Discovery of DS-1971a, a Potent, Selective NaV1.7 Inhibitor

Article abstract of DOI:10.1021/acs.jmedchem.0c00259

A highly potent, selective NaV1.7 inhibitor, DS-1971a, has been discovered. Exploration of the left-hand phenyl ring of sulfonamide derivatives (I and II) led to the discovery of novel series of cycloalkane derivatives with high NaV1.7 inhibitory potency in vitro. As the right-hand heteroaromatic ring affected the mechanism-based inhibition liability of CYP3A4, replacement of this moiety resulted in the generation of 4-pyrimidyl derivatives. Additionally, GSH adducts formation, which can cause idiosyncratic drug toxicity, was successfully avoided by this modification. An additional optimization led to the discovery of DS-1971a. In preclinical studies, DS-1971a demonstrated highly potent selective in vitro profile with robust efficacy in vivo. DS-1971a exhibited a favorable toxicological profile, which enabled multiple-dose studies of up to 600 mg bid or 400 mg tid (1200 mg/day) administered for 14 days to healthy human males. DS-1971a is expected to exert potent efficacy in patients with peripheral neuropathic pain, with a favorable safety profile.

Full text of DOI:10.1021/acs.jmedchem.0c00259

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Drug Annotation
Discovery of DS-1971a, a Potent Selective NaV1.7 Inhibitor
Tsuyoshi Shinozuka, HIROYUKI KOBAYASHI, Sayaka Suzuki, Kyosuke Tanaka, Narayan Karanjule, Noriyuki
Hayashi, Toshifumi Tsuda, Eri Tokumaru, Masahiro Inoue, Kiyono Ueda, Hiroko Kimoto, Yuki Domon, Sakiko
Takahashi, Kazufumi Kubota, Tomihisa Yokoyama, Akiko Shimizugawa, Ryuta Koishi, Chie Fujiwara, Daigo
Asano, Tomoko Sakakura, Kiyoshi Takasuna, Yasuyuki Abe, Toshiyuki Watanabe, and Yutaka Kitano
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00259 • Publication Date (Web): 11 May 2020
Downloaded from pubs.acs.org on May 12, 2020
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Page 1 of 52
Journal of Medicinal Chemistry
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Discovery of DS-1971a, a Potent Selective Na 1.7
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,†
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†
Tsuyoshi Shinozuka,* Hiroyuki Kobayashi, Sayaka Suzuki, Kyosuke Tanaka, Narayan Karanjule,
†
†
†
†
‡
Noriyuki Hayashi, Toshifumi Tsuda, Eri Tokumaru, Masahiro Inoue, Kiyono Ueda, Hiroko
†
†
†
†
†
Kimoto, Yuki Domon, Sakiko Takahashi, Kazufumi Kubota, Tomihisa Yokoyama, Akiko
†
‡
†
†
†
†
Shimizugawa, Ryuta Koishi, Chie Fujiwara, Daigo Asano, Tomoko Sakakura, Kiyoshi Takasuna,
†
†
†
Yasuyuki Abe, Toshiyuki Watanabe, and Yutaka Kitano
†R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
‡R&D Division, Daiichi Sankyo RD Novare Co., Ltd., 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-
8
630, Japan
ABSTRACT
A highly potent, selective Na 1.7 inhibitor, DS-1971a has been discovered. Exploration of the left-hand
V
phenyl ring of sulfonamide derivatives (I and II) led to the discovery of novel series of cycloalkane
derivatives with high Na 1.7 inhibitory potency in vitro. As the right-hand heteroaromatic ring affected
V
the mechanism-based inhibition liability of CYP3A4, replacement of this moiety resulted in the
generation of 4-pyrimidyl derivatives. Additionally, GSH adducts formation, which can cause
idiosyncratic drug toxicity, was successfully avoided by this modification. An additional optimization led
to the discovery of DS-1971a. In preclinical studies, DS-1971a demonstrated highly potent selective in
vitro profile with robust efficacy in vivo. DS-1971a exhibited a favorable toxicological profile, which
enabled multiple-dose studies of up to 600 mg bid or 400 mg tid (1200 mg/day) administered for 14 days
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Page 2 of 52
to healthy human males. DS-1971a is expected to exert potent efficacy in patients with peripheral
neuropathic pain, with a favorable safety profile.
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INTRODUCTION
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Humans experience pain via nociceptive stimuli, such as tissue damage, whereas other types of pain
without meaningful stimulation are often more problematic; this type of pain is termed neuropathic pain
(NP). A variety of pathological conditions, including viral infection, spinal cord injury, and diabetes, are
1
known to induce NP via dysfunction or lesions in the nervous system. The typical symptoms of NP are
spontaneous pain, hyperalgesia, and allodynia, and the duration of such symptoms vary. Peripheral
neuropathic pain (PNP) is a debilitating condition that affects patients spontaneously, and is one of the
2
most common types of NP. Although NP and PNP are not lethal conditions, they affect the quality of life
(QOL) of patients. There are several medications approved for the treatment of such pain, including
calcium channel α δ ligands, tricyclic depressants, and sodium channel blockers. However, there remain
2
substantial unmet medical needs for the treatment of PNP, including diabetic neuropathic pain (DNP),
and patients continue to demand potent analgesic drugs with few adverse effects.3,4
Voltage-gated sodium channels (Na s) play a predominant role in the conduction of action potentials
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in the nervous system. A gain of function mutation in the SCN9A gene, which encodes Na 1.7, leads to
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extremely painful disorders, such as paroxysmal extreme pain disorder (PEPD) and peripheral
erythromelalgia (PE).5,6,7 Conversely, loss of function of the SCN9A gene was shown to result in a rare
genetic condition called congenital insensitivity to pain (CIP).8,9 Patients with CIP present a normal
phenotype, similar to healthy individuals, except for anosmia, which is a lack of olfactory function.9
Deletion of the SCN9A gene in mice, in both sensory and sympathetic neurons was reported to result in
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the same phenotype observed in humans. Thus, genetic evidence suggests that selective inhibition of
Na 1.7 may have potential as a potent analgesic drug with a high safety index. In particular, selective
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Na 1.7 inhibition is expected to demonstrate a high safety margin with regards the central nervous system
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Journal of Medicinal Chemistry
(
CNS), owing to the predominant expression of the Na 1.7 isoform in the peripheral nervous system
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PNS). CNS adverse effects often lead to treatment interruptions with calcium channel α δ ligands.
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High subtype selectivity is crucial for Na 1.7 inhibitors, because the inhibition of several Na s results
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in adverse effects. Na 1.1 is expressed in CNS, and the inhibition of Na 1.1 leads to CNS adverse effects.
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Furthermore, given that Na 1.5 is highly expressed in cardiac myocytes, its inhibition can induce adverse
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cardiovascular (CV) effects. In fact, systemic exposure to non-selective local anesthetic Na blockers
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has been associated with several CNS or CV adverse effects, which limits the use of such drugs.14
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During the course of our research that aimed to acquire novel analgesic agents, we focused on a highly
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potent and subtype-selective Na 1.7 inhibitor I and II reported by the Pfizer group (Figure 1). We
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hypothesized that the derivatization of these compounds would provide an opportunity to obtain an
effective analgesic drug. After the discovery of aryl sulfonamide II, numerous analogs were subsequently
1
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reported. Herein, we describe the discovery of the clinical compound DS-1971a, which showed ideal
analgesic characteristics in preclinical studies. On the basis of our observations that DS-1971a
successfully shows a 10-fold higher potency than compound II using animal models of neuropathic pain,
we believe that DS-1971a has the potential to produce positive results in clinical trials on neuropathic
patients.18
F
O
S
O
N
F
O
S
O
S
N
Cl
Cl
N
H
S
N
H
N
O
O
N
F
Cl
H2N
N
N
N N
H
I
II (PF-05089771)
hNaV1.1 IC50>100000 nM
hNaV1.5 IC50> 75000 nM
hNaV1.7 IC50: 1.9 nM
hNaV1.1 IC50>100000 nM
hNaV1.5 IC50> 100000 nM
hNaV1.7 IC50: 13 nM
mNaV1.7 IC50: 0.55 nM
mNaV1.7 IC50: 4.9 nM
Figure 1. The structure of I and clinical compound II disclosed by the Pfizer group.
RESULTS and DISCUSSION
Chemical Synthesis.
Na 1.7 inhibitors 6 were synthesized as shown in Scheme 1. Sulfonamide 3 was prepared from
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protected amine 2. Synthesis of 4-pyrimidine intermediate 3 proceeded smoothly when DABCO was used
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as a base, whereas LiHMDS was used to prepare 2-thiadiazole analogs 3. Boc-protected intermediates
were used to synthesize the 4-thiazole analog rac-6f (structures not shown).16b An epoxide-opening
reaction with methyl pyrazole yielded racemic alcohol rac-4. Linear alcohol 4a, to prepare 6a (Table 1),
was synthesized in the same manner (structures not shown). Compound 4 was exposed to SNAr reaction
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with 3 to provide rac-5. Final deprotection yielded Na 1.7 inhibitor rac-6.
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a
Scheme 1. Synthesis and Chemical Structures of Na 1.7 Inhibitor 6.
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O
O
Het
S
Het
HN
O
N
a
b or c
Het
F
X
H2N
O
O
O
1
2
3
O
O
O
O
S
Het
S
Het
n(
)
N
N
H
n(
)
n(
)
OH
d
e
O
f
O
N
N
X
X
+
n( )
N
N
N
N
O
O
N
N
O
rac--4
rac-5
rac-6
F
F
O
S
O
N
F
F
O
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O
N
F
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O
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N
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N
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H
*
*
*
*
*
*
*
*
*
*
O
O
O
O
O
O
N
N
N
N
N
N
N
F
N
N
N
N
N
rac-6a
rac-6b
rac-6c
rac-6d
rac-6e
rac-6f
F
O
O
N
F
O
O
N
O
O
N
F
O
O
N
F
O
O
N
F
O
O
N
S
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F
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H
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H
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H
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H
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H
N
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H
N
*
*
*
*
*
*
*
*
*
*
*
*
O
O
O
O
O
F
O
F
F
F
N
N
F
N
N
N
N
N
N
N
N
N
N
rac-6g
rac-6h
rac-6i
rac-6j
rac-6k
rac-6l
F
O
O
N
F
O
O
N
F
O
O
N
F
O
O
N
F
O
O
N
S
S
S
S
S
N
H
N
N
H
N
N
H
N
N
H
N
N
H
N
*
*
*
*
*
*
* *
*
O
F
*
O
O
O
O
Cl
Cl
N
N
N
N
N
N
N
N
N
N
rac-6m
rac-6n
rac-6o
rac-6p
rac-6q
aReagents and conditions: (a) 2,4-dimethoxybenzaldehyde, piperidine, toluene, reflux then NaBH(OAc)3,
EtOH; (b) sulfonyl chloride, LiHMDS, THF, -78°C; (c) sulfonyl chloride, DABCO, THF, 0°C; (d) n-
BuLi, TMEDA, THF, −78°C; (e) 3, NaH, DMF or DMSO; (f) Et SiH, TFA, CH Cl .
3
2
2
In Vitro Activity and in Vitro ADME Profile.
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Journal of Medicinal Chemistry
Initial efforts to modify the sulfonamide moiety were unsuccessful (data not shown). Consequently, we
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aimed to modify the left-hand phenyl ring, which appeared to be the spacer connecting the benzene
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sulfonamide moiety and the pyrazole ring. This hypothesis led us to synthesize ethylene compound 6a,
which presented modest hNa 1.7 activity with poor mNa 1.7 potency (Table 1). Accordingly,
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derivatization of 6a was initiated to enhance human and mouse Na 1.7 activity. As the introduction of
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substituents on the ethylene linker led to poor results (data not shown), cycloalkane analogs were
prepared. When cyclopentane rac-6b and cyclohexane rac-6c were synthesized, both compounds showed
excellent hNa 1.7 potency, with high subtype selectivity over hNa 1.1 and hNa 1.5. Moreover, both
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compounds improved mNa 1.7 activity. Cyclohexane analog rac-6c demonstrated 10-fold higher
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mNa 1.7 potency than cyclopentane rac-6b. Consequently, further optimizations were performed to
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acquire clinical candidates.
Table 1. In Vitro Profile of 6a
F
F
O
S
O
N
N
N
H
S
RO
hNa 1.1 hNa 1.5 hNa 1.7 mNa 1.7
IC50
nM)
V
V
V
V
R
IC50
(nM)
IC50
(nM)
IC50
(
(nM)
0.55
4.9
I
>100000 75000
1.9
II
>100000 >100000 13
>100000 >100000 800
N
6a
N
35300
240
rac-
2
6000
28000
17
12
N
N
6
b
rac-
>
100000 40000
17
6
c
N
N
aData were obtained using a high-throughput electrophysiology system (IonWorks Quattro). Values are
from a single experiment run in quadruplicate.
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As shown in Table 2, in vitro ADME profiling revealed that thiadiazoles rac-6b and rac-6c possessed
high solubility and adequate stability against mice liver microsomes (MLM), whereas both compounds
presented poor membrane permeability and inhibited CYP enzymes. Inhibition of CYP enzymes was
evaluated at 10 μM, and the mean inhibition (%) was determined. As rac-6b and rac-6c showed excellent
selectivity with respect to other ion channels, such as hNa 1.1, hNa 1.5, and hERG, the avoidance of
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CYP inhibition was examined by modifying the right-hand heteroaromatic group.17h
When methyl group was introduced to the thiadiazole ring, hNa 1.7 activity was lost (rac-6d). Although
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rac-6d showed no CYP3A4 inhibition, CYP2C9 inhibitory activity was retained. Replacement with 4-
oxazole, 2-(1,3,4-thiadiazole), or 3-isoxazole yielded compounds with diminished hNa 1.7 potency (IC
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50
= 3.3, 1.7, and 24.2 μM , respectively). 2-Thiazole rac-6e maintained potent hNa 1.7 activity, whereas
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rac-6e showed mechanism-based inhibition (MBI) of CYP3A4 enzyme. The MBI potency of CYP 3A4
was evaluated by reacting the percent remaining activity at 10 μM with CYP3A4 probe substrates after
0.5 h preincubation in human liver microsomes (HLM). Compound rac-6e reduced the remaining
CYP3A4 activity to 41%. Potent CYP2C9 inhibition was also confirmed for rac-6e. Introducing 4-
thiazole group weakened the results. 4-Thiazole rac-6f demonstrated GSH adduct formation in HLMs in
a GSH-trapping assay. GSH adduct formation is often associated with idiosyncratic drug toxicity (IDT),
2
0
which has led to the withdrawal of many drugs from the market. Both thiazoles rac-6e and rac-6f
showed diminished microsomal stability owing to higher lipophilicity. In contrast, molecules with 4-
pyrimidine avoided not only GSH adduct formation but also MBI liability (compounds rac-6g and rac-
6h). Thus, such limitations may be controlled by a right-hand heteroaromatic ring. Further optimization
supported this hypothesis, as none of 4-pyrimidine-based sulfonamide analogs synthesized were
associated with these issues. The fair membrane permeability was observed in 4-pyrimidines rac-6g and
rac-6h. Although 4-pyrimidines rac-6g and rac-6h successfully avoided the aforementioned issues, both
compounds diminished human and mouse Na 1.7 activities by several folds, and still inhibited CYP2C9
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and CYP3A4. Further derivatization was required to enhance in vitro potency and prevent CYP inhibition.
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Journal of Medicinal Chemistry
Table 2. Na 1.7 Potency and In Vitro ADME Profiles of 6
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6
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
F
F
O O
S
Het
N
n(
)
H
O
N
N
PA
MP
A
hNa hNa hNa mNa hER
V1.1 V1.5 V1.7I V1.7I G
Solu MS
bilit (%)
y
DI
(%,
DI
(%,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Log Papp
MBI GSH adduct
Het
n
IC50
IC50
C50
C50
IC50
b
g
h
D
2C9) 3A4)
formation
(
nM) (nM) (nM) (nM) (nM)
(nM) Mou
10−6
f
f
(
a
a
a
a
a
d
e
se
cm/s
c
)
N
S
rac-
6b
2600 2800
>100
000
>190
0
N
N
1
2
17
12
240
17
0.6
0.7
0.3
0.3
91
75
93
97
NT
Negative
NT
0
0
N
S
rac-
6c
>100 4000
000
>100
000
>200
0
87.4 >90 NT
0
rac-
>100 >100 5610
>100
000
102.
N
N
1
1
NT
0.9
2.1
12.6 1800 69
89.2 17.2
4
Negative
6
d
S
000
000
0
N
S
rac-
1300 6300
>100
000
28
100
>50 250
>50 660
54
>90
0
41.2 Negative
6
e
0
0
2
±
5
800
N
rac-
9100 >100 290
9400
0
S
2
1.8
28
66.2 75.1 11.6 Positive
119.
i
6
f
0
000
± 0
00i
rac-
N
N
>100 >100 180
>100
000
1
2
1300
0.3
0.3
7.1
5.0
740
100
86.3 35.3
Negative
N
N
i
i
6
g
000
000
± 30
7
rac-
>100 >100 113
000 000 ± 18
>100
000
400
1900 80
76
50.4 81.0 Negative
6
h
a
Data were obtained with a high-throughput electrophysiology system (IonWorks Quattro). Values are
b
from a single experiment run in quadruplicate. Distribution coefficients (Log D) were measured after
partition between 1-octanol and PBS (pH = 7.4). Parallel artificial membrane permeation assay (PAMPA)
was performed at pH 7.4. Aqueous thermodynamic solubility at pH 6.8. Mouse microsomal stability
c
d
e
f
was assessed based on test compound (%) remaining after 0.5 h of incubation with MLM. Inhibition (%)
g
of CYP enzymes at 10 μM. The % remaining activity at 10 μM reacted with CYP3A4 probe substrates
h
i
after 0.5 h preincubation in HLM. GSH-trapping assay in HLM at 0.5 mM. Values represent at least two
independent experiments run in quadruplicate. Each value represents the mean ± standard error of the
mean (S.E.M).
The introduction of substituents on the central benzene ring was subsequently investigated (Table 3).
Given that 2-fluoro or 3-fluoro derivatives did not show high Na 1.7 potency in vitro (IC = 0.47, 1.7
V
50
μM, respectively), difluoro-substituted analogs were synthesized. 3,5-Difluoro analog rac-6i was found
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to have lost hNa 1.7 activity, whereas 2,3-difluoro analog rac-6j showed diminished activity. Among
V
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
difluoro-substituted compounds, 2,6-difluoro derivative rac-6k showed high potency against human and
mouse Na 1.7 in vitro with diminished CYP3A4 liability. When the saturated ring size of rac-6k was
V
modified, cyclohexane rac-6l enhanced Na 1.7 activities by approximately two fold. Cyclohexane rac-6l
V
also maintained an acceptable in vitro ADME profile with modest CYP inhibition. Although potent
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Na 1.7 activities were observed in cycloheptane rac-6m, rac-6m displayed diminished selectivity with
V
respect to hNa 1.1 and hNa 1.5. Thus, saturated ring was fixed to cyclopentane or cyclohexane for further
V
V
modification. Although 2-fluoro-5-chloro analog rac-6n displayed high in vitro potency against both
species, potent CYP2C9 inhibition remained an issue. Six-membered analog rac-6o showed high Na 1.7
V
potency as well as modest CYP3A4 liability. Similar SAR results were obtained in 2-fluoro-5-methyl
derivatives rac-6p and rac-6q to identify rac-6p with potent activity and an acceptable in vitro ADME
profile.
On the basis of Na activities combined with in vitro ADME profile, rac-6l, rac-6o, and rac-6p were
V
selected for an evaluation of thermal hyperalgesia using Seltzer (partial sciatic nerve ligation [PSL])21
model mice (vide infra) (n = 4); rac-6o was found to be the most potent of the three compounds, and thus
was selected for further pharmacological evaluation.22
Table 3. Na 1.7 Potency and In Vitro ADME Profiles of 6
V
O
O
N
2
S
3
N
N
n(
)
H
6
O
X
5
N
N
PAM
PA
Papp
MS
hNaV hNaV hNaV mNaV
hERG
Solub (%)
DI
(%,
DI
(%,
3A4)
1
.1
1.5
1.7
1.7
Log
D
n
1
X
IC50
ility
b
IC
(
IC50
IC50
IC50
5
0
a
d
f
f
(nM)
(nM) Mous 2C9)
nM)a (nM)a (nM)a (nM)a
(10−6
cm/s)
e
e
c
rac-
g
2-F,
5-F
>100 >100 180 ±
>100
000
1300
0.3
7.1
740
100
86.3
35.3
g
6
000
000
30
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Journal of Medicinal Chemistry
3
5
-F,
-F
>100
000
>100
000
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
rac-6i
1
1
1
2
3
1
2
2
1
2
NT
NT
2800
96
NT
NT
0.4
0.1
0.1
0.6
0.9
0.8
0.8
0.9
NT
5.4
2.2
2.2
6.7
14.3
6.3
5.2
10.3
6.7
NT
NT
82
80
77
97
88
70
92
87
75
NT
NT
2
3
-F,
-F
>100
000
495 ±
>100
000
rac-6j
rac-
84000
550
>90
75
60.3
22.7
45.2
53.9
53.3
56.6
59.5
42.8
52.7
g
35
2-F,
6-F
>100 60 ±
>100
000
52000
1800
1800
1040
240
g
6
k
000
23
2
6
-F,
-F
>100 >100
000 000
>100
000
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
rac-6l
rac-
30
56
65.9
80.4
>90
86.8
70.0
79.8
70.6
2-F,
6-F
>100
000
14000 96000 28
41
6
m
rac-
2-F,
5-Cl
>100 >100
45
>100
000
28
6
n
000
000
rac-
2-F,
5-Cl
>100 >100 24.5 ± 26.5 ± >100
000
>200
0
g
g
6
o
000
8.5
14.5
000
2
5
-F,
-Cl
>100
000
21.7 ± 56.3 ± >100
6o
93000
1500
1200
1800
g
g
4.4
24
6.4
28
000
rac-
2-F,
5-Me
>100
000
>100
000
92000
16000
6
p
rac-
2-F,
5-Me
>100
000
31
15
90000 0.9
6
q
a
Data were obtained with a high-throughput electrophysiology system (IonWorks Quattro). Values are
from a single experiment run in quadruplicate. Distribution coefficients (Log D) were measured after
partition between 1-octanol and PBS (pH = 7.4). Parallel artificial membrane permeation assay (PAMPA)
was performed at pH 7.4. Aqueous thermodynamic solubility at pH 6.8. Mouse microsomal stability
was assessed based on test compound (%) remaining after 0.5 h of incubation with MLM. Inhibition (%)
of CYP enzymes at 10 μM. Values represent at least two independent experiments run in quadruplicate.
Each value represents the mean ± S.E.M.
b
c
d
e
f
g
Preparation of DS-1971a.
Prior to pharmacological assessments, the chiral form of rac-6o was synthesized. The optical resolution
of racemic alcohol rac-4o (n = 2, Scheme 1) was achieved using CHIRALPAK IB, followed by
manipulations to obtain 6o (DS-1971a). The stereochemistry of DS-1971a was determined by X-ray
crystallographic analysis (Figure 2). This synthetic scheme was able to generate DS-1971a at over 200 g.
Note that enantiomer ent-6o lost potent hNa 1.7 potency (IC : 47 μM against hNa 1.7). As indicated in
V
50
V
Table 4, compound 6o showed Na 1.7 activity and an in vitro ADME profile comparable to those of rac-
V
6
o.
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Page 10 of 52
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. An ORTEP view of DS-1971a (CCDC ID: 1989861).
In Vitro Profile of DS-1971a.
The whole-cell manual patch-clamp method was employed to investigate the effects of DS-1971a on
the peak currents of several Na s, as shown in Table 4. DS-1971a inhibited human and mouse Na 1.7
V
V
with IC s 22.8 and 59.4 nM, respectively. Weak inhibitory activity was observed against hNa 1.2 and
5
0
V
hNa 1.6. DS-1971a possessed superior selectivity against hNa 1.1 and hNa 1.5, because the IC s of
V
V
V
50
both hNa 1.1 and hNa 1.5 exceeded 100 μM. The effect of DS-1971a on the potassium currents through
V
V
hERG was evaluated via the whole-cell manual patch-clamp method (Table 4). The rates of inhibition
with 10, 30, and 100 μM of DS-1971a were 2.4, 2.9, and 23.5%, respectively. Thus, DS-1971a
demonstrated low inhibitory activity against the hERG channel. In vitro pharmacological activity of DS-
1971a against 67 receptors, channels, and transporters was evaluated by radioligand binding assays at 10
23
μM to confirm the absence of significant responses (≥ 50% inhibition). These data clearly indicated that
DS-1971a is a highly potent selective Na 1.7 inhibitor in vitro.
V
Table 4. In Vitro Profile of DS-1971aa
IC (nM)
5
0
%
Inhibition at 100 μM
(95% CI)
b
b
hNa 1.1
10.53 ± 4.24
>100000
1960
V
75.40 ± 7.07d
hNa 1.2
V
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Journal of Medicinal Chemistry
(
1010-3820)
1
2
3
4
5
6
7
8
9
b
b
b
hNa 1.3
-0.76 ± 7.45
5.00 ± 3.98
-23.91 ± 2.22
>100000
>100000
>100000
V
hNa 1.4
V
hNa 1.5
V
4
75
190-1190)
2.8
18.4-28.2)
b
86.49 ± 5.81e
95.43 ± 4.25f
hNa 1.6
V
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
2
b
b
hNa 1.7
V
(
hNa 1.8
0.35 ± 7.25
10.39 ± 3.89
-2.51 ± 1.18
>100000
>100000
>100000
V
b
mNa 1.1
V
b
b
mNa 1.5
V
5
9.4
25.7-137)
>100000
mNa 1.7
81.90 ± 0.84f
V
(
hERGc
23.5 ± 2.69
a
Data were obtained via the whole-cell manual patch clamp method. Each value represents the mean ±
S.E.M. n = 3. n = 4. %Inhibition at 10 μM. %Inhibition at 3 μM. %Inhibition at 0.3 μM.
b
c
d
e
f
Blocking Kinetics of Na 1.7 Channel Current by DS-1971a.
V
DS-1971a demonstrated potent and selective inhibition of Na 1.7, as well as unique blocking kinetics.
V
The blocking kinetics of DS-1971a were evaluated using a manual patch-clamp method. Cells expressing
hNa 1.7 or mNa 1.7 were clamped from the prepulse potential of −50 mV for 9 s to the hyperpolarizing
V
V
potential of −120 mV for 1 s to reprime channels not bound by compounds, and then given a 10 ms test
pulse to −10 mV to elicit current. The voltage pulses were run continuously for 20 min (10 min with
perfusion of the test article, and 10 min following washout). A nonselective Na blocker, mexiletine, was
V
used as a control. The time course of hNa 1.7 inhibition by DS-1971a is presented in Figure 3A. While
V
mexiletine inhibited hNa 1.7 within a few seconds, DS-1971a induced a slower onset of inhibition In
V
contrast, the dissociation velocity of DS-1971a was slower than that of mexiletine. These unique blocking
kinetics were observed when mNa 1.7 was utilized (Figure 3B). The association and dissociation
V
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Page 12 of 52
velocities of DS-1971a for human Na 1.7 were slower than those of mice orthologues. The dissociation
V
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
half-lives of DS-171a were estimated as 14 min for hNa 1.7 and 2.8 min for mNa 1.7. Thus, DS-1971a
V
V
showed unique blocking kinetics with Na 1.7, which are expected to result in high in vivo efficacy due
V
to the longer residence time; in vivo studies (vide infra) are required to support this.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A)
B)
Figure 3. Blocking kinetics of Na 1.7 channel current by DS-1971a (A: hNa 1.7, B: mNa 1.7). Data
V
V
V
represent the mean ± S.E.M. (n = 3 or 4).
Effect of DS-1971a on Mechanical Hypersensitivity.
The in vivo pharmacological effects of DS-1971a on mechanical hypersensitivity were investigated
24
using Chung (spinal nerve ligation [SNL]) and PSL model mice, as shown in Figure 4. DS-1971a (1,
1
0, and 100 mg/kg) was administered orally, and the pain threshold in the hind paw was measured with
an electronic von Frey anesthesiometer (n = 8). In the SNL model, the pain threshold was significantly (p
< 0.01) lowered in model animals, indicating the development of a neuropathic pain state (Figure 4A).
The administration of DS-1971a at 1 mg/kg mitigated the pain threshold (p < 0.01) at 60 and 120 min,
and this effect lasted until 180 min. The peak efficacy was observed at 120 min. Further mitigation of
pain threshold was observed in animals administered 10 mg/kg, and this effect was significant until 180
min (p < 0.01). Maximum efficacy was observed in the animals administered 100 mg/kg; this does
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Journal of Medicinal Chemistry
significantly improved the pain threshold to pre-procedure levels (p < 0.01) at 120 and 180 min. This
potent effect was significant until 240 min (p < 0.01). To calculate total analgesic efficacy, the AUC0-4 h
of each curve presented in Figure 4A was calculated. As depicted in Figure 4B, there was a clear dose-
dependent suppression of the pain threshold, and a significant (p < 0.01) improvement of total analgesic
efficacy with all doses. The plasma exposure of DS-1971a at 1 and 3 mg/kg was confirmed through PK
studies with mice (vide infra).
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The mechanical hypersensitivity of PSL mice was then investigated. As shown in Figure 4C and 4D,
the results obtained with PSL mice were comparable to those obtained with SNL mice: the pain threshold
was mitigated in a clear dose-dependent manner. Notably, a significant (p < 0.01) effect was observed for
up to 240 min in PSL mice administered 100 mg/kg (Figure 4C).
A)
B)
C)
D)
Figure 4. Effect of DS-1971a on mechanical hypersensitivity in SNL (A, B) and PSL (C, D) mice models.
The suppression of mechanical hyperalgesia was investigated following oral administration of DS-1971a
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in 0.5% methylcellulose (MC) (n = 8). A) Time course of pain threshold (g) following administration of
a single dose of DS-1971a (1, 10, and 100 mg/kg) in SNL model mice. B) AUC0-4 h (area under the curve
of pain threshold in Figure 4A). C) Time course of pain threshold (g) following administration of a single
dose of DS-1971a (1, 10, and 100 mg/kg) in PSL model mice. D) AUC0-6 h in Figure 4C. Data are presented
as the mean ± S.E.M. Statistical significance compared with vehicle treatment is denoted by *(p < 0.05),
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
*
*(p < 0.01), as determined by the Dunnett multiple comparison test. Statistical significance compared
#
#
with the pre-operation value is denoted by (p < 0.01) as determined by the paired t-test.
Effect of DS-1971a on thermal hyperalgesia
The effect of DS-1971a on thermal hyperalgesia was investigated using PSL mice (Figure 5). DS-1971a
(0.1, 0.3, and 1 mg/kg) was administered orally, and the pain threshold in the hind paw was measured 30,
6
0, 120, 180, and 240 min after administration using a paw thermal stimulator (n = 8). Compared with the
normal control group, the pain threshold was significantly lowered in the model control group, indicating
the development of hyperalgesia. As shown in Figure 5A, normal mice withdrew their paw after
approximately 10 s, whereas hyperalgesia-induced PSL mice withdrew their paws after approximately 3
s. When DS-1971a was administered at 1 mg/kg, the analgesic efficacy peaked at 60 min, and the pain
threshold was significantly improved to the level observed in normal mice (p < 0.01). This response lasted
up to 120 min, and was lost at 180 min. Similarly, administration of DS-1971a at 0.3 mg/kg significantly
lowered the pain threshold from 30 to 120 min (p < 0.05 at 30 min, p < 0.01 at 60 and 120 min), while
administration at 0.1 mg/kg was not effective at lowering the pain threshold. These results clearly indicate
that DS-1971a mitigated thermal hyperalgesia in a dose-dependent manner in PSL mice. At the peak
efficacy (60 min), there was a significant dose-dependent suppression of thermal hyperalgesia (p < 0.01)
in 0.3 and 1 mg/kg administered groups. The ED of DS-1971a at the peak efficacy was 0.32 mg/kg (95%
50
confidence interval: 0.24–0.43 mg/kg). Compound II was similarly evaluated for its efficacy against
22
thermal hyperalgesia in PSL mice, and at peak efficacy, it was determined to have ED of 3.0 mg/kg.
5
0
Consequently, DS-1971a proved to be 10-fold more potent than compound II on a dosage basis.
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Journal of Medicinal Chemistry
The efficacy of DS-1971a against a thermal stimulus in PSL mice was found to be more potent than
1
2
that against a mechanical stimulus. Although the underlying mechanisms remain unclear, these results
may reflect the effects mediated via unmyelinated C-fibers. In this regard, it is conceivable that DS-1971a
disperses more efficiently in unmyelinated C-fibers than in myelinated A-fibers. This contrasting pattern
of DS-1971a distribution in nerve fibers may contribute to difference in the efficacy of impeding thermal
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
and mechanical stimuli. Consistent with this assumption, a higher expression of Na 1.7 in C-fibers than
V
in A-fiber neurons has been reported in rats.25
The AUC0-4 h of each curve was calculated in order determine total analgesic efficacy. As depicted in
Figure 5B, there was a dose-dependent suppression of the pain threshold, and significant (p < 0.01)
improvements in total analgesic efficacy at 0.3 and 1 mg/kg.
To investigate the therapeutic efficacy at higher dose, DS-1971a (1, 3, and 10 mg/kg) was administered
orally to PSL mice (n = 6, Figure 5C). Administration of DS-1971a at 10 mg/kg resulted in higher efficacy
than 1 mg/kg, which was observed for up to 240 min. Notably, administration of DS-1971a at 10 mg/kg
significantly lowered the pain threshold to levels observed in normal mice at 60–180 min (p < 0.01). This
reduced pain threshold was observed for up to 180 min when DS-1971a was administered at 3 mg/kg.
Consequently, a higher dose of DS-1971a improved both the pain threshold and the duration of the
therapeutic effect. To assess total analgesic efficacy, the AUC0-6 h of each curve in Figure 5C was
calculated as indicated in Figure 5D. Significant (p < 0.01) improvement in total thermal-evoked pain
behavior was confirmed following administration of 3 and 10 mg/kg.
A)
B)
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D)
E)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Effect of DS-1971a on thermal hyperalgesia in PSL mice. The suppression of thermal
hyperalgesia was investigated following oral administration of DS-1971a in 0.5% MC. A) Time course
of paw withdraw latency (PWL, s) following administration of a single dose of DS-1971a (0.1, 0.3, and
1
mg/kg, n = 8). B) AUC0-4 h (area under the curve of PWL) in Figure 5A). C) Time course of PWL (s)
following administration of a single dose of DS-1971a (1, 3, and 10 mg/kg, n = 6). D) AUC0-6 h in Figure
C. Data are presented as the mean ± S.E.M. Statistical significance compared with vehicle treatment is
5
denoted by *(p < 0.05), **(p < 0.01), as determined by the Dunnett multiple comparison test.
ADME and PK Profile of DS-1971a
ADME profiling established that DS-1971a showed high stability against rodent and dog liver
microsomes, whereas modest stability was observed in monkey and human microsomes, as indicated in
Table 5. DS-1971a binds to plasma proteins in a reasonable degree. Although the compound inhibited
CYP3A4 and CYP2C9 at micromolar concentration, a sufficient margin is expected due to the low
predicted pharmacologically effective concentrations.
The PK profile was assessed in preclinical studies using four species, as shown in Table 6. DS-1971a
exhibited high clearance (CL) in rodents, whereas better CL was observed in non-rodents. A low
distribution volume (V ) was confirmed in preclinical studies in four species. The compound exhibited
ss
high oral bioavailability in dogs, whereas modest bioavailability was observed in rodents. Overall, the
preclinical profile of DS-1971a in four preclinical species was suitable for proceeding clinical trials.
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To investigate the PK/PD correlation, PK studies were performed in mice (1 and 3 mg/kg) (Table 6).
1
2
The increase in AUC was dose-dependent, whereas the T of plasma DS-1971a was similar following
inf
1/2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
administration of 1 and 3 mg/kg. Thus, the duration of effect was correlated with AUC in the PK study.
inf
In order to determine the ability of the compound to penetrate into the brain, brain Kp value was
assessed. The brain concentration of DS-1971a was determined 30 min after compound administration (1
mg/kg, po), and was found to be below the lower limit of quantification (Kp brain <0.07) (n = 3).
On the basis of the pharmacologically effective dose (ED = 0.32 mg/kg) combined with C at 1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
max
mg/kg in mice (105 nM, Table 6) and the assumption of PK linearity, the pharmacologically effective
plasma concentration was calculated as EC50 = 33.6 nM. If the plasma protein binding ability is
considered, free EC is 1.1 nM. There was a significant disparity between EC values calculated based
5
0
50
on in vivo investigations and in vitro IC value in mice (59.4 nM, Table 4). One plausible reason for this
5
0
disparity is the binding kinetics of DS-1971a, as presented in Figure 3. As DS-1971a has a long residence
time in Na 1.7, this may contribute to the higher efficacy observed in vivo than that predicted from in
V
vitro IC and PK studies. In addition to IC , the dissociation half-life of the target protein is suggested
5
0
50
to be an important factor that determines the pharmacological potency in vivo.26
Table 5. ADME Profile of DS-1971a
MS (%)d
PAMPA
Papp
Solubility
Log Da
c
(nM)
(
10−6
Mouse
92
rat
94
dog
95
monkey human
78 66
b
cm/s)
0
.8
5.2
1500
Plasma protein binding, %
CYP2C9 IC (μM) CYP3A4 IC (μM)
5
0
50
mouse
rat
monkey human
9
9
6.6–
6.8%
97.7–
97.9%
98.9–
99.0%
99.1–
99.2%
4.9
8.7
a
Distribution coefficients (Log D) were measured after partition between 1-octanol and PBS (pH = 7.4).
b
c
Parallel artificial membrane permeation assay (PAMPA) was performed at pH 7.4. Aqueous
thermodynamic solubility at pH 6.8. Microsomal stability was assessed based on test compound (%)
d
remaining after 0.5 h of incubation with corresponding liver microsomes.
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 6. PK Parameters of DS-1971aa
CL
Cmax
AUCinf
Vss
Tmax (h)
T (h)
F (%)
1/2
(mL/min
(nM)
(h·nM)
(L/kg)
/kg)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1570 ± 0.83 ± 3.69 ± 2060
±
6.55 ± 0.150 ±
1.62
Monkeyb 1 mg/kg
35 ± 10
6
48
0.29
0.18
923
0.009
1
708
0.50
4.0
3230
(3630,
8.7
0.57
7
7
Dog
Rat
1 mg/kg
1 mg/kg
(
1
1790,
620)
(0.50,
0.50)
(3.8,
(8.6,
8.9)
(0.49,
0.65)
(
87, 67)c
c
c
c
c
c
c
4.2)
2830)
0
.77
103
1.2
47
1
4
03
0.8
± 0.50
±
16 ± 6.5b
b
b
(
0
0.70,
.85)
(127,
81.6)
(1.4,
1.1)
0.00
(55, 39)c
c
c
c
Moused
Moused
1 mg/kg 105
3 mg/kg 464
0.0833
0.250
2.02
1.54
68.9
277
15
11
78.7
45.3
1.37
0.565
aSee Table 11 for detailed conditions. Total body clearance (CL), distribution volume at steady state
Vss) and F value were calculated following intravenous (1 mg/kg) administration of DS-1971a. Each
(
b
c
d
value represents the mean ± standard deviation (S.D.). n = 3. Individual data (n = 2). n = 3 for each time
point. See SI for the individual data.
In Vivo Safety Pharmacology Studies of DS-1971a.
Preclinical safety pharmacology studies were performed using mice and cynomolgus monkeys. As
shown in Table 7, DS-1971a did not exert any notable effects up to 1000 mg/kg in mice or cynomolgus
monkeys. Furthermore, on the basis on modified Irwin’s multiple observations, spontaneous locomotor
activity, and motor coordination via a rota-rod test, DS-1971a did not appear to affect CNS of male mice,
or their respiratory system. In conscious and unrestrained male cynomolgus monkeys, DS-1971a had no
effects on heart rate, blood pressure (systolic, diastolic, and mean), or electrocardiogram (ECG)
parameters (PR interval, QRS duration, QT interval, and QTc) at any evaluated time point. Moreover, no
occurrences of arrhythmia were observed. Consequently, a single oral dose of DS-1971a is not considered
to affect the cardiovascular system in monkeys at doses up to 1000 mg/kg.
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Table 7. In vivo Safety Pharmacology Studies of DS-1971a.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
n
Species
Age
Dose (mg/kg)
NOEL (mg/kg)
Irwin’s multiple observation
Locomotor activity
6
8
8
8
4
Crl:CD1(ICR) mice
Crl:CD1(ICR) mice
Crl:CD1(ICR) mice
Crl:CD1(ICR) mice
cynomolgus monkeys
7 weeks
7 weeks
7 weeks
7 weeks
5 years
0, 100, 300, 1000 1000
0, 100, 300, 1000 1000
0, 100, 300, 1000 1000
0, 100, 300, 1000 1000
0, 30, 100, 1000 1000
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Motor coordination
Respiratory system
Cardiovascular system
Chronic Toxicity Studies of DS-1971a.
The results of chronic in vivo toxicology studies of DS-1971a are summarized in Table 8. Six- or 9-
month repeated dose toxicity studies were conducted in mice (n = 15/sex/group) at 0, 30, 100, and 1000
mg/kg or in cynomolgus monkeys (n = 4/sex/group). In the mouse study, a satellite group was established
for the toxicokinetic (TK) evaluation (n = 3 for each blood sampling point), and in the monkey study,
additional animals (n = 2/sex) were treated with DS-1971a at a dose level of 1000 mg/kg for 9 months
and were withdrawn from treatment to assess the reversibility of any toxicity following a 1-month
recovery period. No deaths were noted and no treatment-related changes in clinical signs, body weight,
food consumption, ophthalmology, urinalysis, hematology, blood chemistry, necropsy, or histopathology
were observed at any doses of both studies including the recovery period. No toxicity was observed up to
the highest doses, and the no observed adverse effect level (NOAEL) was considered to be the top dose
of 1000 mg/kg/day for both species. In the TK evaluation, the plasma exposure of DS-1971a increased in
a dose-dependent manner. Exposure was higher in female mice than in male mice; however, there were
no clear sex difference in exposure in monkeys. The TK parameters of the highest doses following the
final dosing are shown in Table 8. The pharmacologically effective dose in mice was found to be 0.32
mg/kg. A comparison of exposure at the NOAEL in mice with that at 1 mg/kg (C : 105 nM, AUC :
max
inf
6
8.9 h·nM) in the mouse PK study indicates that DS-1971a showed a favorable safety profile in these
studies.
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This favorable safety profile is consistent the outcome of clinical studies in human, as DS-1971a has
been shown to be well tolerated and safe in clinical phase 1 studies. Among these studies, a single
ascending dose study using DS-1971a revealed good safety and tolerability up to 1500 mg
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
(
https://clinicaltrials.gov/ct2/show/record/NCT02107885), whereas a multiple ascending dose study of up
to 600 mg bid or 400 mg tid (1200 mg/day) for 14 days also found DS-1971a to be safe and tolerable
https://clinicaltrials.gov/ct2/show/NCT02190058).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
(
Table 8. Chronic In Vivo Toxicity Study of DS-1971a.
TK parameters at final dosing
Dose
Dose
mg/kg)
NOAEL
(mg/kg)
Species
Sex Duration
2
(
AUC0-24
(h·nM)
h
Cmax (nM)
Tmax (h)
2
2
2
(mg/kg)
Crl:CD1(ICR)
Mal
0, 30, 100,
1000
1000a
1000a
1000b
1000b
9440
0.5
65900
2
2
6 Months
e
1000
1000
1000
1000
mice
2
Crl:CD1(ICR)
Fem
ale
0, 30, 100,
1000
2
3
9
0
1
6
9
9
Months
Months
Months
105000
6.0
1400000
mice
3
3
3
3
3
3
3
C2 ynomolgus
m3 onkeys
Mal
e
0, 10, 100,
1000
28100
14600
±
146000
±85800
1.2 ± 0.4
1.2 ± 0.4
4
C5 ynomolgus
monkeys
Fem
ale
0, 10, 100,
1000
22500
14600
±
100000 ±
97200
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
b
Each value represents the mean ± S.D. n = 3 for each time point. See SI for the individual data. n = 6.
CONCLUSIONS
We report the discovery of a highly potent and selective Na 1.7 inhibitor, DS-1971a. Derivatization was
V
focused on the avoidance of issues such as CYP inhibition, MBI, and GSH adduct formation. The finding
that the right-hand heteroaromatic ring affects MBI liability and GSH adduct formation led to the
discovery of 4-pyrimidiyl derivatives without such liabilities. Further optimizations provided DS-1971a,
which inhibited human and mouse Na 1.7 in a highly potent and selective manner. DS-1971a
V
demonstrated unique blocking kinetics on Na 1.7: the onset and recovery from DS-1971a block were
V
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very slow compared with those of mexiletine. In vivo pharmacological profiling revealed that DS-1971a
demonstrates potent analgesic efficacy against both thermal and mechanical hypersensitivity induced by
nerve ligation. The results of in vivo safety studies (safety pharmacology studies and chronic toxicity
studies) revealed no concerns about adverse effects with DS-1971a up to 1000 mg/kg. Although DS-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
971a inhibits CYP enzymes, a sufficient margin is expected owing to the low predicted
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
pharmacologically effective concentrations. DS-1971a is expected to be an efficacious analgesic agent
with excellent safety profile.
EXPERIMENTAL SECTION
General Procedures.
Starting reagents were purchased from commercial suppliers, and were used without further
purification, unless otherwise specified. Chromatographic elution was conducted under continuous
monitoring by TLC using silica gel 60F254 (Merck & Co., Inc.) as the stationary phase; and the elution
solvent used in column chromatography as the mobile phase. A UV detector was used for detection. Silica
gel SK-85 (230–400 mesh) or silica gel SK-34 (70–230 mesh), manufactured by Merck & Co., Inc., or
Chromatorex NH (200–350 mesh), manufactured by Fuji Silysia Chemical Ltd., was used as the column-
1
packing silica gel. H NMR spectra were obtained on Varian Unity 400 and 500 MHz spectrometers.
Spectra were recorded in the indicated solvent at ambient temperature; chemical shifts are reported in
ppm (δ) relative to the solvent peak. Resonance patterns are represented with the following notations: br
(broad signal), s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). MS analysis methods were
FAB, EI, or ESI. HRMS was carried out using an LC-MS system composed of a Waters Xevo
Quadropole-ToF MS and an Acquity UHPLC system. Elemental analyses were conducted by using a
Microcorder JM10 and a Dionex ICS-1500. The purity of compounds was confirmed to exceed 95% by
the DAD signal area% performed on an Agilent Infinity 1260 LCMS system. The conditions were as
follows: column: Develosil Combi-RP-5 2.0 mmID × 50 mmL, gradient elution: 0.1% HCO H-H O/0.1%
2
2
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HCO H-MeCN = 98/2 to 0/100 (v/v), flow rate: 1.2 mL/min, UV detection: 254 nm, column temperature:
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
0°C, ionization: APCI/ESI. All assay compounds were ≥ 95% pure. All in vivo experimental procedures
were performed in accordance with the in-house guidelines of the Institutional Animal Care and Use
Committee of Daiichi Sankyo Co., Ltd.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
General Procedure for Epoxide-Opening Reaction (General Procedure A).
n-Butyllithium (1.63 M solution in hexane; 100 mL, 163 mmol) was added to a solution of 1-
methylpyrazole (13.4 g, 163 mmol) in THF (1.0 L) drop-wise at -78°C for 40 min. Epoxide (179 mmol)
was then added at −78°C to the reaction mixture, which was subsequently stirred at room temperature for
20 h. A saturated aqueous solution of NaHCO (100 mL) was added, and the mixture was extracted several
3
times with EtOAc. The combined organic layers were washed with water, dried over anhydrous Na SO ,
2
4
and concentrated under reduced pressure. The crude residue was purified by silica gel column
chromatography to obtain rac-4.
General Procedure for S Ar Reaction to Prepare 9 (General Procedure B).
N
NaH (2.63 mmol) was added to a solution of protected benzenesulfonamide 3 (1.35 mmol) and alcohol
4
(1.36 mmol) in DMSO or DMF (6.0 mL) at 0°C under N , and the mixture was stirred at room
2
temperature for 1 h. Water was added to the cooled mixture, which was then extracted with EtOAc several
times. The combined organic layers were washed with water, dried over anhydrous Na SO , and
2
4
concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography
to obtain compound 5.
General Procedure for Deprotection (General Procedure C).
The solution of 5 (0.449 mmol), Et SiH (0.30 mL), and TFA (3.0 mL) in CH Cl (3.0 mL) was stirred
3
2
2
at room temperature for 1 h. The reaction mixture was concentrated, and the residue was purified via silica
gel chromatography to yield 6.
General Procedure for Imine Formation, Followed by Reduction (General Procedure D).
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A solution of heteroaromatic amine 1 (210 mmol), 2,4-dimethoxybenzaldehyde (69.9 g, 421 mmol),
1
2
and piperidine (2.08 mL, 21.0 mmol) in toluene (1.0 L) was refluxed with azeotropic removal of water
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
for 7 h. After the cooled reaction mixture was diluted with EtOH (500 mL), NaBH (7.96 g, 210 mmol)
4
was added to the mixture at 0°C. After stirring at room temperature for 16 h, water was added, and the
mixture was extracted with EtOAc several times. The combined organic layers were washed with water,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
dried over anhydrous Na SO , and concentrated under reduced pressure. The crude residue was purified
2
4
by silica gel column chromatography to obtain compound 2.
General Procedure for Sulfonamide Formation (General Procedure E).
LiHMDS (1.0 M solution in THF; 4.8 mL, 4.8 mmol) was added to a solution of protected amine 2
(4.00 mmol) in THF (12 mL) dropwise at −78°C. After stirring at −78°C for 10 min, sulfonyl chloride
(
4.42 mmol) was added at −78°C to the reaction mixture, which was then stirred at room temperature for
h. Water was added, and the mixture was extracted with EtOAc several times. The combined organic
layers were washed with water, dried over anhydrous Na SO , and concentrated under reduced pressure.
2
2
4
The crude residue was purified by silica gel column chromatography to obtain 3.
Alternative General Procedure for Sulfonamide Formation (General Procedure F).
Sulfonyl chloride (6.20 mmol) was added to a solution of protected amine 2 (0.76 g, 3.10 mmol) and
1
,4-diazabicyclo[2. 2.2]octane (0.70 g, 6.20 mmol) in MeCN (20 mL) at 0°C, which was then was stirred
at room temperature for 1 h. After the reaction mixture was filtrated, the residue was concentrated, and
the residue was purified with silica gel chromatography to yield compound 3.
N-[(2,4-Dimethoxyphenyl)methyl]-3-methyl-1,2,4-thiadiazol-5-amine (2d)
1
Prepared according to General Procedure D. Yield 60%. H-NMR (500 MHz, CDCl ) δ 2.36 (3H, s),
3
3.78 (3H, s), 3.79 (3H, s), 4.29 (2H, d, J = 5.9 Hz), 6.13 (1H, br), 6.40–6.45 (2H, m), 7.15 (1H, d, J = 8.3
Hz).
N-[(2,4-Dimethoxyphenyl)methyl]pyrimidin-4-amine (2g)
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Prepared according to General Procedure D. Yield 52%. H-NMR (500 MHz, CDCl ) δ 3.80 (3H, s),
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
.84 (3H, s), 4.44 (2H, br), 5.33 (1H, br), 6.34 (1H, d, J = 5.9 Hz), 6.44 (1H, dd, J = 2.4, 8.3 Hz), 6.48
(1H, d, J = 2.0 Hz), 7.18 (1H, d, J = 8.3 Hz), 8.15 (1H ,d, J = 5.4 Hz), 8.55 (1H, s).
N-[(2,4-Dimethoxyphenyl)methyl]-2,4,5-trifluoro-N-(3-methyl-1,2,4-thiadiazol-5-yl)benzene-1-
sulfonamide (3d)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
Prepared according to General Procedure E. Yield 79%. H-NMR (500 MHz, CDCl ) δ 2.45 (3H, s),
3
3
.69 (3H, s), 3.73 (3H, s), 5.29 (2H, s), 6.21 (1H, d, J = 2.4 Hz), 6.34 (1H, dd, J = 2.2, 8.5 Hz), 6.88–6.93
(1H, m), 7.18 (1H, d, J = 8.8 Hz), 7.58–7.63 (1H, m).
N-[(2,4-Dimethoxyphenyl)methyl]-2,4,5-trifluoro-N-(1,3-thiazol-2-yl)benzene-1-sulfonamide (3e)
1
Prepared according to General Procedure F. Yield 54%. H-NMR (400 MHz, CDCl ) δ 3.73 (3H, s),
3
3
.77 (3H, s), 5.19 (2H, s), 6.36–6.39 (2H, m), 7.01–7.07 (2H, m), 7.19 (1H, d, J = 8.2 Hz), 7.42 (1H, d, J
= 3.5 Hz), 7.70–7.76 (1H, m).
N-[(2,4-Dimethoxyphenyl)methyl]-2,4,5-trifluoro-N-(pyrimidin-4-yl)benzene-1-sulfonamide (3g)
1
Prepared according to General Procedure F. Yield 53%. H-NMR (500 MHz, CDCl ) δ 3.78 (3H, s),
3
3
.80 (3H, s), 5.23 (2H, s), 6.42-6.43 (2H, m), 6.99–7.04 (1H, m), 7.13 (1H, d, J = 5.9 Hz), 7.22 (1H, d, J
=
9.3 Hz), 7.91–7.96 (1H, m), 8.48 (1H, d, J = 6.4 Hz), 8.78 (1H, s).
N-[(2,4-Dimethoxyphenyl)methyl]-3,4,5-trifluoro-N-(pyrimidin-4-yl)benzene-1-sulfonamide (3i)
1
Prepared according to General Procedure F. Yield 39%. H-NMR (500 MHz, CDCl ) δ 3.64 (3H, s),
3
3.76 (3H, s), 5.11 (2H, s), 6.35 (1H, d, J = 2.4 Hz), 6.39 (1H, dd, J = 2.4, 8.8 Hz), 7.11–7.15 (2H, m),
7
.49–7.53 (2H, m), 8.52 (1H, d, J = 6.8 Hz), 8.86 (1H, s).
N-[(2,4-Dimethoxyphenyl)methyl]-2,3,4-trifluoro-N-(pyrimidin-4-yl)benzene-1-sulfonamide (3j)
1
Prepared according to General Procedure F. Yield 31%. H-NMR (500 MHz, CDCl ) δ 3.78 (3H, s),
3
3
.80 (3H, s), 5.24 (2H, s), 6.42–6.44 (2H, m), 7.11-7.16 (2H, m), 7.22 (1H, d, J = 7.8 Hz), 7.84–7.89 (1H,
m), 8.48 (1H, d, J = 5.9 Hz), 8.76 (1H, s).
N-[(2,4-Dimethoxyphenyl)methyl]-2,4,6-trifluoro-N-(pyrimidin-4-yl)benzene-1-sulfonamide (3k)
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Prepared according to General Procedure F. Yield 18%. H-NMR (500 MHz, CDCl ) δ 3.73 (3H, s),
3
1
2
3
.78 (3H, s), 5.26 (2H, s), 6.42–6.46 (2H, m), 6.78 (2H, t, J = 8.3 Hz), 7.07 (1H, dd, J = 1.5, 5.9 Hz), 7.24
1H, d, J = 8.8 Hz), 8.46 (1H, d, J = 6.4 Hz), 8.78 (1H, s).
-Chloro-N-[(2,4-dimethoxyphenyl)methyl]-2,4-difluoro-N-(pyrimidin-4-yl)benzene-1-
sulfonamide (3n)
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
Prepared according to General Procedure F. Yield 26%. H-NMR (500 MHz, CDCl ) δ 3.78 (3H, s),
3
3
.79 (3H, s), 5.23 (2H, s), 6.41–6.43 (2H, m), 6.98 (1H, d, J = 9.3 Hz), 7.16 (1H, d, J = 7.3 Hz), 7.22 (1H,
d, J = 8.8 Hz), 8.13 (1H, t, J = 7.3 Hz), 8.49 (1H, d, J = 5.9 Hz), 8.79 (1H, s).
N-[(2,4-Dimethoxyphenyl)methyl]-2,4-difluoro-5-methyl-N-(pyrimidin-4-yl)benzene-1-
sulfonamide (3p)
1
Prepared according to General Procedure F. Yield 79%. H-NMR (500 MHz, CDCl ) δ 2.31 (3H, s),
3
3
7
.77 (3H, s), 3.79 (3H, s), 5.25 (2H, s), 6.40-6.42 (2H, m), 6.83 (1H, t, J = 9.3 Hz), 7.20–7.23 (2H, m),
.89 (1H, t, J = 7.8 Hz), 8.45 (1H, d, J = 5.9 Hz), 8.77 (1H, s).
2
-(1-Methyl-1H-pyrazol-5-yl)ethan-1-ol (4a)
1
Prepared according to General Procedure A. Yield 31%. H-NMR (400 MHz, CDCl ) δ 2.92 (2H, t, J
3
=
6.7 Hz), 3.84 (3H, s), 3.89-3.93 (2H, m), 6.12 (1H, d, J = 2.0 Hz), 7.42 (1H, s).
(1S*,2R*)-2-(1-Methyl-1H-pyrazol-5-yl)cyclopentan-1-ol (rac-4b)
1
Prepared according to General Procedure A. Yield 21%. H-NMR (500 MHz, CDCl ) δ 1.63–-1.91 (4H,
3
m), 2.05-2.12 (1H, m), 2.17-2.24 (1H, m), 3.03 (1H, q, J = 8.3 Hz), 3.86 (3H, s), 4.24 (1H, q, J = 6.4 Hz),
6.03 (1H, s), 7.39 (1H, s).
(1S*,2R*)-2-(1-Methyl-1H-pyrazol-5-yl)cyclohexan-1-ol (rac-4c)
1
Prepared according to General Procedure A. Yield 55%. H-NMR (400 MHz, CDCl ) δ 1.30-1.48 (4H,
3
m), 1.76–1.91 (4H, m), 2.09–2.15 (1H, m), 2.57-2.63 (1H, m), 3.59–3.65 (1H, m), 3.86 (3H, s), 6.08 (1H,
d, J = 2.0 Hz), 7.44 (1H, d, J = 2.0 Hz).
(1S*,2R*)-2-(1-Methyl-1H-pyrazol-5-yl)cycloheptan-1-ol (rac-4m)
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Prepared according to General Procedure A. Yield 13%. H-NMR (500 MHz, CDCl ) δ 1.56–1.89 (9H,
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
m), 1.98–2.05 (1H, m), 2.76–2.82 (1H, m), 3.80–3.86 (1H, m), 3.84 (3H, s), 6.06 (1H, d, J = 2.0 Hz), 7.41
(1H, d, J = 2.4 Hz).
(1S,2R)-2-(1-Methyl-1H-pyrazol-5-yl)cyclohexan-1-ol (4o)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(1S*,2R*)-2-(1-Methyl-1H-pyrazol-5-yl)cyclohexan-1-ol (rac-4c) was optically resolved with
CHIRALPAK IB (Daicel Corp.; hexane/ethanol = 9: 1) to yield the title compound as a colorless oil.
[
α]25 = 33.3 (c 0.916, MeOH).
D
N-[(2,4-Dimethoxyphenyl)methyl]-2,5-difluoro-4-[2-(1-methyl-1H-pyrazol-5-yl)ethoxy]-N-(1,2,4-
thiadiazol-5-yl)benzene-1-sulfonamide (5a)
1
Prepared according to General Procedure B. Yield 16%. H-NMR (400 MHz, CDCl ) δ 3.21 (2H, t, J
3
= 6.3 Hz), 3.73 (6H, s), 3.93 (3H, s), 4.23 (2H, t, J = 6.7 Hz), 5.30 (2H, s), 6.15 (1H, d, J = 2.0 Hz), 6.25
(1H, d, J = 2.0 Hz), 6.36 (1H, dd, J = 2.3, 8.6 Hz), 6.59 (1H, dd, J = 6.5, 10.8 Hz), 7.19 (1H, d, J = 6.7
Hz), 7.44 (1H, d, J = 2.0 Hz), 7.55 (1H, dd, J = 5.9, 9.8 Hz), 8.20 (1H, s).
N-[(2,4-Dimethoxyphenyl)methyl]-2,5-difluoro-4-{[(1S*,2R*)-2-(1-methyl-1H-pyrazol-5-
yl)cyclopentyl]oxy}-N-(1,2,4-thiadiazol-5-yl)benzene-1-sulfonamide (rac-5b)
1
Prepared according to General Procedure B. Yield 51%. H-NMR (400 MHz, CDCl ) δ 1.76–1.99 (4H,
3
m), 2.17–2.24 (1H, m), 2.27–2.35 (1H, m), 3.44–3.50 (1H, m), 3.71 (6H, s), 3.87 (3H, s), 4.57–4.61 (1H,
m), 5.29 (2H, s), 6.06 (1H, d, J = 2.0 Hz), 6.24 (1H, d, J = 2.4 Hz), 6.34 (1H, dd, J = 2.4, 8.2 Hz), 6.45
(1H, dd, J = 6.3, 11.0 Hz), 7.16 (1H, d, J = 8.3 Hz), 8.41 (1H, d, J = 2.0 Hz), 7.53 (1H, dd, J = 6.7, 10.2
Hz), 8.18 (1H, s).
N-[(2,4-Dimethoxyphenyl)methyl]-2,5-difluoro-4-{[(1S*,2R*)-2-(1-methyl-1H-pyrazol-5-
yl)cyclohexyl]oxy}-N-(1,2,4-thiadiazol-5-yl)benzene-1-sulfonamide (rac-5c)
1
Prepared according to General Procedure B. Yield 42%. H-NMR (400 MHz, CDCl ) δ 1.39–1.70 (4H,
3
m), 1.87–1.90 (1H, m), 1.94–1.98 (1H, m), 2.05–2.09 (1H, m), 2.17–2.21 (1H, m), 2.97–3.03 (1H, m),
3
.65 (3H, s), 3.74 (3H, s), 3.90 (3H, s), 4.08 (1H, dt, J = 3.9, 10.2 Hz), 5.21 (1H, d, J = 15.7 Hz), 5.28
(1H, d, J = 15.7 Hz), 6.04 (1H, d, J = 2.0 Hz), 6.19 (1H, d, J = 2.0 Hz), 6.34 (1H, dd, J = 2.4, 8.6 Hz),
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.34-6.39 (1H, m), 7.15 (1H, d, J = 8.2 Hz), 7.36 (1H, d, J = 1.6 Hz), 7.41 (1H, dd, J = 6.7, 10.2 Hz), 8.18
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(1H, s).
N-[(2,4-Dimethoxyphenyl)methyl]-2,5-difluoro-4-{[(1S*,2R*)-2-(1-methyl-1H-pyrazol-5-
yl)cyclopentyl]oxy}-N-(3-methyl-1,2,4-thiadiazol-5-yl)benzene-1-sulfonamide (rac-5d)
1
Prepared according to General Procedure B. Yield 57%. H-NMR (500 MHz, CDCl ) δ 1.74–1.99 (4H,
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
m), 2.14–2.32 (2H, m), 2.43 (3H, s), 3.42–3.46 (1H, m), 3.69 (3H, s), 3.70 (3H, s), 3.84 (3H, s), 4.29 (1H,
d, J = 5.9 Hz), 4.54–4.57 (1H, m), 5.24 (2H, s), 6.02 (1H, d, J = 2.0 Hz), 6.22 (1H, d, J = 2.4 Hz), 6.31
(1H, dd, J = 2.4, 8.3 Hz), 6.40–6.44 (1H, m), 7.15 (1H, t, J = 7.3 Hz), 7.38 (1H, d, J = 2.0 Hz), 7.50 (1H,
dd, J = 6.8, 9.8 Hz).
N-[(2,4-Dimethoxyphenyl)methyl]-2,5-difluoro-4-{[(1S*,2R*)-2-(1-methyl-1H-pyrazol-5-
yl)cyclopentyl]oxy}-N-(1,3-thiazol-2-yl)benzene-1-sulfonamide (rac-5e)
1
Prepared according to General Procedure B. Yield 73%. H-NMR (500 MHz, CDCl ) δ 1.76–1.83 (1H,
3
m), 1.88–1.98 (3H, m), 2.19–2.25 (1H, m), 2.27–2.33 (1H, m), 3.44–3.49 (1H, m), 3.73 (3H, s), 3.75 (3H,
s), 3.86 (3H, s), 4.61–4.64 (1H, m), 5.18 (2H, s), 6.05 (1H, d, J = 2.0 Hz), 6.36–6.38 (2H, m), 6.56 (1H,
dd, J = 6.4, 11.2 Hz), 6.98 (1H, d, J = 3.4 Hz), 7.19 (1H, d, J = 8.3 Hz), 7.38–7.41 (2H, m), 7.58 (1H, dd,
J = 6.4, 10.3 Hz).
tert-Butyl
(2,5-difluoro-4-{[(1S*,2R*)-2-(1-methyl-1H-pyrazol-5-yl)cyclohexyl]oxy}benzene-1-
sulfonyl)1,3-thiazol-4-ylcarbamate (rac-5f)
1
Prepared according to General Procedure B. Yield 57%. H-NMR (400 MHz, CDCl ) δ 1.35 (9H, s),
3
1.44–1.70 (4H, m), 1.88–1.91 (1H, m), 1.96–1.99 (1H, m), 2.06–2.10 (1H, m), 2.26–2.30 (1H, m), 3.00-
3.06 (1H, m), 3.92 (3H, s), 4.18 (1H, dt, J = 3.9, 10.2 Hz), 6.04 (1H, d, J = 2.0 Hz), 6.57 (1H, dd, J = 6.3,
11.0 Hz), 7.36 (1H, d, J = 2.0 Hz), 7.49 (1H, d, J = 2.4 Hz), 7.76 (1H, dd, J = 6.3, 10.2 Hz), 8.78 (1H, d,
J = 2.4 Hz).
N-[(2,4-Dimethoxyphenyl)methyl]-2,5-difluoro-4-{[(1S*,2R*)-2-(1-methyl-1H-pyrazol-5-
yl)cyclopentyl]oxy}-N-(pyrimidin-4-yl)benzene-1-sulfonamide (rac-5g)
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1
Prepared according to General Procedure B. Yield 88%. H-NMR (500 MHz, CDCl ) δ 1.78–1.97 (4H,
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
m), 2.20–2.33 (2H, m), 3.45–3.49 (1H, m), 3.77 (3H, s), 3.79 (3H, s), 3.86 (3H ,s), 4.60–4.64 (1H, m),
5.23 (2H, s), 6.05 (1H, d, J = 2.0 Hz), 6.40–6.42 (2H, m), 6.52 (1H, dd, J = 5.9, 10.7 Hz), 7.18–7.20 (2H,
m), 7.40 (1H, d, J = 2.0 Hz), 7.76 (1H, dd, J = 6.4, 10.3 Hz), 8.45 (1H, d, J = 5.9 Hz), 8.78 (1H, s).
N-[(2,4-Dimethoxyphenyl)methyl]-2,5-difluoro-4-{[(1S*,2R*)-2-(1-methyl-1H-pyrazol-5-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
yl)cyclohexyl]oxy}-N-(pyrimidin-4-yl)benzene-1-sulfonamide (rac-5h)
1
Prepared according to General Procedure B. Yield 80%. H-NMR (500 MHz, CDCl ) δ 1.39–1.68 (4H,
3
m), 1.86–1.96 (2H, m), 2.04–2.07 (1H, m), 2.22–2.25 (1H, m), 2.98–3.03 (1H, m), 3.76 (3H, s), 3.77 (3H,
s), 3.91 (3H ,s), 4.08–4.14 (1H, m), 5.19 (1H, d, J = 17.1 Hz), 5.23 (1H, d, J = 16.6 Hz), 6.02 (1H, d, J =
2
.0 Hz), 6.39–6.40 (2H, m), 6.47 (1H, dd, J = 6.4, 11.2 Hz), 7.17–7.19 (2H, m), 7.33 (1H, d, J = 1.5 Hz),
7.67 (1H, dd, J = 6.4, 9.8 Hz), 8.45 (1H, d, J = 5.9 Hz), 8.78 (1H, s).
N-[(2,4-Dimethoxyphenyl)methyl]-3,5-difluoro-4-{[(1S*,2R*)-2-(1-methyl-1H-pyrazol-5-
yl)cyclopentyl]oxy}-N-(pyrimidin-4-yl)benzene-1-sulfonamide (rac-5i)
1
Prepared according to General Procedure B. Yield 72%. H-NMR (500 MHz, CDCl ) δ 1.71–1.99 (5H,
3
m), 2.27–2.36 (1H, m), 3.38–3.46 (1H, m), 3.65 (3H, s), 3.74 (3H, s), 3.83 (3H, s), 4.80–4.83 (1H, m),
5
.13 (2H, s), 5.98 (1H, d, J = 1.5 Hz), 6.34 (1H, d, J = 2.4 Hz), 6.37 (1H, dd, J = 2.2, 8.5 Hz), 7.11 (1H,
d, J = 8.3 Hz), 7.17 (1H, dd, J = 1.0, 5.9 Hz), 7.34 (1H, d, J = 2.0 Hz), 7.39 (2H, d, J = 7.8 Hz), 8.50 (1H,
d, J = 5.9 Hz), 8.85 (1H, d, J = 1.0 Hz).
N-[(2,4-Dimethoxyphenyl)methyl]-2,3-difluoro-4-{[(1S*,2R*)-2-(1-methyl-1H-pyrazol-5-
yl)cyclopentyl]oxy}-N-(pyrimidin-4-yl)benzene-1-sulfonamide (rac-5j)
1
Prepared according to General Procedure B. Yield 75%. H-NMR (500 MHz, CDCl ) δ 1.77–1.97 (4H,
3
m), 2.22–2.34 (2H, m), 3.44–3.48 (1H, m), 3.76 (3H, s), 3.79 (3H ,s), 3.85 (3H, s), 4.72–4.75 (1H, m),
5
.24 (1H, d, J = 17.1 Hz), 5.28 (1H, d, J = 16.6 Hz), 6.07 (1H, d, J = 2.0 Hz), 6.39–6.42 (2H, m), 6.64
1H, t, J = 8.8 Hz), 7.19–7.21 (2H, m), 7.41 (1H, d, J = 2.0 Hz), 7.71 (1H, t, J = 8.8 Hz), 8.45 (1H, d, J =
.9 Hz), 8.76 (1H, s).
(
5
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Page 29 of 52
Journal of Medicinal Chemistry
N-[(2,4-Dimethoxyphenyl)methyl]-2,6-difluoro-4-{[(1S*,2R*)-2-(1-methyl-1H-pyrazol-5-
1
2
yl)cyclopentyl]oxy}-N-(pyrimidin-4-yl)benzene-1-sulfonamide (rac-5k)
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
Prepared according to General Procedure B. Yield 75%. H-NMR (500 MHz, CDCl ) δ 1.72–1.95 (4H,
3
m), 2.17–2.32 (2H, m), 3.35–3.39 (1H, m), 3.77 (3H, s), 3.82 (6H ,s), 4.62–4.65 (1H, m), 5.27 (2H, s),
6
.04 (1H, d, J = 2.0 Hz), 6.39–6.44 (4H, m), 7.16 (1H, d, J = 7.3 Hz), 7.22 (1H, d, J = 7.3 Hz), 7.41 (1H,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
d, J = 2.0 Hz), 8.44 (1H, d, J = 5.9 Hz), 8.78 (1H, s).
N-[(2,4-Dimethoxyphenyl)methyl]-2,6-difluoro-4-{[(1S*,2R*)-2-(1-methyl-1H-pyrazol-5-
yl)cyclohexyl]oxy}-N-(pyrimidin-4-yl)benzene-1-sulfonamide (rac-5l)
1
Prepared according to General Procedure B. Yield 48%. H-NMR (500 MHz, CDCl ) δ 1.38–1.67 (4H,
3
m), 1.86–1.88 (1H, m), 1.94–1.95 (1H, m), 2.03–2.06 (1H, m), 2.22–2.24 (1H, m), 2.90–2.95 (1H, m),
3
.77 (3H, s), 3.81 (3H, s), 3.86 (3H ,s), 4.10–4.15 (1H, m), 5.24 (2H, s), 5.99 (1H, d, J = 2.0 Hz), 6.29
(
2H, d, J = 10.7 Hz), 6.40–6.44 (2H, m), 7.14 (1H, dd, J = 1.0, 5.9 Hz), 7.21 (1H, d, J = 8.3 Hz), 7.34
(
1H, d, J = 2.0 Hz), 8.44 (1H, d, J = 5.9 Hz), 8.78 (1H, s).
N-[(2,4-Dimethoxyphenyl)methyl]-2,6-difluoro-4-{[(1S*,2R*)-2-(1-methyl-1H-pyrazol-5-
yl)cycloheptyl]oxy}-N-(pyrimidin-4-yl)benzene-1-sulfonamide (rac-5m)
1
Prepared according to General Procedure B. Yield 52%. H-NMR (500 MHz, CDCl ) δ 1.60–1.98 (10H,
3
m), 3.15 (1H, dt, J = 2.9, 9.3 Hz), 3.77 (3H, s), 3.81 (3H, s), 3.85 (3H ,s), 4.36–4.40 (1H, m), 5.25 (2H,
s), 5.98 (1H, d, J = 2.0 Hz), 6.27 (2H, d, J = 10.7 Hz), 6.41 (1H, dd, J = 2.4, 8.3 Hz), 6.44 (1H, d, J = 2.0
Hz), 7.16 (1H, dd, J = 1.5, 5.9 Hz), 7.21 (1H, d, J = 8.3 Hz), 7.33 (1H, d, J = 2.0 Hz), 8.44 (1H, d, J = 5.9
Hz), 8.78 (1H, s).
5-Chloro-N-[(2,4-dimethoxyphenyl)methyl]-2-fluoro-4-{[(1S*,2R*)-2-(1-methyl-1H-pyrazol-5-
yl)cyclopentyl]oxy}-N-(pyrimidin-4-yl)benzene-1-sulfonamide (rac-5n)
1
Prepared according to General Procedure B. Yield 84%. H-NMR (500 MHz, CDCl ) δ 1.73–1.98 (4H,
3
m), 2.17–2.35 (2H, m), 3.48–3.52 (1H, m), 3.76 (3H, s), 3.78 (3H, s), 3.88 (3H ,s), 4.60–4.63 (1H, m),
5
.22 (1H, d, J = 17.1 Hz), 5.26 (1H, d, J = 17.1 Hz), 6.06 (1H, d, J = 1.5 Hz), 6.39–6.41 (2H, m), 6.48
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