Design, synthesis and characterization of novel N-heterocyclic-1-benzyl-1H-benzo[d]imidazole-2-amines as selective TRPC5 inhibitors leading to the identification of the selective compound, AC1903

Article abstract of DOI:10.1016/j.bmcl.2018.12.007

The transient receptor potential cation channel 5 (TRPC5) has been previously shown to affect podocyte survival in the kidney. As such, inhibitors of TRPC5 are interesting candidates for the treatment of chronic kidney disease (CKD). Herein, we report the synthesis and biological characterization of a series of N-heterocyclic-1-benzyl-1H-benzo[d]imidazole-2-amines as selective TRPC5 inhibitors. Work reported here evaluates the benzimidazole scaffold and substituents resulting in the discovery of AC1903, a TRPC5 inhibitor that is active in multiple animal models of CKD.

Full text of DOI:10.1016/j.bmcl.2018.12.007

Accepted Manuscript
Design, synthesis and characterization of novel N-heterocyclic-1-benzyl-1H-
benzo[d]imidazole-2-amines as selective TRPC5 inhibitors leading to the iden-
tification of the selective compound, AC1903
Swagat H. Sharma, Juan Lorenzo Pablo, Monica Suarez Montesinos, Anna
Greka, Corey R. Hopkins
PII:
S0960-894X(18)30941-7
https://doi.org/10.1016/j.bmcl.2018.12.007
BMCL 26176
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 October 2018
29 November 2018
4 December 2018
Please cite this article as: Sharma, S.H., Pablo, J.L., Montesinos, M.S., Greka, A., Hopkins, C.R., Design, synthesis
and characterization of novel N-heterocyclic-1-benzyl-1H-benzo[d]imidazole-2-amines as selective TRPC5
inhibitors leading to the identification of the selective compound, AC1903, Bioorganic & Medicinal Chemistry
Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.12.007
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Design, synthesis and characterization of
novel N-heterocyclic-1-benzyl-1H-
benzo[d]imidazole-2-amines as selective
TRPC5 inhibitors leading to the
identification of the selective compound,
AC1903
Leave this area blank for abstract info.
Swagat H. Sharma, Juan Lorenzo Pablo, Monica Suarez Montesinos, Anna Greka, Corey R. Hopkins

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Design, synthesis and characterization of novel N-heterocyclic-1-
benzyl-1H-benzo[d]imidazole-2-amines as selective TRPC5
inhibitors leading to the identification of the selective compound,
AC1903
Swagat H. Sharmaa,⊥, Juan Lorenzo Pablob,c,⊥, Monica Suarez Montesinosb,c, Anna Grekab,c, Corey R.
Hopkinsa,*
aDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198
bDepartment of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115
cBroad Institute of MIT and Harvard, Cambridge, MA 02142
⊥These authors contributed equally to the manuscript
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The transient receptor potential cation channel 5 (TRPC5) has been previously shown to affect
podocyte survival in the kidney. As such, inhibitors of TRPC5 are interesting candidates for the
treatment of chronic kidney disease (CKD). Herein, we report the synthesis and biological
characterization of a series of N-heterocyclic-1-benzyl-1H-benzo[d]imidazole-2-amines as
selective TRPC5 inhibitors. Work reported here evaluates the benzimidazole scaffold and
substituents resulting in the discovery of AC1903, a TRPC5 inhibitor that is active in multiple
animal models of CKD.
Keywords:
transient receptor potential cation channel 5
TRPC5
2009 Elsevier Ltd. All rights reserved.
inhibitor
chronic kidney disease
AC1903
———
* Corresponding author. Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE
68198, USA; Tel.: +1 402 559 9729; Fax: +1 402 559 5643. Email address: corey.hopkins@unmc.edu

Chronic kidney disease (CKD) is a type of kidney disease
associated with gradual loss of kidney function which affects >300
million people worldwide.1,2 Although many factors contribute to
CKD, the leading diagnosis of CKD is focal segmental
glomerulosclerosis (FSGS) – characterized by proteinuria and
scarring of the glomerulus.3 Although the patient burden of CKD
is large, there has been a dearth of new treatment options
developed over the past several decades. Recently, the TRPC5 ion
channel has emerged as a potential new target for the development
of novel treatments for CKD.4 The mammalian superfamily of
transient receptor potential (TRP) cation channels is comprised of
six subfamilies of receptors: TRPV (vanilloid), TRPC (canonical),
TRPM (melastatin), TRPA (ankyrin), TRPP (polycystin) and
TRPML (mucolipin). The TRPC subfamily is further subdivided
into seven members (TRPC1-7) based on sequence similarities:
TRPC1, TRPC2, TRPC3/6/7, and TRPC4/5.5 Due to their
localization, the TRPC4/5 subfamily has been implicated in a
variety of disease areas, including: drug dependence, anxiety and
kidney filtration.6 In fact, an initial TRPC4/5 inhibitor (ML204,1)
was shown to protect the kidney filter in an in vivo animal model.
Figure 2. Structure and modification points of AC1903, 2.
The synthesis of the desired benzimidazole compounds is
shown in Scheme 1. The initial library centered around keeping
the southern benzyl groups constant while modifying the right-
hand 2-benzimidazole moiety. This was done by reacting the 2-
chlorobenzimidazole (or substituted benzimidazole), 3, with
benzyl bromide under basic conditions (NaH, DMSO, 0 oC to rt).
Next, the resulting compounds, 4 (4, R = Ph, 4a-t, as detailed in
Table 2), were reacted with the appropriate amine (neat) under
o
microwave heating (W, 200 C, 30 min) to yield the desired
compounds 2, 5a-u and 7a-b. The next set of compounds, 6a-t,
were synthesized in a similar manner, except the starting material,
3, was modified with the appropriate alkyl or benzyl bromide to
give 4, followed by reaction with furfuryl amine under microwave
conditions to yield 6a-r.
7
Although ML204 (Figure 1) was an important initial tool
compound, the selectivity profile around this molecule was less
than ideal, (inhibitor of both TRPC4 and 5) and thus, our
laboratories set out to identify a selective TRPC5 inhibitor. As
part of this endeavor, we have recently published on the novel
TRPC5 inhibitor, AC1903, and herein, we report the synthesis and
structure activity-relationship (SAR) studies around the discovery
of AC1903.8
Scheme 1. Synthesis of the target benzimidazoles.
In addition to the benzimidazoles, we also evaluated 3H-
imidazo[4,5-b]pyridines (6s,t) and the synthesis of these analogs
are shown in Scheme 2. The 2-chloro-3-nitropyridine, 8 starting
material was reacted with an appropriate benzyl amine (Cs2CO3,
o
100 C, 2 h) to yield 9.10 The resulting nitropyridine, 9, was
reduced (H2, Pd/C) to yield the dianiline, 10, which was converted
to the imidazo[4,5-b]pyridine-2-one, 11, with N,N’-
carbonyldiimidazole. Compound 11 was converted to the 2-chloro
o
compound (POCl3, PCl5, 90 C) which was then reacted with
o
furfurylamine under microwave heating (W, 200 C, 30 min)
yielding the final targets, 6s,t.
Figure 1. The structure of the quinoline TRPC5 inhibitor,
ML204, 1.
From a high-throughput screen performed at the Johns Hopkins
Ion Channel Center (JHICC) utilizing the >300,000 NIH
Molecular Library Small Molecule Repository (MLSMR)
compound collection, we identified a number of benzimidazole
containing compounds as TRPC4/5 inhibitors for transition into a
lead optimization campaign.9 These molecules afforded an
excellent starting point for a medicinal chemistry effort as they
were low MW (<300), offered multiple points of diversification,
and satisfied many parameters for tool compound development
(e.g., cLogP <3). As exemplified in the optimized compound 2,
the areas for chemical modification were the left-hand phenyl of
the benzimidazole, the 2-position of the benzimidazole, and the
southern benzyl moiety.
Scheme 2. Synthesis of 3H-imidazo[4,5-b]pyridines, 6s,t.
The SAR of the 2-benzimidazole position is outlined in Table
1. The furfuryl amine substituent, 2, was used as the standard
control and the % inhibition at 3 M was normalized to 100%. We
utilized a first triage of % inhibition of the TRPC5 channel using
the Syncropatch instrument in order to have a higher throughput

capacity for SAR, which is then followed up with IC50
determinations of active compounds. Knowing the furan moiety
is prone to oxidation and thus metabolic instability, we set out to
investigate furan replacements. Replacing the furan with 2-pyridyl
compound (5a, -1.8% inhibition) was not a productive change,
neither were any of the other pyridine regioisomers (5h, 5i).
Moving from the furan to the 2-thiophene led to a compound that
was active (5b, 150% inhibition). Replacing the 2-thiophene with
a phenyl bioisostere led to a reduction in activity (5e, 33%
inhibition). In addition, replacing the methylene linker with a
carbonyl (amide) or capping the free NH with a methyl also led to
less active compounds (5c, 5d). In addition, removing the
methylene linker (5f, 5g) or removing the amino substituent (Me,
5j) were not productive. A variety of 5-memebered aryl
heterocyclic replacements were investigated as furan alternative
(5k-u). Unfortunately, all of the thiazole, thiadiazole and oxazole
isomers led to reduced potency compounds. The only two changes
that produced active compounds were the 3-methyl-2-furyl
derivative (5q, 82% inhibition) and the methine branched furan
(5u, 91% inhibition).
5n
5o
5p
19.8 ± 4.6
35.7 ± 7.4
47.0 ± 3.4
5q
5r
5s
5t
82.3 ± 4.9
33.3 ± 4.7
19.1 ± 3.5
42.5 ± 3.5
91.4 ± 6.6
5u
Table 1. SAR evaluation of the 2-benzimidazole moiety.
aThe % inhibition values were recorded in cells (n > 10)
using the Syncropatch (Nanion). Experiments were
recorded (minimum) on one session over two different
assay plates. AC1903 and DMSO were added as control
compounds on every assay plate.
Next, we evaluated the southern benzimidazole substituents by
keeping the furan-2-ylmethyl amine group constant (Table 2).
Moving from the benzyl group to a propyl led to a slight decrease
in activity (6a) and further modification with an ethyl morpholine
provided an inactive compound (6b, 34% inhibition). Additional
moieties that contained basic amines (piperdine) were also not
tolerated (data not shown). Further modification of the alkyl
substituents with cyclic moieties yielded moderately active
compounds, with the exception of the cyclobutene derivative (6d,
117% inhibition). Adding a phenyl ring to the 3-atom spacer
brought back the activity (6f, 91% inhibition). We also evaluated
a number of phenyl substituted analogs (6g-r). Most of these
analogs were of moderate activity (~50-70% inhibition), with a
few highlighted exceptions. The 2-chlorophenyl (6i, 2.6%) and
the 2-chloro-4-fluorophenyl (6r, 24% inhibition) were much less
active than other groups – even less active than the 2-fluoro
substituted analogs (e.g., 6h, 6q). The best compounds from this
evaluation were 4-fluorophenyl (6o, 167% inhibition) and the 4-
chloro-2-fluorophenyl (6q, 128% inhibition). Interestingly,
changing the core scaffold from the benzimidazole to the
azabenzimidazole (6s and 6t) led to compounds with reduced
activity. Lastly, addition of substituents on the phenyl ring of the
benzimidazole (positions 5- and 6-) showed a noticeable trend.
The 5,6-dichlorobenzimidazole (7a, 28% inhibition) was
significantly less active than the 5,6-dimethylbenzimidazole (7b,
123% inhibition) suggesting an electronic effect of the substituent
groups.
% Inhibition
Syncropatch (@ 3 M,
Cmpd
2
R
± SEM)a
100
5a
5b
5c
-1.8 ± 7.9
150 ± 4.0
38.5 ± 3.8
5d
5e
41.7 ± 4.9
32.8 ± 5.4
5f
18.9 ± 6.1
5g
5h
19.9 ± 2.6
12.0 ± 4.4
5i
5j
17.4 ± 3.6
19.0 ± 3.7
Table 2. Evaluation of southern portion.
-CH3
5k
45.0 ± 4.4
5l
18.0 ± 7.0
30.0 ± 4.7
% Inhibition
Syncropatch (@ 3 M,
5m
Cmpd
R1
± SEM)a

2
100
6q
6r
128 ± 4.7
23.6 ± 3.5
6a
81.2 ± 6.4
6b
34.4 ± 4.0
6s
6t
31.2 ± 5.3
21.7 ± 6.4
6c
6d
6e
65.2 ± 8.1
117 ± 5.5
61.6 ± 4.5
Cl
Cl
N
N
O
O
NH
NH
7a
7b
28.2 ± 4.5
123 ± 3.7
6f
91.1 ± 4.7
61.9 ± 4.6
H3C
H3C
N
N
6g
aThe % inhibition values were recorded in cells (n > 10)
using the Syncropatch (Nanion). Experiments were
recorded (minimum) on one session over two different
assay plates. AC1903 and DMSO were added as control
compounds on every assay plate.
6h
6i
54.4 ± 4.5
2.6 ± 10.4
Having identified a number of compounds with activity as
TRPC5 inhibitors at 3 M, we next followed up these compounds
with IC50 determinations. Compound 2 (AC1903) had an IC50
=
4.06 M, as determined using the Syncropatch. This value is
comparable to the manual patch clamp historical value for this
compound.8 Most of the compounds tested had comparable IC50
values to 2. However, there are a few notable exceptions.
6j
50.5 ± 5.8
Compounds 6a (IC50 = 15.5 M), 6p (IC50 = 11.6 M), 6q (IC50
=
6k
6l
87.0 ± 6.4
70.9 ± 6.6
94.6 ± 3.4
18.1 M) and 7b (IC50 = 12.3 M) were less potent than 2. Thus,
the % inhibition values were mostly equivalent to 2 (with a couple
exceptions) and this translated to similar IC50 values. Some of the
minor assay discrepancies may be due to the fact that the %
inhibition assay is run as one concentration in one well versus
multiple concentrations in multiple wells for the IC50
determinations.
None of the synthesized analogs were
significantly improved over 2.
6m
Table 3. IC50 determinations of select compounds.
% inhibition at 3
6n
6o
6p
70.2 ± 3.9
167 ± 3.1
88.4 ± 4.5
TRPC5 IC50 (M ±
Cmpd
M
100
150
82.3
91.4
81.2
87.0
94.6
167
88.4
128
123
SD)
2
4.06 ± 0.91
6.26 ± 1.59
6.93 ± 1.53
4.44 ± 1.12
15.5 ± 5.4
8.42 ± 3.07
4.80 ± 1.14
1.82 ± 0.32
11.6 ± 3.9
18.1 ± 9.7
12.3 ± 7.0
5b
5q
5u
6a
6k
6m
6o
6p
6q
7b

AUC (hr⋅ng/mL)
1130
Again, to progress 2 into in vivo studies we performed an in
vivo pharmacokinetic study (IP dosing, 25 mg/kg, 0 – 24 h). The
results of this study are shown in Table 4. Compound 2 was shown
to have PK values similar to ML204 and based on these values we
progressed this compound into a variety of animal studies of
kidney disease (previously reported).8 In addition, 2 was shown to
be selective for TRPC5 over TRPC4 and TRPC6.8
We herein described the synthesis and biological evaluation of
series of 2-aminobenzimidazole compounds as TRPC5
a
inhibitors. The SAR revelaed that modifications or replacement
of the furan moiety were not tolerated. In addition, the southern
moiety SAR revealed that there is tolerance of many phenyl
analogs as well as non-phenyl ring systems (cyclobutane). Further
evaluation using the Syncropatch for IC50 determination revealed
that 2 (AC1903) was the best compounds that was identified.
Thus, we have revealed the synthesis and biological
characterization of the novel TRPC5 inhibitor 2 (AC1903) which
we’ve previously shown to be active in a number of animal models
of kidney disease. Further modifications of new TRPC5 inhibitors
will be reported in due course.
Table 4. In vivo PK for 2.
IP, 25 mg/kg, vehicle: 5% DMSO, 10% Tween80, 85%
PBS
Cmax (ng/mL)
Tmax (hr)
687
0.11
Acknowledgments
Supplementary Material
The authors thank Dr. Anna L. Blobaum (Vanderbilt
University) for the in vivo PK study of AC1903. The study was
supported by a grant from the NIH (NIDDK: R01DK103658) to
C. R. H.
Supplementary data (synthetic procedures) to this article can be
found online at.
9.
Miller, M.; Shi, J.; Zhu, Y.; Kustov, M.; Tian, J. B.; Stevens,
A.; Wu, M.; Xu, J.; Long, S.; Yang, P.; Zholos, A. V.; Salovich,
J. M.; Weaver, C. D.; Hopkins, C. R.; Lindsley, C. W.;
McManus, O.; Li, M.; Zhu, M. X. J. Biol. Chem. 2011, 286
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Design, synthesis and characterization of
novel N-heterocyclic-1-benzyl-1H-
benzo[d]imidazole-2-amines as selective
TRPC5 inhibitors leading to the
identification of the selective compound,
AC1903
Leave this area blank for abstract info.
Swagat H. Sharma, Juan Lorenzo Pablo, Monica Suarez Montesinos, Anna Greka, Corey R. Hopkins