Improved Cav2.2 channel inhibitors through a gem -dimethylsulfone bioisostere replacement of a labile sulfonamide

Article abstract of DOI:10.1021/ml4002612

We report the investigation of sulfonamide-derived Ca_v2.2 inhibitors to address drug-metabolism liabilities with this lead class of analgesics. Modification of the benzamide substituent provided improvements in both potency and selectivity. However, we discovered that formation of the persistent 3-(trifluoromethyl)benzenesulfonamide metabolite was an endemic problem in the sulfonamide series and that the replacement of the center aminopiperidine scaffold failed to prevent this metabolic pathway. This issue was eventually addressed by application of a bioisostere strategy. The new gem-dimethyl sulfone series retained Ca_v2.2 potency without the liability of the circulating sulfonamide metabolite.

Full text of DOI:10.1021/ml4002612

Letter
pubs.acs.org/acsmedchemlett
Improved Cav2.2 Channel Inhibitors through a gem-Dimethylsulfone
Bioisostere Replacement of a Labile Sulfonamide
Pengcheng P. Shao,*,† Feng Ye,† Prasun K. Chakravarty,† 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, Ion Channels, Drug Metabolism and Pharmacokinetics, and Pharmacology, Merck
Research Laboratories, Rahway, New Jersey 07065, United States
S
* Supporting Information
ABSTRACT: We report the investigation of sulfonamide-
derived Cav2.2 inhibitors to address drug-metabolism liabilities
with this lead class of analgesics. Modification of the
benzamide substituent provided improvements in both
potency and selectivity. However, we discovered that
formation of the persistent 3-(trifluoromethyl)-
benzenesulfonamide metabolite was an endemic problem in
the sulfonamide series and that the replacement of the center
aminopiperidine scaffold failed to prevent this metabolic
pathway. This issue was eventually addressed by application of
a bioisostere strategy. The new gem-dimethyl sulfone series
retained Cav2.2 potency without the liability of the circulating
sulfonamide metabolite.
KEYWORDS: Cav2.2, N-type calcium channel, pain, sulfonamide, bioisostere, sulfone
-Type calcium channels (Cav2.2) are expressed in the
presynaptic termini of primary afferent nociceptors in the
evaluate state-dependent inhibition of Cav2.2 channels, it has
several drug metabolism related issues that confound further
development of the compound. These include moderate
N
spinal cord and are key components of the pain transmission
pathway. We have previously reported the discovery of a series
of aminopiperidine sulfonamide state-dependent Cav2.2
channel inhibitors with potential utility for the treatment of
chronic pain. Members of this class are orally bioavailable and
efficacious in preclinical pain models.1 In particular, 1 (Figure
1) is selective for inhibition of the Cav2.2 channel over the
CYP3A4 inhibition (IC50 = 5.1 μM), PXR activation (EC50
=
0.43 μM), and the formation of the persistent circulating
metabolite 2. The sulfonamide metabolite 2 exhibited
preclinical antinociceptive activity in its own right, although it
was devoid of significant Cav2.2 activity. In order to further
improve this lead class of Cav2.2 inhibitors and address the drug
metabolism issues associated with this series, SAR optimization
studies were carried out on the amide substituent and the
aminopiperidine core, and a bioisostere replacement strategy
was pursued for the sulfonamide moiety.
The first substituent to be investigated for further
optimization was the benzoic amide moiety (Table 1). The
synthesis of piperidine sulfonamide compounds listed in Table
1 was straightforward as described previously.1 This synthetic
route allowed rapid SAR studies modifying substitutions on the
benzamide phenyl ring. In some instances, the desired benzoic
acid partner had to be synthesized. For example, the
intermediate 4-(methylsulfonyl)-2-trifluoromethoxy-benzoic
Figure 1. In vivo metabolism of 1 to afford the primary sulfonamide 2.
cardiovascular ion channels hERG and Cav1.2, with a decent
preclinical PK and biodistribution profile. The compound
exhibits dose-dependent efficacy in preclinical models of
inflammatory hyperalgesia and neuropathic allodynia, but it
has no efficacy in Cav2.2 gene-deleted mice. The compound is
also devoid of ancillary preclinical cardiovascular or CNS
pharmacology. While 1 may be used as a preclinical tool to
Received: July 9, 2013
Accepted: September 8, 2013
Published: September 8, 2013
© 2013 American Chemical Society
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exhibited a 20-fold increase in Cav2.2 potency over 1. A
substantial loss in potency was observed for compounds with
the sulfone in the meta (6) or para (7) positions. However, in
each instance the potency was restored by the addition of an
ortho substituent (8−12). Among ortho substituents, the
lipophilic trifluoromethy and trifluoromethoxy substituents (11
and 12) provided a greater enhancement of Cav2.2 inhibitory
potency than the halogen substituents (8, 9, or 10). PXR
activity was reduced when the methylsulfone group was
introduced at the para or meta position, and the para
substitution has a stronger effect (8 vs 10). Substitution with
other polar functional groups was also explored. With a primary
amide at the para position, compound 13 was significantly less
potent as a Cav2.2 inhibitor. However, again in this instance,
the addition of a CF3 or OCF3 group at the ortho position
restored Cav2.2 inhibitory potency (14 and 15). Many of the
compounds listed in Table 1 were reasonably devoid of MK-
499 binding activity. However, CYP3A4 inhibition was
substantially reduced only for compound 12.
Table 1. Effect of Benzamide Substitutions on Cav2.2
Inhibitory Potency and Selectivity
MK-
CYP
3A4%
inh @
Cav2.2 FLIPR
499%
PXR
EC50
IC50, μM or % inh @
a
b
compd
R
inh @ 1 μM
10 μM
10 μM (μM)
1
2-SO2Me-5-F
2-SO2Me-5-CN
2-SO2Me-4-CN
2-SO2Me-4-CF3
3-SO2Me
0.56
0.67
0.45
0.027
15%
52%
0.34
0.35
0.29
0.021
0.048
12%
0.76
0.39
32%
41%
66%
43%
75%
59%
86%
78%
ND
ND
69%
ND
78%
75%
16%
ND
61%
60%
0.42
5.0
3
4
8.4
5
4.3
c
6
ND
ND
ND
2.7
7
4-SO2Me
ND
8
2-Cl-5-SO2Me
2-F-4-SO2Me
2-Cl-4-SO2Me
2-CF3-4-SO2Me
2-OCF3-4-SO2Me
4-CONH2
49%
78%
32%
58%
63%
ND
9
6.8
10
11
12
13
14
15
10.8
2.8
In a parallel effort, the SAR investigation on the center
aminopiperidine portion of the molecule was undertaken. This
region of the molecule was found to be surprisingly tolerant of
modification while maintaining Cav2.2 inhibitory potency, as
illustrated in Figure 2. The addition of an ipso-methyl group at
1.7
ND
1.9
2-CF3-4-CONH2
2-OCF3-4-CONH2
32%
41%
>10
a
b
IC50 values and % inhibition values are averages of n ≥ 3. hERG
inhibition was measured by binding displacement of [35S]MK-499.
c
ND: not determined.
acid used for 12 was prepared from commercially available 4-
bromo-1-iodo-2-trifluoromethoxy-benzene as indicated in the
Supporting Information.
The resulting compounds were evaluated for their ability to
inhibit Cav2.2 channels using the FLIPR calcium-influx assay
that has been described previously.2 Compounds were screened
for % inhibition of Cav2.2 FLIPR activity at 1 μM
concentration. Compounds showing good activity inhibiting
Cav2.2 channels were titrated to determine their IC50 and were
also counter-screened against the related L-type calcium
channel Cav1.2 using a related FLIPR assay. Potent compounds
were also screened against the hERG K+ channel using a
binding assay that measures the displacement of [35S]MK-499,
a well characterized hERG K+ channel blocker.3 Additionally,
the active Cav2.2 inhibitors were profiled for inhibition of
CYP3A4 and activation of PXR, which may indicate the
likelihood of the compound to induce CYP3A4 expression in
vivo.4 The resulting data are summarized in Table 1.
Compound 1 is a relatively potent PXR activator. Thus,
reducing PXR activity while improving Cav2.2 inhibition was
the focus of our initial SAR optimization. The addition of polar
substituents to specific positions of the molecule (often at the
end region of the molecule) has been shown to reduce PXR
potency in other instances.5 We decided to apply this strategy
to compound 1 by adding more polar functional groups at the
meta or para position of the benzamide phenyl ring while
retaining the methylsulfone group at ortho position. Replacing
the fluoro group in compound 1 with a cyano group (3) had a
minimal impact on Cav2.2 inhibitory potency and resulted in
over 10-fold reduction in PXR potency. The cyano group in the
para position exhibited further reduced PXR activity, with a
slight gain in Cav2.2 potency (4). Interestingly, placing a
lipophilic CF3 group at the para position as in 5 led to a 10-fold
reduction in PXR activity as compared to 1. Compound 5 also
Figure 2. Summary of the center scaffold modifications and SAR
depenence on Cav2.2 potency. IC50 values are averages of n > 3.
the 4-position of the aminopiperidine ring (16) resulted in only
a slight loss of potency as compared to 1. The analogous 3-
aminopyrrolidine (17) exhibited a small improvement in
potency, even as a racemic mixture. Although with azetidine
as the center scaffold compound 18 is less potent in
comparison to its piperidine analogue 1, the addition of the
2-methyl group resulted in a significant increase in potency (19
and 20). In these compounds, the relative stereochemistry has
little effect on potency as both cis- and trans-diastereomers have
similar activity (cis-rac-19 and trans-rac-20). With 2-
azaspiro[4.5]decane as the center scaffold, compound 21 was
3-fold more potent than compound 1 as a Cav2.2 inhibitor.
From this investigation of aminopiperidine replacement
moieties, we concluded that the center substituent of this
lead class provided limited influence on the potency of the
compounds as Cav2.2 inhibitors but rather was acting as a
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scaffold for the presentation of the distal pharmacophores of
the molecules. The compounds in Figure 2 were synthesized
according to the similar procedure used for the synthesis of the
compounds in Table 1. The synthesis of the 4-methylpiperidine
intermediate for the synthesis of 16 is described in the
Supporting Information.
pharmacokinetics studies. Indeed, 25 and 27 gave very high
concentrations of metabolite 2, as illustrated in Figure 4. While
In an earlier report on this lead class,1 we discovered that a
critical issue of compound 1 was formation of the long lasting
sulfonamide metabolite 2 both in vitro and in vivo (Figure 1).
One possible mechanism for formation of metabolite 2 is
through a two-step metabolism sequence involving hydrox-
ylation of the α-carbon of the sulfonamide group, followed by
hydrolysis of hemiamino ketal. Several strategies were pursued
to inhibit the formation of metabolite 2 based on this
hypothesis. These strategies included replacing the piperidine
with more oxidation resisting azetidine,6 making α-carbon
quaternary to prevent hydroxylation, and using the spiro ring
system to provide extra stability. A selection of structurally
diverse compounds that share common 3-CF3-benzene
sulfonamide fragment are illustrated in Figure 3. These
□
△
Figure 4. Blood levels of parent 25 ( ), parent 27 ( ), and
■
▲
metabolite 2 in rats after 3 mg/kg oral dose of 25 ( ) and 27 ( ).
Values are averages for n = 2 rats ( SD).
the normalized AUC for the parent compound 25 in whole
blood was only 0.017 μM·hr·kg/mg after a 3 mg/kg oral dose,
the normalized AUC for metabolite 2 in blood was 64 μM·hr·
kg/mg. Similar results were obtained with 27. The normalized
AUC for the parent and metabolite was 0.022 μM·hr·kg/mg
and 218 μM·hr·kg/mg, respectively, following 3 mg/kg oral
dose of 27. At this point, we were convinced that formation of
the primary sulfonamide metabolite 2 is endemic to the
sulfonamide substituent and that alternative strategies such as
replacing the sulfonamide moiety with a bioisostere would be
required.
We thus shifted our focus to the identification of sulfonamide
replacement bioisosteres, and several of these efforts are
presented in Table 3. Although amide is a common bioisostere
to sulfonamide, replacing sulfonamide in 12 with the
corresponding amide resulted in complete loss of potency
(28). Similarly, replacement of the sulfonamide moiety with
amine, ether, alcohol, and ketone substituents also afforded
compounds lacking significant inhibitory potency for the Cav2.2
channel (data not shown). The sulfone group, however, proved
to be a good replacement for the sulfonamide substituent.
Although sulfone compound 29 was 8-fold less potent than the
analogous sulfonamide 12, the sulfone remained a moderately
potent Cav2.2 inhibitor with an IC50 of 0.36 μM. The addition
of one methyl group to the α-position of the sulfone resulted in
a 4-fold improvement in potency for one enantiomer (30 en-1)
and no change for the other (30 en-2). The gem-dimethyl
analogue 31 retained the potency of the most active
monomethyl enantiomer of 30 and was four times more
potent than the des-methyl analogue 29. The sulfone analogue
bearing the analogous benzamide substitution as compound 1
was synthesized. Compound 32 is equally potent to 1 with
Cav2.2 IC50 of 0.51 μM (Table 3). By comparison of the
sulfonamide analogues (1 and 12) to their corresponding gem-
dimethyl sulfone derivatives (31 and 32), we concluded that
the gem-dimethyl sulfone was an adequate bioisostere for
further investigation. The synthesis of the critical gem-
dimethylsufone piperidine intermediate is described in the
Supporting Information.
Figure 3. Compounds tested for microsomal stability and metabolite 2
formation. Cav2.2 IC50 values are averages of n ≥ 3.
compounds were incubated with human or rat liver microsomes
and analyzed for formation of metabolite 2. To our surprise, for
all the compounds tested in this assay (16 and 22−27), the
formation of metabolite 2 occurred at a greater rate than that of
compound 1 (Table 2).
To determine if metabolite 2 is formed in vivo as well,
compound trans-rac-25 and 27 were evaluated in rat
Table 2. Formation of Metabolite 2 in Microsome
a
Incubation Studies
% of parent remaining
conc of 2 (μM)
compd
rat
40
human
rat
human
1
30
0.01
0.06
0.08
0.08
0.04
0.11
0.18
ND
0.03
0.04
0.95
1.16
0.45
0.83
5.13
ND
b
16
22
23
24
25
26
27
ND
18
ND
26
3.4
72
30
78
4.7
3.8
12
5.3
4.2
8.5
a
Compounds were incubated at 10 μM with 1 mM NADPH and 1
b
Compounds 31 and 32 were further profiled, and the results
are summarized in Table 4. Both compounds maintain excellent
mg/mL rat or human microsomal protein for 1 h. ND: not
determined.
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Table 3. Cav2.2 Inhibitory Potency of Sulfone Compounds
Table 5. Rat PK Profile and Rat CFA Efficacy of 1, 2, 31, and
32
e
rat CFA pain model
a
rat PK profile
% reversal of allodynia
b
c
d
f
compd
AUCN
0.86
CL
F%
dose
1 h
3 h
1
35
99%
10
30
30
10
10
30
43%
66%
53%
21%
36%
57%
38%
57%
44%
30%
49%
54%
2
111
0.97
0.27
0.31
27
46%
95%
24%
31
32
29
a
Dose 3 mg/kg PO, 1 mg/kg IV; PK parameters based on whole
b
blood concentrations. Normalized AUC (PO, μM·h·kg/mg).
c
d
e
Clearance (mL/min/kg). Bioavailability. Average percent reversal
are calculated as (postdose - prdose)/(preinjury predose) for each rat
f
(n = 6). Oral dose in mg/kg.
metabolite 2.1 Sulfone 31 has good rat PK properties with
moderate clearance and half-life (Cl = 27 mL/min/kg; t1/2
=
2.3 h) and excellent bioavailability (F = 95%). Compound 32
has similar moderate clearance (Cl = 29 mL/min/kg) but lower
bioavailability (F = 24%). Compounds 31 and 32 were
evaluated for antinociceptive activity in a rat inflammatory
pain model, and the results are shown alongside the previously
reported results for 1 and the corresponding metabolite 2
(when dosed orally as 2).1 Inflammatory pain was induced by
intraplantar injection of complete Freund’s adjuvant (CFA)
into the hind paws of rats.9 Mechanical allodynia was assessed
at 1 and 3 h post a single oral dose. Although 1 exhibits
significant efficacy in this model, it is impossible to assess the
contribution from the efficacious metabolite 2, which is devoid
of significant Cav2.2 activity and acts via an undetermined
mechanism.1 Compound 31 exhibited modest efficacy (21%
and 30%, respectively) following a 10 mg/kg dose. Compound
32 showed robust activity following a 10 mg/kg oral dose, with
36% and 49% reversal of mechanical allodynia at 1 and 3 h time
points, respectively. This activity increased to 57% and 54%
reversal at 1 and 3 h, respectively, following a 30 mg/kg oral
dose. Therefore, compound 32 achieved similar efficacy to 1 in
the rat CFA model without the confounding contribution of
metabolite 2.
It is interesting that despite the 6-fold greater potency for
compound 31 over 32, and higher oral exposure for 31 in rat
PK studies, 31 was considerably less efficacious in rat CFA
model. Although the full biodistribution for these compounds
into the CSF and spinal cord is not available, we infer that
biodistribution differences may contribute to the discrepancy in
efficacy based on in vitro studies. Sulfone 31 is a more robust
Pgp substrate than 32, which may lead to lower CNS exposure
and thus lower efficacy. In addition, 31 was determined to have
a lower unbound fraction in 100% rat plasma (1.3%) than 32
(6.8%), which may also contribute to lower unbound exposure
of 31 for biodistribution into the CNS.
a
IC50 values and % inhibition values are averages of n ≥ 3.
Table 4. Metabolism Profiles of 31 and 32
a
microsomes %
compd
PXR activation EC50
human
rat
hPgp BA/AB ratio
31
32
>30 μM
2.5 μM
62
64
73
68
4.2
1.8
a
Compounds were incubated at 10 μM with 1 mM NADPH and 1
mg/mL rat or human microsomal protein for 1 h.
selectivity against Cav1.2 and hERG channels (IC50 > 10 μM),
and both are weak CYP3A4 inhibitors with IC50 values of about
10 μM. Although 32 showed significant improvement in PXR
activation over 1, it is still a moderate PXR activator with an
EC50 of 2.5 μM, while 31 is devoid of significant PXR activity. It
is critical that the Cav2.2 channel blockers can access the CNS
and are not substrates for the Pgp efflux mechanism since the
Cav2.2 channel is localized in the synapse of spinal cord
neurons.7 Thus, compounds 31 and 32 were also tested to
determine if they were Pgp substrates. With the same
substitution groups on the benzamide as compound 1, 32
was found not to be a Pgp substrate (human BA/AB ratio:
1.8).8 However, 31 is a Pgp substrate with measured human
BA/AB ratio of 4.2. Both compounds are highly permeable,
with Papp of 35 × 10−6 cm/sec for each compound. Both
compounds exhibit good metabolic stability with percentage of
remaining parent compounds ranging from 62% to 73% after
incubation with human or rat liver microsomes (1 mg/mL
protein and 1 μM substrate) for 1 h.
Although neither 31 nor 32 were suitable to be considered as
a development candidate due to the issues of Pgp substrate and
low in vivo efficacy (for 31) and PXR activity (for 32), these
compounds demonstrated significant metabolic advantage over
sulfonamide series and their potential utility as potent and
selective Cav2.2 inhibitors. With these modifications, we have
demonstrated that the combination of hydrophilic substitutions
such as methylsulfone and lipophilic substitutions of CF3 and
OCF3 on the benzamide phenyl ring resulted in significant
Compounds 31 and 32 were evaluated for their pharmaco-
kinetic and efficacy properties in rats, and the results are shown
in Table 5, along with corresponding data for 1 and the
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(7) Heinke, B.; Balzer, E.; Sandkuhler, J. Pre- and Postsynaptic
Contributions of Voltage-Dependent Ca2+ Channels to Nociceptive
Transmission in Rat Spinal Laminal I Neurons. Eur. J. Neurosci. 2004,
19, 103−111.
(8) Polli, J. W.; Wring, S. A.; Humphreys, J. E.; Huang, L.; Morgan, J.
B.; Webster, L. O.; Serabjit-Singh, C. S. Rational Use of in Vitro P-
Glycoprotein Assays in Drug Discovery. J. Pharmacol. Exp. Ther. 2001,
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(9) Colpaert, F. C. Evidence That Adjuvant Arthritis in the Rat Is
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improvement of Cav2.2 potency, as well as mitigating PXR
activation. In the sulfonamide series, formation of the primary
sulfonamide metabolite 2 in vivo was endemic to the lead class.
This issue was eventually addressed successfully by employing a
bioisostere replacement strategy. The corresponding gem-
dimethyl sulfone compounds exhibited similar Cav2.2 in vitro
potency and efficacy to their sulfonamide analogues, but
without the confounding liability of long lasting and efficacious
sulfonamide metabolite formation. Ongoing work to further
optimize the overall profile and enhance in vivo efficacy in this
new series will be published in the due course.
ASSOCIATED CONTENT
* Supporting Information
■
S
Details for the Cav2.2 FLIPR assay, and the synthesis and
characterization of compounds 12, 31, and 32. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*(P.P.S.) E-mail: pengcheng.shao@merck.com. Phone: (732)
594-2955.
■
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
AUC, area under the pharmacokinetic exposure curve; Cav,
voltage-gated calcium channel; CFA, complete Freund’s
adjuvant; CL, pharmacokinetic clearance; CNS, central nervous
system; conc, concentration; CSF, cerebrospinal fluid; CYP,
cytochrome P450 enzymes; FLIPR, fluorometric imaging plate
reader; hERG, human ether-a-go-go-related gene; IV, intra-
venous; NADPH, nicotinamide adenine dinucleotide phos-
phate hydrogen; Pgp, P-glycoprotein transporter; PK, pharma-
cokinetic; PO, per os or by mouth; PXR, pregnane X receptor;
SAR, structure−activity relationship
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