N-aryl-2-aminobenzimidazoles: Novel, efficacious, antimalarial lead compounds

Article abstract of DOI:10.1021/jm500715u

From the phenotypic screening of the AstraZeneca corporate compound collection, N-aryl-2-aminobenzimidazoles have emerged as novel hits against the asexual blood stage of Plasmodium falciparum (Pf). Medicinal chemistry optimization of the potency against Pf and ADME properties resulted in the identification of 12 as a lead molecule. Compound 12 was efficacious in the P. berghei (Pb) model of malaria. This compound displayed an excellent pharmacokinetic profile with a long half-life (19 h) in rat blood. This profile led to an extended survival of animals for over 30 days following a dose of 50 mg/kg in the Pb malaria model. Compound 12 retains its potency against a panel of Pf isolates with known mechanisms of resistance. The fast killing observed in the in vitro parasite reduction ratio (PRR) assay coupled with the extended survival highlights the promise of this novel chemical class for the treatment of malaria.

Full text of DOI:10.1021/jm500715u

Article
pubs.acs.org/jmc
N‑Aryl-2-aminobenzimidazoles: Novel, Efficacious, Antimalarial Lead
Compounds
†
†,¶
‡
§,¶
†
Sreekanth Ramachandran, Shahul Hameed P., Abhishek Srivastava, Gajanan Shanbhag,
‡
‡
∥
§
§
Sapna Morayya, Nikhil Rautela, Disha Awasthy, Stefan Kavanagh, Sowmya Bharath, Jitendar Reddy,
§
§
§
§
†
○
Vijender Panduga, K. R. Prabhakar, Ramanatha Saralaya, Robert Nanduri, Anandkumar Raichurkar,
†
†
ez-Díaz,○ María Santos Martínez,
Sreenivasaiah Menasinakai, Vijayashree Achar, María Belen
́
Jimen
́
○
○
○
○
#
In
̃
igo Angulo-Barturen, Santiago Ferrer, Laura María Sanz, Francisco Javier Gamo, Sandra Duffy,
#
⊥
▽
▽
▽
Vicky M. Avery, David Waterson, Marcus C. S. Lee, Olivia Coburn-Flynn, David A. Fidock,
Pravin S. Iyer, Shridhar Narayanan, Vinayak Hosagrahara,* and Vasan K. Sambandamurthy*
†
‡
,§
,‡
†
‡
§
Department of Medicinal Chemistry, Department of Biosciences, and DMPK and Animal Sciences, AstraZeneca India Pvt. Ltd.,
Bellary Road, Hebbal, Bangalore 560024, India
∥
Safety Assessment, AstraZeneca, Alderley Park, Macclesfield, U.K.
⊥
Medicines for Malaria Venture, International Center Cointrin, Route de Pre-
́
Bois 20, Post Office Box 1826, 1215 Geneva,
Switzerland
#
Discovery Biology, Eskitis Institute for Drug Discovery, Griffith University, 170 Kessels Road, Nathan, Queensland 4111, Australia
○
Tres Cantos Medicines Development Campus, Diseases of the Developing World (DDW), GlaxoSmithKline, Severo Ochoa 2, Tres
Cantos 28760, Madrid, Spain
▽
Department of Microbiology and Immunology and Division of Infectious Diseases, Department of Medicine, Columbia University
Medical Center, Columbia University, 116th Street and Broadway, New York, New York 10027, United States
*
S Supporting Information
ABSTRACT: From the phenotypic screening of the AstraZeneca corporate compound
collection, N-aryl-2-aminobenzimidazoles have emerged as novel hits against the asexual blood
stage of Plasmodium falciparum (Pf). Medicinal chemistry optimization of the potency against Pf
and ADME properties resulted in the identification of 12 as a lead molecule. Compound 12 was
efficacious in the P. berghei (Pb) model of malaria. This compound displayed an excellent
pharmacokinetic profile with a long half-life (19 h) in rat blood. This profile led to an extended
survival of animals for over 30 days following a dose of 50 mg/kg in the Pb malaria model.
Compound 12 retains its potency against a panel of Pf isolates with known mechanisms of
resistance. The fast killing observed in the in vitro parasite reduction ratio (PRR) assay coupled
with the extended survival highlights the promise of this novel chemical class for the treatment of
malaria.
INTRODUCTION
by the WHO, is an artemisinin-based combination therapy of
■
two or more drugs administered over a period of 3 to 7 days.
The global efforts to treat and eliminate malaria would be
immensely benefitted by introducing novel agents that require a
Malaria caused by the protozoan parasite belonging to the
genus Plasmodium continues to be a global public health
problem with an estimated 219 million cases and 627,000
3
1
single exposure radical cure and prophylaxis (SERCAP). Such
deaths in 2012. Malaria caused by Plasmodium falciparum (Pf)
a treatment regimen would entail a combination of antimalarial
agents that kill the asexual blood stages, sexual gametocyte
accounts for over 95% of all malaria cases occurring in Africa
and South East Asia. The cerebral form of malaria caused by Pf
leads to high mortality among children below 5 years of age.
Malaria is transmitted to humans by the bite of infected female
anopheline mosquitoes. The emergence of Pf strains resistant
to the two former first-line antimalarial drugs, chloroquine
stages (block transmission), and hypnozoites in the liver
3
(
provide radical cure). It is envisaged that a single dose
therapy would greatly reduce the emergence and dissemination
of drug resistance and significantly improve patient compliance.
Substantial progress has been made in the past decade to
advance several novel antimalarials targeting Pf into clinical
(
CQ) and sulphadoxine-pyrimethamine (SP), have seriously
1
impeded efforts to effectively treat malaria. Even more
worrisome is the most recent emergence of Pf strains resistant
to artemisinin derivatives; the frontline agents in the treatment
Received: May 8, 2014
Published: July 9, 2014
2
of malaria. The current treatment for malaria, recommended
©
2014 American Chemical Society
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a
Table 1. Structure−Activity Relationship and Cytotoxicity Selectivity Index of Compounds 1−23
a
a
6
ND : not determined. IC50 against NF54 and K1 strains of Pf were determined using a SYBR-Green based assay as previously reported. The
cytotoxicity of compounds against a mammalian THP-1 cell line was performed as described (see Supporting Information).
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4
development. However, there is a dearth of leads with long
reference drugs in all of the experiments. Chloroquine exhibited
half-life in humans that could be used in combination with
suitable partner drugs to prevent the emergence of drug-
resistant forms of the parasite. The present study describes the
discovery and optimization of an antimalarial series with the
potential for long half-life in humans and activity against a panel
of clinical strains harboring resistance to known antimalarial
agents.
an IC of 17.1 ± 5 nM (NF54 strain) and 347.3 ± 82 nM (K1
strain) and artemisinin an IC50 of 7.1 ± 4 nM (NF54 strain)
and 6.5 ± 2 nM (K1 strain) in this assay.
Structure−Activity Relationship (SAR). Initial efforts to
explore the SAR requirements to improve potency focused on
the optimization of the site 1 ring substitution of the
benzimidazole scaffold (Table 1). The introduction of 2-pyridyl
50
(
4
compound 2) instead of a phenyl group at site 1 resulted in a
-fold improvement in Pf potency (NF54 IC , 53 nM; and K1
RESULTS AND DISCUSSION
■
50
Antiplasmodial Activity and Structure−Activity Rela-
tionship (SAR). A whole cell phenotypic screen of 500,000
compounds from the AstraZeneca corporate library, assayed
against the asexual blood stage of Pf, was carried out using a
IC50, 80 nM). The R position at site 1 turned out to be critical
3
for the observed antimalarial activity, which was evident from
the activity of compounds 3−5, in which lipophilic electron
withdrawing groups displayed better antiplasmodial activity.
The importance of the pyridine-2-ylamino moiety for potency
was further substantiated by compound 6, wherein the 3-
pyridyl derivative was found to be inactive.
On the basis of the encouraging activity observed for the
pyridine-2-ylamino moiety at site 1, this motif was retained
during further medicinal chemistry optimization. The focus was
then shifted to the core benzimidazole; the introduction of
additional nitrogen at the C-5 position of the benzimidazole
core retained the antiplasmodial activity (7) (match pair;
compound 5 versus 7, Table 1). Additionally, chloro
substitution at the C-4 position of benzimidazole was tolerated
for potency (compound 2 versus 8, Table 1). However,
converting the benzimidazole core to 4-aza benzimidazole with
additional fluoro substitution completely abolished the potency
5
high content image-based approach previously reported. This
screen led to the identification of N-aryl-2-aminobenzimida-
zoles 1 as a hit compound suitable for further medicinal
chemistry optimization. Compound 1 displayed moderate
potency against both sensitive (NF54) and resistant (K1)
strains of Pf and a good cytotoxicity index (>1190-fold) against
a mammalian THP-1 cell line. These findings encouraged
further exploration of structure−activity relationship (Table 1)
of this chemical class. The hit compound 1 consists of a core
benzimidazole ring attached to 2-aminophenyl (site 1) and a
basic piperazine ring at the C-6 position (site 2) (Figure 1).
(
compound 2 versus 9, Table 1). This may be due to the
disburbance of electron density of the benzimidazole core.
Efforts were made to identify the best basic side chain at the C-
6
position of benzimidazoles (Table 1). Compounds with basic
side chains having calculated pK ≥ 7 (10, 12−15, and 17−18)
a
Figure 1. Generic structure of the N-aryl-2-amino benzimidazole
scaffold.
retained their antiplasmodial activity, whereas compounds 11
and 16 with weak basic/neutral side chains were found to be
inactive.
All compounds were screened for their antiplasmodial
Having observed a substitution effect on the antiplasmodial
activity at site 1 for these inhibitors, we shifted our attention to
further understand the multisubstitution pattern on the 2-
amino pyridines (Table 1). Toward this objective, compounds
activity against a fully sensitive (NF54) and multidrug resistant
6
(
K1) strain of Pf using a SYBR-Green based readout.
Chloroquine, pyrimethamine, and artesunate were included as
Scheme 1. General Synthetic Scheme for Compounds 1−11, 14, 19−20, and 22−23
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a
Table 2. In Vitro DMPK Profile and hERG Activity for Representative Compounds
aqueous solubility log D at pH human plasma protein binding
human microsomal clearance
rat hepatocyte clearance
hERG IC50
(μM)
6
compd
(μM)
7.4
(% free)
(μL/min/mg)
(μL/min/10 cells)
2
4
8
60
2.7
2.2
3
11.5
18.3
30.1
13.4
27.5
5.1
4.1
4.8
<4
13.7
19.5
9
3.5
1170
>1000
121
12.7
7.3
1
1
2
2
7
2
3
<4
4.7
4.3
>1000
5.6
3.2
3.9
<4
7.5
21.5
8.8
21.8
11.3
a
Aqueous solubility, log D, estimation of free fraction in human plasma, clearance in rat hepatocytes, and inhibition of hERG ion channel were
7
performed as reported earlier. .
Figure 2. Identification and characterization of metabolites of compound 2 incubated with rat and human liver microsomes. (A) UV chromatograms,
(
B) LC-MS extracted ion chromatograms, and (C) MS2 spectra of compound 2 and metabolites M1 and M2.
1
9−23 were synthesized with disubstitutions on the 2-amino
protein binding (Table 2). The series showed good metabolic
stability in human microsomes and rat hepatocytes, while
maintaining excellent aqueous solubility. The observed hERG
inhibition for the series could be attributed to the basic,
lipophilic nature of these compounds.
pyridine group. Within this set, compounds 22 and 23 having a
hydrophobic substituent like a cyclopropyl ring showed
particularly potent Pf IC50 of 17 and 13 nM, respectively.
Chemistry. Synthesis of compounds 1−11, 14, 19−20, 22−
23 followed the general synthetic route as shown in Scheme 1.
Bioactivation Analysis. Compounds containing benzimida-
zole and amino-benzimidazole rings have been shown to
Reaction of appropriately substituted chloro nitro arenes 1a
with piperazines under basic conditions resulted in compound
8
undergo metabolic activation to form reactive metabolites. In
1
b. Reduction of 1b under Pd/C conditions followed by the
such scaffolds, the embedded ortho arrangement of the two
imidazole nitrogen atoms is likely to contribute toward the high
electron density in the ring. This could potentially lead to the
generation of reactive intermediates derived from ortho- and
coupling of the 1,2-diamines 1c with various isothiocyanates 1e
resulted in compounds 1−11, 14, 19−20, 22−23. Alternate
synthetic schemes were applied to the synthesis of compounds
9
12−13, 15−18, and 21 (see Experimental Section).
para-quinone species. Because reactive metabolites have been
DMPK and Safety Profiling. Representative compounds
implicated as causal factors in many drug-associated idiosyn-
10
were profiled for various in vitro ADMET parameters such as
intrinsic clearance, aqueous solubility, log D, and human plasma
cratic toxicities, it was deemed important to evaluate the
propensity of our series to undergo bioactivation at an early
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Scheme 2. Proposed Metabolic Pathways for Compound 2
Scheme 3. Effect of Structural Modification on Reactive Metabolite Formation
stage of drug discovery, while the scope for mitigating this risk
through various chemical modifications remains feasible.
To assess the potential for bioactivation, compound 2 was
incubated with rat and human liver microsomes fortified with
NADPH in the presence and absence of glutathione (GSH) or
N-acetyl cysteine (NAC). As shown in Figure 2A, the UV peak
at 19 min (M1) was only observed in incubation samples
supplemented with NADPH plus GSH but not in the samples
devoid of GSH, indicating this to be a GSH adduct peak.
Further confirmation of bioactivation was obtained by the
observation of NAC adduct peak at 20.3 min (M2) in samples
10
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containing NADPH plus NAC (Figure 2A). However, this peak
was not observed in the absence of NAC.
Table 3. Rat Pharmacokinetics Parameters for Compounds 2
and 12
The extracted ion chromatograms for RLM and HLM are
shown in Figure 2B. M1 produced a protonated molecular ion,
rat PK
compd 2
compd 12
+
+
i.v. (1 mg/kg)
t1/2 (h)
[M + H] at m/z 692, whereas M2 yielded an ion [M + H] at
8.9
3
14.9
6
m/z 548. The chemical formula derived from high-resolution
mass spectrometry (MS) data for M1 and M2 indicated the
addition of GSH (305 Da) and NAC (161 Da) to the parent
molecule (m/z 387), respectively. MS2 spectrum of M1 (Figure
AUC (μM·h)
CL (mL/min/kg)
Vss (L/kg)
p.o. (5 mg/kg)
% F
15.2
10.6
14.9
16.9
2
C) included ions indicative of the generic neutral loss of a
+
100
7.2
79
pyroglutamic acid (m/z 563, [M + H-129] ) and
t1/2 (h)
19.2
0.5
fragmentation at the thioether linkage (m/z 419, [M + H-
+
Cmax (μM)
0.87
2
73] ) from the glutathione moiety. MS2 analysis of M2
showed a major fragment at m/z 419 (Figure 2C), which
corresponds to the characteristic neutral loss of N-acetyldehy-
1
1
droalanine (129 amu) from a mercapturate. This data
confirmed that the UV peaks observed at 19 and 20.3 min
belong to GSH and NAC adducts of 2, respectively. However,
the exact site of this modification could not be located from
MS2 spectra of the parent and metabolites (Figure 2C; Scheme
exhibited a similar effect in terms of parasitemia reduction and
extended survival of treated mice.
On the basis of the efficacy observed in the P. berghei model,
we evaluated the therapeutic efficacy of 2 in the humanized Pf/
14
SCID model. Mice were administered a single oral dose (3,
−1
2
). Another metabolite (M3) with m/z 403 was identified as an
1
0, and 30 mg·kg ) of compound 2 for four consecutive days
oxidative metabolite of 2 (Scheme 2).
followed by monitoring blood parasitemia by flow cytometry.
Two parameters of efficacy were estimated in the study: the
ED and the exposure required for achieving ED (AUCED )
On the basis of the above data, it was proposed that
compound 2 undergoes bioactivation to form an iminium ion
diiminoquinone) type reactive metabolite (Scheme 3, A).
Because the nitrogen of the piperazine ring is para to that of the
imidazole nitrogen, this arrangement can undergo a two-
electron oxidation process by CYP and/or peroxidases to form
a reactive intermediate. This species can be covalently trapped
90
90
90
(
(
Figure 3a,b).
Figure 3a represents the whole blood levels of 2 after the first
dose of treatment. Data represents individual profiles of blood
concentration versus time of 2 (0−23 h). The dotted line
shows the IC in nanomolar. Figure 3b represents parasitemia
5
0
12
0
087/N9
with GSH or NAC (Scheme 3, A).
in peripheral blood of mice infected with Pf 3D7
. Data
To investigate the effect of structural modifications on the
metabolic activation, a variety of amino-benzimidazole
derivatives were selected for testing (Table 1). These were
categorized based on the nature of a linker atom as N-linked
shown in 3b corresponds to individual parasitemia for mice
treated with 2 or the vehicle (n = 2). Under these experimental
conditions, 2 was found to be efficacious against Pf, with an
−1
ED9 of 12.8 mg·kg and an estimated AUCED90 of 5.4 μg·h·
0
−
1
−1
(
1
compounds 2 and 7−10) or C-linked compounds (12, 16, and
mL ·day .
7). In vitro assays were performed with each test compound in
The observed in vivo efficacy of 2 is comparable to those of
marketed antimalarial drugs. Mice infected with Pf 3D7 were
treated with the vehicle and were found to harbor healthy rings,
trophozoites, and mature schizonts. The treatment with 2 leads
to a population of low-intensity DNA staining by flow
cytometry that corresponds to pyknotic parasites by micros-
copy (data not shown).
a rat liver microsomal system in the presence of GSH or NAC.
Efforts were made to reduce the electron density in the ring
by substitution with electron withdrawing groups (compounds
8
and 9) and by incorporating a nitrogen atom in the ring
(
compound 7). However, all N-linked compounds underwent
bioactivation to form GSH or NAC adducts (Scheme 3, A−E).
Replacement of the N-linker with an amide (16) resulted in
the mitigation of bioactivation liability (Scheme 3, H), albeit at
the cost of antiplasmodial potency. Compound 17, with a C-
linker in the form of a secondary carbon produced GSH or
NAC conjugates, potentially via an imine-methide type of
reactive intermediate (Scheme 3, F). Compound 12 (Scheme 3,
G), containing a piperidine ring through a tertiary carbon linker
was able to mitigate the bioactivation issue. Interestingly, this
compound still retained good Pf potency (Pf IC50 of 36 nM).
In Vivo Pharmacokinetic Analysis. Representative com-
pounds 2 and 12 were profiled for pharmacokinetic properties
in rats (Table 3). These compounds displayed a long half-life,
low clearance, large volume of distribution, and good oral
bioavailability.
In Vitro Parasite Reduction Ratio (PRR). We assessed the
rate and extent of kill of the plasmodium parasite by this
chemical class using the in vitro parasite reduction ratio (PRR)
15
assay. This assay uses a limiting dilution technique to quantify
the number of parasites that remain viable after drug treatment.
Pf strain 3D7A (MR4) was treated with compound 2 at a
concentration corresponding to 10× the IC50 concentration.
Samples of parasites were taken from compound treated
cultures after 24 and 48 h. Compound 2 displayed a fast kill
pattern that was similar to the effect seen after 48 h treatment
with the reference drug, chloroquine. (Figure 4).
Novel Mechanism of Action. Representative compounds
were screened against a panel of Pf strains harboring mutations
that confer resistance to known antimalarial agents both curren6t
and under development by employing the SYBR Green assay.
This was done to rule out cross-resistance to existing drugs and
also to ascertain novelty in terms of mechanism of action of this
chemical series. As shown in Table 5, compounds 2, 4, and 19
retained their IC50 values across six different Pf strains. As
reported earlier, chloroquine and pyrimethamine showed a
significant shift (>15-fold) in their IC50 values against strains
In Vivo Efficacy. The excellent pharmacokinetic profiles of
compounds 2 and 12 prompted us to evaluate their in vivo
13
efficacy in a P. berghei model of malaria. Mice treated with
four once-daily doses of 2 at 50 mg/kg displayed a 99.9%
reduction in parasitemia. Strikingly, all mice treated with 2
survived for 30 days, an excellent attribute for a novel
antimalarial agent (Table 4). Compounds 5 and 12 also
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Table 4. N-Aryl-2-amino Benzimidazoles Are Efficacious in a P. berghei Model of Malaria and Prolong the Survival of Treated
a
Mice
in vivo activity following 4 daily oral doses
pharmacokinetic parameters
activity (%) at
9
6 h
survival (days)
Cmax
AUClast
(h·μM)
compd
in vitro IC50 NF54 (μM)
dose (mg/kg)
mean
SD
mean
SD
T1/2 (h)
(μM)
SD
SD
2
2
5
5
5
1
1
1
0.053
0.053
0.083
0.083
0.083
0.039
0.037
0.037
0.013
30 × 4
50 × 4
30 × 4
50 × 4
100 × 4
50 × 4
30 × 4
50 × 4
30 × 4
99.7
99.9
81.3
99.9
99.9
99.9
98.9
99.8
99.9
0.2
14
30
10
15
24
11
25
30
25
1
0
2
2
8
0
6
1
6
7.3
5.1
3.7
5.4
4.9
4.5
9.3
18.5
ND
1.2
0.35
4.83
0.6
12.39
117.12
19.2
2.93
17.53
3.1
0
16.35
3 3
5.0
14.3
0.05
0.08
0.09
1.96
0.06
0.05
2.3
33.7
9.9
6.1
1.2
41.6
11.1
59.9
20.3
4.8
0
2
2
25 5
5.2
11.5
1.4
201.7
54.0
8.8
0 8
ND
130.6
ND
chloroquine
ND
ND
a
ND: not determined.
Figure 4. In vitro parasite reduction ratio (PRR) for compound 2. The
graph depicts the number of viable parasites following the exposure of
Pf 3D7A parasites to 10× IC50 concentrations of compound 2.
resistance results highlight the unique feature of this chemical
class to retain its potency against a panel of Pf isolates with
known mechanisms of resistance. Future genetic studies to
generate spontaneous resistant mutants and characterize the
mutations via whole genome sequencing should provide clues
on the molecular mechanism of resistance as well as the
molecular target for this novel chemical class.
Representative compounds from this class were inactive
against the sexual gametocytes of Pf and against the liver stages
of the parasite (data not shown). Since malaria is treated with a
combination of drugs to prevent the emergence of resistant
forms of the parasites and to kill the various life stages of Pf, it is
imperative that aryl aminobenzimidazoles be evaluated in
combination with newer agents that demonstrate activity
against the sexual as well as liver stages of Pf to treat and
eliminate malaria.
Conclusions. A thorough medicinal chemistry optimization
led to the identification of N-aryl-2-amino benzimidazoles, as a
novel class of antiplasmodial agents, with the potential to
exhibit a long half-life in humans based on attractive rat
pharmacokinetic parameters. The representative compound 12
is orally bioavailable and efficacious in a mouse model of
malaria. Additionally, the series displays a fast killing rate
equivalent to chloroquine in the in vitro parasite reduction ratio
assay. Cross-resistance studies suggest that this class of
compounds acts through a novel mechanism of action. Further
lead optimization efforts coupled with in vivo safety profiling
should enable a novel clinical candidate from this chemical
class.
Figure 3. (a) Pharmacokinetic profile of compound 2 in humanized P.
falciparum SCID mice. (b) Efficacy of compound 2 in a Pf/SCID
mouse model.
K1, 7G8, W2, and Dd2. A significant shift in IC50 values was
observed for atovaquone against the SB1-A6 strain.
Compound 2 was also tested for its activity against transgenic
Pf lines carrying resistance mutations in either PfATP4 or
PfPI4K. Pf strains expressing an extra copy of PfATP4-D1247Y
or PfATP4-I398F/P990R are resistant to the spiroindolone
1
6
NITD609. Pf strains expressing a Pf PI4K-S1320L (engi-
neered into the endogenous gene by ZFN-editing) are resistant
17
to the imidazopyrazines, KAI407. Interestingly, no shift in
IC50 was observed for compound 2 when tested against all
three transgenic lines (data not shown). These data imply that
the antimalarial activity of N-aryl-2-aminobenzimidazoles is not
mediated via the inhibition of PfATP4 or Pf PI4K. The cross-
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Table 5. N-Aryl-2-amino Benzimidazoles Retain Their Antiplasmodial Activity against a Panel of Pf Strains Known to Harbor
a
Mutations Conferring Resistance to Known Antimalarial Agents
IC50 (nM) mean ± SD
compd
NF54
K1
7G8
W2
SB1
DD2
2
4
1
91.4 ± 28
376 ± 110
43.5
152 ± 31
369.7 ± 90
81.8
184.9 ± 7.5
307.1 ± 273
98.8
197 ± 53
437 ± 89
106.5
40.5 ± 0.7
86.2 ± 9
ND
106.8 ± 23
200.6 ± 169
ND
9
artemisinin
7.1 ± 4
6.5 ± 2
1.3 ± 0.1
6.5 ± 1
16.6 ± 5
11.81 ± 4.5
10.9 ± 4
>2000
10.3 ± 2
chloroquine
pyrimethamine
atovaquone
17.1 ± 5
28.3 ± 11
1.4 ± 0.4
347.3 ± 82
8558.4 ± 1983
2.3 ± 0.8
163.9 ± 17
10041.7 ± 1261
2.3 ± 0.7
374.7 ± 228
15379.7 ± 2826
1.2 ± 0.6
251.3 ± 9
13838.6 ± 8619
2 ± 0.02
a
Data represent the IC50 values obtained from each compound tested in duplicate from two independent experiments using a SYBR-Green based
6
method.
EXPERIMENTAL SECTION
to obtain 5-(4-methylpiperazin-1-yl)-2-nitroaniline as a yellow solid.
Yield: 10.0 g (72.8%). LCMS: m/z 237.3 (M+1). H NMR 400 MHz,
■
1
All anhydrous solvents, reagent grade solvents for chromatography and
starting materials were purchased from either Sigma-Aldrich Chemical
Co. or Fisher Scientific. Water was distilled and purified through a
Milli-Q water system (Millipore Corp., Bedford, MA). General
methods of purification of compounds involved the use of silica
cartridges purchased from Grace Purification Systems. The reactions
were monitored by TLC on precoated Merck 60 F254 silicagel plates
and visualized using UV light (254 nm). All compounds were analyzed
DMSO-d : δ 7.80 (d, J = 9.7 Hz, 1H), 7.26 (s, 2H), 6.40 (q, J = 2.4 Hz,
6
1H), 6.22 (d, J = 2.3 Hz, 1H), 3.30−3.34 (m, 4H), 2.40 (t, J = 4.9 Hz,
4H), and 2.21 (s, 3H).
4
-(4-Methylpiperazin-1-yl)benzene-1,2-diamine (1c). To a sol-
ution of 5-(4-methylpiperazin-1-yl)-2-nitroaniline (1b, 15.0 g, 63.48
mmol) in methanol/DCM (300 mL) was added palladium (10%) on
carbon (5 g). The mixture was stirred under hydrogen atmosphere for
16 h. The suspension was filtered through Celite, and the filtrate was
concentrated under reduced pressure to obtain 4-(4-methylpiperazin-
1
for purity by HPLC and characterized by H NMR using Bruker 300
MHz NMR and/or Bruker 400 MHz NMR spectrometers. The purity
of all final derivatives for biological testing was confirmed to be >95%
as determined using the following conditions: a Shimadzu HPLC
instrument with a Hamilton reverse phase column (HxSil, C18, 3 μm,
1
-yl)benzene-1,2-diamine, which was taken to the next step without
further purification. Yield: 9.8 g (75.38%). LCMS: m/z 207.4 (M+1).
1
H NMR 400 MHz, DMSO-d : δ 6.39 (d, J = 8.2 Hz, 1H), 6.21 (d, J =
6
2
.6 Hz, 1H), 6.01 (q, J = 2.6 Hz, 1H), 4.35 (s, 2H), 3.98 (s, 2H), 2.86
t, J = 4.8 Hz, 4H), 2.40 (t, J = 4.7 Hz, 4H), and 2.19 (s, 3H).
-Bromo-4-isothiocyanatobenzene (1e). To the solution of 4-
bromoaniline (1d, 1.0 g, 5.81 mmol) in chloroform (10 mL) was
2
.1 mm × 50 mm (H2). Eluent A, 5% CH CN in H O; eluent B, 90%
3 2
(
CH CN in H O. A flow rate of 0.2 mL/min was used with UV
3
2
1
detection at 254 and 214 nm. The structures of the intermediates and
1
end products were confirmed by H NMR and mass spectroscopy.
added aqueous sodium bicarbonate (5.44 g, 6.4 mmol, 20 mL H O)
Chemical shifts are reported in ppm (δ) relative to the residual solvent
peak in the corresponding spectra; chloroform δ 7.26, methanol δ
2
and then at 0 °C was added thiophosgene (0.45 mL, 5.98 mmol)
dropwise and stirred for 2 h at 0 °C. The reaction mixture was diluted
with cold water and extracted in MDC. The organic layer was
separated, dried over anhydrous sodium sulfate, filtered, and
concentrated under reduced pressure to obtain dark brown liquid,
which was passed through a neutral alumina column using pet ether as
3
.31, DMSO-d δ 3.33, and coupling constants (J) are reported in
6
hertz (Hz) (where s = singlet, bs = broad singlet, d = doublet, dd =
double doublet, bd = broad doublet, ddd = double doublet of doublet,
t = triplet, tt−triple triplet, q = quartet, and m = multiplet) and
analyzed using ACD NMR data processing software. Mass spectra
values are reported as m/z (HRMS).
All reactions were conducted under nitrogen and monitored using
LCMS unless otherwise noted. Solvents were removed in vacuo on a
rotary evaporator.
eluent to obtain 1-bromo-4-isothiocyanatobenzene as a white solid.
1
Yield: 0.6 g (50%). H NMR 300 MHz, DMSO-d
: δ 7.62−7.66 (m,
6
2H), and 7.40 (m, 2H).
N-(5-Bromopyridin-2-yl)-6-(4-methylpiperazin-1-yl)-1H-benzo[d]-
imidazol-2-amine (2). Compound 2 was prepared by a synthetic
route similar to that described earlier. HRMS calculated for
General Synthetic Procedures. N-(4-Bromophenyl)-6-(4-meth-
ylpiperazin-1-yl)-1H-benzo[d]imidazol-2-amine (1). To a solution of
+
+
1
4
-(4-methylpiperazin-1-yl)benzene-1,2-diamine (1b, 0.4 g, 1.93 mmol)
in THF (10 mL) were added 1-bromo-4-isothiocyanatobenze (1e,
.415 g, 2.41 mmol) and EDC·HCl (0.743 g, 3.87 mmol) at room
temperature. The resulting mixture was subjected to microwave
irradiation in a sealed tube at 100 °C for 2 h. Product formation was
confirmed by LCMS. The solvent was evaporated to dryness. The
crude obtained was purified by column chromatography using 1%
[C H BrN ] 387.09329 [M + H] ; found, 387.09325. H NMR
17 19 6
400 MHz, DMSO-d : δ 11.55 (s, 1H), 10.60 (s, 1H), 8.33 (d, J = 2.4
6
0
Hz, 1H), 7.91 (q, J = 2.3 Hz, 1H), 7.32 (d, J = −6.3 Hz, 1H), 7.24 (s,
1H), 6.96 (s, 1H), 6.76 (d, J = 8.9 Hz, 1H), 3.06 (t, J = 4.8 Hz, 4H),
2.51 (t, J = 1.5 Hz, 4H), and 2.23 (s, 3H). HPLC 97.78%.
6-(4-Methylpiperazin-1-yl)-N-(pyridin-2-yl)-1H-benzo[d]imidazol-
2-amine (3). Compound 3 was prepared by using a synthetic route
+
MeOH in DCM saturated with NH to get N-(4-bromophenyl)-6-(4-
similar to that described earlier. HRMS calculated for [C H N ]
3
17 20
6
+
1
methylpiperazin-1-yl)-1H-benzo[d]imidazol-2-amine as a white solid.
309.18277 [M + H] ; found, 309.18215. H NMR(400 MHz, DMSO-
Y i e l d : 0 . 0 7 5
g
( 8 . 5 7 % ) . H R M S c a l c u l a t e d f o r
d ) δ 11.78 (s, 1H), 10.46 (s, 1H), 8.28 (d, J = 4.8 Hz, 1H), 7.68−7.72
6
C H BrN ] 386.09993 [M + H] ; found, 386.09754. 1H NMR
+
+
[
4
(m, 1H), 7.21−7.23 (m, 2H), 6.75−6.93 (m, 2H), 6.73−6.75 (m, 1H),
3.06 (t, J = 4.8 Hz, 4H), 2.47−2.50 (m, 4H), and 2.23 (s, 3H). HPLC
95.74%.
1
8
20
5
00 MHz, DMSO-d : δ 10.72 (s, 1H), 9.48 (d, J = 20.1 Hz, 1H), 7.73
6
(
t, J = 2.7 Hz, 2H), 7.45 (t, J = 1.6 Hz, 2H), 7.21 (d, J = 8.5 Hz, 1H),
.13 (d, J = 8.4 Hz, 1H), 6.95 (s, 1H), 6.86 (d, J = 1.6 Hz, 1H), 6.71
q, J = 6.9 Hz, 1H), 3.05 (t, J = 4.9 Hz, 4H), 2.49 (t, J = 8.2 Hz, 4H),
and 2.23 (s, 3H). HPLC 99.78%.
-(4-Methylpiperazin-1-yl)-2-nitroaniline (1b). To the solution of
-chloro-5-nitropyridin-4-amine (1a10.0 g, 57.9 mmol) in anhydrous
7
(
6-(4-Methylpiperazin-1-yl)-N-(5-methylpyridin-2-yl)-1H-benzo[d]-
imidazol-2-amine (4). Compound 4 was prepared by a synthetic
route similar to that described earlier. HRMS calculated for
+
+
1
5
[C H N ] 323.20373 [M + H] ; found, 323.19767. H NMR 400
18 22 6
2
MHz, DMSO-d : δ 11.71 (s, 1H), 10.31 (s, 1H), 8.11 (s, 1H), 7.55 (s,
6
DMF (100 mL) were added 1-methyl piperazine (12.85 mL, 11.0
mmol) and potassium carbonate (24.03 g, 17.3 mmol). The resulting
mixture was heated at 130 °C for 16 h. The reaction mixture was
poured into cold water. The precipitate formed was filtered and dried
1H), 6.89−7.27 (m, 3H), 6.72 (s, 1H), 3.05 (s, 4H), 2.51 (s, 4H), and
2.24 (s, 6H). HPLC 99.38%.
6-(4-Methylpiperazin-1-yl)-N-(5-(trifluoromethyl)pyridin-2-yl)-1H-
benzo[d]imidazol-2-amine (5). Compound 5 was prepared by using a
6
649
dx.doi.org/10.1021/jm500715u | J. Med. Chem. 2014, 57, 6642−6652

Journal of Medicinal Chemistry
Article
synthetic route similar to that described earlier. HRMS calculated for
2.81 (m, 1H), 2.50 (s, 3H), 2.40−2.42 (m, 1H), and 2.02−2.05 (m,
2H). HPLC purity: 97.87%.
+
+
1
[
C H F N ] 377.17016 [M + H] ; found, 377.16921. H NMR 400
18 19 3 6
MHz, DMSO-d : δ 11.63 (s, 1H), 11.19 (s, 1H), 8.58 (s, 1H), 8.04 (q,
J = 2.1 Hz, 1H), 7.45 (d, J = 8.6 Hz, 1H), 7.28 (d, J = 8.6 Hz, 1H),
6-(Hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl)-N-(5-
6
(trifluoromethyl)pyridin-2-yl)-1H-benzo-[d]imidazol-2-amine (14).
Compound 14 was prepared by using a synthetic route similar to
6.98 (s, 1H), 6.80 (q, J = 2.0 Hz, 1H), 3.07 (t, J = 4.7 Hz, 4H), 2.51 (t,
+
that described for compound 1. HRMS calculated for [C H F N ]
J = 1.6 Hz, 4H), and 2.25 (s, 3H). HPLC 99.07%.
20 21
3
6
+
1
4
d
03.18581 [M + H] ; found, 403.18236. H NMR 400 MHz, DMSO-
: δ 11.70 (s, 1H), 10.92 (s, 1H), 8.58 (s, 1H), 8.05 (t, J = 6.9 Hz,
6
6
-(4-Methylpiperazin-1-yl)-N-(6-(trifluoromethyl) pyridin-3-yl)-1-
H-benzo[d]-imidazol-2-amine (6). Compound 6 was prepared by
using a synthetic route similar to that described earlier. HRMS
1H), 7.45 (d, J = 7.4 Hz, 1H), 7.28 (d, J = −8.4 Hz, 1H), 7.00 (s, 1H),
6.82 (d, J = 8.6 Hz, 1H), 3.62 (s, 1H), 3.55 (d, J = 7.2 Hz, 1H), 3.00−
3.06 (m, 3H), 2.77 (s, 1H), 2.14−2.25 (m, 2H), 1.75−1.86 (m, 3H),
and 1.42 (d, J = 10.8 Hz, 1H). HPLC 98.84%.
+
+
calculated for [C H F N ] 377.17016 [M + H] ; found,
1
8
19
3
6
1
3
77.169170. H NMR: 400 MHz, DMSO-d : δ 8.83 (d, J = 2.4 Hz,
6
1
H), 8.32−8.35 (m, 1H), 7.75 (m, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.10
(d, J = 2.0 Hz, 1H), 6.92−6.95 (m, 1H), 3.31−3.32 (m, 8H), and 2.95
(s, 3H). HPLC 99.27%.
6-(8-Methyl-8-azabicyclo[3. 2. 1]octan-3-yl)-N-(5-
(
trifluoromethyl)pyridin-2-yl)-1H-benzo[d]imidazol-2-amine (15).
+
+
HRMS calculated for [C H F N ] 402.19056 [M + H] ; found,
6
-(4-Methylpiperazin-1-yl)-N-(5-(trifluoromethyl)pyridin-2-yl)-1H-
21 22
3
5
1
4
1
02.18973. H NMR 400 MHz, DMSO-d : δ 11.42 (s, 2H), 8.59 (s,
imidazo[4,5-c]pyridin-2-amine (7). Compound 7 was prepared by
6
H), 8.06 (d, J = 8.5 Hz, 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.32 (t, J = 8.4
using a synthetic route similar to that described earlier. HRMS
+
+
Hz, 2H), 6.99 (d, J = 8.1 Hz, 1H), 3.20 (s, 2H), 3.04 (d, J = 8.2 Hz,
1H), 2.31−2.36 (m, 2H), 2.18 (s, 3H), 2.01 (d, J = 3.9 Hz, 2H), and
calculated for [C H F N ] 378.16541 [M + H] ; found, 378.16239.
17
18
3
7
1
H NMR 400 MHz, DMSO-d : δ 11.64 (s, 1H), 11.37 (s, 1H), 8.60 (s,
6
1
.47 (d, J = 7.0 Hz, 3H). HPLC purity: 94.6%.
1
1
H), 8.28 (s, 1H), 8.10 (dd, J = 2.2, 8.8 Hz, 1H), 7.50 (s, 1H), 6.77 (s,
H), 3.36 (dd, J = 2.6, 13.1 Hz, 4H), 2.45 (s, 4H), and 2.23 (s, 3H).
2
-((5-Bromopyridin-2-yl)amino)-1H-benzo[d]imidazol-6-yl)(4-
methylpiperazin-1-yl)methanone (16). HRMS calculated for
HPLC 99.0%.
+
+
1
[
C H BrN O] 415.0882 [M + H] ; found, 415.0876. H NMR
18 19 6
N-(5-Bromopyridin-2-yl)-4-chloro-6-(4-methylpiperazin-1-yl)-1H-
benzo[d]imidazol-2-amine (8). Compound 8 was prepared by using a
synthetic route similar to that described earlier. HRMS calculated for
4
00 MHz, DMSO-d : δ 11.97 (s, 1H), 10.87 (s, 1H), 8.38 (d, J = 2.2
6
Hz, 1H), 7.96 (dd, J = 2.5, 8.8 Hz, 1H), 7.33−7.53 (m, 3H), 7.10 (s,
1H), 3.54 (s, 4H), 2.40 (s, 4H), and 2.25 (s, 3H). HPLC purity:
+
+
1
[
C H BrClN ] 421.05431 [M + H] ; found, 421.05341. H NMR
17 18 6
9
8.13%.
4
7
00 MHz, DMSO-d : δ 11.85 (s, 1H), 10.94 (s, 1H), 8.34 (s, 1H),
.92−7.94 (m, 1H), 7.18−7.20 (m, 1H), 7.00 (s, 1H), 6.83 (d, J = 2.0
6
6
-((4-Methylpiperazin-1-yl)-N-(5-trifluoromethyl) pyridine-2-yl)-
1
H-benzo[d]imidazol-2-amine (17). HRMS calculated for
Hz, 1H), 3.08 (m, 4H), 2.50 (m, 4H), and 2.23 (s, 3H). HPLC purity
+
+
1
[
C H F N ] 391.18581 [M + H] ; found, 391.18625. H NMR:
19 21 3 6
98%.
4
00 MHz, DMSO-d : δ 8.70 (s, 1H), 8.22 (d, J = 8.0 Hz, 1H), 7.57 (d,
6
N-(5-Bromopyridin-2-yl)-6-fluoro-5-(4-methylpiperazin-1-yl)-3H-
imidazo[4,5-b]pyridin-2-amine (9). Compound 9 was prepared by
J = 7.6 Hz, 2H), 7.44 (d, J = 8.4 Hz, 1H), 7.27 (d, J = 8.0 Hz, 1H),
3
.45 (s, 2H), 3.38−3.44 (m, 4H), 3.08−3.17 (m, 4H), and 2.50 (s,
using a synthetic route similar to that described earlier. HRMS
+
+
3H). HPLC purity; 98.43%.
calculated for [C H BrFN ] 406.07911 [M + H] ; found,
1
6
17
7
1
2-(4-(2-((5-(Trifluoromethyl)pyridin-2-yl)amino)-1H-benzo[d]-
4
06.07849. H NMR (400 MHz, DMSO-d ) δ 9.99 (s, 1H), 8.51 (d,
6
imidazol-6-yl)piperazin-1-yl)ethan-1-ol (18). HRMS calculated for
J = 2.1 Hz, 1H), 8.12 (dd, J = 2.4, 8.8 Hz, 1H), 7.87 (d, J = 11.6 Hz,
+
+
1
[
C H F N O] 407.18072 [M + H] ; found, 407.18021. H NMR
19 21 3 6
1
2
H), 7.24 (d, J = 8.7 Hz, 1H), 3.95−3.98 (m, 2H), 3.55−3.57 (m,
H), 3.22−3.24 (m, 4H), and 2.88 (s, 3H). HPLC purity 99.51%.
N-(5-Bromopyridin-2-yl)-6-(piperazin-1-yl)-1H-benzo[d]imidazol-
-amine Hydrochloride (10). Compound 10 was prepared by using a
4
8
1
3
9
00 MHz, DMSO-d : δ 11.59 (s, 1H), 10.80 (s, 1H), 8.58 (s, 1H),
6
.04 (t, J = 1.9 Hz, 1H), 7.45 (d, J = 8.1 Hz, 1H), 7.27 (d, J = 2.0 Hz,
H), 6.98 (s, 1H), 6.79 (t, J = 2.0 Hz, 1H), 4.48 (d, J = 7.1 Hz, 1H),
.55 (d, J = 4.8 Hz, 2H), 3.08 (s, 4H), and 2.62 (s, 4H). HPLC purity:
5.94%.
2
synthetic route similar to that described earlier. HRMS calculated for
+
+
1
[
C H BrN ] 373.07764 [M + H] ; found, 373.07743. H NMR
16 17 6
N-(5-Fluoro-6-methylpyridin-2-yl)-6-(4-methylpiperazin-1-yl)-1H-
(
400 MHz, DMSO-d ) δ 13.21 (bs, 1H), 13.04 (bs, 1H), 9.36 (bs,
6
benzo[d]imidazol-2-amine (19). Compound 19 was prepared by
using a synthetic route similar to that described for compound 1.
HRMS calculated for [C H FN ] 341.18900 [M + H] ; found,
2
H), 8.52 (d, J = 2.4 Hz, 1H), 8.14 (dd, J = 2.4, 8.7 Hz, 1H), 7.55 (d, J
8.8 Hz, 1H), 7.38 (d, J = 8.7 Hz, 1H), 7.21 (d, J = 1.9 Hz, 1H), 7.11
dd, J = 2.1, 8.8 Hz, 1H), 3.37−3.39 (m, 4H), and 3.25−3.27 (m, 4H).
HPLC 96.81%.
=
(
+
+
18
21
6
1
3
1
6
41.18815. H NMR 400 MHz, DMSO-d : δ 11.33 (s, 1H), 10.33 (s,
H), 7.59 (t, J = 9.0 Hz, 1H), 7.30 (s, 2H), 6.91 (d, J = 2.2 Hz, 1H),
.74 (d, J = 6.1 Hz, 1H), 3.06 (s, 4H), 2.51 (s, 4H), and 2.23 (s, 3H).
6
6
-Morpholino-N-(5-(trifluoromethyl)pyridin-2-yl)-1H-benzo[d]-
imidazol-2-amine (11). Compound 11 was prepared by using a
synthetic route similar to that described earlier. HRMS calculated for
HPLC purity: 99.63%.
N-(5-Chloro-6-methylpyridin-2-yl)-6-(4-methylpiperazin-1-yl)-1H-
imidazo[4,5-c]pyridin-2-amine (20). Compound 20 was prepared by
using a synthetic route similar to that described for compound 1.
+
+
1
[
C H BrN O] 374.06165 [M + H] ; found, 374.06114. H NMR
16 16 5
4
00 MHz, DMSO-d : δ 11.56 (s, 1H), 10.60 (s, 1H), 8.32 (d, J = 2.4
6
+
+
Hz, 1H), 7.90 (q, J = 2.4 Hz, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.26 (d, J
8.4 Hz, 1H), 6.97 (s, 1H), 6.76 (dd, J = 2.0, 8.4 Hz, 1H), 3.75 (t, J =
HRMS calculated for [C H ClN ] 358.15470 [M + H] ; found,
17
20
7
1
=
3
58.15022. H NMR (300 MHz, DMSO-d ) δ ppm 2.23 (s, 3 H) 2.44
6
4.8 Hz, 4H), and 3.03 (t, J = 4.8 Hz, 4H). HPLC 99.07%.
(
7
br. s., 4 H) 2.61 (br. s., 3 H) 6.55−6.98 (m, 1 H) 7.07−7.49 (m, 1 H)
Compounds 12−18 and 21 were synthesized by alternate routes.
The detailed synthetic scheme for each compound is provided in the
Supporting Information.
.66−7.90 (m, 1 H) 8.08−8.42 (m, 1 H) 10.47−11.05 (m, 1 H)
11.32−11.70 (m, 1 H). HPLC purity: 98%.
N-(3-Chloro-5-(trifluoromethyl)pyridin-2-yl)-6-(1-methylpiperi-
din-4-yl)-1H-benzo[d]imidazol-2-amine (21). HRMS calculated for
6
-(1-Methylpiperidin-4-yl)-N-(5-(trifluoromethyl)pyridin-2-yl)-1H-
+
+
+
1
benzo[d]imidazol-2-amine (12). HRMS for C H F N [M + H] :
3
(
7
[C H ClF N ] 410.13594 [M + H] ; found, 410.13504. H NMR
19
20
3
5
19 19 3 5
1
76.17666. H NMR: 400 MHz. MeOD: δ 8.67 (s, 1H), 7.95−7.98
(300 MHz, DMSO-d ) δ ppm 1.55−1.87 (m, 4 H) 1.91−2.08 (m, 2
6
m, 1H), 7.37−7.41 (m, 2H), 7.18 (dd, J = 2.1, 8.0 Hz, 1H), 7.06−
H) 2.14−2.26 (m, 3 H) 2.82−2.95 (m, 2 H) 7.01−7.07 (m, 1 H)
7.15−7.20 (m,1 H) 7.22−7.29 (m, 1 H) 7.92−8.12 (m, 1 H) 8.33−
8.53 (m, 1 H) 12.08−12.47 (m, 2 H). HPLC purity: 98%.
.09 (m, 1H), 3.15−3.18 (m, 2H), 2.68−2.76 (m, 4H), 2.49 (s, 3H),
.41−2.46 (m, 1H), and 1.93−1.99 (m, 4H). HPLC purity: 98%.
2
6
-(1-Methylpyrrolidin-3-yl)-N-(5-(trifluoromethyl)pyridin-2-yl)-
N-(4-Cyclopropyl-5-fluoro-6-methylpyridin-2-yl)-6-(4-methylpi-
perazin-1-yl)-1H-imidazo[4,5-c]pyridin-2-amine (22). Compound 22
was prepared by using a synthetic route similar to that described for
1
[
H-benzo[d]imidazol-2-amine (13). HRMS calculated for
+
+
1
C H F N ] 362.15926 [M + H] ; found, 362.15897. H NMR
18 18 3 5
+
(
8
(
400 MHz), MeOD: δ 8.66 (s, 1H), 7.94−7.97 (m, 1H), 7.40 (d, J =
.0 Hz, 2H), 7.16 (m, 1H), 7.10 (t, J = 1.6 Hz, 1H), 3.67 (s, 1H), 3.55
t, J = 8.8 Hz, 1H), 3.20 (t, J = 8.4 Hz, 1H), 2.97−2.99 (m, 1H), 2.78−
compound 1. HRMS calculated for [C H FN ] 382.21555 [M +
20
24
7
+
1
H] ; found, 382.21254. H NMR (300 MHz, DMSO-d ) δ ppm 0.69−
0.83 (m, 2 H) 1.06−1.17 (m, 2 H) 2.01−2.14 (m, 1 H) 2.17−2.26 (m,
6
6
650
dx.doi.org/10.1021/jm500715u | J. Med. Chem. 2014, 57, 6642−6652

Journal of Medicinal Chemistry
Article
3
8
H) 2.43 (br. s., 4 H) 6.56−6.73 (m, 1H) 6.73−6.95 (m, 1 H) 8.10−
.35 (m, 1 H) 10.16−10.74 (m, 1 H) 11.40−11.70 (m, 1 H). HPLC
pool of reductase or diaphorase enxymes derived from the
mitochondria and cytosol. Therefore, Alamar blue can be used as an
oxidation−reduction indicator in cell viability assays for mammalian
purity: 99%.
1
8
N-(4-Cyclopropyl-5-fluoro-6-methylpyridin-2-yl)-6-(1-methylpi-
peridin-4-yl)-1H-benzo[d]imidazol-2-amine (23). Compound 23 was
prepared by using a synthetic route similar to that described for
compound 1. HRMS calculated for [C H FN ] 380.22505 [M +
H] ; found, 380.22443. H NMR (300 MHz, DMSO-d ) δ ppm 0.72−
cells. THP-1 cells were seeded at 40,000/well in growth medium (95
μL) and 5 μL of compound added immediately at the indicated
concentrations. Five microliters of solvent (5% (v/v) DMSO in
growth medium) was used in the control wells giving a final 0.5% v/v
DMSO. All stock compounds in 50 mM neat DMSO were diluted in
growth medium to 10% (v/v) DMSO and 5 μL used in the assay,
giving a final 0.5% v/v DMSO (final assay volume 100 μL).
Menadione, the standard cytotoxic agent, was used in each experiment
for quality control at a top test concentration of 125 μM. Ninety-six-
well plates containing THP-1 cells with compounds or solvent were
incubated for 24 h under standard cell culture conditions. Eleven
microliters of stock resazurin solution (450 μM; 11.3 mg dissolved in
100 mL of PBS) was added to all wells, mixed, and incubated under
standard cell culture conditions for a further 2 h. Plates were read on
an Envision reader using an excitation λ of 560 nm and emission λ of
590 nm. Data is normalized to the median of the plate controls.
Concentration−effect curves are fitted to the primary (fluorescent)
signal and IC50 values calculated.
+
22
26
5
+
1
6
0.80 (m, 2 H) 1.04−1.15 (m, 2 H) 1.61−1.80 (m, 4 H) 1.91−2.02 (m,
2 H) 2.02−2.10 (m, 1 H) 2.15−2.25 (m,3 H) 2.81−2.95 (m, 2 H)
6.66−6.80 (m, 1 H) 6.82−6.98 (m, 1 H) 7.06−7.46 (m, 2 H) 10.00−
10.34 (m, 1 H) 11.37−11.66 (m, 1 H). HPLC purity: 97%.
Bioactivation Analysis. Chemicals. HLMs were obtained from
BD Biosciences (Singapore). Reduced L-glutathione (GSH), β-
nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium
salt (β-NADPH), formic acid (ultra pure), N-acetyl cysteine (NAC),
dimethyl sulfoxide, and 1-aminobenzotriazole (ABT) were purchased
from Sigma-Aldrich (Bangalore, India). All compounds used in this
article were synthesized internally. Methanol, water, and acetonitrile
were HPLC grade from Thermo Fisher Scientific India (Mumbai,
India). All the other solvents were HPLC grade, and unless otherwise
specified, all of the other reagents were purchased from Sigma-Aldrich.
In Vitro Incubations for the Reactive Metabolite Screen. Each
compound was incubated at a concentration of 10 μM with HLM/
RLM (1 mg/mL protein) in 100 mM phosphate buffer (pH 7.4) in the
presence of NADPH (2 mM). In order to assess the ability of the
microsomes to bioactivate the compounds by trapping the reactive
intermediates, some incubations also contained either GSH or NAC (2
mM). Incubations were performed for 60 min (t ) in a shaking
ASSOCIATED CONTENT
Supporting Information
■
*
S
Details of the synthesis of all compounds and Pf drug resistant
strains. This material is available free of charge via the Internet
at http://pubs.acs.org.
60
incubator at 37 °C. The microsomal reaction was terminated by the
addition of an equal volume of ice-cold acetonitrile, vortexed, and
centrifuged at 13000 rpm for 10 min. The supernatant was transferred
for HPLC-UV/MS analysis. Control incubation samples (500 μL)
AUTHOR INFORMATION
Corresponding Authors
(V.H.) Phone: +1 781-386-7725. E-mail: vinayak.
hosagrahara@astrazeneca.com.
(V.K.S.) Phone: +91-9740089874. E-mail: vasan2005@gmail.
■
*
were quenched at time = 0 (t ) (prior to the addition of NADPH)
0
*
with an equal volume of ice-cold acetonitrile.
LC-MS/MS Analysis. The LC was an Agilent 1100 series system
consisting of an autosampler, vacuum degasser, binary pump system,
column oven, and PDA detector. The metabolites were resolved with a
Phenomenex Gemini C18 column (25 × 0.46 cm, 5-μm particle size).
The mobile phase consisted of a combination of acetonitrile
containing formic acid (0.05%, v/v) and 10 mM ammonium formate
buffer. A sample aliquot of 40 μL was injected onto the column and
eluted at 1 mL/min with a gradient starting at 5% ACN, followed by 5
to 45% ACN from 5 to 30 min and maintaining 45% ACN for 35 min,
and finally returning to 5% ACN in next 5 min. The UV data was
collected for a scan range of 220−320 nm using an in-line Agilent PDA
detector. The HPLC eluent was directly coupled to an Agilent 6520
Q-TOF mass spectrometer with 500 μL/min eluate split-flow to the
LC-MS interface. The instrument was operated in electrospray
ionization (ESI) mode with positive polarity. Nitrogen was used as
collision gas. Capillary voltage was +4000. Drying gas was set at 350
com.
Author Contributions
S.H.P. and A.S. contributed equally to this work.
¶
S.R., S.H.P., and A.R. were responsible for medicinal
chemistry design and analysis. A.S. and V.H. were responsible
for the bioactivation study design and interpretation. S.R. and
G.S. were responsible for synthetic chemistry. S.M. performed
the preparative HPLC. V.A. was responsible for dispensing the
compounds for various biochemical and DMPK assays. N.R.,
D.A., S.M., P.V., S.B., S.R., J.R., V.P., K.R.P., R. N., and V.K.S.
were responsible for biological profiling of compounds in
various assays. S.D. and V.M.A. were responsible for the high
throughput screening and analysis of the AstraZeneca library.
M.C. L. and D. A. F. generated the cross-resistance data with
the PI4K or NITD609-resistant mutant clones. B.J. and M.S.M.
were responsible for generating the efficacy data in the Pf/
SCID model and assessing the kill rates in the in vitro PRR
assay. A.S., S.H.P, S.R., and V.K.S. wrote the manuscript with
comments from all coauthors.
°
C with a flow rate of 13 L/min. Mass analysis was performed in auto-
MSMS mode with mass range (m/z) 100−800 in full scan and mass
range (m/z) 40−800 in MSMS mode.
In Vitro ADME and Pharmacokinetic Studies. All studies were
7
performed as described earlier.
In Vivo P. berghei Efficacy Model. Efficacy studies in the surrogate
13
Notes
P. berghei model was performed as described earlier.
P. falciparum Humanized Mouse Efficacy Model. The therapeutic
The authors declare no competing financial interest.
0
087/N9
efficacy of 2 against P. falciparum Pf 3D7
was studied using a “4-
1
4
ACKNOWLEDGMENTS
day test” protocol as described earlier.
Pharmacokinetic Analysis. The levels of 2 were evaluated in whole
■
We thank the Medicines for Malaria Venture (MMV) for their
financial support of this project (MMV 09/5400). Our heartfelt
thanks are given to Dr. Simon Campbell and Dr. Steve Ward
for their scientific advice during the course of this project. Our
special thanks go to Rajkumar Thimmaiah and Subhash
Rajanna for their help with the in vivo efficacy studies. Special
thanks go to Syngene International, Bangalore for their support
in synthetic chemistry.
1
4
blood using a protocol described earlier.
In Vitro Parasite Reduction Ratio (PRR) Assay. This kill rate of
1
7
compound 2 was assessed using a protocol described earlier.
Assessment of Cytotoxicity against a THP-1 Cell Line. The THP-1
cytotoxicity screen is based upon the determination of the fluorescent
signal generated by the reduction of nonfluorescent resazurin (7-
hydroxy-3H-phenoxazin-3-one 10-oxide) to the fluorescent resorufin
(
Alamar blue assay). Cellular reduction of resazurin is dependent on a
6
651
dx.doi.org/10.1021/jm500715u | J. Med. Chem. 2014, 57, 6642−6652

Journal of Medicinal Chemistry
Article
(
13) Chawira, A. N.; Warhurst, D. C.; Robinson, B. L.; Peters, W.
ABBREVIATIONS USED
■
The effect of combinations of qinghaosu (artemisinin) with standard
antimalarial drugs in the suppressive treatment of malaria in mice.
Trans. R. Soc. Trop. Med. Hyg. 1987, 81, 554−558.
Pf, Plasmodium falciparum; SAR, structure−activity relation-
ship; PRR, parasite reduction ratio; SCID, severe combined
immunodeficiency; PK, pharmacokinetics; GSH, glutathione;
NAC, N-acetyl cysteine
(
14) Angulo-Barturen, I.; Jimenez-Díaz, M. B.; Mulet, T.; Rullas, J.;
ez, E.; Mendoza, A.; Regadera, J.;
́
Herreros, E.; Ferrer, S.; Jimen
́
́
Rosenthal, P. J.; Bathurst, I.; Pompliano, D. L.; Gomez de las Heras, F.;
Gargallo-Viola, D. A murine model of falciparum-malaria by in vivo
selection of competent strains in non-myelodepleted mice engrafted
with human erythrocytes. PLoS One 2008, 3, e2252.
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