Discovery of a novel class of isoxazoline voltage gated sodium channel blockers

Article abstract of DOI:10.1016/j.bmcl.2009.07.125

Analogs of the previously reported voltage gated sodium channel blocker CDA54 were prepared in which one of the amide functions was replaced with aromatic and non-aromatic heterocycles. Replacement of the amide with an aromatic heterocycle resulted in sig

Full text of DOI:10.1016/j.bmcl.2009.07.125

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5329–5333
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a novel class of isoxazoline voltage gated sodium channel blockers
a
a
a
a
a
Pengcheng P. Shao a, , Feng Ye , Ann E. Weber , Xiaohua Li , Kathryn A. Lyons , William H. Parsons ,
Maria L. Garcia b, Birgit T. Priest b, McHardy M. Smith b, John P. Felix b, Brande S. Williams b,
Gregory J. Kaczorowski b, Erin McGowan c, Catherine Abbadie c, William J. Martin c,
Daniel R. McMasters d, Ying-Duo Gao d
*
a Department of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
b Department of Ion Channels, Merck Research Laboratories, Rahway, NJ 07065, USA
c Department of Pharmacology, Merck Research Laboratories, Rahway, NJ 07065, USA
d Department of Molecular Modeling, Merck Research Laboratories, Rahway, NJ 07065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Analogs of the previously reported voltage gated sodium channel blocker CDA54 were prepared in which
one of the amide functions was replaced with aromatic and non-aromatic heterocycles. Replacement of
the amide with an aromatic heterocycle resulted in significant loss of sodium channel blocking activity,
while non-aromatic heterocycle replacements were well tolerated.
Received 6 June 2009
Revised 27 July 2009
Accepted 28 July 2009
Available online 6 August 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Voltage gated sodium channel blocker
Neuropathic pain
Inflammatory pain
Amide replacement
Heterocycles
Isoxazoline
In an earlier communication, we reported our discovery of
CDA54, a cyclopentane dicarboxamide compound, as a potent
and orally bioavailable state-dependent sodium channel blocker.1
Following this work, SAR studies with one of the amide moieties
in CDA54 replaced by alcohol (compound A) were reported.2 In
The use of heterocycles as amide surrogates is a common prac-
tice in medicinal chemistry. This substitution introduces structural
rigidity, which may lead to compounds with improved selectivity,
metabolic stability and enhanced pharmacokinetic properties. It is
well established that the lone electron pair on the amide nitrogen
HN
F3CO
O HO
A
HN
N
F3CO
O
O
H2NO2S
CDA54
HN
F3CO
H
O
Ar
B
*
p orbital of the C@O double bond. This pro-
the present study, we wish to report our findings with heterocycle
replacements of the amide group (compound B).
can delocalize into the
vides the C–N single bond with some double bond character and
* Corresponding author. Tel.: +1 732 594 2955.
E-mail address: pengcheng_shao@merck.com (P.P. Shao).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.125

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P. P. Shao et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5329–5333
H
N
N
O
R
R
R
O
H
H
"extended"
H
N
H
N
H
H
O
R
O
Figure 2. Overlay of N-benzylacetamide (grey carbon atoms) with heterocyclic
"bent"
analogs. Left: extended conformation overlaid with isoxazole (orange carbon
atoms). Right: bent conformation overlaid with isoxazoline (green carbon atoms).
The molecules were superimposed with respect to amide/heterocycle and aromatic
centroid.
Figure 1. Mimicking amide conformation with heterocyles.
slows its rotation. Hence, the amide conformation (the plane de-
fined by OCN) is relatively rigid.3 However, the bond rotational
barrier between the benzylic carbon and the amide nitrogen is rel-
atively small. Rotation of this bond places the phenyl group at dif-
ferent positions relative to the plane defined by OCN in the amide.
We hypothesized that the position of this phenyl ring is important
as it is likely to contribute significantly to its binding to the ion
channels. Among many possible conformations, two relatively
low-energy conformations are illustrated in Figure 1: an ‘extended’
conformation with the phenyl ring centered in the plane defined
by the amide, or a ‘bent’ conformation with the phenyl ring
extending traverse to the amide plane. If the ‘extended’ conforma-
tion is the active conformation, replacing the amide with an aro-
matic heterocyclic moiety should not alter the phenyl position
significantly. On the other hand, if it adopts a ‘bent’ conformation,
typical aromatic heterocycle replacement of the amide should not
be tolerated. To mimic this conformation, non-aromatic heterocy-
les such as oxazoline and isoxazoline may be used. This design fea-
ture becomes more apparent during molecular modeling.4 By
overlaying with respect to both the amide/heterocycle and phenyl
ring, good overall superposition was observed between both pairs:
extended conformation with isoxazole and bent conformation with
isoxazoline (Fig. 2). Therefore, SAR studies were conducted to
determine if state-dependent sodium channel blocking activity
would remain after replacement of one of the amides with either
type of heterocycles.
Our initial effort focused on replacing the sulfamoyl biphenylm-
ethyl amide of CDA54 and retaining the 4-trifluoromethoxybenzyl
amide. The synthesis of 5-phenyl oxazole and oxazoline derivatives
c
a
b
HN
NH
HN
CO2H
F3CO
F3CO
O
O
O
HO2C
F3CO
CO2H
OH
II
I
HN
O
d
HN
O
F3CO
O
N
O
N
6
2
Scheme 1. Reagents and conditions: (a) THF, CDI (1.1 equiv), 25 °C, 1 h; then 4-trifluoromethoxybenzyl amine (1.2 equiv) (67%); (b) THF, EDC (1.2 equiv), HOBt (1.2 equiv),
DIEA (4 equiv), 2-amino-1-phenyl ethanol, 25 °C, 30 min, (92%); (c) CH2Cl2, DAST (1.1 equiv), K2CO3 (1.5 equiv), À78 °C to 25 °C (52%); (d) CHCl3, MnO2 (10 equiv), 90 °C, 16 h
(32%).
a, b
c
NOH
HN
HN
HN
CHO
CO2H
F3CO
F3CO
F3CO
O
O
O
IV
I
III
d, e
d, f
HN
HN
F3CO
F3CO
N
O
O
N
O
O
3
Me
17
Scheme 2. Reagents and conditions: (a) THF, BH3 (2.5 equiv), À78 °C, 15 min (58%); (b) CH2Cl2, DMSO (2 equiv), oxalyl chloride (1.5 equiv), TEA (4 equiv) À78 °C to 25 °C
(89%); (c) CH2Cl2, hydroxylamine hydrochloride (6 equiv), Na2CO3 (2 M, 10 equiv), 40 °C, 16 h (95%); (d) CH2Cl2, NCS (1.2 equiv), 25 °C, 1 h; (e) phenylacetylene (5 equiv), TEA
(2 equiv) slow addition, 25 °C, 16 h (47% -two steps); (f) 2-phenylpropene (5 equiv), TEA (2 equiv) slow addition, 25 °C, 16 h (77% -two steps).

P. P. Shao et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5329–5333
5331
Table 1
2 and 6 is illustrated in Scheme 1. 4-phenyl oxazole and oxazoline
Heterocycle replacement of amide
derivative 1 and 5 were synthesized according to the same scheme
except 2-amino-2-phenyl ethanol was used. Activating ( )-trans-
cyclopentane dicarboxylic acid with one equivalent of CDI, fol-
lowed by addition of excess 4-trifluoromethoxybenzylamine gave
the mono acid intermediate I, along with a small amount of unre-
acted diacid and symmetrical diamide by-product. Intermediate I
was isolated in 67% yield after acid-base work up.5 It was then
coupled with various amino alcohols by a standard EDC coupling
procedure to give diamide II. Intermediate II was cyclized to form
oxazoline derivative such as 6 according to a procedure developed
by Wipf and co-workers.6 Oxazole derivatives such as 2 were
conveniently synthesized from oxazoline compound by treatment
of activated MnO2, although the yield for the step was rather
low.
The synthesis of isoxazole and isoxazoline derivatives was dem-
onstrated with synthesis of compound 3 and 17, outlined in
Scheme 2. A borane reduction of mono acid I, followed by Swern
oxidation of the primary alcohol reduction product, afforded alde-
hyde III. Aldehyde III was treated with hydroxylamine to give
oxime IV in excellent yield. Chlorination of the oxime with NCS
gave hydroximoyl chloride. Without isolation, hydroximoyl chlo-
ride was treated with TEA to form the corresponding nitrile oxide,
which underwent 1,3-dipolar addition with selected alkyne or al-
kene reagents in situ to give isoxazole compound (3) as a pair of
enantiomers or isoxazoline compound (17) as an equal mixture
of four diastereiomers.7 Maintaining a low concentration of nitrile
oxide by slow addition of base (TEA) was the key to achieving a
good yield for this cycloaddition step.
Based on our previous results, majority of compounds in cyclo-
pentane series are state-dependent voltage gated sodium channel
blockers but not subtype selective.1 Most of compounds in this
study were evaluated only for their ability to block the hNav1.7
channel.8 It should be noted that for the initial assay screening,
the oxazoles and isoxazoles such as 1 and 3 were examined as
racemic mixtures. Similarly, the oxazoline and isoxazolines such
as 5 and 17 were initially examined as mixtures of diastereomers.
The extent of the channel block was determined in a functional,
membrane potential-based assay that measures the fluorescence
resonance energy transfer (FRET) between two membrane-associ-
ated dyes.9
Our initial results suggested that the amide may not have an
‘‘extended” conformation since replacing amide with oxazole (1,
2) and isoxazole (3, 4) moieties led to significant loss of potency,
while oxazoline compounds (5, 6) showed better Nav1.7 inhibitory
activity (Table 1). Since oxazoline is prone to hydrolysis in acidic
media, we shifted our focus to the more stable isoxazoline series.
Isoxazoline compound 7 showed potency similar to the oxazoline
compounds (5, 6). Lipophilic substitutions on the phenyl ring are
well tolerated and improve potency slightly regardless of the posi-
tion of substitution (8–12). However, polar substitutions (13–16)
resulted in significant loss of Nav1.7 inhibitory activity. A methyl
group at the 5-position of the isoxazoline did not affect potency
(17). However, polar substitution (hydroxymethyl) at this position
was not tolerated (18). Replacing the phenyl group with pyridine
led to complete loss of activity (19).
HN
F3CO
O
Compound
Nav1.7 VIPR
% inh at 1
*
IC50
(l
M)
lM (%)
H NO S
Me
2
2
N
CDA54
1
0.83
ND
O
N
O
19
0
O
2
ND
N
N
3
ND
38
33
56
71
76
93
92
90
87
75
0
O
4
Cl
ND
N
O
O
N
5
0.97
0.62
0.78
0.50
0.43
0.48
0.34
0.71
ND
O
6
N
N
7
O
O
8
OCF3
Cl
N
9
N
N
O
O
10
11
12
13
14
15
16
17
Cl
CO2Me
N
N
O
O
CO2Me
CO2H
N
N
N
N
N
O
O
O
O
O
CONH2
CONHMe
SO2Me
ND
0
ND
14
26
63
ND
Me
0.62
Once isoxazoline was identified as a good replacement for the
amide, we set out to search for a 4-trifluoromethoxybenzamide
replacements. In order to efficiently modify the amide moiety, an
alternative synthetic scheme was developed. As illustrated in
Scheme 3 for the synthesis of 25, the isoxazoline was installed ear-
lier in the synthetic sequence and the amide coupling reaction was
positioned as the last step. Heating a reaction mixture of methylcy-
clopent-1-ene-1-carboxylate and KCN in DMF with a slight excess
of water at 160 °C resulted in predominantly trans Michael
addition product V. It is noteworthy that under these reaction
HO
O
18
ND
23
0
N
N
N
19
ND
O
*
IC50 and % inhibition data were average of two experiments.

5332
P. P. Shao et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5329–5333
a
e,f
b,c
d
NC
NOH
NC
CO2K
N
CO2Me
NC
NC
CHO
HN
O
V
VI
Me
VII
VIII
g
h
HO2C
F3C
O
N
N
O
O
Me
Me
25
IX
Scheme 3. Reagents and conditions: (a) DMF, KCN (powder, 1.2 equiv), H2O (1.5 equiv), 160 °C, 1 h; (b) THF, ethylchloro formate (1.2 equiv), 0 °C, 1 h, NaBH4 (1.5 equiv),
25 °C, 30 min; (c) CH2Cl2, DMSO (2 equiv), oxalyl chloride (1.5 equiv), TEA (4 equiv) À78 °C to 25 °C; (d) CH2Cl2, hydroxylamine hydrochloride (1.5 equiv), Na2CO3 (2 M,
2 equiv), 50 °C, 0.5 h (55% -four steps); (e) CH2Cl2, NCS (1.2 equiv), 25 °C, 2 h; (f) 2-phenylpropene (5 equiv), TEA (2 equiv) slow addition, 25 °C, 16 h (82% -two steps); (g) HCl
(concd), 90 °C, 2 h (95%); (h) THF, BOP (1.5 equiv), DIEA (3 equiv), 4-trifluoromethylbenzyl amine (2 equiv), 25 °C, 30 min (92%).
Table 2
oxime VII under standard chemical transformation procedures in
good overall yields. Chlorination of the oxime with NCS gave
Modification to 4-trifluoromethoxybenzamide
hydroximoyl chloride. Without isolation, the hydroximoyl chloride
was treated with TEA to form the nitrile oxide, which underwent
Me
1,3-dipolar addition with 2-phenylpropene in situ to give isoxazo-
line derivatives VIII. Nitrile VIII was hydrolyzed under strong
acidic conditions to give carboxylic acid IX. It is noteworthy that
isoxazoline is stable under these harsh reaction conditions. Car-
boxylic acid IX was converted to the desired amide products 25 un-
der a standard amide coupling procedure in excellent yields, again
as mixtures of diastereomers.
As shown in Table 2, it appears that 4-trifluoromethoxy substi-
tution is optimal for conferring Nav1.7 inhibitory activity, and that
we were unable to identify a superior replacement (20–30). It is
also noteworthy that biphenylsulfonamide rendered compounds
completely inactive even though it brought many positive attri-
butes (potency and selectivity) to the diamide series (CDA54).
A series of bisheterocyclic compounds with both amides of
CDA54 replaced with heterocycles were also investigated briefly.
Synthesis of these compounds is illustrated in Scheme 4 for the
synthesis of 31. These compounds (31–34) are generally less po-
tent than the mono amide series (Table 3).
N
O
O
Compound
Nav1.7 VIPR IC50 (lM) or % inh at 1 l
M*
HN
HN
17
0.62
19%
16%
1.5
F3CO
HN
20
21
22
23
24
25
26
MeO
O
HN
HN
26%
15%
2.5
O
HN
Compound 17 has good PK properties (Sprague-Dawley rat, t1/2
:
Cl
2.9 h; Clp: 17.8 mL/min/kg; normalized AUC: 0.82 M h kg/mg and
l
F%: 16%). It was further evaluated in the CFA rat pain model (intra-
dermal injection of complete Freund’s adjuvant).10 It showed good
efficacy with 20% and 48% reversal of mechanic allodynia at 2 h
and 3 h, respectively, after a 3 mg/kg oral dose. As described above,
compound 17 is a diastereomeric mixture of four isomers. In order
to evaluate each diastereomer, they were separated by HPLC with a
chiral column.11 17a and 17c showed slightly higher potency than
17b and 17d in the functional hNav1.7 assay. However, 17a
showed the best efficacy in CFA with 53% reversal of allodynia at
3 h post a 3 mg/kg oral dose. 17a is more efficacious than CDA54
in the CFA rat pain model at the same dose (53% and 31% reversal
of allodynia, respectively). However, 17a is less efficacious in the
rat spinal nerve ligation (SNL) model of neuropathic pain than
CDA54.12 When dosed orally at10 mg/kg, 17a and CDA54 gave
21% and 35% reversal of allodynia, respectively, at 3 h post dose
(Table 4).
In summary, heterocyclic replacement of the amide in CDA54
was explored. Conformational analysis led us to the rational design
of novel oxazoline and isoxazoline voltage gated sodium channel
blockers. Further SAR studies showed that lipophilic substitutions
on phenyl and isoxazoline rings were well tolerated, but not polar
substitutions. Compound 17a was evaluated in rat CFA and SNL
pain model and showed good efficacy in rat CFA model.
HN
F3C
HN
F3CO
13%
SO2Me
SO2NH2
27
0%
0%
HN
Me
SO2NH2
28
29
N
F3CO
30%
17%
N
H
N
30
N
H
*
IC50 and % inhibition data were average of two experiments.
conditions, the methyl ester was hydrolyzed and V was isolated as
the potassium salt. Potassium carboxylate V was elaborated to

P. P. Shao et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5329–5333
5333
a
b,c
d
NC
HON
NOH
NC
N
N
N
N
O
O
O
O
VII
X
XI
31
Scheme 4. Reagents and conditions: (a) CH2Cl2, phenylacetylene (5 equiv), NaClO (10% solution, 2 equiv, slow addition), 25 °C, 8 h, (52%); (b) toluene, DIBAH (1 M toluene
solution, 2 equiv) À78 °C to 0 °C (48%); (c) CH2Cl2, hydroxylamine hydrochloride (1.5 equiv), Na2CO3 (2 M, 2 equiv), 25 °C, 2 h (95%); (d) CH2Cl2, phenyl acetylene (3 equiv),
NaClO (10% solution, 2 equiv, slow addition over 30 min), 25 °C, 16 h, (82%).
on subtype selectivity issues. The authors were also grateful to
Dr. Samer Eid and Mr. Christopher J. Daley for their contribution
Table 3
Compounds with both amide replaced with hetercycles
on Nav1.5 assay.
References and notes
Ph
N
Ar
Nav1.7 VIPR % inh at 1 l
M* (%)
N
O
O
1. (a) Shao, P.; Ok, D.; Fisher, M. H.; Garcia, M. L.; Kaczorowski, G. J.; Li, C.; Lyons,
K. A.; Martin, W. J.; Meinke, P. T.; Priest, B. T.; Smith, M. M.; Wyvratt, M. J.; Ye,
F.; Parsons, W. H. Bioorg. Med. Chem. Lett. 2005, 15, 1901; (b) Brochu, R. M.;
Dick, I. E.; Tarpley, J. W.; McGowan, E.; Gunner, D.; Herrington, J.; Shao, P.; Ok,
D.; Li, C.; Parsons, W. H.; Stump, G. L.; Regan, C. P.; Lynch, J. J., Jr.; Lyons, K. A.;
McManus, O. B.; Clark, S.; Ali, Z.; Kaczorowski, G. J.; Martin, W. J.; Priest, B. T.
Mol. Pharmacol. 2006, 69, 823.
Compound
Ar
Ph
31
32
33
34
13
18
35
43
4-CF3OPh
3,5-Dichloro-Ph
4-MeSO2Ph
*
2. Ok, D.; Li, C.; Abbadie, C.; Felix, J. P.; Fisher, M. H.; Garcia, M. L.; Kaczorowski, G.
J.; Lyons, K. A.; Martin, W. J.; Priest, B. T.; Smith, M. M.; Williams, B. S.; Wyvratt,
M. J.; Parsons, W. H. Bioorg. Med. Chem. Lett. 2006, 16, 1358.
% inhibition data were average of two experiments.
3. Avalos, M.; Babiano, R.; Barneto, J. L.; Bravo, J. L.; Cintas, P. J. Org. Chem. 2001,
66, 7275.
Table 4
In vivo efficacy of the four isomers of compound 17
4. Conformations of N-benzylacetamide, 2-methyl-4-phenyl-1,3-oxazole and 2-
methyl-4-phenyl-4,5-dihydro-1,3-oxazole were optimized using the MMFFs
force field with a distance-dependent dielectric of 4r. For 2-methyl-4-phenyl-
4,5-dihydro-1,3-oxazole, two conformations were found which differed in ring
pucker; the lower-energy conformation was used for the overlay shown in
Figure 2. Molecules were superimposed by minimizing RMSD between the
methyl carbon; the N, C, and O of the heterocycle or amide; and the phenyl ring
centroid.
Compound
Nav1.7 VIPR
Dose
% Reversal of
allodynia 3 h
after dosing
(po, mg/kg)
*
IC50
M)
% inh
CFA (%)
SML (%)
(
l
at 1 l
M* (%)
5. Intermediate I was extracted into basic aqueous layer, while diamide by-
product was washed away with EtOAc. Aqueous portion was then acidified and
17
17a
0.62
0.96
63
64
3
3
10
3
3
3
48
53
intermediate
I was extracted into EtOAc. Since unreacted diacid starting
21
material has higher aqueous solubility, this workup gave essentially pure
intermediate I.
17b
17c
17d
CDA54
ND
1.2
ND
0.83
37
48
35
26
20
17
31
54
6. Phillips, A. J.; Uto, Y.; Wipf, P.; Reno, M. J.; Williams, D. R. Org. Lett. 2000, 2,
1165.
7. Grundmann, C. Synthesis 1970, 7, 344.
8. Selection of compounds were evaluated for blocking hNav1.5 channel and no
selectivity were observed between hNav1.5 and hNav1.7 channels.
9. Felix, J. P.; Williams, B. S.; Priest, B. T.; Brochu, R. M.; Dick, I. E.; Warren, V. A.;
Yan, L.; Slaughter, R. S.; Kaczorowski, G. J.; Smith, M. L.; Garcia, M. L. Assay Drug
Dev. Tech. 2004, 2, 260.
3
10
35
*
IC50 and % inhibition data were average of two experiments.
10. Colpaert, F. C.; Meert, T.; De Witte, P. Life Sci. 1982, 31, 67.
11. Chiral OJ column, 15 Â 250 mm; mobile phase: 12% IPA, 88% heptane; flow
rate: 9 ml/min. Retention time for the four isomers: 17, 23, 31, 37 min.
12. Chaplan, S. R.; Bach, F. W.; Pogrel, J. W.; Chung, J. M.; Yaksh, T. L. J. Neurosci.
Methods 1994, 53, 55.
Acknowledgments
The authors would like to thank Dr. Joseph Duffy for proof-read-
ing the manuscript. The authors would also like to thank Dr. Rich-
ard Hargreaves and Dr. Joseph Duffy for their helpful suggestions