Tetracyclic spirooxindole blockers of hNaV1.7: Activity in vitro and in CFA-induced inflammatory pain model

Article abstract of DOI:10.1007/s00044-012-0180-1

The structure-activity relationship of a new series of tetracyclic spirooxindoles led to the discovery of compound 25a, a potent hNa_V1.7 blocker with improved ADME properties and in vivo efficacy in the CFA-induced inflammatory pain model.

Full text of DOI:10.1007/s00044-012-0180-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:1825–1836
DOI 10.1007/s00044-012-0180-1
ORIGINAL RESEARCH
Tetracyclic spirooxindole blockers of hNaV1.7: activity in vitro
and in CFA-induced inflammatory pain model
•
Sultan Chowdhury Shifeng Liu Jay A. Cadieux Tom Hsieh
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Mikhail Chafeev Shaoyi Sun Qi Jia Jianyu Sun Mark Wood
•
•
Jonathan Langille Serguei Sviridov Jianmin Fu Zaihui Zhang
•
•
•
•
Ray Chui Audrey Wang Xing Cheng Jing Zhong Sazzad Hossain
•
•
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•
•
Kuldip Khakh Ivana Rajlic Henry Verschoof Rainbow Kwan
Wendy Young
Received: 6 January 2012 / Accepted: 21 July 2012 / Published online: 12 August 2012
ꢀ Springer Science+Business Media, LLC 2012
Introduction
Abstract The structure–activity relationship of a new
series of tetracyclic spirooxindoles led to the discovery of
compound 25a, a potent hNaV1.7 blocker with improved
ADME properties and in vivo efficacy in the CFA-induced
inflammatory pain model.
hNaV1.7 is one of the voltage-gated sodium channel sub-
types involved in pain conditions and is considered an
important therapeutic target for pharmaceutical interven-
tion based on compelling genetic evidence (Cummins
et al., 2007). hNaV1.7 is mainly expressed in the peripheral
nervous system (Klugbauer et al., 1995; Toledo-Aral et al.,
1997; Sangameswaran et al., 1997). There are many sci-
entific reports describing the critical role of the hNaV1.7
subtype in the perception of pain sensations, especially in
inflammatory contexts (Goldberg et al., 2007; Cox et al.,
2006; Reimann et al., 2010; Zhang et al., 2010) and may
represent an important target for analgesic/anti-hyper-
algesic drugs (Ghelardini et al., 2010). Studies have shown
that loss-of-function mutations in hNaV1.7 are associated
with congenital insensitivity to painful stimuli (Yang et al.,
2004; Cummins et al., 2004; Fertleman et al., 2006).
Several marketed drugs used in the treatment of neuro-
pathic pain, such as carbamazepine, oxcarbazepine, lido-
caine, lamotrigine, topiramate, and mexiletine, are either
weak sodium channel antagonists or possess alternate
mechanisms of action which limit their clinical efficacy
side effect profiles (Zuliani et al., 2010). Our goal is to
develop more potent, state-dependent hNaV1.7 blockers for
the treatment of pain.
Keywords Sodium channel blockers ꢀ NaV1.7 blockers ꢀ
Pain ꢀ Tetracyclic spirooxindole ꢀ Methylenedioxy ꢀ
Mitsunobu reaction
Abbreviations
hNaV1.7 human sodium channel 1.7 subtype
CYP
DMF
CFA
IP
Cytochrome P450
N,N-Dimethylformamide
Complete Freund’s adjuvant
Intraperitoneal
TMSCl
THF
Chlorotrimethylsilane
Tetrahydrofuran
MOMCl Chloromethoxymethyl ether
tert-BuLi tert-butyllithium
TFA
Trifluoroacetic acid
ADME
Absorption, distribution, metabolism, and
excretion
Recently, we reported the discovery of XEN907 (1),
a novel pentacyclic spirooxindole blocker of hNaV1.7
(Chowdhury et al., 2011). This compound showed high in
vitro hNaV1.7 potency, moderate bioavailability in male
Lewis rats and poor solubility in phosphate-buffered saline
(Fig. 1). XEN907 (1) also showed moderate hepatocyte
stability across species and significant inhibition of
CYP3A4 was observed in a recombinant human enzyme
assay. These results prompted us to direct our medicinal
S. Chowdhury (&) ꢀ S. Liu ꢀ J. A. Cadieux ꢀ T. Hsieh ꢀ
M. Chafeev ꢀ S. Sun ꢀ Q. Jia ꢀ J. Sun ꢀ M. Wood ꢀ J. Langille ꢀ
S. Sviridov ꢀ J. Fu ꢀ Z. Zhang
Department of Medicinal Chemistry, Xenon Pharmaceuticals
Inc., 3650 Gilmore Way, Burnaby, BCV5G 4W8, Canada
e-mail: schowdhury@xenon-pharma.com
R. Chui ꢀ A. Wang ꢀ X. Cheng ꢀ J. Zhong ꢀ S. Hossain ꢀ
K. Khakh ꢀ I. Rajlic ꢀ H. Verschoof ꢀ R. Kwan ꢀ W. Young
Department of Biological Sciences, Xenon Pharmaceuticals Inc.,
3650 Gilmore Way, Burnaby, BCV5G 4W8, Canada
123

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Med Chem Res (2013) 22:1825–1836
Fig. 1 Structure and ADME
properties of XEN907 (1)
O
N
O
O
IC50 (hNaV1.7) = 3 nM
clogP = 3.97
Solubility = 7.3 g/mL (phosphate-buffered saline @ pH 7.4)
µ
PPB (rat) = 97%
Cytotox: > 99%
O
Heptocyte stability = 21% (rat)/34% (human)/46% (dog)
F =13% (po @10 mg/kg)
nPent
XEN907 (1)
chemistry efforts to evaluate tetracyclic analogs lacking the
methylenedioxy moiety which is presumably responsible
for CYP induction (Staack and Maurer, 2005; Castro et al.,
2003; Imperio et al., 2007).
of the spirooxindole scaffold (Table 1). In order to
decrease the electron density of the aryl head group in
an effort to reduce the extent of oxidative metabolism,
a number of electron-withdrawing substituents were
explored. While compounds 8a and 8b bearing fluorine and
methoxy substituents were 20- and 80-fold less potent in
vitro against hNaV1.7 versus compound 1, they did dem-
onstrate reduced CYP3A4 inhibition. A further reduction in
CYP3A4 inhibition could be obtained by introducing a
polar (pyridin-2-yl)methyl substituent at the N-position
(compound 8c). Compared to the 3,4-difluorophenyl
Results and discussion
In order to establish a structure–activity relationship for
surrogates of the methylenedioxy moiety, we designed and
synthesized a series of analogs modifying the head group
Table 1 Structure-activity relationship of spirooxindoles (1, 8a–8n)
O
R1
R2
O
N
R3
Compound
R1
R2
R3
hNaV1.7a
IC50 (lM)
CYP3A4, % inhibition
at 10 lMb
Solubilityc
(lg/mL)
1
–OCH2–
F
–O–
F
n-pentyl
n-pentyl
n-pentyl
0.003
0.24
0.06
0.27
0.008
0.19
0.47
0.23
1.15
0.15
0.01
0.11
0.43
0.70
[1.0
97
50
64
30
29
36
16
43
61
22
35
18
69
75
10
7.3
\5
\5
13
25
15
36
6
8a
8b
8c
8d
8e
8f
OCH3
F
H
F
(Pyridin-2-yl)methyl
(Pyridin-2-yl)methyl
(Pyridin-2-yl)methyl
(Pyridin-2-yl)methyl
(Pyridin-2-yl)methyl
(Pyridin-2-yl)methyl
(Pyridin-2-yl)methyl
OCH3
OCH3
F
H
F
OCH3
CH3
CH3
H
8g
8h
8i
CH3
OCH3
O(CH2)2OCH3
OCH3
OCH3
OCH3
OCH3
F
7
46
14
45
\5
8
8j
H
(Tetrahydro-2H-pyran-2-yl)methyl
((R)-Tetrahydrofuran-2-yl)methyl
(5-(Trifluoromethyl)furan-2-yl)methyl
(5-Chlorothiophen-2-yl)methyl
8k
8l
H
F
8m
8n
OCH3
CN
(3-(Trifluoromethyl)-pyridin-2-yl)
methyl
\5
a
[
14C]guanidinium influx assay adapted for use in HEK-293 cells expressing human NaV1.7 (Reddy et al., 1998)
b
c
Human recombinant fluorogenic assay (Trubetskoy et al., 2005; Cohen et al., 2003) (n = 3)
Solubility at pH 7.4 in phosphate-buffered saline containing 1 % v/v DMSO
123

Med Chem Res (2013) 22:1825–1836
1827
analogs 8a and 8c, the mono-methoxy analogs 8b, 8d, and
8j remained highly potent against hNaV1.7 while main-
taining reduced CYP3A4 activity. For the pyridyl com-
pounds 8c, 8e–i, and 8n, reduction in CYP3A4 inhibition
correlated with reduced hNaV1.7 potency. A combination
of electron-withdrawing and electron-donating substituents
(8e–f, 8l) retained moderate to good potency on hNaV1.7,
suggesting that an H-bond acceptor at either the 3- or
4-positions of the phenyl ring was preferred. Further,
substitution with a 3,4-dimethylphenyl group (8g) retained
potency, but the 3-methyl-4-methoxyphenyl analog (8h)
was less potent. We also replaced the 5-membered meth-
ylenedioxy moiety with an open-chain 2-methoxyethoxy
group (8i) which retained potency (IC50 = 0.15 lM). Also,
we noted that, in general, target potency was quite sensitive
to the nature of the aryl substituents for a given N-sub-
stituent. For instance, compound 8d, bearing an N-(pyridyl)
methyl substituent along with a single methoxy function on
the aryl head was a very potent blocker of hNaV1.7
(IC50 = 8 nM); on the other hand, the analogous com-
pounds 8e (IC50 = 0.19 lM) and 8h (IC50 = 1.15
lM) differing from 8d only by the addition of a fluorine
atom and methyl group, respectively, were significantly
less potent. The 3,4-difluorophenyl compounds 8a and 8c
retained some potency against hNaV1.7, whereas the cor-
responding 3-cyano-4-fluorophenyl compound 8n was
significantly less potent.
These structural modifications were also aimed at
attenuating CYP3A4 inhibition potential and enhancing
aqueous solubility. Only six compounds (8c–e, 8i, 8j, and
8k) met the above criteria. Although we were able to obtain
highly potent compounds with low CYP3A4 inhibition,
such as 8d, this compound was less efficacious than
XEN907 in the complete Freund’s adjuvant (CFA) model
of inflammatory pain. This lack of in vivo efficacy, in
conjunction with the moderate CYP3A4 inhibition and
poor solubility, led us to continue our search for a
replacement for the methylenedioxy moiety.
The above spirooxindole compounds (8a–m) were syn-
thesized in a 5-step process (Scheme 1). Commercially
available isatin 2 was converted in high yield to N-alkylated
isatins 3a–f using different alkylating agents and NaH in
DMF. Following a literature precedent (Hewawasam and
Erway, 1998), the substituted phenols 4a–i were treated with
iso-PrMgCl at 0 ꢁC for 30 min to generate the correspond-
ing alkoxides which were then combined with N-alkyl isa-
tins 3a–f to provide 3-hydroxy-oxindoles 5a–m in high
yield. Dehydroxylation of compounds 5a–m was achieved
by treatment with triethylsilane and trifluoroacetic acid in
dichloromethane to afford compounds 6a–m in excellent
yield (Melch and Williams, 1993; Brueckner et al., 1988).
Intermediates 7a–n were synthesized under Mukaiyama–
aldol conditions (silylenol ether generation with TMSCl and
an organic base in CH2Cl2 at 0 ꢁC for 2 h, followed by
Scheme 1 Reagents and
O
O
conditions: (a) NaH (1.5 eq),
DMF, 0 ꢁC, 0.5 h then R3-X
(1.2 eq) (85–98 %); (b) iso-
PrMgCl (1.5 eq), THF, 0 ꢁC,
0.5 h then 3a-f, 0 ꢁC to rt,
4–16 h (60–85 %); (d) TFA,
Et3SiH, CH2Cl2, 0 ꢁC to rt,
1–4 h (71–94 %);
a
O
O
N
N
R3
H
2
3a-g
HO
HO
H
R1
R2
(e) (i) TMSCl, NEt3, CH2Cl2,
0 ꢁC, 2 h; (ii) (CH2O)n, 37 wt%
in H2O (10 eq), Yb(OTf)3 (cat.),
THF, rt, 48 h; (f) (n-Bu)3P,
diethyl azodicarboxylate, THF,
0 ꢁC to rt, 16 h (80–90 % over
two steps)
R1
R2
HO
HO
R1
b
d
O
O
R2
N
N
R3
R3
6a-m
4a-i
5a-m
e
R1
R2
HO
R1
R2
O
HO
f
O
O
N
R3
N
R3
8a-n
7a-n
123

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Med Chem Res (2013) 22:1825–1836
reaction with aqueous formaldehyde under Yb(OTf)3 catal-
ysis in THF) (Kobayashi and Hachiya, 1992; Kobayashi
et al., 2001). The intramolecular cyclization was achieved
by treating hydroxymethylated compounds 7a–n under
Mitsunobu reaction conditions utilizing diethyl azodicar-
boxylate and (n-Bu)3P in THF at 0 ꢁC to afford the target
spirooxindoles 8a–n in excellent yield (Swamy et al., 2009).
Further SAR studies were focused on the aryl head group
to minimize CYP inhibition and improve solubility. Incor-
poration of heteroatoms in the aryl head group (Table 2) led
to some improvements in both properties. Compounds 17,
18a–c, 24, 25a, and 25e–f have CYP3A4 inhibition values
less than 50 % at 10 lM and maintained hNaV1.7 potency.
Also, compounds 18d and 25b–d are potent blockers of
hNaV1.7 and also potent inhibitors of CYP3A4. Interestingly,
compound 18d showed high CYP3A4 inhibition (74 %),
whereas 25a gave only 26 % inhibition with the only differ-
ence in structure being the regioisomeric position of the
nitrogen in the head group. Although 25a no longer had a
CYP3A4 liability, it was a less potent inhibitor of hNaV1.7
than 18d. The (R)-(tetrahydrofuran-2-yl)methyl N-substitu-
tion for compounds 8d (Table 1) and 18a exhibited very
low CYP3A4 inhibition (18 and 14 %, respectively). Simi-
larly, incorporating the (tetrahydro-2H-pyran-2-yl)methyl
N-substituent also reduced the CYP3A4 liabilities for com-
pounds 8e and 18c without any loss of potency. These results
suggested that a combination of nitrogen substitution at the
head group and a polar N-substituent was required for low
CYP3A4 inhibition.
To our satisfaction, analogs 17, 18b–c, and 24 (Table 2)
had significantly improved solubility due to the introduction
of the additional heteroatom. As shown in Table 2, a non-
polar N-substituent led to reduced solubility and increased
CYP3A4 inhibition. For instance, compounds such as 18d
and 25b–c had poor solubility and increased CYP3A4
inhibition. The exception was compound 25a which main-
tained low CYP3A4 inhibition (26 % at 10 lM).
Of the set of potent blockers of hNaV1.7 in Table 2, 18b
(IC50 = 20 nM) had minimal CYP3A4 inhibition and good
solubility in phosphate-buffered saline; however, 18b dis-
played poor metabolic stability. Compound 18b was
extensively metabolized in rat liver microsomes (RLM)
following a 30-min incubation with only 8 % of the parent
compound remaining. Furthermore, analog 18b did not
show in vivo efficacy in the CFA-induced inflammatory
pain model at doses of 10 and 40 mg/kg (IP and oral,
respectively). As previously mentioned, the lack of in vivo
efficacy was likely due to poor metabolic stability.
Table 2 Structure-activity relationship of regioisomeric methoxypyridine spirooxindoles (17, 18a-d, 24, 25a-e)
O
X
OCH3
Y
O
N
R
Compound
X
Y
R
hNaV1.7a
IC50 (lM)
CYP3A4, % inhibition
at 10 lMb
Solubilityc
(lg/mL)
17
N
N
N
N
N
C
C
C
C
C
C
C
C
C
C
C
C
N
N
N
N
N
N
N
H
0.35
0.05
13
14
22
22
74
27
26
73
70
63
42
49
52
18a
18b
18c
18d
24
(R)-Tetrahydrofuran-2-yl)methyl
(S)-Tetrahydrofuran-2-yl)methyl
(Tetrahydro-2H-pyran-2-yl)methyl
(5-(Trifluoromethyl)furan-2-yl)methyl
H
\5.0
61
0.02
0.10
67
0.004
[1.0
0.13
\5.0
52
25a
25b
25c
25d
25e
25f
a
(5-(Trifluoromethyl)furan-2-yl)methyl
(5-Chlorothiophen-2-yl)methyl
(2-(Isopropyl)thiazole-4-yl)methyl
\5.0
\5.0
5
0.06
0.06
(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)methyl
(Pyridin-2-yl)methyl
0.03
5
0.18
\5.0
\5.0
(3-(Trifluoromethyl)pyridin-2-yl)methyl
[0.25
[
14C]guanidinium influx assay adapted for use in HEK-293 cells expressing human NaV1.7 (Reddy et al., 1998)
b
c
Human recombinant fluorogenic assay (Trubetskoy et al., 2005; Cohen et al., 2003) (n = 3)
Solubility at pH 7.4 in phosphate-buffered saline containing 1 % v/v DMSO
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Med Chem Res (2013) 22:1825–1836
1829
MOMO
HO
N
Br
OH
OMOM
OCH3
c
b
a
O
N
N
N
N
R
OCH3
9
OCH3
10
OCH3
11
13
R = benzhydryl
d
O
HO
N
HO
HO
N
N
OCH3
OCH3
e
O
OCH3
f
O
N
R
O
N
R
N
16
R
15
14
g
O
O
N
N
OCH3
OCH3
h
O
O
N
H
N
R1
17
18a-d
Scheme 2 Reagents and conditions: (a) (i) nBuLi (1.1 eq), THF,
-78 ꢁC, 0.5 h, (ii) B(OCH3)3 (1.25 eq), -78 ꢁC to rt, 9 h, (iii) 35 %
aq. H2O2 (2.0 eq), 3 h, then acidic workup (91 % over three steps);
(b) (i) NaH (3.0 eq), 0 ꢁC, THF, 0.5 h, then MOMCl (1.2 eq), 16 h
(89 %); (c) i. tert-BuLi (1.0 eq), THF, -78 ꢁC, 0.5 h, then
N-benzhydrylisatin (12) (1.2 eq), -78 ꢁC to rt, 16 h (64 %);
(d) TFA/CH2Cl2 (1:1), rt, 16 h (98 %); (e) (i) SOCl2, NEt3, DCE,
0 ꢁC to reflux, 1 h, (ii) Zn dust, THF/HOAc (7:1), rt, 16 h (34 % over
two steps); (f) ICH2Cl, Cs2CO3, THF, rt, 16 h (40 %); (g) TFA,
Et3SiH (5 eq), reflux, 8 h (50 %); (h) RX or ROTs (1.5–5 eq),
Cs2CO3 (3 eq), MEK or acetone, reflux, 1.5 h (89–95 %)
Target compounds 17 and 18a–d (Table 2) were syn-
thesized as shown in Scheme 2. Treatment of the com-
mercially available halide 9 with nBuLi for 30 min at
-78 ꢁC followed by quenching with trimethylborate and
oxidation of the borate ester to the corresponding phenol
with H2O2 afforded intermediate 10 in 91 % yield. The
5-hydroxypyridine 10 was converted to the methoxy
methyl ether (MOM) derivative using NaH and MOMCl to
afford the protected derivative 11 in 89 % yield. Regio-
selective ortho-lithiation of compound 11 was achieved
using tert-BuLi at -78 ꢁC for 0.5 h followed by the
addition of N-benzhydrylisatin 12 to afford intermediate 13
in moderate yield (64 %). The MOM group was removed
using TFA in CH2Cl2 to obtain intermediate 14 in high
yield.
Treating 14 with HI did not give any desired product,
instead resulting in decomposition of the starting material.
Having failed to obtain the desired product 15, we tried to
remove the 3-hydroxyl group by conditions employed by
Vedejs in the total synthesis of Diazonamide A (Zajac and
Vedejs, 2004). Treating compound 14 with SOCl2 and
Et3N in dichloroethane at reflux afforded the corresponding
chloride which was then subjected to Zn dust reduction to
afford the desired product 15 in 34 % yield over 2 steps.
Compound 15 was treated with chloroiodomethane and
Cs2CO3 to afford the target compound 16 in 40 % yield.
The benzhydryl-protecting group at the N-position was
removed using Et3SiH in TFA to provide compound 17 in
low yield (38–50 %). Having obtained the key intermediate
17, we were in a position to explore the substitution at the
N-position of the spirooxindole. Compounds 18a–d were
obtained in high yield by treating 17 with Cs2CO3 and the
corresponding alkyl or benzyl halides.
When compound 14 was subjected to similar conditions
as depicted in Scheme 1 (i.e., Et3SiH and TFA in CH2Cl2),
the expected dehydroxylated product 15 was not formed.
To our surprise, we recovered starting material 14 in
quantitative yield. Attempts to remove the 3-hydroxyl
group of compound 14 turned out to be problematic.
As shown in Scheme 3, compounds 24 and 25a–f were
prepared following a different synthetic pathway than
Scheme 2 to improve synthetic feasibility and yields.
123

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Med Chem Res (2013) 22:1825–1836
HO
OH
O
O
OH
HO
Br
HO
Br
Br
N
O
N
N
a
b, c
OH
d
N
O
N
O
O
N
H
N
H
O
OH
22
20
21
10
e
O
N
O
O
Br
O
N
O
O
g
O
f
N
O
N
O
N
H
23
N
H
R
25a-f
24
Scheme 3 Reagents and conditions: (a) iso-PrMgCl (1.5 eq), THF,
0 ꢁC, 0.5 h then 4-bromoisatin (19), rt, 4–16 h (60–90 %); (b) SOCl2
(2.0 eq), Et3N (3.0 eq), CH2Cl2, 0 ꢁC, 1 h (quant.); (c) Zn dust
(10 eq), THF–AcOH (9:1), rt, 16 h (80–95 %); (d) NaOH (aq.)
(4 eq), (CH2O)n, 37 wt% in H2O, dioxane, rt, 48 h (quant.);
(e) (n-Bu)3P, di-tert-butyl azodicarboxylate, THF, 0 ꢁC to rt, 0.5 h
(80–90 %); (f) Pd(PPh3)4 (10 mol %), HCO2H (2 eq), Et3N (2 eq),
dioxane, reflux, 16 h (90–95 %); (g) RX (1.2 eq), NaH or Cs2CO3
(1.5 eq), DMF, 0 ꢁC to rt, 2–4 h (80–90 %)
Having failed to obtain the product of addition of 10 to
isatin 2 in high yield, we investigated the feasibility of this
reaction with other substituted isatins. We found that the
addition of 10 to 4-substituted isatins gave the best yields.
Hence, 5-hydroxypyridine 10 was treated with iso-PrMgCl at
0 ꢁC for 0.5 h to generate the alkoxide which was in turn
added to 4-bromoisatin 19 to afford intermediate 20 in high
yield. The addition of 10 to isatin can take place at both the
1
4- and 6-positions of the methoxypyridine. The H NMR
spectrum of the methoxypyridine adduct contained two
doublets assignable to the pyridine ring and was hence
diagnostic for structure 20. In turn, compound 20 was con-
verted to the corresponding chloride using SOCl2 and Et3N in
CH2Cl2 and subsequently transformed to compound 21 with
10 equivalents of Zn dust in excellent yield. When compound
21 was treated with aqueous NaOH and para-formaldehyde
in THF, the diol 22 was obtained in quantitative yield.
Compound 22 was subjected to Mitsunobu reaction condi-
tions followed by addition of aqueous NaOH solution to
hydrolyze the aminal, affording spirooxindole 23 in 80–90 %
yield. Standard palladium-catalyzed dehalogenation using
Pd(PPh3)4, formic acid and Et3N at reflux in dioxane afforded
compound 24 in 95 % yield (Chowdhury et al., 1994). With
compound 24 in hand, we introduced different substituents at
the N-position of the spirooxindole using either NaH or
Cs2CO3 and an alkylating agent in DMF to obtain compounds
25a–f in high yield. The regioselectivity of the aforemen-
tioned phenol-isatin coupling reaction was confirmed by
obtaining a single X-ray crystal structure of compound 25a
(Fig. 2). This crystal structure demonstrated that the addition
of the phenol compound 10 to 4-bromoisatin (19) occurred at
Fig. 2 Single crystal X-ray structure of 25a
the 6-position (C16 in Fig. 2) rather than at the 4-position
(C18). By means of the route depicted in Scheme 3, we were
able to prepare large amounts of compound 25a for further
biological evaluation.
Compound 25a (Table 3) was not cytotoxic and had
favorable hepatocyte metabolic stability in both human and
dog. When subjected to rat, human, and dog liver micro-
some incubations for 60 min (Di et al., 2003), 25a had 18,
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Med Chem Res (2013) 22:1825–1836
1831
Table 3 ADME properties of
25a
Property
Value for 25a
CYP3A4, IC50 (lM)a
hERG (% Inhibition at 10 lM)b
[10
7.5
Solubility (phosphate-buffered saline, pH 7.4) (lg/mL)
Plasma protein binding (rat)
\5.0
95 %
a
Human recombinant
fluorogenic assay (Hewawasam
Cytotoxicity (HepG2, % viable after 16 h)
Permeability (CaCo-2), Papp a ? b/b ? a (10-6 cm/s)
Hepatocyte stability, % remaining after 2 h (rat/human/dog)
[96 %
9.1/8.8
18/29/49 %
and Erway, 1998) (n = 3)
b
Rb efflux assay (Terstappen,
1998)
29, and 49 %, respectively, of parent compound remaining.
Importantly, no meaningful inhibition of CYP3A4
(IC50 [ 10 lM) was observed in a recombinant human
enzyme assay. In vivo rat pharmacokinetic data for 25a
are presented in Table 4. When Male Sprague–Dawley
rats were dosed iv with 1 mg/kg of 25a, the Cmax was
884 ng/mL, compared to very low Cmax (12.7 ng/mL) for oral
administration at 10 mg/kg. The clearance for 25a is high as
reflected by a relatively short terminal half-life of about 2 h.
Compound 25a also displayed poor oral bioavailability at
10 mg/kg and the overall pharmacokinetic profile for this
compound was not improved over that obtained with our
initial spirooxindole hNaV1.7 blocker XEN907 (1).
p = 0.001
Pre-dose
1 h Post-dose
15
10
5
0
Vehicle
25a 10 mg/kg
The in vivo efficacy of compound 25a was evaluated in
a CFA-induced inflammatory pain model after oral dosing
of male rats at 10 mg/kg. The results are illustrated in
Fig. 3. Compound 25a had a significant analgesic effect at
this dose level with a 59 % change from baseline
(p \ 0.001). This level of in vivo efficacy was surprising
given its poor pharmacokinetic profile. One possibility was
that 25a might distribute particularly well to peripheral and
central nerve tissues. Although further tissue distribution
experiments would be required for a definitive confirma-
tion, this possibility was consistent with the steady-state
volume of distribution for this compound, which was sig-
nificantly higher than the rat plasma volume.
Fig. 3 Mean SD von Frey paw withdrawal thresholds in grams for
both baseline (i.e., pre-treated) and 1 h post-treatment (n = 4/group)
with either vehicle or 25a
Conclusions
In conclusion, we have shown that the tetracyclic spiro-
oxindole compounds 8f and 18d are potent hNaV1.7
blockers. During this medicinal chemistry effort, we also
identified several compounds, such as 8d, 8f, 8k, 17,
18a–c, 24, and 25a, that have improved CYP inhibition
profiles by virtue of the replacement of the methylenedioxy
moiety. Moreover, 25a was identified as a potent hNaV1.7
blocker with improved ADME profile and significant in
vivo efficacy in an inflammatory pain model. Future work
in this series of spirooxindoles will focus on further
improvements to the pharmacokinetic/pharmacodynamic
relationship and will be reported in due course.
Table 4 Mean pharmacokinetic parameters of 25a after administra-
tion to male Sprague–Dawley rats (n = 2/group)
Parameter
po (10 mg/kg)a
iv (1 mg/kg)b
Cmax (ng/mL)
12.7
77.1
ND
4.0
–
884
244
1.9
–
AUClast (h•ng/mL)
t
(h)
‘
Tmax (h)
Vss (L/kg)
Cl (L/h/kg)
F (%)
Experimental
4.1
4.0
–
–
Chemistry
3.2
a
All reagents were obtained from commercial sources and
were either used as supplied or purified by the reported
methods. Melting points were determined on a Bu¨chi hot-
stage apparatus and are uncorrected. NMR spectra were
Vehicle: labrasol: water: solutol (60:39.5:0.5), dose vol-
ume = 5 mL/kg
b
Vehicle: NMP: solutol: saline (5:20:75), dose volume = 0.5 mL/kg
ND not determined
123

1832
Med Chem Res (2013) 22:1825–1836
Synthesis of 4-bromo-3-(3-hydroxy-6-methoxypyridin-
recorded on a Bruker Avance 300 spectrometer with
chemical shifts (d) reported in parts-per-million (ppm)
relative to the residual signal of the deuterated solvent.
Mass spectra were obtained using a Waters 2795/ZQ LC/
MS system (Waters Corporation, Milford, MA). HPLC
analyses were performed at 25 ꢁC on Agilent 1100 or 1200
systems (Agilent Technologies, Santa Clara, CA) using an
EMD Chromolith SpeedROD RP-18e column (4.6 mm
i.d. 9 50 mm length) (Merck KGaA, Darmstadt, Ger-
many). The mobile phase consisted of a gradient of com-
ponent ‘‘A’’ (0.1 % v/v aqueous trifluoroacetic acid) and
component ‘‘B’’ (acetonitrile) at a flow rate of 1 mL/min.
The gradient program used was as follows: initial condi-
tions 5 % B, hold at 5 % B for 1 min., linear ramp from 5
to 95 % B over 5 min, 100 % B for 3 min., and return to
initial conditions for 1 min. Peaks were detected at a
wavelength of 254 nm using an Agilent photodiode array
detector. All compounds reported herein exhibited spectral
data consistent with their proposed structures and had
HPLC purities in excess of 96 % a/a.
2-yl)-1,3-dihydro-2H-indol-2-one (21)
HO
Br
H
O
N
O
N
H
To a solution of 4-bromo-3-hydroxy-3-(3-hydroxy-6-
methoxypyridin-2-yl)-1,3-dihydro-2H-indol-2-one (20) (4.20 g,
12.0 mmol) in anhydrous dichloromethane (100 mL) and
anhydrous tetrahydrofuran (10 mL) was added triethyl-
amine (3.64 g, 36.0 mmol) followed by thionyl chloride
(4.28 g, 36.0 mmol) at 0 ꢁC. The resultant solution was stir-
red for 0.5 h at 0 ꢁC and 10 % aqueous hydrochloric acid
solution (50 mL) was added. The mixture was extracted with
ethyl acetate (3 9 50 mL). The combined organic extracts
were washed with saturated ammonium chloride (3 9
50 mL) and brine (50 mL), dried over anhydrous sodium
sulfate, and filtered. The filtrate was concentrated in vacuo to
dryness. The residue was dissolved in tetrahydrofuran
(70 mL) and acetic acid (30 mL) and zinc dust (7.84 g,
120 mmol) was added. The reaction mixture was stirred at
ambient temperature for 3 h and filtered. The filtrate was
concentrated in vacuo to dryness. The residue was dissolved
in ethyl acetate (100 mL), washed with saturated ammo-
nium chloride (3 9 50 mL) and brine (50 mL), dried over
anhydrous sodium sulfate, filtered, and concentrated in
vacuo to dryness. The residue was purified by column
chromatography eluting with ethyl acetate to afford the title
compound (21) (2.58 g, 64 %) as a pale yellow solid: mp
Synthesis of 4-bromo-3-hydroxy-3-(3-hydroxy-6-
methoxypyridin-2-yl)-1,3-dihydro-2H-indol-2-one (20)
HO
Br
HO
N
O
O
N
H
To a pale yellow solution of 5-hydroxy-2-methoxypyr-
idine (10) (7.16 g, 57.2 mmol) in anhydrous tetrahydrofu-
ran (100 mL) isopropylmagnesium chloride (28.6 mL,
57.2 mmol, 2.0 M solution in tetrahydrofuran) was added
at 0 ꢁC. The resultant solution was stirred for 0.5 h and
4-bromoisatin (10.3 g, 45.8 mmol) was added in portions.
The reaction mixture was stirred at ambient temperature for
2 days and 10 % aqueous hydrochloric acid (100 mL) was
added. The mixture was extracted with ethyl acetate
(3 9 100 mL). The combined organic extracts were dried
over anhydrous sodium sulfate, filtered, and concentrated
in vacuo to dryness. The residue was triturated with diethyl
ether to afford the title compound (20) (10.7 g, 67 %) as a
1
149–152 ꢁC; H NMR (300 MHz, DMSO-d6) d 10.61 (s,
1H), 9.34 (br, 1H), 7.15–7.06 (m, 2H), 6.99 (d, J = 7.8 Hz,
1H), 6.81 (d, J = 7.4 Hz, 1H), 6.55 (d, J = 8.5 Hz, 1H),
4.90 (s, 1H), 3.52 (s, 3H); 13C NMR (75 MHz, DMSO-d6) d
176.2, 156.5, 147.4, 147.2, 145.7, 138.9, 130.1, 127.7,
124.7, 118.5, 109.7, 108.7, 53.3, 49.4; MS (ES?) m/z 337.2
(M ? 1), 335.2 (M ? 1).
Synthesis of 4-bromo-3-(3-hydroxy-6-methoxypyridin-
2-yl)-3-(hydroxymethyl)-1,3-dihydro-2H-indol-2-one
(22)
1
pale yellow solid: mp 172–175 ꢁC; H NMR (300 MHz,
O
DMSO-d6) d 10.61 (s, 1H), 9.41 (s, 1H), 7.10 (d,
J = 8.0 Hz, 2H), 6.99 (d, J = 8.1 Hz, 1H), 6.81 (d,
J = 7.6 Hz, 1H), 6.77 (br s, 1H), 6.68 (d, J = 8.6 Hz, 1H),
3.77 (s, 3H); 13C NMR (75 MHz, DMSO-d6) d 176.5,
155.5, 145.9, 139.4, 131.6, 128.9, 127.2, 125.6, 118.9,
111.8, 111.1, 109.3, 53.8; MS (ES?) m/z 352.2 (M ? 1),
350.2 (M ? 1).
N
Br
OH
OH
O
N
OH
123

Med Chem Res (2013) 22:1825–1836
1833
To a mixture of 4-bromo-3-(3-hydroxy-6-methoxypyridin-
J = 8.8 Hz, 1H), 7.18 (dd, J = 7.6, 7.6 Hz, 1H), 7.10 (d,
J = 8.2 Hz, 1H), 6.91 (d, J = 7.5 Hz, 1H), 6.64 (d,
J = 8.8 Hz, 1H), 4.80 (d, J = 9.9 Hz, 1H), 4.76 (d,
J = 9.9 Hz, 1H), 3.54 (s, 3H); 13C NMR (75 MHz, DMSO-
d6) d 177.4, 159.7, 151.1, 144.9, 143.9, 131.2, 130.1, 126.0,
121.4, 119.1, 110.4, 109.8, 76.7, 60.3, 53.9; MS (ES?) m/
z 349.2 (M ? 1), 347.2 (M ? 1).
2-yl)-3-(hydroxymethyl)-1,3-dihydro-2H-indol-2-one
(21)
(1.67 g, 5.00 mmol) and para-formaldehyde (0.60 g,
20 mmol) in tetrahydrofuran (20 mL) a solution of sodium
hydroxide (0.80 g, 20 mmol) in water (10 mL) was added at
0 ꢁC. The reaction mixture was stirred for 1 h at 0 ꢁC and 10 %
aqueous hydrochloric acid (50 mL) was added. The mixture
was extracted with ethyl acetate (3 9 50 mL). The combined
organic extracts were washed with saturated aqueous ammo-
nium chloride (3 9 50 mL) and brine (50 mL), dried over
anhydrous sodium sulfate, and filtered. The filtrate was con-
centrated in vacuo to dryness. The residue was purified by
column chromatography eluting with 50 % ethyl acetate in
hexanes to afford the title compound (22) (1.83 g, 81 %) as a
colorless solid: mp 142–145 ꢁC; 1H NMR (300 MHz, DMSO-
d6) d 9.08 (s, 1H), 7.11 (d, J = 7.8 Hz, 1H), 7.05 (d,
J = 6.7 Hz, 1H), 7.00 (dd, J = 7.8, 1.0 Hz, 1H), 6.93 (d,
J = 8.7 Hz, 1H), 6.55 (d, J = 8.6 Hz, 1H), 6.23 (br, 1H), 5.09
(d, J = 10.8 Hz, 1H), 5.01 (d, J = 10.6 Hz, 1H), 4.64 (br, 1H),
4.75 (d, J = 10.5 Hz, 1H), 4.29 (d, J = 10.4 Hz, 1H), 3.76
(s, 3H); 13C NMR (75 MHz, DMSO-d6) d 176.8, 155.5, 147.2,
146.1, 139.5, 129.6, 129.5, 127.2, 125.8, 117.7, 109.7, 108.2,
78.3, 63.5, 63.3, 53.3; MS (ES?) m/z 419.2 (M ? 23), 417.2
(M ? 23).
Synthesis of 5-methoxyspiro[furo[3,2-b]pyridine-3,30-
indol]-20(10H)-one (24)
O
O
N
O
N
H
A mixture of 40-bromo-5-methoxyspiro[furo[3,2-b]pyri-
dine-3,30-indol]-20(10H)-one (23) (1.10 g, 3.18 mmol), tetra-
kis(triphenylphosphine)palladium (0) (0.92 g, 0.79 mmol),
formic acid (1.92 g, 41.6 mmol), triethylamine (4.21 g,
41.6 mmol), and anhydrous p-dioxane (40 mL) was heated at
reflux for 2 h. The hot reaction solution was filtered through a
pad of diatomaceous earth. The pad of diatomaceous earth was
washed with ethyl acetate (50 mL). The filtrate was concen-
trated in vacuo to dryness. The residue was dissolved in ethyl
acetate (50 mL) washed with 10 % aqueous hydrochloric acid
(25 mL), saturated ammonium chloride (3 9 15 mL) and
brine (3 9 10 mL), dried over anhydrous sodium sulfate, and
filtered. The filtrate was concentrated in vacuo to dryness. The
residue was purified by column chromatography eluting with
ethyl acetate to afford the title compound (0.59 g, 69 %) (24) as
a colorless solid: mp 199–200 ꢁC; 1H NMR (300 MHz,
DMSO-d6) d 10.62 (s, 1H), 7.38 (d, J = 8.8 Hz, 1H), 7.21 (dd,
J = 7.7, 7.7 Hz, 1H), 7.08 (d, J = 7.2 Hz, 1H), 6.94 (d,
J = 7.5 Hz, 1H), 6.89 (d, J = 8.0 Hz, 1H), 6.63 (d,
J = 8.8 Hz, 1H), 4.83 (d, J = 9.5 Hz, 1H), 4.71 (d,
J = 9.5 Hz, 1H), 3.53 (s, 3H); 13C NMR (75 MHz, DMSO-d6)
d 177.8, 159.9, 149.8, 146.5, 142.6, 132.1, 129.3, 124.3, 122.7,
121.8, 110.3, 110.2, 79.4, 59.0, 53.9; MS (ES?) m/z 269.3
(M ? 1), 241.3 (M-17).
40-Bromo-5-methoxyspiro[furo[3,2-b]pyridine-3,30-
indol]-20(10H)-one (23)
O
Br
N
O
O
N
H
To a solution of 4-bromo-3-(3-hydroxy-6-methoxypyridin-
2-yl)-3-(hydroxymethyl)-1,3-dihydro-2H-indol-2-one (22)(3.48 g,
8.83 mmol) in anhydrous tetrahydrofuran (80 mL) was
added tri-n-butylphosphine (2.23 g, 11.000 mmol) followed
by a solution of di-tert-butyl azodicarboxylate (2.54 g,
11.0 mmol) in anhydrous tetrahydrofuran (10 mL) at 0 ꢁC.
The mixture was stirred for 1 h at 0 ꢁC, concentrated
ammonium hydroxide (5 mL) was added, and stirring was
continued for a further 0.5 h at 0 ꢁC. The mixture was con-
centrated in vacuo to dryness. The residue was dissolved in
ethyl acetate (100 mL), washed with 10 % aqueous hydro-
chloric acid (2 9 50 mL), saturated ammonium chloride
(30 mL) and brine (30 mL), dried over anhydrous sodium
sulfate, and filtered. The filtrate was concentrated in vacuo to
dryness. The residue was purified by column chromatogra-
phy eluting with ethyl acetate to afford the title compound
(23) (1.28 g, 42 %) as a pale yellow solid: mp 265–268 ꢁC;
1H NMR (300 MHz, DMSO-d6) d 10.86 (s, 1H), 7.33 (d,
General procedure A: synthesis of 5-methoxy-10-{[5-
(trifluoromethyl)-2-furyl]methyl}spiro[furo[3,2-
b]pyridine-3,30-indol]-20(10H)-one (25a)
O
N
OCH3
CF3
O
N
O
123

1834
Med Chem Res (2013) 22:1825–1836
A mixture of 5-methoxyspiro[furo[3,2-b]pyridine-3,30-in-
Following general procedure A, the title compound
dol]-20(10H)-one (24) (0.27 g, 1.0 mmol), 4-2-(chloromethyl)
-5-(trifluoromethyl)furan (0.18 g, 1.0 mmol) and cesium
carbonate (0.32 g, 1.0 mmol) in acetone (2 mL) was stirred at
ambienttemperaturefor16 h.Themixturewasfilteredandthe
solid was washed with ethyl acetate (5 mL). The filtrate was
concentrated in vacuo to dryness. The residue was purified by
column chromatography eluting with 30 % ethyl acetate in
hexanes to afford the title compound (25a) (0.35 g, 83 %) as a
colorless solid: mp 145–147 ꢁC; 1H NMR (300 MHz,
CD3CN) d 7.41 (d, J = 8.888 Hz, 1H), (7.30 (dd, J = 7.7,
7.7 Hz, 1H), 7.20–7.12 (m, 3H), 7.03 (dd, J = 7.4, 7.4 Hz,
1H), 6.67–6.21 m, 2H), 5.15 (d, J = 16.1 Hz, 1H), 4.98 (d,
J = 16.2 Hz, 1H), 4.87 (d, J = 9.7 Hz, 1H), 4.78 (d,
J = 9.7 Hz, 1H), 3.56 (s, 3H); 13C NMR (75 MHz, DMSO-
d6) d 176.0, 159.9, 153.8, 153.7, 149.7, 146.0, 142.5, 139.8
(m),131.2,129.4,124.3,123.7,122.2,114.5(m),110.8,109.8,
109.7, 79.1, 58.5, 53.6, 37.0; MS (ES?) m/z 417.2 (M ? 1).
(25c) (0.13 g, 64 %) was obtained as a colorless solid: mp
1
136–138 ꢁC; H NMR (300 MHz, DMSO-d6) d 7.42 (d,
J = 8.8 Hz, 1H), 7.29–7.23 (m, 2H), 7.19 (d, J = 6.8 Hz,
1H), 7.06–6.99 (m, 2H), 6.65 (d, J = 8.8 Hz, 1H), 5.07 (d,
J = 16.4 Hz, 1H), 4.95 (d, J = 16.6 Hz, 1H), 4.89 (d,
J = 9.8 Hz, 1H), 4.81 (d, J = 9.7 Hz, 1H), 3.54 (s, 3H),
3.27–3.18 (m, 1H), 1.28 (d, J = 6.9 Hz, 6H); 13C NMR
(75 MHz, DMSO-d6) d 178.3, 176.1, 159.9, 150.7, 149.7,
146.2, 142.9, 131.3, 129.4, 124.2, 123.6, 122.2, 114.2,
110.8, 109.9, 79.1, 58.6, 53.9, 40.7, 33.0, 23.2; MS (ES?)
m/z 408.3 (M ? 1).
Synthesis of 10-(2,3-dihydro-1,4-benzodioxin-6-
ylmethyl)-5-methoxyspiro[furo[3,2-b]pyridine-3,30-
indol]-20(10H)-one (25d)
O
N
O
Synthesis of 10-[(5-chloro-2-thienyl)methyl]-5-
methoxyspiro[furo[3,2-b]pyridine-3,30-indol]-20(10H)-
one (25b)
O
N
O
O
O
Following general procedure A, the title compound
(25d) (0.17 g, quantitative yield) was obtained as a color-
less solid: mp 144–146 ꢁC (methanol/water); 1H NMR
(300 MHz, CDCl3) d 7.23–7.14 (m, 3H), 7.02 (dd, J = 7.5,
7.5 Hz, 1H), 6.94–6.89 (m, 2H), 6.79–6.74 (m, 2H), 6.56
(d, J = 8.7 Hz, 1H), 5.26 (d, J = 15.8 Hz, 1H), 5.09 (d,
J = 9.3 Hz, 1H), 4.81 (d, J = 9.3 Hz, 1H), 4.57 (d,
J = 15.8 Hz, 1H), 4.21 (s, 4H), 3.73 (s, 3H); 13C NMR
(75 MHz, CDCl3) d 176.5, 160.4, 149.3, 145.8, 143.9,
143.1, 142.5, 131.8, 129.0, 128.6, 123.6, 123.3, 121.3,
120.1, 117.6, 116.0, 110.7, 109.7, 79.2, 64.4, 59.1, 54.0,
43.5; MS (ES?) m/z 416.9 (M ? 1).
O
N
O
N
S
Cl
Following general procedure A, compound (25b) (0.31 g,
78 %) was obtained as a colorless solid: mp 130–132 ꢁC; 1H
NMR (300 MHz, DMSO-d6) d 7.41 (d, J = 8.8 Hz, 1H), 7.28
(dd, J = 7.7, 7.7 Hz, 1H), 7.18 (d, J = 4.3 Hz, 1H), 7.16 (d,
J = 4.5 Hz, 1H), 7.07 (d, J = 3.8 Hz, 1H), 7.01 (d, J =
7.6 Hz, 1H), 6.94 (d, J = 3.8 Hz, 1H), 6.65 (d, J = 8.8 Hz,
1H), 5.15 (d, J = 16.1 Hz, 1H), 4.98 (d, J = 16.2 Hz, 1H),
4.87 (d, J = 9.7 Hz, 1H), 4.78 (d, J = 9.7 Hz, 1H), 3.52 (s,
3H); 13C NMR (75 MHz, DMSO-d6) d 176.0, 159.9, 149.7,
146.1, 142.3, 138.4, 131.3, 129.4, 128.1, 127.1, 126.8, 124.257,
123.7, 122.1, 110.8, 109.9, 79.0, 58.5, 53.7, 38.9; MS (ES?)
m/z 401.2 (M ? 1), 399.2 (M ? 1).
Synthesis of 5-methoxy-10-(pyridin-2-
ylmethyl)spiro[furo[3,2-b]pyridine-3,30-indol]-20(10H)-
one (25e)
O
Synthesis of 10-[(2-isopropyl-1,3-thiazol-4-yl)methyl]-
5-methoxyspiro[furo[3,2-b]pyridine-3,30-indol]-
20(10H)-one (25c)
O
N
O
N
N
O
O
N
O
Following general procedure A, the title compound (25e)
(0.08 g, 75 %) was obtained as a colorless solid: mp
N
N
1
168–171 ꢁC; H NMR (300 MHz, DMSO-d6) d 8.57 (d,
J = 4.8 Hz, 1H), 7.72 (dt, J = 7.7, 1.8 Hz, 1H), 7.46 (d,
S
123

Med Chem Res (2013) 22:1825–1836
1835
J = 8.8 Hz, 1H), 7.37 (d, J = 7.8 Hz, 1H), 7.33–7.23
(m, 3H), 7.05 (dt, J = 7.5, 0.8 Hz, 1H), 6.94 (d, J = 7.7 Hz,
1H), 6.70 (d, J = 8.8 Hz, 1H), 5.19 (d, J = 16.7 Hz, 1H),
4.97–4.86 (m, 3H), 3.62 (s, 3H); 13C NMR (75 MHz,
DMSO-d6) d 175.8, 159.4, 155.4, 149.4, 149.1, 145.7, 142.5,
137.0, 130.8, 128.9, 123.7, 123.0, 122.6, 121.7, 120.3, 110.2,
109.3, 78.5, 58.1, 53.3, 44.9; MS (ES?) m/z 360.4 (M ? 1).
The plate was incubated for 2 h at ambient temperature, after
which time the plates were washed with LHNBSS ? 10 mM
guanidinium chloride. Residual volumes were removed and
air dried overnight. Plates were read the following day on a
Wallac MicroBeta TriLux liquid scintillation counter (Perk-
inElmer, Waltham, MA).
CFA assay
Synthesis of 5-methoxy-10-((3-(trifluoromethyl)pyridin-
2-yl)methyl)-2H-spiro[furo[3,2-b]pyridine-3,30-
indolin]-20-one (25f)
Complete Freund’s adjuvant (CFA) (Sigma-Aldrich Canada,
Oakville, ON) was suspended in a mineral oil/saline (1:1 v/v)
emulsion at a concentration of 0.5 mg/mL. Under light isoflu-
rane anesthesia, 150 lL of the CFA emulsion was injected
subcutaneously into the plantar surface of the rat’s left hind
paw. At 7 days post-injection, animals were screened for CFA-
induced inflammatory mechanical allodynia by the von Frey
test as described below. Only animals with significant allodynia
were enrolled in the study. The paw withdrawal threshold of
animals to mechanical tactile stimuli was measured using the
Model 2290 Electrovonfrey anesthesiometer (IITC Life Sci-
ence, Woodland Hills, CA). Animals were placed inan elevated
acrylic enclosure set on a wire mesh surface. After 20 min of
acclimation, a #13 Von Frey tip was applied perpendicularly to
the plantar surface of the ipsilateral hind paw of the animals
with sufficient force to elicit a crisp flickering response of the
paw, indicating a withdrawal response from pain that defined
the experimental endpoint. A total of five readings were taken
from each animal with an interval of 1 min between the read-
ings. The force (in grams) required to elicit the withdrawal
response was recorded both before (baseline measurement) and
1 h after test article dosing.
O
N
O
O
N
N
F3C
Following general procedure A, the title compound (25f)
(0.15 g, 76 %) was obtained as a colorless solid: mp
1
195–198 ꢁC; H NMR (300 MHz, DMSO-d6) d 8.68 (d,
J = 4.4 Hz, 1H), 8.23 (d, J = 7.2 Hz, 1H), 7.54 (dd,
J = 7.8, 4.9 Hz, 1H), 7.45 (d, J = 8.8 Hz, 1H), 7.30 (ddd,
J = 8.2, 8.2, 5.8 Hz, 1H), 7.23 (d, J = 6.6 Hz, 1H), 7.04
(dd, J = 7.5 Hz, 1H), 6.94 (d, J = 7.8 Hz, 1H), 6.69 (d,
J = 8.8 Hz, 1H), 5.25 (s, 2H), 4.86 (d, J = 9.5 Hz, 2H),
3.61 (s, 3H); 13C NMR (75 MHz, DMSO-d6) d 176.2,
159.3, 152.6, 152.4, 152.3, 149.5, 145.3, 143.5, 134.7
4
(q, JC–F = 4.9 Hz), 130.3, 128.9, 128.8, 125.6 (q,
1JC–F = 274 Hz), 123.7, 122.8, 121.4, 109.9, 109.2, 79.2,
58.0, 53.3, 42.2; MS (ES?) m/z 427.2 (M ? 1), 449.2
(M ? 23).
Human recombinant CYP fluorogenic assay
This assay is based on the cytochrome P450 inhibition kit
described by Vivid CYP450 Screening kit protocol, 2005,
Invitrogen Corporation (Invitrogen Corporation, 1600
Faraday Avenue, Carlsbad, California, 92008, USA).
Pharmacology
Guanidinium influx assay
Rb efflux assay
All IC50 data reported herein are means of no fewer than two
replicate experiments. Briefly, cells expressing hNaV1.7 were
plated on a PDL-coated Scintiplate (PerkinElmer, Waltham,
MA) at a density of *106 cells/well and incubated overnight
(37 ꢁC, 5 % CO2). Cells were washed with low-sodium
HEPES-buffered saline solution (LHNBSS) assay buffer
(150 mM choline chloride, 20 mM HEPES, 1 mM CaCl2,
5 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with
Tris to pH 7.4). Test compounds were added in DMSO (final
assay DMSO concentration 0.5 %), followed by [14C]
guanidinium chloride (0.05 lCi/well, American Radiola-
beled Chemicals, St. Louis, MO) and aconitine (final con-
centration 10 lM, Cedarlane Laboratories, Burlington, ON).
HEK293 cells stably expressing the hERG potassium channel
were plated on a PDL-coated 96-well plate (Becton–Dick-
inson, 1 Becton Drive, Franklin Lakes, New Jersey, 07417,
USA) at a density of 106 cells/well and incubated overnight
(37 ꢁC, 5 % CO2). Cells were loaded with Rb loading buffer
(5.4 mM RbCl, 150 mM NaCl, 2 mM CaCl2, 0.8 mM
NaH2PO4, 1 mM MgCl2, 5 mM glucose, 25 mM HEPES
adjusted with Tris to pH 7.4) and incubated for 3 h (37 ꢁC,
5 % CO2). Test compounds were added in DMSO (final assay
DMSO concentration 0.5 %) during the last 30 min of
loading. The Rb loading buffer was then removed and cells
were washed twice with 200 uL of wash buffer (5.4 mM KCl,
123

1836
Med Chem Res (2013) 22:1825–1836
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Acknowledgments The authors are grateful to Drs. Michael
Winther and Rostam Namdari of ADME group for their support.
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