Aminoazabenzimidazoles, a Novel Class of Orally Active Antimalarial Agents

Article abstract of DOI:10.1021/jm500535j

Whole-cell high-throughput screening of the AstraZeneca compound library against the asexual blood stage of Plasmodium falciparum (Pf) led to the identification of amino imidazoles, a robust starting point for initiating a hit-to-lead medicinal chemistry effort. Structure-activity relationship studies followed by pharmacokinetics optimization resulted in the identification of 23 as an attractive lead with good oral bioavailability. Compound 23 was found to be efficacious (ED₉₀ of 28.6 mg·kg^-1) in the humanized P. falciparum mouse model of malaria (Pf/SCID model). Representative compounds displayed a moderate to fast killing profile that is comparable to that of chloroquine. This series demonstrates no cross-resistance against a panel of Pf strains with mutations to known antimalarial drugs, thereby suggesting a novel mechanism of action for this chemical class.

Full text of DOI:10.1021/jm500535j

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Article
Amino Azabenzimidazoles, a Novel Class of Orally Active Antimalarial agents
Shahul Hameed P, Murugan Chinnapattu, Gajanan Shanbag, Praveena Manjrekar, Krishna Koushik,
Anandkumar Raichurkar, Vikas Patil, Sandesh Jatheendranath, Suresh Rudrapatna, Shubhada
Barde, Nikhil Rautela, Disha Awasthy, Sapna Morayya, Chandan Narayan, Stefan Kavanagh,
Ramanatha Saralaya, Sowmya Bharath, Pavithra Viswanath, Kakoli Mukherjee, Balachandra
Bandodkar, Abhishek Srivastava, Vijender Panduga, Jitender Reddy, K R Prabhakar, Achyut
Sinha, Maria Belén Jimenez-Díaz, María Santos Martinez, Inigo Angulo-Barturen, Santiago Ferrer,
Laura María Sanz, Francisco-Javier Gamo, Sandra Duffy, Vicky M. Avery, Pamela A Magistrado,
Amanda K Lukens, Dyann F Wirth, David Waterson, V Balasubramanian, Pravin S Iyer, Shridhar
Narayanan, Vinayak Hosagrahara, Vasan K. Sambandamurthy, and Sreekanth Ramachandran
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 10 Jun 2014
Downloaded from http://pubs.acs.org on June 13, 2014
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Lukens, Amanda; Harvard School of Public Health,
Wirth, Dyann; Harvard School of Public Health,
Waterson, David; Medicines for Malaria Venture,
Balasubramanian, V; AstraZeneca India Pvt Ltd, Bioscience
Iyer, Pravin; AstraZeneca India Pvt Ltd, Medicinal Chemistry
Narayanan, Shridhar; AstraZeneca India Pvt Ltd, Bioscience
Hosagrahara, Vinayak; AstraZeneca Pharmaceuticals, Drug Metabolism and
Pharmacokinetics
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Sambandamurthy, Vasan; AstraZeneca, Bioscience
Ramachandran, Sreekanth; AstraZeneca India Pvt Ltd, Medicinal Chemistry
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Amino Azabenzimidazoles, a Novel Class of Orally
Active Antimalarial agents
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Shahul Hameed P.†, Murugan Chinnapattu†, Gajanan Shanbag†, Praveena Manjrekar†,
Krishna Koushik†, Anandkumar Raichurkar†, Vikas Patil†, Sandesh Jatheendranath† Suresh
Rudrapatna†, Shubhada Barde†, Nikhil Rautela‡, Disha Awasthy‡, Sapna Morayya‡, Chandan
Narayan‡, Stefan KavanaghΩ, Ramanatha Saralaya§, Sowmya Bharath§, Pavithra
Viswanath‡, Kakoli Mukherjee‡, Balachandra Bandodkar†, Abhishek Srivastava§, Vijender
Panduga§, Jitender Reddy§, KR Prabhakar§, Achyut Sinha‡, María Belén JiménezꢀDíaz±,
María Santos Martínez±, Iñigo AnguloꢀBarturen±, Santiago Ferrer±, Laura María Sanz±,
Francisco Javier Gamo±, Sandra Duffy¥, Vicky M Avery¥, Pamela A. Magistrado∞, Amanda
K. Lukens∞, Dyann F. Wirth∞, David Waterson£, V. Balasubramanian†, Pravin S. Iyer†,
Shridhar Narayanan‡, Vinayak Hosagrahara§, Vasan K. Sambandamurthy‡*, Sreekanth
Ramachandran†*
†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 PréꢁBois 20, Post Office Box 1826, 1215
Geneva, Switzerland; ¥ Discovery Biology, Eskitis Institute for Drug Discovery, Griffith
University, Queensland 4111, Australia; ± Tres Cantos Medicines Development Campus.
Diseases of developing Word (DDW), GlaxoSmithKline, Severo Ochoa 2, Tres Cantos
28760, Madrid, Spain; ∞Harvard School of Public Health, Boston, MA02115.
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KEYWORDS: Malaria, cross resistance, SCID model, structure activity relationship,
pharmacokinetics, efficacy
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Abstract
Whole cell high throughput screening of the AstraZeneca compound library against the
asexual blood stage of Plasmodium falciparum (Pf), led to the identification of amino
imidazoles, a robust starting point for initiating a hitꢁtoꢁlead medicinal chemistry effort.
Structureꢁactivity relationship studies followed by pharmacokinetics optimization resulted in
the identification of 23 as an attractive lead with good oral bioavailability. Compound 23
was found to be efficacious (ED90 of 28.6 mg/kg) in the humanized P. falciparum mouse
model of malaria (Pf/SCID model). Representative compounds displayed a moderate to fast
killing profile that is comparable to chloroquine. This series demonstrates no crossꢁresistance
against a panel of Pf strains with mutations to known antimalarial drugs, thereby suggesting a
novel mechanism of action for this chemical class.
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Introduction
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Malaria continues to be a major global public health challenge with an estimated 627, 000
deaths in 2012.1 The African subcontinent had the highest incidence (80%) and death toll
(90%) from malaria, and most deaths (77%) occurred in children under 5 years of age.1
Among the five distinct Plasmodium species that cause malaria, Plasmodium falciparum (Pf)
and Plasmodium vivax account for more than 95% of clinical cases and deaths due to this
disease. The rapid emergence and spread of malarial parasites resistant to currently available
antimalarial drugs, including artemisinin derivatives, threaten to derail the global efforts to
treat and prevent malaria.2 Globally, with an estimated 3.4 billion people being at risk of
contracting malaria, there is an urgent need for the discovery and development of novel and
chemically distinct classes of compounds leading to effective and affordable drugs against
malaria.3
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Over the last decade, whole cell screening efforts have led to greater success in delivering
new molecular entities approved by the FDA compared to classical targetꢁbased approaches.4
A number of novel chemical entities currently under preclinical and clinical development
against protozoan parasites have emerged from whole cell screening efforts.5 ꢁ 8 In particular,
phenotypic highꢁthroughput screening campaigns have resulted in the identification of novel
chemotypes active against the erythrocytic stage of Pf parasites, which is primarily
responsible for the clinical manifestations of malaria.9 ꢁ 12 A phenotypic high throughput
screen of 500,000 compounds from the AstraZeneca corporate library, against the asexual
blood stage of Pf, was carried out using a high content imageꢁbased approach previously
reported.13 The screening cascade began with singleꢁpoint determination of growth inhibition
at 1.6 µM, followed by a dose response study of actives (compounds displaying >50%
growth inhibition). A hit triage exercise by applying filters for leadꢁlike properties such as
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molecular weight (<500), lipophilicity, number of hydrogen bond donors/acceptors led to the
identification of several attractive starting points.
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Results and Discussion
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Antiplasmodial Activity and Structure−Activity Relationship (SAR). Following the hit
triage, the 2ꢁamino imidazole 1 was selected as a promising hit for structure activity
exploration. This prioritized scaffold consists of a 2ꢁamino imidazole core with an Nꢁlinked
phenylethyl tether (Fig 1). Analysis of the activities of close analogs from the corporate
collection screened revealed that the primary amine and the benzylic hydroxyl (1, 4ꢁamino
alcohol motif) groups were critical for the antimalarial activity. Hence, the initial SAR
exploration in the synthetic program focused on identifying the best substituents on the
phenyl ring for antimalarial potency while monitoring two selectivity readouts, i.e.,
cytotoxicity toward A549 mammalian cell line14 and effect on the hERG ion channel (Table
1). All compounds were screened for their antimalarial activity against a sensitive (NF54) and
multidrug resistant (K1) strain of Pf using a SYBRꢁGreen based readout.15,16 Chloroquine,
pyrimethamine, and artesunate were included as reference drugs in all of the experiments.
A chloro substitution at R2 position of the phenyl ring was found to be critical with 2,3ꢁ
dichloro and 2,4ꢁdichloro being the best for antiplasmodial activity of (1 – 4 in Table 1). On
the imidazole ring, hydrophobic groups at R and R1 were well tolerated (5 ꢁ 8). These
compounds were not cytotoxic (displayed good selectivity index of >50), and a majority of
them were weakly active against the hERG ion channel (hERG IC50 > 22µM).
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Fig 1: 2ꢁamino imidazole
Compound
R
H
NF54
Cytotoxicity
index (A 549)
hERG
R1
H
R2
Cl
R3
R4
K1 IC50
(ꢀM)
IC50 (ꢀM)
IC50 (ꢀM)
NDa
60
NDa
22
>33.3
>33.3
4.5
H
Cl
0.38
0.28
>263
1
H
H
H
H
F
Me
H
H
Cl
Cl
>2
>2
>2
>2
>50
>50
2
3
H
Me
Me
H
H
Me
H
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
Cl
Cl
Cl
0.35
0.38
0.3
0.12
0.14
0.23
0.25
0.2
0.07
0.06
>285
131
220
241
72
4
5
6
7
8
Cyclopropyl
4ꢁ
Fluorophenyl
H
25
CF3
Table 1: In vitro antiplasmodial activity, cytotoxicity and hERG inhibition profile of
compounds 1 – 8. Selectivity index against A549 (human lung carcinoma cell line) was
calculated by dividing the Pf IC50 by the IC50 generated in the A549 cytotoxicity assay. NDa:
not determined
Fig 2: Structure activity features of amino substituted azabenzimidazole
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Having established that 4, 5ꢁ lipophilic group substitution on the imidazole ring was
tolerated, further SAR explorations were continued with the 2ꢁamino benzimidazoles Fig 2.
Unsubstituted 2ꢁamino benzimidazole 9 (Table 2) was one of the best compounds in the
series with a potency of 28 nM against the sensitive Pf NF54 strain. However, compound 9
had a high clearance and was rapidly metabolized in mouse microsomal and rat hepatocytes,
while moderate clearance was observed in human microsomes. This is likely to be due to the
hydroxylation of electronꢁrich benzimidazoles followed by phase II elimination. Hence,
medicinal chemistry efforts were directed towards blocking the metabolic soft spots in
molecules to address their high metabolic clearance.
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Compound
R
X
Y
NF54
Cytotoxicity
Rat Hep
Hu mics
Mu mics
hERG
R1
R2
R3
K1 IC50
(ꢀM)
selectivity (ꢀl/min/1E6) (ꢀl/min/mg) (ꢀl/min/mg)
index (A549)
IC50 (ꢀM)
IC50 (ꢀM)
9
10
H
CN
C
C
C
C
Cl
Cl
H
Cl
Cl
H
0.028
0.65
0.007
0.3
607
0.86
155
53
25
96
213
NDa
NDa
NDa
62
10.3
>33.3
11
12
13
14
15
16
C
C
C
C
N
N
C
C
C
N
C
C
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
>2
0.32
0.053
>2
>2
>50
2.3
94
44
22
68
49
9
>33.3
>33.3
>10.5
NDa
>33.3
SO2Me
Cl
0.04
0.01
>0.5
0.18
0.085
31
CF3
H
C3
Cl
H
H
>25
26
14
5
41
NDa
102
H
Cl
H
0.34
0.089
110
106
69
21
348
12.2
CF3
Cl
N
N
17
N
C
Cl
H
0.04
0.014
375
198
17
87
3.5
NDa
NDa
18
19
N
N
C
C
Cl
Cl
H
H
Cl
Cl
0.096
>2
0.022
>0.5
>260
>10
94
47
>130
NDa
NDa
NDa
20
N
C
Cl
H
0.043
0.01
232
36
45
21
>33.3
CF3
21
22
N
N
C
C
Cl
Cl
H
H
0.025
>2
0.018
>0.5
800
>50
105
14
76
<4
90
NDa
19
16.6
CF3
CF3
>33.3
23
N
C
Cl
H
0.04
0.005
>325
43
32
>33.3
CF3
Table 2: Inꢀvitro antiplasmodial activity, cytotoxicity and hERG inhibition profile of
compounds 9 – 23. Selectivity index against A549 (human lung carcinoma cell line) was
calculated by dividing the Pf IC50 by the IC50 generated in the A549 cytotoxicity assay.
NDa: not determined
The 5ꢁsubstituted 2ꢁaminobenzimidazoles 10 ꢁ 13 showed good microsomal stability with 13
being the best in terms of stability and potency. The introduction of nitrogen at 6ꢁposition of
benzimidazole 14 seemed unfavorable for antiplasmodial potency, while the nitrogen at 4ꢁ
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position 15 was tolerated. Since 5ꢁsubstitution appeared to provide the potential for metabolic
stability, other functional groups at this position were explored as follows. The Nꢁlinked
compounds 16 – 17 and the Cꢁlinked compound 18 retained antiplasmodial activity whereas
the amide 19 turned out to be inactive. Finally, the Oꢁlinked compounds 20 – 23 provided the
balance of potency and microsomal stability with 23 being the best with adequate potency (Pf
IC50 0.04µM) and metabolic stability in different species. The compound 23 also showed
good cytotoxicity selectivity index (>325ꢁfold) and was inactive in the hERG inhibition assay
(IC50 >33 µM).
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Having developed the SAR and the essentiality of the 1,4ꢁamino alcohol moiety for
antiplasmodial activity, we undertook a limited scaffold hopping strategy to find an alternate
heterocyclic core by synthesizing compounds 24 ꢁ 28 (Table 3). While the 2ꢁamino triazole
24 and 5ꢁamino tetrazole 25 were inactive, the 2ꢁamino pyridine 26, the phenolic linked 2ꢁ
aminobenzimidazole 27 and the 2ꢁamino quinazoline 28 retained antiplasmodial activity.
These diverse heterocyclic cores could provide an alternate avenue for multiple hit to lead
campaigns for these 1, 4ꢁamino alcohol inhibitors.
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Compound
Structure
Cytotoxicity selectivity
NF54 IC50 (ꢀM) K1 IC50 (ꢀM)
index (A549)
5
>2
>2
>50
24
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>2
>2
>50
294
25
26
0.18
0.05
0.084
0.177
0.054
0.025
1190
141
27
28
N
N
NH2
OH
Table 3: Scaffold hopping of 1,4ꢁamino alcohols to generate compounds 24 – 28 and their
antiplasmodial, cytotoxicity and hERG activity. Selectivity index against A549 (human lung
carcinoma cell line) was calculated by dividing the Pf IC50 by the IC50 generated in the A549
cytotoxicity assay.
Chemistry: The 2ꢁamino imidazoles 1 – 4 were synthesized by the base promoted Nꢁ
alkylation of 2ꢁnitro imidazoles with phenacyl bromides, followed by the sequential reduction
of keto and the nitro group as depicted in Scheme 1. Compounds 5 – 8 were synthesized by
alternate routes (see supporting information).
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Scheme 1: General synthetic scheme for 2ꢁamino imidazoles
The general synthesis of 5ꢁsubstitutedꢁ4ꢁazabenzimidazoles 16 ꢁ 17 and 20 ꢁ 23 is shown in
Scheme 2. Initial displacement of 6ꢁchloro of 6ꢁchloroꢁ3ꢁnitropyridinꢁ2ꢁamine with various
alcohols and amines under basic condition yielded the 6ꢁsubstituted 3ꢁnitropyridineꢁ2ꢁamines
(16a). This was then subjected to reduction under Pd/C to provide the diamines (16b), which
was then cyclized to 2ꢁamino benzimidazoles (16c) using cyanogen bromide. Alkylation of
16c with phenacyl bromides using K2CO3 provided the Nꢁsubstituted ketoamines 16d which
were reduced with NaBH4 to the corresponding 1, 4ꢁamino alcohols 16 ꢁ 17, 20 – 23.
Compounds 9 ꢁ 15, 18, 19, 24 – 28 were synthesized by alternate routes (see supporting
information).
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Scheme 2: General scheme for the synthesis of 5ꢁsubstitutedꢁ4ꢁ azabenzimidazoles
In vitro and in vivo DMPK profile. Representative compounds were profiled for various in
vitro DMPK properties, and CYP inhibition (Table 4).
CYP IC50 (ꢀM)
compound
Aq.
Solubility
(ꢀM)
LogD Hu PPB CYP3A4 CYP2D6 CYP2C9 CYP2C19 CYP1A2
(% ) free
3
>4.6
3
<1
>30
>30
26.3
>30
27.9
>30
2.7
29.4
5.2
13
15
26
11.8
10.2
118
160
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2.8
3.7
23.4
2.6
<1
14.1
2.4
27.9
12.5
28.3
>30
>30
22.9
5
>30
>30
>30
16
21
23
0.78
4.7
>4.5
20.3
Table 4: In vitro DMPK profile of representative compounds. Aqueous solubility, logD,
estimation of free fraction in human plasma and inhibition of CYP isozymes were performed
using protocols reported earlier.17
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There appears to be an inverse correlation between logD and aqueous solubility for this
series. A direct correlation between logD and extent of binding to human plasma proteins
was observed. The series showed low to moderate inhibition against a human CYP P450
panel (5 isoforms) (Table 4).
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In order to understand the pharmacokinetic behavior of this series, a study was performed in
Wistar rats with compound 23 being administered orally (5 mg/kg) and via the intravenous
route (1 mg/kg). The in vivo blood clearance following intravenous dosing was found to be
moderate clearance (23 mL/min/kg) and a large volume of distribution (2.5 L/kg).
Compound 23 showed excellent oral bioavailability (100%) (Table 5).
Rat PK
Compound
23
Intravenous (1 mg/kg)
1.76
t
1/2 (h)
AUC (ꢀM.h)
CL (ml/min/kg)
Vss (l/Kg)
1.66
22.6
2.49
Oral (5 mg/kg)
% F
100
7.16
t1/2 (h)
Cmax (ꢀM)
2.04
Table 5: Rat in vivo pharmacokinetic profile for compound 23. Two rats were used for each
of the dose groups. The reported values are the average PK parameters (n=2).
In vivo efficacy studies. Given the attractive antiplasmodial activity of this series against
NF54 and K1 strains of P. falciparum along with favorable pharmacokinetic properties, in
vivo proof of concept study in a murine P. berghei model of malaria18 was undertaken with
compound 23. Following four onceꢁdaily doses of 23 at 50 mg/kg, a 94% reduction in
parasitemia was observed. At this dose, 23 was not curative in this efficacy model (mean
survival time of 12 ± 2 days). Chloroquine dosed at 30mg/kg for four days resulted in a
99.9% reduction in parasitemia and mice survived for 27 days (mean survival time of 27 ± 3).
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This efficacy data provided the first in vivo proof of target engagement following oral
administration of an amino azabenzimidazole in a murine model of malaria. Based on the
encouraging efficacy obtained in the P. berghei model, we embarked on determining the
therapeutic efficacy of this compound in the highly relevant humanized Pf/SCID efficacy
model.19 Efficacy was assessed following administration of a single oral dose per day (3, 10,
30 and 50 mg·kgꢁ1) of compound 23 for four consecutive days, and by measuring the effect
on blood parasitemia by flow cytometry. Two parameters of efficacy were estimated in the
study – the ED90 and the exposure required for achieving an ED90 (AUCED90) (Figure 3a,
3b).
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A
B
Treatm ent
1 00 0 0
10 0 0
1 0 0
1 0
1
0
ED 9 0
ꢁ1
ꢁ2
0
3
10
30
50
mg/kg
IC50
1
0
1
2
3
4
5
6 7
0
6
12
18
24
Days postꢁinfection
H ours postꢁdo sage
C
Vehicle
50 m g/kg
SYT Oꢁ16
SYT Oꢁ16
Figure 3. Efficacy of compound 23 in a humanized P. falciparum mouse model.
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3a represents parasitemia in peripheral blood of mice infected with P. falciparum
Pf3D70087/N9 corresponds to individual parasitemia for mice treated with 23 (n=4, where each
individual was treated at different dose level) or vehicle (n=2). 3b represents the whole blood
levels of 23 after the first dose of treatment in the very same individuals of the efficacy
experiment. Data represents individual profiles of blood concentration versus time of 23 (0ꢁ
23 hours). The dotted line shows the in vitro IC50 in ng·mlꢁ1. 3c represents the phenotype of
parasites remaining in the peripheral blood of mice after 4 days of exposure to compound 23.
Under these experimental conditions, 23 was found to be efficacious against Pf, with an ED90
of 28.6 mg·kgꢁ1 and an estimated AUCED90 of 6.55 ꢂg·h·mlꢁ1·dayꢁ1.
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The therapeutic efficacy of 23 against P. falciparum 3D7 in a ‘4ꢁday test’ is shown in Figure
3a. The blood levels of 23 were measured on day 1 following oral administration of the
compound to P. falciparumꢁinfected NODꢁSCID ILꢁ2Rγnull mice. The experimental design
was optimized for analysis of efficacy parameters (ED90, AUCED90). The pharmacokinetic
parameters estimated following oral administration of 23 are shown in Table 6. The
compound blood concentration versus time data profiles are plotted in Figure 3b.
Cmax / Dose
tmax
AUC (0ꢁ23)
DNAUCa
(0ꢁ23)
(ꢀg/ml per mg/kg) (hours) (ꢀg.h/ml) (ꢀg.h/ml per mg/kg)
Dose (mg/kg) Cmax (ꢀg/ml)
3
0.6
0.019
0.035
0.022
0.021
0.5
2
6
0.48
2.7
6.93
10.45
0.16
0.9
2.31
3.48
10
30
50
0.36
0.68
1.07
4
aDNAUC, dose normalised value of AUC
Table 6. Pharmacokinetic parameters estimated in blood after oral gavage administration of
23 to Pf3D70087/N9ꢁinfected humanized SCID mice.
Nonꢁlinear fitting to a sigmoid doseꢁresponse curve of log10 of % parasitemia at day 7 after
infection versus the dose was used to estimate the ED90. Nonꢁlinear fitting to a sigmoid doseꢁ
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response curve of log10 of % parasitemia at day 7 after infection versus the area under the
curve of levels of compound obtained during the first 23 hours after the first administration
was used to estimate the potency of 23. The potency was expressed as the daily exposure that
reduced parasitemia from peripheral blood at day 7 after infection by 90% with respect to
vehicleꢁtreated mice (AUCED90) was 6.55 ꢂg·h·mlꢁ1·dayꢁ1. This parameter of efficacy was
estimated by assuming that the drug accumulation during treatment had no significant effects
on parasite reduction. Microscopic examination of Pf3D70087/N9ꢁinfected mice treated with
vehicle showed erythrocytes harboring healthy rings, trophozoites and mature schizonts. At
day 7 postꢁinfection, an accumulation of trophozoites were observed in mice treated with 23.
This data demonstrates the value of compound 23 in reducing the parasite burden following
infection with Pf.
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Amino alcohols retain their activity against P. falciparum strains with known mechanism
of resistance: The SAR was optimized based on the antiplasmodial activity of compounds
against the fully sensitive NF54 strain, and the chloroquine resistant K1 strain of P.
falciparum. To rule out crossꢁresistance to other antimalarial agents both current and under
development, representative compounds were screened against a panel of wellꢁcharacterized
strains of P. falciparum using the SYBR Green assay described earlier.15,16
As shown in Table 7, compounds 9, 13, 16, 20, 26, 27 retained their IC50 values across six
different Pf strains (see supporting information Table 1 for the Pf strain details) in contrast to
antimalarial agents currently in use. Chloroquine and pyrimethamine showed a large shift to
higher IC50’s against strains K1, 7G8, W2 and DD2. Atovaquone, a clinically used
antimalarial agent targeting mitochondrial metabolism showed a specific increase in IC50
against SB1 strain.
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IC50 (nM)
Compound
NF54
18.6 ± 11.6
29.2 ± 24.1
K1
7G8
W2
SB1
42.5 ± 12
121.3 ± 11
DD2
30 ± 7
48.7 ± 6
10.14 ± 6.06
12.21 ± 9.6
4.09 ± 2.3
3.25 ± 2.2
11.57 ± 0.1
24.1 ± 4
9
13
16
20
26
141.2 ± 123.9 220.48 ± 116.43 37.88 ± 35.35 156.04 ± 92.21 219.6 ± 42
96 ± 98.5 50.48 ± 39.34 12.04 ± 15.44 61.96 ± 39.5 177.8 ± 30
263.1 ± 289.6 109.03 ± 85.52 29.25 ± 39.66 110.49 ± 44.82 471.9 ± 39
100.3 ± 66.5 252.38 ± 174.03 66.07 ± 52.04 259.62 ± 190.81 163.6 ± 18
369.6 ± 45
134.8 ± 28
410.2 ± 12
328.9 ± 35
10.26 ± 2
251.34 ± 9
27
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23
24
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7.05 ± 4
17.09 ± 5
28.26 ± 11
1.36 ± 0.4
6.47 ± 2
347.29 ± 82
1.3 ± 0.1
163.91 ± 17
6.52 ± 1
374.66 ± 228 11.81 ± 4.5
16.60 ± 5
Artemisinin
Chloroquine
Pyrimethamine
Atovaquone
8558.42 ± 1983 10041.72 ± 1261 15379.7 ± 2826 10.85 ± 4 13838.59 ± 8619
2.26 ± 0.8 2.32 ± 0.7 1.16 ± 0.6 >2000 2 ± 0.02
Table 7. Amino azabenzimidazoles retain their potency against a panel of Pf strains known to
harbor mutations conferring resistance to known antimalarial agents. Data represent the IC50
values obtained from each compound tested in duplicate from two independent experiments.
To further eliminate overlapping mode of action with known targets, crossꢁresistance testing
against drug resistant lines with wellꢁcharacterized mutations in Pf dihydroorotate
dehydrogenase (PfDHODH)20, Pf heat shock protein 90 (PfHSP90) (personal communication
with U.E. Ribacke), Pf chloroquine resistance transporter (PfCRT)21 or Pf cytochrome b
reduction site (PfCTYbꢁQi) (personal communication with A.K. Lukens) was performed (see
supporting information for the Pf strain details). Mutations in these genes contribute to
resistance to antimalarial agents under development, namely 5ꢁbenzimidazolylꢁNꢁ
alkylthiopheneꢁ2ꢁcarboxamide (Genz-669178), geldanamycin, 1,4ꢁdibenzothiazepene amides
(IDI-3783 and IDI-5918), respectively21 (personal communication with U. Ribacke and A.K.
Lukens). Table 8 shows that compound 20 remained equally potent to both parental and
mutant DD2 strains, in contrast to the corresponding control inhibitors (see also
Supplementary Figure 1). This indicates that the target of compound 20 is distinct from
PfDHODH, PfHSP90, PfCRT or PfCYTbꢁQi. Artesunate was included as an additional
control compound showing no significant difference in IC50s among all of the strains tested.
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IC50 (nM)
Compound
DD2
DD2-DHODH
19.72 ± 3
1.81 ± 0.3
76.8 ± 10
257 ± 40
DD2-HSP90
34.13 ± 1
1.29 ± 0.2
16.2 ± 2
658 ± 30
8.42 ± 1
ND
DD2-PfCRT
16.45 ± 1
1.84 ± 0.3
5.07 ± 4
DD2-CYTbQi
11.79 ± 2
1.26 ± 0.4
9.94 ± 0.9
169 ± 40
20
24.6 ± 8
1.51 ± 0.2
5.79 ± 1
176 ± 30
7.30 ± 7
2.60 ± 0.6
Artesunate
29 (Genz-669178)
Geldanamycin
30 (ID-3783)
31 (IDI-5918)
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23
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204 ± 20
16.9 ± 9
1045 ± 700
1.95 ± 1*
3.16 ± 0.2
618 ± 90
2.37 ± 0.5
*value reflects mean of two replicate assays ± standard deviation; ND = not determined.
Table 8. Compound 20 retains its potency against a panel of Pf strains known to harbor
mutations conferring resistance to antimalarial agents under development. Values are the
mean of three replicate assays ± standard deviation. IC50s of control compounds against their
specific resistantꢁmutant strain are highlighted in bold. *value reflects mean of two replicate
assays ± standard deviation; ND = not determined.
In order to identify the mode of action of compound 20 and confirm that it is novel, a
chemogenomic approach was used, whereby resistant parasites are generated by in vitro
selection under drug pressure and whole genome sequencing is employed to identify the
genetic basis of resistance22. However, in vitro resistance generation failed after multiple
attempts at various parasite density and compound concentrations (see Supporting
Information).
In vitro parasite reduction ratio (PRR). We employed the previously published in vitro PRR
methodology23 to understand the rate and extent of kill of this chemical class. Representative
compounds (20 & 21) when tested at 10X IC50 displayed a fast killing profile and an effect
similar to chloroquine after 48 hours of compound treatment.
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Figure 4. In vitro parasite reduction ratio (PRR) for compound 20 and 21. The graph depicts
the number of viable parasites following exposure of Pf 3D7A parasites to 10X IC50
concentrations of the compounds.
Most of the currently available antimalarial agents are active against the asexual blood stages
of Pf that are primarily responsible for the clinical symptoms associated with malaria and
control the emergence of drug resistance24. Given the global goal of eradicating malaria,
newer antimalarial agents that kill the sexual forms of the parasite (transmission blocking) or
target the quiescent hepatic forms of the parasite in addition to the bloodꢁstage forms are
desirable. Representative compounds from this series lacked activity against the male/female
gametocytes24 and were inactive in the P. berghei liver stage assay25. Contrary to some of the
newer antimalarial agents in clinical development that target multiple life stages of Pf, the
amino azabenzimidazoles lack activity against the liver and sexual stages of Pf. Given the
wide spread prevalence of drug resistant forms of Pf, amino azabenzimidazoles owing to their
novel mechanism of action and fast kill against the asexual forms could potentially be used in
combination with other novel agents in clinical development to treat and eradicate malaria.
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Conclusion: A detailed medicinal chemistry optimisation of a sub micromolar 2ꢁamino
imidazole screening hit has led to the identification of 2ꢁamino azabenzimidazoles, a novel
class of antiplasmodial agent, exhibiting nanomolar inhibitory activity against Pf. The
representative compound 23 is orally bioavailable and efficacious in the humanised Pf/SCID
model of malaria. The series displayed a fast rate of kill equivalent to chloroquine in the in
vitro PRR assay. Representative compounds of this series retained their IC50 values against a
panel of drug resistant strains of Pf suggesting a novel mechanism of action. Additionally,
three novel chemotypes (compounds 24 ꢁ 28) derived by scaffold hopping provide further
avenues to diversify this class of inhibitors to build in desirable properties suitable for
progression towards a candidate selection. In conclusion, the chemotype described in this
study has desirable attributes for a novel antimalarial agent, such as fast kill, lack of cross
resistance, low frequency of resistance emergence, excellent oral bioavailability and potential
for low cost of goods based on fewer number of synthetic steps. Further studies are
warranted to improve potency, solubility and evaluating in vivo safety profile in suitable
preclinical models to progress this series towards candidate selection to treat malaria.
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EXPERIMENTAL SECTION
5
6
7
8
9
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 MilliꢁQ 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 preꢁcoated Merck
60 F254 silica gel plates and visualized using UV light (254 nm). All compounds were
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1
analyzed for purity by HPLC and characterized by H NMR using Bruker 300 MHz NMR
and/or Bruker 400 MHz NMR spectrometers. Chemical shifts are reported in ppm (δ) relative
to the residual solvent peak in the corresponding spectra; chloroform δ 7.26, methanol δ 3.31,
DMSOꢁd6 δ 3.33 and coupling constants (J) are reported in 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, 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. All the compounds exemplified in
this work are either racemic mixtures or diastereomically pure compounds.
General synthetic procedures for compounds 1 - 4:
1-(2,4-Dichlorophenyl)-2-(2-nitro-1H-imidazol-1-yl)ethanone (1a) : To a stirred solution
of 2ꢁNitroꢁ1Hꢁimidazole (0.5 g, 4.424 mmoles,) in acetonitrile (5 mL) phenacyl bromide (1.1
g, 4.424 mmoles) was added followed by diisopropyl ethylamine (1.1 g, 8.848 mmoles). The
reaction mixture was heated under microwave conditions at 150 °C for 2 h. After confirming
the absence of the starting material, acetonitrile was removed under reduced pressure. The
crude residue obtained was purified by column chromatography to obtain 0.8 g (60%) of 1ꢁ(2,
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4ꢁdichlorophenyl)ꢁ2ꢁ(2ꢁnitroꢁ1Hꢁimidazolꢁ1ꢁyl)ethanone (1a). LCMS [M+1]: 301.8. 1H
NMR 400 MHz, DMSOꢁd6: δ ppm 7.96 (dd, J = 8.00 Hz, 1H), 7.86ꢁ7.87 (m, 1H), 7.70 (m,
1H), 7.66 (m, 1H), 7.28 (m, 1H), and 6.02 (s, 2H).
9
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21
22
23
24
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1-(2,4-Dichlorophenyl)-2-(2-nitro-1H-imidazol-1-yl)ethanol (1b): To a stirred solution of
1a (0.8 g, 2.666 mmoles) in MeOH (15 mL) sodium borohydride (0.2 g, 5.332 mmoles) was
added portion wise slowly at 0 °C under N2 atm. The reaction mixture was then stirred at
room temperature for overnight. After confirming the absence of the starting material the
reaction mixture was cooled and quenched with water. Then it was extracted with ethyl
acetate and washed with water and brine solution. The combined organic layer was
1
concentrated under vaccum to obtain 0.7 g (86 %) of 1b. LCMS [M+1] : 303. H NMR, 400
MHz, DMSOꢁd6: δ ppm 7.58 ꢁ 7.57 (m, 1H), 7.46ꢁ7.38 (m, 3H), 7.11 (d, J = 4.00 Hz, 1H),
6.09 (d, J = 8.00 Hz, 1H), 5.26ꢁ5.21 (m, 1H), and 4.59ꢁ4.57 (m, 2H).
2-(2-Amino-1H-imidazol-1-yl)-1-(2,4-dichlorophenyl)ethanol (1) : To a stirred solution of
1b (0.7 g, 2.3 mmol) in MeOH (10 mL) activated zinc powder (1 g, 16.22 mmol) and
ammonium chloride (1.2 g, 23 mmol) were successively added. The resulting mixture was
stirred at room temperature for overnight. After confirming the absence of the starting
material the reaction mixture was filtered through celite and washed with MeOH. The
organic layer was concentrated and purified by column chromatography to obtain 433 mg
(69%) of 2ꢁ(2ꢁaminoꢁ1Hꢁimidazolꢁ1ꢁyl)ꢁ1ꢁ(2,4ꢁdichlorophenyl)ethanol 1.
HRMS for
C11H11Cl2N3O [M+H]+: 272.03541. 1H NMR 400 MHz, DMSOꢁd6: δ ppm 8.27 (s, 1H),
7.62ꢁ7.60 (m, 2H), 7.50ꢁ7.48 (m, 1H), 7.10 (s, 2H), 6.68 (s, 1H), 6.63ꢁ6.62 (m, 1H), 5.16 –
5.13 (m, 1H), and 3.96ꢁ3.94 (m, 2H). HPLC 98.91 %.
2-(2-Amino-1H-imidazol-1-yl)-1-(4-chloro-2-fluorophenyl)ethan-1-ol (2) : Compound 2
was prepared using similar synthetic scheme as described above for compound 1 . HRMS for
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C11H11ClFN3O [M+H]+: 256.06496. 1HꢁNMR (400 MHz, DMSOꢁd6): δ ppm 7.56ꢁ7.58 (m,
3H), 7.40ꢁ7.43 (m, 1H), 7.32ꢁ7.35 (m, 1H), 6.82ꢁ6.86 (m, 2H), 6.18 (s, 1H), 5.10ꢁ5.13 (m,
1H), 4.16ꢁ4.17 (m, 1H), and 4.12ꢁ4.14 (m, 1H). HPLC 99.72 %.
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2-(2-Amino-1H-imidazol-1-yl)-1-(4-chloro-2-methylphenyl)ethan-1-ol (3): Compound 3
was prepared using similar synthetic scheme as described above for for compound 1. HRMS
1
for C12H14ClN3O [M+H]+: 252.08925; H NMR (400 MHz, DMSOꢁd6) δ ppm 7.45ꢁ7.52 (m,
3H), 7.26ꢁ7.28 (m, 2H), 6.89ꢁ6.90 (m, 2H), 5.88 (d, J = 4.36 Hz, 1H), 5.00ꢁ5.01 (m, 1H),
3.94ꢁ4.05 (m, 2H), and 2.33 (s, 3H). HPLC 93.59 %.
2-(2-Amino-1H-imidazol-1-yl)-1-(2,3-dichlorophenyl)ethanol (4): Compound 4 was
prepared using similar synthetic scheme as described above for compound 1. HRMS for
C11H11Cl2N3O [M+H]+: 272.0353. 1H NMR (300 MHz, DMSOꢁd6) δ ppm 3.59ꢁ3.83 (m, 1H),
3.83ꢁ3.97 (m, 1H), 5.15 (m, 3H), 6.11 (d, J=4.33 Hz, 1H), 6.32 (d, J=1.32 Hz, 1H), 6.43 (d,
J=1.32 Hz,1H), 7.32ꢁ7.46 (m, 1H), 7.48ꢁ7.67 (m, 2H). HPLC purity: 98.74%.
2-(2-Amino-4-methyl-1H-imidazol-1-yl)-1-(2,4-dichlorophenyl)ethanol (5): Compound 5
was prepared by an alternate procedure (see supporting information).
HRMS for
1
C12H13Cl2N3O [M+H]+: 286.05079. H NMR 400 MHz, DMSOꢁd6: δ ppm 7.60 (d, J = 8.00
Hz, 1H), 7.49 (d, J = 2.00 Hz, 1H), 7.39 (dd, J = 2.00, 6.60 Hz, 1H), 6.36 (s, 1H), 5.30ꢁ5.31
(m, 1H), 3.99 (m, 2H) and 2.09 (s, 3H). HPLC: 99.04%.
2-(2-Amino-4,5-dimethyl-1H-imidazol-1-yl)-1-(2,4-dichlorophenyl)-ethan-1-ol
(6):
Compound 6 was prepared by an alternate scheme (see supporting information). HRMS for
1
C13H15Cl2N3O [M+H]+: 300.06686. HꢁNMR (400 MHz, MeOD): δ ppm 7.58 (d, J = 8.40
Hz, 1H), 7.48 (d, J = 2.04 Hz, 1H), 7.36ꢁ7.38 (m, 1H), 5.27ꢁ5.30 (m, 1H), 3.97 (d, J = 2.92
Hz, 1H), 3.93 (d, J = 2.96 Hz, 1H), 1.94 (s, 3H), and 1.82 (s, 3H). HPLC purity: 99.63%.
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2-(2-Amino-4-cyclopropyl-1H-imidazol-1-yl)-1-(2,4-dichlorophenyl)ethanol
(7):
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Compound 7 was prepared using an alternate scheme (see supporting information) HRMS for
C14H15Cl2N3O [M+H]+: 312.06302. 1H NMR (400 MHz, CD3OD) δ ppm 7.60 (d, J = 8.40
Hz, 1H), 7.49 (d, J = 1.92 Hz, 1H), 7.39 (dd, J = 1.88, 8.40 Hz, 1H), 6.34 (s, 1H), 5.33 (t, J =
5.20 Hz, 1H), 3.96ꢁ4.02 (m, 2H), 1.68ꢁ1.72 (m, 1H), 0.89ꢁ0.93 (m, 2H), and 0.59ꢁ0.62 (m,
2H). HPLC purity: 98 %.
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2-(2-Amino-4-(4-fluorophenyl)-1H-imidazol-1-yl)-1-(2-chloro-4-
(trifluoromethyl)phenyl)ethanol (8) : Compound 8 was prepared by an alternate scheme
1
(see supporting information). HRMS for C18H14ClF4N3O [M+H]+: 400.08366. H NMR (400
MHz, DMSOꢁd6) δ ppm 7.7ꢁ7.86 (m, 3H), 7.58ꢁ7.61 (m, 2H), 7.06ꢁ7.10 (m, 2H), 6.96 (s,
1H), 6.19ꢁ6.20 (d, 1H), 5.37 (s, 2H), 5.22 (s, 1H), 3.92ꢁ3.97 (m, 1H), and 3.79ꢁ3.85 (m, 1H).
HPLC purity 93.76 %.
Compounds 9 – 15 were synthesized by alternate routes. The detailed synthetic scheme for
each compound is provided in the supporting information.
2-(2-Amino-1H-benzo[d]imidazol-1-yl)-1-(2,4-dichlorophenyl)ethanol (9) : HRMS for
1
C15H13Cl2N3O [M+H]+: 322.05057. H NMR (300 MHz, DMSOꢁd6) δ ppm 3.96 ꢁ 4.19 (m, 2
H) 5.14 ꢁ 5.31 (m, 1 H) 6.04 ꢁ 6.11 (m, 1 H) 6.12 ꢁ 6.31 (m, 1 H) 6.75 ꢁ 6.88 (m, 1 H) 6.88 ꢁ
6.96 (m, 1 H) 6.95 ꢁ 6.96 (m, 1 H) 6.99 (d, J=7.54 Hz, 1 H) 7.06 ꢁ 7.18 (m, 1 H) 7.40 ꢁ 7.51
(m, 1 H) 7.56 ꢁ 7.63 (m, 1 H) 7.68 ꢁ 7.78 (m, 1 H). ES+MS m/z: 322.30 (M+1).
2-Amino-1-(2-(2,3-dichlorophenyl)-2-hydroxyethyl)-1H-benzo[d]imidazole-6-
1
carbonitrile (10): HRMS from C16H12Cl2N4O [M+H]+: 347.04688. H NMR (300 MHz,
DMSOꢁd6) δ ppm 4.25 (d, 2 H) 5.25 ꢁ 5.39 (m, 1 H) 6.02 ꢁ 6.19 (m, 1 H) 6. 81 (s, 2 H) 7.20 ꢁ
7.35 (m, 1 H) 7.36 – 7.40 (m, 2 H) 7.45 ꢁ 7.50 (m, 1 H) 7.61 ꢁ 7.63 (m, 1 H) 7.75 ꢁ 7.79 (m, 1
H). HPLC purity: 99%.
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2-(2-Amino-6-(methylsulfonyl)-1H-benzo[d]imidazol-1-yl)-1-(2,3-
1
dichlorophenyl)ethanol (11) : HRMS for C16H15ClN3O3S [M+H]+: 400.02941. H NMR
(300 MHz, DMSOꢁd6) δ ppm 3.06 (s, 3H), 4.24 (d, J=5.65 Hz, 2H), 5.32 (q, J=5.46 Hz, 1H),
6.11 (d, J=4.52 Hz, 1H), 6.80 (s, 2H), 7.25 (d, J=8.29 Hz, 1H), 7.35ꢁ7.53 (m, 3H), 7.58 (dd,
J=7.91, 1.32 Hz, 1H), 7.62ꢁ7.74 (m, 1H). HPLC purity: 99.29%
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2-(2-Amino-6-chloro-1H-benzo[d]imidazol-1-yl)-1-(2,3-dichlorophenyl)ethanol (12)
:
HRMS for C15H12Cl3N3O [M+H]+: 356.00879. 1H NMR (300 MHz, DMSOꢁd6) δ ppm 4.05 ꢁ
4.23 (m, 2 H) 5.18 ꢁ 5.36 (m, 1 H) 6.04 ꢁ 6.16 (m, 1 H) 6.29 ꢁ 6.49 (m, 2 H) 6.85 ꢁ 6.98 (m, 2
H) 7.01 ꢁ 7.14 (m, 1 H) 7.33 ꢁ 7.45 (m, 1 H) 7.52 ꢁ 7.61 (m, 1 H) 7.62 ꢁ 7.70 (m, 1 H). HPLC
purity: 99%.
2-(2-Amino-6-(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)-1-(2-chloro-4-
(trifluoromethyl)phenyl)ethanol (13): HRMS for C17H12ClF6N3O [M+H]+: 424.06424. H
1
NMR (300 MHz, DMSOꢁd6) δ ppm 4.17 ꢁ 4.38 (m, 2 H) 5.29 ꢁ 5.40 (m, 1 H) 6.12 ꢁ 6.22 (m,
1 H) 6.62 ꢁ 6.80 (m, 2 H) 6.91 ꢁ 7.03 (m, 1 H) 7.12 ꢁ 7.25 (m, 2 H) 7.62 ꢁ 7.79 (m, 2 H) 7.81 ꢁ
7.91 (m, 1 H). HPLC purity: 97 %.
2-(2-Amino-1H-imidazo[4,5-c]pyridin-1-yl)-1-(2,4-dichlorophenyl)ethanol (14): HRMS
for C14H12Cl2N4O [M+H]+: 323.04563. H NMR (300 MHz, DMSOꢁd6) δ ppm 4.09 ꢁ 4.22
1
(m, 2 H) 5.15 ꢁ 5.32 (m, 1 H) 6.08 (d, J=4.52 Hz, 1 H) 6.57 (s, 2 H) 6.97 (d, J=5.27 Hz, 1
H),7.46 (dd, J=8.38, 2.17 Hz, 1 H) 7.57 (d, J=2.07 Hz, 1 H) 7.69 (d, J=8.48 Hz, 1 H) 7.97 (d,
J=5.27 Hz, 1 H) 8.34 (s, 1 H). HPLC purity: 99%.
2-(2-Amino-3H-imidazo[4,5-b]pyridin-3-yl)-1-(2,3-dichlorophenyl)ethanol (15): HRMS
for C14H12Cl2N4O [M+H]+: 323.04731. 1H NMR (300 MHz, DMSOꢁd6) δ ppm 4.15ꢁ4.32 (m,
2H), 5.45 (m, 1H), 6.20 (s, 1H), 6.62 (bs, 2H), 6.92 (m,1H), 7.35 (m, 2H), 7.55 (m, 1H), 7.65
(m, 1H), 7.80 (m, 1H). HPLC purity: 99.17%.
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General synthetic procedures for compounds 16 – 17, 20 - 23:
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2-(2-Amino-5-(4-methylpiperazin-1-yl)-3H-imidazo[4,5-b]pyridin-3-yl)-1-(2-chloro-4-
(trifluoromethyl)phenyl)ethan-1-ol (16):
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6-(4-Methylpiperazin-1-yl)-3-nitropyridin-2-amine (16a):
To the solution of 6ꢁchloroꢁ3ꢁnitropyridinꢁ2ꢁamine (2.0 g, 17.0 mmol) in anhydrous DMF (20
mL) were added 1ꢁmethyl piperazine (3.46 g, 50.70 mmol) and potassium carbonate (4.77 g,
50 mmol). The resulting mixture was heated at 50°C for 16 h. The reaction mixture was
poured in to ice cold water and solid precipitated was filtered and dried to obtain 6ꢁ(4ꢁ
methylpiperazinꢁ1ꢁyl)ꢁ3ꢁnitropyridinꢁ2ꢁamine as a yellow solid. Yield: 1.6 g (59.25%).
LCMS: m/z 238.4 (M+H)+. 1H NMR 300 MHz, DMSOꢁd6: δ ppm 8.02ꢁ8.05 (m, 3H), 6.33 (d,
J = 9.54 Hz, 1H), 3.69 (s, 4H), 2.34 (t, J = 4.98 Hz, 4H), and 2.19 (s, 3H).
6-(4-Methylpiperazin-1-yl)pyridine-2,3-diamine (16b): To
a
solution of 6ꢁ(4ꢁ
methylpiperazinꢁ1ꢁyl)ꢁ3ꢁnitropyridinꢁ2ꢁamine (1.6 g, 6.74 mmol) in methanol : DCM (50
mL) was added palladium (10%) on carbon (0.5 g). The mixture was stirred under hydrogen
atmosphere for 1 h. The suspension was filtered through celite and filtrate was concentrated
under reduced pressure to obtain 6ꢁ(4ꢁmethylpiperazinꢁ1ꢁyl)pyridineꢁ2,3ꢁdiamine which was
taken to next step without further purification. Yield: 0.8 g (57.5%). LCMS: m/z 208.7
(M+H)+.
5-(4-Methylpiperazin-1-yl)-3H-imidazo[4,5-b]pyridin-2-amine (16c): To the solution of
6ꢁ(4ꢁmethylpiperazinꢁ1ꢁyl)pyridineꢁ2,3ꢁdiamine (0.54 g, 2.60 mmol) in MeOH : H2O (1 : 1)
(50mL) was added cyanogen bromide (0.551 g, 5.21 mmol) at 0°C. The dark green mixture
was stirred at RT for 16 h. The solvent was evaporated to dryness and the residue was
triturated with acetonitrile to obtain brown residue which was purified by column
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chromatography using 10% MeOH in DCM saturated with ammonia as eluent to obtain 5ꢁ(4ꢁ
methylpiperazinꢁ1ꢁyl)ꢁ3Hꢁimidazo[4,5ꢁb]pyridinꢁ2ꢁamine as brown solid. LCMS: m/z 233.4
(M+H)+. 1H NMR 400 MHz, DMSOꢁd6: δ ppm 10.62 (s, 1H), 7.21 (d, J = 8.36 Hz, 1H), 6.29
(d, J = 8.36 Hz, 1H), 6.15 (s, 2H), 3.30 (t, J = 4.88 Hz, 4H), 2.41 (t, J = 4.80 Hz, 4H), and
2.21 (s, 3H).
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2-(2-Amino-5-(4-methylpiperazin-1-yl)-3H-imidazo[4,5-b]pyridin-3-yl)-1-(2-chloro-4-
(trifluoromethyl)phenyl)ethan-1-one (16d) : To the solution of 5ꢁ(4ꢁmethylpiperazinꢁ1ꢁyl)ꢁ
3Hꢁimidazo[4,5ꢁb]pyridinꢁ2ꢁamine (0.171 g, 0.73 mmol) in acetonitrile (5 mL) was added 2ꢁ
bromoꢁ1ꢁ(2ꢁchloroꢁ4ꢁ(trifluoromethyl)phenyl)ethanꢁ1ꢁone (0.221 g, 0.73 mmol) and DIPEA
(0.243 mL, 1.47 mmol). The resulting mixture was subjected to microwave irradiation in a
sealed tube at 150°C for 2 h. The product formation was confirmed by LCMS. The solvent
was evaporated to dryness and the residue obtained was purified by column chromatography
using 5% MeOH in DCM saturated with ammonia as eluent to obtain 2ꢁ(2ꢁaminoꢁ5ꢁ(4ꢁ
methylpiperazinꢁ1ꢁyl)ꢁ3Hꢁimidazo[4,5ꢁb]pyridinꢁ3ꢁyl)ꢁ1ꢁ(2ꢁchloroꢁ4ꢁ
1
(trifluoromethyl)phenyl)ethanꢁ1ꢁone as off white solid. Yield: 0.048 g (14.44%). H NMR
400 MHz, DMSOꢁd6: δ ppm 8.14 (d, J = 8.04 Hz, 1H), 8.08 (s, 1H), 7.98 (d, J = 8.04 Hz,
1H), 7.32 (d, J = 8.36 Hz, 1H), 6.45 (t, J = 8.48 Hz, 3H), 5.41 (s, 2H), 3.32 (t, J = 5.32 Hz,
4H), 2.38 (t, J = 4.80 Hz, 4H), and 2.20 (s, 3H).
2-(2-Amino-5-(4-methylpiperazin-1-yl)-3H-imidazo[4,5-b]pyridin-3-yl)-1-(2-chloro-4-
(trifluoromethyl)phenyl)ethan-1-ol (16): To the solution of 2ꢁ(2ꢁaminoꢁ5ꢁ(4ꢁ
methylpiperazinꢁ1ꢁyl)ꢁ3Hꢁimidazo[4,5ꢁb]pyridinꢁ3ꢁyl)ꢁ1ꢁ(2ꢁchloroꢁ4(trifluoromethyl)phenyl)
ethanꢁ1ꢁone (0.043 g, 0.094 mmol) in methanol (1 mL) was added sodium borohydride
(0.0036 g, 0.094 mmol) at 0°C. The mixture was stirred at RT for 1 h. The reaction mixture
was quenched with EtOAc and evaporated to dryness and the residue was purified by column
chromatography using 4% MeOH in DCM as eluent to obtain 2ꢁ(2ꢁaminoꢁ5ꢁ(4ꢁ
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methylpiperazinꢁ1ꢁyl)ꢁ3Hꢁimidazo[4,5ꢁb]pyridinꢁ3ꢁyl)ꢁ1ꢁ(2ꢁchloroꢁ4(trifluoromethyl)phenyl)
ethanꢁ1ꢁone as off white solid. Yield: 0.013 g (37.20%). HRMS for C20H22ClF3N6O [M+H]+:
455.15743. 1H NMR 400 MHz, CD3OD: δ ppm 7.66 (t, J = 8.28 Hz, 2H), 7.44 (d, J = 7.96
Hz, 1H), 7.33 (d, J = 8.48 Hz, 1H), 6.44 (d, J = 8.52 Hz, 1H), 5.58 (t, J = 5.12 Hz, 1H), 4.33ꢁ
4.45 (m, 2H), 3.41 (s, 4H), 2.60 (t, J = 4.96 Hz, 4H), and 2.39 (s, 3H). HPLC 97.31 %.
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2-(2-Amino-5-(2,2,2-trifluoroethylamino)-3H-imidazo[4,5-b]pyridin-3-yl)-1-(2,4-
dichlorophenyl)ethanol (17) : Compound 17 was prepared using similar synthetic scheme as
described above for compound 16. HRMS for C16H14Cl2F3N5O [M+H]+: 420.06022. 1H NMR
(300 MHz, DMSOꢁd6) δ ppm 3.88 ꢁ 4.26 (m, 4 H) 5.28 ꢁ 5.43 (m, 1 H) 6.02 ꢁ 6.12 (m, 2 H)
6.13 ꢁ 6.19 (m, 1 H) 6.19 ꢁ 6.26 (m, 1 H) 6.55 ꢁ 6.70 (m, 1 H) 7.14 ꢁ 7.28 (m, 1 H) 7.38 ꢁ 7.47
(m, 1 H) 7.54 ꢁ 7.60 (m, 1 H) 7.63 ꢁ 7.71 (m, 1 H). HPLC 98.5 %.
Compounds 18 and 19 were synthesized by alternate routes. The detailed synthetic scheme
for each compound is provided in the supporting information
2-(2-Amino-5-cyclopropyl-3H-imidazo[4,5-b]pyridin-3-yl)-1-(2,4-
1
dichlorophenyl)ethanol (18): HRMS for C17H16Cl2N4O [M+H]+: 363.07717. H NMR (300
MHz, DMSOꢁd6) δ ppm 7.63 (d, J = 8.48 Hz, 1H), 7.54 (d, J = 2.07 Hz, 1H), 7.41 (dd, J =
2.07, 8.48 Hz, 1H), 7.18 ꢁ 7.28 (m, 1H), 6.85 (d, J = 7.72 Hz, 1H), 6.46 (s, 2H), 6.11 (d, J =
4.52 Hz, 1H), 5.35 (dd, J = 3.96, 7.91 Hz, 1H), 3.97 ꢁ 4.23 (m, 2H), 1.96 (t, J = 6.03 Hz, 1H),
0.72 ꢁ 0.90 (m, 4H). HPLC purity: 95.3 %.
2-Amino-N-cyclopropyl-3-(2-(2,4-dichlorophenyl)-2-hydroxyethyl)-3H-imidazo[4,5-
1
b]pyridine-5-carboxamide (19): HRMS for C18H17Cl2N5O2 [M+H]+: 406.08353. H NMR
(300 MHz, DMSOꢁd6) δ ppm 0.48 ꢁ 0.66 (m, 2 H) 0.72 ꢁ 0.86 (m, 2 H) 2.75 ꢁ 2.91 (m, 1 H)
4.15 ꢁ 4.39 (m, 2 H) 5.31 ꢁ 5.47 (m, 2 H) 6.41 ꢁ 7.07 (bs,2 H) 7.31 ꢁ 7.46 (m, 2 H) 7.47 ꢁ 7.56
(m, 1 H) 7.58 ꢁ 7.72 (m, 2 H) 7.76 ꢁ 7.96 (m, 1 H). HPLC purity: 98 %.
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2-(2-Amino-5-(2,2,2-trifluoroethoxy)-3H-imidazo[4,5-b]pyridin-3-yl)-1-(2-chloro-4-
(trifluoromethyl)phenyl)ethanol (20): Compound 20 was prepared using similar synthetic
scheme as described above for compound 16. HRMS for C17H13ClF6N4O2 [M+H]+:
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455.07107. H NMR (300 MHz, DMSOꢁd6) δ ppm 4.02ꢁ4.31 (m, 2H), 4.67ꢁ4.91 (m, 2H),
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5.47 (d, J=4.33 Hz, 1H), 6.26 (d, J=4.33 Hz, 1H), 6.35ꢁ6.52 (m, 3H), 7.42 (d, J=8.10 Hz,
1H), 7.67 (d, J=9.04 Hz, 1H), 7.72ꢁ7.86 (m, 2H). HPLC purity: 96.95%.
2-(2-Amino-5-(tetrahydrofuran-3-yloxy)-3H-imidazo[4,5-b]pyridin-3-yl)-1-(2-chloro-4-
(trifluoromethyl)phenyl)ethanol (21): Compound 21 was prepared using similar synthetic
scheme as described above for compound 16. HRMS for C19H18ClF3N4O3 [M+H]+: 443.1089.
1H NMR (300 MHz, DMSOꢁd6): δ ppm 7.57 ꢁ 7.88 (m, 3H), 7.35 (dd, J = 1.51, 8.10 Hz, 1H),
6.21 ꢁ 6.45 (m, 3H), 5.47 (br. s., 1H), 5.10 ꢁ 5.29 (m, 1H), 4.10 ꢁ 4.30 (m, 2H), 3.59 ꢁ 3.94 (m,
5H), 2.03 ꢁ 2.26 (m, 1H), 1.87 ꢁ 2.02 (m, 1H). HPLC purity: 95.5%.
2-(2-Amino-5-(azetidin-3-yloxy)-3H-imidazo[4,5-b]pyridin-3-yl)-1-(2-chloro-4-
(trifluoromethyl)phenyl)ethanol (22) : Compound 22 was prepared using similar synthetic
scheme as described above for compound 16. HRMS for C18H17ClF3N5O2. HCl [M+H]+:
428.10923. 1H NMR (300 MHz, DMSOꢁd6) δ ppm 1.89 (s, 3H), 3.40ꢁ3.60 (m, 2H), 3.68ꢁ
3.84 (m, 2H), 3.99ꢁ4.30 (m, 2H), 5.07 (m, 1H), 5.45 (t, J=5.37 Hz, 1H), 6.21ꢁ6.40 (m, 3H),
7.36 (d, J=8.29 Hz, 1H), 7.60ꢁ7.75 (m, 1H), 7.76ꢁ7.88 (m, 2H). HPLC purity: 99.45%.
2-(2-Amino-5-(1,1,1-trifluoropropan-2-yloxy)-3H-imidazo[4,5-b]pyridin-3-yl)-1-(2-
chloro-4-(trifluoromethyl)phenyl)ethanol (23): Compound 23 was prepared using similar
synthetic scheme as described above for compound 16. HRMS for C18H15ClF6N4O2 [M+H]+:
469.08154. 1H NMR (300 MHz, DMSOꢁd6) δ ppm 1.39 (d, J=6.59 Hz, 3 H) 4.15 ꢁ 4.24 (m, 2
H) 5.41 ꢁ 5.51 (m, 1 H) 5.58 ꢁ 5.70 (m, 1 H) 6.25 ꢁ 6.32 (m, 1 H) 6.39 ꢁ 6.44 (m, 1 H) 6.45 ꢁ
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