Identification of (R)-(2-Chloro-3-(trifluoromethyl)phenyl)(1-(5-fluoropyridin-2-yl)-4-methyl-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (JNJ 54166060), a Small Molecule Antagonist of the P2X7 receptor

Article abstract of DOI:10.1021/acs.jmedchem.6b00989

The synthesis and SAR of a series of 4,5,6,7-tetrahydro-imidazo[4,5-c]pyridine P2X7 antagonists are described. Addressing P2X7 affinity and liver microsomal stability issues encountered with this template afforded methyl substituted 4,5,6,7-tetrahydro-imidazo[4,5-c]pyridines ultimately leading to the identification of 1 (JNJ 54166060). 1 is a potent P2X7 antagonist with an ED₅₀ = 2.3 mg/kg in rats, high oral bioavailability and low-moderate clearance in preclinical species, acceptable safety margins in rats, and a predicted human dose of 120 mg of QD. Additionally, 1 possesses a unique CYP profile and was found to be a regioselective inhibitor of midazolam CYP3A metabolism.

Full text of DOI:10.1021/acs.jmedchem.6b00989

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Article
The Identification of (R)-(2-chloro-3-(trifluoromethyl)phenyl)
1-(5-fluoropyridin-2-yl)-4-methyl-6,7-dihydro-1H-
(
imidazo[4,5-c]pyridin-5(4H)-yl)methanone (JNJ 54166060),
a small molecule antagonist of the P2X7 receptor
Devin Maun Swanson, Brad M. Savall, Kevin J Coe, Freddy Schoetens, Tatiana
Koudriakova, Judith Skaptason, Jessica Wall, Jason C. Rech, Xiahou Deng, Meri De
Angelis, Anita Everson, Brian Lord, Qi Wang, Hong Ao, Brian Scott, Kia Sepassi, Timothy
W Lovenberg, Nicholas I. Carruthers, Anindya Bhattacharya, and Michael A Letavic
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00989 • Publication Date (Web): 22 Aug 2016
Downloaded from http://pubs.acs.org on August 23, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
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produced by employees of any Commonwealth realm Crown government in the course
of their duties.

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The Identification of (R)-(2-chloro-3-(trifluoromethyl)phenyl)(1-(5-fluoropyridin-2-yl)-4-
methyl-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (JNJ 54166060), a small
molecule antagonist of the P2X7 receptor
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†,
†
†
†
Devin M. Swanson, * Brad M. Savall, Kevin J. Coe, Freddy Schoetens, Tatiana
†
†
†
†
†
‡
Koudriakova, Judith Skaptason, Jessica Wall, Jason Rech, Xiahou Deng, Meri De Angelis,
†
†
†
†
†
†
Anita Everson, Brian Lord, Qi Wang, Hong Ao, Brian Scott, Kia Sepassi, Timothy W.
†
†
†
†
Lovenberg, Nicholas I. Carruthers, Anindya Bhattacharya, and Michael A. Letavic
†
Janssen Pharmaceutical Research & Development, LLC, 3210 Merryfield Row, San Diego, CA,
92121 USA
‡
Janssen Research & Development, Discovery Sciences, A Division of Janssen-Cilag, Jarama
7
5, 45007 Toledo, Spain
Abstract
The synthesis and SAR of a series of 4,5,6,7ꢀtetrahydroꢀimidazo[4,5ꢀc]pyridine P2X7
antagonists are described. Addressing P2X7 affinity and liver microsomal stability issues
encountered with this template afforded methyl substituted 4,5,6,7ꢀtetrahydroꢀimidazo[4,5ꢀ
c]pyridines ultimately leading to the identification of 1 (JNJ 54166060). 1 is a potent P2X7
antagonist with an ED = 2.3 mg/kg in rat, high oral bioavailability and lowꢀmoderate clearance
5
0
in preꢀclinical species, acceptable safety margins in rat, and a predicted human dose of 120 mg
QD. Additionally, 1 possesses a unique CYP profile and was found to be a regioselective
inhibitor of midazolam CYP3A metabolism.
Introduction
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The P2X7 receptor is an ATP gated, purinergic ion channel that controls the release of
pro inflammatory cytokines (ILꢀ1β, ILꢀ18) in both the periphery and the central nervous system.
The P2X7 receptor belongs to the P2X family of ionotropic receptors and is expressed
peripherally in hematopoietic cells such as lymphocytes, monocytes, and macrophages and
1
0
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centrally in microglia and astrocytes. Of the seven subtypes, the P2X7 receptor possesses the
lowest affinity for ATP, and thus under normal circumstances, the receptor remains silent. Upon
increasing ATP concentrations the P2X7 receptor is activated and triggers a proinflammatory
2
+
3, 4
cascade via Ca influx and subsequent ILꢀ1β release. In addition to ILꢀ1β signaling, P2X7
5ꢀ8
dependent release of glutamate, GABA, cathepsins, and chemokines have all been reported.
The therapeutic importance of a P2X7 antagonist in humans is currently being assessed.
At least two peripherally restricted compounds and one centrally penetrant compound have
progressed into clinical trials. Pfizer reported their clinical candidate was tolerated but failed to
9
show efficacy for the treatment of rheumatoid arthritis, Astra Zeneca demonstrated efficacy in a
1
0
proof of concept study in Crohn’s disease, and GSK published safety and tolerability data for
11
GSK 1482160. Our interest in the P2X7 receptor is focused on its central role in
neuroinflammation, specifically the relationship between P2X7 antagonism, ILꢀ1β suppression,
and improvements in the symptoms of depression. Indeed several laboratories have shown that
ILꢀ1β mediates stress induced depression and anhedonia in mice while P2X7 knockouts are
1
2, 13
protected from depression like behavior.
Recently, GSK and our own laboratories have disclosed a series of 5,6,7,8ꢀtetrahydroꢀ
1,2,4]triazolo[4,3ꢀa]pyrazines (1,2,4ꢀtriazolopyrazines), 4,5,6,7ꢀtetrahydroꢀ1Hꢀ
[
[
1,2,3]triazolo[4,5ꢀc]pyridines (1,2,3ꢀtriazolopyridines), and related P2X7 antagonists with
1
4ꢀ19
reasonable physiochemical properties and demonstrated target engagement in rat (Figure 1).
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The 1,2,4ꢀtriazolopyrazines (2,4,5) and 1,2,3ꢀtriazolopyridine (3) were shown to be potent
human P2X7 antagonists that found utility as in vivo tool compounds for the program. The SAR
2
0
from these series are summarized in a recent review. As an extension of this work the 4,5,6,7ꢀ
tetrahydroꢀ1Hꢀimidazo[4,5ꢀc]pyridine (imidazopyridine) core was explored to advance novel
P2X7 chemotypes with an improved ADME and safety profile. Herein we report the synthesis
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and SAR of the imidazopyridine series and the preclinical profile of 1 (JNJ 54166060).
Figure 1. 1 (JNJ 54166060) and previously disclosed triazolopyrazines and triazolopyridines (2ꢀ
).
5
Cl
O
R*
F3C
N
N
N
N
F
1
(JNJ 54166060)
Cl
O
Cl
O
Cl
O
Cl
O
F3C
F3C
N
N
N
Cl
N
N
N
F3C
N
S
N
N
N
N
S
N
N
N
N
N
N
N
N
N
N
2
F
3
5
4
hP2X7 IC50 = 14 nM
rP2X7 IC50 = 6400 nM
hP2X7 IC50 = 4 nM
rP2X7 IC50 = 7 nM
hP2X7 IC50 = 0.7 nM
rP2X7 IC50 = 79 nM
hP2X7 IC50 = 8 nM
rP2X7 IC50 = 8 nM
Results and Discussion
Our initial exploration into the imidazopyridine core involved first replacing the triazole
in compounds 2 and 3 with a 1Hꢀimidazole. Starting from commercially available 1Hꢀ
imidazo[4,5ꢀc]pyridine (6), we arylated the imidazole nitrogen using CꢀN coupling conditions to
provide 7a and 7b as a 1:1 mixture of regioisomers which were separable by silica gel
22
chromatography (Scheme 1). From 7a we reduced the imidazopyridine ring by forming an Nꢀ
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alkylpyridinium ion with BnBr followed by NaBH reduction to obtain 8. Hydrogenolysis of the
4
benzyl group and subsequent peptide coupling provided the final compounds, 10ꢀ17.
Scheme 1. Synthesis of imidazopyridine compounds 10-17.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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9
0
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0
1
2
3
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0
1
2
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4
5
6
7
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9
0
2
Reagents & Conditions: a) R ꢀX, Cu O, 8ꢀOH quinoline, Cs CO , DMSO, microwave, 1 h, 140 °C, 17ꢀ40% b)
2
2
3
1
BnBr, NaBH , DCM, MeOH, rt, 68ꢀ87% c) H , EtOH, Pd/C, rt, 24 h, 75ꢀ83% d) R COOH, HATU, Et N, DMF, rt,
4
2
3
22ꢀ96%
Results from our initial analogs established that changing the core from a triazolopyridine
to an imidazopyridine was tolerated with respect to hP2X7 affinity (Table 1). Thus compounds
10ꢀ17 all possessed a similar profile, with a hP2X7 IC < 100 nM and rP2X7 IC > 500 nM.
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1
2
Compound 12, where R = 2ꢀClꢀ3ꢀCF phenyl and R = 2ꢀpyridyl, provided the best combination
3
of human and rat P2X7 affinity with a hP2X7 IC = 4 nM and a rP2X7 IC = 609 nM. The
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0
50
measured disconnect between human and rat P2X7 affinity has been previously observed in the
triazolopyrazine and triazolopyridine templates as highlighted by compound 2. Additionally,
1
steep SAR was observed for the R space where small changes to the 2ꢀClꢀ3ꢀCF phenyl
3
substituent resulted in diminished hP2X7 affinity as seen with compounds 15ꢀ17. Further
profiling of these compounds identified microsomal stability as a liability for this template as
measured extraction ratios (ERs) from human and rat liver microsomes (HLM/RLM) were all
greater than 0.8 indicative of metabolic instability.
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Table 1. Human and rat P2X7 IC s and microsomal data for compounds 10-17.
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21
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hP2X7
rP2X7
HLM/
RLM
1
2
Compd
R
R
c
a
b
IC50 (nM)
IC50 (nM)
89
4006
2909
609
0.93/>0.92
0.94/0.89
0.91/>0.92
0.9/0.87
1
1
1
1
0
1
2
3
1
2
4
9
1782
2
8
2476
4872
0.84/0.87
1
4
1
8
14
80
ꢀꢀ
ꢀꢀ
15
>10,000
1
1
6
7
3
538
>10,000
ꢀꢀ
a
All compounds were found to be P2X7 antagonists using a human peripheral blood monocyte (HPBMC) assay
prior to testing in FLIPR for confirmation of activity and IC determination. The protocols for the HPBMC and
50
2+
b
FLIPR assays can be found in the Experimental Section. Human FLIPR pIC measured in a Ca flux assay. Rat
FLIPR measured in a Ca flux assay. Stability in rat and human liver microsomes as measured by extraction
50
2
+
c
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ratio(s). Primary assays run in triplicate with the mean value reported. Standard deviation in all cases was less than
twoꢀfold
.
In the 1,2,4ꢀtriazolopyrazine series (4), methyl substitution on the core improved rat
1
5
P2X7 affinity. As such we wished to explore the impact of similar substitution on the
imidazopyridine core and chose substitution on the 4ꢀ position due to synthetic accessibility. To
synthesize these analogs we utilized the route shown in Scheme 2 and kept the 2ꢀchloroꢀ3ꢀ
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trifluoromethylaryl group (R ) constant due the SAR observed in Table 1. Here, we added a
methyl Grignard to an Nꢀacylpyridinium ion, formed by 7a and an acid chloride (18), to affect a
2
3
1
,2 addition which gave intermediate 19. This intermediate could then be reduced under
hydrogenation conditions to provide the desired compounds, 20-27, as racemic mixtures.
Racemates of interest were then separated by chiral chromatography.
Scheme 2. Synthesis of imidazopyridine compounds 1 and 20-30.
Cl
O
Cl
O
F3C
N
N
R2
F3C
N
N
R2
N
N
R2
N
a
N
b
N
0-27
c
7a
19
2
Cl
O
Cl
O
S
R
F3C
F3C
N
N
N
N
N
+
N
R2
R2
1, 28-30
Reagents & Conditions: a) 2ꢀClꢀ3ꢀCF3PhCOCl (18), MeMgBr, THF, rt, 3 h, 33ꢀ54% b) H , EtOH, Pd/C, 24 h, 20ꢀ
2
62% c) SFC chiral chromatography, 40ꢀ42%
Shown in Table 2 are the human and rat P2X7 IC s and microsomal stability data for
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compounds 20-27. Compound 21 provided the first indication of increased microsomal stability
within the series. Thus going from the unsubstituted phenyl (20) to a 4ꢀF phenyl (21) saw
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measurable improvement in stability with 21 possessing a HLM/RLM ER = 0.32/0.59. The 4ꢀF
pyrimidine derivative (23) and 4ꢀF pyridine derivative (27) were also equally stable in vitro and
both compounds possessed improved target affinity with a hP2X7 IC = 10 and 14 nM,
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respectively. Modest gains in rat affinity were observed with 4ꢀMe substitution on the
imidazopyridine core. Compounds 20, 25, and 27 were two to four fold more active at the rat
P2X7 receptor over their des methyl equivalents (11, 12, and 14) and prompted additional
investigation of the single enantiomers.
Table 2. Human and rat P2X7 IC s and liver microsomal stability data for compounds 20-27.
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hP2X7
rP2X7
HLM/
RLM
2
Compd
R
c
a
b
IC50 (nM)
IC50 (nM)
5
766
0.75/0.73
0.32/0.59
2
2
0
1
7
8
1182
3
1
0
0
113
0.79/0.80
<0.3/0.54
22
3828
2
3
77
1225
295
0.77/0.84
0.74/0.89
2
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2319
592
0.81/0.81
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4
<0.3/0.52
27
0
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0
a
2+
b
2+
c
Human FLIPR pIC measured in a Ca flux assay. Rat FLIPR measured in a Ca flux assay. Stability in rat and
human liver microsomes reported as hepatic extraction ratio(s). Primary assays have been run in triplicate with the
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mean value reported. Standard deviation in all cases was less than twoꢀfold
.
Four of the racemic imidazopyridine analogs (21, 23, 25, 27) were purified by chiral
HPLC and the enantiomers were submitted for testing (Table 3). One enantiomer from each
racemate was responsible for P2X7 affinity (1, 28-30) with the other enantiomer possessing
human and rat P2X7 IC s > 1000 nM (data not shown). Importantly, the rat affinity for the
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active enantiomers was improved over the racemates with rP2X7 IC s now in the 100 to 300 nM
50
range. Mouse P2X7 IC s and human P2X7 K s were generated for 1, 28-30 and were found to
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i
2+
be in good agreement with human Ca mobilization data. The active enantiomer of 27, 1 (JNJ
54166060), possessed single digit potency in both human functional and binding assays with an
hP2X7 IC = 4 nM and hP2X7 K = 7 nM.
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i
Table 3. Human, rat, and mouse P2X7 data for compounds 1, 28ꢀ30.
hP2X7
rP2X7
hP2X7
2
mP2X7 c
IC50 (nM)
Compd
R
a
b
d
IC50 (nM)
IC50 (nM)
K (nM)
i
6
7
303
45
40
2
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34
300
38
22
3
0
a
2+
b
2+
c
Human FLIPR pIC measured in a Ca flux assay. Rat FLIPR measured in a Ca flux assay. Mouse FLIPR
measured in a Ca flus assay. Human K measured with [ H]ꢀ5 as the binding ligand. All assay data has been run in
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2+
d
3
i
triplicate with the mean value reported. Standard deviation in all cases was less than twoꢀfold
.
To confirm the structural assignment of our compounds we submitted 1 and its
enantiomer (31) for absolute stereochemical determination via Xꢀray crystallography. The single
crystal Xꢀray structures for the two compounds are shown in Figure 2. The absolute
2
4
stereochemistry of the compounds was determined as depicted with the Flack parameter
=
ꢀ0.004 and ꢀ0.015 for 1 and 31, respectively. There were no hydrogen bond interactions or
solvates within the crystals with the pyridine and phenyl substituents off the imidazopyrazine
core adopting planar geometry. The asymmetric unit contained one disordered molecule of 1 and
one fully ordered molecule of 31.
Figure 2. Single crystal structures and stereo chemical assignment of 1 and 31.
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2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
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9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
Further in vitro characterization of 1 and 28ꢀ30 are shown in Table 4. The compounds
were assessed for liver microsomal stability (HLM/RLM), plasma protein binding (PPB),
cytochrome P450 (CYP) inhibition (5 isoforms), solubility (pH2/pH7), and counter screened
against the hERG channel. The compounds tested possessed no CYP or hERG liabilities,
measurable solubility, and moderate free fraction in both human and rat plasma. Improvements
in microsomal stability were observed with 4ꢀF substitution at the R2 substituent with the
nitrogen containing heterocycles enhancing solubility across physiologically relevant pH ranges.
Indeed a metabolite identification study on 1 in liver microsomes revealed no oxidation on the
fluoropyridine substituent with the major metabolite in human being oxidation on the
imidazopyridine core. Subsequent isozyme mapping in recombinant human CYPs determined
the metabolism of 1 is primarily driven by the CYP3A isoform. There was no evidence of
CYP3A time dependent inhibition (TDI) for 1 up to concentrations of 10 ꢁM. In combination
with in vitro potency, 1 was identified as a lead candidate for the P2X7 program due to a number
of favorable properties including metabolic stability in HLMs, high permeability with no
evidence of efflux in Cacoꢀ2 cells, moderate free fractions in plasma, no hERG liability,
measurable solubility, and P2X7 selectivity as assessed by a CEREP panel and Upstate® kinase
panel (EurofinsꢀCEREP, www.eurofins.com, EMD Millipore, www.emdmillipore.com, no
inhibition >50% @ 1 uM). The full in vitro profile of 1 is shown in Figure 3.
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Table 4. ADME data for compounds 1, 28-30.
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Solub.
pH2/pH7
2
HLM/
RLM
b
CYPs IC50
(ꢁM)
hERG dofet
IC50 (ꢁM)
Compd
R
a
h/r PPB
c
d
(ꢁM)
0
.36/0.40
96/97.5
> 10
> 10
165/14
2
8
0
0
.63/0.85
.35/0.64
95.3/97.4
94.5/94.9
> 10
> 10
> 10
> 10
>400/20
>400/29
29
1
<
0.3/0.73
95.2/96.2
2C19 = 7.8
> 10
>400/4
30
a
b
Stability in rat and human liver microsomes reported as hepatic ratio(s). Human and rat plasma protein
binding reported as % bound. Screening CYP inhibition data obtained from human liver microsomes. hERG IC
c
d
50
3
as measured in an [ H]astemizole competition binding assay in HEKꢀ293 cells expressing the hERG channel.
Figure 3. In vitro profile of 1.
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0
To gauge the viability of 1 for a CNS indication, central target engagement was assessed
via ex vivo P2X7 occupancy in rat brain hippocampal tissue sections using tritiated 5 as the
2
5
radiotracer. Following oral administration, 1 exhibited dose dependent occupancy in rat brain
with an ED = 2.3 mg/kg corresponding to an EC = 125 ng/ml and 362 ng/ml in the plasma
5
0
50
and brain, respectively. Although the brain concentrations are ~3 fold greater than plasma levels,
free plasma and free brain concentrations are similar after correcting for brain binding (1.5%
free) and plasma protein binding (5.5% free) with an unbound brain to plasma partition ratio (Kp
u,u) of 0.84. Such a Kpu,u suggests a limited impact of efflux, in line with Cacoꢀ2 in vitro data.
Additionally, the free IC s from this study match in vitro P2X7 potency where the unbound
5
0
plasma IC = 6.4 ng/mL (15 nM) and the unbound brain IC = 5.4 ng/mL (12 nM) (Figure 4).
5
0
50
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Figure 4. Ex vivo P2X7 occupancy with compound 1 in rat brain; dose dependency following
oral administration (n = 3 per dose ± SEM). P2X7 occupancy was measured 30 min after drug
administration using tritiated 5 as the radiotracer.
1
00
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0
ED = 2.3 mg/kg, p.o.
5
0
80
EC (plasma) = 125 ng/mL
50
7
6
0
0
EC (brain) = 362 ng/mL
5
0
50
4
3
2
1
0
0
0
0
0
0
.001
0.01
0.1
1
10
100
Dose (mg/kg)
The pharmacokinetic parameters for 1 in rat, dog, and monkey are shown in Table 5.
Across the three species, there is a strong correlation between predicted and measured clearance
values. The predicted in vitro clearance from liver microsomal data for 1 was within 2ꢀfold from
the clearance observed in vivo: 35 mL/min/kg vs. 30 mL/min/kg measured for rat; 10 mL/min/kg
vs. 5.5 ml/min/kg measured for dog; and 18 ml/min/kg vs. 14 ml/min/kg measured for
cynomolgus monkey. High oral bioavailability was observed across three species (%F > 50) with
the Cmax achieved at 1 h from oral administration. Given the high degree of concordance between
in vitro and in vivo clearance values across three preꢀclinical species, allometric scaling was
considered suitable to predict the human dose of 1. Since the human and rat P2X7 K and PPB
i
values were similar (r/h P2X7 K = 7 nM, r/h PPB ~ 95%) we used the plasma EC from the rat
i
50
dose response experiment of 125 ng/mL as the trough for human plasma concentration. To
maintain the plasma concentration above 125 ng/mL a dose of 120 mg QD was predicted for 1 in
human with a CL = 2.8 mL/min/kg, Vss = 2.0 L/kg, t1/2 = 8.3 hr, and F = 85%.
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1
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1
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1
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1
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2
2
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2
2
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3
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3
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3
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6
Table 5. Pharmacokinetic parameters of 1.
In vitro
IV
PO
from LMs
a
a
b
c
d
e
CL
CL
Vss
(L/kg)
3.8
3.6
2.1
t1/2
Cmax
tmax
(hr)
1
F
(%)
55
>100
54
(ml/min/kg) (ml/min/kg)
(hr)
1.7
11.9
4.2
(ng/mL)
375
1249
389
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Rat
Dog
Monkey
Human
predicted)
=
36.8
10.3
18.2
30
5.5
14
0.5
2.3
7.3
2.8
2.0
8.3
ꢀꢀ
ꢀꢀ
85
(
a
b
c
d
e
Clearance, = Volume of Distribution at steady state = IV half live, = Maximum concentration = Time of
maximum concentration
As part of the preclinical toxicology package a single dose and repeat dose toleration
study were run in male rats with 1 (Figure 5). To meet the compound requirements for the
studies additional material was synthesized using Scheme 2 and the route was found to be
scalable. In the repeat dose study, animals were administered 1 at 62.5, 125 or 250 mg/kg/day for
4
days with necropsy performed on day 5. 1 was well tolerated in both the single and repeat dose
studies with adequate exposures and no noted CNS side effects. In the single dose experiment
increasing exposures were observed up to the 1000 mg/kg dose with a tmax of 24 hours for all
doses tested. In the repeat dose study no conclusive trends could be extrapolated from the 1 and 4
day exposure data. The histology findings in both studies were considered mild with doseꢀrelated
increases in total serum cholesterol observed at the 125 and 250 mg/kg/day repeat doses. In
addition, one rat dosed at 250 mg/kg/day showed minimal central lobular hepatocellular
degeneration and necrosis. Overall, the safety profile observed in the toleration studies supported
further development of the molecule.
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Figure 5. Single dose and repeat dose toleration studies of 1 in male rats.
1
Rat PO SD Toleration
Vehicle: 0.5% HPMC Suspension (10 mL/kg)
20% HPbCD Solution (10 mL/kg)
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1
00000
125 mg/kg Solution
50 mg/kg Suspension
00 mg/kg Suspension
1000 mg/kg Suspension
2
5
1
0000
1000
0
4
8
12
Time (hr)
16
20
24
1
Rat PO RD Toleration
Vehicle: 20%HPbCD (10 mL/kg) for 62.5 and 125 mg/kg doses
.5% HPMC (10 mL/kg) for 250 mg/kg dose
0
100000
62.5 mg/kg Day 1
62.5 mg/kg Day 4
1
0000
125 mg/kg Day 1
125 mg/kg Day 4
250 mg/kg Day 1
250 mg/kg Day 4
1000
100
0
4
8
12
Time (hr)
16
20
24
With the preclinical package for 1 nearing completion a discrepancy was identified in a
definitive CYP inhibition study. In this study, 1 was found to be a potent inhibitor of CYP3A
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with an IC = 2 ꢁM. This data was at odds with the original screening CYP inhibition data,
5
0
where 1 showed no significant CYP3A inhibition up to a concentration of 10 µM. Further
investigation of the screening CYP inhibition assay revealed that 1ꢀOHꢀmidazolam and 4ꢀOHꢀ
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midazolam, the two metabolites mediated by CYP3A, coꢀeluted in the LCꢀMS assay. When
these metabolites were chromatographically resolved, 1 was found to only inhibit the formation
of 1ꢀOHꢀmidazolam (IC = 2.2 ꢁM) and not 4ꢀOHꢀmidazolam (IC > 10 ꢁM) (Table 6). Thus
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the lack of inhibition of 4ꢀOHꢀmidazolam masked 1’s CYP3A inhibition when these metabolites
coꢀeluted in the screening assay. The CYP3A enzyme contains a large active site that bears
multiple binding orientations, such that the use of multiple CYP3A probes (e.g. midazolam,
nifedipine, and testosterone) are required to adequately assess the inhibitory potential of drugs.
However, to our knowledge this is the first report of the regioselective inhibition of midazolam
27
by a potential drug candidate. In contrast, ketoconazole, a widely recognized CYP3A inhibitor,
exhibited similar inhibition potency at 1ꢀOH midazolam and 4ꢀOH midazolam with IC values
50
of 9.1 and 7.4 nM, respectively. Overall, given the drugꢀdrug interaction risk, both as perpetrator
and as victim, with progressing a potent CYP3A inhibitor whose primary route of clearance is
CYP3A mediated; development of 1 was stopped in favor of alternative compounds.
Table 6. Percent inhibition of 1ꢀOH and 4ꢀOH midazolam fromation, 1 vs. Ketoconazole
1
Ketoconazole (CYP3A4 positive control)
1
-OH-
4-OH-
Midazolam
% inhibition
1-OH-
Midazolam
% inhibition
4-OH-
Midazolam
% inhibition
Concentration
ꢀM)
Concentration
Midazolam
% inhibition
(
(ꢀM)
0.313
.625
.25
.5
20
29
41
52
63
74
0.1
-5
0.005
0.01
0.02
0.04
0.08
0.16
34
53
70
82
90
94
37
57
74
85
92
95
0
1
-10
-13
-13
-4
2
5
10
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Conclusion
In summary, we have demonstrated that this series of novel 4,5,6,7ꢀtetrahydroꢀ
imidazo[4,5ꢀc]pyridines are potent and selective P2X7 antagonists. Optimization of the
imidazopyridine core led to compounds with improved liver microsomal stability and P2X7 rat
affinity. Compound 1 (JNJ 54166060) was identified as a lead candidate for the P2X7 program
with an excellent in vitro profile. In conjunction with ex vivo autoradiography and
pharmacokinetic parameters across three preꢀclinical species, we were able to predict the human
PK profile for 1 and this analysis suggested that 1 would have a human PK profile suitable to
treat depressive disorders (i.e. 24 h coverage, oral administration, brain penetration). Preclinical
toxicology established that 1 was well tolerated in rats with no behavioral side effects and no
major histopathology findings related to drug treatment. Finally, 1 was found to possess a unique
CYP profile and was determined to be a regioselective inhibitor of CYP3A; inhibiting the
formation of 1ꢀOH midazolam but not 4ꢀOH midazolam.
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Experimental Section
In obtaining the compounds described the following experimental and analytical protocols were
followed unless otherwise indicated. Anhydrous solvents were obtained from a GlassContour
solvent dispensing system. Reagents were purchased from commercial suppliers and were used
without purification. The reaction mixtures were magnetically stirred at room temperature (rt)
under a nitrogen atmosphere. Where solutions were “dried,” they were generally dried over a
drying agent such as Na SO or MgSO . Where mixtures, solutions, and extracts were
2
4
4
“concentrated”, they were typically concentrated on a rotary evaporator under reduced pressure.
Reactions under microwave irradiation conditions were carried out in a Biotage Initiator or CEM
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6
Discover instrument. Normalꢀphase silica gel column chromatography (sgc) was performed on
silica gel (SiO ) using prepackaged cartridges, eluting with 2 M NH /MeOH in CH Cl or EtOAc
2
3
2
2
in hexanes unless otherwise indicated. Nuclear magnetic resonance (NMR) spectra were
0
1
2
3
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0
1
obtained on Bruker model DRX spectrometers. The format of the H NMR data below is:
chemical shift in ppm downfield of the tetramethylsilane reference (multiplicity, coupling
constant J in Hz, integration). High resolution mass spectrometry data were obtained with a
Bruker ꢁTOf detector in positive mode, a Zorbax SBꢀC18 3.5 µM, 2.1 x 50 mm column at 40 °C,
an acetonitrile/water with 0.05% formic acid gradient over 7 min, and a flow rate of 0.3 mL/min.
Calculated (calcd.) mass corresponds to the exact mass. Chemical names were generated using
ChemDraw Ultra 6.0.2 (CambridgeSoft Corp., Cambridge, MA) or ACD/Name Version 9
(Advanced Chemistry Development, Toronto, Ontario, Canada). All compounds tested were of a
minimum of 98% purity as determined by HPLC. The HPLC methods used for purity
determinations are as follows: Analytical LCMS was obtained on an Agilent 1100/1200 series
LCMS with ESI in positive mode and a scan range of 100ꢀ700 amu. Samples were run on an
Halo C18 column (2.7um, 50 x 3 mm), with a mobile phase of 5ꢀ99% ACN in 0.05% TFA over
3.5 min and then held at 99% ACN for 0.5 min, at a flow rate of 1 mL/min (Temperature = 50
°
C). Analytical MS was obtained in flow injection mode on an Agilent 1100/1200 Series ESIꢀ
SQD in positive mode with a scan range of 100ꢀ1000 amu using a mobile phase of 75% ACN:
24.9% H2O: 0.1% TFA at a flow rate of 0.4 mL/min (Temperature = 50 °C). Chiral compounds
were analyzed by chiral HPLC or chiral SFC and demonstrated to have ≥ 98% ee.
Representative Procedure A. Step 1. 1-(pyridin-2-yl)-1H-imidazo[4,5-c]pyridine
(7a). A solution of 5ꢀazabenzimidazole (1.00 g, 8.40 mmol), 2ꢀbromopyridine (1.30 g, 8.40
mmol), copper (I) oxide (0.13 g, 0.84 mmol), 8ꢀhydroxyquinoline (0.24 g, 1.68 mmol), and
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Cs CO (5.50 g, 16.8 mmol) in DMSO (4 mL) was irradiated in a microwave apparatus for 1
2
3
hour at 140 °C. The reaction was diluted with H O (100 mL) and extracted with EtOAc (75 mL x
2
3
). The organic layers were combined, dried (Na SO ), and concentrated. Chromatography of the
2 4
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11
12
13
14
15
16
17
18
19
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1
resulting residue (SiO ; MeOH (NH ):DCM) gave the title compound (0.45 g, 25%). H NMR
2
3
(600 MHz, DMSO) δ 9.18 – 9.04 (m, 2H), 8.71 – 8.63 (ddd, J = 4.7, 2.0, 0.9 Hz, 1H), 8.54 –
.44 (d, J = 5.6 Hz, 1H), 8.32 – 8.23 (dd, J = 5.6, 1.0 Hz, 1H), 8.20 – 8.07 (ddd, J = 8.3, 7.4, 1.9
8
Hz, 1H), 8.05 – 7.97 (dt, J = 8.2, 1.0 Hz, 1H), 7.56 – 7.43 (ddd, J = 7.4, 4.9, 0.9 Hz, 1H). MS
+
(ESI): mass calculated for C H N , 196.07; m/z found 197.1 [M+H] .
1
1
8
4
Step 2. 5-Benzyl-1-(pyridin-2-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine (8).
To a solution of 7a (0.20 g, 1.02 mmol) in DCM (25 mL) was added benzyl bromide (0.12 g,
1.02 mmol). The reaction was let stir for 4 h then concentrated. The resulting solid was dissolved
in MeOH (10 mL) and NaBH (0.05 g, 1.4 mmol) was added slowly. After 5 h, the reaction was
4
quenched with a small amount of water and concentrated. Chromatography of the resulting
1
residue (SiO ; MeOH (NH ):DCM) gave the title compound (0.20 g, 68%). H NMR (500 MHz,
2
3
CDCl ) δ 8.76 – 8.66 (m, 1H), 8.55 – 8.39 (m, 2H), 8.14 – 8.03 (m, 1H), 7.32 (ddt, J = 22.0,
3
1
1.6, 7.5 Hz, 6H), 3.89 (s, 1H), 3.78 (d, J = 5.4 Hz, 2H), 3.63 (s, 1H), 2.99 (t, J = 5.5 Hz, 1H),
.83 (tt, J = 26.6, 5.6 Hz, 3H). MS (ESI): mass calculated for C H N , 290.2; m/z found 291.2
2
1
8
18
4
+
[
M+H] .
Step 3. 1-(Pyridin-2-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine (9). To a
solution of 5ꢀbenzylꢀ1ꢀ(pyridinꢀ2ꢀyl)ꢀ4,5,6,7ꢀtetrahydroꢀ1Hꢀimidazo[4,5ꢀc]pyridine (0.13 g, 0.45
mmol) in DCE (5 mL) was added 1ꢀchloroethyl chloroformate (0.10 mL , 0.90 mmoL). The
reaction was let stir for 15 min then heated at reflux for 4 h. The reaction was let cool,
concentrated, dissolved in MeOH and heated again at 60 °C for 1 h. The reaction was
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5
5
5
5
5
5
5
6
concentrated and the product was used in the next step without further purification (0.075 g,
1
8
3%). H NMR (400 MHz, DMSO): δ 9.17 (s, 2H), 9.04 (d, J = 4.9 Hz, 1H), 7.72 ꢀ7.28 (m, 2H),
4.46 (t, J = 1.3 Hz, 2H), 3.49 (t, J = 6.0 Hz, 2H), 3.38 (s, 2H). MS (ESI): mass calculated for
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
C H N , 200.1; m/z found 201.2 [M+H] .
11
12
4
Step 4. (2-Chloro-3-(trifluoromethyl)phenyl)(1-(pyridin-2-yl)-6,7-dihydro-1H-
imidazo[4,5-c]pyridin-5(4H)-yl)methanone (12). A solution of 1ꢀ(pyridinꢀ2ꢀyl)ꢀ4,5,6,7ꢀ
tetrahydroꢀ1Hꢀimidazo[4,5ꢀc]pyridine (9) (0.050 g, 0.25 mmol), 2ꢀchloroꢀ3ꢀtrifluoromethyl
benzoic acid (0.056 g, 0.25 mmol), HATU (0.10 g, 0.26 mmol, and DIPEA (0.09 mL, 0.50
mmol) in DMF (2 mL) was stirred for 30 min. The reaction was diluted with EtOAc (15 mL) and
washed with H O (3 x 10 mL). The organic layers were combined, dried (Na SO ), and
2
2
4
concentrated. Chromatography of the resulting residue (SiO ; MeOH (NH ):DCM) gave the title
2
3
1
compound (22 mg, 22%). H NMR (400 MHz, CDCl ) δ 8.52 (dd, J = 4.9, 1.8 Hz, 1H), 8.02 (d,
3
J = 18.1 Hz, 1H), 7.92 – 7.71 (m, 2H), 7.58 – 7.40 (m, 2H), 7.37 – 7.28 (m, 2H), 5.12 – 4.78 (m,
1H), 4.51 – 4.19 (m, 2H), 3.97 (ddd, J = 13.4, 12.7, 9.9 Hz, 1H), 3.61 – 3.45 (m, 1H), 3.27 – 2.82
+
(
m, 2H). HRMS calc. for C H ClF N O [M+H] 407.0881, found 407.0869.
1
9
14
3
4
Representative Procedure B. Step 1. 2-Chloro-3-(trifluoromethyl)benzoyl chloride
(18). To a suspension of 2ꢀchloroꢀ3ꢀ(trifluoromethyl)benzoic acid (15 g, 67 mmol) and catalytic
DMF (0.06 mL, 0.67 mmol) in DCM (150 mL) was added oxalyl chloride (6.8 mL, 80 mmol)
dropwise. The reaction was let stir (vigorous bubbling) for 4 h and concentrated to an oily solid
which became solid after overnight drying on high vacuum.
Step 2. (2-chloro-3-(trifluoromethyl)phenyl)(1-(5-fluoropyridin-2-yl)-4-methyl-1H-
imidazo[4,5-c]pyridin-5(4H)-yl)methanone (19). To a solution of 7a (0.70 g, 3.27 mmol) in
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THF (20 mL) was added 18 (0.87 g, 3.60 mmol) dropwise. The reaction was let stir for 1 h then
cooled to – 78 °C. To the cooled solution was added 3M MeMgBr in Et O (1.31 mL, 3.92
2
mmoL) and the reaction was let come to room temperature. The mixture was then quenched with
10
11
12
13
14
15
16
17
18
19
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60
1
N NaOH (50 mL) and extracted with EtOAc (3 x 30 mL). The organic layers were combined,
dried (Na SO ), and concentrated. Chromatography of the resulting residue (SiO ; MeOH
2
4
2
1
(NH ):DCM) gave the title compound (770 mg, 54%). H NMR (400 MHz, CDCl ) δ 8.43 – 8.34
3 3
(m, 1H), 7.92 – 7.73 (m, 2H), 7.70 – 7.33 (m, 4H), 6.08 (dtd, J = 19.7, 11.7, 8.0 Hz, 3H), 1.54 (t,
J = 7.0 Hz, 3H). MS (ESI): mass calculated for C H ClF N O, 436.07; m/z found 437.1
2
0
13
4
4
+
[
M+H] .
Step 3. (2-chloro-3-(trifluoromethyl)phenyl)(1-(5-fluoropyridin-2-yl)-4-methyl-6,7-
dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (27). To a solution of 19 (0.80 g, 1.83
mmol) in degassed EtOH (25 mL) was added palladium on carbon (0.20 mg, 0.19 mmol). The
reaction was placed under an atmosphere of hydrogen and let stir for 48 h. The reaction was
diluted with DCM and filtered through a pad of Celite ©. The solvent was concentrated and
chromatography of the resulting residue (SiO ; MeOH (NH ):DCM) gave the title compound
2
3
(500 mg, 62%). 1H NMR (500 MHz, CDCl3) δ 8.45 – 8.30 (m, 1H), 7.94 (dd, J = 18.2, 10.7 Hz,
1
H), 7.76 (d, J = 5.7 Hz, 1H), 7.67 – 7.43 (m, 3H), 7.43 – 7.30 (m, 1H), 5.81 (dd, J = 13.3, 6.7
Hz, 1H), 5.07 (d, J = 5.6 Hz, 1H), 4.52 (d, J = 6.7 Hz, 1H), 3.61 – 3.31 (m, 1H), 3.08 – 2.69 (m,
+
1H), 1.63 ꢀ 145 (m, 3H). HRMS calc. for C H ClF N O [M+H] 439.0943, found 439.0957.
2
0
13
4
4
Step 4. (R)-(2-chloro-3 (trifluoromethyl)phenyl)(1-(5-fluoropyridin-2-yl)-4-methyl-
,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (1). The title compound was
6
obtained as a single enantiomer by Chiral SFC purification performed using CHIRALCEL ODꢀH
(5 µm, 250x20 mm) and a mobile phase of 72% CO , 28% 1:1 EtOH:iPrOH. The enantiomeric
2
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purity was confirmed by analytical SFC using Whelkꢀal (S,S) (250x4.6 mm) and a mobile phase
of 60% CO , 40% MeOH over 7 minutes (100% single enantiomer, 4.03 min retention time)
2
1
(
0.16 g, 41%). H NMR (500 MHz, CDCl ) δ 8.43 – 8.32 (m, 1H), 7.94 (dd, J = 18.3, 11.2 Hz,
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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9
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8
9
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1H), 7.80 – 7.70 (m, 1H), 7.65 – 7.29 (m, 4H), 5.91 – 5.74 (m, 1H), 5.14 – 4.46 (m, 1H), 3.60 –
1
3
3.30 (m, 1H), 3.30 – 3.04 (m, 1H), 3.06 – 2.68 (m, 1H), 1.76 – 1.35 (m, 3H). C NMR (126
MHz, CDCl ) δ 165.95 – 165.79 (s), 139.33 – 139.10 (d, J = 8.8 Hz), 137.50 – 137.15 (m),
3
135.54 – 135.31 (m), 131.24 – 131.08 (s), 130.68 – 130.52 (s), 130.42 – 130.26 (s), 128.08 –
127.83 (t, J = 5.7 Hz), 127.50 – 127.13 (m), 126.23 – 125.85 (dd, J = 20.4, 7.6 Hz), 123.69 –
1
23.54 (s), 122.56 – 122.40 (s), 116.00 – 115.72 (m), 52.32 – 52.16 (s), 47.41 – 47.25 (s), 41.25
41.09 (s), 35.57 – 35.41 (s), 24.49 – 24.19 (m), 19.96 – 18.59 (m). HRMS calc. for
–
+
20
D
C H ClF N O [M+H] 439.0943, found 439.0957. Specific rotation: [α] – 52.6 (c 0.5,
20
13
4
4
CHCl3).
The following intermediates were prepared in a manner analogous to Representative
Procedure A. Step 1.
1
-Phenyl-1H-imidazo[4,5-c]pyridine. (40%). MS (ESI): mass calculated for C H N ,
12 9 3
+
1
95.08; m/z found 196.1 [M+H] .
1-(Pyrazin-2-yl)-1H-imidazo[4,5-c]pyridine. (24%). MS (ESI): mass calculated for
+
C H N , 197.07; m/z found 198.1 [M+H] .
10
7
5
1
-(5-Fluoropyridin-2-yl)-1H-imidazo[4,5-c]pyridine. (37%). MS (ESI): mass
+
calculated for C H FN , 214.07; m/z found 215.1 [M+H] .
11
7
4
1-(4-fluorophenyl)-1H-imidazo[4,5-c]pyridine. (34%) MS (ESI): mass calculated for
+
C H FN , 213.07; m/z found 214.1 [M+H] .
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8
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-(pyrimidin-2-yl)-1H-imidazo[4,5-c]pyridine. (24%) MS (ESI): mass calculated for
+
C H N , 197.07; m/z found 198.1 [M+H] .
10
7
5
1-(5-fluoropyrimidin-2-yl)-1H-imidazo[4,5-c]pyridine. (17%) MS (ESI): mass
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
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59
60
+
calculated for C H FN , 215.06; m/z found 216.1 [M+H] .
1
0
6
5
1
-(3-fluoropyridin-2-yl)-1H-imidazo[4,5-c]pyridine. (20%) MS (ESI): mass calculated
+
for C H FN , 214.07; m/z found 215.1 [M+H] .
1
1
7
4
The following intermediates were prepared in a manner analogous to Representative
Procedure A. Step 2.
5
-Benzyl-1-phenyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine. (89%). MS (ESI):
+
mass calculated for C H N , 289.16; m/z found 290.0 [M+H] .
1
9
19
3
5
-Benzyl-1-(pyrazin-2-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine. (69%). MS
+
(
ESI): mass calculated for C H N , 291.15; m/z found 292.0 [M+H] .
17 17
5
5-Benzyl-1-(5-fluoropyridin-2-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine.
+
(
87%). MS (ESI): mass calculated for C H FN , 308.14; m/z found 309.2 [M+H] .
1
8
17
4
The following intermediates were prepared in a manner analogous to Representative
Procedure A. Step 3.
1
-Phenyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine. (79%). MS (ESI): mass
+
calculated for C H N , 199.11; m/z found 200.1 [M+H] .
1
2
13
3
1
-(Pyrazin-2-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine. (75%). MS (ESI):
+
mass calculated for C H N , 201.10; m/z found 202.1 [M+H] .
1
0
11
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5
5
5
5
6
1
-(5-Fluoropyridin-2-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine. (78%). MS
+
(
ESI): mass calculated for C H FN , 218.10; m/z found 219.1 [M+H] .
11 11
4
The following compounds were prepared in a manner analogous to Representative
Procedure A. Step 4.
0
1
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8
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0
(2,3-Dichlorophenyl)(1-phenyl-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-
1
yl)methanone (10). (75%). H NMR (400 MHz, CDCl ) δ 7.67 (s, 0.4H), 7.60 (s, 0.6H), 7.56 –
3
7
.39 (m, 4H), 7.33 – 7.20 (m, 5H), 4.96 – 4.90 (m, 1H) , 4.46 – 4.20 (m, 2H), 4.03 – 3.92 (m,
H), 3.60 – 3.43 (m, 1H), 2.67 (ddd, J = 20.9, 15.5, 6.8 Hz, 1H). HRMS calc. for C H Cl N O
1
1
9
15
2
3
+
[M+H] 372.0665, found 372.0682.
(2-Chloro-3-(trifluoromethyl)phenyl)(1-phenyl-6,7-dihydro-1H-imidazo[4,5-
1
c]pyridin-5(4H)-yl)methanone (11). (90%). H NMR (400 MHz, CDCl ) δ 7.79 – 7.74 (m, 1H),
3
7.68 (s, 0.4H), 7.60 (s, 0.6H), 7.56 – 7.39 (m, 5H), 7.34 – 7.28 (m, 2H), 4.99 – 4.90 (m, 1H) ,
4
.45 – 4.27 (m, 2H), 3.93 (ddd, J = 12.8, 7.2, 5.2 Hz, 1H), 3.60 – 3.45 (m, 1H). HRMS calc. for
+
C H ClF N O [M+H] 406.0929, found 406.0933.
20 15
3
3
(2-Chloro-3-(trifluoromethyl)pheny1)(1-(pyrazin-2-yl)-6,7-dihydro-lH-imidazo[
1
4
,5-c]pyridin-5(4H)-yl) methanone (13). (47%) H NMR (400 MHz, CDCl ) δ 8.81 – 8.75 (m,
3
1
H), 8.64 – 8.42 (m, 2H), 8.16 – 8.02 (m, 1H), 7.83 – 7.71 (td, J = 7.5, 1.9 Hz, 1H), 7.60 – 7.40
(m, 2H), 5.08 – 4.80 (m, 1H), 4.50 – 4.23 (m, 2H), 4.10 – 3.94 (ddd, J = 13.0, 6.6, 5.6 Hz, 1H),
.67 – 3.46 (m, 1H), 3.21 – 2.91 (m, 2H), 2.11 – 1.76 (m, 2H). HRMS calc. for C H ClF N O
3
1
8
13
3
5
+
[
M+H] 408.0833, found 408.0819.
(2-Chloro-3-(trifluoromethyl)phenyl)(1-(5-fluoropyridin-2-yl)-6,7-dihydro-lH-
1
imidazo[4,5-c]pyridin-5(4H)-yl)methanone (14). (45%) H NMR (400 MHz, DMSO) δ 8.59 –
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.51 (m, 1H), 8.28 – 8.17 (m, 1H), 8.04 – 7.92 (m, 2H), 7.84 – 7.63 (m, 3H), 4.91 – 4.54 (dd, J =
2.1, 16.0 Hz, 2H), 4.23 – 4.14 (d, J = 3.0 Hz, 1H), 3.51 – 3.36 (q, J = 5.9, 5.4 Hz, 2H), 3.08 –
+
+
.79 (m, 2H). [M+H] . HRMS calc. for C H CIF N O [M+H] 425.0787, found 425.0781.
1
9
13
4
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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31
32
33
34
35
36
37
38
39
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41
42
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46
47
48
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50
51
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(
1-(5-Fluoropyridin-2-yl)-4,5,6,7-tetrahydro-1H-benzo[d]imidazol-5-yl)(2-methyl-3-
1
(
trifluoromethyl)phenyl)methanone (15). (51%) H NMR (400 MHz, DMSO) δ 8.59 – 8.50
(
m, 1H), 8.28 – 8.16 (m, 1H), 8.06 – 7.94 (m, 1H), 7.85 – 7.74 (dt, J = 9.1, 4.7 Hz, 2H), 7.63 –
7
.46 (m, 2H), 4.95 – 4.48 (m, 1H), 4.22 – 4.06 (m, 1H), 3.49 – 3.37 (q, J = 4.8, 4.3 Hz, 2H), 2.43
+
– 2.20 (m, 3H). HRMS calc. for C H F N O [M+H] 405.1333, found 405.1332.
2
0
16
4
4
(2,4-Dichlorophenyl)(1-(5-fluoropyridin-2-yl)-6,7-dihydro-1H-imidazo[4,5-
1
c]pyridin-5(4H)-yl)methanone (16). (74%) H NMR (400 MHz, DMSO) δ 8.59 – 8.49 (m, 1H),
8
.29 – 8.14 (m, 1H), 8.05 – 7.95 (ddd, J = 6.8, 3.9, 2.2 Hz, 1H), 7.81 – 7.73 (m, 2H), 7.59 – 7.40
(
m, 2H), 4.85 – 4.51 (d, J = 16.0 Hz, 1H), 4.37 – 4.08 (m, 1H), 4.08 – 3.87 (m, 1H), 3.60 – 3.41
+
(
m, 1H), 3.07 – 2.83 (m, 2H). HRMS calc. for C H Cl FN O [M+H] 391.0523, found
1
8
13
2
4
391.0533.
(2-Fluoro-3-(trifluoromethy l)pheny1)(1-(5-fluoropyri- din-2-yl)-6,7-dihydro- lH-
1
imidazo[ 4,5-c]pyridin-5 (4H)-yl)methanone (17). (96%) H NMR (400 MHz, DMSO) δ 8.62
– 8.51 (m, 1H), 8.29 – 8.16 (m, 1H), 8.05 – 7.72 (m, 4H), 7.62 – 7.48 (m, 1H), 4.78 – 4.60 (s,
1
H), 4.36 – 4.23 (s, 1H), 4.06 – 3.97 (t, J = 5.7 Hz, 1H), 3.61 – 3.48 (t, J = 5.6 Hz, 1H), 2.96 (m,
+
2
H). HRMS calc. for C H F N O [M+H] 409.1082, found 409.1095.
1
9
13
5
4
The following intermediates were prepared in a manner analogous to Representative
Procedure B. Step 3.
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1
2
2
2
2
2
2
2
2
2
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6
(
2-chloro-3-(trifluoromethyl)phenyl)(4-methyl-1-phenyl-1H-imidazo[4,5-c]pyridin-
5
(4H)-yl)methanone. (52%) MS (ESI): mass calculated for C H ClF N O, 417.09; m/z found
21 15 3 3
+
4
18.1 [M+H] .
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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1
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1
2
3
4
5
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7
8
9
0
(2-chloro-3-(trifluoromethyl)phenyl)(1-(4-fluorophenyl)-4-methyl-1H-imidazo[4,5-
c]pyridin-5(4H)-yl)methanone. (49%) MS (ESI): mass calculated for C H ClF N O, 435.08;
21 14
4
3
+
m/z found 436.1 [M+H] .
(2-chloro-3-(trifluoromethyl)phenyl)(4-methyl-1-(pyrimidin-2-yl)-1H-imidazo[4,5-
c]pyridin-5(4H)-yl)methanone. (35%) MS (ESI): mass calculated for C H ClF N O, 419.08;
1
9
13
3
5
+
m/z found 420.1 [M+H] .
(2-chloro-3-(trifluoromethyl)phenyl)(1-(5-fluoropyrimidin-2-yl)-4-methyl-1H-
imidazo[4,5-c]pyridin-5(4H)-yl)methanone. (37%) MS (ESI): mass calculated for
+
C H ClF N O, 437.07; m/z found 438.1 [M+H] .
1
9
12
4
5
(2-chloro-3-(trifluoromethyl)phenyl)(4-methyl-1-(pyrazin-2-yl)-1H-imidazo[4,5-
c]pyridin-5(4H)-yl)methanone. (35%) MS (ESI): mass calculated for C H ClF N O, 419.08;
1
9
13
3
5
+
m/z found 420.1 [M+H] .
(2-chloro-3-(trifluoromethyl)phenyl)(4-methyl-1-(pyridin-2-yl)-1H-imidazo[4,5-
c]pyridin-5(4H)-yl)methanone. (33%) MS (ESI): mass calculated for C H ClF N O, 418.08;
2
0
14
3
4
+
m/z found 419.1 [M+H] .
(2-chloro-3-(trifluoromethyl)phenyl)(1-(3-fluoropyridin-2-yl)-4-methyl-1H-
imidazo[4,5-c]pyridin-5(4H)-yl)methanone. (38%) MS (ESI): mass calculated for
+
C H ClF N O, 436.07; m/z found 437.1 [M+H] .
20
13
4
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9
The following compounds were prepared in a manner analogous to Representative
Procedure B. Step 3.
(2-Chloro-3-(trifluoromethyl)phenyl)(4-methyl-1-phenyl-6,7-dihydro-1H-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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37
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41
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44
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54
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58
59
60
1
imidazo[4,5-c]pyridin-5(4H)-yl)methanone (20). (66%) H NMR (500 MHz, CDCl ) δ 7.76
3
(
dd, J = 5.1, 2.0 Hz, 1H), 7.65 (d, J = 15.1 Hz, 1H), 7.59 – 7.39 (m, 5H), 7.35 – 7.28 (m, 2H),
5
.83 (dd, J = 13.5, 6.7 Hz, 1H), 5.07 (dd, J = 12.3, 10.6 Hz, 1H), 4.19 – 3.99 (m, 1H), 3.61 – 2.93
+
(
m, 1H), 2.75 – 2.35 (m, 1H), 1.99 – 1.90 (m , 3H). HRMS calc. for C H ClF N O [M+H]
21 17 3 3
420.1085, found 420.1072.
(2-Chloro-3-(trifluoromethyl)phenyl)(1-(4-fluorophenyl)-4-methyl-6,7-dihydro-1H-
1
imidazo[4,5-c]pyridin-5(4H)-yl)methanone (21). (40%) H NMR (500 MHz, CDCl ) δ 7.79 –
3
7
.73 (m, 1H), 7.62 (d, J = 1.3 Hz, 1H), 7.58 – 7.35 (m, 2H), 7.33 – 7.25 (m, 2H), 7.25 – 7.15 (m,
2
H), 5.94 – 5.47 (m, 1H), 5.07 – 4.59 (m, 1H), 4.10 – 3.73 (m, 1H), 3.59 – 2.81 (m, 1H), 2.74 –
+
2
.23 (m, 1H), 1.69 – 1.50 (m, 3H). HRMS calc. for C H ClF N O [M+H] 438.0991, found
2
1
16
4
3
438.0973.
(
2-Chloro-3-(trifluoromethyl)phenyl)(4-methyl-1-(pyrimidin-2-yl)-6,7-dihydro-1H-
1
imidazo[4,5-c]pyridin-5(4H)-yl)methanone (22). (58%) H NMR (500 MHz, CDCl ) δ 8.75 –
3
8.49 (m, 3H), 7.76 (dt, J = 9.6, 4.8 Hz, 1H), 7.59 – 7.42 (m, 2H), 7.21 (ddd, J = 17.7, 8.0, 4.8 Hz,
1
H), 5.91 – 5.67 (m, 1H), 5.16 – 4.55 (m, 1H), 3.60 – 2.87 (m, 4H), 1.72 – 1.56 (m, 3H). HRMS
+
calc. for C H ClF N O [M+H] 422.0990, found 422.0976.
1
9
15
3
5
(
2-Chloro-3-(trifluoromethyl)phenyl)(1-(5-fluoropyrimidin-2-yl)-4-methyl-6,7-
1
dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (23). (52%) H NMR (500 MHz,
CDCl ) δ 8.61 – 8.38 (m, 3H), 7.82 – 7.70 (m, 1H), 7.58 – 7.32 (m, 2H), 5.90 – 5.60 (m, 1H),
3
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Journal of Medicinal Chemistry
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6
5
.19 – 4.54 (m, 1H), 3.66 – 2.82 (m, 3H), 1.81 – 1.29 (m, 3H). HRMS calc. for C H ClF N O
1
9
14
4
5
+
[
M+H] 440.0896, found 440.0886.
(2-Chloro-3-(trifluoromethyl)phenyl)(4-methyl-1-phenyl-6,7-dihydro-1H-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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9
0
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9
0
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2
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9
0
1
imidazo[4,5-c]pyridin-5(4H)-yl)methanone (24). (44%) H NMR (500 MHz, CDCl ) δ 8.65 –
3
8.45 (m, 3H), 7.80 – 7.70 (m, 1H), 7.58 – 7.42 (m, 2H), 7.24 – 7.18 (m, 1H), 5.91 – 5.71 (m,
1H), 5.10 – 4.70 (m, 1H), 3.60 – 2.88 (m, 4H), 1.70 – 1.57 (m, 3H). HRMS calc. for
+
C H ClF N O [M+H] 422.0990, found 422.0992.
19
15
3
5
(2-chloro-3-(trifluoromethyl)phenyl)(4-methyl-1-(pyridin-2-yl)-6,7-dihydro-1H-
1
imidazo[4,5-c]pyridin-5(4H)-yl)methanone (25). (40%) H NMR (500 MHz, CDCl ) δ 8.58 –
3
8
7
1
4
.44 (m, 1H), 7.99 (dt, J = 10.8, 8.7 Hz, 1H), 7.93 – 7.80 (m, 1H), 7.80 – 7.72 (m, 1H), 7.59 –
.43 (m, 2H), 7.41 – 7.24 (m, 2H), 5.91 – 5.72 (m, 1H), 5.20 – 4.58 (m, 1H), 3.63 – 3.29 (m,
+
H), 3.29 – 2.76 (m, 2H), 1.74 – 1.60 (m, 3H). HRMS calc. for C H ClF N O [M+H]
1
9
14
3
4
21.1037, found 421.1039.
(2-Chloro-3-(trifluoromethyl)phenyl)(1-(3-fluoropyridin-2-yl)-4-methyl-6,7-dihydro-
1
1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (26). (20%) H NMR (500 MHz, CDCl ) δ
3
8
.43 – 8.28 (m, 1H), 7.94 (dd, J = 23.2, 20.8 Hz, 1H), 7.81 – 7.61 (m, 2H), 7.59 – 7.29 (m, 3H),
5.82 (dt, J = 12.9, 6.5 Hz, 1H), 5.06 (dd, J = 13.5, 6.7 Hz, 1H), 4.76 – 4.37 (m, 1H), 3.63 – 2.62
+
(m, 2H), 1.86 – 1.55 (m, 3H). HRMS calc. for C H ClF N O [M+H] 439.0943, found
2
0
14
4
4
439.0940.
(2-Chloro-3-(trifluoromethyl)phenyl)(1-(5-fluoropyrimidin-2-yl)-4-methyl-6,7-
1
dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (27). (62%) H NMR (500 MHz,
CDCl ) δ 8.61 – 8.38 (m, 3H), 7.82 – 7.70 (m, 1H), 7.58 – 7.32 (m, 2H), 5.90 – 5.60 (m, 1H),
3
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Journal of Medicinal Chemistry
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.19 – 4.54 (m, 1H), 3.66 – 2.82 (m, 3H), 1.81 – 1.29 (m, 3H). HRMS calc. for C H ClF N O
2
0
14
4
4
+
[
M+H] 439.0943, found 439.0940.
The following compounds were prepared in a manner analogous to Representative
Procedure B. Step 4.
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
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(R)-(2-chloro-3-(trifluoromethyl)phenyl)(1-(4-fluorophenyl)-4-methyl-6,7-dihydro-
1
1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (28). (40%) H NMR (500 MHz, CDCl ) δ
3
7
.76 (dd, J = 5.1, 3.2 Hz, 1H), 7.62 (s, 1H), 7.58 – 7.36 (m, 2H), 7.33 – 7.24 (m, 2H), 7.24 – 7.14
(m, 2H), 5.89 – 5.78 (m, 1H), 5.07 (dt, J = 12.3, 6.0 Hz, 1H), 4.77 – 4.39 (m, 1H), 3.62 – 2.86
+
(m, 1H), 2.77 – 2.28 (m, 1H), 1.80 – 1.10 (m, 3H). HRMS calc. for C H ClF N O [M+H]
2
1
16
4
3
438.0991, found 438.0988.
(R)-(2-chloro-3-(trifluoromethyl)phenyl)(4-methyl-1-(pyridin-2-yl)-6,7-dihydro-1H-
1
imidazo[4,5-c]pyridin-5(4H)-yl)methanone (29). (44%) H NMR (600 MHz, CDCl ) δ 8.59 –
3
8
6
4
.45 (m, 1H), 8.01 (dd, J = 20.6, 15.5 Hz, 1H), 7.86 (tdd, J = 8.1, 5.7, 3.8 Hz, 1H), 7.76 (dd, J =
.3, 4.7 Hz, 1H), 7.58 – 7.28 (m, 4H), 5.82 (dd, J = 14.3, 7.4 Hz, 1H), 4.58 (d, J = 6.5 Hz, 1H),
.03 (dt, J = 12.2, 6.1 Hz, 1H), 3.34 – 2.80 (m, 2H), 1.69 – 1.44 (m, 3H). HRMS calc. for
+
C H ClF N O [M+H] 421.1037, found 421.1039.
19
14
3
4
(R)-(2-chloro-3-(trifluoromethyl)phenyl)(1-(5-fluoropyrimidin-2-yl)-4-methyl-6,7-
1
dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (30). (42%) H NMR (500 MHz,
CDCl ) δ 8.61 – 8.38 (m, 3H), 7.82 – 7.70 (m, 1H), 7.58 – 7.32 (m, 2H), 5.90 – 5.60 (m, 1H),
3
5.19 – 4.54 (m, 1H), 3.66 – 2.82 (m, 3H), 1.81 – 1.29 (m, 3H). HRMS calc. for C H ClF N O
19 14 4 5
+
[
M+H] 440.0896, found 440.0901.
P2X7 Antagonism in Human Peripheral Blood Mononuclear Cells (PBMCs) and Mouse
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