Synthesis and Pharmacology of (Pyridin-2-yl)methanol Derivatives as Novel and Selective Transient Receptor Potential Vanilloid 3 Antagonists

Article abstract of DOI:10.1021/acs.jmedchem.6b00287

Transient receptor potential vanilloid 3 (TRPV3) is a Ca²⁺- and Na⁺-permeable channel with a unique expression pattern. TRPV3 is found in both neuronal and non-neuronal tissues, including dorsal root ganglia, spinal cord, and keratinocytes. Recent studies suggest that TRPV3 may play a role in inflammation, pain sensation, and skin disorders. TRPV3 studies have been challenging, in part due to a lack of research tools such as selective antagonists. Herein, we provide the first detailed report on the development of potent and selective TRPV3 antagonists featuring a pyridinyl methanol moiety. Systematic optimization of pharmacological, physicochemical, and ADME properties of original lead 5a resulted in identification of a novel and selective TRPV3 antagonist 74a, which demonstrated a favorable preclinical profile in two different models of neuropathic pain as well as in a reserpine model of central pain.

Full text of DOI:10.1021/acs.jmedchem.6b00287

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Article
Synthesis and Pharmacology of (Pyridin-2-yl)methanol Derivatives as Novel
and Selective Transient Receptor Potential Vanilloid 3 (TRPV3) Antagonists
Arthur R Gomtsyan, Robert G Schmidt, Erol K Bayburt, Gregory A. Gfesser, Eric A. Voight, Jerome
F Daanen, Diana L Schmidt, Marlon D. Cowart, Huaqing Liu, Robert J. Altenbach, Michael E.
Kort, Bruce Clapham, Phil B. Cox, Anurupa Shrestha, Rodger Henry, David N. Whittern, Regina M
Reilly, Pamela S Puttfarcken, Jill-Desiree Brederson, Ping Song, Bin Li, Susan M Huang, Heath A
McDonald, Torben R Neelands, Steve P. McGaraughty, Donna M Gauvin, Shailen K Joshi, Patricia N
Banfor, Jason A Segreti, Mohamad Shebley, Connie R. Faltynek, Michael J. Dart, and Philip R. Kym
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00287 • Publication Date (Web): 14 Apr 2016
Downloaded from http://pubs.acs.org on April 15, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
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Synthesis and Pharmacology of (Pyridin-2-yl)methanol Derivatives as
Novel and Selective Transient Receptor Potential Vanilloid 3 (TRPV3)
Antagonists
Arthur Gomtsyan,* Robert G. Schmidt, Erol K. Bayburt, Gregory A. Gfesser, Eric A. Voight,
Jerome F. Daanen, Diana L. Schmidt, Marlon D. Cowart, Huaqing Liu, Robert J. Altenbach,
Michael E. Kort, Bruce Clapham, Phil B. Cox, Anurupa Shrestha, Rodger Henry, David N.
Whittern, Regina M. Reilly, Pamela S. Puttfarcken, Jill-Desiree Brederson, Ping Song, Bin Li,
Susan M. Huang, Heath A. McDonald, Torben R. Neelands, Steve P. McGaraughty, Donna M.
Gauvin, Shailen K. Joshi, Patricia N. Banfor, Jason A. Segreti, Mohamad Shebley, Connie R.
Faltynek, Michael J. Dart, Philip R. Kym
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Research & Development, AbbVie Inc., 1 North Waukegan Road, North Chicago, Illinois 60064, United States
Abstract
2+
+
Transient receptor potential vanilloid 3 (TRPV3) is a Ca - and Na - permeable channel with a
unique expression pattern. TRPV3 is found in both neuronal and non-neuronal tissues, including
dorsal root ganglia, spinal cord and keratinocytes. Recent studies suggest that TRPV3 may play a
role in inflammation, pain sensation and skin disorders. TRPV3 studies have been challenging, in
part due to a lack of research tools such as selective antagonists. Herein we provide the first
detailed report on the development of potent and selective TRPV3 antagonists featuring a
pyridinyl methanol moiety. Systematic optimization of pharmacological, physicochemical and
ADME properties of original lead 5a resulted in identification of a novel and selective TRPV3
antagonist 74a, which demonstrated a favorable preclinical profile in two different models of
neuropathic pain as well as in a reserpine model of central pain.
Introduction
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Transient receptor potential channel, subfamily V, member 3 (TRPV3) is a nonselective cation
°
channel on neuronal and non-neuronal cells that responds to both thermal (31-37 C) and
1
,2
chemical stimuli. Although its physiological functions have yet to be fully elucidated, unique
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attributes of TRPV3 distinguish it from other vanilloid family members. Unlike other vanilloid
1
,3
TRPs, TRPV3 is sensitized dramatically by repeated stimulation. Predominant expression is
2
,4,5
observed in human spinal cord, dorsal root ganglia, and keratinocytes.
While numerous plant-
6
derived natural products have been shown to activate TRPV3, they show little selectivity. The
synthetic small molecule, 2-aminoethyl diphenylborinate (2-APB) is most often used to
7
characterize TRPV3 function. Activation of TRPV3 elicits release of multiple molecules
implicated in barrier formation, hair growth, itch, and pain perception, suggesting the potential
8
-10
for neuronal and non-neuronal functions of this channel. In addition, multiple TRPV3 gain-of-
function mutations have been linked to Olmsted syndrome, a rare but debilitating skin
1
1,12
disease.
Its distinctive pharmacology and tissue distribution suggest that pursuit of TRPV3 antagonists
13,14
for the treatment of chronic pain and skin disease represents an attractive opportunity.
Nevertheless, TRPV3 is one of the least studied TRP subtypes, a consequence of lack of efficient
research tools such as selective antagonists. We have identified a small molecule lead series that
combines the favorable attributes of high potency and selectivity for TRPV3, desirable drug-like
physiochemical properties, and efficacy in models of pathological pain. Here we report on
identification and characterization of a novel class of pyridinol antagonists of TRPV3.
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Results and Discussion
As a starting point, the project team identified compound 5a originating from medicinal
chemistry efforts following a high throughput screen (HTS) of the AbbVie compound collection.
Although a relatively lipophilic compound (cLogP 3.9), this (S)-enantiomer of the racemic hit
was singled out for its potency at TRPV3 (human Kb = 0.08 µM; IC = 0.16 µM), structural
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compactness, relatively low molecular weight of 308 and, therefore, the potential to access
desirable drug-like space. Chirality was found to be critically important, since the enantiomeric
counterpart (R)-5a was virtually inactive.
The first set of investigations we undertook was aimed at reducing the high unbound clearance of
5a in both rat (rClint u = 150 L/h/kg) and human liver microsomes (hClint u = 40 L/h/kg) (Table
1
).
a, b
Table 1. SAR and microsomal stability of 5a and its analogs modified at aromatic sites
OH
R1
N
X
R2
hTRPV3Kb RLM / HLM Clint u
1
2
Compound
R
R
X
(µM)
0.08
0.08
0.14
0.55
0.61
0.64
0.91
0.37
1.54
(L/h/kg)
151/41
103/133
63/20
5
5
5
a
b
c
3,4-di-Cl
H
H
H
H
CH
CH
CH
CH
CH
CH
CH
N
3-OCF
4-OCF
2-OCF
3
3
3
(±)-5d
110/74
180/50
231/32
190/46
50/16
(±)-5e
3,4-di-Cl 3-F
3,4-di-Cl 4-F
3,4-di-Cl 5-F
(±)-5f
±)-5g
(
5
h
3,4-di-Cl
4-OCF
H
H
(±)-5i
3
N
29/6
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Cl
Cl
OH
N
3
.4
380/100
(±)-5j
a
All experiments to determine Kb values were performed with at least duplicates at each
compound dilution and all Kb values were averaged when determined in two or more
independent experiments. In vitro microsomal stability of compounds was determined using a
high-throughput automated 384-well plate assay using published protocols. Microsomal
intrinsic clearance (Clint) values were corrected for non-specific microsomal binding using
experimentally measured unbound fraction (f mic) according to published methods, and results
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b
1
5
1
6
u
were reported as unbound Clint (Clint u).
Compound 5a was incubated in rat liver microsomes for 1 h at 37°C, metabolite distribution was
1
analyzed by HPLC (Figure 1) and the structures of the metabolites were elucidated by H NMR.
Both pyridine and benzene rings as well as alcohol and cyclobutane moieties were determined to
be sites of metabolic oxidation resulting in approximately equal ratio of corresponding
metabolites. Blocking different positions of the pyridine fragment with fluorine [(±)-5e-g] did
not offer any gains in metabolic stability, but replacement of the pyridine ring with pyrimidine
produced compound 5h with 2.5-3-fold diminished microsomal clearance in rats (Table 1).
Alternatively, the 4-position on the other aromatic site, phenyl ring, was found to be slightly
favored over the 3- and 2-positions in regard to stability (5bc, (±)-5d). Taking advantage of these
observations, we synthesized (±)-5i that combined a pyrimidine fragment on its left-hand side
with a 4-trifluoromethoxyphenyl group on the right-hand side. Compound (±)-5i did exhibit
improved metabolic stability over 5a (rClint u 29 L/h/kg vs. 151 L/h/kg) but also substantially
weaker TRPV3 potency (1.54 µM for (±)-5i vs 0.08 µM for 5a, 0.14 µM for 5c and 0.37 µM for
5h) even factoring in its racemic nature.
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mA
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Cl
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-
Oxidation at
cyclobutane
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Oxidation of
alcohol
5a
Oxidation at
aromatic sites
-
2
2
3
3
4
4
5 mi
Figure 1. Composition of metabolites of 5a (HPLC UV trace)
Exploration of SAR and structure-stability relationship around the alcohol functionality in 5a
indicated that TRPV3 potency is highly sensitive to modifications in this region. Structural
manipulations such as O-alkylation, homologation to hydroxymethyl group, removal of OH or its
replacement with an amine led to a significant reduction in potency. In order to block the
oxidation of the alcohol to the ketone, a methyl group was incorporated at the carbon bearing an
OH group. However, the resulting tertiary alcohol (±)-5j was over 40-fold less potent than the
parent 5a (Table 1).
A tertiary OH group was better tolerated in molecules with modified topology of the central
moiety such as cyclopentanes 7-10 (Table 2). Unlike the compounds from the original series,
these new analogs showed no prohibitive dependency on the stereochemical disposition at the
benzylic carbon as all four diastereomers displayed comparable TRPV3 potency in the range of
0
.1-0.6 µM. However, the two most potent diastereomers 7 and 8 were also the ones that
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displayed the highest values of intrinsic unbound clearance in microsomes (rClint u 151 and 460
L/h/kg, respectively). By introducing polarity into the cyclopentane ring we expected to reduce
the number of potential sites of oxidative metabolism and improve
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Table 2. SAR and microsomal stability of tertiary alcohols 7-10 and 15-18 with
cyclopentane and tetrahydrofuran core
a, b
HO
O
5
HO
N
1 3
3
N
CF3
OCF3
7
-10
15-18
RLM/HLM Clint u
L/h/kg)
Compound
Chirality
hTRPV3 Kb (µM)
(
7
8
9
1
1
1
1S, 3R
1R, 3R
1R, 3S
1S, 3S
3S, 5R
3R, 5S
3S, 5S
3R, 5R
0.24
0.13
0.54
0.61
0.42
1.12
1.71
2.46
151/9
460/23
86/20
21/24
10/7
20/4
23/13
9/7
0
5
6
17
1
8
a
All experiments to determine Kb values were performed with at least duplicates at each
compound dilution and all Kb values were averaged when determined in two or more
b
independent experiments. In vitro microsomal stability of compounds was determined using a
1
5
high-throughput automated 384-well plate assay using published protocols. Microsomal
intrinsic clearance (Clint) values were corrected for non-specific microsomal binding using
16
experimentally measured unbound fraction (f mic) according to published methods, and results
u
were reported as unbound Clint (Clint u).
drug-like properties. Indeed, the tetrahydrofuran moiety in 15-18 reduced cLogP by about 0.7
and resulted in up to 50-fold improvement in metabolic stability compared with cyclopentane
analogues 7-10 (Table 2). Although tetrahydrofuran 15 exhibited a significant advantage over the
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carbon analog 7 with respect to metabolic stability, we were concerned about the low TRPV3
potency of this compound when its free fraction in plasma was factored in. According to the free
drug hypothesis, the pharmacological effects in vivo are elicited by free (unbound) drug
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concentration. On that measure, 15 exhibited plasma adjusted potency of 12 µM calculated
from its total potency in FLIPR assay (Kb = 0.42 µM) and free fraction of 0.03 in plasma. Such a
high concentration of the compound required for pharmacological effects was undesirable based
on pharmacokinetic and toxicological considerations.
In addition to the two aromatic rings and an alcohol moiety in 5a, the cyclobutane ring was also
found to undergo oxidation in rat liver microsomes, representing another major metabolic
pathway for 5a. The initial strategy to mitigate that liability was to replace the cyclobutane ring
18-20
with heterocycles. The 3-oxetane replacement improved the metabolic stability as expected,
(compound 24), but at the same time it led to a complete erosion in TRPV3 potency (Table 3).
Replacing the ether oxygen with
Table 3. SAR and microsomal stability of oxetanes 24 and 37ab, azetidines 29ab and
a, b
tetrahydrofurans 40 and 46ab
OH
R
1
N
2
X
n
Y
hTRPV3 RLMHLM Clint
Kb (µM) u (L/h/kg)
Compound
n
R
X
Y
Chirality
2
2
4
9a
0
0
0
0
0
1
3,4-di-Cl CH
4-CF3
4-CF3
3,4-di-Cl
3,4-di-Cl
2
2
2
O
NH
racemate
racemate
7.37
6.68
6.63
0.38
0.15
0.63
25/8
3/2
7/3
60/13
3 / 4
59/25
CH
CH
O
29b
NMe racemate
3
3
4
7a
7b
0
CH
CH
O
2
1,2-trans
1,2-cis
racemate
O
2
3,4-di-Cl CH
2
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6b
1
1
3,4-di-Cl
3,4-di-Cl
O
O
CH
CH
2
1,2-trans
1,2-cis
0.043
0.47
179/23
not determined
4
2
a
All experiments to determine Kb values were performed with at least duplicates at each
compound dilution and all Kb values were averaged when determined in two or more
independent experiments. In vitro microsomal stability of compounds was determined using a
b
15
high-throughput automated 384-well plate assay using published protocols. Microsomal
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15
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intrinsic clearance (Clint) values were corrected for non-specific microsomal binding using
1
6
experimentally measured unbound fraction (f mic) according to published methods, and results
u
were reported as unbound Clint (Clint u).
nitrogen resulted in azetidine analogs 29a and 29b with profiles similar to that of 3-oxetanes.
TRPV3 potency rebounded for 2-oxetanes 37ab. The tetrahydrofurans 40 and 46ab also had
improved potency for TRPV3, but lower microsomal stability. Replacement of C-H bonds in the
cyclobutane ring with C-F bonds yielded compounds with good potency and moderate to good
stability, as shown by gem-difluoro analogs 55a and 55c (Table 4). However, further exploration
of gem-difluoro-cyclobutane
a, b
Table 4. SAR and microsomal stability of gem-difluorocyclobutane and diol analogs
OH
X
Y
N
R3
R1 R2
hTRPV3 Kb RLM/HLM Clint
1
2
3
Compound
R
R
R
X
Y
(µM)
0.08
0.04
0.04
0.09
2.24
0.08
0.21
1.87
0.28
0.33
u (L/h/kg)
51/15
12/11
7/9
5
5
6
6
5a
5c
0b
0e
F
F
F
F
3,4-di-Cl
4-OCF
3-OCF
3-CF
4-CF
3-OCF
3-CF
4-CF
3-OCF
4-OCF
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
3
H
H
H
OH
OH
OH
OH
OH
OH
H
H
H
3
11/8
3/3
3
60f
61b
3
13/7
6/3
3/2
3
6
6
6
6
1e
1f
2b
2c
3
3
OH Me
OH Me
12/10
8/4
3
3
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3
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
0.30
0.84
1.31
1.51
1.55
4.79
19/9
9/3
3 / 7
6/3
4/2
6/2
3
3
3
3e
3f
3-CF
4-CF
3
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
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OCF
3
OH
N
>10.4
6/3
OH
64
7
7
73a
73b
7
7
7
7
2a
2b
H
H
OH
OH
OH
OH
H
3-OCF
3-OCF
3-OCF
3-OCF
3-OCF
3-OCF
3-OCF
3-OCF
3
3
3
3
3
3
3
3
N
CH
N
CH
N
CH
N
CH
N
CH
N
CH
N
CH
N
0.27
0.06
0.32
0.53
0.56
0.50
0.73
4.27
3/2
3/2
3/2
12/5
6/2
5/4
7/2
3/4
H
4a
4b
5a
5b
OH Me
OH Me
Me OH
Me OH
CH
OH
N
N
CF
3
>9.1
6/2
HO
7
a
6
All experiments to determine Kb values were performed with at least duplicates at each
compound dilution and all Kb values were averaged when determined in two or more
independent experiments. In vitro microsomal stability of compounds was determined using a
high-throughput automated 384-well plate assay using published protocols. Microsomal
intrinsic clearance (Clint) values were corrected for non-specific microsomal binding using
experimentally measured unbound fraction (f mic) according to published methods, and results
were reported as unbound Clint (Clint u).
b
15
1
6
u
analogs was plagued by consistent cardiovascular side-effects in rat studies. Thus, 55c at plasma
concentration of 4.7 µg/mL produced a statistically significant increase in cardiac contractility
(
dP/dt ) to a maximum of 29% above vehicle in anesthetized rats. This effect was also
50
accompanied by a 16% increase in mean arterial pressure above vehicle. Additionally, compound
5c was found to be strong activator of the human pregnan X receptor (PXR) (EC = 5.7 µM), a
5
5
0
2
1
nuclear receptor playing a key role in regulating the expression of metabolizing enzymes. One
of the most significant implications of PXR activation could be drug-drug-interaction (DDI) due
22
to the increased expression of CYP450 enzymes. Therefore, the gem-difluoro-cyclobutane
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series was abandoned and SAR design strategy shifted towards targeting diols such as 60b and
6
1b where the introduction of the second hydroxyl group at the cyclobutane would preempt the
formation of possible products of metabolic oxidation.
10
11
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52
53
54
55
56
57
58
59
60
Having reached favorable in vitro unbound clearance for the first two diols 60b and 61b (rClint u
= 7 and 13 L/h/kg respectively), combined with excellent TRPV3 potency, we began a rigorous
examination of this series (Table 4). Three key structural elements that influenced the activity of
compounds against TRPV3 in the diol series were: 1) stereochemistry at the benzylic carbon
adjacent to the pyridine ring, 2) syn/anti-isomerism at the cyclobutane ring and 3) the nature of
secondary versus tertiary hydroxyl group at the cyclobutane ring. Analogous to 5a, TRPV3
potency in diols is highly sensitive to the stereo-configuration of the benzylic carbon with (S)-
isomers being significantly more potent than their (R)-counterparts (for example, compare 74a
vs. 76 and 63c vs. 64). A syn-relationship between cyclobutane hydroxyl and pyridinyl-methanol
fragment is favored over anti-configuration, although the potency difference is less pronounced
in the case of sec-cyclobutanols (60b vs. 61b or 73a vs. 72a) than in the case of tert-
cyclobutanols (74b vs. 75b or 62f vs. 63f). Finally, a sec-OH group on the cyclobutane ring
generally provides higher potency than a tert- OH group, however, the magnitude of the
difference is dependent on the identity of the right-hand side fragment and, as mentioned above,
syn/anti isomerism. Thus, sec-alcohol 72b in the anti-series featuring a 4-pyridyl fragment was
70-fold more potent than the corresponding tert-alcohol 75b (Kb = 0.06 µM vs. 4.3 µM), yet syn-
isomeric analogs 73b and 74b with the same 4-pyridyl fragment were virtually equipotent with
Kb value about 0.5 µM. These effects were substantially muted for other pairs of sec/tert-OH
cyclobutanol analogs. For example, with a 2-pyridyl instead of a 4-pyridyl group, there was only
a modest 2.7- and 1.5-fold reduction in potency going from secondary to tertiary alcohols in both
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2
2
2
2
2
2
2
2
2
3
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3
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3
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3
4
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4
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4
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5
5
5
5
5
5
5
5
6
anti- (72a vs 75a) and syn-series (73a vs. 74a). Importantly, unlike the gem-difluoro series, the
compounds in the diol series were largely devoid of cardiovascular side-effects and did not
activate PXR.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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8
9
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
Based on the most balanced combination of pharmacological, ADME and physicochemical
properties, compound 74a was chosen for more advanced characterization. Despite its modest
potency in the FLIPR assay (human Kb = 0.56 µM; IC50 = 0.93 µM), compound 74a was
calculated to exhibit Kb = 0.62 µM when free fraction of 0.89 in human plasma protein was
factored in. Based on this measure, 74a was superior to a number of its analogs and represented
one of the most potent TRPV3 antagonists in the series. This lead compound was also potent in a
patch-clamp electrophysiological assay, where it blocked TRPV3 agonist 2-APB-activated
currents in a stable recombinant HEK293 cell line expressing human TRPV3 with IC = 0.38
5
0
µM. In addition, 74a was highly selective for TRPV3 as screening at 10 µM in CEREP and
AbbVie internal panels of biological targets (including TRPV1, Na 1.5, Na 1.7 Na 1.8 Ca 1.2
v
v
v
v
Ca 2.2 Ca 3.2, hERG) showed no evidence of binding >50% (see Supporting Information).
v
v
A number of molecular and structural parameters define compound 74a as more drug-like than
the parent compound 5a and lead 15 from the tetrahydrofuran series (Figure 2). For example, the
3
advantages of 74a over 5a and 15 were manifested in lower lipophilicity, higher fraction of sp -
hybridized carbon atoms and higher lipophilic efficiency parameter LLE. Additionally, 74a
exhibits an attractive CNS MPO of 4.5 (out of maximal desirable score of 6) based on six
23
fundamental molecular properties.
O
Cl
Cl
OH
N
HO
OH
N
N
C
F3
N
OCF3
HO
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9
Properties
MW
5a
308
4.2
33
15
339
3.7
74a
338
2.0
logD (calcd)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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44
45
46
47
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50
51
52
53
54
55
56
57
58
59
60
TPSA
52
66
LLE (pKb – LogD
LLE (pKb – LogD (based on
plasma adjusted pKb)
2.9
2.68
4.25
0
.7
1.41
4.2
3
fSP
0.31
6
0.35
5
0.41
3
3
Ar - SP
PFI (LogD – NAR)
Human Clint u (L/h/kg) hep
Human Fu plasma
CNS MPO
6.2
14
5.7
16
4.0
2
0.007
4
0.06
5.0
0.89
4.5
a
a
Figure 2. Properties of initial lead 5a and more advanced leads 15 and 74a . PFI, property
forecast index; NAR, number of aromatic rings
The improved unbound microsomal stability of 74a in vitro (rClint u = 6 L/h/kg) translated well
to the in vivo setting where unbound clearance of rClp u = 2.5 L/h/kg (iv) was recorded in rat PK
studies (Table 5). For comparison, the unbound clearance for tetrahydrofuran analog 15 after iv
administration in rats was 7-fold higher, correlating with its higher microsomal clearance in
vitro.
a
Table 5. Pharmacokinetic parameters of 15 and 74a in rats
Total
Unbound
T1/2
(iv)
(h)
3.8
0.5
AUC0-8
(po)
(µg*h/mL) (µg*h/mL)
0.93
0.38
Unbound
AUC0-8 (po) (po)
F
Vss
L/kg)
Cmax
(µg/mL)
b
Compound Clp (iv) Clp (iv)
(
(
L/h/kg) (L/h/kg)
(%)
56
26
1
7
5
4a
0.6
1.6
17.6
2.5
3.2
1.4
0.13
0.19
0.03
0.24
a
iv doses were 1 mg/kg for both compounds and po doses were 1 mg/kg for 15 and 2 mg/kg for
4a. As a reference, rat hepatic blood flow is 3.3 L/h/kg and human hepatic blood flow is 1.2
b
7
L/h/kg
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2
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4
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Following ADME and PK characterization, compound 74a was assessed in rat behavioral
models of neuropathic pain (Figure 3). Dose-dependent efficacy with an estimated EC value
50
around 11-12 µmol was observed in both the chronic constriction injury (CCI) and sciatic nerve
0
1
2
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9
0
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8
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0
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8
9
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ligation (SNL) models of neuropathic pain.
A
B
Figure 3. Compound 74a (10, 30,100 mg/kg po) was anti-allodynic in both the CCI (A) and
SNL (B) models of neuropathic pain. In both models, the compound dose-dependently attenuated
mechanical allodynia induced by von Frey hair stimulation to the ipsilateral (injured) hind paw.
A response threshold of 15 g is considered to be the maximal possible effect (MPE). The blue
symbols represent %MPE in the neuropathic models, and the red symbols represent the plasma
concentrations at the specific doses for these studies. For comparison, gabapentin (Gaba) was
also administered at 150 mg/kg. Veh = vehicle (10% DMSO in Polyethylene glycol), n = 6 per
group. **p<0.01 vs. the vehicle group.
Additionally, 74a exhibited anti-nociceptive activity in the reserpine model of central pain by
2
4, 25
increasing muscle pressure threshold (see Supporting Information).
During these
experiments, no overt impairments or other changes to animal behavior were observed. Efficacy
of 74a in reserpine model of central pain can be attributed to its attractive CNS MPO (Figure 2)
and favorable plasma to brain ratio of 1.
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Chemistry
Synthesis of 5a depicted in Scheme 1 represents a general synthetic strategy for preparation of
pyridyl-methanol-containing TRPV3 antagonists. Installation of a cyclobutyl group on 3,4-
dichlorophenylacetonitrile (1a) was accomplished via alkylation with 1,3-dibromopropane
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
58
59
60
a
Scheme 1. Synthesis of parent 5a and its analogs 5b-j
Cl
OH
N
Cl
5
j
h
O
OH
d
R1
R1
c
R1
R1
a
N
N
NC
NC
-
2a d
-
-
a
g
a
1
4
−
d
-
±
a
g
( )
5
R2
R2
b
f
e
OH
,
R1
R
e
g
N
OHC
X
a
R2
3
b
1
=
, - - ,
2
= , =
,
=
= , =
a
5
R 3 4 di Cl R H X CH
-
=
1
=
,
2
5
b R 3 OCF R H X CH
3
1
=
-
,
2
c
5
R 4 OCF R H X CH
3
-
= -
R1
1= - -
=
,
=
,
2
±
2 OC
F R H X
C
H
5
5
5
5
d
e R
(
)
)
3
-
-
-
= -
,
=
,
=
,
,
2
±
±
3 4 di C
l R
F X
H
H
3
C
(
=
,
- -
,
2
= -
R1
3 4 di C
l R
4
F X
f
C
( )
±
=
,
- -
,
2
2
= -
,
=
R1
3 4 di C
l R
F X
g
5
C
H
(
)
-
-
=
,
=
1
= ,
,
5
h R 3 4 di Cl R H X N
-
= -
R1
=
,
=
,
2
±
4 OC N
F R H X
5i
(
)
3
a
o
Reagents and conditions: (a) 1,3-dibromopropane, NaH, DMSO, Et O, 15 C, 16 h, 84% for 2a; (b)
diisobutylaluminum hydride, CH Cl , -78 °C, 55% for 3b; (c) 2-bromopyridine, THF, n-butyl lithium, -78 °C, then
H SO , 5-50 °C, 56% for 4a; (d) NaBH , MeOH, 5°C, 42% for 4a; (e) chiral separation on Chiralcel OJ-H column;
f) (HCO H, Et N, (S,S)-N-(p-touenesulfonyl)-1,2-diphenylethanediamine(chloro)(p-cumene)ruthenium (II), 35° C,
2
2
2
2
4
4
(
2 3
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1
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1
1
1
1
2
2
2
2
2
2
2
2
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
o
1
8 h, 98%, 96% ee for 5a; (g) 2-(tributylstannyl)pyrimidine, THF, n-butyl lithium, -90 C, 9% for (±)-5i; (h)
MeMgBr, THF, room temperature, 0.5 h, 68%
followed by the in situ intramolecular cyclization to give 2a. Nucleophilic addition of pyridyl
lithium to 2a followed by treatment of the transient ketimine intermediate with sulfuric acid
provided ketone 4a. The latter was reduced to the mixture of racemic alcohols, from which the
desired (S)-enantiomer 5a was isolated by chiral HPLC. Alternatively, optically pure 5a was
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
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
6
prepared by ruthenium-catalyzed asymmetric transfer hydrogenation of the ketone 4a. The
absolute configuration of the chiral center in 5a was determined from single crystal X-ray
27
diffraction data by anomalous dispersion analysis . Modified procedures were used for the
synthesis of pyrimidine analogs 5h and (±)-5i. Pyrimidin-2-yllithium nucleophile was prepared
from corresponding 2-(tributylstannyl)pyrimidine derivatives, while aldehydes 3ab were used as
electrophiles instead of nitrile in the synthesis of pyridine analogs.
For the synthesis of tertiary alcohols 7-10 featuring a cyclopentane core (Scheme 2), we took
advantage of the protocol developed by Hayashi et al. for rhodium-catalyzed asymmetric
a
Scheme 2. Synthesis of tertiary alcohols with cyclopentane core
O
HO
HO
O
a
N
N
b
+
a
6
CF3
7
8
CF3
CF3
O
HO
HO
O
c
N
b
N
+
6b
CF3
9
10
CF3
CF3
a
Reagents and conditions: (a) 4-(trifluoromethyl)phenylboronic acid, Rh(acac)(C H ) (R)-BINAP, dioxane-water,
00°C, 5 h, 89%; (b) 2-bromopyridine, THF, n-butyl lithium, -78 C - room temperature, 35% for 7 and 30% for 8;
0% for 9 and 15% for 10 (c) 4-(trifluoromethyl)phenylboronic acid, Rh(acac)(C H ) (S)-BINAP, dioxane-water,
2
4
o
1
1
1
2
4
00 °C, 5 h, 79%.
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1
,4-addition of arylboron compounds to enones. Enantiopure (R)- and (S)-cyclopentanone
intermediates 6a and 6b prepared by this procedure were treated with pyridyl-2-lithium followed
by chiral HPLC separation of the resulting diastereomeric mixtures to afford target molecules 7-
10. The absolute stereochemistry of the newly formed chiral center at the secondary carbon atom
on the cyclopentane ring was established indirectly by 2D NMR experiments confirming cis- or
trans- relationship between two aromatic rings in the molecule (see Supporting Information).
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
58
59
60
Preparation of tertiary alcohols 15-18 containing tetrahydrofuran central ring is shown in Scheme
29
3
. Alkynylation of acetophenone 11 with propargyl-lithium followed by gold-catalyzed
a
Scheme 3. Synthesis of tertiary alcohols with tetrahydrofuran core
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1
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1
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1
1
1
1
1
2
2
2
2
2
2
2
2
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
O
O
HO
a
b
c
O
OCF3
OCF3
OCF3
11
12
13
O
O
HO
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
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HO
N
N
OCF3
OCF3
-
-
c s
trans 14
i 14
d
d
O
O
O
O
HO
H
O
HO
HO
N
N
N
N
OCF3
OCF3
OCF
OCF3
3
15
16
17
18
,
c
b
O
OH
e
OCF3
OCF3
-
R 12
( )
1
1
a
Reagents and conditions: (a) propargyl bromide, n-butyl lithium, TMEDA, Et O, 53%; (b) 3,5-dichloropyridine 1-
oxide, PPh AuNTf , MeOH - CH Cl , 53%; (c) 2-bromopyridine, lanthanum trichloride-lithium chloride complex,
n-butyl lithium, AcOH, THF, 55% for trans-14 and 27% for cis-14; (d) chiral separation on ChiralPak IA by SFC;
e) allenyl-dioxaborolane, (R)-3,3'-dibromo-1,1'-bi-2-naphthol, 165 °C, 16 h, 98%.
2
3
2
2
2
(
3
0
oxidative intramolecular cyclization of the resulting homopropargylic alcohol 12 led to
dihydrofuran-3-one derivative 13. The latter was then reacted with pyridyl-2-lithium in the
31
presence of LaCl ·2LiCl , and each of the resulting trans- and cis-racemic mixtures of trans-14
3
and cis-14, obtained in overall 83% yield, were separated by chiral preparative supercritical fluid
chromatography (SFC) to provide enantiomerically pure target molecules 15-18. More efficient
access to the lead compound 15 was achieved by using enantiomerically pure homopropargylic
alcohol (R)-12, which was prepared by asymmetric propargylation of 11 with allenylboronate in
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the presence of catalytic amounts of 3,3’-dibromo-BINOL. Stereochemical assignment of
chiral centers in 15 was confirmed by X-ray crystallography.
Preparation of 3-oxetane 24 (Scheme 4) was initiated via transformation of aldehyde 20 to triol
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
58
59
60
33
34
2
1 via Tollens condensation. Cyclization of 21 followed by oxidation of the resulting alcohol
a
Scheme 4. Synthesis 3-oxetane 24
Cl
Cl
Cl
Cl
Cl
a
b
c
O
HO
HO
HO
Cl
Cl
H
OH OH
1
9
2
0
21
C
l
Cl
Cl
OH
O
a
d
N
C
l
Cl
H
O
O
O
2
2
23
24
a
Reagents and conditions: (a) Dess-Martin periodinane, CH Cl , room temperature, 4 h; (b) paraformaldehyde,
2
2
°
°
Ca(OH) , THF, 60°C, 120 h; (c), (EtO) CO, KOH, 95 C, 4 h with removal of EtOH, then 190 C, 1 h; (d) 2-
2
2
°
bromopyridine, THF, n-butyl lithium, -75 C, 1 h, 46%.
2
2 to aldehyde 23 and subsequent reaction with pyridyl-2- lithium generated the final compound
4. Nitrogen analogs of 24, azetidines 29ab, were synthesized from cyanoazetidine intermediate
2
25 (Scheme 5). Nucleophilic addition of 1-fluoro-4-trifluoromethylbenzene afforded 26, which
was subjected to a combination of protective group manipulations and standard synthetic
protocol described for the preparation of 5a to obtain 29ab.
a
Scheme 5. Synthesis of azetidines 29ab
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8
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
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
CF3
CF3
CF
3
O
OH
N
CN
N
NC
N
N
a
c
b
N
oc
N
B
oc
B
oc
B
Boc
25
2
6
27
28
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
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CF3
CF3
OH
OH
N
N
e
d
N
H
N
Me
a
2
9
2
9b
a
Reagents and conditions: (a) 1-fluoro-4-(trifluoromethyl)benzene, KHMDS, toluene, 60°C, 24 h, 27%; (b) 2-
bromopyridine, THF, n-butyl lithium, -78°C, then 1 N H SO , 5-50 °C, 0.5 h, 5%; (c) NaBH , MeOH, room
temperature, 1 h, 29%; (d) HCl in MeOH, room temperature, 16 h, 32%; (e) HCO H, HCHO, reflux, 16 h, 13%.
2
4
4
2
2
-Oxetane analogs 37ab were prepared as shown in Scheme 6. Starting with the ketoester 30,
addition of tributylallyltin gave intermediate 31. The stepwise ozonolysis of the double bond of
31 followed by oxidation of the resulting aldehyde 32 to alcohol 33 and its tosylation gave the
ester 34. The latter was cyclized under basic conditions to afford the key 2-oxetane intermediate
3
5 in moderate yield. Addition of pyridyl-2-lithium provided the ketone 36, thus setting the stage
for ruthenium-catalyzed asymmetric reduction to generate a separable mixture of diastereomers
7ab with fixed (R)-configuration of the secondary alcohol. Stereochemistry at the second chiral
3
center was determined by X-ray analysis of 37a.
a
Scheme 6. Synthesis of 2-oxetanes 37ab
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9
O
O
OH
OH
Cl
Cl
Cl
Cl
b
EtO
Cl
Cl
a
EtO
EtO
O
O
O
3
0
31
32
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
58
59
60
s
HO
EtO
T O
O
OH
OH
e
Cl
Cl
c
Cl
Cl
d
Cl
Cl
EtO
EtO
O
O
O
3
5
3
3
34
Cl
Cl
Cl
Cl
Cl
Cl
O
OH
OH
f
g
N
N
+
N
O
O
O
a
3
6
37
37b
a
Reagents and conditions: (a) allyltributyltin, TiCl , CH Cl , 0°C – room temperature, 16 h, 77%; (b) O , CH Cl , -
78 °C, 4 h, then dimethylsulfide, -78°C to room temperature, 9 h, used crude; (c), tetramethylammonium
triacetoxyborohydride, AcOH, MeCN, -40 C, used crude; (d) TsCl, DBU, CH Cl , room temperature, 24% for three
steps; (e) t-BuOK, 18-crown-6, THF, room temperature, 2 h, 54 %; (f) 2-bromopyridine, diethyl ether, n-butyl
4
2
2
3
2
2
°
2
2
°
lithium,
-75°C
-
0 C,
2.5
h,
65%;
(g)
HCO H,
Et N,
3
(S,S)-N-(p-toluenesulfonyl)-1,2-
2
diphenylethanediamine(chloro)(p-cymene)ruthenium(II), 35°C, 16 h, 33% of 37a and 40% of 37b.
Synthesis of 3- and 2-tetrahydrofuran analogs 40 and 46ab (Scheme 7) was accomplished by
employing the synthetic strategy used for the preparation of 5a shown in Scheme 1. 3-
Tetrahydrofurancarbononitrile
38
was
generated
by
treatment
of
2-(3,4-
dichlorophenyl)acetonitrile (1a) with 1-chloro-2-(chloromethoxy)ethane under basic conditions
and then elaborated to the target compound 38 in two-step standard protocol. Unlike a simple
one-step preparation of key intermediate 38 in 3-tetrahydrofuran series, its regioisomeric
counterpart 44 in 2-tetrahydrofuran series was synthesized in 4 steps. Weinreb amide 41 was
converted to cyclopropyl ketone 42, which then underwent ring opening reaction with pyridine
hydrochloride to provide chlorobutanone 43 in moderate yield. Cyclization of the ketone 43 in
the presence of potassium cyanide then furnished desired 2-tetrahydrofurancarbononitrile
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
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
intermediate 44. Reaction with pyridyl-2-lithium and subsequent reduction of the ketone with
sodium borohydride yielded trans- and cis- mixture of racemic compounds 46ab.
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
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 7. Synthesis of 3- and 2-tetrahydrofurans
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
O
OH
a
N
NC
b
c
N
NC
O
O
O
a
1
3
8
39
40
Cl
Cl
Cl
C
l
Cl
Cl
Cl
C
l
d
e
f
NC
N
Cl
O
O
O
O
4
O
42
43
44
1
Cl
Cl
Cl
Cl
Cl
Cl
OH
O
OH
N
N
c
N
b
+
O
O
O
a
46
46b
45
a
Reagents and conditions: (a) 1-chloro-2-(chloromethoxy)ethane, NaH, N-methylmorpholine N-oxide, THF, -20°C -
room temperature, 24 h, 17%; (b) 2-bromopyridine, THF, n-butyl lithium, -78 °C, then 1 N H SO , 5-50°C, 0.5 h,
2
4
1
0
5
3%; (c) NaBH , MeOH, room temperature, 1 h, 46% (d) cyclopropanecarbonylmagnesium bromide (2 N in THF),
4
°C – room temperature, 4 h, quantitative; (e) TsOH.H O, pyridine hydrochloride, MeCN, 160°C, 3 h, microwave,
2
°
0%; (f) KCN, MeOH, 35 C, 48 h, 90% for 40.
Difluorocyclobutane analogs 55ac were prepared as shown in Scheme 8. Starting with 3,4-
dichlorophenylacetonitrile 1a, di-alkylation with 1,3-dibromo-2,2-dimethoxypropane gave the
ketal 47, which was subsequently treated with sulfuric acid in EtOH to form ketoester 48.
Fluorination of the cyclobutanone with diethylaminosulfur trifluoride (DAST) afforded 49,
a
Scheme 8. Synthesis of gem-difluorocyclobutane and diol analogs
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9
Cl
c
Cl
Cl
Cl
Cl
Cl
Cl
d
b
O
O
O
F
O
O
N
EtO
Cl
EtO
H N
2
47
48
49
5
0
F
F
F
O
O
e
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
58
59
60
a
OH
R
R
N
NC
-
,
,
,
a c e
1
a c
55
f
F
F
f
e
OCF3
OCF3
c
g
d
O
R
R
NC
NC
N
NC
, , ,
c e
, , ,
c e
5
1b
f
52b
f
5
3
54
F
F
F
F
O
HO
h
R
d
O
O
i
OH
j
OH
NC
R
R
R
N
N
N
O
O
, , ,
c e
O
, , ,
c e
5
6b
f
57b
f
O
N
O
, , ,
c e
f
, , ,
c e
f
58b
5
9b
O
k
OH
OH
i
OH
H
OH
R
R
R
R
N
N
N
+
+
HO
OH
O
H
HO
H
,
e
,
, , ,
c e
,c,e,
63b
, ,
e
60b f
62b
f
f
6
1b f
OCF3
OH
=
=
=
=
, - -
a
R 3 4 di Cl
N
-
b R 3 OCF3
-
c
e
R 4 OCF3
-
R 3 CF3
OH
=
-
f R 4 CF3
6
4
a
Reagents and conditions: (a) 1,3-dibromo-2,2-dimethoxypropane, KOtBu, DMSO, H O, 120°C for 1 h, then room
temperature for 20 h, 47%; (b) EtOH, H SO , reflux, 2 h, 57%; (c) diethylaminosulfur trifluoride, CH Cl , 0°C –
room temperature, 16 h, 72% for 49; (d) 2-bromopyridine, diethyl ether, n-butyl lithium, -75 °C- 0°C, 2.5 h, 89% for
50; (e) HCO H, Et N, (S,S)-N-(p-toluenesulfonyl)-1,2-diphenylethanediamine(chloro)(p-cymene)ruthenium(II),
2
2
4
2
2
2
3
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1
2
2
2
2
2
2
2
2
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
3
5°C, 16 h, 83% for 55a; (f) MeLi in diethyl ether, epibromohydrin in THF, then MeMgBr in THF, -78°C to room
temperature, 16 h, 91% for 51b; (g) Dess-Martin periodinane, CH Cl , room temperature, 16 h, 95% for 52b; (h)
1
MeOH, room temperature, 16 h, 99% for 58b; (j) aq. HCl, acetone-water, room temperature, 16 h, then chiral
separation on a Chiralpak AD-H column, 39% for 59b; (k) MeLi, 2-methyltetrahydrofuran, -78°C to room
temperature, 16 h, 45% for 62b and 8% for 63b.
2
2
,2-bis(trimethylsilyloxy)ethane, TMSOTf, CH Cl , room temperature, 16 h, 97% for 56b; (i) NaBH , CH Cl -
2
2
4
2
2
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
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
and subsequent utilization of two-step protocol involving addition of pyridyl-2-lithium and
2
6
asymmetric transfer hydrogenation of the resulting ketone 50, furnished compound 55a with
>
95% ee. Carbononitrile 51 was used as a key intermediate for the preparation of 4-
trifluoromethoxyphenyl analog 55c as opposed to ester 49 for the synthesis of dichlorophenyl
analog 55a. One pot two-step alkylation of 4-trifluoromethoxyphenylacetonitrile 1c with
epibromohydrin by stepwise use of methyl lithium and methylmagnesium bromide as bases
afforded 51c in excellent yield. Oxidation of alcohol with Dess-Martin periodinane and
subsequent fluorination of the resulting ketone 52c with DAST yielded carbononitrile
intermediate 53. Similar to the synthesis of 55a from 49, compound 53 was converted to the
target 55c through installation of the pyridine moiety followed by asymmetric reduction of the
ketone 54.
Diol analogs 60-64 were prepared starting from the cyclobutanones 52 (Scheme 8).
Intermediates 52 were carried through five steps starting with ketal formation and installation of
the pyridine moiety to give 57. It should be noted that the formation of the ketal 56 using 1,2-
3
5
bis(trimethylsilyloxy)ethane and catalytic trimethylsilyl trifluoromethanesulfonate
was
significantly superior to more traditional use of ethyleneglycol resulting in 97% yield of
compound 56. Ketone reduction in 57 followed by hydrolysis of the ketal and chiral separation
gave enantiopure ketoalcohols 59. The latter were reduced with sodium borohydride to provide
diols 60 and 61 with secondary alcohol on the cyclobutane ring
a
Scheme 9. Synthesis of bis-pyridyl diols
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9
X
Y
O
O
X
Y
CN
CN
CN
NC
O
N
c
a
b
CF3
d
e
CF3
O
O
O
O
O
69ab
6
5
66
67
68 b
a
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
58
59
60
OH
X
Y
OH
X
Y
N
f
N
CF3
CF3
O
O
O
a
0 b
a
7
71 b
h
g
OH
X
Y
OH
X
Y
OH
X
Y
OH
X
Y
N
N
N
N
CF3
CF3
CF3
+
CF3
+
1
7
18
HO
H
HO
OH
H
OH
=
=
, =
, =
a
= , =
= , =
a
=
, =
, =
a
2 X N Y CH
73a X N Y CH
3b X CH Y N
74 X N Y CH
75 X N Y CH
, =
7
=
, =
=
=
74b X CH Y N
75b X CH Y N
72b X CH Y N
7
OH
N
OH
N
N
N
CF3
h
CF3
O
HO
-
a
R 71
76
(
)
a
Reagents and conditions: (a) ruthenium (III) chloride trihydrate, NaIO , CH Cl -MeCN-H O, 0°C – room
temperature, 89%; (b) HOCH CH OH, TsOH.H O, toluene, reflux with Dean-Stark trap, 1 h, 73%; (c) 2-fluoro-4-
(trifluoromethyl)pyridine, KHMDS, toluene, room temperature to 60°C, 2.5 h, 84% for 68a; (d) 2-bromopyridine,
Et O, n-butyl lithium, -78°C, then 1 N HCl, 0°C – room temperature, 0.75 h, 79% for 69a; (e) NaBH , MeOH -
CH Cl , room temperature, 1 h, 84% for 70a; (f) HCl, acetone, H O , room temperature, 16 h, 97%, then chiral
chromatography on Chiralpac AD-H, 43% for 71a; (g) NaBH4, MeOH - CH Cl , room temperature, 16 h, 81%, then
SFC on Chiralpac AD-H, 24% for 72a and 39% for 73a; (h) MeLi (1.6 M in Et O), THF, -78°C – 0°C, 16 h, 40%
for 74a and 14% for 75a
4
2
2
2
2
2
2
2
4
2
2
2
2
2
2
or treated with methyl lithium to yield diols 62 and 63 containing tertiary alcohol on the
cyclobutane ring.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
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
Diols 72-76 flanked by pyridine fragments in each side of the molecule were prepared in largely
similar way as their mono-pyridine analogs with most notable difference being the synthesis of
the key intermediate 68 (Scheme 9). Oxidation of 3-methylenecyclobutanecarbonitrile (65) with
sodium periodate in the presence of ruthenium (III) chloride afforded ketone 66 in good yield.
Ketalization followed by nucleophilic addition of 2-fluoro-4-(trifluoromethyl)- pyridine or other
pyridyl halides afforded 68. The latter were elaborated to the target compounds 72-76 by a
similar set of reactions described above for the conversion of 56 to 60-64.
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
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Conclusions
This manuscript represents the first detailed report on the development of potent and selective
TRPV3 antagonists. Initial lead compound 5a was a potent TRPV3 antagonist, but it also
exhibited poor metabolic stability in liver microsomes. In an effort to address this primary
deficiency, we systematically modified the structural fragments identified as sites of metabolic
activation. Blockade of the metabolic hot spots with fluorine atoms or replacement of the
oxidation-prone carbon atoms in the cyclobutane moiety with oxygen or nitrogen atoms either
did not lead to the desired outcome or resulted in loss of TRPV3 potency. However, the
introduction of an hydroxyl group into the cyclobutane ring proved to be critical for increased
metabolic stability and improved plasma adjusted TRPV3 potency. This approach, in
conjunction with some manipulations of the right hand side aromatic group, afforded 74a, a
molecule with improved logD, LLE and other druglike properties that demonstrated anti-
nociceptive activity in rat models of pathological pain.
Experimental Section
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9
General methods . Unless otherwise noted, all materials were obtained from commercial
suppliers and used without further purification. Anhydrous solvents were obtained from Aldrich
1
(
Milwaukee, WI) and used directly. H NMR data were recorded using Varian Mercury 300 Mz,
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
58
59
60
Varian Inova 500 Mz or Agilent 400 MR spectrometers. The data are reported as follows:
chemical shift in ppm from internal tetramethylsilane standard on the δ scale, multiplicity (s =
singlet, d = doublet, t = triplet, q = quartet, qn = quintet, m = multiplet), coupling constants (Hz),
and integration. Mass spectral data were collected using Finnigan DCI/MS SSQ7000, Thermo
Scientific DSQ II (DCI) or Thermo Finnigan LCQ Deca (ESI) spectrometers. Analytical LC-MS
was performed on a Thermo MSQ-Plus mass spectrometer and Agilent 1100/1200 HPLC system
running Xcalibur 2.0.7, Open-Access 1.4, and custom login software. The mass spectrometer
was operated under positive APCI or ESI ionization conditions. The HPLC system comprised an
Agilent Binary pump, degasser, column compartment, autosampler and diode-array detector,
with a Polymer Labs ELS-2100 evaporative light-scattering detector. The column used was a
Phenomenex Kinetex C8, 2.6 μm 100Å (2.1mm × 30mm), at a temperature of 65 °C. A gradient
of 5-100% acetonitrile (A) and 0.1% trifluoroacetic acid in H O (B) was used, at a flow rate of
2
1.5 mL/min (0-0.05 min 5% A, 0.05-1.2 min 5-100% A, 1.2-1.4 min 100% A, 1.4-1.5 min 100-
5
% A. 0.25 min post-run delay). Preparative reverse phase HPLC purification was done on a
Phenomenex Luna C8(2) 5 µm 100Å AXIA column (30 mm × 75 mm) by using a gradient of
acetonitrile and 0.1% trifluoroacetic acid in H O. A Biotage initiator was used for microwave
2
reactions. HRMS data was collected using an Agilent 6550 Q-TOF using an electrospray ion
source. The purity for all compounds tested in bioassays was 95+%.
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3
3
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5
5
5
5
5
6
1
-(3,4-Dichlorophenyl)cyclobutanecarbonitrile (2a). To a suspension of sodium hydride (6.45 g, 269
mmol) in DMSO (250 mL) was added a solution of 2-(3,4-dichlorophenyl)acetonitrile (1a) (10
g, 53.8 mmol) and 1,3-dibromopropane (11.94 g, 59.1 mmol) in diethyl ether (60 mL) over 30
min at < 30°C.The reaction mixture was stirred at 15°C for 16 h and then diluted with 250
mL of ether and 150 mL of H O. The organic layer was separated and washed with H O
0
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and brine. After drying (Na
2
SO
4
), filtering, and concentrating, the residue was purified by
column chromatography on silica gel (petroleum ether: EtOAc= 10:1) to give 2a (10.2 g,
1
8
4 % yield). H NMR (300 MHz, DMSO-d ) δ 7.51 (m, 2H), 7.22 (m, 2H), 2.89 – 2.02 (m, 6H).
6
+
MS (ESI) m/z 227 (M + H) .
1
-(4-(Trifluoromethoxy)phenyl)cyclobutanecarbaldehyde (3b). To a solution of 2b (4.82 g,
o
0
.02 mol) in dry CH Cl (100 mL) at -78 C under an argon atmosphere was added
2
2
diisobutylaluminum hydride (24 mL, 1 M solution in toluene). The reaction mixture was stirred
o
at –78 C for 1 h and then quenched by dropwise addition of potassium sodium tartrate (10%
solution in H O). The resulting mixture was warmed to room temperature, stirred vigorously for
2
4
0 min and then diluted with CH Cl . The organic phase was separated and the aqueous phase
2 2
extracted with CH Cl . The combined organic layers were washed with brine, dried over Na SO
4
2
2
2
and the solvent was evaporated under reduced pressure. The crude product was purified by silica
gel column chromatography (petroleum ether:EtOAc = 100:0 to 30:1) to give 3b as a viscous oil
+
(2.7 g, yield 55.3%). MS (ESI) m/z 245 (M + H) .
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Page 29 of 88
Journal of Medicinal Chemistry
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(
1-(3,4-Dichlorophenyl)cyclobutyl)(pyridin-2-yl)methanone (4a). To a cooled to -78°C
solution of 2-bromopyridine (0.968 mL, 9.95 mmol) and THF (15 mL) was added n-butyllithium
(4.0 mL, 2.5 M solution, 9.95 mmol). After 10 min, a solution of 2a (1.5 g, 6.63 mmol) in THF
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(
10 mL) was added dropwise and after another 10 min 10 mL 2 N H SO was added. The
2
4
reaction mixture was stirred at 50-60 °C for 30 min, after which the aqueous phase was separated
and extracted with EtOAc. The combined organic phases were washed with H O, brine, dried
2
over Na SO , and filtered. After concentration in vacuo, the crude product was purified by
2
4
column chromatography on silica gel (EtOAc:hexane = 0:20) to give 4a (1.12 g, 55.5 % yield).
1
H NMR (400 MHz, CD CN) δ 8.47 (ddd, J = 4.8, 1.6, 0.9 Hz, 1H), 7.93 (dt, J = 7.9, 1.0 Hz,
3
1
H), 7.83 (td, J = 7.7, 1.7 Hz, 1H), 7.64 (d, J = 2.1 Hz, 1H), 7.43 - 7.32 (m, 3H), 3.02 - 2.91 (m,
+
2H), 2.66 - 2.57 (m, 2H), 2.02 - 1.95 (m, 1H), 1.91 - 1.79 (m, 1H). MS (ESI) m/z 307 (M + H) .
(S)-(1-(3,4-Dichlorophenyl)cyclobutyl)(pyridin-2-yl)methanol (5a) (by chiral separation of
racemic mixture (±)-5a). To solution of 4a (1.19 g, 3.9 mmol) in methanol (50 mL) was added
NaBH (0.45 g, 11.8 mmol) in portions, and the mixture was stirred overnight at room
4
temperature. After removal of the solvent, the pH of the remainder was adjusted to 7-8 by
addition of 1 N HCl and then extracted with EtOAc. The organic phase was dried over Na SO
2
4
and after concentration in vacuo, the residue was purified by column chromatography on silica
gel (petroleum ether: EtOAc=10: 1) to give racemic alcohol (±)-5a (0.54 g, 45% yield). MS
+
(ESI) m/z 309 (M + H) . The latter (88 mg) was separated on chiral column Chiralcel OJ-H
(isopropanol : hexane = 2:98) to obtain 5a (34 mg, 35% yield, 98% ee), [α]D = -57.4° (c 0.5,
MeOH) and (R)-5a) (40 mg, 41%, 94% ee), [α] = 51.8° (c 0.5, MeOH). Analytical data for 5a:
D
1
H NMR (300 MHz, DMSO-d6) δ ppm 8.42 (ddd, J = 4.8, 1.8, 0.9 Hz, 1H), 7.55 (d, J = 7.7, 1.8
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