Structure-activity relationship studies and discovery of a potent transient receptor potential vanilloid (TRPV1) antagonist 4-[3-chloro-5-[(1 S)-1,2-dihydroxyethyl]-2-pyridyl]-N-[5-(trifluoromethyl)-2-pyridyl]-3, 6-dihydro-2 H-pyridine-1-carboxamide (V116517) as a clinical candidate for pain management

Article abstract of DOI:10.1021/jm500818a

A series of novel tetrahydropyridinecarboxamide TRPV1 antagonists were prepared and evaluated in an effort to optimize properties of previously described lead compounds from piperazinecarboxamide series. The compounds were evaluated for their ability to block capsaicin and acid-induced calcium influx in CHO cells expressing human TRPV1. The most potent of these TRPV1 antagonists were further characterized in pharmacokinetic, efficacy, and body temperature studies. On the basis of its pharmacokinetic, in vivo efficacy, safety, and toxicological properties, compound 37 was selected for further evaluation in human clinical trials.

Full text of DOI:10.1021/jm500818a

Article
pubs.acs.org/jmc
Structure−Activity Relationship Studies and Discovery of a Potent
Transient Receptor Potential Vanilloid (TRPV1) Antagonist 4‑[3-
Chloro-5-[(1S)‑1,2-dihydroxyethyl]-2-pyridyl]‑N‑[5-(trifluoromethyl)-
2-pyridyl]-3,6-dihydro‑2H‑pyridine-1-carboxamide (V116517) as a
Clinical Candidate for Pain Management
Laykea Tafesse,*,† Toshiyuki Kanemasa,‡ Noriyuki Kurose,‡ Jianming Yu,† Toshiyuki Asaki,‡ Gang Wu,†
Yuka Iwamoto,‡ Yoshitaka Yamaguchi,‡ Chiyou Ni,† John Engel,† Naoki Tsuno,‡ Aniket Patel,†
Xiaoming Zhou,† Takuya Shintani,‡ Kevin Brown,† Tsuyoshi Hasegawa,‡ Manjunath Shet,†
Yasuyoshi Iso,‡ Akira Kato,‡ and Donald J. Kyle†
†Discovery Research, Purdue Pharma LP, 6 Cedar Brook Drive, Cranbury, New Jersey 08512, United States
‡Shionogi Research Laboratories, Shionogi & Co., Ltd., 3-1-1 Futaba-chow, Toyonaka, Osaka, Japan
S
* Supporting Information
ABSTRACT: A series of novel tetrahydropyridinecarboxamide TRPV1 antagonists were
prepared and evaluated in an effort to optimize properties of previously described lead
compounds from piperazinecarboxamide series. The compounds were evaluated for their
ability to block capsaicin and acid-induced calcium influx in CHO cells expressing human
TRPV1. The most potent of these TRPV1 antagonists were further characterized in
pharmacokinetic, efficacy, and body temperature studies. On the basis of its
pharmacokinetic, in vivo efficacy, safety, and toxicological properties, compound 37 was
selected for further evaluation in human clinical trials.
role in the initiation of neurogenic inflammatory responses and
may be a target for the treatment of other disorders such as
cough, urinary incontinence, irritable bowel syndrome, and
pruritus.
INTRODUCTION
■
Capsaicin, the active ingredient of chili peppers, has been
known for decades to specifically activate mammalian
nociceptors and cause pain.1 The cloning of the capsaicin
receptor, TRPV1, transient receptor potential cation channel,
subfamily V, member 1, by Julius and co-workers in 1997 has
stimulated basic research on the transduction of noxious stimuli
by sensory neurons at the molecular level and has facilitated the
identification of novel drugs to treat pain by targeting this
channel.2−5 TRPV1 is a nonselective cation channel that is
polymodal, being activated by multiple unrelated stimuli such
as capsaicin, heat, low pH, and certain endogenous substances
produced by inflamed tissues. Furthermore, TRPV1 is highly
expressed by nociceptive sensory neurons. The expression is
up-regulated in the presence of persistent inflammation. A
critical role of TRPV1 in the development of inflammatory pain
is supported by genetic inhibition of the channel in animals. In
particular, TRPV1 knockout mice are devoid of experimentally
induced thermal hypersensitivity with a pro-inflammatory
substance.6,7 Taken together, those features make TRPV1 to
be an attractive molecular target for treating pain, especially
pain associated with inflammation. In addition to pain
transduction, TRPV1 has also been shown to play a central
Identification of TRPV1 antagonists has been the focus of
drug discovery efforts over the past several years by a number
of pharmaceutical companies. A number of compounds have
reached clinical development, including 1,8 2,9 and 310 (Figure
1). In a phase I clinical trial with 2, dose-limiting hyperthermia
occurred at subtherapeutic doses during postoperative dental
pain studies.9 Compound 3 also induced hyperthermia in
healthy humans, but the magnitude was lower than what was
reported for 2.10,11 It is worth pointing out that hyperthermia
was not described as one of the adverse effects for compound
1.4,8 An additional unwanted side effect during clinical use of
TRPV1 antagonists is their effect in altering temperature
sensation. Clinical candidate 3 was reported to impair thermal
sensation by elevating skin and oral heat pain thresholds.10 This
has raised concerns about a potential risk of burn injury to the
skin or mouth for people who, for instance, take a hot water
Received: May 28, 2014
Published: July 24, 2014
© 2014 American Chemical Society
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Figure 1. Structures of selected TRPV1 antagonists.
shower or drink hot coffee/tea. These unwanted side effects
associated with the use of TRPV1 antagonists (i.e., hyper-
thermia and impairment of noxious thermal sensation) became
the main dose-limiting factors in current clinical develop-
ments.3,5 The underlying mechanisms behind these two
unwanted side effects is unclear. In addition, the magnitude
and duration of the side effects seems to be different for
different molecules. Despite unclear mechanisms, pharmaceut-
ical research to identify compounds with improved safety
margin is underway by integrating body temperature and
thermal sensation measurements in preclinical development
strategy. The identification of novel TRPV1 antagonists with a
wider safety margin will allow clinical trials designs to include
higher dose groups for proof-of-concept studies that the field of
TRPV1 research still awaits.
During the past decade, various structural classes of TRPV1
antagonists have been reported in the literature.12 We
previously described SAR study results of piperazinecarbox-
amides analogues such as 4 as potent TRPV1 antagonists.13
Lead compounds from this series were highly potent and
efficacious across a range of preclinical pain models. Despite
this, development of lead compounds from this series was
hindered due to a variety of issues including poor aqueous
solubility and oral bioavailability. We, therefore, initiated our
follow-up SAR effort with the aim of identifying novel potent
TRPV1 antagonists with improved aqueous solubility,
pharmacokinetic properties, and overall pharmaceutical profile.
Our strategy to achieve this goal was based on a systematic
exploration of polar, water solubilizing substitutions on pyridine
ring A and aniline C. Our initial SAR exploration effort has led
to the identification of tetrahydropyrine ring as a bioisosteric
replacement for piperazine ring B (Figure 2). Therefore, in this
article, we used tetrahydropyridine as the core ring B and
describe SAR study results for rings A and C with emphasis on
potency, aqueous solubility, and metabolic stability. This SAR
study effort has culminated with the identification of compound
37, a potent TRPV1 antagonist that is currently being evaluated
in human clinical trials for treatment of pain.
CHEMISTRY
■
The synthetic route employed to prepare analogues 9−16 is
shown in Scheme 1. Commercially available N-Boc-1,2,3,6-
tetrahydropyridine-4-boronic acid pinacol ester (6) was reacted
with 2,4-dichloropyridine (5) by Suzuki coupling to give the
corresponding N-Boc-tetrahydropyridine (7) in 79% yield. The
Boc protecting group was removed by treatment with 4 N HCl
in dichloromethane, leading to 8 as a hydrochloride salt in 95%
yield. Intermediate 8 was then treated with different substituted
and unsubstituted isocyanates in the presence of diisopropy-
lethylamine (DIEA) as the base to give the corresponding
targets (9−16) in good yields. Analogues (20−29) were
prepared in a similar manner by using 5-substituted pyridines
(17a−j) as starting materials instead of 5 (Scheme 2).
The synthesis of enantiomers (S)-35 and (R)-36 is outlined
in Scheme 3. The 5-formyl group in 2,3-dichloro-5-formylpyr-
idine (30) was converted to a vinyl group by reacting with
methyltriphenylphosphonium bromide via Wittig reaction in
the presence of t-BuOK in toluene to afford 31 in 70% yield.
Subsequent asymmetric dihydroxylation of the vinyl group with
either AD-mix-α or AD-mix-β by using t-BuOH and water as
co-solvents gave the corresponding (S)-diol 32a (>95% ee) or
(R)-diol 32b (>95% ee) in 80% yield. Then Suzuki coupling of
32a and 32b with pinacole ester 6 afforded 33a and 33b in 80%
yields. Deprotection of the boc protecting group with 4 N HCl
followed by coupling with 4-(trifluoromethyl)phenyl isocyanate
in the presence of diisopropylethylamine (DIEA) as the base
gave analogues (S)-35 and (R)-36.
The synthesis of analogues 37−39 is outlined in Scheme 4.
Coupling reaction of 34a with phenyl(5-(trifluoromethyl)-
pyridin-2-yl)carbamate or phenyl(6-(trifluoromethyl)pyridin-2-
yl)carbamate in the presence of diisopropylethylamine (DIEA)
in CH2Cl2 afforded the corresponding ureas (S)-37 and (S)-38
in 74% and 70% yield, respectively. Similarly, coupling of 34b
with phenyl(5-(trifluoromethyl)pyridin-2-yl)carbamate gave
(R)-39 in good yield.
RESULTS AND DISCUSSION
■
The compounds were evaluated for TRPV1 antagonist activity
based on their ability to block capsaicin (CAP) or low pH-
induced activation of human TRPV1 channel in CHO cell line.
The tests were carried out in fluorescence-based calcium influx
assays using FLIPR (Molecular Devices, CA) for CAP assay
and FDDS (Hamamatsu Photonics, Japan) for pH assay.
We began our investigation by replacing the piperazine ring
of our previous lead molecules with tetrahydropyridine as a B-
Figure 2. SAR approach based on piperazine carboxamides.
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a
Scheme 1. Synthesis of C-Ring Modified Tetrahydropyridine Analogues 9−16
a
Reagents and conditions: (a) Pd(PPh3)2Cl2, K2CO3, DME/EtOH/H2O (2:1:2), 90 °C; (b) 4 M HCl in Et2O, DCM, 45 °C; (c) substituted phenyl
isocyanate or substituted N-phenyl-1H-imidazole-1-carboxamide, DIEA, DCM, 0 °C → rt.
a
Scheme 2. Synthesis of A-Ring Modified Analogues 20−29
a
Reagents and conditions: (a) Pd(PPh3)2Cl2, K2CO3, DME/EtOH/H2O (2:1:2), 90 °C; (b) 4 M HCl in Et2O, DCM, 45 °C; (c) 4-
(trifluoromethyl)phenyl isocyanate, DIEA, DCM, 0 °C → rt.
a
Scheme 3. Synthesis of Dihydroxyl Analogues (S)-Diol 35 and (R)-Diol 36
a
Reagents and conditions: (a) PPh3CH3Br, t-BuOK, toluene; (b) AD-mix-α, t-BuOH/H2O (1:1), 0 °C → rt, then Na2SO3; (c) AD-mix-β, t-BuOH/
H2O (1:1), 0 °C → rt, then Na2SO3; (d) Pd(PPh3)2Cl2, K2CO3, 6, DME/EtOH/H2O (2:1:2), 90 °C; (e) 4 M HCl in Et2O, DCM, 45 °C; (f) 4-
(trifluoromethyl)phenyl isocyanate, DIEA, DCM, 0 °C → rt.
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a
Scheme 4. Synthesis of Pyridine Analogues 37−39
a
Reagents and conditions: (a) phenyl(5-(trifluoromethyl)pyridin-2-yl)carbamate, DIEA, DCM, −20 °C → rt; (b) phenyl(5-(trifluoromethyl)-
pyridin-2-yl)carbamate, DIEA, DCM, 0 °C → rt.
Table 1. Effect of Aryl Group Modification on in Vitro Potency and ADME Properties
metabolic stability
a
in vitro potency, IC50 (nM)
(% remaining)
solubility (μM)
pH: 1.2 pH: 6.8
no.
R1
t-Bu
R2
CAP
pH
RLM
HLM
BCTC
9
H
H
H
H
H
2.9 0.1
135.9 33.0
211.2 13.4
103.7 17.4
469.4 138.2
7633.8 2690.5
156.6 6.2
>10000
0.4 0.1
31.8 (n: 1)
59.7 5.9
29.3 4.8
0
59
57
62
0
19
68
106
103
2
24
0
0
0
CF3
10
OCF3
SO2CF3
N(Et)2
CF3
>50
>50
>50
>50
>50
>50
>50
2
11
4
12
40
10
13
13
2-OCH3
47
37
15
0
73
17
29
0
14
CF3
3-OCH3
213.9 31.7
17.6 3.4
15
CF3
2-N(CH3)2
3-N(Et)2
0.97
0.8
16
CF3
124.2 11.9
a
IC50 values are the mean SEM of at least three determinations unless otherwise stated.
ring. This bioisosteric replacement was proven to be feasible as
evidenced by favorable in vitro potency of compound 9.
Compound 9 was a good starting point for our lead
optimization effort because of its favorable in vitro potency
and metabolic stability. While the in vitro potency for
compound 9 was acceptable, the compound showed very
poor aqueous solubility and pharmacokinetic properties. In an
effort to improve the aqueous solubility of such analogues, we
initiated a detailed SAR study with the goal of introducing
water solubilizing groups to pyridine ring-A and aniline ring-C.
We initially focused on the examining the effect of
introducing polar substitutions on ring C (Table 1).
Replacement of the lipophilic substitution CF3 group at the
4-position of ring-C with OCF3 or SO2CF3, as in compounds
10 and 11, resulted in analogues that retained similar potency
and metabolic stability to the lead compound 9. Compounds
10 and 11 also showed significant improvements in aqueous
solubility in acidic pH, however, their poor aqueous solubility in
neutral pH made them less desirable for follow-up studies. A
significant improvement in aqueous solubility was achieved
when the CF3 group on ring-C of 9 was replaced with an amino
group, as in 12. The resulting analogue, however, showed a
dramatic decrease in metabolic stability and a slight decrease in
potency. Having explored few possible replacements for CF3
group, we then focused our attention to study the influence of
additional substitution on C-ring. We quickly learned that the
position of the substitution on the aniline ring has a significant
effect on potency. For example, while introduction of both
amino and methoxy groups at 2-position of the ring-C, as in 13
and 15, showed slight improvements in aqueous solubility, it
resulted in a dramatic loss of potency. On the other hand,
analogues with similar substitutions at 3-position of the aniline
ring-C showed acceptable potency as demonstrated by
compounds 14 and 16. These 3-substituted analogues,
however, did not provide good metabolic stability and aqueous
solubility to justify advancement to additional studies.
We next examined the effect of introducing polar groups to
ring-A of the lead compound 9 (Table 2). Most of the
exploration was focused on the 5-position of the A-ring because
that position was identified to be of tolerant of substitutions
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Table 2. Effect of Pyridine Ring Substitution on in Vitro Potency and ADME Properties
metabolic stability
(% remaining)
a
in vitro potency, IC50 (nM)
solubility (μM)
no.
R1
R2
CAP
pH
RLM HLM
pH: 1.2
pH: 6.8
9
Cl
Cl
Cl
F
H
135.9 33.0
923.5 54.0
3166.7 606.7
>25000
31.8 (n: 1)
59
6
68
71
66
28
96
96
81
94
87
88
94
91
97
0
>50
>50
>50
29
0
>50
>50
>50
>50
>50
9
20
21
22
23
24
25
26
27
28
29
35
36
5-CH2N(CH3)2
5-(CH2)2N(CH3)2
4-CH2N(CH3)2
5-CO2H
5
0
Cl
Cl
Cl
Cl
Cl
F
>25000
95
101
9
5-CH2CO2H
>25000
>50
27
5-CH2CN
265.2 69.0
128.6 26.0
130.5 20.0
7584.5 1109.3
471.3 127.3
75.8 12.4
37.1 14.8
165 (n: 1)
41.7 4.4
5-CH2OH
89
74
85
84
81
88
>50
>50
>50
17
>50
10
5-(CH2)2OH
185.3 16.0
4-CH2OH
24
Cl
Cl
Cl
5-CH2NHSO2CH3
5-S-CH(OH)CH2OH
5-R-CH(OH)CH2OH
0.5
11.6 0.5
38.3 4.0
>50
>50
>50
>50
a
IC50 values are the mean SEM of at least three determinations unless otherwise stated.
Table 3. Effect of Switching Ring-C from Aniline to Pyridine
metabolic stability
(% remaining)
a
in vitro potency, IC50 (nM)
solubility (μM)
no.
X
Y
CAP
pH
RLM
HLM
pH: 1.2
pH: 6.8
35
37
38
39
CH
N
CH
CH
N
75.8 12.4
35.1 8.8
11.6 0.5
39.5 (n: 2)
81
85
94
90
91
97
>50
>50
>50
>50
>50
>50
>50
>50
CH
N
800.4 63.3
161.1 41.7
5156.0 (n: 2)
223.3 14.0
102
99
CH
a
IC50 values are the mean SEM of at least three determinations unless otherwise stated.
based on previous SAR experience.13 Introduction of basic
center on ring-A for salt formation site, as in 20 and 21,
resulted in analogues that showed significant improvements on
aqueous solubility in both acidic and neutral media. However,
the in vitro potency as well as the metabolic stability of the
resulting analogues was significantly reduced. We prepared
compound 22 with the basic center moved to the 4-position of
ring-A to explore the possibility of regaining in vitro potency.
The resulting compound retained good aqueous solubility
profile as expected, however, the substitution at this position
was found to be detrimental for in vitro potency. We next
explored the effect of introducing acidic groups at the 5-
position of the A-ring. The introduction of acidic functionality
to ring-A, as in 23 and 24, offered much improved aqueous
solubility and metabolic stability profile. However, the resulting
compounds did not show potency at TRPV1. On the basis of
these results, the possible use of acidic or basic functionality on
ring-A was abandoned. We then prepared compound 25 to
explore the effect of incorporating less polar substitutions on
ring-A. Compound 25 showed about a 2-fold loss of potency in
CAP assay and a 5-fold loss of potency in potency in pH assay.
This result suggested that it may be possible to introduce a
substitution with some polarity at this position and still
maintain potency. We next explored the use of small
monohydroxyl groups such as methanol and ethanol, 26 and
27, on ring-A. This modification resulted in significant
improvements in aqueous solubility while also maintaining
good in vitro potency and metabolic stability. The mono-
hydroxyl compounds, 26 and 27, showed very similar in vitro
potencies when compared to compound 9 in CAP assay. In pH
assay, the methanol substituted analogue 26 was about 4-fold
more potent than the ethanol substituted analogue 27. We also
moved the hydroxyl group substitution to the 4-position on
ring-A, compound 28, and found that this was not tolerated for
in vitro potency. We next explored the effect of introducing
methylsulfonamide group, compound 29, as a bioisosteric
replacement for the hydroxyl group on compound 27. This
replacement did not seem to provide benefit, as the resulting
compound showed about 4-fold less potency and also lower
aqueous solubility neutral pH. Encouraged by the results from
the monohydroxyl groups, we next explored the effect of
introducing dihydroxyl group at the 5-position of ring-A, as in
35 and 36. Both dihydroxyl substituted compounds showed
excellent aqueous solubility and metabolic stability. In addition,
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both compounds exhibited good in vitro potencies. The
chirality of the dihydroxyl group did not seem to have an
effect on the in vitro potency and metabolic stability of the
initial two analogues.
that the hydrogen-bond acceptor moiety of the ligand may
interact with Tyr 667 of the channel. The ring domain within
the proposed antagonist pharmacophore fits within a hydro-
phobic space formed by the side chains of four Tyr 667 residues
of the four monomers.17
Having identified an optimal substitution for pyridine ring-A
that maintains good in vitro potency and metabolic stability, we
then focused our attention on fine-tuning the ring-C (Table 3).
It has previously been shown that the use of pyridine as a ring-
C instead of aniline improves pharmaceutical properties of
carboxamide analogues.14 We, therefore, prepared dihydroxyl
analogues using pyridine ring-C, as in 37, 38, and 39. All of the
resulting analogues showed optimal metabolic stability and
aqueous solubility profiles. Unlike the analogues 35 and 36 with
an aniline ring-C, chirality of the dihydroxyl substitution on A-
ring had a significant effect on the in vitro potency (e.g., 37 vs
39). The potency of R-enantiomer dihydroxyl compound 39 is
about 5-fold less than its corresponding S-enantiomer
compound 37 in both CAP and low pH assays. We have also
found that the position of the pyridine nitrogen on ring-C
pyridine nitrogen has significant effect on in vitro potency as
shown by compounds 37 vs 38. Compound 37 with a pyridine
nitrogen at 2-position is about 20-fold more potent in CAP
assay and over 100-fold more potent in pH assay when
compared to the compound with a pyridine nitrogen at 3-
position, 38.
During our SAR investigation of this work, a reference
describing the introduction of polar groups such as carboxylic
acid and hydroxyl groups was published on a piperazine-
benzimidazole class of TRPV1 antagonists.15 In a similar
manner to our findings, the reference described that hydroxyl
groups on ring-A were well tolerated in the piperazine-
benzimidazole series. The article described the synthesis and
profile of a racemic dihydroxyl substitution in the piperazine-
benzimidazole series. In our studies, we have found that the
chirality of the dihydroxyl substitution can have significant
effects on in vitro potency and pharmacokinetic properties of
the resulting analogues. The article also describes that
carboxylic acid substitutions are tolerated on pyridine ring-A
in the piperazine-benzimidazole series. In our series of
analogues, however, we have observed that the incorporation
of a carboxylic acid at the 5-position of ring-A, as in 23 and 24,
can be detrimental to in vitro potency.
Potent compounds, such as compound 26, 27, 35, 36, 37,
and 39, that contain a substitution on A-ring, may conform to
the known antagonist pharmacophore and therefore may
interact comparably with other reported TRPV1 antagonists
because they contain the hydrogen bond donor hydroxyl group,
the hydrogen bond acceptor (trifluoromethyl), and the ring
functionalities. We have not conducted specific homology
modeling and docking simulations to evaluate this possibility
directly, but we presume that such compounds would fit the
proposed model given that it contains the three important
pharmacophore elements of other known antagonists and given
further a consideration of its overall molecular size, shape, and
flexibility. Of interest in future molecular modeling studies
would be an analysis of our surprising result showing that the
diol-containing molecules block TRPV1 activation, while
carboxylate- or amine-containing molecules do not. There are
pKa and steric differences between these functional groups that
likely contribute to the explanation, but these differences may
also reveal nuance in the character of the hydrogen bond donor
functionality that is optimal for channel modulation.
The compounds that demonstrated acceptable in vitro
potency, aqueous solubility, and metabolic stability were
advanced into our preliminary pharmacokinetic screening
studies (Table 4). Compounds 22, 26, 27, 35, 36, 37, and
Table 4. Rat Pharmacokinetic Parameters for Selected
a
Compounds
T1/2 (h)
(iv)
CL (L/h/kg)
(iv)
Vd
(L/kg)
Cmax (ng/mL)
F
(%)
no.
(po)
26
27
35
36
37
39
0.2
0.7
1.6
0.8
1.9
0.5
1.7
0.5
0.4
1.5
0.2
1.5
0.5
0.4
0.8
1.2
0.52
0.8
BQL
13
0
4
76
26
15
108
23
25
427
74
a
Compounds dosed in DMA/PG (1:1) for iv (0.5 mg/kg), and 0.5%
methylcellulose for po (1 mg/kg).
Although the molecular structures of our compounds differ
from capsaicin and other vanilloids, they are highly similar to
our initial lead molecule 4, which was shown by Schild analysis
to compete with capsaicin binding at the TRPV1 receptor.16
On that basis, we propose that the mode of TRPV1 binding for
our compounds is likely similar to 4 and thus interact
significantly with key amino acid residues at or near the
capsaicin binding site, although experimental proof of
competitive binding remains to be obtained. Capsaicin is
highly lipophilic and is presumed to penetrate the lipid bilayer
then bind to the TRPV1 channel near the interface of the
intracellular plasma membrane. Three important binding sites
have been proposed for capsaicin binding to TRPV1 including
two polar contacts (Arg114, Glu761) at the intracellular plasma
membrane and a hydrophobic contact (described as the TM3
region) buried within the lipid bilayer domain of the
channel.17,18 Most TRPV1 antagonists, including our new
compounds, contain three essential moieties in the pharmaco-
phore, specifically a hydrogen bond donor, a hydrogen bond
acceptor, and a ring. Recently published homology modeling of
the TRPV1 channel−TRPV1 antagonist interaction proposes
39 were dosed to rats intravenously (0.5 mg/kg) and orally (1
mg/kg). The monohydroxyl analogues 26 and 27 exhibited
very short half-lives following intravenous administration and
subsequently poor bioavailabilities after oral administration.
The dihydroxyl analogues 35 and 36 showed higher systemic
exposure after oral administration when compared to
monohydroxyl analogues. It is interesting to note the effect
the chirality of the dihydroxyl substitution has on the
pharmacokinetic properties of the dihydroxyl analogues 35
and 36. The S-enantiomer analogue 35 demonstrated about 4-
fold lower clearance after intravenous administration and 3-fold
higher exposure after oral administration when compared to the
R-enantiomer 36. This chirality effect on pharmacokinetic
properties was consistent even when ring-C is switched to
pyridine, e.g. 37 vs 39. The S-enantiomer dihydroxyl analogue
37 showed about 8-fold lower clearance after intravenous
administration when compared to its corresponding R-
enantiomer analogue 39. Compound 37 showed significantly
improved plasma exposure (Cmax: 427 ng/mL) and bioavail-
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ability (108%) after oral administration, whereas the corre-
sponding enantiomer, 39, showed lower exposure (Cmax: 74 ng/
mL) and bioavailability (23%) after oral administration.
Because of its favorable in vitro potency, metabolic stability,
and pharmacokinetic properties, compound 37 was selected as
a lead candidate for pharmacokinetic, efficacy, and safety
studies.
Compound 37 was screened for selectivity at 10 μM in a
panel of 66 ion channels, receptors, transporters, and enzymes
(NovaScreen). Compound 37 had no interaction with any of
the targets in the panel with the exception of a weak interaction
with adenosine transporter (51.1%). In a follow-up experiment,
the activity of compound 37 at adenosine transporter was
determined to be 8.7 μM. Selectivity of compound 37 versus
other closely related TRP channels such as TRPV3 and TRPV4
was also evaluated. Compound 37 was highly selective for
TRPV1 and did not show potency up to 10 μM in both TRPV3
and TRPV4 assays (see Supporting Information).
concentration was determined to be 0.09, indicating that
compound 37 is primarily restricted in periphery.
A comparative pharmacokinetic study was also conducted
across preclinical species rat, dog, and monkey (Table 7).
Table 7. Pharmacokinetic Parameters for Compound 37
after Intravenous (1 mg/kg) and Oral (3 mg/kg)
a
Administration to Rats, Dogs, And Monkeys
T1/2 (h)
(iv)
CL (L/h/kg)
(iv)
Vd
(L/kg)
Cmax (ng/mL)
species
(po)
F (%)
rat
3.3
3.6
18
0.24
0.28
0.36
0.68
1.2
1380
1120
459
74
100
107
dog
monkey
6.0
a
Data are expressed as the mean of three animals.
Compound 37 showed very low clearance across species after
intravenous administration at 1 mg/kg. After oral admin-
istration at 3 mg/kg, good plasma exposures were obtained in
all three species. The oral bioavailabilities were 74% with a Cmax
of 1380 ng/mL for rat, 100% with a Cmax of 1120 ng/mL for
dog, and 107% with a Cmax of 459 ng/mL for monkey.
Prior to in vivo evaluations in rat pain models, we studied the
ability of compound 37 to block the native TRPV1 channel in
rat dorsal root ganglion (DRG) neurons. Whole cell patch
clamp current recordings were performed to directly measure
the channel activity. In this assay, compound 37 showed potent
activity in inhibiting both CAP and low pH induced activation
of native TRPV1 in a CAP-sensitive population of rat sensory
A thorough examination of pharmacokinetic properties of
compound 37 was conducted to determine its oral bioavail-
ability and dose proportionality. As shown in Table 5,
Table 5. Pharmacokinetic Parameters of Compound 37 after
a
Single Oral Administration to Nonfasted Rats
dose
pharmacokinetic parameters 0.3 mg/kg 1 mg/kg 3 mg/kg 10 mg/kg
Cmax (ng/mL)
Tmax (h)
131
1.0
1.0
74
447
1.7
4.2
92
1380
1.3
5630
2.7
c
AUCinf (μg·h/mL)
bioavailability (%)
10.1
74
59.1
83
neurons [IC50 = 423.2 39.1 nM (n = 3) for CAP; IC50
=
b
180.3 54.3 nM (n = 3) for acid] (Figure 3). Consistent with
calcium influx data from recombinant TRPV1 (Table 2), this
suggests that 37 belongs to the group of TRPV1 antagonists
that block both CAP and low pH activations of TRPV1.19,20
Furthermore, in the patch clamp assay it is apparent that the
channel activity recovered quickly upon washout of the
compound in both capsaicin and proton modes (Figure 4),
suggesting that compound 37 has fast-off kinetics for
antagonism of both mode activations of TRPV1.
In addition to in vitro electrophysiology (EP) study, we
studied in vivo target engagement using capsaicin induced eye-
wipe model in rats. In this model, compound 37 dose
dependently reduced the number of eye-wipes induced by
capsaicin. A significant reversal was demonstrated at 10 and 30
mg/kg oral doses for up to 5 h, proving that compound 37 has
a long duration of action and that the in vivo efficacy is on
target.
Compound 37 was then evaluated in acute inflammatory
CFA model for its ability to reverse thermal hyperalgesia. In
this model, compound 37 showed dose-dependent reversal of
thermal hyperalgesia with an ED50 of 2 mg/kg (PO). The
strong effect of compound 37 on inflammatory pain may be
attributed to its high potency for blocking proton activation of
TRPV1 in inflamed tissue (Figure 5). This supports the notion
that the ability to block proton activation of TRPV1 is required
for anti-inflammatory effects of TRPV1 antagonists.21
a
b
Data are expressed as the mean of three rats. Bioavailability (%) =
c
dose(iv)/dose(po) × AUC∞(po)/AUC∞(iv) × 100. Using AUCiv 1
mg/kg for BA of 0.3, 1, and 3 mg/kg, and using AUCiv 10 mg/kg for
BA of 10 mg/kg.
bioavailability and plasma levels were high after oral
administration of 37 at 0.3, 1, 3, and 10 mg/kg doses. For
example, oral bioavailability of 1 mg/kg dose was 92% with Cmax
of 447 ng/mL and the oral bioavailability of 3 mg/kg dose was
74% with Cmax of 1380 ng/mL. Dose-proportionality of
compound 37 was also maintained between doses 0.3−3 mg/
kg. Compound 37 showed low clearance and volume of
distribution after intravenous administration at 1, 3, and 10 mg/
kg (Table 6). In addition to plasma exposure studies,
compound 37 was also evaluated for its ability to penetrate
into CNS. The ratio of brain-to-plasma was assessed in rats at 3
h after oral administration of compound 37 at 3 mg/kg. The
plasma concentration was 954 ng/mL and the brain
concentration was 87 ng/mL. The ratio of brain-to-plasma
Table 6. Pharmacokinetic Parameters of Compound 37 in
Plasma after Single Intravenous Administration to
a
Nonfasted Rats
dose
pharmacokinetic parameters
1 mg/kg
3 mg/kg
10 mg/kg
Several reports indicate that TRPV1 block is associated with
an increase in core body temperature. We, therefore, evaluated
the effects of compounds 37 on core body temperature after
oral administration to rats implanted with telemetry devices
(Figure 6). Similar to other previously described TRPV1
antagonist molecules, compound 37 caused dose-dependent
increase in body temperature in rats after acute administration.
AUCinf (μg·h/mL)
CLtot (L/h/kg)
t1/2 (h)
4.6
14.4
0.21
2.9
71.6
0.14
2.7
0.24
3.3
Vdss (L/kg)
0.68
0.56
0.45
a
Data are expressed as the mean of three rats.
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Figure 3. Inhibition by compound 37 of capsaicin (CAP)- and acid (pH 5)-induced currents in rat DRG neurons expressing native TRPV1. CAP
(blue bar) induced inward currents in a population of rat DRG neurons, and the currents were inhibited by coapplication of compound 37 (red bar).
Acid (pH 5, blue bar)-induced sustained inward currents were also inhibited by co-application of compound 37 (red bar) in a population of rat DRG
neurons (responsive to CAP, not shown). Concentration−inhibition curves are plotted. Data are presented as the mean SEM.
study is left shifted compared to the dose−response curve for
its analgesic efficacy. As a result, a significant increase in body
temperature was observed for 2 even at subanalgesic doses. In
contrast, compound 37 did not exhibit a leftward shift on the
dose−response curve for its effects on body temperature when
compared to its analgesic efficacy. It has previously been
reported that the body temperature risk with TRPV1 block can
only be minimized by designing compounds that show
differential pharmacology, i.e., blocking CAP mode of activation
and not pH.22 However, in this case we have discovered a
compound that blocks both CAP and pH modes of activation
and yet shows a reduced risk for body temperature elevation
when compared to comparator 2. The mechanism behind such
differences in the magnitude of body temperature change is not
yet clearly understood, however, it could be due to a
combination of factors including receptor kinetics. Compound
37 showed fast off-rate in receptor kinetics study. This property
Figure 4. Effect of compound 37 in the eye-wipe test model after oral
administration. Treatment with compound 37 dose dependently
reduces the number of eye-wipes induced by capsaicin. Data are
presented as the mean SEM; (*) p < 0.05, (**) p < 0.01 vs vehicle.
of compound 37 is unique compared to previously reported
clinical compounds such as 2 and 3, which exhibited slow-off
rates. Additional details on our receptor kinetics study will be
reported elsewhere.
A second undesirable clinical finding in the development of
TRPV1 antagonist compounds was their effect of TRPV1 block
on thermal sensitivity. In clinical studies using healthy subjects,
compound 3 has previously been reported to cause impairment
in thermosensation.10 The effect was sustained over the dosing
period, but recovery was fast after washout period. We used a
hot plate test in order to assess the effect of compound 37 on
thermal sensation in a preclinical setting.23 The results from
this study were compared with previous TRPV1 antagonist
clinical compounds 2 and 3 (Figure 8). In our studies, we
found that oral administration of all three compounds showed a
dose-dependent reduction in thermal sensitivity. There was a
very slight but favorable separation in dose−response curve
between efficacy and temperature sensation for all three
compounds. In this assay, compound 37 did not show any
favorable advantage when compared to 2 or 3. A detailed
analysis of the effect of compound 37 on thermosensation in a
clinical setting will be the subject of future publication.
In this study, the maximum body temperature increase was
capped at about 1.0 °C above vehicle controls even when 37
was dosed at exposures severalfold higher than analgesic doses.
We also evaluated the effect of compound 37 on body
temperature after repeated dosing. Compound 37 was dosed
twice a day for 4 days, and rectal temperatures were measured
by using a rectal probe thermistor. In agreement with previous
reports, the increase in body temperature was attenuated after
repeated administration of compound 37 (Table 8).
We next conducted body temperature and analgesic efficacy
studies for 2 and compared its effects with compound 37.
Compound 2 is withdrawn from clinical trials due to dose-
limiting hyperthermia effects during postoperative dental pain
studies. In our study, we found that compound 2 shows a large
degree of separation between the dose−response curves for
body temperature and analgesic efficacy (Figure 7). For
compound 2, the dose−response curve for body temperature
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Figure 5. Efficacy of compound 37 in the CFA inflammatory pain model after oral administration to rats. Compound 37 dose-dependently reverses
inflammatory thermal hyperalgesia. Data are presented as the mean SEM; (*) p < 0.05, (**) p < 0.01 vs vehicle.
Figure 6. Body temperature changes as compared to vehicle following oral administration of compound 37 in rats. Compound 37 was administered
at 13:00.
S-enantiomer of a dihydroxyl substituent at the 5-position of A-
ring as an optimal group for improving aqueous solubility and
pharmacokinetic properties while maintaining excellent in vitro
potency.
Table 8. Maximal Body Temperature Changes As Compared
to Vehicle Following Repeated Oral Administration of
Compound 37 (10 mg/kg, bid) for 4 Days in Naive Rats
̈
day 1
day 2
day 3
day 4
The development of TRPV1 antagonists have been
hampered by dose-limiting side effects such as hyperthermia
and effects on temperature sensation. To this end, we have
demonstrated that analgesic efficacy can be separated from the
unwanted side effects by profiling 37 alongside the clinical
comparator 2 in preclinical studies. Although compound 37
does not show mode-selective antagonism of TRPV1 for
capsaicin and proton, its pharmacology is unique in that it
shows fast-off kinetics in EP assay using CAP and pH as
stimulants when compared to 2. The results of this receptor
kinetics study will be the subject of future publication.
In summary, compound 37 showed good in vitro potency,
aqueous solubility, metabolic stability, oral bioavailability in
multiple species, and excellent efficacy in a variety of animal
pain models. After extensive safety and toxicology studies,
a
ΔBT
0.95 0.20
0.71 0.16
0.64 0.11
0.41 0.12
a
ΔBT values are the mean SEM of eight animals.
SUMMARY
■
In conclusion, we have conducted SAR studies with the goal of
identifying a novel development candidate with acceptable in
vitro potency and overall pharmaceutical profile. This was
achieved through systematic modifications of the different
regions of the lead molecule: rings A, B, and C. The SAR study
was initiated by replacing the piperazine ring portion of the lead
molecules with a tetrahydropyridine ring. This was followed by
detailed SAR with the goal of improving aqueous solubility,
pharmacokinetic properties, and overall pharmaceutical profile
of the lead compounds. This has led to the identification of the
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Figure 7. Plot comparing the effect of 37 or 2 on body temperature and pain in rats. Maximal body temperature changes (as compared to time-
matched vehicle) and maximal percent reversal of CFA inflammatory pain are plotted against the oral dose.
Figure 8. Plot comparing the effect of 37, 2, and 3 on pain and thermal sensation in rats. Maximal percent reversal of CFA inflammatory pain and
maximal percent inhibition of thermal sensation in the hot plate test are plotted against oral dose.
mM D-glucose (pH 7.4), supplemented with 0.1% BSA and 5 mM
CaCl2.
compound 37, named as V116517, was selected to advance into
clinical trials.
Sensory Neuron Assay. Whole cell patch clamp recordings and
primary culture of sensory neurons from dorsal root ganglia (DRGs)
were conducted according to previously described methods.6,24 In
brief, DRG were harvested from postnatal (P2−P4) Sprague−Dawley
rats. Following trypsinization (2.5%; Invitrogen, Carlsbad, CA) in
Ham’s F-12 media, the digested tissue was rinsed with F-12 media
containing 10% fetal calf serum. The tissue was triturated and plated
on PDL/laminin coated coverslips (BD Biosciences, San Jose, CA) in
neurobasal media supplemented with B27 (Invitrogen, Carlsbad, CA)
and 50 ng/mL 2.5S mouse NGF (Sigma, St. Louis, MO). The plated
neurons were incubated at 37 °C in 5% CO2 for up to 3 days during
which whole-cell voltage-clamp recordings were made using an
Axopatch 200B amplifier and the pClamp software (Axon Instruments
Inc., Union City, CA) using conventional patch-clamp techniques at
room temperature (22−24 °C). Currents were obtained at a hold
potential of −70 mV with Borosilicate patch pipettes (resistance: 1.5−
3 MΩ), low pass filtered at 2−5 kHz and digitized at 1 kHz. The
external solution contained (mM): NaCl (145), KCl (5), CaCl2 (2),
MgCl2 (1), glucose (10), HEPES (10), pH 7.3. The internal solution
was (mM): KCl (140), CaCl2 (1), MgCl2 (2), EGTA (11), pH 7.4.
For proton activation, HEPES was replaced with MES (2-(N-
morpholino)ethanesulfonic acid, 4-morpholineethanesulfonic acid) in
the external solution, and the solution was adjusted to pH 5 with HCl.
Acidic pH, capsaicin, and test compounds were applied to cells using
an array of glass pipettes together with the fast-switching solution
exchange system (SF-77B; Warner Instruments, Hamden, CT) .
Eye-Wipe Test. Male Sprague−Dawley rats (6 weeks, 180−280 g)
were used. Eye-wiping behavior was measured before (0 h) and at 1, 3,
and 5 h of after oral dosing with vehicle or the compounds. Rats were
acclimated for 10−20 min in 10 × 20 × 15 cm3 acrylic glass chambers
(Neuroscience, Japan) before testing at each time point. Capsaicin
solution (100 μM in 10% ethanol/PBS) or the eye solution vehicle
(10% ethanol/PBS) was applied into one eye (3 μL/eye) with a pipet
EXPERIMENTAL SECTION
■
TRPV1 Functional Assay. Cell-based fluorescence assays of
calcium influx upon TRPV1 activation were performed according to
previous described methods with modifications.16,21 In brief, CHO
cells stably overexpressing the human TRPV1 (hTRPV1) channel
(GenBank accession AJ277028) were cultured in DMEM supple-
mented with 2 mM L-glutamine and 10% fetal bovine serum in an
incubator (5% CO2) at 37 °C.
For capsaicin activation assay, cells were seeded in 96-well plates 24
h before measurements of Ca2+ influx were performed using
Fluorescence Imaging Plate Reader (FLIPR; Molecular Devices).
Plates were washed with Hank’s Solution (Life Technologies)
containing 1.6 mM CaCl2 and 20 mM HEPES (pH 7.4) before
loading of cells with Fluo-4 (3 μM × 1 h at 37 °C) in the presence of
2.5 mM probenecid. Cells were washed and then transferred to the
FLIPR. After a 15 s baseline reading, cells were incubated with test
compounds for 2 min before addition of the agonist (100 nM
capsaicin), and fluorescence was read for additional 3 min.
In the pH assay, cells were seeded in 96-well plates for 2 or 3 days
before measurements of Ca2+ influx performed using FDSS-3000
(Hamamatsu Photonics, Japan). After washout of culture medium,
cells were loaded with Fura-2 AM (5 μg/mL) and 0.5% Pluronic F127
in the loading buffer for 50−60 min at 37 °C. The loading buffer was
composed of 20 mM HEPES, 115 mM NaCl, 5.4 mM KCl, 0.8 mM
MgCl2, 1.8 mM CaCl2, 13.8 mM D-glucose, and 2.5 mM probenecid
(pH 7.4). Cells were washed and incubated with test compounds in
measuring buffer at 4 °C for 15−20 min before being transferred to
the FDSS-3000. After a baseline collection, acid pH solution was
added to cells to activate hTRPV1 (final pH 5.0−5.1 by titration with
1 N H2SO4). The measuring buffer was composed of 20 mM HEPES,
115 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 1.8 mM CaCl2, and 13.8
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at each time point. The number of forelimb eye-wiping movements
during 2 min was counted immediately after application of capsaicin or
the solution control. Bilateral eye wiping (moving both fore limbs
simultaneously) and wiping of the opposite, untreated eye were not
counted. Data from any animal that continued eye wiping with
grooming for more than 2 min at each time point, or that showed 40
or greater eye wipes during 2 min were excluded because eye wiping in
response to capsaicin-induced pain does not normally continue
beyond 1 min.25 Statistical analysis was performed by Windows SAS
program. The statistical tests (p value) were performed at a 2-sided
0.05 level.
CFA Efficacy Test. Male Sprague−Dawley rats (6 weeks, 180−280
g) were used. Pharmacodynamic response was measured in terms of
withdrawal latency to a thermal stimulus applied to the left hind paw
using Plantar Test Instrument (Ugo-Basil, Italy). CFA (50 μL of 50%
FCA) was injected into the left hind paw. Withdrawal latency was
measured just before FCA injection to provide a pre-CFA value.
Approximately 22 h after the CFA injection, the withdrawal latency
was measured before the compound administration, and 1, 3, 5, and 8
h after.
measured once at 3 h after the compound administration. The %
inhibition of thermal sensation was calculated as follows; 100 × (Lc −
Lv)/(30 − Lv), where Lc and Lv are average response latency in the
compound and vehicle administered groups, respectively. The 0.5 w/v
% methylcellulose aqueous solution was prepared as vehicle.
Pharmacokinetic Studies. Male Sprague−Dawley rats were used.
The intravenous doses were formulated as N,N-dimethylacetamide
(DMA)/propylene glycol (PG) = 1/1(v/v) solution while the oral
doses 0.5% methyl cellulose suspension. Blood samples were collected
from the cannula inserted into jugular vein using a syringe containing
anticoagulant. Male beagle dogs and female cynomolgus monkeys
were administered at doses of 1 mg/kg iv or 3 mg/kg po, formulated
as DMA/PG/25% hydroxylpropyl-β-cyclodextrin solution = 1/1/2 (v/
v/v) solution for iv and as 0.5% methyl cellulose suspension for po.
Blood samples were collected from the limb vein using a syringe
containing anticoagulant. Their blood samples were centrifuged to
obtain plasma for LC/MS/MS analysis. The pharmacokinetic
parameters were calculated by WinNonlin (version 5.0.1, Pharsight
Corporation) based on a noncompartment model with uniform
weighting.
The efficacy data comprised the % of reversal value at 1, 3, 5, and 8
h after the compound administration calculated as follows; % of
reversal = (withdrawal latency for the CFA-injected left paw at each
time point after the compound administration − withdrawal latency
for the CFA-injected left paw before the compound administration)/
(withdrawal latency for the left paw before CFA injection −
withdrawal latency for CFA-injected left paw before the compound
administration) × 100. The 0.5 w/v% methylcellulose aqueous
solution was prepared as vehicle. Statistical analysis was performed
by Windows SAS program. The statistical tests (p value) were
performed at a 2-sided 0.05 level.
Chemistry. General Information. All reagents and solvents
including anhydrous solvents were of commercial quality and used
without further purification. All reactions were carried out under a
static atmosphere of nitrogen or argon and stirred magnetically unless
otherwise stated. All flash chromatographic separations were
performed using ISCO RediSep disposable silica gel column eluting
with EtOAc/hexanes or methanol/CH2Cl2. All final compounds were
1
purified to >95% purity as determined by two LC/MS methods. H
NMR spectra were recorded on either Bruker 400 or 500 MHz.
Chemical shifts are specified in ppm referenced to deuterated solvent
peaks.
Body Temperature Test (Telemetry). Male Sprague−Dawley
rats (7 weeks, 180−280 g) were used. Transmitters (G2 E-Mitter,
Philips Respironics) were implanted in the intra-abdominal cavity
under anesthesia with isoflurane. After the surgery, the operation sites
were disinfected with an antiseptic solution (Isodine solution 10%,
Meiji Seika Pharma, Japan). Following a recovery period of 5−7 days,
rats were used in the experiment after they were confirmed to be
devoid of any abnormalities in their general condition (i.e., only
healthy animals were used). The rats were treated for acclimation in a
plastic cage (1 animal/cage) beginning several days before the
compound administration. Mean body temperature (BT) for each 5
min recording was automatically recorded by a data computer system
(Vital View, Philips Respironics) via telemetry signals from each
animal. BT was continuously recorded from 24 h before to 24 h after
the compound administration. Mean BT were calculated for each
group at each time point. Differences from mean of the vehicle group
(ΔBT) were calculated at each time point. Each time point was
representative of data averaged over 1 h. The 0.5 w/v%
methylcellulose aqueous solution was prepared as vehicle.
Body Temperature Test (Thermistor). Male Sprague−Dawley
rats (7 weeks, 180−280 g) were used. A digital probe thermistor
(TD300, Shibaura Electronics, Japan) was used for this study. A day
before the actual study period, rats were habituated to handling for
rectal probe insertion and oral dosing procedures. On evaluation days
(days 1 to 4), rectal body temperatures were measured at 9:00, 10:00,
10:30, 11:00, 12:0, 13:00, 14:00, 15:00, 16:00, 17:00, 17:30, and 18:00.
Compound 37 (10 mg/kg) or vehicle was orally administered at 10:00
and 17:00 for 4 days. A 0.5 w/v% methylcellulose aqueous solution
was used as vehicle. Mean BT were calculated for each group at each
time point on each day. Differences from mean of the vehicle group
(ΔBT) were calculated at each time point on each day.
tert-Butyl 3-Chloro-5′,6′-dihydro-(2,4′-bipyridine)-1′(2′H)-car-
boxylate (7). To a mixture of 5 (1.45 g, 10 mmol), pinacol ester 6
(3.09 g, 10 mmol), and potassium carbonate (2.07 g, 15 mmol) in
DME/EtOH/H2O (2/1/2, 50 mL) at room temperature was added
Pd(PPh3)2Cl2 (702 mg, 1.0 mmol). The resulting reaction mixture was
degassed and heated at 95 °C for 10 h. After this period, the reaction
mixture was cooled, diluted with water, extracted with CH2Cl2 (50 mL
× 2), dried over Na2SO4, and concentrated under reduced pressure to
provide a crude product as a yellow oil. The crude sample was
chromatographed by silica gel column chromatography eluting with a
gradient of 20−50% ethyl acetate in hexanes to provide 7 (2.32 g, 79%
yield) as a pale-yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.53 (d, J =
4.5 Hz, 1H), 7.81 (d, J = 7.9 Hz, 1H), 7.24 (m, 1H), 6.18 (m, 1H),
4.15 (m, 2H), 3.69 (t, J = 5.3 Hz, 2H), 2.63 (m, 2H), 1.52 (s, 9H). MS
m/z 317.2 [M + Na]+.
3-Chloro-1′,2′,3′,6′-tetrahydro-2,4′-bipyridine (8). To a solution
of 7 (0.90 g, 3.0 mmol) in CH2Cl2 (10 mL) at 0 °C was added 4 M
HCl in Et2O (10 mL). The ice bath was removed and the resulting
mixture was stirred at 40 °C for 20 h. After this period, the reaction
mixture was allowed to cool to room temperature and the resulting
solid was filtered, washed with Et2O (20 mL), and dried under reduced
pressure to provide 8 as an HCl salt (0.66 g, 95% yield) as a white
solid. 1H NMR (400 MHz, CD3OD) δ 8.40 (dd, J = 4.7, 1.3 Hz, 1H),
7.84 (dd, J = 8.2, 1.4 Hz, 1H), 7.27 (dd, J = 8.2, 4.7 Hz, 1H), 6.08 (m,
1H), 3.79 (m, 2H), 3.37 (t, J = 6.1 Hz, 2H), 2.71 (m, 2H). MS m/z
195 [M + H]+.
3-Chloro-N-[4-(trifluoromethyl)phenyl]-3′,6′-dihydro-(2,4′-bipyri-
dine)-1′(2′H)-carboxamide (9). To a suspension of 8 (199 mg, 0.86
mmol, HCl salt) in anhydrous CH2Cl2 (10 mL) at 0 °C was added
diisopropylethylamine (DIEA, 1 mL) and 1-isocyanato-4-
(trifluoromethyl)benzene (161 mg, 0.86 mmol). The reaction mixture
was warmed to room temperature and stirred for an additional 2 h.
The resulting mixture was concentrated under reduced pressure, and
the residue was chromatographed by silica gel column chromatography
eluting with a gradient of 0−20% MeOH in CH2Cl2 to provide 9 (306
Thermal Sensation Test. Male Sprague−Dawley rats (6 weeks,
180−280 g) were used. Rats were placed on a hot plate heated to 50
°C, and the time interval to flinching of their hind paws (response
latency) was recorded by stop watch. If no response was obtained
within 30 s (designated as the “cutoff time”), the rats were removed
from the hot plate to avoid tissue injury. Prior to the compound
administration, the response latency was measured on a single
occasion to familiarize the animals. The response latency was
1
mg, 80% yield) as a white solid. H NMR (400 MHz, DMSO-d6) δ
8.98 (s, 1H), 8.53 (dd, J = 4.7, 1.4 Hz, 1H), 7.97 (dd, J = 8.1, 1.3 Hz,
1H), 7.73 (d, J = 7.5 Hz, 2H), 7.60 (d, J = 8.8 Hz, 2H), 7.36 (dd, J =
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8.1, 4.6 Hz, 1H), 6.23 (m, 1H), 4.27−4.13 (m, 2H), 3.70 (t, J = 5.6 Hz,
2H), 2.64−2.52 (m, 2H). MS m/z 382.2 [M + H]+.
J = 1.7 Hz, 1H), 7.72 (d, J = 8.7 Hz, 2H), 7.60 (d, J = 8.7 Hz, 2H),
6.22 (m, 1H), 4.20 (m, 2H), 3.72−3.65 (m, 2H), 2.57 (m, 2H). MS
m/z 439.7 [M + H]+.
3-Chloro-5-(cyanomethyl)-N-[4-(trifluoromethyl)phenyl]-3′,6′-di-
hydro-(2,4′-bipyridine)-1′(2′H)-carboxamide (25). 1H NMR (400
MHz, DMSO-d6) δ 8.96 (s, 1 H), 8.52 (s, 1H) 7.97 (s, 1H), 7.72 (d, J
= 8.0 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 6.25 (m, 1H), 4.31 (m, 2H),
4.12 (s, 2H), 3.70 (t, J = 8.0 Hz, 2H), 2.58 (m, 2H). MS m/z 420.8 [M
+ H]+.
3-Chloro-N-[4-(trifluoromethoxy)phenyl]-3′,6′-dihydro-(2,4′-bi-
1
pyridine)-1′(2′H)-carboxamide (10). H NMR (400 MHz, CDCl3) δ
8.50 (dd, J = 4.6, 1.5 Hz, 1H), 7.73 (dd, J = 8.1, 1.5 Hz, 1H), 7.46−
7.38 (m, 2H), 7.22−7.13 (m, 3H), 6.40 (s, 1H), 6.20 (dt, J = 3.3, 1.7
Hz, 1H), 4.23 (m, 2H), 3.77 (t, J = 5.6 Hz, 2H), 2.77−2.68 (m, 2H).
MS m/z 398.1 [M + H]+.
3-Chloro-N-{[4-[(trifluoromethyl)sulfonyl]phenyl}-3′,6′-dihydro-
(2,4′-bipyridine)-1′(2′H)-carboxamide (11). 1H NMR (400 MHz,
CDCl3) δ 8.50 (dd, J = 4.7, 1.4 Hz, 1H), 7.95 (d, J = 9.0 Hz, 2H),
7.80−7.61 (m, 3H), 7.20 (dd, J = 8.1, 4.6 Hz, 1H), 6.80 (s, 1H), 6.21
(dt, J = 3.2, 1.7 Hz, 1H), 4.27 (m, 2H), 3.80 (t, J = 5.7 Hz, 2H), 2.80−
2.69 (m, 2H). MS m/z 446.1 [M + H]+.
3-Chloro-5-(hydroxymethyl)-N-[4-(trifluoromethyl)phenyl]-3′,6′-
1
dihydro-(2,4′-bipyridine)-1′(2′H)-carboxamide (26). H NMR (400
MHz, CD3OD) δ 8.45 (d, J = 2.0 Hz, 1H), 7.96−7.85 (m, 1H), 7.74−
7.49 (m, 4H), 6.15 (m, 1H), 4.67 (s, 2H), 4.27 (m, 2H), 3.81 (t, J =
5.6 Hz, 2H), 2.75−2.55 (m, 2H). MS m/z 412.2 [M + H]+.
3-Chloro-5-(hydroxyethyl)-N-[4-(trifluoromethyl)phenyl]-3′,6′-di-
hydro-(2,4′-bipyridine)-1′(2′H)-carboxamide (27). 1H NMR (400
MHz, CD3OD) δ 8.97 (S, 1H), 8.38 (d, J = 1.8 Hz, 1H), 7.82 (d, J =
1.8 Hz, 1H), 7.73 (d, J = 8.1 Hz, 2H), 7.60 (d, J = 8.8 Hz, 2H), 6.22−
6.17 (m, 1H), 4.20 (m, 2H), 3.73−3.60 (m, 4H), 2.74 (t, J = 6.5 Hz,
2H), 2.56 (m, 2H). MS m/z 426.5 [M + H]+.
3-Chloro-N-[4-(diethylamino)phenyl]-3′,6′-dihydro-(2,4′-bipyri-
1
dine)-1′(2′H)-carboxamide (12). H NMR (400 MHz, CD3OD) δ
8.87−8.77 (m, 1H), 8.74−8.66 (m, 1H), 8.05−7.97 (m, 1H), 7.75 (d, J
= 9.0 Hz, 2H), 7.53 (d, J = 8.8 Hz, 2H), 6.48 (s, 1H), 4.40 (s, 2H),
3.88 (t, J = 5.3 Hz, 2H), 3.77−3.54 (m, 4H), 2.75 (s, 2H), 1.17 (t, J =
7.2 Hz, 6H). MS m/z 385.2 [M + H]+.
3-Chloro-N-[2-methoxy-4-(trifluoromethyl)phenyl]-3′,6′-dihydro-
(2,4′-bipyridine)-1′(2′H)-carboxamide (13). 1H NMR (400 MHz,
CD3OD) δ 8.82−8.71 (m, 1H), 8.68−8.56 (m, 1H), 8.01 (d, J = 9.0
Hz, 1H), 7.97−7.56 (m, 1H), 7.29−7.22 (m, 2H), 6.43 (s, 1H), 4.38
(m, 2H), 3.99 (s, 3H), 3.85 (t, J = 5.5 Hz, 2H), 2.73 (d, J = 2.0 Hz,
2H). MS m/z 412.2 [M + H]+.
3-Fluoro-4-(hydroxymethyl)-N-[4-(trifluoromethyl)phenyl]-3′,6′-
1
dihydro-(2,4′-bipyridine)-1′(2′H)-carboxamide (28). H NMR (400
MHz, CD3OD) δ 8.37 (d, J = 4.8 Hz, 1H), 7.68−7.53 (m, 4H), 7.49
(t, J = 5.0 Hz, 1H), 6.53 (m, 1H), 4.77 (s, 2H), 4.30 (m, 2H), 3.80 (t, J
= 5.6 Hz, 2H), 2.76 (m, 2H). MS m/z 396.1 [M + H]+.
3-Chloro-5-(methylsulfonamidomethyl)-N-[4-(trifluoromethyl)-
phenyl]-3′,6′-dihydro-(2,4′-bipyridine)-1′(2′H)-carboxamide (29).
1H NMR (400 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.49 (d, J = 2.0
3-Chloro-N-[3-methoxy-4-(trifluoromethyl)phenyl]-3′,6′-dihydro-
(2,4′-bipyridine)-1′(2′H)-carboxamide (14). 1H NMR (400 MHz,
CD3OD) δ 8.37 (dd, J = 4.8, 1.5 Hz, 1H), 7.87 (dd, J = 8.1, 1.5 Hz,
1H), 7.38−7.31 (m, 2H), 7.24 (dd, J = 8.1, 4.6 Hz, 1H), 6.97 (dd, J =
8.6, 1.1 Hz, 1H), 6.04 (m, 1H), 4.16 (m, 2H), 3.79 (s, 3H), 3.70 (t, J =
5.6 Hz, 2H), 2.61−2.51 (m, 2H). MS m/z 412.2 [M + H]+.
3-Chloro-N-[2-(dimethylamino)-4-(trifluoromethyl)phenyl]-3′,6′-
Hz, 1H), 7.90 (d, J = 1.8 Hz, 1H), 7.78−7.64 (m, 4H), 7.60 (d, J = 8.8
Hz, 3H), 6.27−6.20 (m, 1H), 4.27−4.17 (m, 6H), 3.70 (t, J = 5.6 Hz,
3H), 2.96 (m, 4H), 2.58 (d, J = 1.5 Hz, 3H). MS m/z 489.1 [M + H]+.
2,3-Dichloro-5-vinylpyridine (31). Potassium-t-butoxide (3.14 g, 28
mmol) was added portionwise to a cooled (0 °C) slurry of
methyltriphenylphosphonium bromide (10 g, 28 mol) in toluene
(200 mL). The reaction mixture was stirred for 1 h and was cooled to
−20 °C. After this period, a solution of 30 (4.0 g, 22.7 mmol) in
tetrahydrofuran (10 mL) was added dropwise to produce a purple-
colored slurry. The resulting mixture was then heated to 100 °C and
stirred for additional 1 h. The reaction mixture was then cooled to
room temperature, diluted with ethyl acetate (200 mL), washed with
aqueous brine (150 mL), dried over Na2SO4, and concentrated under
reduced pressure to give a crude sample. The crude sample was
purified by column chromatography eluting with a gradient of 0−10%
ethyl acetate in hexanes to provide 31 (2.77 g, 70% yield) as a colorless
oil. 1H NMR (400 MHz, CDCl3) δ 8.30 (d, J = 2.2 Hz, 1H), 7.80 (d, J
= 2.2 Hz, 1H), 6.63 (dd, J = 17.8, 11.0 Hz, 1H), 5.86 (d, J = 17.8 Hz,
1H), 5.45 (d, J = 11.0 Hz, 1H). MS m/z 174 [M + H]+.
1
dihydro-(2,4′-bipyridine)-1′(2′H)-carboxamide (15). H NMR (400
MHz, CD3OD) δ 8.37 (dd, J = 4.8, 1.5 Hz, 1H), 7.73 (d, J = 1.8 Hz,
1H), 7.57−7.41 (m, 4H), 6.01 (m, 1H), 4.15 (m, 2H), 3.69 (t, J = 5.6
Hz, 2H), 2.78−2.71 (m, 2H), 2.56−2.45 (m, 4H), 2.22 (s, 6H). MS
m/z 425.1 [M + H]+.
3-Chloro-N-[3-(dimethylamino)-4-(trifluoromethyl)phenyl]-3′,6′-
1
dihydro-(2,4′-bipyridine)-1′(2′H)-carboxamide (16). H NMR (400
MHz, CDCl3) δ 8.49 (dd, J = 4.6, 1.5 Hz, 1H), 7.73 (dd, J = 8.1, 1.3
Hz, 1H), 7.60−7.50 (m, 2H), 7.23−7.12 (m, 2H), 6.58 (s, 1H), 6.19
(dt, J = 3.2, 1.7 Hz, 1H), 4.24 (m, 2H), 3.78 (t, J = 5.6 Hz, 2H), 2.96
(m, 4H), 2.80−2.68 (m, 2H), 1.00 (t, J = 7.1 Hz, 6H). MS m/z 453.1
[M + H]+.
3-Chloro-5-[(dimethylamino)methyl]-N-[4-(trifluoromethyl)-
phenyl]-3′,6′-dihydro-(2,4′-bipyridine)-1′(2′H)-carboxamide (20).
1H NMR (400 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.42 (s, 1H), 7.82
(S)-1-(5,6-Dichloropyridin-3-yl)ethane-1,2-diol (32a). To a stirred
slurry of AD-mix-α (8.95 g) in water (30 mL) and t-butanol (30 mL)
at 0 °C was added a solution of 31 (0.91 g, 5.25 mmol) in t-butanol (5
mL). The resulting mixture was stirred at 0 °C for 2 h and at room
temperature for 22 h. After this period, solid sodium sulfite (9.57 g)
was added and the resulting slurry was allowed to stir for 30 min. The
mixture was extracted with ethyl acetate (50 mL × 3). The organic
layers were combined, washed with brine, dried over Na2SO4, and
concentrated under reduced pressure to give a crude sample. The
crude sample was purified by column chromatography eluting with a
gradient of 50−100% ethyl acetate in hexanes to provide 32a (0.75 g,
70% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.29 (dd, J
= 1.9, 0.4 Hz, 1H), 7.87 (dd, J = 2.2, 0.6 Hz, 1H), 4.87 (m, 1H), 3.84
(m, 1H), 3.66 (m, 1H), 2.83 (d, J = 5.9 Hz, 1H), 2.11 (t, J = 5.9 Hz,
1H). MS m/z 208 [M + H]+.
(s, 1H), 7.73 (d, J = 8.0 Hz, 2H), 7.59 (d, J = 8.0 Hz, 2H), 6.23 (m,
1H), 4.20 (m, 2H), 3.70 (d, J = 4.0 Hz, 2H), 3.43 (m, 2H), 2.58 (m,
2H), 2.16 (s, 6H). MS m/z 439.1 [M + H]+.
3-Chloro-5-[(2-(dimethylamino)ethyl]-N-[4-(trifluoromethyl)-
phenyl]-3′,6′-dihydro-(2,4′-bipyridine)-1′(2′H)-carboxamide (21).
1H NMR (400 MHz, CD3OD) δ 8.26 (d, J = 1.5 Hz, 1H), 7.73 (d,
J = 1.8 Hz, 1H), 7.57−7.41 (m, 4H), 6.01 (m, 1H), 4.15 (m, 2H), 3.69
(t, J = 5.6 Hz, 2H), 2.78−2.71 (m, 2H), 2.56−2.45 (m, 4H), 2.22 (s,
6H). MS m/z 453.1 [M + H]+.
3-Fluoro-4-[(dimethylaminomethyl)]-N-[4-(trifluoromethyl)-
phenyl]-3′,6′-dihydro-(2,4′-bipyridine)-1′(2′H)-carboxamide (22).
1H NMR (400 MHz, CD3OD) δ 8.35 (d, J = 4.8 Hz, 1H), 7.66−
7.53 (m, 4H), 7.39 (t, J = 5.0 Hz, 1H), 6.55 (s, 1H), 4.30 (m, 2H),
3.80 (t, J = 2.6 Hz, 2H), 3.64 (m, 2H), 2.77 (m, 2H), 2.32 (s, 6H). MS
m/z 423.1 [M + H]+.
(R)-1-(5,6-Dichloropyridin-3-yl)ethane-1,2-diol (32b). The inter-
3-Chloro-1′-{[4-(trifluoromethyl)phenyl]carbamoyl}-1′,2′-3′,6′-
1
mediate 32b was prepared following a similar dihydroxylation
tetrahydro-(2,4′-bipyridine)-5-carboxylic acid (23). H NMR (400
1
procedure as 32a by using AD-mix-β as oxidizing agent. H NMR
MHz, CD3OD) δ 8.96 (d, J = 1.8 Hz, 1H), 8.31 (d, J = 1.8 Hz, 1H),
7.69−7.54 (m, 5H), 6.21 (s, 1H), 4.28 (m, 2H), 3.82 (t, J = 5.6 Hz,
2H), 2.68 (m, 2H). MS m/z 426.1 [M + H]+.
(400 MHz, CDCl3) δ 8.29 (dd, J = 1.9, 0.4 Hz, 1H), 7.87 (dd, J = 2.2,
0.6 Hz, 1H), 4.87 (m, 1H), 3.84 (m, 1H), 3.66 (m, 1H), 2.83 (d, J =
5.9 Hz, 1H), 2.11 (t, J = 5.9 Hz, 1H). MS m/z 208 [M + H]+.
(S)-3-Chloro-5-(1,2-dihydroxyethyl)-3′,6′-dihydro-2′H-[2,4′]-
bipyridinyl-1′-carboxylic Acid tert-Butyl Ester (33a). A 50 mL round-
2-{3-Chloro-1′-[(4-(trifluoromethyl)phenyl)carbamoyl]-1′,2′,3′,6′-
tetrahydro-(2,4′-bipyridin)-5-yl}-acetic Acid (24). 1H NMR (400
MHz, DMSO-d6) δ 12.71 (brs, 1H), 8.41 (d, J = 1.7 Hz, 1H), 7.87 (d,
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Article
bottomed flask was charged with 32a (0.70 g, 3.37 mmol), pinacol
ester 6 (1.25 g, 4.04 mmol), Pd(PPh3)2Cl2 (0.19 g, 0.27 mmol),
potassium carbonate (0.88 g, 6.40 mmol), and a mixture of DME/
EtOH/H2O (2/1/2, 20 mL). The reaction mixture was degassed and
heated at 90 °C with vigorous stirring. After 2 h, the reaction mixture
was cooled to room temperature and diluted with ethyl acetate (50
mL). The organic layer was washed with brine, dried over Na2SO4, and
concentrated under reduced pressure. The residue was chromato-
graphed by silica gel column chromatography eluting with a gradient
of 50−100% ethyl acetate in hexanes to provide 33a (0.96 g, 80%
yield) as a colorless oil. 1H NMR (400 MHz, CD3OD) δ 8.47 (s, 1H),
7.93 (s, 1H), 6.06 (m, 1H), 4.74 (t, J = 5.9 Hz, 1H), 4.12 (m, 2H),
3.67 (m, 4H), 2.54 (m, 2H), 1.52 (s, 9H). MS m/z 355 [M + H]+.
(R)-3-Chloro-5-(1,2-dihydroxyethyl)-3′,6′-dihydro-2′H-[2,4′]-
bipyridinyl-1′-carboxylic Acid tert-butyl Ester (33b). The intermedi-
ate 33b was prepared following a similar procedure as 33a by using
sample that was purified by silica gel column chromatography eluting
with a gradient of 0−20% MeOH in CH2Cl2 to provide 37 (10.51 g,
74% yield) as a white solid. 1H NMR (500 MHz, DMSO-d6) δ 9.84 (s,
1H), 8.63 (dd, J = 2.4, 1.0 Hz, 1H), 8.48 (d, J = 1.8 Hz, 1H), 8.13−
8.04 (m, 1H), 8.03−7.97 (m, 1H), 7.84 (d, J = 1.8 Hz, 1H), 6.19 (m,
1H), 5.55 (d, J = 4.8 Hz, 1H), 4.86 (t, J = 5.8 Hz, 1H), 4.62 (q, J = 5.6
Hz, 1H), 4.22 (d, J = 2.8 Hz, 2H), 3.72 (t, J = 5.6 Hz, 2H), 3.56 (m,
1H), 3.51−3.43 (m, 1H), 2.58 (m, 2H). MS m/z 443.1 [M + H]+.
Anal. Calcd for C19H18ClF3N4O3: C 51.53; H 4.10; N 12.65. Found: C
51.58: H 3.83; N 12.71.
(S)-4-[3-Chloro-5-(1,2-dihydroxyethyl)pyridin-2-yl]-N-[6-
(trifluoromethyl)pyridin-3-yl]-3′,6′-dihydropyridine-1′(2′H)-carbox-
amide (38). The analogue 38 was prepared following a similar
procedure as 37 by coupling intermediate 34a with phenyl(6-
(trifluoromethyl)pyridin-2-yl)carbamate. 1H NMR (400 MHz,
CD3OD) δ 8.69 (d, J = 2.4 Hz, 1H), 8.37 (d, J = 1.5 Hz, 1H), 8.06
(dd, J = 8.6, 2.2 Hz, 1H), 7.83 (d, J = 1.5 Hz, 1H), 7.62 (d, J = 8.8 Hz,
1H), 6.03 (m, 1H), 4.66 (t, J = 5.7 Hz, 1H), 4.18 (m, 2H), 3.71 (t, J =
5.6 Hz, 2H), 3.63−3.53 (m, 2H), 2.58−2.52 (m, 2H). MS m/z 443.1
[M + H]+.
1
33b as pyridine starting material. H NMR (400 MHz, CD3OD) δ
8.47 (s, 1H), 7.93 (s, 1H), 6.06 (m, 1H), 4.74 (t, J = 5.9 Hz, 1H), 4.12
(m, 2H), 3.67 (m, 4H), 2.54 (m, 2H), 1.52 (s, 9H). MS m/z 355 [M +
H]+.
(R)-4-[3-Chloro-5-(1,2-dihydroxyethyl)pyridin-2-yl]-N-[5-
(trifluoromethyl)pyridin-2-yl]-3′,6′-dihydropyridine-1′(2′H)-carbox-
amide (39). 1H NMR (500 MHz, DMSO-d6) δ 9.84 (s, 1H), 8.63 (dd,
J = 2.4, 1.0 Hz, 1H), 8.48 (d, J = 1.8 Hz, 1H), 8.13−8.04 (m, 1H),
8.03−7.97 (m, 1H), 7.84 (d, J = 1.8 Hz, 1H), 6.19 (m, 1H), 5.55 (d, J
= 4.8 Hz, 1H), 4.86 (t, J = 5.8 Hz, 1H), 4.62 (q, J = 5.6 Hz, 1H), 4.22
(d, J = 2.8 Hz, 2H), 3.72 (t, J = 5.6 Hz, 2H), 3.56 (m, 1H), 3.51−3.43
(m, 1H), 2.58 (m, 2H). MS m/z 443.1 [M + H]+.
(S)-1-(3-Chloro-1′,2′,3′,6′-tetrahydro-[2,4′]bipyridinyl-5-yl)-
ethane-1,2-diol (34a). To a solution of 33a (0.90 g, 2.54 mmol) in
CH2Cl2 (10 mL) at 0 °C was added 4 M HCl in Et2O (10 mL). The
ice bath was removed, and the resulting mixture was stirred at 40 °C
for 20 h. After this period, the reaction mixture was allowed to cool to
room temperature and the resulting solid was filtered, washed with
Et2O (20 mL), and dried under reduced pressure to provide 34a as an
HCl salt (0.70 g, 95% yield). 1H NMR (400 MHz, CD3OD) δ 8.74 (s,
1H), 8.52 (s, 1H), 6.38 (m, 1H), 4.91 (m, 1H), 4.00 (m, 2H), 3.75 (m,
4H), 3.54 (t, J = 5.9 Hz, 2H), 2.89 (m, 2H). MS m/z 255 [M + H]+.
(R)-1-(3-Chloro-1′,2′,3′,6′-tetrahydro-[2,4′]bipyridinyl-5-yl)-
ethane-1,2-diol (34b). 1H NMR (400 MHz, CD3OD) δ 8.74 (s, 1H),
8.52 (s, 1H), 6.38 (m, 1H), 4.91 (m, 1H), 4.00 (m, 2H), 3.75 (m, 4H),
3.54 (t, J = 5.9 Hz, 2H), 2.89 (m, 2H). MS m/z 255 [M + H]+.
(S)-3-Chloro-5-(1,2-dihydroxyethyl)-N-[4-(trifluoromethyl)-
phenyl]-3′,6′-dihydro-(2,4′-bipyridine)-1′(2′H)-carboxamide (35).
General procedure for syntheses of 35 and 36. To a suspension of
34a (800 mg, 2.45 mmol) in anhydrous CH2Cl2 (20 mL) at 0 °C was
added diisopropylethylamine (DIEA, 2 mL) and 1-isocyanato-4-
(trifluoromethyl)benzene (462 mg, 2.45 mmol). The reaction mixture
was warmed to room temperature and further stirred for 2 h. After
concentrated under reduced pressure, the residue was chromato-
graphed by silica gel chromatography column eluting with a gradient
of 0−20% MeOH in CH2Cl2 to provide 35 (0.60 g, 56% yield) as a
white solid. 1H NMR (400 MHz, CD3OD) δ 8.49 (d, J = 1.5 Hz, 1H),
7.94 (dd, J = 1.3 Hz, 1H), 7.71−7.51 (m, 4H), 6.14 (m, 1H), 4.78 (t, J
= 5.6 Hz, 1H), 4.35−4.23 (m, 2H), 3.82 (t, J = 5.6 Hz, 2H), 3.73−3.64
(m, 2H), 2.65 (m, 2H). MS m/z 442.1 [M + H]+.
ASSOCIATED CONTENT
* Supporting Information
■
S
Pharmacological selectivity profile of compound 37, effect of
compound 37 on body temperature after repeated admin-
̈
istration to naive and CFA treated rats, results of PK/PD
relationship study for compound 37. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 1-202-905-6936. E-mail: laykea@chemist.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank members of the Purdue−Shionogi research
collaboration for the support of this program.
■
(R)-3-Chloro-5-(1,2-dihydroxyethyl)-N-[4-(trifluoromethyl)-
phenyl]-3′,6′-dihydro-(2,4′-bipyridine)-1′(2′H)-carboxamide (36).
1H NMR (400 MHz, CD3OD) δ 8.49 (d, J = 1.5 Hz, 1H), 7.94
(dd, J = 1.3 Hz, 1H), 7.71−7.51 (m, 4H), 6.14 (m, 1H), 4.78 (t, J = 5.6
Hz, 1H), 4.35−4.23 (m, 2H), 3.82 (t, J = 5.6 Hz, 2H), 3.73−3.64 (m,
2H), 2.65 (m, 2H). MS m/z 442.1 [M + H]+.
ABBREVIATIONS USED
■
TRPV1, transient receptor potential vanilloid 1; SAR,
structure−activity relationship; BCTC, 4-(3-chloro-2-pyridin-
yl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide;
CAP, capsaicin; CHO cell, Chinese hamster ovary cell; FLIPR,
fluorometric imaging plate reader; CFA, complete Freund’s
adjuvant; EP, electrophysiology
(S)-4-[3-Chloro-5-(1,2-dihydroxyethyl)pyridin-2-yl]-N-[5-
(trifluoromethyl)pyridin-2-yl]-3′,6′-dihydropyridine-1′(2′H)-carbox-
amide (37). General procedure for syntheses of 37−39. To a stirred
solution of 2-amino-5-(trifluoromethyl)pyridine (12.1 g, 74.1 mmol)
at 0 °C in CH2Cl2 (50 mL) was added phenylchloroformate (12.7 g,
81.5 mmol). Then pyridine (6.3 mL, 81.5 mmol) was added dropwise.
The reaction mixture was stirred for 2 h at 0 °C. The resulting solid
was filtered off and washed with CH2Cl2 to afford phenyl(5-
(trifluoromethyl)pyridin-2-yl)carbamate (16.7 g). In a separate flask,
to a suspension of 34a as its HCl salt (9.36 g, 32.27 mmol) in
anhydrous CH2Cl2 (50 mL) at −20 °C was added phenyl(5-
(trifluoromethyl)pyridin-2-yl)carbamate (9.10 g, 32.27 mmol) in one
portion. Then diisopropylethylamine (DIEA 14 mL, 80.65 mmol) was
added dropwise to the mixture over 20 min. The resulting reaction
mixture was stirred for 2 h at −20 °C. After this period, the reaction
mixture was concentrated under reduced pressure to give a crude
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