Phenethyl nicotinamides, a novel class of NaV1.7 channel blockers: Structure and activity relationship

Article abstract of DOI:10.1016/j.bmcl.2012.08.031

The Na_V1.7 ion channel is an attractive target for development of potential analgesic drugs based on strong genetic links between mutations in the gene coding for the channel protein and inheritable pain conditions. The (S)-N-chroman-3-ylcarboxamide series, exemplified by 1, was used as a starting point for development of new channel blockers, resulting in the phenethyl nicotinamide series. The structure and activity relationship for this series was established and the metabolic issues of early analogues were addressed by appropriate substitutions. Compound 33 displayed acceptable overall in vitro properties and in vivo rat PK profile.

Full text of DOI:10.1016/j.bmcl.2012.08.031

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6108–6115
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Phenethyl nicotinamides, a novel class of NaV1.7 channel blockers: Structure
and activity relationship
Inger Kers a, , Istvan Macsari a, Gabor Csjernyik a, Martin Nylöf a, Karin Skogholm a, Lars Sandberg a,
⇑
Alexander Minidis a, Tjerk Bueters b, Jonas Malmborg b, Anders B. Eriksson c, Per-Eric Lund d,
Elisabet Venyike d, Lei Luo d, Jan-Erik Nyström c, Yevgeni Besidski a
a Medicinal Chemistry, CNSP iMed Science, AstraZeneca R&D, Innovative Medicines, SE-15185 Södertälje, Sweden
b DMPK, CNSP iMed Science, AstraZeneca R&D, Innovative Medicines, SE-15185 Södertälje, Sweden
c CNSP iMed Project Management, AstraZeneca R&D, Innovative Medicines, SE-15185 Södertälje, Sweden
d Neuroscience, CNSP iMed Science, AstraZeneca R&D, Innovative Medicines, SE-15185 Södertälje, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
The NaV1.7 ion channel is an attractive target for development of potential analgesic drugs based on
strong genetic links between mutations in the gene coding for the channel protein and inheritable pain
conditions. The (S)-N-chroman-3-ylcarboxamide series, exemplified by 1, was used as a starting point for
development of new channel blockers, resulting in the phenethyl nicotinamide series. The structure and
activity relationship for this series was established and the metabolic issues of early analogues were
addressed by appropriate substitutions. Compound 33 displayed acceptable overall in vitro properties
and in vivo rat PK profile.
Received 3 July 2012
Revised 6 August 2012
Accepted 8 August 2012
Available online 14 August 2012
Keywords:
Phenethyl nicotinamides
NaV1.7 blocker
Ó 2012 Elsevier Ltd. All rights reserved.
Pain
Sodium channel blockers
Voltage-gated channel
NaV1.7 is a voltage-gated sodium channel with an important
role in the progression of electrical signals in the nervous system
caused by variations in membrane potentials of nerve cells.1 This
ion channel is an attractive target for development of potential
analgesic drugs based on strong genetic links between mutations
in the gene coding for the NaV1.7 protein and inheritable pain
conditions.2 Hereditary loss of function mutations of NaV1.7 in
humans were reported to cause congenital inability to experience
pain (CIP).3,4 Importantly, although CIP is a severe condition by
itself, it is not accompanied by other major physiological problems.
In addition, gain of function mutations have been connected to the
inheritable pain conditions erythromelalgia and familial rectal
pain.5 Tolerability issues limit the use of sodium channel blocker
therapies that are currently available. Finding new compounds
with increased potency for NaV1.7 and higher subtype selectivity
against other NaV channels could greatly improve quality of life
for a large population of patients suffering from pain.6
selectivity over NaV1.5, as measured in whole-cell voltage clamp
electrophysiology assays using physiologically relevant activation
protocols, were generally achieved within the series including
compound 1.8,9 The NaV1.5 channel is widely expressed in heart
muscle and inhibition leads to ventricular arrhythmia. High selec-
tivity against this subtype is therefore an important requirement
for the progression of a potential drug candidate.10,11 Close ana-
logues to compound 1 showed low hERG activity, as measured in
a whole-cell voltage-clamp electrophysiology assay.12 In addition,
this series displayed good in vitro metabolic stability in rat and hu-
man liver microsomes and showed no reactive metabolite forma-
tion. Compound 1 demonstrated low solubility however. We
have found that this shortcoming can be addressed by modifying
the heteroaromatic ring attached to the chromane in combination
with the introduction of a methyl trifluoroethoxy side chain.
O
NaV1.7 pIC50 7.1
NaV1.5 pIC50 5.4
clogP 4.18
hERG pIC50 4.9
O
During the course of our NaV1.7 channel blocker program
we have established the structure-activity relationship in the
(S)-N-chroman-3-ylcarboxamide series, represented by 1 (Fig. 1).7
High potency on the NaV1.7 channel and good functional
F
N
H
F
F
N
O
Solubility 3 µM
N
N
RLM Clint <10 µL/min/µg
HLM Clint <10 µL/min/µg
GSH in HLM: -
1
O
⇑
Corresponding author. Tel.: +46 8 553 28609.
E-mail address: inger.kers@astrazeneca.com (I. Kers).
Figure 1. Profile of chromane 1.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.031

I. Kers et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6108–6115
6109
Table 1
SAR of unsubstituted phenethyl compounds 2–12
O
NaV1.7
pIC50
NaV1.5
pIC50
hERG
pIC50
Solubility
RLM/HLM Clint (
ll/
GSH trapping in
HLM
R1
R2
cLogP
R2
N
H
(lM)
min/ g)
l
R1
1
ꢀ
ꢀ
4.18
7.1
6.9
5.4
4.9
4.9
5.0
3
<10/<10
>500/17
ꢀ
*
*
F
F
F
F
F
N
N
N
O
O
O
2
3
3.87
4.08
57
NM
N
N
N
O
*
*
*
F
F
7.6
7.6
5.2
5.2
4.7
5.2
12
4
129/124
270/210
++
N
O
N
F
*
F
F
N
4
4.52
NM
F
O
*
*
*
F
F
F
F
5
6
3.87
3.42
7.4
7.2
5.2
5.3
5.2
4.9
11
35
>500/160
>500/11
++
++
N
N
O
O
N
*
F
F
N
N
*
*
F
F
F
F
N
O
N
7
3.29
7.0
<4.9
<4.6
310
130/10
NM
HO
N
*
*
*
*
*
*
*
*
*
*
F
F
O
8
9
2.29
3.19
3.69
2.76
1.98
5.7
6.4
6.0
6.6
5.5
<4.5
<4.5
<4.5
5.2
NM
4.5
376
360
150
89
<10/<10
<10/23
NM/NM
<10/11
<10/15
++
N
N
N
N
N
N
N
N
F
F
F
F
F
O
O
NM
NM
+++
NM
O
O
N
N
N
N
F
F
10
11
12
NM
4.7
H
N
F
F
N
N
O
H
N
F
F
<4.5
NM
390
F
NM, not measured; ꢀ, no GSH formation detected; +, GSH formation detected; ++, GSH formation abundant; +++, GSH formation extensive.
Core replacements of the chromane ring were limited to tetralin
due to synthetic challenges. We were interested in exploring a dif-
ferent chemical space by opening up the chromane aliphatic ring
as this would allow variation of the substituents on the resulting
monocyclic core more easily. To this end a number of analogues
were designed and prepared by varying the position and length
of the linker between the phenyl core and the amido substituent.
Generally, these analogues showed low affinity to the NaV1.7 chan-
nel. Therefore we were pleased to see that in compound 2 (see
Table 1) the –OCH2– fragment of the chromane ring could be
removed without major influence on the NaV1.7 potency.
Structurally related to amidochromane 1, compound 2 had a sim-
ilar profile except for a diminished metabolic stability in rat liver
microsomes and improved solubility. Encouraged by this finding
we prepared close analogues to 2, and the results are summarized
in Table 1.
The replacement of the pyrimidine ring with pyrazine in 3 fur-
ther increased the potency on NaV1.7. In case of 4 the NaV1.7
potency was retained (cf. 3), but because cLogP was increased from
3.87 to 4.52 solubility and human metabolic stability were reduced
compared to 2. The use of unsubstituted pyridine (5), methyl-
pyrimidine (6) and hydroxymethyl pyridine (7) rings also retained

6110
I. Kers et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6108–6115
Table 2
SAR of substituted phenethyl analogues 13–21
Compound
cLogP
NaV1.7 pIC50
7.1
NaV1.5 pIC50
5.4
hERG pIC50
4.9
Solubility
M)
RLM/HLM Clint
l/min/ g)
GSH trapping
in HLM
(
l
(l
l
1
4.18
3
<10/<10
ꢀ
O
F
F
F
F
N
H
F
F
13
14
4.32
4.01
7.5
7.2
5.2
5.5
5.2
5.1
2
32/65
ꢀ
N
N
N
N
O
O
O
O
N
N
N
N
N
N
N
O
O
O
F
O
O
O
N
H
F
F
30
13/<10
<10/17
17/45
++
Cl
N
H
F
F
15
16
4.58
3.91
7.5
6.4
5.3
5.2
5.4
<4.5
++
O
N
H
F
F
NM
15
NM
N
O
O
N
O
F
N
H
F
F
17
3.91
<5
4.7
5.2
2
150/40
NM
N
O
N
O
N
CN
O
F
N
H
F
F
18
19
20
21
3.45
4.36
4.21
4.23
5.2
<4.5
5.1
6.1
5.6
NM
5.4
NM
4.9
17
24/<10
190/<10
230/100
35/74
ꢀ
N
O
N
N
O
O
OCHF2
O
F
N
F
F
H
<5
NM
NM
NM
ꢀ
N
O
N
F
F
O
F
N
F
H
7.5
7.6
17
N
O
F
N
N
N
O
O
N
H
F
F
40
N
O
N
F
O
NM, not measured; ꢀ, no GSH formation detected; +, GSH formation detected; ++, GSH formation abundant; +++, GSH formation extensive.

I. Kers et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6108–6115
6111
Table 3
SAR of substituted phenethyl analogues 22–26
Compound
cLogP
NaV1.7 pIC50
7.1
NaV1.5 pIC50
5.4
hERG pIC50
4.9
Solubility
RLM/HLM Clint
l/min/ g)
GSH trapping
in HLM
(lM)
(
l
l
1
4.18
3
<10/<10
ꢀ
F
O
F
N
F
F
H
O
22
3.30
7.4
6.0
4.7
240
88/61
+
N
N
N
O
F
F
O
O
F
F
F
N
H
F
O
23
24
25
3.24
2.80
2.64
7.3
6.9
6.7
5.8
5.8
NM
4.7
14
120
3
45/44
<10/21
67/46
ꢀ
N
N
F
N
O
F
N
H
F
F
<4.5
ꢀ
O
N
N
N
F
O
F
O
N
H
F
F
O
NM
NM
N
N
N
O
Cl
O
N
F
F
F
H
26
3.67
<5.0
<4.5
NM
19
NM/72
NM
O
N
N
N
O
NM, not measured; ꢀ, no GSH formation detected; +, GSH formation detected; ++, GSH formation abundant; +++, GSH formation extensive.
the high NaV1.7 potency whilst decreasing lipophilicity. Introduc-
tion of a homologous ether chain in 8 resulted in a substantial
decrease in potency, cf. 6. The chain elongation in compound 9 in-
creased the potency (cf. 8) but the introduction of a methyl group
on the nicotinic acid moiety lowered it again (10, cf. 9). Worth
noticing is the unexpectedly high solubility for compounds 7 and
9. Introduction of amido and amino groups in 11 and 12 gave only
moderately potent compounds.
In general the structural changes of the heteroaromatic part of
the molecules in 2–7 were well tolerated in terms of NaV1.7
potency, but the resulting compounds were prone to high meta-
bolic turnover particularly in rat liver microsomes. In contrast, 8,
9, 11 and 12 showed good metabolic stability, which may be par-
tially due to reduced lipophilicity, but also suggested that meta-
bolic stability, could be modulated by optimising the right-hand
chain. The potency for NaV1.5 and hERG was low throughout the
sub-series and NaV1.7 selectivity versus these channels was
approximately 100 fold. In general, we observed that a decrease
in cLogP was accompanied by an increase in solubility.
logues suggested that glutathione adducts were localised on the
core phenyl moiety. Further evidence of this was obtained from
6-fluorinated analogues, for which we observed that fluorine was
replaced by hydroxyl (e.g. 14, vide infra). The most plausible mech-
anism behind the reactive metabolite formation, based on this
observation, is via an epoxidation of the phenyl ring.
In order to block the reactive metabolite formation and improve
metabolic stability we focused our attention on substitutions on
the phenyl core. The 6-position in the chromanes is blocked by
an alkoxy moiety and therefore we started our investigation with
introducing substituents in this position. The SAR data is shown
in Table 2 and Table 3. Compound 13 was substituted with a
methyl group which improved the metabolic stability in rat, but
lowered it in human liver microsomes (cf. 2). No glutathione ad-
ducts were observed. The incorporation of halogens such as fluo-
rine in 14 and chlorine in 15 at the same position increased the
stability, but failed to attenuate the reactive metabolite formation.
On the other hand, the methoxy analogue (16) showed only
moderate stability. In 17 the methoxy-moiety was placed in the
4-position which significantly decreased the metabolic stability
in rats compared to that observed for compound 16, indicating that
the 6-position is important for maintaining stability, particularly in
We also found evidence for the production of reactive metabolic
intermediates using a glutathione trapping assay in cases of 3, 5, 6,
8 and 11.13 Initial analysis of the biotransformation of these ana-

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I. Kers et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6108–6115
O
O
i
iv
ii, iii
Ar-B(OH)2
+
+
NH2
NBn2
NH2
N
H
Ar
HO
Ar
C
Br
Br
Ar
Ar
A
B
D
E
F
ii, vii
NHBoc
v
vi
+
Ar-Br
B
NHBoc
O
O
Br
G
H
I
Scheme 1. Reagents and conditions: (i) Benzylbromide, K2CO3, KI, MeCN; (ii) PdCl2ꢁdppf, 2 M K2CO3, iPrOH, heating; (iii) 10% Pd/C, ammonium formate, MeOH, heating; (iv)
HBTU, NEt3, DMF or 1,10-carbonyldiimidazole, EtOAc, 60 °C; (v) di-tert-butyl dicarbonate, NEt3, DCM, 0 °C; (vi) bis(pinacolato)diboron, PdCl2ꢁdppf, KOAc, dioxane, heating;
(vii) TFA, DCM.
rats. Substituents with strong electron withdrawing character gave
moderately (18) and highly (19) unstable compounds in rats. Con-
trarily, the human intrinsic clearance was low for 18 and 19 which
indicates that different metabolic pathways may be activated in
rats compared to humans.
compounds 17 and 26 with no observed NaV1.7 activity in the
measured concentration range. Worth to notice is compound 16
with high apparent similarity to chromane 1, but lower potency.
Placing electron withdrawing substituents in 6-position substan-
tially lowered the NaV1.7 potency (compounds 18 and 19). As we
have previously seen for the unsubstituted analogues (Table 1)
the low potency for NaV1.5 and hERG was retained throughout
the sub-series. The solubility was generally lower though, with
exception of compound 22. In conclusion, introduction of appropri-
ate substituents on the phenyl core was found to improve
metabolic stability and reduce formation of reactive metabolites
while certain types of modifications also were well tolerated from
a potency perspective.
The general synthesis of phenethyl nicotinamides is outlined in
Scheme 1. The starting materials were either commercially avail-
able or could readily be prepared according to published meth-
ods.14–17 Thus, the amino groups of A were dibenzylated and the
formed B subsequently reacted with appropriate arylboronic acids
C. Deprotection afforded D which were coupled with carboxylic
acids E, yielding final products F. The commercial availability of
heteroaryl boronic acids or esters (C) was limited and therefore
we devised an alternative route to D which allowed the use of
heteroaryl halides as coupling partners in the Suzuki reaction.
Boc-protection of A afforded G which was converted to boronic
ester H in a palladium promoted reaction. The formed intermediate
H could subsequently be reacted with suitable arylbromides I,
The fluorine atom at the 6-position improved the metabolic sta-
bility of 14 (cf. 2), but addition of a methyl group at the 3-position
as in 20 abolished the improvement in metabolic stability. An ana-
logue to compound 20 with the shorter trifluoroethoxy substituent
(21) showed a similar overall profile with the exception of clearly
improved metabolic stability. In 22 the replacement of the pyrim-
idine ring with pyrazine and a homologous alkoxy side chain
somewhat improved the stability (cf. 20). Upon replacing the
3-methyl with fluorine in 23 the stability was somewhat improved.
In case of 24 the replacement of the methoxy group on the pyra-
zine led to further increased metabolic stability of the compound.
When the 3-methyl in 22 was replaced by a methoxy group (25) a
marginally more stable compound was obtained. These 3- and
6-disubstituted analogues produced, however, only little or no
reactive metabolites, which is an advantage over 14. Chlorine at
the 5-position in 26, which was not substituted in the chromanes,
did not have major influence on the metabolic stability.
In general, the compounds with halogen or methyl substituents
at the 6-position (13–15) exhibited high potency on NaV1.7 chan-
nel, also with additional substituent in the 3-position (20–25).
However, introducing substituents on the 4 or 5-positions afforded
Method 1
Method 2
O
O
OH
Br
R
R
R
R
R
R
NHBoc
i
NO2
NBn2
ii, iii, iv, v
H
vii, viii
vi
H
Br
Br
Cl
Cl
Cl
Method 3
Method 4
N
R
R
R
N
N
x
iv, v
N
N
N
ix
iv, v
CN
CN
CN
NHBoc
NHBoc
F
Br
Br
Br
Br
Br
Br
Method 5
X
O
X
X
X
O
iv, v
xi
xii
I
O
NH2
NHBoc
F
F
F
F
F
F
Br
Br
Br
Br
X = CH, N, CF
Scheme 2. Reagents and conditions: (i) Sodium borohydride, THF; (ii) thionyl chloride, THF; (iii) NaCN, NEt3, DMSO; (iv) BH3-tetrahydrofuran complex, THF; (v) 10% NaOH,
di-tert-butyl dicarbonate, THF; (vi) ammonium acetate, MeNO2, heating; (vii) LiAlH4, diethyl ether, 0 °C; (viii) benzylbromide, K2CO3, KI, MeCN; (ix) NaHMDS, MeCN, toluene,
0 °C; (x) MeI, NaH, DMF; (xi) ethyl bromodifluoroacetate, Cu bronze, DMSO, heating; (xii) NH3 in methanol.

I. Kers et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6108–6115
6113
Table 4
SAR of compounds 27–34
Compound
cLogP NaV1.7
NaV1.5
pIC50
hERG
pIC50
Solubility
RLM/HLM Clint (
ll/
GSH trapping in
HLM
pIC50
(l
M)
min/ g)
l
1
4.18
7.1
5.4
4.9
3
<10/<10
ꢀ
N
O
F
N
H
F
F
27
2.48
<5.0
<4.5
NM
360
<10/<10
ꢀ
ꢀ
N
O
N
N
N
O
O
N
H
F
F
F
28
2.98
<5.0
<4.5
NM
140
<10/<10
N
O
N
N
O
N
N
O
F
N
F
F
H
O
29
30
31
32
33
1.85
2.88
3.07
3.34
3.55
<5.0
<5.0
6.0
<4.5
<4.5
NM
4.7
NM
NM
NM
4.7
260
370
64
<10/<10
<10/<10
<10/<10
13/<10
NM
NM
NM
ꢀ
N
N
O
N
O
F
F
F
F
N
H
F
F
N
N
N
N
O
O
N
N
N
N
N
N
O
O
O
O
O
O
O
N
H
F
F
N
N
H
F
F
F
F
F
O
O
6.2
190
N
N
H
F
F
F
6.6
4.5
4.6
86
16/<10
ꢀ
N
F
O
F
N
H
F
F
F
F
O
34
3.69
6.7
5.0
4.7
52
24/<10
ꢀ
N
N
N
O
NM, not measured.; ꢀ, no GSH formation detected; +, GSH formation detected; ++, GSH formation abundant; +++, GSH formation extensive.
affording common intermediates D. Using the procedure described
in Scheme 1 and starting from commercially available phenethyl-
amines compounds 2–12, 14 and 15 were prepared.
Many of the substituted phenethylamines were not commer-
cially available and were prepared using one of the methods
outlined in Scheme 2. The prepared phenethylamines of type B
or G were then used to synthesise the desired compounds accord-
ing to Scheme 1. Synthesis of the phenethylamines required for the
preparation of compounds 18–24 and 26 is described in Method 1.
For compounds 18 and 19 the starting materials were prepared
according to literature procedures.18,19 The synthesis of the phen-
ethylamines required for compounds 13, 16, 17 and 25 is described

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I. Kers et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6108–6115
in Method 2. The phenethylamines used in the synthesis of com-
pounds 27–29 is described in Method 3. For compound 30 synthe-
sis of the required phenethylamine followed Method 4. For
compounds 31–34 the synthesis of phenethylamines used is de-
scribed in Method 5.
At this point of our investigation we decided to introduce a pyr-
idine ring in the core, aiming to improve metabolic stability and to
avoid reactive metabolite formation. Therefore close analogues to 2
were designed and prepared and their data is summarized in
Table 4. Unfortunately compounds 27–30 did not show activity
on the NaV1.7 channel in the measured concentration range, prob-
ably in part due to their low lipophilicity, but showed high meta-
bolic stability in both rat and human liver microsomes and
reactive metabolites were absent. Compound 31, that combine a
100
80
60
40
20
0
0
0.9
2.7
9
Compound 33 (µM)
Figure 2. Concentration-dependent effects of compound 33 on ectopic firing from
injured DRG neurons. Buffer or compound at 0.9, 2.7 and 9 M were perfused
cumulatively on DRGs excised from SNL-operated rats, 15 min for each concentra-
tion, and the effects during the last 3 min perfusion were analysed.
l
pyridine core structure with
a difluorinated ethylene linker,
showed a moderate potency (cf. 27–30) most likely due to in-
creased cLogP in combination with finding a permitted region for
introducing substituents.
intestinal microsomes, rat S9 fraction and rat intestinal fluid. Per
oral PK was also performed at a higher dose (22 at 100 lmol/kg)
The important finding that difluoro substitution on the ethylene
linker is accepted from a potency perspective prompted us to turn
our attention back to unsubstituted phenyl analogues, such as 2, to
further study this substitution with respect to metabolic stability.
Therefore, compound 32 was prepared and tested (Table 4). We
were particularly pleased to see that low intrinsic clearance in rats
was measured suggesting acceptable pharmacokinetic properties
in vivo in rat. For this series in general, the in vivo clearance could
be predicted within a 2 fold based on the in vitro metabolic stabil-
ity in rat. Furthermore, the compound retained moderate NaV1.7
potency. The pyrazine analogue 33 and fluorine substituted 34
both showed further increased potency, similar metabolic profile
and no reactive metabolite formation. In addition these com-
pounds displayed good solubility and 30–100 fold selectivity over
NaV1.5 and hERG.In vivo rat PK data for compounds 1, 13, 22, 23,
24, 32 and 33 is presented in Table 5.20 The (S)-N-chroman-
3-ylcarboxamide series generally displayed a low to moderate
clearance and the bioavailability of the compounds could mainly
be explained by the hepatic first-pass clearance. Compound 1
showed low clearance but a moderate bioavailability. When we
opened up the chromane ring we initially found high clearance
and consequently low bioavailability, exemplified by 13. This im-
peded in vivo pharmacological testing of these initial analogues.
The systemic clearance in rat was markedly improved by substitut-
ing the 3- and 6-positions of the central phenyl ring and/or by
using a methyl trifluoroethyl ether chain as in 22, 23 and 24. In
spite of this, the bioavailability remained less than 10%. Introduc-
tion of a difluoro substitution on the ethylene linker restored the
bioavailability to that of the chromane series as shown by 32 and
33.
but bioavailability remained unchanged, indicating that no satura-
ble enzymes or transporters were involved. Oral administration of
the non-selective CYP inhibitor 1-aminobenzotriazole (ABT) im-
proved the bioavailability of compound 24 from 7% to 40%. This
improvement correlated well with the estimated hepatic first-pass
effect for compound 24 based on the systemic clearance, which
was observed to decrease ꢂ30% using ABT after intravenous
administration.21 We concluded from these combined results that
the low bioavailability in the rat was not majorly due to metabo-
lism, although first-pass metabolism did contribute. Subsequently,
precipitation tests of 33 in fasted state simulated intestinal fluid
(FaSSIF) using different formulations were performed. Precipita-
tion of a fine particle suspension occurred immediately, which
did not dissolve after 12 h. This suggested that the low bioavail-
ability may be related to low solubility in physiological solutions.
Low bioavailability mainly impeded in vivo pharmacological
analysis of analogues in the described phenethyl nicotinamide ser-
ies. Instead, efficacy of compound 33 was investigated in a rat DRG
ex vivo preparation of SNL-induced ectopic activity.22 Test concen-
trations of the compound was selected based on the in vitro po-
tency for the rat NaV1.7 channel; IC50 = 0.9 lM, using the same
steady-state protocol (Vhold ꢀ65 mV) as for the human channel.9
The average basal firing frequency of the tested fibres was
9.02 1.86 Hz (mean SEM, n = 10). Compound 33 applied cumu-
latively on the DRGs caused progressive inhibition of ectopic firing
of all fibres. At 0.9, 2.7 and 9 lM of compound 33 the ectopic activ-
ity was inhibited by 51.5 7.9%, 84.5 5.4% and 96.1 2.2%
(mean SEM), respectively (Fig. 2). Thus, the estimated EC50 for
ex vivo inhibition of SNL-induced ectopic activity correlated very
well to the in vitro NaV1.7 potency of the compound. Similar corre-
lation between in vitro Nav1.7 potency and ex vivo inhibition of
SNL-induced ectopic activity has been established for clinically
used sodium channel blockers such as lidocaine, carbamazepine
and lamotrigine (Kalezic et al., in preparation), as well as for a com-
pound from a novel series of 3-Oxoisoindoline-1-carboxamides.23
In summary, we established the structure-activity relationship
for a novel phenethyl nicotinamide series of NaV1.7 blockers. We
showed that metabolic issues could be addressed by introduction
of appropriate substituents on the central phenyl ring and difluori-
nation on the ethylene linker restored the bioavailability observed
in the chromane series while retaining potency and selectivity
versus NaV1.5 and hERG at acceptable levels. As a result of our
studies we found compound 33 that displayed good solubility
and NaV1.7 potency in addition to a 100 fold selectivity to hERG
and NaV1.5 channels. In contrast to earlier analogues in the phen-
ethyl nicotinamide series compound 33 displayed good DMPK
properties. Finally, we demonstrated efficacy in an ex vivo nerve
Based on systemic clearance a much higher bioavailability was
anticipated after oral administration (e.g. 22, 23). Potential
contribution of intestinal metabolism in rat was dismissed using
Table 5
Rat in vivo PK for compounds 1, 13, 22, 23, 24, 32 and 33
Compound
1
13
22
23
24
32
33
AUC/dosea (h kg/L)
Vss (L/kg)
1.2
7.2
6.4
14
0.32
0.23
4.8
7.6
26
0.33
2.4
0.98
50
0.002
0.02
0.25
2.4
0.91
1.5
0.90
19
0.03
0.53
1.5
3.6
4
1.3
0.68
1.74
5.9
24.6
0.05
0.25
0.50
2.3
0.71
1.4
2.0
1.5
0.72
1.2
11
0.41
1.4
0.33
2.8
27
a
0.85
0.90
13
0.06
0.16
0.67
3.0
a
t
(h)
½
CLa (mL/min/kg)
23
AUC/doseb (h kg/L)
0.24
0.84
0.75
2.4
b
Cmax
(
l
mol/L)
b
tmax (h)
b
t
(h)
½
Fb (%)
0.6
5
7
34
a
Intravenous (iv), dose 3
l
mol/kg.
b
Per oral (po), dose 10
lmol/kg.

I. Kers et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6108–6115
6115
8. Harmer, A. R.; Abi-Gerges, N.; Easter, A.; Woods, A.; Lawrence, C. L.; Small, B. G.;
Valentin, J.-P.; Pollard, C. E. J. Pharmacol. Toxicol. Methods 2008, 57, 30.
9. Measurements of NaV1.7 currents in whole-cell voltage clamp
electrophysiology described in Supplementary data.
injury model for compound 33 with good correlation to in vitro
NaV1.7 potency.
10. Goldin, A. L. Annu. Rev. Physiol. 2001, 63, 871.
11. Tfelt-Hansen, J.; Winkel, B. G.; Grunnet, M.; Jespersen, T. J. Cardiovasc.
Electrophysiol. 2010, 21, 107.
Acknowledgments
The authors wish to thank colleagues at Physical Chemistry
Characterization Team for providing solubility data, and the Safety
Screening Centre for providing NaV1.5 and hERG data. In addition,
we would like to acknowledge NAEJA Pharmaceuticals Inc., Canada
for the supply of synthesized compounds.
12. Bridgland-Taylor, M. H.; Hargreaves, A. C.; Easter, A.; Orme, A.; Henthorn, D. C.;
Ding, M.; Davis, A. M.; Small, B. G.; Heapy, C. G.; Abi-Gerges, N.; Persson, F.;
Jacobson, I.; Sullivan, M.; Albertson, N.; Hammond, T. G.; Sullivan, E.; Valentin,
J. P.; Pollard, C. E. J. Pharmacol. Toxicol. Methods 2006, 54, 189.
13. Evans, D. C.; Watt, A. P.; Nicoll-Griffith, D. A.; Baillie, T. A. Chem. Res. Toxicol.
2004, 17, 3.
14. Besidski, Y.; Claesson, A.; Csjernyik, G.; Gravenfors, Y.; Kers, I.; Skogholm, K.;
Sohn, D. PCT Int. Appl., WO2008130319, 2008.
15. Besidski, Y.; Kers, I.; Nylöf, M.; Sandberg, L.; Skogholm, K. PCT Int. Appl.,
WO2008130320, 2008.
Supplementary data
16. Besidski, Y.; Kers, I.; Macsari, I.; Nylöf, M.; Rotticci, D. PCT Int. Appl.,
WO2008130321, 2008.
17. Clayton, J.; Ma, F.; Van Wagenen, B.; Ukkiramapandian, R.; Egle, I.; Empfield, J.;
Isaac, M.; Slassi, A.; Steelman, G.; Urbanek, R. PCT Int. Appl., WO200602087,
2008.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.08.
031.
18. Attardo, G.; Tripathy, S. PCT Int. Appl., WO2010132999, 2010.
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