, 11, 1226−1254.
(5) Cabrera, D. G.; F. Douelle, F.; Feng, T.-S.; Nchinda, A. T.;
Younis, Y.; White, K. L.; Wu, Q.; Q. Ryan, E.; Burrows, J. N.;
Waterson, D.; Witty, M. J.; Wittlin, S.; Charman, S. A.; Chibale, K.
Novel orally active antimalarial thiazoles. J. Med. Chem. 2011, 54,
7713−7719.
NMR (300 MHz, DMSO-d6) δ 9.15 (d, J = 2.0 Hz, 1H), 8.77 (s, 1H),
8.46 (dd, J = 2.0, 8.0 Hz, 1H), 8.22 (d, J = 8.6 Hz, 2H), 8.04 (d, J = 8.0
Hz, 1H), 7.98 (d, J = 8.6 Hz, 2H), 7.44 (m, 1H), 6.85 (br s, 2H), 2.46
(d, J = 5.0 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 153.7, 150.1,
146.6, 146.0, 144.1, 142.8, 141.2, 139.6, 138.6, 138.4, 136.8, 134.5,
127.0, 125.9, 123.0, 120.0, 29.4. Anal. RP-HPLC tR = 12.1 min (purity
95.1%). Calcd m/z [M + H]+ for C17H14F3N5O2S, 409.39; found,
409.1.
(6) Charman, S. A.; Arbe-Barnes, S.; Bathurst, I. C.; Brun, R.;
Campbell, M.; Charman, W. N.; Chiu, F. C. K.; Chollet, J.; Craft, J. C.;
Creek, D. J.; Dong, Y.; Matile, H.; Maurer, M.; Morizzi, J.; Nguyen, T.;
Papastogiannidis, P.; Scheurer, C.; Shackleford, D., M.; Sriraghavan,
K.; Stingelin, L.; Tang, Y.; Urwyler, H.; Wang, X.; White, K. L.;
Wittlin, S.; Zhou, L.; Vennerstrom, J. L. Synthetic ozonide drug
candidate OZ439 offers new hope for a single-dose cure of
uncomplicated malaria. Proc. Natl. Acad. Sci.U.S.A. 2011, 108, 4400−
4405.
(7) Dubar, F.; Egan, T. J.; Pradines, B.; Kuter, D.; Ncokazi, K. K.;
Forge, D.; Paul, J.-F.; Pierrot, C.; Kalamou, H.; Khalife, J.; Buisine, E.;
Rogier, C.; Vezin, H.; Forfar, I.; Slomianny, C.; Trivelli, X.;
Kapishnikov, S.; Leiserowitz, L.; Dive, D.; Biot, C. The antimalarial
ferroquine: Role of the metal and intramolecular hydrogen bond in
activity and resistance. ACS Chem. Biol. 2011, 6, 275−287.
(8) Wein, S.; Maynadier, M.; Tran Van Ba, C.; Cerdan, R.; Peyrottes,
S.; Fraisse, L.; Vial, H. Reliability of antimalarial sensitivity tests
depends on drug mechanisms of action. J. Clin. Microbiol. 2010, 48,
1651−1660.
ASSOCIATED CONTENT
* Supporting Information
Additional details of the characterization of selected com-
pounds and the procedures used for the in vitro and in vivo
antimalarial studies as well as metabolism and PK studies. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: +27-21-6502553; Fax: +27-21-6505195; E-mail: Kelly.
Chibale@uct.ac.za.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
(9) Chigorimbo-Murefu, N. T. L.; Njoroge, M.; Nzila, A.; Louw, S.;
Masimirembwa, C.; Chibale, K. Biotransformation and biocatalysis:
Roles and applications in the discovery of antimalarials. Future Med.
Chem. 2012, 4, 2325−2336.
(10) Biot, C.; Chibale, K. Novel approaches to antimalarial drug
discovery. Infect. Disord.: Drug Targets 2006, 2, 173−204.
́
(11) Younis, Y.; Douelle, F.; Feng, T.-S.; Gonzalez Cabrera, D.; Le
■
We thank Medicines for Malaria Venture (project MMV09/
0002) and the South African Technology Innovation Agency
(TIA) for financial support of this research. 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 acknowl-
edged for support (to K.C.). We thank Christian Scheurer,
Petros Papastogiannidis, and Jolanda Kamber for assistance in
performing the antimalarial assays and Kasiram Katneni, Thao
Pham, Eileen Ryan, Oliver Montagnat, and Alison Gregg for
assistance in running the in vitro ADME studies.
Manach, C.; Nchinda, T. A.; Duffy, S.; White, K. L.; Shackleford, D.
M.; Morizzi, J.; Mannila, J.; Katneni, K.; Bhamidipati, R.; Zabiulla, K.
M.; Joseph, J. T.; Bashyam, S.; Waterson, D.; Witty, M. J.; Hardick, D.;
Wittlin, S.; Avery, V.; Charman, S. A.; Chibale, K. 3,5-Diaryl-2-
aminopyridines as a novel class of orally active antimalarials
demonstrating single dose cure in mice and clinical candidate
potential. J. Med. Chem. 2012, 55, 3479−3487.
́
(12) Gonzalez Cabrera, D.; Douelle, F.; Younis, Y.; Feng, T.-S.; Le
Manach, C.; Nchinda, A. T.; Street, L. J.; Scheurer, C.; Kamba, J.;
White, K. L.; Montagnat, O. D.; Ryan, E.; Katneni, K.; Zabiulla, K. M.;
Joseph, J. T.; Bashyam, S.; Waterson, D.; Witty, M. J.; Charman, S. A.;
Wittlin, S.; Chibale, K. Synthesis and structure-activity relationship
studies of antimalarial 3,5-substituted 2-aminopyridines. J. Med. Chem.
2012, 55, 11022−11030.
ABBREVIATIONS USED
■
ADME, absorption, distribution, metabolism, and excretion;
AUC, area under the curve; CQ, chloroquine; DMF, N,N-
dimethylformamide; EH, hepatic extraction ratio; GSK,
GlaxoSmithKline; GNF, Genomics Institute of the Novartis
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(13) Miyaura, N.; Suzuki, A. Palladium-catalyzed cross-coupling
reactions of organoboron compounds. Chem. Rev. 1995, 95, 2457−
2483.
(14) Daab, J.; Bracher, F. Total syntheses of the alkaloids
ipalbidinium and clathryimine B. Monatsh. Chem. 2003, 134, 573−583.
(15) Grant, T. G.; Shahbaz, M. Ethoxyphenylmethyl inhibitors of
SGLT2. WO Patent 2010048358 A2, 2010.
(16) Pettit, M.-R.; Stacey, M.; Tatlow, J.-C. Trifluoroacetic acid. VIII.
Diazotizations of aromatic amines in aqueous trifluoroacetic acid and
other perhalocarboxylic acids. J. Chem. Soc. 1953, 8, 3081−3084.
(17) Chen, Q.-Y.; Wu, S.-W. Methyl(fluorosulfonyl)difluoroacetate; a
new trifluoromethylating agaent. J. Chem. Soc., Chem. Commun. 1989,
11, 705−706.
(18) De Wael, F.; Jeanjot, P.; Moens, C.; Verbeuren, T.; Cordi, A.;
Bouskela, E.; Rees, J.-F.; Marchand-Brynaert, J. In vitro and in vivo
studies of 6,8-(diaryl)imidazo[1,2-a]pyrazin-3(7H)-ones as new
antioxidants. Bioorg. Med. Chem. 2009, 17, 4336−4344.
(19) Charrier, J.-D.; Durrant, S.; Golec, J.; Kay, D.; Knegtel, R.;
MacCormick, S.; Mortimore, M.; O’Donnell, M.; Pinder, J.; Reaper, P.;
Rutherford, A.; Wang, P.; Young, S.; Pollard, J. Discovery of potent
and selective inhibitors of ataxia telangiectasia mutated and Rad3
related (ATR) protein kinase as potential anticancer agents. J. Med.
Chem. 2011, 54, 2320−2330.
(20) Berg, S.; Bergh, M.; Hellberg, S.; Hogdin, K.; Lo-Alfredsson, Y.;
̈
Soderman, P.; Berg, S.; Weigelt, T.; Ormo, M.; Xue, Y.; Tucker, J.;
̈
̈
Neelissen, J.; Jerning, E.; Nilsson, Y.; Bhat, R. Discovery of novel
potent and highly selective glycogen synthase kinase-3β (GSK3β)
inhibitors for Alzheimer’s disease: Design, synthesis, and character-
ization of pyrazines. J. Med. Chem. 2012, 55, 9107−9119.
(21) Gamo, F.-J.; Sanz, L. M. S.; Vidal, J.; de Cozar, C.; Alvarez, E.;
Lavandera, J.-L.; Vanderwall, D. E.; Green, D. V. S.; Kumar, V.; Hasan,
S.; Brown, J. R.; Peishoff, C. E.; Cardon, L. R.; Garcia-Bustos, J. F.
Thousands of chemical starting points for antimalarial lead
identification. Nature 2010, 465, 305−310.
(22) Plouffe, D.; Brinke, A.; McNamara, C.; Henson, K.; Kato, N.;
́
Kuhen, K.; Nagle, A.; Adrian, F.; Matzen, J.; Anderson, P.; Nam, T-G;
Gray, N.; Chatterjee, A.; Janes, J.; Frank Yan, S.; Trager, R.; Caldwell,
J.; Schultz, P.; Zhou, Y.; Winzeler, E. In silico activity profiling reveals
the mechanism of action of antimalarials discovered in a high-
throughput screen. Proc. Natl Acad. Sci.U.S.A. 2008, 105, 9059−9064.
(23) Meister, S.; Plouffe, D.; Kuhen, K.; Bonamy, G.; Wu, T.; Barnes,
S. W.; Bopp, S.; Borboa, R.; Bright, A. T.; Che, J.; Cohen, S.; Dharia,
N.; Gagaring, K.; Gettayacamin, M.; Gordon, P.; Groess, T.; Kato, N.;
Lee, M.; McNamara, C.; Fidock, D.; Nagle, A.; Nam, T.; Richmond,
W.; Roland, J.; Rottmann, M.; Zhou, B.; Froissard, P.; Glynne, R.;
Mazier, D.; Sattabongkot, J.; Schultz, P.; Tuntland, T.; Walker, J.;
Zhou, Y.; Chatterjee, A.; Diagana, T.; Winzeler, E. Imaging of
plasmodium liver stages to drive next-generation antimalarial drug
discovery. Science 2011, 334, 1372−1376.
(24) Liu, B.; Chang, J.; Gordon, W.; Isbell, J.; Zhou, Y.; Tuntland, T.
Snapshot PK: A rapid rodent in vivo preclinical screening approach.
Drug Discovery Today 2008, 13, 360−367.
8871
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