Aminopiperidine sulfonamide Cav2.2 channel inhibitors for the treatment of chronic pain

Article abstract of DOI:10.1021/jm301056k

The voltage-gated calcium channel Ca_v2.2 (N-type calcium channel) is a critical regulator of synaptic transmission and has emerged as an attractive target for the treatment of chronic pain. We report here the discovery of sulfonamide-derived, state-dependent inhibitors of Ca _v2.2. In particular, 19 is an inhibitor of Ca_v2.2 that is selective over cardiac ion channels, with a good preclinical PK and biodistribution profile. This compound exhibits dose-dependent efficacy in preclinical models of inflammatory hyperalgesia and neuropathic allodynia and is devoid of ancillary cardiovascular or CNS pharmacology at the doses tested. Importantly, 19 exhibited no efficacy in Ca_v2.2 gene-deleted mice. The discovery of metabolite 26 confounds further development of members of this aminopiperidine sulfonamide series. This discovery also suggests specific structural liabilities of this class of compounds that must be addressed.

Full text of DOI:10.1021/jm301056k

Article
pubs.acs.org/jmc
Aminopiperidine Sulfonamide Cav2.2 Channel Inhibitors for the
Treatment of Chronic Pain
Pengcheng P. Shao,*,† Feng Ye,† Prasun K. Chakravarty,† Deepu J. Varughese,† James B. Herrington,‡
Ge Dai,‡ Randal M. Bugianesi,‡ Rodolfo J. Haedo,‡ Andrew M. Swensen,‡ Vivien A. Warren,‡
McHardy M. Smith,‡ Maria L. Garcia,‡ Owen B. McManus,‡ Kathryn A. Lyons,§ Xiaohua Li,§
Mitchell Green,§ Nina Jochnowitz,⊥ Erin McGowan,⊥ Shruti Mistry,⊥ Shu-Yu Sun,⊥ Catherine Abbadie,⊥
Gregory J. Kaczorowski,‡ and Joseph L. Duffy†
†Departments of Medicinal Chemistry, Merck Research Laboratories, Rahway, New Jersey 07065, United States
‡Ion Channels, Merck Research Laboratories, Rahway, New Jersey 07065, United States
§Drug Metabolism and Pharmacokinetics, Merck Research Laboratories, Rahway, New Jersey 07065, United States
⊥Pharmacology, Merck Research Laboratories, Rahway, New Jersey 07065, United States
ABSTRACT: The voltage-gated calcium channel Cav2.2 (N-type calcium channel) is a critical
regulator of synaptic transmission and has emerged as an attractive target for the treatment of
chronic pain. We report here the discovery of sulfonamide-derived, state-dependent inhibitors
of Cav2.2. In particular, 19 is an inhibitor of Cav2.2 that is selective over cardiac ion channels,
with a good preclinical PK and biodistribution profile. This compound exhibits dose-dependent
efficacy in preclinical models of inflammatory hyperalgesia and neuropathic allodynia and is devoid of ancillary cardiovascular or
CNS pharmacology at the doses tested. Importantly, 19 exhibited no efficacy in Cav2.2 gene-deleted mice. The discovery of
metabolite 26 confounds further development of members of this aminopiperidine sulfonamide series. This discovery also
suggests specific structural liabilities of this class of compounds that must be addressed.
The Cav2.2−/− mice are healthy and can live a normal life span.
However, they show significantly reduced sensitivity to spinal
INTRODUCTION
■
Neuropathic pain is a chronic, debilitating state that results from
injury to the peripheral or central nervous system. It can be
triggered by a variety of events or conditions, including traumatic
injury, diabetes, shingles, and chemotherapies. It is estimated that
nerve ligation (SNL)-induced mechanical allodynia and thermal
hyperalgesia.10 Clinical validation of Cav2.2 as a nonopioid
analgesic target has been established by the registration of
intrathecal ziconotide, a 25 amino acid synthetic peptide based
neuropathic pain affects 4 million people in the U.S. Despite
on ω-conotoxin MVIIA from marine snail Conus magus that is
aggressive treatment, some patients remain refractory.1 Intra-
approved for the treatment of chronic intractable pain.11
thecal administration of analgesics has proven to be effective in
severe cases, providing relief by dispensing the therapeutic agent
proximal to the pharmacologic target in the central nervous
However, ziconotide is a state-independent Cav2.2 blocker and
therefore inhibits the activity of the channel in both the
system.2 N-Type calcium channels (Cav2.2) are one such target,
hyperpolarized state (closed) and the depolarized state (open
or inactivated).12 Intrathecal administration of the peptide is
as the expression of these channels is high in the presynaptic
associated with dizziness and sedation, and systemic admin-
termini of primary afferent nociceptors in the dorsal horn of the
istration leads to profound hemodynamic effects.11,13
spinal chord.3 The Cav2.2 channels control the influx of
We sought to discover state-dependent small molecule
Cav2.2 antagonists that inhibit the channel preferentially
extracellular calcium into primary afferent termini, which results
in the release of the neurotransmitters glutamate, substance P,
under the conditions of hyperexcitability associated with
and calcitonin gene-related peptide (CGRP) into the synaptic
cleft.4 Therefore, blocking Cav2.2 may lead to decreased neuro-
chronic pain syndromes (depolarized, open, and inactivated
state), while sparing low frequency signaling associated with
transmitter release and suppression of pain signals to the brain
sensory centers.5
The involvement of Cav2.2 in analgesia is substantiated by
normal nociceptive function (hyperpolarized, closed state). We
hypothesize that, by inhibiting the channel in a state-dependent
manner, a small molecule Cav2.2 inhibitor may achieve equal
several lines of evidence. Preclinical support is provided by
analgesia with minimal neuronal or hemodynamic side effects.14
rodent pain models. In rat models of both neuropathic and
inflammatory pain, the expression of the α subunit of Cav2.2 is
Additionally, a small molecule antagonist may provide the
up-regulated in dorsal horn.6,7 Spinal administration of Cav2.2
additional advantage of oral administration rather than the
intrathecal administration required of the petide ziconotide.
blocking peptides has been shown to ameliorate painful behavior
in rodent models of neuropathic and inflammatory pain.8 Genetic
support for the involvement of Cav2.2 in pain signaling is pro-
Received: July 19, 2012
Published: October 25, 2012
vided by three independently derived strains of Cav2.2−/− mice.9
© 2012 American Chemical Society
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a
Previously we reported the discovery of indole-derived Cav2.2
inhibitors and an oxindoline-based Cav2 inhibitor that afford
significant efficacy in rodent pain models while exhibiting
minimal ancillary pharmacology.7,15 In addition, there have been
several recent reports of the discovery of a variety of chemotypes
of small molecule Cav2.2 inhibitors that include varying levels
of in vitro and in vivo characterization.16,17 In the present study,
we report the discovery and full characterization of a novel series
of aminopiperidine sulfonamide state-dependent Cav2.2 inhib-
itors.18 During the course of these investigations, additional
aminopiperidine sulfonamide derivatives have independently
appeared in the patent literature as Cav2.2 blockers, indicating
the possible generality of this class of compounds as
analgesics.19−22
Scheme 1
a
Reagents and conditions: (a) NaBH(OAc)3, AcOH, 2,6-dimethox-
ybenzaldehyde, THF, 25 °C. (b) ArCO2H, Bop reagent, DIEA, DMF,
25 °C.
RESULTS AND DISCUSSION
■
and amide derivatives 3 may be synthesized in one step from
commercially available N-cyclopropyl-N-(piperidin-4-yl)-3-
(trifluoromethyl)benzenesulfonamide via a reductive amination
or Bop-mediated amide coupling reaction in good yield.
To investigate the effect of substitutions on the aryl
sulfonamide, an alternative synthetic scheme was pursued that
adds the sulfonamide moiety in the final step (Scheme 2).
A high-throughput screen of our internal sample collection was
performed using a calcium-influx assay detected with a
fluorescent calcium indicator dye measured on a fluorometric
imaging plate reader (FLIPR) with HEK-293 cells selectively
expressing Cav2.2, as described in the Experimental Section.23
One of the lead compounds identified from this screen was
the aminopiperidine sulfonamide derivative 1 (Table 1). This
a
Scheme 2
Table 1. Aminopiperidine Sulfonamide Screening Hit
Modification
Cav2.2 FLIPR
Cav1.2 FLIPR
hERG % inh @
ab
a
,
compound
R
IC50, μM3
IC50, μM
1 μM
1
2
n-Pr
cPr
0.12 (0.07)
0.06 (0.03)
3.2 (0.7)
ND
91 (5)
70 (2)
a
IC50 values and % inhibition values are averages of n ≥ 3. Standard
b
deviation is provided in parentheses. hERG inhibition was measured by
binding displacement of 35S-labeled MK-0499, a known hERG blocker.
a
Reagents and conditions: (a) 5-fluoro-2-(methylsulfonyl)benzoic
compound provided an advantageous starting point for SAR
optimization as a state-dependent channel inhibitor. It exhibits
more potent Cav2.2 inhibition of the calcium influx signal under
assay conditions in which the cells were depolarized (30 mM
potassium, IC50 of 0.12 μM) than when cells are hyperpolarized
(4 mM potassium, IC50 = 4.8 μM). For comparison, the state-
independent Cav2.2 blocking peptide ω-conotoxin GVIA was
also examined in our FLIPR assay.5,8 This peptide exhibited
similar inhibition under depolarizing conditions (30 mM
potassium, IC50 of 0.04 μM) as under hyperpolarizing conditions
(4 mM potassium, IC50 of 0.02 μM).
The sulfonamide 1 is not selective against ion channels that
mediate cardiovascular function, particularly L-type calcium
channels (Cav1.2), and hERG, which are known to regulate
blood pressure and QT intervals, respectively. In addition, the
compound exhibits a poor pharmacokinetic profile when dosed
in rats (oral normalized AUC = 0.007 μM·h·kg/mg dose,
Clp = 169 mL/min/kg, bioavailability = 4%). Initial modification
of the sulfonamide alkyl substituent afforded the cyclopropyl
derivative 2, which provided improved potency in the Cav2.2
FLIPR assay. This cyclopropyl aminopiperidine sulfonamide
was utilized as the core structure for further modification.
Chemistry. The synthesis of piperidine sulfonamide com-
pounds is illustrated in Scheme 1. Both the amine derivative 2
acid, Bop reagent, DIEA, DMF, 25 °C. (b) i. Cyclopropylamine,
MeOH, 45 °C, 1 h; ii. NaBH4, 25 °C, 1 h. (c) ArSO2Cl, DIEA, DMAP
(cat.), CH2Cl2, 25 °C.
Bop-mediated amide coupling reaction between piperidin-
4-one hydrochloride and 5-fluoro-2-(methylsulfonyl)benzoic acid
gave 4, which underwent reductive amination with cyclopropyl
amine to afford 5. Reacting 5 with various arylsulfonyl chlorides
afforded the desired sulfonamide derivatives 6.
Inhibition of Ion Channels. Once synthesized, these
compounds were evaluated for their ability to inhibit Cav2.2
using the FLIPR calcium-influx assay. Compounds were initially
screened for % inhibition of Cav2.2 FLIPR activity at 1 μM
compound concentration. Compounds showing good activity
inhibiting Cav2.2 channels were titrated to determine the IC50
and were also counter screened against Cav1.2 using a related
FLIPR assay and against the hERG K+ channel using a binding
assay that measures the displacement of 35S-labeled MK-499, a
well characterized hERG K+ channel blocker.24
Our initial SAR effort was focused on reducing hERG
binding activity while maintaining the Cav2.2 potency of this
class. Although incorporation of the cyclopropyl substituent in
2 afforded improved selectivity for Cav2.2 potency over hERG,
2 retained strong potency as an hERG channel modulator.
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It is well documented that lipophilic compounds with a basic
tertiary amine tend to have high affinity for the hERG
channel.25,26 To reduce hERG binding activity, we decided to
replace the basic amine with an amide substituent, and the results
are summarized in Table 2. The benzamide derivative 7 was
Table 3. Effect of Sulfonamide Substitutions on Cav2.2
potency
Table 2. Effect of Benzamide Substitutions on Cav2.2
Potency and Selectivity
a
compound
R
Cav2.2 FLIPR IC50, μM or% inh. @ 1 μM
19
20
21
22
23
24
3-CF3
0.56 (0.24)
17 (7)%
1 (13)%
2.5 (0.1)
0 (12)%
1.1 (0.2)
0 (15)%
2-CF3
4-CF3
2-F-5-CF3
3-SO2Me
3-OCF3
4-OCF3
MK-0499% - Cav1.2 FLIPR-
inh @
% inh @
25
Cav2.2 FLIPR %
a
IC50 values and % inhibition values are averages of n ≥ 3. Standard
inh. @ 1 μM or
a
a,
b
a
deviation is provided in parentheses.
compound
R
IC50, μM
1 μM 10 μM
1 μM 3 μM
c
7
H
42 (3)%
0.12 (0.06)
0.37(0.15)
0.30 (0.08)
0.38 (0.04)
0.24(0.17)
15 (5)%
ND
ND
ND
8
2-OCF3
21
4
75
72
77
66
51
which the CF3 group was in the ortho or para position exhibited
a significant loss of Cav2.2 potency (20 and 21). Adding a fluoro
group to the ortho position resulted in a 4-fold loss of potency
(22). The replacement of the CF3 group with a polar methyl
sulfone moiety resulted in complete loss of potency (23). In
fact, only the OCF3 was found to be comparable to CF3, as this
substitution resulted in only a moderate reduction of potency
(24). However, the OCF3 group in the para position again
resulted in a significant loss of Cav2.2 potency (25).
9
3-OCF3
60
73
78
88
10
11
12
13
14
15
16
17
18
19
4-OCF3
0
3-CO2Me
3-NMe2
2.4
0
ND
ND
40
11
63
17
2-CONH2
3-CONH2
4-CONH2
2-SO2Me
3-SO2Me
4-SO2Me
ND
ND
ND
0 (10)%
12 (11)%
0.66 (0.09)
15 (25)%
52 (5)%
0
0
1
67
32
19
39
ND
ND
ND
ND
Pharmacokinetic and Efficacy Profiling. The most
promising candidates 8, 16, and 19 were evaluated further for
microsomal stability by determining the percentage of
remaining parent compound following incubation (1 μM) in
human or rat liver microsome preparations (1 mg/mL) for 1 h,
and the pharmacokinetic profile of the compounds in rats was
also determined. The in vivo efficacy was evaluated using the
rat complete Freund’s adjuvant (CFA)-induced inflammatory
pain model.27 The results are summarized in Table 4. The tri-
fluoromethoxy derivative 8 exhibited poor human and modest
rat microsome stability with 1% and 21% of the parent
compound remaining, respectively, after 1 h of microsomal
incubation. The compound exhibited a modest PK profile with
relatively high clearance rate and moderate bioavailability.
Compounds 16 and 19 exhibited improved stability in both
human and rat microsomes. These compounds had an improved
rat PK profile, exhibiting a moderate clearance rate and excellent
bioavailability when dosed at 1 mg/kg iv and 3 mg/kg po. The
half-life is fairly short for these compounds (calculated from an iv
dosing study); however, the po arm of the study provided a later
2-SO2Me-5-F 0.56 (0.24)
1.6
18
34
a
IC50 values and % inhibition values are averages of n ≥ 3. Standard
b
deviation is provided in parentheses. hERG inhibition was measured
by binding displacement of 35S-labeled MK-0499, a well characterized
c
hERG blocker. ND: not determined.
significantly less potent as a Cav2.2 inhibitor. By introducing a
lipophilic moiety on the phenyl ring, regardless of electronic
nature and position of the substituent, the Cav2.2 inhibitory
activity was retained (8−12). However, these ring substitutions
afforded little significant improvement in selectivity against
hERG for 8−12 in comparison to 2. In addition, selectivity
against Cav1.2 was poor for the compounds in which it was
measured (9, 10, and 12). The incorporation of a hydrogen
bond donor substituent on the benzamide substituent afforded
a reduction in Cav2.2 activity. For example, the addition of a
primary amide caused complete loss of potency regardless of
substitution position (13−15). The effect of the polar
methylsulfone substitution varies depending on the substitution
position (16−18). The ortho-methylsulfone substitution was well
tolerated (16), however similar substitution in the meta or para
position afforded lower Cav2.2 potency (17−18). Introduction
of the polar methylsulfone group at the ortho position in 16 also
enhanced selectivity against Cav1.2. Addition of a fluoro group to
the 5-position of the methylsulfone-substituted phenyl ring (19)
afforded slightly enhanced Cav2.2 potency and also showed good
selectivity against hERG and Cav1.2 channels.
t
max of 1.7 h, which is consistent with slow oral absorption of 19.
These compounds were evaluated in the rat CFA pain model
dosed orally at 30 mg/kg. Compound 19 showed the best
efficacy among the three compounds with 66% and 57% reversal
of allodynia at 1 and 3 h time points, respectively, after dosing.
Because of its significant in vivo efficacy, compound 19 was
profiled further as a potential development candidate.
Profile of Compound 19. In Vitro Electrophysiology
Characterization. Compound 19 was evaluated for its ability
to inhibit hCav2.2 using conventional patch clamp electro-
physiology, and the results are shown in Figure 1. At a de-
polarized membrane potential (−40 mV), application of 3 μM
of compound 19 inhibited Cav2.2 channel current by 84 5%
(n = 4) (Figure 1A). From a limited titration at four compound
concentrations (Figure 1B), the compound achieves >50% block
Because the 2-methylsulfonyl-5-fluorobenzamide substituent
in 19 exhibited desirable potency and selectivity, this substituent
was retained and the further optimization of substitutions on the
sulfonamide phenyl ring was attempted. It appeared that a CF3
group at the meta position of the sulfonamide phenyl ring, as in
19, was optimal for Cav2.2 potency (Table 3). Compounds in
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Table 4. Microsome Stability, Rat PK Profile, and Rat CFA Efficacy of Selected Compounds
ef
,
rat CFA pain model
% reversal of
allodynia hours after
dosing
microsome stability, %
remaining after 1 h
rat pharmacokinetic profile
a
b
c
d
compound
human
rat
F%
AUCN
Clp
tl/2
dose (mg/kg)
1 h
3 h
plasma exposure 3 h
g
8
1
21
45
40
47
100
99
0.23
65
36
35
1.6
0.91
1.3
3 (iv)
23
47
66
17
45
57
ND
h
16
19
73
30
1.06
30 (po)
2.4 (0.8)
1.6 (1.1)
h
0.86
30 (po)
a
b
c
d
e
Bioavailability. Normalized AUC (po, μM·h·kg/mg). Clearance (mL/min/kg). Half-life (h). Average percent reversal is calculated as (postdose
f
g
− predose)/(preinjury-predose) for each rat (n = 6). Plasma exposure (μM) satellite rats (n = 2, standard deviation), ND: not determined. Dose
1.8 mg/kg po, 0.6 mg/kg iv. Dose 3 mg/kg po, 1 mg/kg iv;.
h
Figure 1. In vitro electrophysiology characterization of compound 19.
Figure 2. In vivo analgesic efficacy of compound 19 (A) in rat CFA
(A) Electrophysiological measurement of peak hCav2.2 current versus
inflammatory pain model and (B) in rat SNL neuropathic pain model.
time. Compound 19 (3 μM) was applied by bath perfusion for the time
All experimental and control groups contained at least six animals per
indicated by the solid bar. The holding membrane potential was
group, and data are expressed as mean + SEM. Percent reversal are
calculated as (postdose − predose)/(preinjury-predose) for each rat.
Significance was calculated using a two-way ANOVA followed by
Bonferroni posthoc tests for multiple comparisons with vehicle values
at each time point (*, p < 0.05).
○
−40 mV. (Inset) Representative traces are shown prior to ( ) and after
●
( ) addition of 19. (B) Plot of the percent block of Cav2.2 versus
concentration of 19. Inhibition was measured at a depolarized (−40 mV;
□
) and a hyperpolarized potential (−90 mV, Δ). Symbols represent the
average SEM of two, three, four, and one measurement(s) at 0.3, 1, 3,
and 10 μM, respectively, for −40 mV and four and six measurements at
3 and 10 μM, respectively, for −90 mV.
with a maximal effect 1 h after dosing of 65% reversal for a
30 mg/kg oral dose, and 45% reversal for a 10 mg/kg dose
(Figure 2A). We were unable to reproducibly dose the Cav2.2
blocking peptide ziconotide in these rat experiments, due to the
need for intrathecal administration for this agent. However, the
level of efficacy exhibited by 19 compares favorably to the effect
found with naproxen, which was utilized as a positive control
(47% reversal at 1 h following a 10 mg/kg dose, p < 0.05, n = 6).
In separate CFA-treated rats, the plasma exposure of a 10 mg/kg
oral dose was found to be 1.66 μM after 3 h. The efficacy of 19
was significantly lower at the 8 h time point and diminished at
24 h postdose in this model. A similar dose-dependent efficacy
was observed in the rat spinal nerve ligation (SNL) model28 of
neuropathic pain, with maximal effect of 59% reversal of
mechanical allodynia at 3 h after dosing for a 30 mg/kg dose, and
49% reversal for a 10 mg/kg dose (Figure 2B). The level of
for depolarized Cav2.2 channels at concentrations of <1 μM.
To assess whether inhibition of Cav2.2 was state-dependent, 19
was applied to cells held at a hyperpolarized membrane potential
(−90 mV) where channels are largely in the resting (closed) state.
At −90 mV, application of 10 μM 19 inhibited Cav2.2 channels
only 43 9% (n = 6), indicating that 19 is a state-dependent
Cav2.2 inhibitor.
In Vivo Efficacy. Compound 19 was evaluated for its anti-
nociceptive activity in both inflammatory and neuropathic pain
models. Inflammatory pain was induced by intraplantar
injection of complete Freund’s adjuvant (CFA) into the hind
paws of rats.27 Mechanical hyperalgesia was assessed at 1, 3, 4,
8, and 24 h following a single oral dose of 1, 3, 10, and 30 mg/kg.
Compound 19 dose-dependently reversed mechanical allodynia
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efficacy of 19 in neuropathic allodynia compared favorably with
the effect observed with the positive control pregabalin (58%
reversal at 3 h following a 10 mg/kg dose, p < 0.05, n = 6). In
separate SNL-treated rats, the plasma exposure of a 10 mg/kg
oral dose was found to be 1.26 μM after 3 h.
The target-mediated efficacy of 19 was established by
measuring the antinoccipetive activity of the compound in
Cav2.2-deficient mice as compared to wild-type mice (Figure 3).
In Vitro Selectivity and in Vivo Side-Effect Profile.
Sulfonamide 19 was counter-screened against a panel of 166
additional targets (a collection of representative drug and known
toxicity-causing targets) at Ricerca Laboratories and exhibited
no significant activity (>50% inhibition) at 10 μM compound
concentration in any of these assays. The compound was
evaluated for deleterious CNS effect in the rat rota-rod model
and showed no effect on rat motor coordination 3 h following a
100 mg/kg oral dose. The plasma and brain exposure of 19
following this experiment was 9.5 μM and 11.9 μM, respectively,
indicating a broad preclinical therapeutic window for Cav2.2-
mediated analgesia.
As discussed above, the state-independent Cav2.2 blocking
peptide ziconotide exhibits profound hemodynamic effects
when administered systemically, and we have observed mild
to moderate preclinical cardiovascular effects with unrelated
chemotypes of state-dependent Cav2.2 inhibitors.7,15 To further
evaluate any potential cardiovascular liabilities of blocking
Cav2.2 channels, compound 19 was administrated with
intravenous infusion in sequential rising doses at 1, 3, and
10 mg/kg (cumulative) to anesthetized, vagotomized dogs.
Treatment with compound 19 did not show any effect on the
evaluated CV parameters (MAP, HR, QRS, PR, and QTc) up to
a maximal plasma exposure of 20.4 μM (Figure 4), which is over
Figure 3. Efficacy of 19 (30 mg/kg p.o., 2 h.) in Cav2.2+/+ and
Cav2.2−/− mice . (A) CFA sensitization model, hindpaw withdraw
latency to thermal stimuli. (B) SNL pain model, 50% hindpaw
withdraw thresholds to stimulation with von Frey filaments.
Significance was calculated using a two-way ANOVA followed by
Bonferroni posthoc tests for multiple comparisons with vehicle values
(*, p < 0.05).
Figure 4. Cardiovascular effect of 19 administered in rising continuous
infusion to anesthetized, vagotomized dogs. Plasma exposure at the
a
indicated time point (μM, n = 3).
The mice were made hypersensitive to heat by intraplantar
injection of CFA, and the thermal threshold latency was com-
pared to that in the absence of injury (non-CFA) (Figure 3A).
While 19 showed significant efficacy reversing thermal threshold
in the CFA assay with wild type mice 1 h after a 30 mg/kg oral
dose, the compound exhibited no effect on Cav2.2−/− mice in
the same pain model It is noteworthy that the Cav2.2−/− mice
remain responsive to the noccieptive sensitization of CFA,
indicating the utilization of multiple pain pathways in this
model.29 As a positive control, naproxen reversed the CFA
sensitivity in both strains 1 h following a 30 mg/kg oral dose
(60% reversal in −/− mice, 67% in +/+ mice, p < 0.05, n = 7),
which indicates the appropriateness of this model to investigate
selective Cav2.2-mediated analgesia. Sulfonamide 19 exhibited
similar efficacy 2 h following a 30 mg/kg oral dose in the
reversal of mechanical allodynia in the SNL assay in +/+ mice,
while this activity was ablated in −/− mice (Figure 3B). In
this instance, both strains of mice remained responsive to
the positive control gabapenin (48% reversal in −/− mice, 71%
reversal in +/+ mice 2 h following a 30 mg/kg oral dose).
10× higher than the exposure associated with significant efficacy
in the rat CFA assay (Table 4).
Drug Metabolism Profile. The favorable exposure and
preclinical analgesic efficacy of 19, coupled with the lack of
ancillary pharmacology, lead us to investigate the full drug
metabolism profile of this compound as a potential development
candidate. The compound has a low free fraction in plasma from
rat (2.0%) and human (1.4%). Additionally, the compound is a
modest CYP3A4 inhibitor with IC50 of 5.1 μM and a relatively
strong PXR activator (EC50 of 0.43 μM, maximal activation
124% at 10 μM), which indicates the likelihood of compound-
mediated CYP3A4 induction in vivo.30 By evaluating the in vitro
metabolism of 19 in the presence of selective CYP inhibitors, we
found that CYP3A4 was the primary CYP isoform responsible
for the metabolism of the compound. These findings raised
the concern for potential drug−drug interactions and made pre-
diction of a human PK profile more difficult. Furthermore, meta-
bolite identification studies revealed formation of 3-trifluorome-
thylbenzenesulfonamide (26) as a major metabolite (Figure 5).
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EXPERIMENTAL SECTION
■
Experimental procedures for fluorescent Cav1.2 and Cav2.2 assays,
electrophysiology characterization of compound 19, rat CNS (rota-rod)
and CV dog studies, and in vivo pharmacology efficacy studies were
similar to previously published work.7
Cav2.2 FLIPR Assay. A fluorescent assay was used to measure
compound-mediated inhibition of calcium influx with a calcium-
sensitive fluorescent dye on a FLIPR Tetra instrument (Molecular
Devices, Sunnyvale, CA). The assay is similar to that described
previously,23 except that a different Cav2.2 cell line (labeled CBK) was
used in the present work. CBK cells express three calcium channel
subunits (α1B‑1, α2-δ, and β3a) along with an inwardly rectifying
potassium channel (Kir2.3) that enabled the cell membrane potential to
be controlled by bath potassium concentration. Incubation of CBK cells
in elevated potassium solution leads to depolarization of cell membrane
potential. Exposing CBK cells to modestly elevated concentrations
(30 mM) of potassium caused partial inactivation of the Cav2.2 channel
population. In comparison, incubating cells in a bath solution
containing 4 mM potassium maintained a negative membrane potential
(−90 mV), at which voltage most Cav2.2 channels are in resting
conformations. After compound incubation in 30 mM potassium, to
evaluate blocking of open/inactivated channels, channel opening was
initiated with 1:1 (by volume) addition of 140 mM potassium solution.
All compounds were tested in quadruplicate, and average SD values
were calculated for 10 point titrations (0.001−30 μM).
Chemistry. All procedures were carried out under a nitrogen
atmosphere using commercially available solvents that were used
without further purification. Starting materials were obtained from
commercial sources and were used without further purification. Normal
phase column chromatography was carried out using prepacked silica
gel cartridges on a Combiflash Rf separation system by Teledyne ISCO.
Reverse phase HPLC purification was carried out using a YMC C18
column on a Gilson HPLC system utilizing a gradient of 0.1% TFA in
water/acetonitrile as the eluent. NMR spectra were recorded using
Varian Inova500 spectrometers. The reported compounds are of ≥95%
purity, unless otherwise noted. Purity analysis was carried out by
LC-Mass analysis using an Agilent 1100 system fitted with Waters
Micromass.
N-Cyclopropyl-N-[1-(2,6-dimethoxybenzyl)piperidin-4-yl]-3-
(trifluoromethyl)benzenesulfonamide (2). To a vial were added
N-cyclopropyl-N-(piperidin-4-yl)-3-(trifluoromethyl)benzenesulfonamide
(50 mg, 0.144 mmol), acetic acid (8.2 μL, 0.144 mmol), 2,6-
dimethoxybenzaldehyde (28.6 mg, 0.172 mmol), sodium triacetoxybor-
ohydride (91 mg, 0.43 mmol), and 2 mL of THF. The resulting reaction
mixture was stirred at room temperature for 2 h. It was acidified with
TFA, diluted with DMSO and water, and purified on Gilson HPLC using
a reverse phase column to give the desired product (TFA salt) as a white
solid (80 mg, 91%). 1H NMR (CDCl3): δ: 8.11 (s, 1H), 8.05 (d, J = 8.1
Hz, 1H), 7.90 (d, J = 7.6 Hz, 1H), 7.71 (t, J = 7.9 Hz, 1H), 7.41 (t, J =
8.4 Hz, 1H), 6.62 (d, J = 8.4 Hz, 2H), 4.29 (s, 2H), 4.1 (m, 1H), 3.86 (s,
6H), 3.65 (m, 2H), 2.75 (m, 2H), 2.6 (m, 2H), 1.7 (m, 3H), 0.9 (m, 2H)
0.8 (m, 2H). [M + H+]: m/z 499.
1-{[5-Fluoro-2-(methylsulfonyl)phenyl]carbonyl}piperidin-4-
one (4). To a 250 mL round-bottom flask were added piperidin-4-one
hydrochloride (0.5 g, 3.69 mmol), 5-fluoro-2-(methylsulfonyl)benzoic
acid (0.96 g, 4.43 mmol), BOP reagent (1.9 g, 4.43 mmol), N,N-
diisopropylethylamine (1.92 mL, 11.07 mmol), and THF (50 mL).
The resulting solution was stirred at ambient temperature for 1 h.
LC-MS indicated complete conversion to product. The reaction was
quenched by the addition of 50 mL of 1 N HCl solution, and the
product was extracted with EtOAc. Organics were dried over sodium
sulfate, filtered, and concentrated. The crude product was purified by
flash chromatography with EtOAc/hexane 1:1 as the eluent to afford
compound 4 (0.95 g, 86%) as white solid. 1H NMR (CDCl3): δ: 8.17
(m, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.16 (d, J = 7.9 Hz, 1H), 4.62
(m, 1H), 3.7 (m, 1H), 3.56 (m, 2H), 4.7 (m, 1H), 3.3 (s, 3H), 2.88
(m, 2H), 2.55 (m, 1H), 2.32 (m, 1H). [M + H+]: m/z 300.
Figure 5. Concentration of parent 19 and metabolite 26 in whole
blood and brain following 10 mg/kg oral dose of 19 in rats.
Although 26 formed at low concentration during in vitro
incubation with both human and rat liver microsomes, it was
identified as a significant circulating species in vivo. When male
Sprague−Dawley rats are administered an oral dose of 19 at
10 mg/kg, the concentration of metabolite 26 in whole blood
and brain is substantially higher than parent compound 19
(Figure 5). At 8 h postdose, the concentration of compound 19
in whole blood was only 0.31 μM, while the concentration of the
metabolite was 6.1 μM. The metabolite 26 clears slowly in whole
blood (clearance rate: 0.31 mL/min/kg in rat), and the half-life
could not be accurately measured in 24 h. This metabolite is
efficacious in both the CFA and SNL pain models,31 even
though it does not appreciably block Cav2.2 channels in vitro at
concentrations up to 10 μM. The antinociceptive mechanism of
26 is currently unknown. In fact, an in vitro screen of 26 against
a collection of 48 known and proprietary putative pain targets
revealed no activity (>50% inhibition or activation) of these
targets at 10 μM compound concentration. The abundance of the
metabolite in both the CNS and peripheral circulation contributes
to an unknown effect to the preclinical efficacy ascribed to 19.
The presence of an efficacious metabolite of undetermined
mechanism would introduce additional complexity and risk to the
development of 19 or closely related sulfonamide derivatives. Due
to these drug metabolism related issues, development effort on
compound 19 was terminated.
CONCLUSIONS
■
We have identified the aminopiperidine sulfonamide derivative
1 as a state-dependent inhibitor of Cav2.2. Incorporation of the
benzamide substituent significantly improved the off-target
profile of this class of compounds, The lead class was optimized
to provide 19, a potent Cav2.2 inhibitor with significant efficacy
in preclinical assays of both neuropathic and inflammatory pain,
and a clean profile in ancillary CNS and CV evaluation.
However the compound has several drug metabolism related
issues including inhibition of CYP3A4 enzyme, activation of
PXR, and the significant formation of the circulating metabolite
26 in vivo. The metabolic liability of the exocyclic tertiary
sulfonamide will have to be addressed in order for members of
the lead class outlined herein to be developed as candidates for
analgesia, and overcoming this liability represents the focus of
our forthcoming work in this series of compounds.
[4-(Cyclopropylamino)piperidin-1-yl][5-fluoro-2-
(methylsulfonyl)phenyl]methanone (5). Compound 4 (0.60 g,
2 mmol) and cyclopropylamine (0.137 g, 2.4 mmol) were dissolved in
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MeOH (5 mL) in a 100 mL round-bottom flask. The solution was
heated to 45 °C for 2 h. The solution was cooled, to this solution was
added NaBH4 (0.3 g, 8 mmol) in portions at ambient temperature, and
resulting reaction mixture was stirred overnight. The volatiles were
removed by rotary evaporation, and the residue was redissolved in
EtOAc. The solution was washed with water, dried over sodium
sulfate, filtered, and concentrated to afford 0.6 g compound 5 as white
7.78 (t, J = 8.2 Hz, 1H), 7.58 (t, J = 8.0 Hz, 1H), 7.5 (t, J = 8.0 Hz,
1H), 7.3(m, 1H), 6.2 (b, 1H), 4.85 (b, 1H), 4.18 (b, 1H), 3.45 (m,
1H), 3.15 (m, 1H), 2.8 (m, 1H), 1.8 (m, 1H), 1.5 (m, 1H), 1.0 (m,
2H), 0.8 (m, 2H). [M + H+]: m/z 496.
3-{[4-(Cyclopropyl{[3-(trifluoromethyl)phenyl]sulfonyl}-
amino)piperidin-1-yl]carbonyl}benzamide (14). Compound 14
was synthesized with the same procedure as that used for compound
1
1
19. H NMR (CD3OD): δ: 8.2 (d, J = 8.0 Hz, 1H), 8.18 (s, 1H), 8.0
solid. It was used for the next step without further purification. H
(d, J = 8.1 Hz, 1H), 7.86 (m, 3H), 7.5 (d, J = 8.4 Hz, 2H), 4.7 (b, 1H),
4.2 (m, 1H), 3.7 (b, 1H), 3.2 (b, 1H), 2.85 (b, 1H), 2.2 (m, 1H),
2.0 (b, 2H), 1.75 (b, 1H), 1.6 (b, 1H), 1.0 (m, 2H), 0.8 (m, 2H).
[M + H+]: m/z 496.
NMR (CDCl3): δ: 8.13 (m, 1H), 7.22 (t, J = 7.8 Hz, 1H), 7.05 (m,
1H), 4.58 (m, 1H), 4.42 (m, 1H), 3.75 (m, 1H), 3.42 (m, 1H), 3.25 (s,
3H), 3.15 (m, 2H), 3.0 (m, 1H), 2.85 (m, 2H), 2.15 (m, 2H), 1.95 (m,
1H), 1.45 (m, 2H), [M + H+]: m/z 341.
4-{[4-(Cyclopropyl{[3-(trifluoromethyl)phenyl]sulfonyl}-
amino)piperidin-1-yl]carbonyl}benzamide (15). Compound 15
was synthesized with the same procedure as that used for compound
N-Cyclopropyl-N-[1-(phenylcarbonyl)piperidin-4-yl]-3-
(trifluoromethyl)benzenesulfonamide (7). Compound 7 was
synthesized with the same procedure as that used for compound 19.
1H NMR (CDCl3): The compound exists as a pair of equal amount
1
19. H NMR (CDCl3): δ: 8.18 (s, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.9
(m, 1H), 7.88 (d, J = 8.5 Hz, 2H), 7.78 (t, J = 8.2 Hz, 1H), 7.5 (d, J =
8.0 Hz, 2H), 6.1(b, 1H), 4.2 (m, 1H), 3.78 (b, 1H), 3.1 (b, 1H), 2.8
(b, 1H), 2.0 (m, 1H), 1.0 (m, 2H), 0.8 (m, 2H). [M + H+]: m/z 496.
N-Cyclopropyl-N-(1-{[2-(methylsulfonyl)phenyl]carbonyl}-
piperidin-4-yl)-3-(trifluoromethyl)benzenesulfonamide (16).
Compound 16 was synthesized with the same procedure as that
rotamers at ambient temperature. δ: 8.18 (s, 1H), 8.08 (d, J = 8.0 Hz,
1H), 7.9 (d, J = 8.0 Hz, 1H), 7.7 (t, J = 8.2 Hz, 1H), 7.42 (m, 5H), 4.8
(b, 1H), 4.18 (m, 1H), 3.8 (b, 1H), 3.2 (m, 1H), 2.8 (m, 1H), 2.0 (m,
1H), 1.8 (m, 1H), 1.7 (m, 1H), 1.0 (m, 2H), 0.8 (m, 2H). [M + H+]:
m/z 453.
N-Cyclopropyl-N-(1-{[2-(trifluoromethoxy)phenyl]carbonyl}-
piperidin-4-yl)-3-(trifluoromethyl)benzenesulfonamide (8).
Compound 8 was synthesized according to the same procedure as
1
used for compound 19. H NMR (CDCl3): The compound exists as a
pair of rotamers at ambient temperature. δ: 8.18 (s, 1H), 8.08 (m, 1H),
8.15 (m, 1H), 7.7 (m, 2H), 7.6 (m, 1H), 7.38 (d, J = 6.5 Hz, 1H), 4.83
(b, 1H, major rotamor), 4.75 (b, 1H, minor rotamor), 4.2 (m, 1H, major
rotamer), 4.15 (m, 1H, minor rotamer), 3.45 (b, 1H, major rotamer),
3.35 (m, 1H, minor rotamer), 3.25 (s, 3H, major rotamor), 3.2 (s, 3H,
minor rotamer), 3.1 (m, 2H, major rotamer), 2.8 (m, 2H, minor
rotamer), 2.3 (m, 1H, major rotamor), 2.18 (m, 1H, minor rotamor),
2.1 (m, 1H), 1.9 (m, 1H), 1.8 (m, 1H), 1.65 (b, major rotamor), 1.6
(b, 1H, minor rotamor), 1.35 (b, 1H), 1.0 (m, 2H), 0.8 (m, 2H). [M + H+]:
m/z 531.
1
that used for compound 19. H NMR (CD3OD): δ: 8.18 (d, J = 7.8
Hz, 1H), 8.15 (s, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.83 (t, J = 8.0 Hz,
1H), 7.6 (m, 1H), 7.4 (m, 3H), 4.7 (m, 1H), 4.2 (m, 1H), 3.4 (m,
1H), 3.2 (m, 1H), 2.8 (m, 1H), 3.5 (m, 1H), 1.5−2.1 (m, 5H), 0.8−
1.0 (m, 4H). [M + H+]: m/z 537.
N-Cyclopropyl-N-(1-{[3-(trifluoromethoxy)phenyl]carbonyl}-
piperidin-4-yl)-3-(trifluoromethyl)benzenesulfonamide (9).
Compound 9 was synthesized with the same procedure as that used
1
for compound 19. H NMR (CDCl3): δ: 8.18 (s, 1H), 8.10 (d, J =
N-Cyclopropyl-N-(1-{[3-(methylsulfonyl)phenyl]carbonyl}-
piperidin-4-yl)-3-(trifluoromethyl)benzenesulfonamide (17).
Compound 17 was synthesized with the same procedure as that
8.0 Hz, 1H), 7.89 (d, J = 7.8 Hz, 1H), 7.72 (t, J = 7.7 Hz, 1H), 7.49
(t, J = 8.0 Hz, 1H), 7.35−7.25 (m, 3H), 4.8 (b, 1H), 4.2 (m, 1H), 3.8
(b, 1H), 3.1 (b, 1H), 2.8 (b, 1H), 2.0 (m, 1H), 1.85 (m, 1H), 1.75
(m, 4H), 1.0 (b, 2H), 0.85 (m, 2H).[M + H+]: m/z 537.
1
used for compound 19. H NMR (CDCl3): δ: 8.18 (s, 1H), 8.10
(d, J = 7.8 Hz, 1H), 8.02 (d, J = 7.4 Hz, 1H), 8.0 (s, 1H), 7.87 (d, J =
7.7 Hz, 1H), 7.6−7.8 (m, 3H), 4.8 (b, 1H), 4.2 (m, 1H), 3.65 (b, 1H),
3.15 (b, 1H), 3.1 (s, 3H), 2.8 (b, 1H), 2.0 (m, 1H), 1.85 (m, 1H), 1.65
(m, 1H), 1.0 (b, 2H), 0.85 (m, 2H).[M + H+]: m/z 531.
N-Cyclopropyl-N-(1-{[4-(trifluoromethoxy)phenyl]carbonyl}-
piperidin-4-yl)-3-(trifluoromethyl)benzenesulfonamide (10).
Compound 10 was synthesized with the same procedure as that
used for compound 19. 1H NMR (CDCl3): δ: 8.84 (s, 1H), 8.2
(s, 1H), 8.1 (t, J = 8.0 Hz, 2H), 7.9 (d, J = 8.4 Hz, 1H), 7.8 (d, J =
8.4 Hz, 1H), 7.7 (t, J = 8.4 Hz, 1H), 4.85 (b, 1H), 4.2 (m, 1H), 4.0
(b, 1H), 3.15(b, 1H), 3.2 (b, 1H), 2.8 (b, 1H), 2.0 (m, 2H), 1.8
(b, 1H), 1.65 (b, 1H), 1.0 (m, 2H), 0.8 (m, 2H). [M + H+]: m/z 537.
Methyl 3-{[4-(Cyclopropyl{[3-(trifluoromethyl)phenyl]-
sulfonyl}amino)piperidin-1-yl]carbonyl}benzoate (11). Com-
pound 11 was synthesized with the same procedure as that used for
compound 19. 1H NMR (CDCl3): δ: 8.18 (s, 1H), 8.1 (d, J = 8.0 Hz,
1H), 8.08 (d, J = 8.0 Hz, 1H), 8.05 (s, 1H), 7.85 (d, J = 8.4 Hz, 1H),
7.7 (t, J = 8.2 Hz, 1H), 7.6 (d, J = 8.4 Hz, 1H), 7.5 (t, J = 8.5 Hz, 1H),
5.2 (b, 1H), 4.8 (b, 1H), 4.18 (m, 1H), 3.92 (s, 3H), 3.8 (b, 1H),
3.15(b, 1H), 2.8 (b, 1H), 2.0 (m, 1H), 1.8 (b, 1H), 1.6 (b, 1H), 1.0
(m, 2H), 0.8 (m, 2H). [M + H+]: m/z 511.
N-Cyclopropyl-N-(1-{[3-(dimethylamino)phenyl]carbonyl}-
piperidin-4-yl)-3-(trifluoromethyl)benzenesulfonamide (12).
Compound 12 was synthesized with the same procedure as that
used for compound 19. 1H NMR (CDCl3): The compound exists as a
pair of equal amount rotamers at ambient temperature. δ: 8.18 (s, 1H),
8.08 (d, J = 8.0 Hz, 1H), 7.9 (d, J = 8.0 Hz, 1H), 7.7 (t, J = 8.2 Hz,
1H), 7.4 (t, J = 8.2 Hz, 1H), 7.1 (m, 2H), 6.9 (d, J = 8.0 Hz, 1H), 4.8
(b, 1H), 4.18 (m, 1H), 3.8 (b, 1H), 3.2 (m, 1H), 3.1 (s, 6H), 2.8
(m, 1H), 2.0 (m, 1H), 1.8 (m, 1H), 1.7 (m, 1H), 1.6 (b, 1H), 1.0 (m, 2H),
0.8 (m, 2H). [M + H+]: m/z 496.
N-Cyclopropyl-N-(1-{[4-(methylsulfonyl)phenyl]carbonyl}-
piperidin-4-yl)-3-(trifluoromethyl)benzenesulfonamide (18).
Compound 18 was synthesized with the same procedure as that
1
used for compound 19. H NMR (CDCl3): δ: 8.18 (s, 1H), 8.10 (d,
J = 8.0 Hz, 1H), 8.02 (d, J = 8.3 Hz, 2H), 7.89 (d, J = 7.8 Hz, 1H), 7.73
(t, J = 7.8 Hz, 1H), 7.6 (d, J = 8.3 Hz, 2H), 4.8 (b, 1H), 4.2 (m, 1H),
3.65 (b, 1H), 3.15 (b, 1H), 3.1 (s, 3H), 2.8 (b, 1H), 2.0 (m, 1H), 1.85
(m, 1H), 1.65 (m, 1H), 1.0 (b, 2H), 0.85 (m, 2H).[M + H+]: m/z 531.
N-Cyclopropyl-N-(1-{[5-fluoro-2-(methylsulfonyl)phenyl]-
c a r b o n y l } p i p e r i d i n - 4 - y l ) - 3 - ( t r i fl u o r o m e t h y l ) -
benzenesulfonamide (19). To a 100 mL round-bottom flask were
added 5-fluoro-2-(methylsulfonyl)benzoic acid (0.702 g, 3.22 mmol), N-
cyclopropyl-N-(piperidin-4-yl)-3-(trifluoromethyl)benzenesulfonamide
(1.12 g, 3.22 mmol), Bop reagent (1.57 g, 3.54 mmol), DIEA (1.68 mL,
9.66 mmol), and 10 mL of DMF. The resulting reaction mixture was
stirred at ambient temperature for 1 h. It was diluted with 60 mL of
EtOAc and washed sequentially with 60 mL of 1 N HCl, 30 mL of 5%
KOH, and 20 mL of brine. Organics were dried over Na2SO4, filtered,
and concentrated. The residue was purified on prepacked silica gel
column eluted with 2:3 ethyl acetate/hexane to give the desired product
as a fluffy white solid after lyophilization (1.5 g, 85%). 1H NMR
(CDCl3): The compound exists as a pair of rotamers at rt (ratio: 2.4:1).
The major rotamer: δ: 8.16 (s, 1H), 8.1 (m, 2H), 7.86 (d, J = 7.9 Hz,
1H), 7.7 (d, J = 8.1 Hz, 1H), 7.3 (m, 1H), 7.07 (dd, J = 2.5, 7.9 Hz, 1H),
4.8 (m, 1H), 4.2 (m, 1H), 3.5 (m, 1H), 3.26 (s, 3H, major rotamer), 3.1
(m, 1H), 2.8 (m, 1H), 2.25 (m, 1H), 2.15 (m, 1H), 1.95 (m, 1H), 1.65
(m, 1H), 1.35 (m, 1H), 1.0 (m, 2H), 0.8 (m, 2H); The minor rotamer:
many peaks overlap with the major rotamer. Only peaks that are
clearly separated from the major rotamer are listed: δ: 4.7 (m, 1H),
2-{[4-(Cyclopropyl{[3-(trifluoromethyl)phenyl]sulfonyl}-
amino)piperidin-1-yl]carbonyl}benzamide (13). Compound 13
was synthesized with the same procedure as that used for compound
19. 1H NMR (CDCl3): The compound exists as a pair of equal
amount rotamers at ambient temperature. δ: 8.18 (s, 1H), 8.08
(d, J = 8.0 Hz, 1H), 7.9 (d, J = 8.0 Hz, 1H), 7.8 (d, J = 8.2 Hz, 1H),
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4.05 (m, 1H), 3.35 (m, 1H), 2.05 (m, 2H), 1.6 (m, 1H); [M + H+]: m/z
549. HRMS calcd for C23H25F4N2O5S2 (MH+) 549.1136, found
549.1149.
3.35 (m, 1H, minor rotamer), 3.26 (s, 3H, major rotamor), 3.22
(s, 3H, minor rotamer), 3.1 (m, 1H), 2.8 (m, 1H), 1.8 - 2.3 (m, 3H),
1.66 (d, J = 13.2, 1H, major rotamor), 1.60 (m, 1H, minor rotamor),
1.37 (d, J = 12.4 Hz, 1H, major rotamor), 1.0 (m, 2H), 0.8 (m, 2H).
[M + H+]: m/z 565.
N-Cyclopropyl-N-(1-{[5-fluoro-2-(methylsulfonyl)phenyl]-
c a r b o n y l } p i p e r i d i n - 4 - y l ) - 2 - ( t r i fl u o r o m e t h y l ) -
benzenesulfonamide (20). Compound 20 was synthesized with the
same procedure as that used for compound 21. 1H NMR (CDCl3): δ:
8.32 (m, 1H), 8.15 (m, 1H), 7.85 (m, 1H), 7.75 (m, 2H), 7.3(m, 1H),
7.05 (d, J = 8.0 Hz, 1H), 4.85 (b, major rotamer, 1H), 4.75 (b, minor
rotamer, 1H), 4.4 (m, major rotamer, 1H), 3.5 (b, major rotamer, 1H),
3.4(b, 1H, minor rotamer), 3.25 (s, 3H, major rotamer), 3.2 (s, 3H,
minor rotamer), 3.2 (m, 1H), 2.85 (m, 1H), 2.4 (m, 1H), 2.25 (m,
1H), 1.85 (b, 1H), 1.5 (s, 3H), 0.7 (m, 2H), 0.5 (m, 2H). [M + H+]:
m/z 549.
N-Cyclopropyl-N-(1-{[5-fluoro-2-(methylsulfonyl)phenyl]-
c a r b o n y l } p i p e r i d i n - 4 - y l ) - 4 - ( t r i fl u o r o m e t h y l ) -
benzenesulfonamide (21). To a 25 mL round-bottom flask were
added compound 4 (167 mg, 0.491 mmol), 4-(trifluoromethyl)-
benzene-1-sulfonyl chloride (100 mg, 0.409 mmol), DIEA (0.107 mL,
0.613 mmol), DMAP (5 mg, 0.041 mmol), and 5 mL of THF. The
resulting reaction mixture was stirred at ambient temperature for
10 min and heated at 50 °C for an additional 2 min. Analysis by
LC-MS indicated a complete conversion. The mixture was acidified
with TFA and purified on Gilson HPLC using a reverse phase column
to give the desired product as a fluffy white solid after lyophilization
(120 mg, 53%). 1H NMR (CDCl3): The compound exists as a pair of
rotamers at ambient temperature. δ: 8.1 (m, 1H), 8.0 (d, J = 8.3 Hz,
2H), 7.8 (t, J = 7.2 Hz, 2H), 7.25 (m, 1H), 7.05 (d, J = 8.3 Hz, 1H),
4.8 (b, 1H, major rotamor), 4.7 (b, 1H, minor rotamor), 4.2 (m, 1H,
major rotamer), 4.05 (m, 1H, minor rotamer), 3.45 (b, 1H, major
rotamer), 3.35 (m, 1H, minor rotamer), 3.25 (s, 3H, major rotamor),
3.2 (s, 3H, minor rotamer), 3.15 (m, 2H, major rotamer), 2.8 (m, 2H,
major rotamer), 2.3 (m, 1H, major rotamor), 2.2 (m, 1H, minor
rotamor), 2.0 (m, 1H), 1.85 (m, 1H), 1.7 (b, 1H, major rotamor), 1.6
(b, minor rotamor), 1.4 (b, 1H), 1.0 (m, 2H), 0.8 (m, 2H). [M + H+]:
m/z 549.
N-Cyclopropyl-N-(1-(5-fluoro-2-(methylsulfonyl)benzoyl)-
piperidin-4-yl)-4-(trifluoromethoxy)benzenesulfonamide (25).
Compound 25 was synthesized with the same procedure as that used
for compound 21. 1H NMR (CDCl3): The compound exists as a pair
of rotamers at RT. δ: 8.16 (m, 1H), 7.93 (m, 2H), 7.38 (m, 2H), 7.4
(t, J = 8.0 Hz 2H), 7.25 (m, 1H), 7.1 (m, 1H), 4.82 (m, 1H, major
rotamer), 4.7 (m, 1H, minor rotamer), 4.2 (m, 1H, major rotamer),
4.05 (m, 1H, minor rotamer), 3.45 (m, 1H, major rotamer), 3.35 (m,
1H, minor rotamer), 3.25 (s, 3H, major rotamor), 3.2 (s, 3H, minor
rotamer), 3.15 (m, 2H, major rotamer), 2.8 (m, 2H, major rotamer),
2.3 (m, 1H, major rotamor), 2.2 (m, 1H, minor rotamor), 2.0 (m, 1H),
1.85 (m, 1H), 1.7 (m, 1H, major rotamor), 1.6 (m, minor rotamor),
1.4 (m, 1H), 1.0 (m, 2H), 0.8 (m, 2H). [M + H+]: m/z 565.
AUTHOR INFORMATION
Corresponding Author
*Phone: (732) 594-2955. E-mail: pengcheng.shao@merck.com.
■
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
AcOH, acetic acid; AUC, area under the pharmacokinetic
exposure curve; b, broad; Bop reagent, Bbenzotriazol-1-
yloxytris(dimethylamino)phosphonium hexafluorophosphate;
Cav, voltage-gated calcium channel; CFA, complete Freund’s
adjuvant; CLp, plasma pharmacokinetic clearence; CNS, central
nervous system; CV, cardiovascular; CYP, cytochrome P450
enzymes; d, doublet; DIEA, diisopropylethylamine; DMAP,
4-dimethylaminopyridine; DMF, N,N-dimethylformamide;
DMSO, dimethyl sulfoxide; EtOAc, ethyl acetate; HEK cells,
human embrionic kidney; hERG, human ether-a-go-go-related
gene; HPLC, high performance liquid chromatography; HR,
heart rate; LC-MS, liquid-chromatography−mass spectroscopy;
m, multiplet; MAP, mean arterial pressure; MeOH, methanol;
NMR, nuclear magnetic resonance; PK, pharmacokinetic; PXR,
pregnane X receptor; s, singlet; SAR, structure−activity relation-
ship; SNL, spinal nerve ligation; t, triplet; THF, tetrahydrofuran;
TFA, trifluoroacetic acid
N-Cyclopropyl-2-fluoro-N-(1-{[5-fluoro-2-(methylsulfonyl)-
phenyl]carbonyl}piperidin-4-yl)-5-(trifluoromethyl)-
benzenesulfonamide (22). Compound 22 was synthesized with the
same procedure as that used for compound 21. 1H NMR (CDCl3): δ:
8.25 (m, 1H), 8.18 (m, 1H), 7.85 (m, 1H), 7.38 (m, 1H), 7.3 (m, 1H),
7.05 (d, J = 8.0 Hz, 1H), 4.85 (b, major rotamer, 1H), 4.75 (b, minor
rotamer, 1H), 4.4 (m, major rotamer, 1H), 4.2 (m, minor rotamer),
3.5 (b, major rotamer, 1H), 3.4(b, 1H, minor rotamer), 3.25 (s, 3H,
major rotamer), 3.2 (s, 3H, minor rotamer), 3.2 (m, 1H), 2.85 (m,
1H), 2.4 (m, 1H), 2.25 (m, 1H), 1.85 (b, 1H), 1.6 (m, 1H), 0.85 (m,
2H), 0.8 (m, 2H). [M + H+]: m/z 567.
N-Cyclopropyl-N-(1-{[5-fluoro-2-(methylsulfonyl)phenyl]-
c a r b o n y l } p i p e r i d i n - 4 - y l ) - 3 - ( m e t h y l s u l f o n y l ) -
benzenesulfonamide (23). Compound 23 was synthesized with the
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