Structure-activity relationship studies of orally active antimalarial 3,5-substituted 2-aminopyridines

Article abstract of DOI:10.1021/jm301476b

In an effort to address potential cardiotoxicity liabilities identified with earlier frontrunner compounds, a number of new 3,5-diaryl-2-aminopyridine derivatives were synthesized. Several compounds exhibited potent antiplasmodial activity against both the multidrug resistant (K1) and sensitive (NF54) strains in the low nanomolar range. Some compounds displayed a significant reduction in potency in the hERG channel inhibition assay compared to previously reported frontrunner analogues. Several of these new analogues demonstrated promising in vivo efficacy in the Plasmodium berghei mouse model and will be further evaluated as potential clinical candidates. The SAR for in vitro antiplasmodial and hERG activity was delineated.

Full text of DOI:10.1021/jm301476b

Article
pubs.acs.org/jmc
Structure−Activity Relationship Studies of Orally Active Antimalarial
3,5-Substituted 2‑Aminopyridines
Diego Gonzalez Cabrera,† Frederic Douelle,† Yassir Younis,† Tzu-Shean Feng,† Claire Le Manach,†
́
Aloysius T. Nchinda,† Leslie J. Street,† Christian Scheurer,‡,# Jolanda Kamber,‡,# Karen L. White,§
Oliver D. Montagnat,§ Eileen Ryan,§ Kasiram Katneni,§ K. Mohammed Zabiulla,∥ Jayan T. Joseph,∥
Sridevi Bashyam,∥ David Waterson,⊥ Michael J. Witty,⊥ Susan A. Charman,§ Sergio Wittlin,‡,#
and Kelly Chibale*,†,▽
†Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
‡Swiss Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel, Switzerland
§Centre for Drug Candidate Optimisation, Monash University (Parkville campus), 381 Royal Parade, Parkville, VIC 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, Route de Pre-
#University of Basel, 4002 Basel, Switzerland
́
Bois 20, P.O. Box 1826, 1215 Geneva, Switzerland
▽Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
S
* Supporting Information
ABSTRACT: In an effort to address potential cardiotoxicity liabilities
identified with earlier frontrunner compounds, a number of new 3,5-
diaryl-2-aminopyridine derivatives were synthesized. Several com-
pounds exhibited potent antiplasmodial activity against both the
multidrug resistant (K1) and sensitive (NF54) strains in the low
nanomolar range. Some compounds displayed a significant reduction
in potency in the hERG channel inhibition assay compared to
previously reported frontrunner analogues. Several of these new
analogues demonstrated promising in vivo efficacy in the Plasmodium
berghei mouse model and will be further evaluated as potential clinical
candidates. The SAR for in vitro antiplasmodial and hERG activity was
delineated.
INTRODUCTION
■
Malaria, a parasitic disease caused in humans primarily by
Plasmodium falciparum and Plasmodium vivax,1 has been recently
listed as the fifth of the top ten causes of death in low-income
countries. According to the World Health Organization 2011
report, malaria was responsible for 216 million clinical cases and
655000 deaths in 2010, especially among children and pregnant
women.2
Despite the effectiveness that standard antimalarial agents
have had in recent years,3 rapid widespread emergence of drug
resistance has dramatically compromised the therapeutic options
for the treatment of malaria.4 Thus, the search for novel,
Figure 1. Chemical Structures of 1 and 2.
structurally diverse, and affordable drugs has become an urgent
necessity to control and eventually eradicate this parasitic
disease.1
(IC50 = 5.5 μM) in the IonWorks patch clamp electrophysiology
Recently, we demonstrated the potential of 3,5-diaryl-2-
aminopyridines 1 and 2 (Figure 1) as clinical candidate
antimalarials based on mice in vivo efficacy data. One of the
liabilities identified with the aminopyridine 1 is potential
cardiotoxicity risks as signaled by the moderate hERG activity
assay.5
Received: October 11, 2012
Published: November 29, 2012
© 2012 American Chemical Society
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a
Scheme 1. Synthetic Approaches to 3,5-Diaryl-2-aminopyridines Analogues
a
Reagents and conditions: (a) (i) I2, DMSO, 100 °C, 4 h, rt, 12 h, 38%; (ii) 6-methoxypyridin-3-yl boronic acid, Pd(PPh3)2Cl2, K2CO3, 1,4-dioxane,
90 °C, 14 h, 72%; (iii) R = appropriate boronic acid, Pd(PPh3)2Cl2, K2CO3, 1,4-dioxane or DMF, 90 °C, 14 h, 28−76%; (b) (i) I2, DMSO, 100 °C, 4
h, rt, 12 h, 50%; (ii) 4-(morpholine-4-carbonyl)phenylboronic acid, Pd(PPh3)2Cl2, K2CO3, 1,4-dioxane, 90 °C, 14 h, 63%; (iii) R1 = appropriate
boronic acid, Pd(PPh3)2Cl2, K2CO3, 1,4-dioxane, 90 °C, 14 h, 62% and 76%; (iv) 4-carboxyphenylboronic acid, Pd(PPh3)2Cl2, K2CO3, DMF, 90 °C,
14 h, 59%; (v) SOCl2, CH2Cl2, rt, 3h, 1-methyl piperazine, triethylamine, 0 °C to rt, 6h, 94%; (vi) appropriate boronic acid, Pd(PPh3)2Cl2, K2CO3,
1,4-dioxane or DMF, 90 °C, 14 h, 45% and 42%; (vii) 1-Boc piperazine, EDCI, HOBt, DMF, rt, 12 h, 59%, appropriate boronic acid, Pd(PPh3)2Cl2,
K2CO3, 1,4-dioxane or DMF, 90 °C, 14 h, 58% and 50%; (viii) trifluoroacetic acid, CH2Cl2, rt, 2 h, Amberlyst A-21, CH2Cl2/CH3OH, 30 min, 90%.
Drug-induced blockade of the hERG potassium channel is
associated with prolongation of the length of time between the
start of the Q wave and the T wave on an electrocardiogram
which may, under certain circumstances, lead to potentially life-
threatening arrhythmia.6 Thus accordingly we embarked on a
campaign aimed at identifying compounds that would either
minimize or eliminate the hERG liability associated with 1 while
retaining high in vitro potency along with good ADME
properties and in vivo efficacy.
key intermediate 32. This was in turn subjected to a second
Suzuki reaction to afford analogues 33 and 33a. Derivatives 34−
35a were accessed via amidation of intermediate 36 in the
presence of the relevant piperazine amine followed by a Suzuki
cross-coupling reaction with commercially available boronic
acids. Deprotection of the N-Boc moiety of precursors to 35 and
35a was accomplished using trifluoroacetic acid followed by its
removal with Amberlyst A-21.8
Biology. In Vitro Antiplasmodial Activity. Table 1
summarizes the IC50 values against multidrug resistant (K1)
and sensitive (NF54) strains of P. falciparum9 along with
physicochemical properties. In general, the dose−response
curves for the highly active compounds were steep and the
IC90 values ∼2× higher than the IC50 values. These compounds
were designed with a view to investigating the structural and
electronic properties of the 5-aryl substituent necessary for
modulating the antiplasmodial, ADME, and hERG activity. As
previously noted, potent activity was associated with the
presence of a methylsulfonyl group at the para position.5 Potent
RESULTS AND DISCUSSION
■
Chemistry. Target compounds (1a and 3−29) in which the
5-aryl substituent was systematically varied, were prepared using
key intermediate 30 and the appropriate commercially available
boronic acid as shown in Scheme 1a. The synthesis described in
Scheme 1b was then utilized to optimize the 3-aryl substituent of
the most potent 5-aryl-2-aminopyridine derivatives. Suzuki
cross-coupling7 between starting material 315 and commercially
available 4-(morpholine-4-carbonyl)phenylboronic acid yielded
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Table 1. In Vitro Activity against Sensitive and Multidrug-Resistant Strains of Plasmodium falciparum, Solubility, and Partition
Coefficients Values
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Table 1. continued
a
b
c
Mean from n values of ≥2 independent experiments. Estimated using nephelometry. Estimated using the chromatographic glogD technique.
d
e
Data from Gonzalez Cabrera et al.11 Data from Younis et al.5
Table 2. In Vitro Activity against Sensitive and Multidrug-Resistant Strains of Plasmodium falciparum, Solubility and Partition
Coefficients Values
a
b
c
Mean from n values of ≥2 independent experiments. Estimated using nephelometry. Estimated using the chromatographic glogD technique.
d
Data from Gonzalez Cabrera et al.11
activity was retained with other polar, electron-withdrawing
groups such as N-substituted sulphonamides (3, 4, and 7 but not
the primary sulphonamide 5), and primary, secondary, or tertiary
carboxamides (22, 24−29, and 23, respectively). Small polar
amides such as the primary carboxamide or morpholino,
piperazine, or methyl piperazine amides gave compounds with
excellent potency, e.g., 28 (IC50 NF54 = 52 nM) and 29 (IC50
NF54 = 74 nM), which were followed up for further optimization
(see below).
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While other polar groups such as MeSO2NH− (9) and
−CO2H (21) retained some activity, lipophilic electron
withdrawing groups such as −OCF3 (12) or −CF3 (13) were
much less active.
Table 3. In Vitro Metabolic Stability Assessed in Human Liver
Microsomes
degradation half-
life (min)
in vitro CLint
(μL/min/mg protein)
microsome-
a
compd
predicted EH
As was observed in the original screening of the BioFocus DPI
library set, substitution at the para position was strongly favored
over the corresponding meta and/or ortho position as was
confirmed in some of the most active compounds (compare 1
with 1a and 22 with 22a).5 Introduction of substituent groups
onto the aryl ring ortho or meta to the 4-methanesulfonyl group
also reduced the activity 10 (IC50 NF54 = 107 nM) and 11 (IC50
NF54 = 980 nM). However, some examples retained activity
when the adjacent substituent was sterically constrained as part
of an electron deficient bicycle, e.g., 18 and 20 with IC50 104 nM
(NF54) and 463 nM (NF54), respectively.
On the basis of the previously reported SAR for the
frontrunner analogues 1 and 2 (Figure 1),5 the 2-methoxypyr-
idine substituent in the 3-aryl group of the most active 5-
substituted amide analogues was replaced by 6-trifluoromethyl-
pyridin-3-yl or 4-trifluoromethylphenyl moieties. The critical
role played by these molecular fragments in the antiplasmodial
activity can be observed in Table 2. In general, IC50 values of the
prepared analogues were 20 or below 20 nM and represent the
most potent compounds of the series.
In Vitro ADME Profiling. All compounds were assessed for
their physicochemical properties and in vitro metabolic stability
in human microsomal preparations.10 The estimated spartition
coefficients were generally low to moderate, with LogD7.4 values
ranging from 0.8 to 4.3 (Tables 1 and 2).
The kinetic solubility at pH 6.5 was quite varied, with 17
analogues displaying poor solubility and the remaining
compounds showing moderate solubility. Good solubility was
only observed for 23, 26, 27, 28, and 34. Under acidic conditions
(pH 2), all the compounds displayed improved solubility, which
supports their predicted basic characteristics.
1a
3
227
17.5
181
7.6
98.8
9.6
<7
15
0.3
0.85
0.45
4
b
5
>250
114
<0.28
6
0.46
0.59
0.66
7
67.2
49.1
>250
>250
>250
76
25.8
35.3
<7
<7
<7
22.8
6.1
19.9
8
8
b
9
<0.28
b
10
11
12
13
14
15
16
17
18
19
20
21
22
22a
23
24
25
26
27
28
29
33
33a
34
34a
35
35a
<0.28
b
<0.28
0.56
0.25
0.53
0.32
0.32
0.81
0.4
285
87
210
203
9
23
76
146
12
143
12
0.4
181
10
0.35
b
>250
>250
>250
156
<7
<7
<7
11.1
<7
<7
12
<0.28
b
<0.28
b
<0.28
0.38
b
>250
>250
151
<0.28
b
<0.28
0.39
b
>250
>250
>250
>250
186
<7
<7
<7
<7
9
<0.28
b
<0.28
b
<0.28
b
<0.28
0.34
0.45
0.63
120
14
Generally, most of the derivatives were metabolically stable in
human liver microsomes (Table 3) and based on the in vitro
intrinsic clearance values, low or intermediate in vivo hepatic
clearance would be expected. In addition, the microsome-
predicted hepatic extraction ratios (EH) of each compound were
comparable across three species: human, rat, and mouse (data
not shown).
56
31
b
>250
123
<7
14
<0.28
0.44
a
Predicted hepatic extraction ratio based on in vitro intrinsic clearance.
No measurable degradation of the parent compound was observed,
hence, the clearance parameters could not be determined. EH
b
considered to be <0.28.
In Vitro hERG Activity. Several derivatives of the series were
tested for their activity against the hERG potassium channel
using in vitro IonWorks patch clamp electrophysiology. As can
be observed in Table 4, compounds displayed varying degrees of
activity. Derivatives, which displayed poor solubility, as
evidenced by precipitation under the assay conditions, were
tested at a maximum concentration of 11 μM. Analogues with
better solubility were tested at 33 μM. Compounds where the
methanesulfonyl group is replaced by sulphonamide or N,N-
dimethylacetamide groups showed an improved hERG profile in
comparison to the parent compound 1 (IC50 = 5.5 μM),
excluding 6. Furthermore, when more polar substituents were
introduced in the 5-aryl group, as in for example methyl
piperazine 28, an improvement in the hERG profile was observed
(IC50 = 20.5 μM). Improvements in the hERG liability were also
observed by the introduction of 6-trifluoromethylpyridin-3-yl
groups at the 3-aryl position. However, derivatives containing the
4-trifluoromethylphenyl moiety, 33a (IC50 = 3 μM) and 35a
(IC50 = 5.5 μM), showed no improvement relative to the parent
compound 1 (IC50 = 5.5 μM).
In Vivo Efficacy Studies. On the basis of in vitro potency,
metabolic stability, and structural diversity, nine compounds
were tested for in vivo oral activity in the P. berghei infected
mouse model.9 Parasitemia reduction and mean survival days
(MSD) for single- or multi-dose regimens are reported in Table
5.
The in vivo antimalarial effect for compounds 22 and 23 at the
4 × 50 mg/kg multidose was high (99.6% and 99.7% inhibition),
albeit no cure was observed as evidenced by a MSD of 12 days.
On the other hand, the in vivo efficacy was modest at a single oral
dose of 30 mg/kg (73% inhibition for 22). In the multi (4 × 50
mg/kg) oral dosing regimen, differences between 29 (MSD =
13), 33 (MSD = 24), and 33a (MSD = 12) became more
apparent, where 33 proved to be superior.
Despite the potent in vitro activity against P. falciparum,
compounds 34, 34a, and 35 started to lose in vivo activity at the 1
× 10 mg/kg oral dose (73%, 92%, and 43%). Only 35a showed
better efficacy, where dosing of 3, 10, and 30 mg/kg were able to
suppress parasitemia by 71%, 98%, and 99.2%, respectively.
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Table 4. hERG Inhibition Data
Table 5. In Vivo Antimalarial Efficacy Using Single- and
Multidose of Selected Compounds in Plasmodium berghei-
a
Infected Mice
a
No hERG inhibition at the highest measured concentration (11 μM).
Average of two independent experiments.
b
a
Compounds were dissolved or suspended in 70/30 Tween 80/
Unfortunately, none of the compounds produced a complete
cure of mice. This is in contrast to previously reported 1 and 2
derivatives.5
ethanol and diluted 10× with water, except for 34, 34a, 35, and 35a,
which were dosed in a nonsolubilizing, standard suspension vehicle
b
c
(HPMC). MSD = mean survival time (in days). Mice were
euthanized on day 3 in order to prevent death otherwise occurring at
Mouse Exposure Studies. In an attempt to rationalize the
observed in vivo efficacy, mouse exposure studies were
conducted on five representative analogues. These analogues
were chosen on the basis of their varying degrees of in vivo
efficacy. The plasma concentrations in mice were measured after
a single oral dose of 30 mg/kg and followed for 24 h using a
limited sampling schedule. As observed in Table 6, the plasma
concentrations of all tested compounds, with the exception of
33a, were higher than those for 33. It was also shown that this
analogue exhibited a relatively short duration of exposure
(plasma concentrations were not detectable 24 h after
administration), but when administered orally to mice at
multiple doses of 4 × 50 mg/kg, it displayed 99.7% inhibition
of P. berghei and prolonged mean mouse survival to 24 days
compared to 12, 12, 13, and 12 days exhibited by 22, 23, 29, and
day 6. Data from Gonzalez Cabrera et al.11
d
33a, respectively. Thus, even the relatively low systemic exposure
of 33 was sufficient to significantly clear parasites in blood given
the significantly higher in vitro potency (IC50 = 12 nM (K1), 15
nM (NF54)) and the dose regime utilized. Moreover, the lower
in vivo activity exhibited by 33a compared to 33, regardless of its
apparently longer duration of exposure in vivo and similar
antiplasmodial activity, may be rationalized on the basis of the
lower overall plasma concentrations achieved for 33a.
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as follows: br s = broad singlet, d = doublet, m = multiplet, q = quartet,
Table 6. Plasma Concentrations of Compounds 22, 23, 29, 33,
and 33a in Mice Following Administration of a Single 30 mg/
kg Oral Dose
quint. = quintet, s = singlet, t = triplet. In many cases. DMSO-d6 was
1
used as a solvent, and the H was referenced to 2.500 ppm for the
quintuplet downfield methyl signal. 13C was reference 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. LC purity
traces were performed using one of the methods shown in Supporting
Information.
Purity was determined by HPLC, and all compounds were confirmed
to have >95% purity.
General Procedure 1 for the Synthesis of 3-(6-Methoxypyr-
idin-3-yl)-5-(3-(methylsulfonyl)phenyl)pyridin-2-amine 1a. To a
solution of 5-bromo-3-(6-methoxypyridin-3-yl)pyridin-2-amine (100
mg, 0.36 mmol)5 in 1,4-dioxane (2 mL), 3-(methylsulfonyl)-
phenylboronic acid (79 mg, 0.39 mmol) was added. The mixture was
thoroughly degassed with nitrogen for 15 min, at which time
Pd(PPh3)2Cl2 (13 mg, 0.02 mmol) was added under an atmosphere
of nitrogen, followed by (1M) aqueous K2CO3 (0.38 mL, 0.38 mmol).
The reaction mixture was stirred at 90 °C for 14 h, poured into H2O (15
mL), and extracted with ethyl acetate (3 × 10 mL). The combined
organic layers were washed with brine, dried over MgSO4, and
concentrated in vacuo. The remaining residue was subjected to column
chromatography on silica gel using hexane/ethyl acetate in a 8:2 v/v
ratio as eluent to furnish 1a as a white solid (78 mg, 62%); mp 216−218
°C. 1H NMR (400.0 MHz, DMSO-d6): δ 8.42 (d, J = 2.4 Hz, 1H), 8.31
(d, J = 2.4 Hz, 1H), 8.15 (t, J = 1.7 Hz, 1H), 8.03 (dt, J = 1.7 Hz, 7.8 Hz,
1H), 7.86 (dd, J = 2.4 Hz, 8.4 Hz, 1H), 7.82−7.78 (m, 1H), 7.77 (d, J =
2.4 Hz, 1H), 7.68 (t, J = 8.4 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 5.97 (br s,
2H), 3.92 (s, 3H), 3.28 (s, 3H). 13C NMR (100.6 MHz, DMSO-d6): δ
163.4, 157.5, 147.1, 146.1, 142.2, 140.0, 139.5, 136.6, 130.9, 130.4, 127.4,
125.0, 124.2, 123.9, 117.7, 111.0, 53.7, 43.9. Anal. RP-HPLC tR = 11.90
min (method 1A, purity 95.53). LRMS (APCI): m/z = 356.2 [(M +
H)+] (anal. calcd for C18H17N3O3S+: m/z = 355.10).
a
b
Values are the mean from two animals. Sample time postdose in
c
hours. ND = not detected. Based on the lower limit of quantitation.
d
Avarage plasma concentration over the time period (t) calculated as
AUC0−t/t. c.n.c. = could not be calculated as plasma concentrations at
24h were not available.
CONCLUSION
General Procedure 2 for the Synthesis of (4-(6-Amino-5-(6-
(trifluoromethyl)pyridin-3-yl)pyridin-3-yl)phenyl)-
(morpholino)methanone 33. To a solution of 32 (0.25 g, 0.70
mmol) in 1,4-dioxane (5 mL), 2-(trifluoromethyl)pyridine-5-boronic
acid pinacol ester (0.21 g, 0.76 mmol) was added. The mixture was
thoroughly degassed with nitrogen for 15 min, at which time
Pd(PPh3)2Cl2 (0.03 g, 0.04 mmol) was added under an atmosphere of
nitrogen, followed by (1M) aqueous K2CO3 (0.73 mL, 0.73 mmol). The
reaction mixture was stirred at 90 °C for 14 h and extracted with ethyl
acetate (4 × 10 mL). The combined organic layers were washed with
brine (3 × 5 mL), dried over Na2SO4, and concentrated in vacuo. The
remaining residue was subjected to column chromatography on silica gel
using dichloromethane/methanol in a 9.7:0.3 v/v ratio as eluent to
furnish 33 as a white solid. Yield 0.19 g, 62%; mp 122−124 °C. 1H NMR
(300.1 MHz, DMSO-d6): δ 8.90 (d, J = 1.8 Hz, 1H), 8.43 (d, J = 2.4 Hz,
1H), 8.23 (dd, J = 2.4 Hz, 8.1 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.81 (d, J
= 2.4 Hz, 1H), 7.75 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 6.06 (br
s, 2H), 3.62 (br s, 4H), 3.52 (br s, 4H). 13C NMR (100.6 MHz, DMSO-
d6): δ 168.9, 156.5, 150.0, 146.6, 138.6, 138.3, 137.4, 136.5, 133.5, 127.8,
125.4, 124.2, 123.2, 120.7, 115.7, 66.1, 47.8. Anal. RP-HPLC tR = 4.90
min (method 1B, purity 99.2%). LR-MS (APCI): m/z = 429.1 [(M +
■
A series of 3,5-diaryl-2-aminopyridine derivatives has been
synthesized and evaluated for SAR in vitro against K1 and NF54
strains of P. falciparum. Most of the synthesized compounds
showed IC50 values in the low nanomolar range and good to
moderate metabolic stability in human liver microsomes. From
among the several derivatives synthesized, 28, 33, 34a, and 35
also exhibited a dramatic reduction in hERG activity compared to
the parent compound 1.5 However, improvements of in vitro
antiplasmodial and hERG activity was not accompanied by
exceptional curative oral efficacy in the P. berghei mouse model.
Despite the improved in vitro antiplasmodial and hERG
activities that some of these compounds exhibited, further
optimization to ameliorate in vivo efficacy against P. berghei is
essential prior to selection of a clinical candidate.
EXPERIMENTAL SECTION
■
General Comments on Experimental Data. Chloroform
(CHCl3) and tetrahydrofuran (THF) solvents were analytical grade,
without stabilizer; ethyl acetate, hexane, and dichloromethane were
distilled. Unless stated otherwise, all other reagents were purchased from
commercial sources and used without further 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.
+
H)+] (anal. calcd for C22H19F3N4O2 : m/z = 428.15).
General Procedure 3 for the Synthesis of (4-(6-Amino-5-(6-
(trifluoromethyl)pyridin-3-yl)pyridin-3-yl)phenyl)(4-methylpi-
perazin-1-yl)methanone 34. Thionyl chloride (0.74 mL, 10.23
mmol) was slowly added to a solution of 36 (0.6 g, 2.05 mmol) in dry
dichloromethane (5 mL) and refluxed for 2 h. The reaction mixture was
concentrated under reduced pressure and dissolved in dichloromethane
(2 mL). A solution of 1-methyl piperazine (0.25 mL, 2.25 mmol) in
triethylamine (0.86 mL, 6.14 mmol) was then added to the reaction
mixture at 0 °C and allowed to stir for 6 h. The mixture was extracted
with dichloromethane (2 × 5 mL), dried over anhydrous Na2SO4, and
concentrated under reduced pressure. The remaining residue was
subjected to column chromatography on silica gel using dichloro-
methane/methanol in a 9.7:0.3 v/v ratio as eluent to furnish (4-(6-
amino-5-bromopyridin-3-yl)phenyl)(4-methylpiperazin-1-yl)-
Routine H and 13C NMR spectra were recorded on either a Varian
1
Mercury-300 (1H 300.1, 13C 75.5 MHz) or 400 MHz on a Bruker AV
400 (1H 400.0, 13C 100.6 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 referenced to
residual solvent. Standard abbreviations indicating multiplicity are used
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methanone as a colorless solid (0.65 g, 94%), which was used without
further purification. To a solution of (4-(6-amino-5-bromopyridin-3-
yl)phenyl)(4-methylpiperazin-1-yl)methanone (0.25 g, 0.67 mmol) in
1,4-dioxane (4 mL), 2-(trifluoromethyl)pyridine-5-boronic acid pinacol
ester (0.20 g, 0.73 mmol) was added. The mixture was thoroughly
degassed with nitrogen for 15 min, at which time Pd(PPh3)2Cl2 (23 mg,
0.03 mmol) was added under an atmosphere of nitrogen, followed by
(1M) aqueous K2CO3 (0.70 mL, 0.70 mmol). The reaction mixture was
stirred at 90 °C for 14 h and extracted with ethyl acetate (4 × 10 mL).
The combined organic layers were washed with brine (3 × 5 mL), dried
over Na2SO4, and concentrated in vacuo. The remaining residue was
subjected to column chromatography on silica gel using dichloro-
methane/methanol in a 9.7:0.3 v/v ratio as eluent to furnish 34 as a
ASSOCIATED CONTENT
* Supporting Information
Additional details of the characterization of selected compounds
and the procedures used for the in vitro and in vivo antimalarial
studies as well as in vitro metabolism and mouse exposure
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.
■
1
white solid (0.13 g, 45%); mp 82−84 °C. H NMR (400.0 MHz,
Notes
DMSO-d6): δ = 8.92 (s, 1H), 8.43 (d, J = 2.4 Hz, 1H), 8.25 (d, J = 8.4 Hz,
1H), 7.99 (d, J = 8.4 Hz, 1H), 7.82 (d, J = 2.4 Hz, 1H), 7.75 (d, J = 8.4
Hz, 2H), 7.43 (d, J = 8.2 Hz, 2H), 6.22 (s, 2H), 3.60 (br s, 4H), 2.33 (br
s, 4H), 2.20 (s, 3H). 13C NMR (100.6 MHz, CDCl3): δ 168.8, 156.5,
150.0, 146.6, 138.5, 138.3, 137.4, 136.6, 133.9, 127.6, 125.4, 123.2, 120.7,
115.7, 54.5, 46.9, 45.5, 41.6. Anal. RP-HPLC tR = 12.63 min (method
2A, purity 96%). LRMS (APCI): m/z = 442.2 [(M + H)+] (anal. calcd
for C23H22F3N5O+: m/z = 441.18).
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Medicines for Malaria Venture (MMV) for financial
support for this research (project MMV09/0002). We thank Dr
Jeremy N. Burrows (MMV) for helpful discussions. 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 (KC).
General Procedure 4 for the Synthesis of (4-(6-Amino-5-(6-
(trifluoromethyl)pyridin-3-yl)pyridin-3-yl)phenyl)(piperazin-1-
yl)methanone 35. EDCI (1.24 g, 6.49 mmol) was added in small
batches to a solution of 36 (1.73 g, 5.90 mmol) and HOBt (0.88 g, 6.49
mmol) in dry DMF (15 mL). The reaction mixture was stirred for 10
min, at which time a solution of N-Boc piperazine (1.21 g, 6.49 mmol)
and triethylamine (2.46 mL, 17.71 mmol) in dry DMF (2 mL) was
added and the solution was stirred for an additional 12 h at room
temperature. H2O (10 mL) was added to the mixture and extracted with
ethyl acetate (4 × 10 mL). The combined organic layers were washed,
dried over Na2SO4, and concentrated in vacuo. The remaining residue
was subjected to column chromatography on silica gel using ethyl
acetate to yield (4-(6-amino-5-bromopyridin-3-yl)phenyl)(N-Boc
piperazin-1-yl)methanone (1.61 g, 59%), which was used without
further purification. To a solution of (4-(6-amino-5-bromopyridin-3-
yl)phenyl)(N-Boc piperazin-1-yl)methanone (0.25 g, 0.54 mmol) in
1,4-dioxane (4 mL), 2-(trifluoromethyl)pyridine-5-boronic acid pinacol
ester (0.16 g, 0.60 mmol) was added. The mixture was thoroughly
degassed with nitrogen for 15 min, at which time Pd(PPh3)2Cl2 (19 mg,
0.03 mmol) was added under an atmosphere of nitrogen, followed by
aqueous K2CO3 (0.57 mL, 0.57 mmol). The reaction mixture was stirred
at 90 °C for 14 h, poured into H2O (10 mL), and extracted with ethyl
acetate (4 × 10 mL). The combined organic layers were washed with
brine (3 × 5 mL), dried over Na2SO4, and concentrated in vacuo. The
remaining residue was subjected to column chromatography on silica gel
using dichloromethane/methanol in a 9.7:0.3 v/v ratio as eluent to
furnish (4-(6-amino-5-(6-(trifluoromethyl)pyridin-3-yl)pyridin-3-yl)-
phenyl)(N-Boc piperazin-1-yl)methanone (0.17 g, 58%) as a white
solid, which was used without further purification. To a suspension of 4-
(6-amino-5-(6-(trifluoromethyl)pyridin-3-yl)pyridin-3-yl)phenyl)(N-
Boc piperazin-1-yl)methanone (0.17 g, 0.32 mmol) in dichloromethane
(2 mL) was added trifluoroacetic acid (0.12 mL, 1.61 mmol). The pale-
yellow solution was stirred vigorously for 12 h. The reaction mixture was
concentrated under reduced pressure to give a pale-yellow solid. The
resulting powder was stirred with Amberlyst A-21 resin in a
dichloromethane/methanol 1:1 v/v ratio (15 mL) for 1 h. The reaction
mixture was then filtered, and the solvent was removed under reduced
pressure to yield 35 as hygroscopic powder (0.12 g, 90%); mp 92−94
°C. 1H NMR (400.0 MHz, DMSO-d6): δ 8.91 (d, J = 1.8 Hz, 1H), 8.42
(d, J = 2.4 Hz, 1H), 8.24 (dd, J = 1.9 Hz, 8.4 Hz, 1H), 7.98 (d, J = 8.4 Hz,
1H), 7.81 (d, J = 2.4 Hz, 1H), 7.74 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.4
Hz, 2H), 6.19 (s, 2H), 3.38 (br s, 2H), 3.26 (br s, 2H), 2.67 (br s, 4H).
13C NMR (100.6 MHz, DMSO-d6): δ 168.8, 156.5, 150.1, 146.6, 138.6,
ABBREVIATIONS USED
■
DMF, dimethylformamide; po, oral administration; HPLC, high
pressure liquid chromatography; HPMC, hydroxypropylmethyl
cellulose; MSD, mean survival time; SAR, structure−activity
relationship; PK, pharmacokinetics; TLC, thin layer chromatog-
raphy; TMS, tetramethylsilane; MMV, Medicines for Malaria
Venture
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