Development of New Benzenesulfonamides As Potent and Selective Nav1.7 Inhibitors for the Treatment of Pain

Article abstract of DOI:10.1021/acs.jmedchem.6b01918

By taking advantage of certain features in piperidine 4, we developed a novel series of cyclohexylamine- and piperidine-based benzenesulfonamides as potent and selective Na_v1.7 inhibitors. However, compound 24, one of the early analogs, failed to reduce phase 2 flinching in the mouse formalin test even at a dose of 100 mpk PO due to insufficient dorsal root ganglion (DRG) exposure attributed to poor membrane permeability. Two analogs with improved membrane permeability showed much increased DRG concentrations at doses of 30 mpk PO, but, confoundingly, only one of these was effective in the formalin test. More data are needed to understand the disconnect between efficacy and exposure relationships.

Full text of DOI:10.1021/acs.jmedchem.6b01918

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Article
Development of new benzenesulfonamides as potent
v
and selective Na1.7 inhibitors for the treatment of pain
Yong-Jin Wu, Jason Guernon, Jianliang Shi, Jonathan Ditta, Kevin J. Robbins, Ramkumar
Rajamani, Amy Easton, Amy E. Newton, Clotilde Bourin, Kathleen Mosure, Matthew
G. Soars, Ronald J. Knox, Michele Matchett, Rick L. Pieschl, Debra J. Post-Munson,
Shuya Wang, James Herrington, John Graef, Kimberly Newberry, Linda J. Bristow,
Nicholas A. Meanwell, Richard E. Olson, Lorin A. Thompson, and Carolyn D. Dzierba
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01918 • Publication Date (Web): 24 Feb 2017
Downloaded from http://pubs.acs.org on February 26, 2017
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Development of new benzenesulfonamides as potent and selective
Nav1.7 inhibitors for the treatment of pain
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YongꢀJin Wu,* Jason Guernon, Jianliang Shi, Jonathan Ditta, Kevin J. Robbins, Ramkumar Rajamani,
Amy Easton, Amy Newton, Clotilde Bourin, Kathleen Mosure, Matthew G. Soars, Ronald J. Knox,
Michele Matchett, Rick L. Pieschl, Debra J. PostꢀMunson, Shuya Wang, James Herrington, John Graef,
Kimberly Newberry, Linda J. Bristow, Nicholas A. Meanwell, Richard Olson, Lorin A. Thompson, and
Carolyn Dzierba
BristolꢀMyers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06492ꢀ7660,
USA
Supporting Information Placeholder
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ABSTRACT: By taking advantage of certain features in piperidine 4, we developed a novel series of cyꢀ
clohexylamineꢀ and piperidineꢀbased benzenesulfonamides as potent and selective Nav1.7 inhibitors. Howꢀ
ever, compound 24, one of the early analogs, failed to reduce phase 2 flinching in the mouse formalin test
even at a dose of 100 mpk PO due to insufficient DRG exposure attributed to poor membrane permeability.
Two analogs with improved membrane permeability showed much increased DRG concentrations at doses
of 30 mpk PO but, confoundingly, only one of these was effective in the formalin test. More data are needꢀ
ed to understand the disconnect between efficacy and exposure relationships.
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INTRODUCTION
The voltageꢀgated sodium channel Nav1.7, which is encoded by the SCN9A gene, has been shown
to play an important role in painful disorders in humans. For example, gainꢀofꢀfunction mutations in the
SCN9A gene lead to painful conditions such as inherited erythromelalgia and paroxysmal extreme pain
disorder1,2 whereas lossꢀofꢀfunction mutations in the SCN9A gene result in congenital insensitivity to pain,
a rare disorder characterized by a complete inability to sense painful stimuli.3ꢀ5 Apart from anosmia,
humans and mice deficient in Nav1.7 are apparently normal,6,7 suggesting that Nav1.7 inhibitors would not
incur safetyꢀrelated, onꢀtarget toxicity. The Nav1.7 isoform is expressed predominantly in the peripheral
nervous system (PNS) and, therefore, a nonꢀbrain penetrant inhibitor would be expected to have reduced
potential for eliciting central nervous system (CNS)ꢀrelated adverse effects.8 Thus, Nav1.7 is viewed as a
promising analgesic drug target for acute, inflammatory and neuropathic pain.9ꢀ12 As the blocking of the
cardiac sodium channel Nav1.5 may lead to arrhythmia,13 recent efforts have focused on the development of
isoformꢀselective inhibitors. These efforts have led to the discovery of the sulfonamide GNEꢀ131 (1)14 and
benzenesulfonamides 215 and 316 as potent and isoformꢀselective Nav1.7 inhibitors that demonstrate
efficacy in various rodent pain models. These compounds bind to a unique site in the voltage sensor
domain 4 (VSD4) of Nav1.7 that is distinct from the pore binding sites of tetrodotoxin or local anesthetics.17
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The crystal structure of a chimera consisting of human Nav1.7 VSD4 and a bacterial pore domain in
complex with an aryl sulfonamide has been recently disclosed.18 Recently, we embarked on identifying
new leads from within the heteroaryl sulfonamide class of inhibitors that exhibit high levels of potency and
selectivity over the hNav1.5 cardiac channel.19 More specifically, we sought to discover potent Nav1.7
inhibitors that combine favorable pharmacokinetic properties, with oral efficacy in rodent pain models.
This report describes the development of these new benzenesulfonamides.
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Figure 1. Structures of Nav1.7 inhibitors
RESULTS AND DISCUSSION
The inhibitory activity of test compounds towards hNav1.7 and hNav1.5 channels was assessed
using an automated 384ꢀwell IonWorks Barracuda® patchꢀclamp assay designed to measure potency on the
inactivated channel. The Barracuda data generally correlated well with manual patch clamp
electrophysiology results (data not shown). The inhibitory activity of these compounds toward cytochrome
P450 isoforms was also monitored closely since heteroaryl sulfonamides are known to exhibit potent CYP
inhibition, especially for CYP3A4.16,19
In 2014, Sun et al. reported the discovery of piperidine 4 as a potent and selective Nav1.7 isoform
inhibitor (IC50 = 1.1 nM in a PatchXpress assay).19 As the sulfonamide functionality and the thiadiazole
ring that form the acidic element are essential for binding to the protein via a salt bridge to a positively
charged arginine in the domain 4 voltage sensor,17,18 we undertook modification of 4 by relocating the
aryloxymethyl group from C3 to C4 of the piperidine ring to form the 4,4ꢀdisubstituted piperidine 5 (Figure
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2). This compound exhibited an IC50 value of 0.9 µM, significantly less potent than 4 (IC50 = 2 nM in the
Barracuda assay). Nevertheless, we felt that it could serve as a useful new lead based on the reasoning that
the linkage between the benzenesulfonamide and 4ꢀchlorophenyl moieties in 5 might be spatially too close.
Indeed, extension of the linker by just one carbon as in 6 increased channel inhibitory activity by 12ꢀfold
(IC50 = 78 nM). With the spatial orientation between the benzenesulfonamide moiety and 4ꢀchlorophenyl
established, we investigated the impact of the position of the piperidine nitrogen, an effort that led to
identification of the cis (amine vs. the ethylene linker) cyclohexylamine 7 as a potent (IC50 = 8 nM)
hNav1.7 inhibitor with more than 3000ꢀfold selectivity over the hNav1.5 isoform. As the 4ꢀchlorophenyl
analog 7 was analogous to the unsubstituted 4ꢀphenyl counterpart 9 (IC50 = 15 nM) (Table 1), structureꢀ
activity relationship studies focused on the more readily accessible 4ꢀphenyl analogs. Monoalkyl
homologues of the primary amine in 11 (IC50 = 10 nM) with methyl as in 13 (IC50 = 64 nM) and
cyclopropyl as in 15 (IC50 = 105 nM) decreased potency by 6ꢀ and 10ꢀfold, respectively, indicating a tight
pocket in this area of the channel. The 2,5ꢀdifluoroꢀ and 2ꢀfluoroꢀ5ꢀchloro benzenesulfonamides 9 and 11
were equally potent in hNav1.7 activity, but both also suffered from CYP3A4 inhibition with
submicromolar IC50 values. Thus, replacing the 5ꢀfluoro with chloro made little impact on both Nav1.7
activity and CYP3A4 inhibition. The amineꢀlinked Nꢀ(thiazolyl)benzenesulfonamide 17 (IC50 = 9 nM)
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displayed
comparable
hNav1.7
potency
as
the
corresponding
etherꢀlinked
Nꢀ
(thiadiazolyl)benzenesulfonamide 9 (IC50 = 15 nM), but its liability for CYP3A4 inhibition was
significantly ameliorated (IC50 = 6.7 µM for 17 vs. 0.43 µM for 9). As the favorable contribution from the
5ꢀchloro substituent of 17 was likely to be limited, we concluded that the thiazole heterocycle together with
the amine linkage contributed to the favorable CYP3A4 inhibition profile of 17. In an effort to reduce
molecular weight, we replaced the phenyl in 9 with a methyl group, and the resulting analog 19 still
maintained moderate activity (IC50 = 78 nM), thus disproving our long held belief that an aryl or heteroaryl
moiety besides the Nꢀ(heteroaryl)benzenesulfonamide core was essential for Nav1.7 inhibitory activity.20
Next, we eliminated the phenyl ring of the amineꢀlinked Nꢀ(thiazolyl)benzenesulfonamide 17, which was
chosen over the ether linked Nꢀ(thiadiazolyl)benzenesulfonamide 9 because of its reduced potential for
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CYP3A4 inhibition. The resulting cis and trans isomers 21 (IC50 = 8 nM) and 22 (IC50 = 14 nM) were
nearly as potent as the progenitor 17 (IC50 = 9 nM) while preserving the favorable CYP3A4 inhibition
profile of the latter compound. Interestingly, in the 4ꢀdesphenyl benzenesulfonamide series, both two
carbon (as in 21 and 22) and one carbon (as in 23 and 24) linkages resulted in comparable Nav1.7 inhibitory
activity, a trend contrary to that observed with the 4ꢀphenyl piperidineꢀbased chemotype. With respect to
the 4,4ꢀdisubstituted cyclohexylamine series 7ꢀ20, the cis (amine vs. linker) isomers were consistently more
potent than the trans isomers. Within the 4ꢀmonosubstituted series 21, 22, 24, and 25, stereochemical
recognition was dependent on the linker: a methylene linker resulted in comparable activity for both
isomers 21 and 22, while an ethylene linker showed a strong preference for the cis isomer 24 (IC50 = 6 nM)
over the trans isomer 25 (IC50 = 398 nM). In terms of CYP3A4 inhibition, no significant difference was
observed between cis and trans isomers.
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Figure 2. Structures of Nav1.7 inhibitors
Table 1. hNav1.7, hNav1.5 and CYP3A4 inhibition IC50 data of 7-25a
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Compd.
R1
R2
R3
Y
X
n
Config.f
hNav1.7 IC50
(nM)b
hNav1.5 IC50
(µM)c
CYP3A4 IC50
(ꢀM)d
4ꢀClꢀPh
4ꢀClꢀPh
Ph
H
H
F
F
O
O
O
O
O
O
O
O
O
O
N
N
N
N
N
N
N
N
N
N
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1
1
1
1
1
1
1
1
0
0
0
cis
trans
cis
8.0 ± 1.8
388e
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
4.9
0.28
NT
0.43
0.68
0.27
0.58
0.45
NT
0.09
NT
6.7
7
8
H
F
15 ± 8
9
Ph
H
F
trans
cis
473 ± 111
10 ± 1
10
11
12
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17
18
19
20
21
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23
24
25
Ph
H
Cl
Cl
F
Ph
H
trans
cis
458 ± 45
64 ± 26
963e
Ph
Me
Me
cꢀPr
cꢀPr
H
Ph
F
trans
cis
Ph
F
105 ± 93
1820e
Ph
F
trans
cis
Ph
Cl
Cl
F
NH CH
NH CH
9.0 ± 1.2
164 ± 15
78 ± 21
383 ± 114
8.0 ± 2.0
14 ± 7
Ph
H
trans
cis
9.8
Me
Me
H
H
O
O
N
N
0.78
3.0
H
F
trans
cis
H
Cl
Cl
Cl
Cl
Cl
NH CH
NH CH
20
H
H
trans
cis
NT
9.0
H
H
NH
N
16e
2.6
2.3
H
H
NH CH
NH CH
cis
6.0 ± 1.3
398 ± 194
1.9
20
H
H
trans
>30
NT
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aAll values are the mean of at least two separate assay determinations. hNav1.7 data were collected using
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an Ion Works Barracuda® automated electrophysiology platform using a protocol where cells were held at ꢀ
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4
5
6
c
60 mV. hNav1.5 data were collected on the same platform using a protocol where cells were held at ꢀ50
7
8
d
f
mV. For assay conditions, see Ref. 21. eTested only once. Relative stereochemistry between NHR2 and
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(CH2)nY moiety. NT: not tested.
The discovery of cyclohexylamine 24 via phenyl truncation of 17 prompted us to explore the 4ꢀdesꢀ
aryl piperidine benzenesulfonamides (Figure 3). Thus, we prepared 4ꢀmonosubstituted piperidine 26 (IC50
= 42 nM), which was 2ꢀfold more potent than the 4ꢀarylꢀsubstituted etherꢀlinked counterpart 6 (IC50 = 78
nM). Again, compound 26 was 200ꢀfold less potent than 6 in CYP3A4 inhibition as the result of
combining the amine linkage with a thiazole moiety. Both the 3ꢀ and the 2ꢀsubstituted piperidines 27 and
29 showed IC50 values in the 10 nM range, 4ꢀfold more potent than the 4ꢀsubstituted counterpart 26 (IC50 =
42 nM). The 3ꢀsubstituted piperidine 27 (IC50 = 12 nM) and pyrrolidine 31 (IC50 = 14 nM) were 2ꢀfold
more potent than azetidine 32 (IC50 = 29 nM). Most of the analogs in Table 2 were prepared as racemates
and evaluation of the two pairs of enantiomers 33 and 34 or 37 and 38 revealed no stereochemical
preference. With respect to the linkage between the cyclic amine and benzenesulfonamide core, ethylene
as in 27 and 32 was preferred over methylene as in 33, 34 and 40. Interestingly, the directꢀlinked sterically
hindered piperidine 41 (IC50 = 27 nM) was 2ꢀfold more active than the ethylene linked homolog 30 (IC50 =
70 nM). The introduction of an additional oxygen to form a morpholine, as in 35 (IC50 = 69 nM), was
tolerated. Methylation, hydroxylation and acetylation of the piperidine nitrogen as in 36, 37, 38 and 39
brought about a significant decrease in potency. Finally, we examined the impact of attaching a ring
nitrogen directly to the linker, an effort that led to pyrrolidine 42 with an IC50 value of 11 nM.
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Figure 3. Structures of hNav1.7 inhibitors
Table 2. hNav1.7, hNav1.5 CYP3A4 inhibition IC50 data of 26ꢀ42a
Compd.
R
X
hNav1.7 IC50
(nM)b
hNav1.5 IC50
(µM)c
CYP3A4 IC50
(ꢀM)d
CH
CH
N
42 ± 6
12 ± 5
15 ± 6
>30
NT
NT
20
6.7
5.9
26
27
28
CH
8.4 ± 0.5
NT
20
29
CH
CH
70 ± 17
14 ± 3
>30
>30
20
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CH
CH
29 ± 2
>30
>30
20
20
32
33
49 ± 16
5
6
7
8
CH
CH
CH
CH
CH
CH
57e
NT
>30
>30
NT
NT
>30
20
20
34
35
36
37
38
39
69 ± 2
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38
264 ± 74
186 ± 6
207 ± 148
324 ± 177
NT
7.9
17
NT
CH
CH
339 ± 55
27 ± 6
>30
>30
20
20
40
41
CH
11 ± 6
5.1
20
42
a,b,c,d,e see footnotes in Table 1.
As a result of its potent cellular activity in the Barracuda assay, compound 24 was tested in a
manual patch clamp electrophysiology (EP) assay. The results showed that 24 potently inhibited
recombinant Nav1.7 channel activity across species with EP IC50 values of 4.0, 7.5 and 12.3 nM for the
human, mouse and rat Nav1.7 isoforms, respectively, at −70 mV holding potential (partially inactivated
state), which is thought to be representative of pain states.12 However, when the holding potential was set
at −120 mV (resting closed state), the IC50 value at human Nav1.7 was shifted rightwards by >2,000ꢀfold to
9.4 µM (Figure 4) and, as such, 24 was determined to be a stateꢀdependent inhibitor of Nav1.7 function.
Compound 24 was also tested for inhibition of sodium channel currents recorded from cell bodies of
individual sensory neurons dissociated from rat dorsal root ganglia (DRG). In this assay, 24 potently
inhibited fastꢀinactivating, tetrodotoxinꢀsensitive (TTXꢀS) sodium currents measured in adult mouse DRG
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sensory neurons with an IC50 of 6.0 nM. Finally, using the Maestro microelectrode array (MEA) platform,
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4
24 was shown to potently block spontaneous firing of rat embryonic DRG cells (IC50 = 1.2 nM).
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6
100
-70
-120
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0
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0.1
1
10
100
1000
10000
Concentration (nM)
Figure 4. Compound 24 shows stateꢀdependent inhibition of hNav1.7 at ꢀ70 mV (blue, IC50 = 4.0 nM) and ꢀ
120 mV (red, IC50 = 9.4 µM).
Compound 24 was then examined in the formalin test which measures spontaneous, nociceptiveꢀ
like paw flinching/licking responses after peripheral nociceptor activation. This model was chosen for in
vivo efficacy screening since formalinꢀinduced nociceptive behaviors are abolished in global Nav1.7
knockout mice.6 Male CD1 mice were injected subcutaneously in the dorsal surface of the right hind paw
with 20 µL of 5% formalin solution and nociceptive behavior recorded for 45 min using an automated
system (Automated Nociception Analyzer, UCSD, La Jolla, CA, USA). Formalin injection elicits a
biphasic behavioral response; phase I occurs during the first 5 min and results from direct nociceptor
activation whereas phase II occurs 15ꢀ45 min after injection and is thought to involve spinal sensitization.
Treatment with compound 2 (60 mpk, PO) 60 minutes prior to formalin injection had no effect on phase I
responses (data not shown) but abolished phase II nociceptive behaviors (Figure 5). In contrast, no
significant effect on either phase I or phase II responses was seen in mice treated with 24 at 100 mpk PO
(Figure 5). Plasma exposures, measured in a subset (n = 3/group) of experiment subjects on completion of
testing (i.e. ~ 110 minutes post dose), were similar for both compounds (mean ± SD total drug
concentration: compound 2 = 1.2 ± 0.37 µM; compound 24 = 2.8 ± 0.15 µM).
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Figure 5. Effect oral administration of 2 (60 mpk) or 24 (100 mpk) on phase II formalinꢀinduced
nociceptive behaviors. Results are presented as the mean ± SEM response (n=12ꢀ14/group) and were
analyzed by ANOVA followed by Dunnett’s postꢀhoc tests; ***P<0.001 compared to vehicle/formalin
group.
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To further examine tissue drug concentrations we treated male CD1 mice with compound 2 (60 mpk
PO) or 24 (100 mpk PO) and collected plasma and DRG samples 60 minutes later. The Nav1.7 channel is
highly expressed in the DRG and inhibition of channel function in this region may be a primary site of
action for analgesic effect. Consistent with our previous results, the total plasma drug concentrations were
similar for both agents (mean ± SD: compound 2 = 5.3 ± 1.8 µM; compound 24 = 4.0 ± 0.9 µM).
Determination of mouse plasma protein binding, however, showed that the unbound fraction for 24 was 3ꢀ
fold lower than 2 (8% versus 24%; Table 3) resulting in a lower average free plasma drug concentration
(0.32 µM versus 1.27 µM respectively; Table 3). Compound 24 also showed ~ 5 fold lower total drug
concentration in DRG (Table 3). While we did not measure protein binding specifically in mouse DRG
tissue; if the differences observed in plasma protein binding extend to DRG then free DRG concentrations
of 24 may be further reduced compared to compound 2. Compound 24 was also 2ꢀfold weaker at inhibiting
recombinant mouse Nav1.7 currents determined by manual EP (Table 3). Using these results we calculated
the free DRG concentration/mouse Nav1.7 IC50 ratio (using plasma protein binding as an estimate of tissue
binding) and showed that the ratio was >20ꢀfold lower for 24 (ratio = 0.8 versus 21 for 24 and 2
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respectively). Taken together, we concluded that free DRG concentrations of 24 were insufficient to
3
4
achieve the level of Nav1.7 inhibition required for efficacy in the formalin test.
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We speculated that the poor DRG exposure of 24 resulted from its poor membrane permeability as
shown by the PAMPA data (Papp: 0 at both pH 5.5 and 7.4 compared to 8.0 and 142 nm/sec for 2). To test
this hypothesis, we evaluated two analogs 41 and 42 with improved membrane permeability based on
PAMPA data (Table 3) in the formalin model. In this study mice were treated with 30 mpk PO of 41 or 42,
30 minutes prior to formalin injection, and nociceptive behaviors monitored for 45 min as previously
described. On completion of testing (i.e. ~ 75 minutes postꢀtreatment) plasma and DRG samples were
collected from a subset of animals to determine exposure. Both compounds achieved DRG concentrations
of 1.5 µM when dosed at 30 mpk orally in mice, a 19ꢀfold improvement compared to 100 mpk 24 (Table
3). These results are consistent with our hypothesis that improving membrane permeability can enhance
DRG exposure. Intriguingly, compound 42 fully reversed formalinꢀinduced phase II nociceptive behaviors
at the 30 mpk dose whereas 41 was not effective (Figure 6). The explanation for these discrepant results is
unclear given that Nav1.7 potency, mouse plasma protein binding and exposures in plasma and DRG were
similar for both compounds (Table 3). Nevertheless similar discrepancies between PK and efficacy have
also been reported with aminotriazine Nav1.7 inhibitors in the formalin test.22 A direct measure of target
occupancy may be helpful in understanding the inactivity of 41.
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Figure 6. Effect of oral administration of 41 (30 mpk) or 42 (30 mpk) on phase II formalinꢀinduced
nociceptive behaviors. Results are presented as the mean ± SEM response (n=10ꢀ14/group) and were
analyzed by ANOVA followed by Dunnett’s postꢀhoc tests; **P<0.01, ***P<0.001.
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Table 3. Miscellaneous Data for 2, 24, 41 and 42
a
hNav1.7
EP IC50
mNav1.7
EP IC50
Papp pH
Fu
Dose
PO
Plasma
Conc.b
Free Plasma
Conc.
DRG
Active in
Formalin
Conc.b
Compd.
5.5/7.4c
Test
2
2.4 nM
4.0 nM
17 nM
12 nM
3.9 nM
7.5 nM
30 nM
51 nM
8.0/142
0/0
24%
8%
60 mpk
100 mpk
30 mpk
30 mpk
1.27 ± 0.43
0.32 ± 0.07
0.28 ± 0.23
0.24 ± 0.14
Yes
No
5.3 ± 1.8 ꢀM
4.0 ± 0.9 ꢀM
1.1 ± 1.0 ꢀM
1.2 ± 0.7 ꢀM
0.34 ± 0.19 ꢀM
24
41
42
72 ± 33 nM
23/241
47/949
23%
20%
No
1.5 ± 0.4 ꢀM
1.5 ± 0.6 ꢀM
Yes
b
aUnbound fraction in mouse plasma. Compound dosed PO to male mice in 0.5% Methocel/0.1%
Tween/99.4% water (10 mL/kg). Results are presented as the mean (SD), N = 3–9/group. Cnm/sec.
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CHEMISTRY
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Scheme 1 describes a general synthesis of benzenesulfonamides 5, 6 and 21−42 from 44a/b/c,
which were readily available through reductive amination of 2,4ꢀdimethoxybenzaldehyde with 2ꢀ
aminothiazole or 2ꢀaminothiadiazole. Treatment of 44 with benzenesulfonyl chloride 43 under basic
conditions afforded the benzenesulfonamides 45a/b/c.15,19 A nucleophilic aromatic substitution reaction
of an amine or alcohol with 45a/b using K2CO3 as the base gave 46, which underwent deprotection with
TFA to afford final analogs.
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Scheme 1. Reagents and conditions: (a) LHMDS, THF, ꢀ78 °C, 80% (45a), 91% (45b), 55% (45c); (b)
RYH, K2CO3, DMF, rt, 40ꢀ85%; (c) TFA, DCM, rt, 50ꢀ95%.
Scheme 2 describes the synthesis of analogs 7, 8, 19 and 20. Treatment of the electron deficient
2,4,5ꢀtrifluorobenzenesulfonamides 45a/b with 2ꢀ(methylsulfonyl)ethanol in the presence of excessive
potassium tertꢀbutoxide in DMSO or Cs2CO3 in DMF, respectively, brought about a regioselective
nucleophilic aromatic substitution followed by spontaneous elimination of vinyl sulfoxide to give 4ꢀ
hydroxyꢀ2,5ꢀdifluorobenzenesulfonamide 47a/b on a multiple gram scale in good yields.23 Mitsunobu
reaction of alcohols 48 and 49 with phenol 47a gave 50 and 51, respectively, and subsequent
deprotection with trifluoroacetic acid provided compounds 7, 8, 19 and 20.
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Scheme 2. Reagents and conditions: (a) 2ꢀ(methylsulfonyl)ethanol, tꢀBuOK, DMSO, rt, 1 h, 62%; (b) 2ꢀ
(methylsulfonyl)ethanol, Cs2CO3, DMF, rt, 1 h, 70%; (c) 47a, DEAD, Ph3P, THF, rt, 85% (50), 75%
(51); (d) TFA, 7ꢀ51%.
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Scheme 3 describes the synthesis of benzenesulfonamides 9−16. Aldehyde 52 underwent Wittig
reaction with methyl triphenylphosphonium bromide and n-BuLi, and the resulting vinyl compound was
converted to the primary alcohol 53 via hydroboration. Treatment of 53 with 45a and K2CO3 gave 54a,
and Mitsunobu reaction of 53 with phenol 47b (Scheme 2) furnished 54b. Compounds 54a/b
underwent global deprotection with TFA to afford ketones 55a/b, and reductive amination with
appropriate amines generated compounds 9ꢀ16.
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Scheme 3. Reagents and conditions: (a) triphenylphosphonium bromide and n-BuLi, THF, ꢀ78 °C to 0
°C, 54%; (b) BH3·THF, THF, 0 °C; NaOH, H2O2, 65%; (c) LHMDS, 45a/b, K2CO3, DMF, rt, 73%; (d)
DEAD, Ph3P, THF, rt, 30%; (e) 1N HCl, acetone, reflux, 73% (55a), 100% (55b); (f) NH4OAc or
amine, NaBH3CN, methanol, 6ꢀ37%.
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Scheme 4 describes the synthesis of analogs 17 and 18. Alcohol 53 was converted to amine 56
in two steps: Mitsunobu reaction with phthalimide and hydrazinolysis of the resulting phthalimide
moiety. The transformation of amine 56 was to 17 and 18 was analogous to that of 53 to 9ꢀ16 (Scheme
3).
Scheme 4. Reagents and conditions: (a) phthalimide, Ph3P, DEAD, THF, rt; (b) hydrazine,
THF/methanol; (c) 45c, K2CO3, DMF, rt, 12% for 3 steps; (d) HCl, acetone, refluxt, 100%; (e)
NH4OAc, NaBH3CN, rt, 6% (17) and 16% (18).
CONCLUSIONS
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We recognized certain features in 4 and used them to develop new benzenesulfonamides, an
approach that culminated in the discovery of a novel series of cyclohexylamineꢀ and piperidineꢀbased
benzenesulfonamides as potent and selective Nav1.7 inhibitors. One of the early analogs, compound 24,
was ineffective in the mouse formalin test which was attributed to insufficient DRG exposure. We then
identified two analogs, compounds 41 and 42, with much improved DRG exposure; however, only
compound 42 demonstrated efficacy in the formalin model. While the reason for this disconnect is
unclear, a direct measure of occupancy at the VSD4 binding site on Nav1.7 would improve our
understanding of the efficacy/exposure relationship and potentially explain the different results for these
compounds.
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EXPERIMENTAL SECTION
Patch clamp electrophysiology. Nav1.7 currents were recorded using wholeꢀcell patch clamp
of HEK293 cells stably expressing recombinant human or mouse Nav1.7. Glass microelectrodes filled
with internal solution containing (in mM) 50 CsCl, 90 CsF, 10 NaF, 2 MgCl2, 10 EGTA, 10 HEPES, pH
7.2 with CsOH had resistances of 1ꢀ4 MOhms. The external solution was (in mM): 150 NaCl, 4 KCl, 1
MgCl2, 1.8 CaCl2, 10 HEPES, 10 glucose, pH 7.4 with NaOH. Series resistance was kept below 10
MOhms and electronically compensated at least 50%. Currents were acquired at 50 kHz via an EPCꢀ10
amplifier with Patchmaster software (HEKA Instruments Inc., Bellmore, NY). Leakage subtraction was
performed with the P/N method. Solution exchange was performed with a 16ꢀwell Dynaflow Resolve
glass chip (Cellectricon, Sweden). The holding potential was ꢀ70 mV unless otherwise indicated. Folꢀ
lowing a 100 msec prepulse to ꢀ130 mV, Nav1.7 current was elicited by a 10 msec step to +10 mV.
Peak inward current was measured with PatchMaster software and percent inhibition calculated. IC50
values were obtained by fits of the Hill equation to the average percent inhibition data. Data are preꢀ
sented as mean ± S.E.M.
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Automated Electrophysiology Methods: Ion Works Barracuda population patch clamp
(PPC). Population patch clamp (PPC) measurements from HEK 293 cells expressing Nav1.7 and
Nav1.5 channels were performed using an IonWorks Barracuda instrument (Molecular Devices
Corporation, Union City, CA) using a proprietary PPC PatchPlate™ substrate (Molecular Devices
Corporation) with 64 patch clamp apertures per well. The ability to average Nav1.x currents from 64
recordings from each well greatly improves data consistency and recording success rates in the
measurement of Nav1.x mediated ionic currents. Calculated leak current was digitally subtracted from
the total cell Nav1.x current for each sample data point acquired.
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Nav1.7 currents were elicited by a voltage clamp protocol designed to strongly bias the Nav1.7
channels toward their inactivated state as follows. From a holding potential of –60 mV cells were briefly
hyperpolarized to ꢀ100 mV for 1.25 sec, then stepped to ꢀ20 mV for 20 sec to inactivate the channels.
This was followed by a relatively brief hyperpolarization to ꢀ100 mV for 300 ms, then a 20 msec test
pulse to ꢀ20 mV to elicit the Nav1.7 current used to measure the pharmacology of all test compounds.
Compounds were incubated for 600 sec between the preꢀ and postꢀcompound reads. The external reꢀ
cording solution used was (in mM) 137 NaCl, 4 KCl, 1 MgCl2, 1.8 CaCl2, 10 Hepes, 10 glucose, pH to
7.4 with NaOH, and the internal solution used was (in mM) 100 Kꢀgluconate, 40KCl, 3.2 MgCl2, 5
EGTA, 10 HEPES pH to 7.2 with KOH. The same solutions were used to record Nav1.5 currents, with
the following voltage clamp protocol. Nav1.5 currents were elicited by a voltage clamp protocol deꢀ
signed to bias the Nav1.5 channels to their inactivated state as follows. From holding potential of –40
mV cells were briefly hyperpolarized to ꢀ100 mV for 300 ms, then stepped to ꢀ10 mV for 20 sec to inacꢀ
tivate the channels. This was followed by a relatively brief hyperpolarization to ꢀ100 mV for 30 ms,
then a 20 msec test pulse to ꢀ10 mV to elicit the Nav1.5 current used to measure the pharmacology of all
test compounds.
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HEK 293 cells expressing Nav1.7 and Nav1.5 channels, were used (Essen Biosciences, Ann Arꢀ
bor, Michigan). Cells were cultured in Tꢀ175 flasks and passaged every 2 to 3 days at 1:3 to 1:6 seeding
density dilutions. Cells were grown to 70% to 90% confluence in a flask and removed from the incubaꢀ
tor (37° C, 5% CO2) 1 to 3 days after plating. Growth medium was aspirated from the culture flasks.
Cells were gently rinsed with 10 mL of Phosphate buffered saline (PBS) to remove residual media. Next
a total of 2 mL TrypLE (Gibco) solution was added, and the flasks containing cells sat for 3 min at rt,
after which, the cells became visibly rounded and were easily dislodged from the bottom of the flask
with a few brief taps on a solid surface. A total of 8 mL of media was added to the flask to inactivate the
TrypLE, and the mixture was centrifuged at 910 rpm for 4 min. The cell supernatant was decanted, and
the cell pellets were resuspended in 5ꢀ6 mL of external solution followed by gentle trituration using a 10
mL pipette, and transferred to a 15 mL conical tube and immediately brought to the IW Barracuda inꢀ
strument. The cell suspension had a final concentration of ∼2 to 3 million cells per mL; this corresponds
to 10,000 cells added per well.
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Peak membrane currents were analyzed with IW Barracuda software and exported to Excel for
further analysis. Concentration response curve fitting was performed with BMS inꢀhouse software. IC50
values were obtained by fits of the Hill equation to the average percent inhibition data plotted versus
compound concentration. Concentrationꢀresponse curves for all test compounds were fitted to a 4ꢀ
parameter equation: % of control = 100 (1 + ([drug]/IC50)p)–1, where IC50 is the concentration of drug
required to inhibit current by 50% and p is the Hill slope.
Formalin test. Persistent pain was induced with an injection of formalin in the dorsum of one
hind paw as previously described.24 Formalinꢀinduced flinching was quantified with an automated sysꢀ
tem (Automated Nociception Analyzer, UCSD, La Jolla, CA, USA). A metal band was glued on the
right hind paw, the mouse was then acclimated for 60 min in an individual Plexiglas test chamber. Each
mouse was dosed (10 mL/kg PO) orally with the compound or vehicle, 30 min before formalin injecꢀ
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tion. The right hind paw was injected with 20 µL of 5% formalin (SC of formalin w/v in saline, Sigma),
and the animal was immediately placed back into the chamber. Each time the band breaks the electroꢀ
magnetic field located under the mouse a signal was generated and nociceptive behavior, paw flinches
and licking, were recorded with the automated system for 45 minutes.
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Chemistry. Proton magnetic resonance (1H NMR) spectra were recorded on either a Bruker
Avance 400 or a JEOL Eclipse 500 spectrometer and are reported in ppm relative to the reference
solvent of the sample in which they were run. HPLC and LC−MS analyses were conducted using a
Shimadzu SCLꢀ10A liquid chromatograph and a SPD UV−vis detector at 220 nm with the MS detection
performed with either a Micromass Platform LC spectrometer or a Waters Micromass ZQ spectrometer.
All flash column chromatography was performed on EM Science silica gel 60 (particle size of 40−60
ꢁm). All reagents were purchased from commercial sources and used without further purification unless
otherwise noted. All reactions were performed under an inert atmosphere. HPLC analyses were
performed using the following conditions. All final compounds had an HPLC purity of ≥ 95% unless
specifically mentioned.
HPLC Methods. Analytical HPLC analyses were carried out following methods A and B, and
preparatory reverseꢀphase scale purifications were performed using methods C and D.
Method A. A linear gradient using 5% acetonitrile, 95% water, and 0.05% TFA (solvent A) and
95% acetonitrile, 5% water, and 0.05% TFA (solvent B) (t = 0 min, 10% B; t = 15 min, 100% B) was
employed on a SunFire C18 3.5 ꢁm 3.5 mm × 150 mm column. Flow rate was 0.5 mL/min, and UV
detection was set to 220 nm. The LC column was maintained at ambient temperature.
Method B. A linear gradient using 5% acetonitrile, 95% water, and 0.05% TFA (solvent A) and
95% acetonitrile, 5% water, and 0.05% TFA (solvent B) (t = 0 min, 10% B; t = 15 min, 100% B (20
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min)) was employed on a XBridge Ph 3.5 ꢁm 3.0 mm × 150 mm column. Flow rate was 0.5 mL/min,
and UV detection was set to 220 nm. The LC column was maintained at ambient temperature.
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Method C. Column: Waters XBridge C18, 19 x 200 mm, 5 ꢁm particles; Guard Column:
Waters XBridge C18, 19 x 10 mm, 5 ꢁm particles; Mobile Phase A: water with 20 mM ammonium
acetate; Mobile Phase B: 95:5 methanol:water with 20 mM ammonium acetate; Gradient: 25ꢀ65% B
over 40 minutes, then a 5ꢀminute hold at 100% B; Flow: 20 mL/min.
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4-((4-(4-Chlorophenyl)piperidin-4-yl)methoxy)-2,5-difluoro-N-(1,2,4-thiadiazol-5-
yl)benzenesulfonamide
(5).
To
a
solution
of
tertꢀbutyl
4ꢀ(4ꢀchlorophenyl)ꢀ4ꢀ
(hydroxymethyl)piperidineꢀ1ꢀcarboxylate25 (35 mg, 0.11 mmol) in THF (0.54 mL) at rt was added
LHMDS in THF (0.12 mL, 0.12 mmol), and the reaction mixture was stirred at rt for 30 min. A soluꢀ
tion of 45a (48 mg, 0.11 mmol) in THF (0.54 mL) was added and the reaction mixture was stirred at rt
for 1 h. Water was added and the aqueous layer was extracted with EtOAc (x3). The combined organic
layers were washed with brine, dried over anhydrous sodium sulfate, and filtered, and the filtrate was
evaporated to give the crude product. This crude product was dissolved in DCM (0.54 mL), TFA (0.49
mL, 6.4 mmol) was added, and the reaction mixture was stirred at rt for 1 h. The solvents were reꢀ
moved, and the product was purified via preparative HPLC (Method C) to give 5 (15 mg, 56% over 2
1
steps). H NMR (500 MHz, DMSOꢀd6) δ 7.95 (s, 1H), 7.87 (s, 1H), 7.55 ꢀ 7.40 (m, 5H), 7.18 ꢀ 7.08 (m,
1H), 4.10 (s, 2H), 3.23 (br d, J = 12.8 Hz, 2H), 2.81 (br t, J = 10.5 Hz, 2H), 2.33 (br d, J = 15.4 Hz,
2H), 2.13 (br t, J = 11.0 Hz, 2H). LC/MS m/z: (M + H)+ calcd for C20H20ClF2N4O3S2, 501.06; found
501.2.
4-(2-(4-(4-Chlorophenyl)piperidin-4-yl)ethoxy)-2,5-difluoro-N-(1,2,4-thiadiazol-5-
yl)benzenesulfonamide (6). Prepared from tertꢀbutyl 4ꢀ(4ꢀchlorophenyl)ꢀ4ꢀ(2ꢀhydroxyethyl)piperidineꢀ
1
1ꢀcarboxylate26 in 72% yield using the procedures described for 5. H NMR (500 MHz, DMSOꢀd6) δ
7.88 (s, 1H), 7.48 ꢀ 7.34 (m, 5H), 6.97 (dd, J = 11.0, 7.0 Hz, 1H), 3.79 (t, J = 6.2 Hz, 2H), 3.22 (br. s.,
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2H), 2.90 ꢀ 2.77 (m, 2H), 2.25 (br. s., 2H), 2.15 ꢀ 1.97 (m, 4H). LC/MS m/z: (M + H)+ calcd for
C21H22ClF2N4O3S2, 515.08; found 515.2.
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4-(2-((1R,4R)-4-Amino-1-(4-chlorophenyl)cyclohexyl)ethoxy)-2,5-difluoro-N-(1,2,4-
8
9
thiadiazol-5-yl)benzenesulfonamide
(7)
and
4-(2-((1S,4S)-4-amino-1-(4-
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chlorophenyl)cyclohexyl)ethoxy)-2,5-difluoro-N-(1,2,4-thiadiazol-5-yl)benzenesulfonamide (8). To
a solution of 50 (45 mg, 0.06 mmol) in DCM (0.5 mL) was added TFA (0.09 mL, 1.2 mmol). The mixꢀ
ture was stirred at rt for 3 h and then concentrated. The residue was purified by preparative HPLC
(Method C) to give 8 (2 mg, 7%) and 7 (16 mg, 51%). Compound 8: 1H NMR (500 MHz, DMSOꢀd6) δ
7.87 (s, 1H), 7.47 ꢀ 7.37 (m, 5H), 6.93 (dd, J = 11.4, 6.6 Hz, 1H), 3.80 (t, J = 6.8 Hz, 2H), 2.85 ꢀ 2.72
(m, 1H), 2.45 ꢀ 2.30 (m, 2H), 1.87 (2H, m), 1.70 ꢀ 1.46 (m, 5H). LC/MS m/z: (M + H)+ calcd for
C22H24ClF2N4O3S2, 529.09; found 529.1. Compound 7: 1H NMR (500 MHz, DMSOꢀd6) δ 7.96 (s, 1H),
7.87 (s, 1H), 7.50 ꢀ 7.31 (m, 5H), 6.99 (dd, J = 11.0, 7.0 Hz, 1H), 3.75 (t, J = 6.6 Hz, 2H), 3.01 (br. s.,
1H), 2.22 ꢀ 2.07 (m, 4H), 1.84 ꢀ 1.53 (m, 6H). LC/MS m/z: (M + H)+ calcd for C22H24ClF2N4O3S2,
529.09; found 529.1.
4-(2-((1R,4R)-4-Amino-1-phenylcyclohexyl)ethoxy)-2,5-difluoro-N-(1,2,4-thiadiazol-5-
yl)benzenesulfonamide (9) and 4-(2-((1S,4S)-4-amino-1-phenylcyclohexyl)ethoxy)-2,5-difluoro-N-
(1,2,4-thiadiazol-5-yl)benzenesulfonamide (10). A mixture of 55a (100 mg, 0.20 mmol), NH4OAc
(156 mg, 2.0 mmol), NaBH3CN (19 mg, 0.30 mmol) and 4Å MS (200 mg) in methanol (1.4 mL) was
stirred at rt for 2 h. The crude material was purified via preparative HPLC (Method C) to give 9 (6 mg,
6%) and 10 (20 mg, 20%) and Compound 9: 1H NMR (500 MHz, DMSOꢀd6) δ 7.88 (s, 1H), 7.47 ꢀ 7.37
(m, 3H), 7.34 (t, J = 7.5 Hz, 2H), 7.24 ꢀ 7.18 (m, 1H), 6.94 (dd, J = 10.6, 6.6 Hz, 1H), 3.75 (t, J = 6.6
Hz, 2H), 3.06 (br. s., 1H), 2.17 (br. s., 4H), 1.86 ꢀ 1.57 (m, 6H). LC/MS m/z: (M + H)+ calcd for
1
C22H25F2N4O3S2, 495.13; found 495.2. Compound 10: H NMR (500 MHz, DMSOꢀd6) δ 7.96 (s, 1H),
7.87 (s, 1H), 7.49 ꢀ 7.29 (m, 5H), 7.22 (t, J = 7.0 Hz, 1H), 6.88 (dd, J = 11.2, 6.8 Hz, 1H), 3.79 (t, J =
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6.8 Hz, 2H), 3.36 (br. s., 2H), 3.11 ꢀ 3.01 (m, 1H), 2.57 ꢀ 2.52 (m, 2H), 1.88 (t, J = 6.4 Hz, 2H), 1.76 (d,
J = 11.7 Hz, 2H), 1.58 (t, J = 13.4 Hz, 2H), 1.30 ꢀ 1.14 (m, 2H). LC/MS m/z: (M + H)+ calcd for
C22H25F2N4O3S2, 495.13; found 495.2.
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4-(2-((1R,4R)-4-Amino-1-phenylcyclohexyl)ethoxy)-5-chloro-2-fluoro-N-(1,2,4-thiadiazol-5-
yl)benzenesulfonamide (11) and 4-(2-((1S,4S)-4-Amino-1-phenylcyclohexyl)ethoxy)-5-chloro-2-
fluoro-N-(1,2,4-thiadiazol-5-yl)benzenesulfonamide (12). A mixture of 55b (60 mg, 0.12 mmol),
NH4OAc (91 mg, 1.176 mmol), NaBH3CN (11 mg, 0.18 mmol) and 4Å MS (100 mg) in methanol (0.78
mL) was stirred at rt for 12 h. This reaction mixture was diluted with methanol and then filtered, and
the filtrate was purified by preparative HPLC (Method C) to give 11 (4 mg, 6%) and 12 (12 mg, 20%).
Compound 11: 1H NMR (500 MHz, DMSOꢀd6) δ 7.88 (s, 1H), 7.66 (s, 1H), 7.64 (s, 1H), 7.43 ꢀ 7.38 (m,
2H), 7.34 (t, J = 7.7 Hz, 2H), 7.25 ꢀ 7.19 (m, 1H), 6.92 (d, J = 11.0 Hz, 1H), 3.52 ꢀ 3.38 (m, 2H), 3.04
(br. s., 1H), 2.29 ꢀ 2.14 (m, 4H), 1.85 ꢀ 1.50 (m, 6H).LC/MS m/z: (M + H)+ calcd for C22H25ClFN4O3S2,
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511.10; found 511.2. MS: 511.2 (M + H)+. Compound 12: H NMR (500 MHz, DMSOꢀd6) δ 7.88 (s,
1H), 7.64 (d, J = 7.3 Hz, 1H), 7.47 ꢀ 7.34 (m, 5H), 7.22 (s, 1H), 6.86 (d, J = 11.0 Hz, 1H), 3.79 (br t, J
= 6.8 Hz, 2H), 3.05 (br s, 1H), 1.93 ꢀ 1.84 (m, 3H), 1.76 (br d, J = 9.9 Hz, 2H), 1.60 (br t, J = 12.7 Hz,
2H), 1.22 (br d, J = 12.8 Hz, 2H). LC/MS m/z: (M + H)+ calcd for C22H25ClFN4O3S2, 511.10; found
511.2.
2,5-Difluoro-4-(2-((1R,4R)-4-(methylamino)-1-phenylcyclohexyl)ethoxy)-N-(1,2,4-
thiadiazol-5-yl)benzenesulfonamide (13) and 2,5-Difluoro-4-(2-((1S,4S)-4-(methylamino)-1-
phenylcyclohexyl)ethoxy)-N-(1,2,4-thiadiazol-5-yl)benzenesulfonamide (14). A solution of methylꢀ
amine (122 µL, 0.12 mmol) (1M in THF) and 55a (30 mg, 0.06 mmol) in methanol (304 µl) was heated
at 65 °C for 1 h. After cooling, sodium borohydride (4.6 mg, 0.12 mmol) was added. The reaction mixꢀ
ture was stirred at rt for 2 h. The crude material was purified via preparative HPLC (Method C) to give
13 (7 mg, 24%) and 14 (9 mg, 28%). 1H NMR (500 MHz, DMSOꢀd6) δ 7.87 (s, 1H), 7.46 ꢀ 7.30 (m, 5H),
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7.22 (t, J = 7.2 Hz, 1H), 6.94 (dd, J = 11.2, 6.8 Hz, 1H), 3.74 (t, J = 6.8 Hz, 2H), 3.55 (br. s., 3H), 2.93
(br. s., 1H), 2.25 ꢀ 2.08 (m, 4H), 1.90 ꢀ 1.82 (m, 2H), 1.74 ꢀ 1.55 (m, 4H). LC/MS m/z: (M + H)+ calcd
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for C23H27F2N4O3S2, 509.15; found 509.1. Compound 14: H NMR (500 MHz, DMSOꢀd6) δ 7.87 (s,
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1H), 7.47 ꢀ 7.34 (m, 6H), 7.22 (br t, J = 7.2 Hz, 1H), 6.88 (dd, J = 11.0, 7.0 Hz, 1H), 3.79 (br t, J = 6.4
Hz, 2H), 3.06 ꢀ 2.94 (m, 1H), 2.56 (br d, J = 12.8 Hz, 2H), 1.93 ꢀ 1.85 (m, 4H), 1.55 (br t, J = 12.7 Hz,
2H), 1.30 ꢀ 1.09 (m, 3H). 13C NMR (126 MHz, DMSOꢀd6) δ 185.8, 162.8, 159.4, 156.0, 154.0, 149.4 (t,
J = 11.0 Hz, 1C), 147.5, 145.5, 143.2, 129.2, 127.4, 126.5, 123.8 (dd, J = 19.1, 3.8 Hz, 1C), 115.8 (br
dd, J = 22.4, 2.4 Hz, 1C), 104.1, 103.9, 66.7, 57.6, 44.0, 40.9, 40.6, 36.3, 33.4, 31.3, 30.2, 25.2. LC/MS
m/z: (M + H)+ calcd for C23H27F2N4O3S2, 509.15; found 509.1.
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4-(2-((1R,4R)-4-(Cyclopropylamino)-1-phenylcyclohexyl)ethoxy)-2,5-difluoro-N-(1,2,4-
thiadiazol-5-yl)benzenesulfonamide
(15)
and
4-(2-((1S,4S)-4-(cyclopropylamino)-1-
phenylcyclohexyl)ethoxy)-2,5-difluoro-N-(1,2,4-thiadiazol-5-yl)benzenesulfonamide (16). A soluꢀ
tion of cyclopropylamine (8 µL, 0.12 mmol) (1M in THF) and 55a (30 mg, 0.06 mmol) in ethanol (0.3
mL) was heated at 65 °C for 2 h. After cooling, sodium borohydride (5 mg, 0.12 mmol) was added, and
the reaction mixture was stirred at rt for 2 h. The crude material was purified via preparative HPLC
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(Method C) to give 15 (4 mg, 13%) and 16 (12 mg, 37%). H NMR (500 MHz, DMSOꢀd6) δ 7.96 (s,
1H), 7.87 (s, 1H), 7.46 ꢀ 7.30 (m, 5H), 7.22 (t, J = 7.5 Hz, 1H), 7.00 ꢀ 6.88 (m, 1H), 3.75 (br. s., 2H),
3.39 ꢀ 3.27 (m, 2H), 3.11 (br. s., 1H), 2.66 (br. s., 1H), 2.26 ꢀ 2.10 (m, 4H), 1.95 (br. s., 2H), 1.77 ꢀ 1.53
(m, 4H), 0.73 (d, J = 9.5 Hz, 4H). LC/MS m/z: (M + H)+ calcd for C25H29F2N4O3S2, 535.16; found
535.0. Compound 16: 1H NMR (500 MHz, DMSOꢀd6) δ 7.96 (s, 1H), 7.87 (s, 1H), 7.45 ꢀ 7.39 (m, 3H),
7.34 (br t, J = 7.3 Hz, 2H), 7.22 (br t, J = 7.5 Hz, 1H), 6.99 ꢀ 6.88 (m, 1H), 3.81 ꢀ 3.73 (m, 2H), 3.41 ꢀ
3.25 (m, 1H), 3.11 (br s, 1H), 2.66 (br s, 1H), 2.24 ꢀ 2.13 (m, 4H), 2.01 ꢀ 1.90 (m, 2H), 1.79 ꢀ 1.52 (m,
4H), 0.73 (br d, J = 9.5 Hz, 4H). 13C NMR (126 MHz, DMSOꢀd6) δ 185.4, 172.1, 162.3, 158.9, 155.5,
153.6, 149.0 (t, J = 11.4 Hz, 1C), 147.0, 145.1, 145.0, 143.6, 128.6, 127.0, 125.8, 123.2 (dd, J= 19.1,
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4.8 Hz, 1C), 115.3 (br dd, J = 21.9, 2.9 Hz, 1C), 103.6, 103.4, 66.3, 57.1, 40.4, 40.3, 35.8, 33.5, 30.8,
27.7, 27.6, 21.1, 5.0. LC/MS m/z: (M + H)+ calcd for C25H29F2N4O3S2, 535.16; found 535.0.
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4-(2-((1R,4R)-4-Amino-1-phenylcyclohexyl)ethoxy)-5-chloro-2-fluoro-N-(thiazol-2-
yl)benzenesulfonamide (17) and 4-(2-((1S,4S)-4-amino-1-phenylcyclohexyl)ethoxy)-5-chloro-2-
fluoro-N-(thiazol-2-yl)benzenesulfonamide (18). A mixture of 58 (50 mg, 0.10 mmol), NH4OAc (76
mg, 0.98 mmol), NaBH3CN (12 mg, 0.20 mmol) and 4Å MS (100 mg) in methanol (0.7 mL) was stirred
at rt for 2 h. This reaction mixture was diluted with methanol and then filtered. The crude material was
purified via preparative HPLC (Method C) to give 18 (8 mg, 16%) and 17 (3 mg, 6%). Compound 17:
1H NMR (500 MHz, DMSOꢀd6) δ 7.51 (d, J = 7.3 Hz, 1H), 7.44 ꢀ 7.35 (m, 4H), 7.29 ꢀ 7.21 (m, 1H),
7.02 (d, J = 4.0 Hz, 1H), 6.55 (d, J = 3.7 Hz, 1H), 6.03 ꢀ 5.96 (m, 2H), 3.05 (br. s., 1H), 2.74 (d, J = 4.0
Hz, 2H), 2.13 (br. s., 2H), 1.94 ꢀ 1.75 (m, 4H), 1.72 ꢀ 1.58 (m, 4H). LC/MS m/z: (M + H)+ calcd for
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C23H27ClFN4O2S2, 509.12; found 509.2. Compound 18: H NMR (500 MHz, DMSOꢀd6) δ 7.51 ꢀ 7.38
(m, 1H), 7.27 (t, J = 7.0 Hz, 1H), 6.93 (d, J = 4.0 Hz, 1H), 6.45 (d, J = 3.7 Hz, 1H), 5.81 ꢀ 5.75 (m, 1H),
5.70 (d, J = 12.5 Hz, 1H), 3.06 (br. s., 1H), 2.87 ꢀ 2.78 (m, 2H), 1.77 (d, J = 10.6 Hz, 2H), 1.67 ꢀ 1.59
(m, 2H), 1.51 (t, J = 12.5 Hz, 2H), 1.22 (d, J = 12.8 Hz, 2H). LC/MS m/z: (M + H)+ calcd for
C23H27ClFN4O2S2, 509.12; found 509.2.
4-(2-((1S,4S)-4-Amino-1-methylcyclohexyl)ethoxy)-2,5-difluoro-N-(1,2,4-thiadiazol-5-
yl)benzenesulfonamide (19) and 4-(2-((1R,4R)-4-amino-1-methylcyclohexyl)ethoxy)-2,5-difluoro-
N-(1,2,4-thiadiazol-5-yl)benzenesulfonamide (20). A solution of 51 (50 mg, 0.07 mmol) TFA (0.11
mL, 1.5 mmol) in DCM (0.5 mL) was stirred at rt for 3 h. DCM was removed, and the crude material
was purified via preparative HPLC (Method C) to give 19 (11 mg, 35%) and 20 (3 mg, 9%). Compound
19: 1H NMR (500 MHz, DMSOꢀd6) δ 7.88 (s, 1H), 7.51 ꢀ 7.44 (m, 1H), 7.24 (dd, J = 11.2, 7.2 Hz, 1H),
4.14 (t, J = 7.0 Hz, 2H), 2.95 (br. s., 1H), 1.82 ꢀ 1.65 (m, 4H), 1.62 ꢀ 1.43 (m, 4H), 1.27 ꢀ 1.09 (m, 2H).
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LC/MS m/z: (M + H)+ calcd for C17H23F2N4O3S2, 433.12; found 433.0. Compound 20: H NMR (500
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MHz, DMSOꢀd6) δ 7.88 (s, 1H), 7.45 (dd, J = 10.8, 6.4 Hz, 1H), 7.21 (dd, J = 10.8, 6.8 Hz, 1H), 4.15 (t,
J = 6.8 Hz, 2H), 2.9 (br s, 1H), 1.76 ꢀ 1.61 (m, 4H), 1.55 ꢀ 1.39 (m, 4H), 1.31 (d, J = 13.9 Hz, 2H).
LC/MS m/z: (M + H)+ calcd for C17H23F2N4O3S2, 433.12; found 433.0.
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4-((2-((1S,4S)-4-Aminocyclohexyl)ethyl)amino)-5-chloro-2-fluoro-N-(thiazol-2-
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yl)benzenesulfonamide (21). Prepared using the procedures described for 26. H NMR (500 MHz,
DMSOꢀd6) δ 7.55 (d, J = 7.3 Hz, 1H), 6.97 (d, J = 3.7 Hz, 1H), 6.50 (s, 1H), 6.48 ꢀ 6.43 (m, 1H), 5.98
(br s, 1H), 3.20 ꢀ 3.11 (m, 2H), 1.71 ꢀ 1.37 (m, 13H). LC/MS m/z: (M + H)+ calcd for
C17H23ClFN4O2S2, 433.09.; found 433.0.
4-((2-((1R,4R)-4-Aminocyclohexyl)ethyl)amino)-5-chloro-2-fluoro-N-(thiazol-2-
1
yl)benzenesulfonamide (22). Prepared using the procedures described for 26. H NMR (500 MHz,
DMSOꢀd6) δ 7.54 (d, J = 7.0 Hz, 1H), 6.96 (d, J = 3.7 Hz, 1H), 6.49 (d, J = 3.5 Hz, 1H), 6.48 ꢀ 6.41
(m, 1H), 5.89 (br s, 1H), 3.18 ꢀ 3.12 (m, 2H), 2.95 ꢀ 2.88 (m, 1H), 1.93 ꢀ 1.87 (m, 3H), 1.86 ꢀ 1.76 (m,
2H), 1.42 (q, J = 6.8 Hz, 2H), 1.31 ꢀ 1.20 (m, 3H), 0.97 (q, J = 11.4 Hz, 2H). LC/MS m/z: (M + H)+
calcd for C17H23ClFN4O2S2, 433.09; found 433.0.
4-((((1S,4S)-4-Aminocyclohexyl)methyl)amino)-5-chloro-2-fluoro-N-(1,2,4-thiadiazol-5-
yl)benzenesulfonamide (23). Prepared using the procedures described for 26. 1H NMR (500 MHz,
DMSOꢀd6) δ 7.88 (s, 1H), 7.71 (br s, 2H), 7.52 (d, J = 7.3 Hz, 1H), 6.51 (d, J = 12.8 Hz, 1H), 6.20 (br t,
J = 5.5 Hz, 1H), 3.20 (br s, 1H), 3.09 (t, J = 6.6 Hz, 2H), 1.76 (br s, 1H), 1.63 (q, J = 5.9 Hz, 4H), 1.55
ꢀ 1.41 (m, 4H). LC/MS m/z: (M + H)+ calcd for C15H20ClFN5O2S2, 420.07; found 420.0.
4-((((1S,4S)-4-Aminocyclohexyl)methyl)amino)-5-chloro-2-fluoro-N-(thiazol-2-
yl)benzenesulfonamide (24). Prepared using the procedures described for 26. 1H NMR (500 MHz,
DMSOꢀd6) δ 7.54 (d, J = 7.3 Hz, 1H), 6.94 (d, J = 3.8 Hz, 1H), 6.50 ꢀ 6.41 (m, 2H), 6.03 (br t, J = 5.3
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Hz, 1H), 3.17 ꢀ 3.12 (m, 1H), 3.11 ꢀ 3.02 (m, 2H), 1.75 (br d, J = 6.0 Hz, 1H), 1.65 ꢀ 1.56 (m, 4H), 1.56
ꢀ 1.39 (m, 4H). LC/MS m/z: (M + H)+ calcd for C16H21ClFN4O2S2, 419.08; found 419.1.
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yl)benzenesulfonamide (25). Prepared using the procedures described for 26. H NMR (500 MHz,
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DMSOꢀd6) δ 7.54 (d, J = 7.3 Hz, 1H), 6.97 (d, J = 3.7 Hz, 1H), 6.60 ꢀ 6.44 (m, 2H), 6.03 (br. s., 1H),
3.18 (s, 1H), 3.01 (t, J = 6.4 Hz, 2H), 2.92 (t, J = 11.9 Hz, 1H), 1.98 ꢀ 1.88 (m, 2H), 1.78 (d, J = 12.5
Hz, 2H), 1.53 (br. s., 1H), 1.31 ꢀ 1.17 (m, 2H), 1.10 ꢀ 0.83 (m, 2H). LC/MS m/z: (M + H)+ calcd for
C16H21ClFN4O2S2, 419.08; found 419.1.
5-Chloro-2-fluoro-4-((2-(piperidin-4-yl)ethyl)amino)-N-(thiazol-2-yl)benzenesulfonamide
(26). To a mixture of tertꢀbutyl 4ꢀ(2ꢀaminoethyl)piperidineꢀ1ꢀcarboxylate (53 mg, 0.23 mmol) and 5ꢀ
chloro-N-(2,4ꢀdimethoxybenzyl)ꢀ2,4ꢀdifluoro-N-(thiazolꢀ2ꢀyl)benzenesulfonamide (45c) (107 mg, 0.23
mmol) in DMF (2.3 mL) was added cesium carbonate (151 mg, 0.46 mmol). The resulting mixture was
stirred at rt for 1 h. The mixture was then diluted with EtOAc, washed with water, brine, dried over
MgSO4, filtered and concentrated to give tertꢀbutyl 4ꢀ(2ꢀ((2ꢀchloroꢀ4ꢀ(Nꢀ(2,4ꢀdimethoxybenzyl)-N-
(thiazolꢀ2ꢀyl)sulfamoyl)ꢀ5ꢀfluorophenyl)amino)ethyl)piperidineꢀ1ꢀcarboxylate as a crude mixture which
was used in the next step without further purification. The crude product was dissolved in DCM (2 mL),
and TFA (0.4 mL 5.2 mmol) was added. The resulting mixture was stirred at rt for 15 min. The mixꢀ
ture was then concentrated. The crude material was purified via preparative HPLC (Method C) to give
1
26 (17 mg, 12%). H NMR (500 MHz, DMSOꢀd6) δ 7.55 (d, J = 7.3 Hz, 1H), 7.00 (br d, J = 3.8 Hz,
1H), 6.57 ꢀ 6.44 (m, 2H), 5.97 (br t, J = 5.1 Hz, 1H), 3.24 (br d, J = 12.7 Hz, 2H), 3.21 ꢀ 3.14 (m, 2H),
2.83 (td, J = 12.6, 2.5 Hz, 2H), 1.85 (br d, J = 12.4 Hz, 2H), 1.70 ꢀ 1.54 (m, 1H), 1.49 (q, J = 6.9 Hz,
2H), 1.38 ꢀ 1.19 (m, 2H). LC/MS m/z: (M + H)+ calcd for C16H21ClFN4O2S2, 419.08; found 418.9.
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5-Chloro-2-fluoro-4-((2-(piperidin-3-yl)ethyl)amino)-N-(thiazol-2-yl)benzenesulfonamide
(27). Prepared using the procedures described for 26. 1H NMR (500 MHz, DMSOꢀd6) δ 7.56 (d, J = 7.3
Hz, 1H), 6.99 (d, J = 4.0 Hz, 1H), 6.53 (s, 1H), 6.52 ꢀ 6.49 (m, 1H), 6.07 ꢀ 5.94 (m, 1H), 3.28 ꢀ 3.15 (m,
3H), 2.81 ꢀ 2.64 (m, 1H), 2.57 (s, 1H), 2.55 (s, 2H), 1.86 (br d, J = 12.8 Hz, 1H), 1.81 ꢀ 1.70 (m, 2H),
1.63 ꢀ 1.41 (m, 3H), 1.15 (br d, J = 9.5 Hz, 1H). LC/MS m/z: (M + H)+ calcd for C16H21ClFN4O2S2,
419.08; found 418.9.
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5-Chloro-2-fluoro-4-((2-(piperidin-2-yl)ethyl)amino)-N-(thiadizol-2-yl)benzenesulfonamide
1
(28). Prepared using the procedures described for 26. H NMR (500 MHz, DMSOꢀd6) δ 7.85 (s, 1H),
7.54 (d, J = 7.0 Hz, 1H), 6.58 (d, J = 12.8 Hz, 1H), 6.25 (s, 1H), 3.33 ꢀ 3.17 (m, 1H), 3.03 (br. s., 1H),
2.92 ꢀ 2.79 (m, 1H), 1.97 (d, J = 11.7 Hz, 1H), 1.87 ꢀ 1.63 (m, 4H), 1.52 (d, J = 13.2 Hz, 1H), 1.43 (d, J
= 12.5 Hz, 1H), 1.36 ꢀ 1.28 (m, 1H). LC/MS m/z: (M + H)+ calcd for C15H20ClFN5O2S2, 420.07; found
420.1.
5-Chloro-2-fluoro-4-((2-(piperidin-2-yl)ethyl)amino)-N-(thiazol-2-yl)benzenesulfonamide
1
(29). Prepared using the procedures described for 26. H NMR (500 MHz, DMSOꢀd6) δ 7.56 (d, J =
7.3 Hz, 1H), 6.98 (d, J = 4.0 Hz, 1H), 6.55 ꢀ 6.48 (m, 2H), 6.21 (br t, J = 5.1 Hz, 1H), 3.25 (br dd, J =
5.9, 3.0 Hz, 3H), 3.09 ꢀ 2.93 (m, 1H), 2.81 (td, J = 12.5, 2.8 Hz, 1H), 1.95 ꢀ 1.88 (m, 1H), 1.85 ꢀ 1.77
(m, 1H), 1.77 ꢀ 1.61 (m, 3H), 1.56 ꢀ 1.25 (m, 3H). LC/MS m/z: (M + H)+ calcd for C16H21ClFN4O2S2,
419.08; found 419.1.
5-Chloro-2-fluoro-4-((2-(1-methylpiperidin-4-yl)ethyl)amino)-N-(thiazol-2-
1
yl)benzenesulfonamide (30). Prepared using the procedures described for 26. H NMR (500 MHz,
DMSOꢀd6) δ 7.56 (d, J = 7.3 Hz, 1H), 7.10 (d, J = 4.0 Hz, 1H), 6.65 (d, J = 4.0 Hz, 1H), 6.53 (d, J =
12.8 Hz, 1H), 6.10 (br s, 1H), 3.18 (q, J = 6.4 Hz, 2H), 3.14 ꢀ 2.99 (m, 2H), 2.47 (s, 3H), 2.43 (br s,
2H), 1.79 (br d, J = 13.2 Hz, 2H), 1.51 ꢀ 1.38 (m, 3H), 1.38 ꢀ 1.19 (m, 2H). LC/MS m/z: (M + H)+ calcd
for C17H23ClFN4O2S2, 433.09; found 433.0.
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Journal of Medicinal Chemistry
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5-Chloro-2-fluoro-4-((2-(pyrrolidin-3-yl)ethyl)amino)-N-(thiazol-2-yl)benzenesulfonamide
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(31). Prepared using the procedures described for 26. H NMR (500 MHz, DMSOꢀd6) δ 7.56 (d, J = 7.3
Hz, 1H), 6.99 (d, J = 4.0 Hz, 1H), 6.59 ꢀ 6.48 (m, 2H), 6.07 (br. s., 1H), 3.26 ꢀ 3.15 (m, 2H), 3.12 ꢀ 3.01
(m, 1H), 2.70 (dd, J = 11.4, 9.2 Hz, 1H), 2.26 ꢀ 2.08 (m, 2H), 1.72 ꢀ 1.41 (m, 3H). LC/MS m/z: (M +
H)+ calcd for C15H19ClFN4O2S2, 405.06; found 405.0.
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4-((2-(Azetidin-3-yl)ethyl)amino)-5-chloro-2-fluoro-N-(thiazol-2-yl)benzenesulfonamide
1
(32). Prepared using the procedures described for 26. H NMR (500 MHz, DMSOꢀd6) δ 7.59 (d, J =
7.3 Hz, 1H), 7.26 (d, J = 4.4 Hz, 1H), 6.83 (d, J = 4.4 Hz, 1H), 6.69 (d, J = 13.2 Hz, 1H), 6.43 (t, J =
5.9 Hz, 1H), 4.11 ꢀ 3.84 (m, 2H), 3.75 ꢀ 3.53 (m, 2H), 3.18 (q, J = 6.6 Hz, 2H), 2.83 (dt, J = 15.7, 8.1
Hz, 1H), 1.83 (q, J = 6.7 Hz, 2H). LC/MS m/z: (M + H)+ calcd for C14H17ClFN4O2S2, 391.05; found
391.1.
(S)-5-Chloro-2-fluoro-4-((piperidin-3-ylmethyl)amino)-N-(thiazol-2-yl)benzenesulfonamide
1
(33). Prepared using the procedures described for 26. H NMR (500 MHz, DMSOꢀd6) δ 7.65 (d, J =
8.4 Hz, 1H), 6.99 (d, J = 3.7 Hz, 1H), 6.75 (d, J = 12.8 Hz, 1H), 6.61 ꢀ 6.40 (m, 2H), 3.52 (d, J = 10.6
Hz, 1H), 3.08 ꢀ 3.07 (m, 1H), 3.27 ꢀ 3.04 (m, 2H), 3.29 ꢀ 3.00 (m, 2H), 2.83 ꢀ 2.76 (m, 1H), 2.66 (t, J =
11.9 Hz, 1H), 2.00 ꢀ 1.93 (m, 1H), 1.86 ꢀ 1.75 (m, 2H), 1.63 ꢀ 1.48 (m, 1H), 1.28 ꢀ 1.17 (m, 1H). LC/MS
m/z: (M + H)+ calcd for C15H19ClFN4O2S2, 405.06; found 405.0.
(R)-5-Chloro-2-fluoro-4-((piperidin-3-ylmethyl)amino)-N-(thiazol-2-
yl)benzenesulfonamide (34). Prepared using the procedures described for 33. 1H NMR and MS idenꢀ
tical to those of 33.
5-Chloro-2-fluoro-4-((morpholin-2-ylmethyl)amino)-N-(thiazol-2-yl)benzenesulfonamide
1
(35). Prepared using the procedures described for 26. H NMR (500 MHz, DMSOꢀd6) δ 7.57 (d, J =
7.3 Hz, 1H), 7.10 (d, J = 4.0 Hz, 1H), 6.68 ꢀ 6.60 (m, 2H), 6.19 ꢀ 6.04 (m, 1H), 3.84 (d, J = 8.4 Hz, 1H),
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