Substituted Indazoles as Nav1.7 Blockers for the Treatment of Pain

Article abstract of DOI:10.1021/acs.jmedchem.6b00063

The genetic validation for the role of the Na_v1.7 voltage-gated ion channel in pain signaling pathways makes it an appealing target for the potential development of new pain drugs. The utility of nonselective Na_v blockers is often li

Full text of DOI:10.1021/acs.jmedchem.6b00063

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Article
V
Substituted Indazoles as Na1.7 Blockers for the Treatment of Pain
Jennifer M Frost, David A. DeGoey, Lei Shi, Rebecca J Gum, Meagan M Fricano, Greta L Lundgaard,
Odile F El-Kouhen, Gin C Hsieh, Torben R Neelands, Mark A Matulenko, Jerome F Daanen,
Madhavi V Pai, Nayereh Ghoreishi-Haack, Cenchen Zhan, Xu-Feng Zhang, and Michael E. Kort
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00063 • Publication Date (Web): 25 Mar 2016
Downloaded from http://pubs.acs.org on March 27, 2016
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Substituted Indazoles as Na 1.7 Blockers for the Treatment of Pain
v
*
Jennifer M. Frost, † David A. DeGoey, Lei Shi, Rebecca J. Gum, Meagan M. Fricano, Greta L.
†
†
†
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†
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Lundgaard, Odile F. El-Kouhen, Gin C. Hsieh, Torben Neelands, Mark A. Matulenko, Jerome F.
†
†
††
†
†
Daanen, Madhavi Pai, Nayereh Ghoreishi-Haack, Cenchen Zhan, Xu-Feng Zhang, and Michael
E. Kort †
†
AbbVie, Research and Development, 1 North Waukegan Road, North Chicago, IL 60064, USA
††Aptinyx, 1801 Maple Ave. #4300 Evanston, IL 60201
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Abstract
The genetic validation for the role of the Na 1.7 voltageꢀgated ion channel in pain signaling pathways
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makes it an appealing target for the potential development of new pain drugs. The utility of nonꢀ
selective Na blockers is often limited due to adverse cardiovascular and CNS side effects. We sought
v
more selective Na 1.7 blockers with oral activity, improved selectivity and good drugꢀlike properties.
v
The work described herein focused on a series of 3 and 4ꢀsubstituted indazoles. SAR studies of 3ꢀ
substituted indazoles yielded analog 7 which demonstrated good in vitro and in vivo activity, but poor
rat pharmacokinetics. Optimization of 4ꢀsubstituted indazoles yielded two compounds, 27 and 48, that
exhibited good in vitro and in vivo activity with improved rat pharmacokinetic profiles. Both 27 and 48
demonstrated robust activity in the acute rat monoꢀiodoacetateꢀinduced osteoarthritis model of pain and
subꢀchronic dosing of 48 showed a shift to a lower EC over seven days.
5
0
Introduction
Voltageꢀgated sodium channels are involved in the initiation and propagation of action potentials in
1
,2
excitable tissues such as nerve and muscle.
Na 1.7, one of nine sodium channel isoforms, is of
v
particular interest as a target for the treatment of pain based on strong genetic evidence. Specifically,
lossꢀofꢀfunction mutations in SCN9A, the gene that encodes Na 1.7, result in congenital insensitivity to
v
pain (CIP), a condition characterized by the total absence of the ability to perceive any kind of noxious
3ꢀ6
stimulus as painful.
In contrast, gainꢀofꢀfunction SCN9A mutations can result in painful conditions
7
,8
such as inherited erythromelalgia and paroxysmal extreme pain disorder.
Furthermore, knockꢀout
studies in which Na 1.7 was ablated in both sensory and sympathetic neurons resulted in the ablation of
v
9
both inflammatory and neuropathic pain. Clinically, nonꢀselective sodium channel blockers approved
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for use as local anesthetics, anticonvulsants or tricyclic antidepressants have proven useful in the
management of pain although their utility may be hindered by their lack of subtype selectivity leading to
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unwanted side effects. Given the high level of validation, there have been significant efforts towards
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11ꢀ16
identifying selective Na 1.7 blockers for the treatment of pain.
In addition to subtype selectivity,
v
sodium channel blockers can bind preferentially to the inactivated state of the channel which is thought
1
7,18
to be of particular importance in the hyperexcitable neurons present in chronic pain.
Identification
of state dependent blockers that can target channels in different kinetic states and tissues could thus
impart improved functional selectivity.
Our efforts described herein were aimed at discovering novel Nav1.7 blockers that demonstrated
robust efficacy in pain models while lacking CNS and cardiovascular side effects and also exhibiting
good drugꢀlike properties (cLogP, MW, pharmacokinetics). Our initial work focused on a 3ꢀsubstituted
indazole series which led to several active analogs with moderate to poor selectivity versus the cardiac
sodium channel, Na 1.5 and generally poor pharmacokinetics profiles (Figure 1). We then focused on 4ꢀ
v
substituted indazole analogs which exhibited improved selectivity versus Na 1.5 and improved
v
pharmacokinetic properties. Select compounds were interrogated in the rat monoꢀiodoacetateꢀinduced
osteoarthritis model of pain as we viewed the treatment of osteoarthritis pain as a significant unmet
need. This clinical target of Na 1.7 in osteoarthritis pain is supported by the reported knockout studies
v
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,19,20
and additional literature.
Figure 1. General Structures of 3 and 4-substituted indazoles
Chemistry
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The preparation of the 3ꢀhydroxy substituted indazoles is shown in Schemes 1 and 2 and began with
the coupling of commercially available acid 1 and hydrazine 2 in the presence of carbonyl dimidazole
(
CDI) and Hünig’s base to give the intermediate hydrazide which when treated with NaNO and HCl at
2
0
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6 °C gave 3ꢀhydroxyꢀindazole 3 as shown in Scheme 1. Intermediate 3 was then alkylated with
commercially available 2ꢀbromoꢀ1ꢀmorpholinoethanone or 1ꢀ(2ꢀchloroethyl)ꢀ1,3ꢀdihydroꢀ2Hꢀimidazolꢀ
ꢀone in the presence of NaH to give 4 and 8, respectively. Alkylation of 3 with methyl 2ꢀ
2
bromopropanoate followed by base hydrolysis gave carboxylic acid 6. Acid 6 was treated with oxalyl
chloride to form the intermediate acid chloride which was then coupled with morpholine to give analog
5
.
Scheme 1. Preparation of 3-Hydroxyindazoles
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The 3ꢀhydroxyꢀindazole 3 was also subjected to Mitsunobu coupling conditions in the presence of
commercially available 1ꢀ(2ꢀhydroxyethyl)ꢀ2ꢀimidazolidinone or 1ꢀ(2ꢀhydroxyethyl)imidazole to give 7
or 9, respectively. Analog 7 was then alkylated with methyl iodide and NaH to form methylated analog
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
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2
2
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7
8
9
0
1
0. Mitsunobu coupling of 3 and commercially available 2ꢀ(3ꢀoxazolidine)ethanol gave intermediate 11
which was then cyclized in the presence of carbonyldiimidazole (CDI) and 1,8ꢀ
diazabicyclo[5.4.0]undecꢀ7ꢀene (DBU) to generate oxazolidinone 12.
Scheme 2. Preparation of 3-Hydroxyindazoles 15 and 16
Analogs 15 and 16 were obtained as shown in Scheme 2. The 3ꢀhydroxyꢀindazole intermediate 13
was obtained upon treatment of 1 with NaNO and Na SO in the presence of acid. Bocꢀprotection of
2
2
3
the resulting indazole nitrogen followed by Mitsunobu coupling with 1ꢀ(2ꢀhydroxyethyl)ꢀ2ꢀ
2
2,23
imidazolidinone and removal of the Boc group resulted in intermediate 14. Buchwald coupling
4 with 2,4ꢀdifluoroꢀ1ꢀiodobenzene and 1ꢀfluoroꢀ4ꢀiodobenzene gave 15 and 16, respectively.
The synthetic routes to the 4ꢀsubstituted indazole analogs are shown in Schemes 3ꢀ8. As shown in
of
1
Scheme 3, the coupling of commercially available 17 with 1ꢀfluoroꢀ2ꢀiodobenzene in the presence of
CuI gave the intermediate Nꢀarylated product which was then debenzylated with ammonium formate
and palladium on carbon to give 18. Alkylation of 18 with t-butyl (2ꢀbromoethyl)carbamate and NaH
resulted in Bocꢀamine 19.
Scheme 3. Preparation of 4-Substituted Indazole 19
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As shown in Scheme 4, imidazolidinone analogs 24, 26 and 32-34 were prepared by first coupling
commercially available 2ꢀbromoꢀ6ꢀfluorobenzaldehyde (20) and (2ꢀfluorophenyl)hydrazine (2) in the
presence of Cs CO in Nꢀmethylꢀ2ꢀpyrrolidinone at 140 °C to give intermediate 21. Aryl bromide 21
2
3
then underwent Pdꢀcatalyzed coupling with imidazolidinꢀ2ꢀone giving 22 which was then alkylated with
various alkyl halides in the presence of NaH to give analogs 24, 26, 32-34 in moderate to good yield.
Intermediate 22 was also Bocꢀprotected giving 23 and was Nꢀarylated with 4ꢀiodopyridine in the
presence of CuI to give analog 35. Intermediate 22 was also treated with t-butylbromoacetate and NaH
and the resulting tꢀbutyl ester was then hydrolyzed with TFA to give carboxylic acid intermediate 30.
The acid was then coupled with various amines using HATU conditions to give amides 25, 27-29 and 31
in moderate to good yield.
Scheme 4. Preparation of 4-Substituted Indazoles
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F
F
N
O
N
O
F
N
N
F
F
+
a
N
b
O
f
N
N
H N NH
N
2
Br
NH
22
N
10
11
12
13
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17
18
19
20
21
22
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24
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Br
30
2
0
2
21
O
OH
c
g
e
d
H
NH2
N
F
N
N
25: R =
27: R =
2
4: R =
1
N
O
F
N
O
F
N
N
N
N
26: R =
N
1
O
F
N
F
N
O
N
O
O
O
NR1
N
N
28: R =
29: R =
F
N
32: R =
1
N
N
N
O
OH
33: R =
N
N
1
O
O
N
NR2
O
N
N
N
31: R =
34: R =
1
F
2
3
35
(
a) Cs CO , NMP, 140 °C, 91% yield (b) imidazolidin-2-one, Cs CO , Xantphos, Pd (dba) , DME, 80 °C, 51% yield; (c) alkyl halide, NaH, DMF,
2
3
2
3
2
1
3
2
24-76% yield; (d) Boc O, DMAP, CH CN, 78% yield (e) 4-iodopyridine, CuI, trans-N ,N -dimethylcyclohexane-1,2-diamine, K PO , 1,4-dioxane,
2
3
3
4
83% yield; (f) (i) tert-butyl 2-bromoacetate, NaH, DMF, 81% yield (ii) TFA, CH Cl , 99% yield; (g) amine, HATU, i-Pr EtN, THF 54-96% yield
2
2
2
Piperazinone analog 38 was prepared as described in Scheme 5. Coupling of piperazinꢀ2ꢀone with
bromoꢀindazole intermediate 21 using Pd (dba) and Nolan’s catalyst gave 36. Alkylation with ethyl 2ꢀ
2
3
iodoacetate in the presence of NaH followed by hydrolysis resulted in carboxylic acid 37. Amide
formation with HATU and Hünig’s base gave piperazinone analog 38.
Scheme 5. Preparation of 4-Substituted Indazole 38
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Bicyclic analog 42 was prepared as described in Scheme 6 starting from commercially available
materials 39 and 40. Cyclization of 39 and 40 occurred in the presence of catalytic TFA and was
followed by removal of the Bocꢀprotecting group to give 41 in good yield. Intermediate 41 then
underwent the CuIꢀcatalyzed Buchwald coupling with 21 to generate analog 42.
Scheme 6. Preparation of 4-Substituted Indazole 42
F
N
H
N
N
O
O
N
O
N
a
b
+
N
N
O
Si
O
O
N
Ph
3
9
40
41
Ph
42
(
a) (i) trifluoroacetic acid (cat), CH Cl , 86% yield; (ii) trifluoroacetic acid (8 eq), CH Cl , 73% yield (b)
2 2 2 2
1
2
2
1, CuI, trans-N ,N -dimethylcyclohexane-1,2-diamine,K PO , 1,4-dioxane, 83% yield
3 4
The chiral bicycle 48 was also prepared starting with 39 and using commercially available αꢀ
methylbenzyl analog 43 in the presence of catalytic TFA to give an approximate 1:1 mixture of isomers
4
4 and 45 which was separated via silica gel chromatography (Scheme 7). The resulting single
enantiomer 45 next underwent Bocꢀremoval in the presence of excess TFA and the resulting lactam was
coupled with indazole bromide 21 using CuIꢀBuchwald conditions to give 46. The αꢀmethylbenzyl
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analog 46 was then treated with chloroethyl chloroformate to give free amine 47 which then underwent
amide coupling with 3ꢀhydroxyꢀ3ꢀmethylbutanoic acid to give desired analog 48.
Scheme 7. Preparation of 4-Substituted Indazole 48
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
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2
2
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3
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0
O
O
O
O
N
N
N
N
O
O
H
H
H
H
(R)
O
N
O
a
b
N
+
Si
O
O
39
43
44
45
F
F
N
O
N
N
F
N
N
O
N
N
N
N
c
d
O
N
H
H
H
H
H
H
N
OH
N
H
O
46
47
48
(a) trifluoroacetic acid, CH Cl , 40% yield 45 separated from ~1:1 mixture of 44 and 45; (b) (i) TFA, CH Cl , quantitative
2
2
2
2
1
2
yield; (ii) 21, CuI, trans-N ,N -dimethylcyclohexane-1,2-diamine, K PO , 1,4-dioxane, 87% yield; (c) 1-chloroethyl
3
4
chloroformate, CH Cl , 50 °C; MeOH, reflux, 73% yield; (d) 3-hydroxy-3-methylbutanoic acid, HATU, i-Pr EtN, THF,
2
2
2
68% yield
24
Related analog 55 was prepared as shown in Scheme 8. Literature precedence describes the racemic
synthesis of intermediate 52 with the literature analog having an N-benzyl group in place of the αꢀ
methylbenzyl group shown. Butꢀ3ꢀenꢀ1ꢀol (49) was treated with MsCl to give the intermediate mesylate
which then underwent displacement with (S)ꢀ1ꢀphenylethanamine followed by alkylation with methyl
bromoacetate to give 50. Sequential treatment of 50 with lithium diisopropyl amide and zinc bromide
followed by iodine gave the cyclized iodoꢀintermediate which was reacted with sodium azide to give 51.
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2
2
2
2
2
2
2
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3
3
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3
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4
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5
6
Staudinger reduction of the azide to the corresponding amine resulted in lactam formation. To facilitate
purification, the lactam was Bocꢀprotected and the isomers were separated via silica gel chromatography
which, after removal of the Bocꢀgroup, gave the desired single enantiomer 52 in good yield. The lactam
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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0
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9
0
1
2
3
4
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7
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9
0
5
2 was then coupled with indazole bromide 21 again using CuIꢀBuchwald conditions to give 53 which
underwent hydrogenation to provide amine 54. Amide coupling with 3ꢀhydroxyꢀ3ꢀmethylbutanoic acid
resulted in formation of desired analog 55.
Scheme 8. Preparation of 4-Substituted Indazole 55
N3
H
a
b
c
d
CO Me
NH
2
N
(
CO Me
N
2
OH
N
S)
H
O
Ph
Ph
Ph
49
50
51
52
H
F
N
F
N
N
N
N
N
e
f
H
Ph
N
O
N
N
O
N
H
O
H
H
O
F
H
N
NH
OH
53
54
55
(
a) (i) MsCl, Et N, CH Cl , 92% yield; (ii) (S)-1-phenylethanamine, CH CN, 85 °C, 20% yield; (iii) methyl bromoacetate, i-
3
2
2
3
Pr EtN, DMSO, 85% yield; (b) (i) LDA, Et O, -78 °C; ZnBr , Et O, -74 °C to r.t.; iodine, Et O, 0 °C, 96% yield; (ii) NaN , DMF,
2
2
2
2
2
3
5
0 °C, quantitative yield; (c) (i) Ph P, 1:1 MeTHF/H O, 74 °C; (ii) Boc O, DMAP (cat), i-Pr EtN, DMF; minor isomer separated
3 2 2 2
1
2
out; 71% yield; (iii) TFA, CH Cl , 99% yield; (d) 21, CuI, trans-N ,N -dimethylcyclohexane-1,2-diamine, K PO , 1,4-dioxane,
2
2
3
4
1
00 °C, 77% yield; (e) 20% Pd(OH) on carbon (wet), 30 psi, trifluoroethanol, quantitative yield; (f) 3-hydroxy-3-
2
methylbutanoic acid, HATU, Et N, DMF, 62% yield.
3
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Biological Evaluation
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
The indazole analogs were first evaluated in a Fluorescence Resonance Energy Transfer (FRET)ꢀbased
membrane potential functional assay using HEK293 cells that were stably expressing recombinant
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
human Na 1.7. Selected analogs were additionally tested in an electrophysiology (EP) assay using
v
HEK293 cell lines stably expressing human Na 1.7 utilizing a voltage protocol to assess activity at
v
resting and partially inactivated states of the channel (holding potentials of ꢀ100 mV and ꢀ65 mV,
respectively) to evaluate the stateꢀdependence of compounds. Data shown in the Tables 1 and 2 depict
1
8
the ꢀ65 mV value which is thought to be representative of pain states.
Virtually no activity was
observed at the resting state (ꢀ100 mV) for all analogs and, as such, all were determined to be stateꢀ
dependent. The Nav1.7 FRET and electrophysiology data generally correlated; the expected variations
that were observed may be attributable to significant differences in the in vitro assay conditions.
Compounds were further evaluated for selectivity versus the cardiac sodium channel, Na 1.5 via an
v
electrophysiology assay using HEK293 cell lines stably expressing human Na 1.5 from a holding
v
2
5
potential of ꢀ90 mV and a voltage protocol designed to mimic cardiac myocyte physiological activity.
ADME properties were gauged using metabolic stability in human and rat liver microsomes and in rat
PK on select compounds. The Pꢀgp efflux ratio was determined using MDCK cells transfected with the
MDR1 gene which encodes human Pꢀgp grown on transwell inserts and directional differences in cell
monolayer permeability of test compounds are reported as a ratio. Finally, select analogs were tested in
the rat monoꢀiodoacetateꢀinduced osteoarthritis model (MIAꢀOA, grip force endpoint) wherein an intraꢀ
articular injection of monoꢀiodoacetate causes osteoarthritisꢀlike knee joint lesions. Analogs were then
26
administered orally and the rats were tested for grip force deficit of hind limbs.
Results and Discussion
Our work began with the 3ꢀhydroxy indazole series (Table 1) with 4 as the lead compound, which was
27
identified from a high throughput screen. While similar analogs have been reported in the literature,
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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
the analogs described here lack the basic amine side chain pharmacophore of the previously reported
structures. Although 4 showed reasonable in vitro activity at Na 1.7 in both the FRET (IC = 0.76 ꢀM)
v
50
and electrophysiology assays (IC = 1.4 ꢀM) along with moderate rat and human microsomal stability,
5
0
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
its bioavailability in rat PK (F = 22%) was low primarily due to plasma instability (1% remaining, 4h in
plasma). Our goal for this series was to improve plasma stability while maintaining Na 1.7 activity and
v
good microsomal stability. We also sought improved selectivity against the cardiac sodium channel,
Na 1.5. Analog 5 with an additional methyl group on the glycolate amide, demonstrated improved
v
Na 1.7 activity (FRET IC = 0.11 ꢀM; EP IC = 0.62 ꢀM) and stability in plasma (>80% remaining, 4h
v
50
50
in plasma) but poor metabolic stability (hClint,u = 19 L/h/Kg). Replacement of the glycolate amide with
a cyclic urea, provided compound 7 which exhibited moderate activity at Na 1.7 in FRET (IC = 1.9
v
50
ꢀ
M) and electrophysiology assays (IC = 2.7 ꢀM) and had moderate metabolic stability (hClint,u = 11
5
0
L/h/Kg). Rat PK studies demonstrated that 7 had similar bioavailability (F = 17%) to 4 (F = 22%) but
was stable in plasma (>80% remaining, 4h in plasma) although 7 did exhibit a short halfꢀlife in rat (t1/2
.4 h). Metabolite identification studies in human and rat liver microsomes suggested that metabolism
=
0
was primarily occurring at the 4ꢀposition of the Nꢀaryl group with additional metabolites resulting from
oxidation of the cyclic urea ring being observed. Cyclic urea 7 did exhibit robust activity in our
osteoarthritis model of pain (vida infra) and, as such, efforts were undertaken to improve the
pharmacokinetic properties of the series. Most attempts to modify the cyclic urea moiety resulted in
analogs with good to moderate activity at Na 1.7, but generally poor metabolic stability. For example,
v
unsaturated analog 8 maintained activity at Na 1.7 (FRET IC = 0.96 ꢀM) and was somewhat selective
v
50
versus Na 1.5 (IC = 5.1 ꢀM), however, the halfꢀlife in rat (t = 0.4 h) was not improved. Imidazole 9
v
50
1/2
also showed an insufficient improvement in PK properties relative to 7. Methylated analog 10 and
oxazolidinone 12 exhibited acceptable activity at Na 1.7, but both analogs had poor metabolic stability.
v
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The identification of the 4ꢀposition of the Nꢀphenyl group as the primary site of metabolism for 7 led to
an investigation of fluorination as a blocking strategy with analogs 15 and 16. Both analogs
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
demonstrated only moderate Na 1.7 activity however, 15 did exhibit somewhat improved rat PK
v
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
properties (F = 71%, t1/2 = 0.94 h).
Table 1: In Vitro Activity of 3-Hydroxy Indazoles
EP
FRET
d
hNa 1.7 hNa 1.5
hCl,int,u
rCl,int,u
t1/2
(h)
v
v
hNa 1.7
%F
v
c
IC , -65 IC50 (ꢀM) (L/h/Kg) (L/h/Kg)
a
50
IC50 (ꢀM)
b
mV (ꢀM)
R1
R2
tetracaine
0
.056 ±
0.32
0.65 ± 0.3
(manual)
0
.002
0
.76 ±
4
5
7
8
9
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2,4-F
1.4
0.62
2.7
1 ± 0.24
7
11
43
22
27
17
35
28
1.6
2
0
.05
0.11 ±
0.009
19
11
12
9
1.9 ± 0.16
2.8 ± 1.0
5.1 ± 1.2
2 ± 0.04
83
0.4
0.4
1.1
0.96 ±
47
0
.32
1.1 ± 0.14
7
69
0.32 ±
0.002
10
1.2
60
41
9
432
149
37
12
2.6 ± 0.09
2 ± 0.30
15
12
3.4 ± 0.92
71 0.94
a
Na 1.7 IC50 values were determined by leastꢀsquares fitting of a logistic equation to data from full eightꢀ
v
point, halfꢀlog concentration–response curves using a FRETꢀmembrane potential assay as described in the
Experimental Section. Data is shown with standard error (SEM) and represent the mean of two or more
b
separate determinations. Na 1.7 EP, data were collected using a slow inactivated state QPatch protocol with
v
c
V1/2= ꢀ65 mV. Na 1.5, data were collected using an inactivated state protocol with V1/2= ꢀ90 mV with
v
d
standard error (SEM) representing the mean of two or more separate determinations. Human and rat liver
microsomal stability data, rat bioavailability and halfꢀlife were obtained as described in the Experimental
Section.
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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
With consistently poor metabolic stability for the 3ꢀsubstituted indazole series and narrow SAR,
adequate progress was not made towards achieving the desired potency and pharmacokinetic profile.
We postulated that other indazole connectivities may allow for similar binding to the receptor but also
tolerate increased functional group diversity, particularly the 4ꢀposition of the indazole which would
project the side chain with a similar vector to the 3ꢀposition. We discovered that the 4ꢀsubstituted
indazoles did indeed provide more tolerant SAR in increased metabolic stability and we thus shifted our
focus to this series (Table 2).
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
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7
8
9
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1
2
3
4
5
6
7
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
We first looked at linear side chain analog 19 which we were pleased to note maintained activity at
Na 1.7 (FRET IC = 0.58 ꢀM), but exhibited poor microsomal stability in human (hClint,u = 43
v
50
L/h/Kg) and rat liver microsomes (hClint,u = 513 L/h/Kg). Cyclization of the side chain to the cyclic
urea resulted in 23 which again maintained activity at Na 1.7 (FRET IC = 0.73 ꢀM; EP IC50 = 2.3
v
50
ꢀM). Importantly, the metabolic stability of 23 was significantly better than linear analog 19 and the PK
properties of 23 were also markedly improved relative to 3ꢀhydoxyindazole 7.
We next made a series of related amides (24-29, 31) which all demonstrated good selectivity for
Na 1.7 versus Na 1.5. In the series of amides described, the (S)ꢀ3ꢀfluoropyrrolidine 27 exhibited good
v
v
activity at Na 1.7 (FRET IC = 0.37 ꢀM, EP IC = 0.48 ꢀM; manual EP IC = 0.57 ꢀM), good
v
50
50
50
selectivity versus Na 1.5 (>33 ꢀM) and good pharmacokinetic properties (F = 83%; t = 1.7 h). This
v
1/2
analog was further tested in vivo (vida infra). It was also noted that the corresponding (R)ꢀ3ꢀ
fluoropyrrolidine 28, the unsubstituted pyrrolidine 26 and (S)ꢀ3ꢀhydroxypyrrolidine 29 offered no
improvement in Na 1.7 activity or overall properties relative to 27. The (S)ꢀ3ꢀfluoropiperidine 31,
v
although active at Na 1.7 (FRET IC = 1.1 ꢀM), had weaker potency than 27 and a shorter halfꢀlife in
v
50
rats (t1/2 = 0.83 h). The acetamide 24 maintained Na 1.7 activity (FRET IC = 0.84 ꢀM; EP IC = 2.5
v
50
50
ꢀM) and selectivity but showed unfavorable rat PK properties.
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5
Reported Nav1.7 knockout studies found that ablation of sensory neurons was adequate to abolish
mechanical pain and inflammatory pain, while ablation of both sensory and sympathetic neurons was
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
9
required for the abolishment of neuropathic pain. This observation suggests that analogs with CNS
penetration would be beneficial for neuropathic pain modulation, but CNS penetration may not be
necessary for treatment of osteoarthritis (viewed as our primary indication) which may be peripherally
driven. While both centrally penetrant and peripherally restricted analogs were of interest, we
postulated that the CNS penetrant analogs may exhibit broader efficacy in pain models as suggested by
9
,28
the reported knockꢀout studies
and that peripherally restricted analogs may also be active in
peripheral pain models, offering the opportunity to limit potential CNS side effects.
Interestingly, we observed that the 4ꢀindazole substituent influenced the extent to which an analog
was a substrate for Pꢀgp mediated efflux. We further noted that the Pꢀgp efflux ratio (ratio ≥ 2
suggested a Pꢀgp efflux substrate) correlated well with observed brain impairment (BI)
([plasma] /[brain]
≥ 3) (Table 2). We observed that while carbamate 23 was not an Pꢀgp efflux
free
free
substrate, all of the amides tested (24-29, 31, 38, 42, 48, 55) were efflux substrates. We also noted that
analogs with ketone or aryl substituents on the cyclic urea were not Pꢀgp efflux substrates (32-35) (Table
2).
Ketone 32 was of interest in that it showed good Na 1.7 activity in the FRET assay (IC = 0.57 ꢀM).
v
50
Notably, 32 was not an efflux substrate and as such was not brain impaired. A series of aryl substituted
cyclic ureas (33-35) was investigated and these analogs were also found not to be Pꢀgp efflux substrates.
While all aryl analogs were active at Na 1.7, methyl pyrazine 34 was the most potent (FRET IC = 0.27
v
50
ꢀM) and was significantly more selective versus Na 1.5 than oxazole 33 or pyridine 35. Once again,
v
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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
however, the rat pharmacokinetic profile was deemed too unfavorable for further in vivo
characterization.
Finally, we looked at replacements of the cyclic urea with other conformationally constrained analogs
such as 38, 42, 48, and 55 (Table 2). Piperazinone 38 exhibited robust activity at Na 1.7 (FRET IC =
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
v
50
0.19 ꢀM; EP IC50 = 0.54 ꢀM) but selectivity verus Na 1.5 was diminished relative to analog 27.
v
Bicyclic analog 42 also demonstrated good activity at Na 1.7 (FRET IC = 0.58 ꢀM) but had relatively
v
50
poor selectivity versus Na 1.5 (IC = 2.5 ꢀM) and very poor human and rat metabolic stability. Further
v
50
investigation of the bicyclo[3.3.0] core led to analogs 48 and 55. Although 48 demonstrated more
moderate activity at Na 1.7 in the FRET assay (FRET IC = 3.9 ꢀM), it exhibited no activity at Na 1.5,
v
50
v
excellent metabolic stability and good PK properties. As such, 48 was tested in Na 1.7 manual
v
electrophysiology where 48 gave an IC50 of 0.34 ꢀM (Table 2). Given the activity, the promising in
vitro metabolism and PK data, 48 was further tested in vivo (vida infra). The regiomeric analog 55 was
also synthesized, and although 55 exhibited improved activity at Na 1.7 in the FRET assay (IC = 1.35
v
50
ꢀM) relative to 48, increased activity at Na 1.5 was noted as was decreased metabolic stability. We
v
further noted that, as was the case with amides in the cyclic urea series, both 48 and 55 were efflux
substrates and as such exhibited decreased CNS drug levels as indicated by the brain impairment ratio in
Table 2.
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Table 2: In Vitro Activity of 4-Hydroxy Indazoles
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
EP
hNa
FRET
P-gp
BI
d
1.7 hNav1.5
rCl,int,u
F
v
hCl,int,u
N
hNav1.7
a
IC50 (ꢀM)
%F
t1/2 (h)
efflux ([plasma]free/[
c
IC50, -65 IC50 (ꢀM) (L/h/Kg) (L/h/Kg)
N
brain]free)
ratio
b
mV (ꢀM)
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
R
0
.056 ±
0.32
tetracaine
0.65 ± 0.3
(manual)
0.002
0.58 ±
1
9
43
5.9
513
7.2
0.98
22
1.6
13.6
10.9
2.2
0.010
0.73 ±
23
2.3
2.5
5 ± 1.6
19*
100
55
73
94
83
2
0.090
0.84 ±
2
4
5
8.2
11.8
<3.6
<3.8
<2.9
<3.3
<3.8
14
0.6
1.4
1.4
1.7
11.4
6
0.06
0.92 ±
2
1.1
>33
<2.2
<1.9
<1.7
<2.0
<2.2
4.1
0.054
0
.81 ±
.16
26
0.99
>33
15
5.2
0
0
.37 ± 0.48 (0.57
.027 manual)
27
28
29
>33
0
0.90 ±
0.024
2.72 ±
9
8.8
1.1
1.3
1.1
0.72
23
5.8
1.8
1.2
2.0
3.3
10.5
0.20
31
1.1 ± 0.57
23*
15 ± 5.6
14.2 ± 3.5
>33
77
82
37
21
0.83
1.4
0.57 ±
32
33
34
5.1
3.9
11.4
6
0.095
1.5 ± 0.21
0.27 ±
>10
<2.0
2.5
1.1
12.7
0.41
0.064
35
1.5 ± 0.48
0.33
0.54
3.1*
4.4 ± 0.70
2.5
3.8
<1.8
54
13.7
6.9
28
84
1.3
1.3
0.19 ±
3
8
2
0.18
0.58 ±
4
171
21
10.4
13.9
0.099
0.34
48
3.9 ± 0.42
>33
2.6
<2.8
55
1.7
17.6
(
manual)
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2
2
2
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6
a
Na 1.7 IC50 values were determined by leastꢀsquares fitting of a logistic equation to data from full eightꢀpoint, halfꢀlog
v
concentration–response curves using a FRETꢀmembrane potential assay as described in the Experimental Section. Data is
shown with standard error (SEM) and represent the mean of two or more separate determinations. Na 1.7 EP, data were
collected using a slow inactivated state QPatch protocol with V1/2= ꢀ65 mV. Na 1.5, data were collected using an inactivated
state protocol with V1/2= ꢀ90 mV with standard error (SEM) representing the mean of two or more separate determinations. An
b
v
c
v
d
(*) denotes an n=1 where no SEM can be reported. Human and rat liver microsomal stability data, rat bioavailability and
halfꢀlife, Pꢀgp efflux and brain and plasma levels were obtained as described in the Experimental Section.
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
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3: Selectivity versus Na 1.2, Na 1.3 and Na 1.6
v
v
v
FRET hNa 1.7 FRET hNa 1.2 FRET hNa 1.3 FRET hNa 1.6
v
v
v
v
IC (ꢀM)
IC (ꢀM)
50
IC (ꢀM)
50
IC (ꢀM)
50
5
0
tetracaine
0.056
1.9
0.49
2.6
0.74
3.4
0.52
5.0
7
27
0.37
3.9
4.0
7.1
8.6
4
8
>20
>20
>20
a
Na 1.7, 1.2, 1.3 and 1.6 IC50 values were determined by leastꢀsquares fitting of a logistic equation to data from full eightꢀpoint, halfꢀ
v
log concentration–response curves using a FRETꢀmembrane potential assay as described in the Experimental Section.
On the basis of activity at Na 1.7, selectivity versus Na 1.5 and overall drugꢀlike properties (metabolic
v
v
stability, rat PK), analogs 27 and 48 were selected for further testing in the rat monoꢀiodoacetateꢀ
induced osteoarthritis model (MIAꢀOA, grip force endpoint). Centrally penetrant analog 7 did not have
a favorable rat PK profile but it was chosen to exemply the 3ꢀhydroxyindazole series. Although we
were primarily focused on selectivity in vivo (pain model activity vs cardiovascular and CNS side
effects), these analogs were also tested in the Na 1.2, Na 1.3 and Na 1.6 FRET assays to better assess
v
v
v
their potential CNS liabilities (Table 3). While the 3ꢀsubstituted indazole 7 exhibited moderate to poor
selectivity, the peripherally restricted 4ꢀsubstituted indazole analogs 27 and 48, demonstrated better
selectivity relative to 7.
In this model, an intraꢀarticular injection of monoꢀiodoacetate causes osteoarthritisꢀlike knee joint
lesions with analogs being tested in these rats approximately three weeks after injection of the monoꢀ
2
6
iodoacetate. The rats were then tested for grip force deficit of hind limbs. The NSAID diclofenac was
used as a positive control and typically produced about an 80% increase in grip force relative to vehicle.
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Figure 2: Effects of 7, 27 and 48 in MIA-OA Pain Model
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Figure 2 (A) Oral acute dosing of 7 in the rat monoꢀiodoacetateꢀinduced osteoarthritis model (MIAꢀOA) with a
grip force end point. The vehicle control group was assigned a value of 0% whereas a naïve group was assigned a
value of 100%. The percent effect for each doseꢀgroup were expressed as % return to normal grip force as found
in the naïve group, and calculated using the formula: (% return to normalcy = [(Treatment CFmax ꢀ vehicle
CFmax) / (naïve CFmax ꢀ vehicle CFmax)] X 100%) where CFmax is maximum compressive force (see
Supplemental section for complete procedure). (B) Oral acute doing of 27 in the MIAꢀOA model as described
above. (C) Oral acute and subꢀchronic dosing of 48 in the MIAꢀOA model with solid line representing acute
dosing and dashed line subꢀchronic dosing as described in the Experimental Section. Dicolfenac (30 mg/kg p.o.)
was administered as a positive control in all acute experiments.
Centrally active, 3ꢀhydroxyindazole analog 7 (BI = 0.83) exhibited a robust dose response effect in
this model at 3, 10 and 30 mg/kg (Figure 2A). Similarly, analog 27 also demonstrated robust activity in
the MIAꢀOA model (Figure 2B) with significant effects at 3, 10 and 30 mg/kg. Interestingly, the
efficacious plasma levels (total) for the more peripherally restricted analog 27 (EC of 2.2 ꢀg/mL) were
5
0
higher than the centrally active analog 7 (EC = 500 ng/mL). Finally, [3.3.0] bicyclic diamine analog
5
0
4
8, also exhibited good activity in the acute rat monoꢀiodoacetateꢀinduced osteoarthritis model at 10 and
3
0 mg/kg (EC = 2.5 ꢀg/mL) as shown in the solid line in Figure 2C. Again, we observed that the
50
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peripherallyꢀrestricted analog 48 required higher plasma concentrations than the centrally active analog
. Based on our observations, CNS penetration was not required for efficacy in the MIAꢀOA model of
7
OA pain, however higher plasma levels of peripherally restricted agents were required for full efficacy.
Interestingly, when 48 was tested with subꢀchronic dosing twice a day for seven days, we observed a
shift in efficacious plasma levels. On day one, results were consistent with acute administration but on
day seven (shown in dashed line in Figure 2C) there was a significant shift in efficacy. In this subꢀ
chronic dosing study, significant effects were observed at 0.3 mg/kg and the EC shifted to 800 ng/mL.
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We hypothesized that, in this subchronic dosing paradigm, drug levels achieved a steadyꢀstate at the site
of action (neurons) resulting in improved efficacy. Importantly, none of the compounds tested exhibited
any CNS side effects as assessed by highꢀdose locomotor activity and rotorod performance assays in
rodents (data not shown).
Table 4: Plasma concentrations in the OAꢀGF pain model.
OA-GF plasma conc. OA-GF plasma conc. OA-GF plasma conc.
@ 3 mg/Kg (ꢀg/mL) @ 10 mg/Kg (ꢀg/mL) @ 30 mg/Kg (ꢀg/mL)
7
0.05
2.7
0.38
6.8
1.05
10.5
6.3
2
7
8
4
0.69
3.3
To better understand the efficacy of these analogs in the OAꢀGF model, plasma levels were obtained
and are shown in Table 4. For analog 7, robust efficacy was observed at 1.05 ꢀg/mL or 2.9 ꢀM which
was sufficient to cover the FRET IC of 1.9 ꢀM. Higher drug levels were obtained with both 27 and
5
0
4
8, consistent with their improved rat PK profiles relative to 7 and also exceed their FRET IC values.
50
Plasma levels for the lower doses from the subꢀchronic study with 48 were 0.96 ꢀg/mL and 0.19 ꢀg/mL
for 1 mg/kg and 0.3 mg/kg, respectively.
Conclusion
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In summary, we discovered two series of indazole analogs that exhibited activity at Na 1.7. The 3ꢀ
v
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6
hydroxyindazole series was observed to have reasonable Na 1.7 activity and moderate selectivity versus
v
Na 1.5. Analog 7 in this series exhibited robust activity in the monoꢀiodoacetate induced osteoarthritis
v
0
1
2
3
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0
model of pain with a comparatively low efficacious plasma concentration (500 ng/mL). Unfortunately,
despite good activity in an acute pain model, the suboptimal pharmacokinetic profile of 7 and related
analogs precluded further development.
A related 4ꢀsubstituted indazole series was also investigated. Analogs in this series exhibited good to
moderate activity at Na 1.7 as measured by the FRETꢀmembrane potential assay and electrophysiology
v
and showed moderate to good selectivity versus the cardiac sodium channel, Na 1.5. Furthermore, this
v
series demonstrated improved pharmacokinetic profiles with good rat bioavailabilities and halfꢀlives. In
this series, we noted that the 4ꢀindazole side chain influenced the Pꢀgp efflux activity and thus
determined the extent of CNS penetration. Amides such as 27 or 48 were Pꢀgp efflux substrates and, as
such, were minimally present in the CNS while a ketone substituent (32) or aryl group (33-35) yielded
analogs that were centrally active. Two analogs (27 and 48) in this series were tested in the monoꢀ
iodoacetate induced osteoarthritis pain model. Both analogs demonstrated robust activity in acute
testing with higher plasma levels required for activity relative to analog 7. However, upon subꢀchronic
dosing of 48 for seven days, a favorable shift towards higher efficacy was observed at the same plasma
levels. Both 27 and 48 remain compounds of interest for potential clinical development.
Experimental Section
Procedures for all biological assays are included in the Supplemental Section.
Chemistry. Proton NMR spectra were obtained on a Varian Inova 500 (500 MHz), Varian Inova
4
00 (400 MHz), Varian 400ꢀMR (400 MHz), Varian UNITY plus 300 (300 MHz) with chemical shifts
(δ) reported relative to tetramethylsilane as an internal standard. Column chromatography was carried
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6
out on silica gel 60 (230ꢀ400 mesh). Dry organic solvents (CH Cl , CH CN, DMF, etc) were purchased
2
2
3
from Aldrich packaged under nitrogen SureꢀSeal bottles. Preparative reversed phase HPLC (Gilson
33/334 pump system and Gilson 215 liquid handler, custom packed YMC TriArt™ C18 20 ꢁm
3
0
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9
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column, 50 x 150 mm, flow rate 80 mL/min with solvents as described in individual experiments. Thinꢀ
layer chromatography was performed using 250 mm silica gel 60 glassꢀbacked plates with F254 as
indicator. The purity of all final compounds was assessed to be ≥95% as determined by HPLC (data is
included in the Supplemental Section). All starting materials were commercially available and were
obtained from Aldrich unless otherwise specified.
2-((5-Fluoro-1-(2-fluorophenyl)-1H-indazol-3-yl)oxy)-1-morpholinoethanone (4). Step 1: To a
solution of 2ꢀaminoꢀ5ꢀfluorobenzoic acid (Matrix Scientific, 21.7 g, 140 mmol) in THF (500 mL) at 0
ºC was added carbonyldiimidazole (23.8 g, 147 mmol) using a mechanical stirrer. The iceꢀbath was
removed after the addition was complete and the mixture was stirred at ambient temperature for 1.5 h.
Hünig's base (58.6 mL, 336 mmol) was added and the solids dissolved completely. The 2ꢀ
fluorophenylhydrazine hydrochloride (25.0 g, 154 mmol) was added portionwise over 15 min then the
mixture was allowed to stir at ambient temperature for 18 h. The mixture was quenched with aqueous
HCl (1N) to pH 1 then aqueous NaOH (50%) was added until pH 7. The layers were separated and the
aqueous layer was extracted with ethyl acetate (2 X 100 mL). The combined organics were washed with
water (100 mL), saturated, aqueous NaHCO (100 mL) and brine (100 mL) then were dried over
3
anhydrous Na SO , filtered, concentrated under reduced pressure and the resulting 2ꢀaminoꢀ5ꢀfluoroꢀN'ꢀ
2
4
(2ꢀfluorophenyl)benzohydrazide (40 g, 129 mmol, 92% yield) was carried on without further
+
purification to step 2. MS (DCI) m/z 264 [M+H] . Step 2: A mixture of 2ꢀaminoꢀ5ꢀfluoroꢀN'ꢀ(2ꢀ
fluorophenyl)benzohydrazide (from Step 1) (37 g, 141 mmol) in ethanol (450 mL) and aqueous HCl
(1N, 422 mL, 422 mmol) was warmed to 76 ºC and was allowed to stir until all solids were dissolved.
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Then sodium nitrite (29.1 g, 422 mmol) in water (50 mL) was added dropwise via addition funnel over 1
h. The mixture was allowed to stir at 76 ºC for 1 h then was allowed to cool to ambient temperature and
4
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1
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6
was stirred for 16 h. The solids were isolated via filtration and washed with water (2 X 20 mL) to give
0
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5
ꢀfluoroꢀ1ꢀ(2ꢀfluorophenyl)ꢀ1Hꢀindazolꢀ3ꢀol, 3 (20.5 g, 83 mmol, 59% yield). H NMR (300 MHz,
DMSOꢀd ) δ 11.39 – 10.89 (m, 1H), 7.60 (td, J = 7.8, 1.6 Hz, 1H), 7.55 – 7.41 (m, 3H), 7.41 – 7.23 (m,
6
+
3
H); MS (DCI) m/z 247 [M+H] . Step 3: To a solution of 5ꢀfluoroꢀ1ꢀ(2ꢀfluorophenyl)ꢀ1Hꢀindazolꢀ3ꢀol
(3, from Step 2) (2.25 g, 9.14 mmol) in DMF (35 mL), NaH (60 wt%, 1.1 g, 27.4 mmol) was added
portionwise at ambient temperature and stirred for 15 min. To this dark brown suspension was added 2ꢀ
bromoꢀ1ꢀmorpholinoethanone (2.38 g, 11.42 mmol) in DMF (1 mL) and the reaction mixture was stirred
for another 1 hr. Water was added and extracted with CH Cl . The organic layer was dried over
2
2
anhydrous Na SO , filtered, concentrated under reduced pressure and purified via column
2
4
chromatography (SiO , 30% ethyl acetate/hexanes to 100% ethyl acetate) to give 2ꢀ((5ꢀfluoroꢀ1ꢀ(2ꢀ
2
1
fluorophenyl)ꢀ1Hꢀindazolꢀ3ꢀyl)oxy)ꢀ1ꢀmorpholinoethanone, 4, (2.58 g, 6.9 mmol, 76% yield). H NMR
(400 MHz, Chloroformꢀd) δ 7.54 (td, J = 7.6, 1.6 Hz, 1H), 7.44 – 7.40 (m, 1H), 7.35 (dddq, J = 9.5, 4.9,
2
3
.4, 1.8 Hz, 1H), 7.31 – 7.24 (m, 2H), 7.22 – 7.17 (m, 2H), 5.10 (s, 2H), 3.76 – 3.67 (m, 6H), 3.60 –
+
.53 (m, 2H); MS (APCI) m/z 374 [M+H] .
2-((5-Fluoro-1-(2-fluorophenyl)-1H-indazol-3-yl)oxy)-1-morpholinopropan-1-one (5). Step 1: A
mixture of 5ꢀfluoroꢀ1ꢀ(2ꢀfluorophenyl)ꢀ1Hꢀindazolꢀ3ꢀol (3, from synthesis of 4, step 2), methyl 2ꢀ
bromopropanoate (0.713 g, 4.27 mmol), and Cs CO (1.45 g, 4.46 mmol) in acetonitrile (12 mL) was
2
3
stirred for 16 h. The mixture was then diluted with ethyl acetate (10 mL) and washed with 1N NaOH
10 mL) and brine (10 mL). The organics were dried over Na SO and purified via column
(
2
4
chromatography (SiO , 100% hexanes to 50% ethyl acetate/hexanes) to give methyl 2ꢀ((5ꢀfluoroꢀ1ꢀ(2ꢀ
2
1
fluorophenyl)ꢀ1Hꢀindazolꢀ3ꢀyl)oxy)propanoate (methyl ester of 6) (1.03 g, 3.1 mmol, 76% yield). H
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2
2
2
2
2
2
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6
NMR (300 MHz, DMSOꢀd ) δ 7.60 – 7.50 (m, 4H), 7.46 – 7.30 (m, 3H), 5.30 (q, J = 6.8 Hz, 1H), 3.69
6
+
(
s, 3H), 1.64 (d, J = 6.9 Hz, 3H); MS (APCI) m/z 333 [M+H] . Step 2: A mix of methyl 2ꢀ(5ꢀfluoroꢀ1ꢀ
2ꢀfluorophenyl)ꢀ1Hꢀindazolꢀ3ꢀyloxy)propanoate (methyl ester of 6 from Step 1, 1.03 g, 3.10 mmol) and
(
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
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1
N NaOH (3.8 mL, 3.80 mmol) in methanol (3 mL) and THF (9 mL) was allowed to stir for 1h then was
diluted with ethyl acetate (5 mL). The mixture was acidified with 1N HCl (4 mL) and the layers were
separated. The organic layer was washed with water (5 mL) and brine (5 mL), dried over anhydrous
Na SO and concentrated under reduced pressure to give 2ꢀ(5ꢀfluoroꢀ1ꢀ(2ꢀfluorophenyl)ꢀ1Hꢀindazolꢀ3ꢀ
2
4
1
yloxy)propanoic acid, 6 (926 mg, 2.91 mmol, 94% yield). H NMR (300 MHz, DMSOꢀd ) δ 13.00 (s,
6
1
H), 7.62 – 7.45 (m, 4H), 7.44 – 7.30 (m, 3H), 5.21 (q, J = 7.0 Hz, 1H), 1.62 (d, J = 7.0 Hz, 3H); MS
+
(
APCI) m/z 319 [M+H] . Step 3: A mixture of 2ꢀ(5ꢀfluoroꢀ1ꢀ(2ꢀfluorophenyl)ꢀ1Hꢀindazolꢀ3ꢀ
yloxy)propanoic acid (Step 2, 0.926 g, 2.91 mmol) and oxalyl chloride (0.33 mL, 3.78 mmol) in CH Cl
2
2
(10 mL) with catalytic DMF (0.1 mL) was stirred at ambient temperature for 90 min then was
concentrated under reduced pressure. The material was dissolved in CH Cl (10 mL) then morpholine
2
2
(0.28 mL, 3.21 mmol) and pyridine (0.35 mL, 4.33 mmol) were added and the mixture was allowed to
stir for 16 h. The reaction mixture was then diluted with ethyl acetate (10 mL), washed with saturated,
aqueous NaHCO (5 mL) and brine (mL) and dried over anhydrous Na SO . The material was then
3
2
4
filtered, concentrated under reduced pressure and purified via column chromatography (SiO 18% ethyl
2
,
acetate/CH Cl ) to give 2ꢀ(5ꢀfluoroꢀ1ꢀ(2ꢀfluorophenyl)ꢀ1Hꢀindazolꢀ3ꢀyloxy)ꢀ1ꢀmorpholinopropanꢀ1ꢀone,
2
2
1
5
(1.06 g, 2.75 mmol, 94% yield). H NMR (300 MHz, DMSOꢀd ) δ 7.62 – 7.46 (m, 4H), 7.44 – 7.30
6
(m, 3H), 5.70 (q, J = 6.6 Hz, 1H), 3.68 – 3.46 (m, 7H), 3.47 – 3.35 (m, 1H), 1.54 (d, J = 6.6 Hz, 3H);
+
MS (APCI) m/z 388 [M+H] .
1
-(2-(5-Fluoro-1-(2-fluorophenyl)-1H-indazol-3-yloxy)ethyl)imidazolidin-2-one, (7). To a solution
of 5ꢀfluoroꢀ1ꢀ(2ꢀfluorophenyl)ꢀ1Hꢀindazolꢀ3ꢀol (3, from synthesis of 4, step 2, 2.5 g, 10.15 mmol), 1ꢀ(2ꢀ
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hydroxyethyl)ꢀ2ꢀimidazolidinone (Alfa Aesar, 2.11 g, 16.3 mmol) and triꢀNꢀbutylphosphine (4.01 mL,
6.3 mmol) in toluene (60 mL) was added 1,1’ꢀ(azodicarbonyl)dipiperidine (4.10 g, 16.3 mmol)
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
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1
portionwise over 5 min. The mixture was warmed to 80 ºC and was allowed to stir for 5 h. The mixture
was then allowed to cool to ambient temperature and was stirred for 16 h. The reaction mixture was
diluted with toluene (10 mL) and filtered through Celite, rinsing the residue with toluene. The filtrate
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
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4
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7
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9
0
was concentrated under reduced pressure and purified via column chromatography (SiO , 5% ethyl
2
acetate/hexanes to 75% ethyl acetate/hexanes) to give 1ꢀ(2ꢀ(5ꢀfluoroꢀ1ꢀ(2ꢀfluorophenyl)ꢀ1Hꢀindazolꢀ3ꢀ
1
yloxy)ethyl)imidazolidinꢀ2ꢀone, 7 (2.9 g, 8.09 mmol, 80% yield). H NMR (300 MHz, Methanolꢀd ) δ
4
7
3
.60 (td, J = 7.7, 1.7 Hz, 1H), 7.50 – 7.31 (m, 4H), 7.28 – 7.21 (m, 2H), 4.57 (t, J = 5.2 Hz, 2H), 3.71 –
+
.62 (m, 4H), 3.43 – 3.35 (m, 2H); MS (DCI) m/z 359 [M+H] .
1
-(2-(5-Fluoro-1-(2-fluorophenyl)-1H-indazol-3-yloxy)ethyl)-1H-imidazol-2(3H)-one, (8). To a
solution of 5ꢀfluoroꢀ1ꢀ(2ꢀfluorophenyl)ꢀ1Hꢀindazolꢀ3ꢀol (3, from synthesis of 4, step 2, 4 g, 16.25
mmol) in DMF (40 mL) at ambient temperature was added NaH (60 wt%, 2.60 g, 65.0 mmol). This
mixture was stirred at ambient temperature for 30 min then 1ꢀ(2ꢀchloroethyl)ꢀ1,3ꢀdihydroꢀ2Hꢀimidazolꢀ
2
ꢀone (Chembridge, 4.76 g, 32.5 mmol) in DMF (10 mL) was added. The mixture was warmed to 50 °C
and was allowed to stir for 16 h. Thre reaction mixture was quenched with saturated, aqeuous NaHCO3
(5 mL) and diluted with ethyl acetate (10 mL). The layers were separated and the aqueous layer was
extracted with ethyl acetate (3 X 5 mL). The combined organics were dried over anhydrous Na SO ,
2
4
filtered, concentrated and purified via column chromatography (SiO , 1% ethyl acetate/heptanes to 30%
2
ethyl acetate/heptanes) to give 1ꢀ(2ꢀ(5ꢀfluoroꢀ1ꢀ(2ꢀfluorophenyl)ꢀ1Hꢀindazolꢀ3ꢀyloxy)ethyl)ꢀ1Hꢀ
1
imidazolꢀ2(3H)ꢀone, 8 (0.95 g, 2.67 mmol, 16% yield). H NMR (300 MHz, Methanolꢀd ) δ 7.59 (td, J
4
=
7.7, 1.6 Hz, 1H), 7.49 – 7.30 (m, 4H), 7.28 – 7.20 (m, 2H), 6.57 (d, J = 3.0 Hz, 1H), 6.38 (d, J = 3.0
+
Hz, 1H), 4.64 (dd, J = 5.8, 4.6 Hz, 2H), 4.17 – 4.09 (m, 2H); MS (DCI) m/z 357 [M+H] .
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-(2-(1H-Imidazol-1-yl)ethoxy)-5-fluoro-1-(2-fluorophenyl)-1H-indazole (9). To a solution of 5ꢀ
fluoroꢀ1ꢀ(2ꢀfluorophenyl)ꢀ1Hꢀindazolꢀ3ꢀol (3, from synthesis of 4, step 2, 2.3 g, 9.34 mmol), 1ꢀ(2ꢀ
hydroxyethyl)imidazole (Oakwood, 1.05 g, 9.34 mmol) and triꢀNꢀbuylphosphine (2.305 mL, 9.34 mmol)
in toluene (60 mL) was added 1,1’ꢀ(azodicarbonyl)dipiperidine (2.357 g, 9.34 mmol) portionwise over 5
min. The mixture was warmed to 80 ºC and was allowed to stir for 5 h. The mixture was allowed to
cool to ambient temperature then was stirred for 16 h. The reaction mixture was diluted with toluene
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(10 mL) and filtered through Celite, rinsing the residue with toluene. The filtrate was concentrated
under reduced pressure and the material was purified via column chromatography (SiO , 5% ethyl
2
acetate/hexanes to 75% ethyl acetate/hexanes) to give 3ꢀ(2ꢀ(1Hꢀimidazolꢀ1ꢀyl)ethoxy)ꢀ5ꢀfluoroꢀ1ꢀ(2ꢀ
1
fluorophenyl)ꢀ1Hꢀindazole, 9 (2.4 g, 7.05 mmol, 75% yield). H NMR (300 MHz, Methanolꢀd ) δ 7.76
4
(d, J = 1.2 Hz, 1H), 7.58 (td, J = 7.7, 1.5 Hz, 1H), 7.51 – 7.30 (m, 4H), 7.27 – 7.22 (m, 3H), 6.98 (t, J =
+
1
.1 Hz, 1H), 4.71 (dd, J = 5.6, 4.3 Hz, 2H), 4.55 (dd, J = 5.5, 4.5 Hz, 2H); MS (DCI) m/z 341 [M+H] .
1
-(2-(5-Fluoro-1-(2-fluorophenyl)-1H-indazol-3-yloxy)ethyl)-3-methylimidazolidin-2-one, (10). To
a solution of 1ꢀ(2ꢀ(5ꢀfluoroꢀ1ꢀ(2ꢀfluorophenyl)ꢀ1Hꢀindazolꢀ3ꢀyloxy)ethyl)imidazolidinꢀ2ꢀone, 7 (0.20 g,
.56 mmol) in DMF (5 mL) at ambient temperature was added NaH (60 wt%, 0.089 g, 2.23 mmol).
0
This mixture was stirred at ambient temperature for 1 h then iodomethane (0.042 mL, 0.670 mmol) was
added. The mixture was allowed to stir at ambient temperature for 3 h then the mixture was quenched
with saturated, aqeuous NaHCO (5 mL) and diluted with ethyl acetate (10 mL). The layers were
3
separated and the aqueous layer was extracted with ethyl acetate (3 X 5 mL). The combined organics
were dried over anhydrous Na SO , filtered, concentrated and purified via column chromatography
2
4
(SiO , 5% ethyl acetate/heptanes to 100% ethyl acetate to 10% methanol in ethyl acetate) to give 1ꢀ(2ꢀ
2
(5ꢀfluoroꢀ1ꢀ(2ꢀfluorophenyl)ꢀ1Hꢀindazolꢀ3ꢀyloxy)ethyl)ꢀ3ꢀmethylimidazolidinꢀ2ꢀone, 10 (0.17 g, 0.46
1
mmol, 82% yield). H NMR (300 MHz, Methanolꢀd ) δ 7.60 (td, J = 7.6, 1.7 Hz, 1H), 7.50 – 7.31 (m,
4
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H), 7.28 – 7.22 (m, 2H), 4.61 – 4.53 (m, 2H), 3.72 – 3.64 (m, 2H), 3.59 – 3.50 (m, 2H), 3.37 – 3.31 (m,
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+
H), 2.74 (s, 3H); MS (DCI) m/z 373 [M+H] .
3-(2-(5-Fluoro-1-(2-fluorophenyl)-1H-indazol-3-yloxy)ethyl)oxazolidin-2-one, (12). Step 1: To a
solution of 5ꢀfluoroꢀ1ꢀ(2ꢀfluorophenyl)ꢀ1Hꢀindazolꢀ3ꢀol (3, from synthesis of 4, step 2, 1.34 g, 5.43
mmol), 2ꢀ(3oxazolidine)ethanol (Frinton Labs, 0.633 mL, 5.98 mmol) and triꢀNꢀbuylphosphine (1.47
mL, 5.98 mmol) in toluene (20 mL) was added 1,1’ꢀ(azodicarbonyl)dipiperidine (1.51 g, 5.98 mmol)
portionwise over 5 min. The mixture was warmed to 80 ºC and was allowed to stir for 5 h. The mixture
was allowed to cool to ambient temperature and was stirred for 16 h. The mixture was filtered through
Celite and the filtrate was concentrated under reduced pressure and purified via column chromatography
0
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2
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8
9
0
1
2
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7
8
9
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9
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(SiO , 10% ethyl acetate/hexanes to 100% ethyl acetate to 9:1:0.1 ethyl acetate : methanol :
2
triethylamine) to give 2ꢀ(2ꢀ(5ꢀfluoroꢀ1ꢀ(2ꢀfluorophenyl)ꢀ1Hꢀindazolꢀ3ꢀyloxy)ethylamino)ethanol, 11
1
(0.762 g, 2.286 mmol, 42% yield). H NMR (300 MHz, Methanolꢀd ) δ 7.60 (td, J = 7.6, 1.6 Hz, 1H),
4
7
.50 – 7.31 (m, 4H), 7.28 – 7.23 (m, 2H), 4.60 – 4.54 (m, 2H), 3.77 – 3.68 (m, 2H), 3.20 – 3.12 (m, 2H),
+
2.89 – 2.81 (m, 2H); MS (DCI) m/z 334 [M+H] . Step 2: To a solution of 2ꢀ(2ꢀ(5ꢀfluoroꢀ1ꢀ(2ꢀ
fluorophenyl)ꢀ1Hꢀindazolꢀ3ꢀyloxy)ethylamino)ethanol (from Step 1, 0.21 g, 0.630 mmol) in THF (5
mL) was added 1,8ꢀdiazabicyclo[5.4.0]undecꢀ7ꢀene (DBU, 9.40 µL, 0.063 mmol) followed by N,N’ꢀ
carbonyldimidazole (CDI, 0.102 g, 0.630 mmol). The mixture was allowed to stir at ambient
temperature for 4 h. The mixture was quenched with saturated, aqueous NaHCO (5 mL) and diluted
3
with ethyl acetate (5 mL). The layers were separated and the aqueous layer was extracted with ethyl
acetate (3 X 5 mL). The combined organics were dried over anhydrous Na SO , filtered, concentrated
2
4
and purified via column chromatography (SiO , 5% ethyl acetate/hexanes to 75% ethyl acetate/hexanes)
2
to give 3ꢀ(2ꢀ(5ꢀfluoroꢀ1ꢀ(2ꢀfluorophenyl)ꢀ1Hꢀindazolꢀ3ꢀyloxy)ethyl)oxazolidinꢀ2ꢀone, 12 (0.215 g, 0.598
1
mmol, 95% yield). H NMR (300 MHz, Methanolꢀd ) δ 7.61 (td, J = 7.7, 1.7 Hz, 1H), 7.51 – 7.31 (m,
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H), 7.29 – 7.22 (m, 2H), 4.62 (t, J = 5.1 Hz, 2H), 4.34 (dd, J = 9.1, 7.1 Hz, 2H), 3.85 – 3.73 (m, 4H);
+
MS (DCI) m/z 360 [M+H] .
1-(2-(1-(2,4-Difluorophenyl)-5-fluoro-1H-indazol-3-yloxy)ethyl)imidazolidin-2-one (15). Step 1:
Sodium nitrite (6.67 g, 97 mmol) in water (25 mL) was added dropwise to a solution of 2ꢀaminoꢀ5ꢀ
fluorobenzoic acid (15 g, 97 mmol) in 2N HCl (100 mL) at 0 ºC. The mixture was stirred for 30 min
then sodium sulfite (30.5 g, 242 mmol) in water (120 mL) was added in one portion. The cooling bath
was removed and the reaction was stirred for 2 h. Concentrated HCl (31.8 mL, 387 mmol) was added
slowly and the mixture was stirred at ambient temperature for 18 h. The mixture was then heated to 80
ºC for 4 h. After cooling to ambient temperature, the pH was adjusted to 5 ꢀ 5.5 with 2N NaOH (125ꢀ
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1
30 mL). The mixture was filtered to obtain the precipitate, 5ꢀfluoroꢀ1Hꢀindazolꢀ3ꢀol (10 g, 65.7 mmol,
1
6
8% yield). H NMR (300 MHz, DMSOꢀd ) δ 11.55 (s, 1H), 10.52 (s, 1H), 7.39 – 7.27 (m, 2H), 7.18
6
+
(td, J = 9.1, 2.7 Hz, 1H); MS (DCI) m/z 170 [M+NH ] . Step 2: To a solution of 5ꢀfluoroꢀ1Hꢀindazolꢀ
4
3
ꢀol (from Step 1, 10 g, 65.7 mmol) and Boc O (30.5 mL, 131 mmol) in acetonitrile (200 mL) was
2
added 4ꢀdimethylaminopyridine (DMAP, 0.803 g, 6.57 mmol). This mixture was allowed to stir at
ambient temperature for 18 h. The mixture was concentrated under reduced pressure and the residue
was stirred with 7N NH /methanol (10 mL) for 2 h. The material was again concentrated under reduced
3
pressure and dried under vacuum. The crude tertꢀbutyl 5ꢀfluoroꢀ3ꢀhydroxyꢀ1Hꢀindazoleꢀ1ꢀcarboxylate,
1
1
3 (18 g, 71.4 mmol, >100% yield) was carried forward without further purification. H NMR (300
MHz, DMSOꢀd ) δ 8.00 (dd, J = 9.0, 4.3 Hz, 1H), 7.57 – 7.39 (m, 2H), 3.32 (s, 1H), 1.60 (s, 9H); MS
6
+
(DCI) m/z 170 [M+NH ] . Step 3: To a solution of tertꢀbutyl 5ꢀfluoroꢀ3ꢀhydroxyꢀ1Hꢀindazoleꢀ1ꢀ
4
carboxylate (13, from Step 2, 16.6 g, 65.7 mmol), 1ꢀ(2ꢀhydroxyethyl)ꢀ2ꢀimidazolidinone (Alfa Aesar,
1
3.7 g, 105 mmol) and triꢀNꢀbutylphosphine (25.9 mL, 105 mmol) in toluene (200 mL) was added 1,1’ꢀ
(azodicarbonyl)dipiperidine (26.5 g, 105 mmol) portionwise over 5 min. The mixture was warmed to 80
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ºC and was allowed to stir for 5 h. The mixture was allowed to cool to ambient temperature then was
allowed to cool to ambient temperature and was stirred for 16 h. The mixture was diluted with toluene
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(10 mL) and filtered through Celite, rinsing the residue with toluene. The filtrate was concentrated
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3
4
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2
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under reduced pressure and the material was purified via column chromatography (SiO , 5% ethyl
2
acetate/hexanes to 75% ethyl acetate/hexanes) to give tertꢀbutyl 5ꢀfluoroꢀ3ꢀ(2ꢀ(2ꢀoxoimidazolidinꢀ1ꢀ
1
yl)ethoxy)ꢀ1Hꢀindazoleꢀ1ꢀcarboxylate (6 g, 16.5 mmol, 25% yield). H NMR (300 MHz, DMSOꢀd ) δ
6
8
.05 – 7.95 (m, 1H), 7.59 – 7.45 (m, 2H), 6.34 (s, 1H), 4.50 (t, J = 5.3 Hz, 2H), 3.58 – 3.44 (m, 4H),
+
3.26 – 3.17 (m, 2H), 1.63 (s, 9H); MS (DCI) m/z 382 [M+NH ] . Step 4: To a solution of tertꢀbutyl 5ꢀ
4
fluoroꢀ3ꢀ(2ꢀ(2ꢀoxoimidazolidinꢀ1ꢀyl)ethoxy)ꢀ1Hꢀindazoleꢀ1ꢀcarboxylate (from Step 3, 1.5 g, 4.12 mmol)
in CH Cl (25 mL) at 0 ºC was added TFA (12.7 mL, 165 mmol). The mixture was allowed to warm to
2
2
ambient temperature and was stirred for 2 h. The mixture was concentrated under reduced pressure and
purified via column chromatography (SiO , 10% ethyl acetate/hexanes to 100% ethyl acetate to 9:1:0.1
2
ethyl acetate:methanol:triethylamine) to give 1ꢀ(2ꢀ(5ꢀfluoroꢀ1Hꢀindazolꢀ3ꢀyloxy)ethyl)imidazolidinꢀ2ꢀ
1
one, 14 (0.71 g, 2.69 mmol, 65% yield). H NMR (300 MHz, DMSOꢀd ) δ 12.03 (s, 1H), 7.39 (dd, J =
6
9
.0, 4.2 Hz, 1H), 7.31 (dd, J = 8.8, 2.4 Hz, 1H), 7.23 (td, J = 9.1, 2.4 Hz, 1H), 6.33 (s, 1H), 4.39 (t, J =
+
5
.5 Hz, 2H), 3.52 – 3.43 (m, 4H), 3.22 (tt, J = 7.5, 1.1 Hz, 2H); MS (DCI) m/z 265 [M+H] . Step 5: To
a pressure tube was added 1ꢀ(2ꢀ(5ꢀfluoroꢀ1Hꢀindazolꢀ3ꢀyloxy)ethyl)imidazolidinꢀ2ꢀone (14 from Step 4,
0
.3 g, 1.135 mmol), copper (I) iodide (10.8 mg, 0.057 mmol) and potassium phosphate tribasic (0.506 g,
.38 mmol). This mixture was degassed three times with a nitrogen backflush each time. The 2,4ꢀ
2
difluoroꢀ1ꢀiodobenzene (0.163 mL, 1.362 mmol) was added followed by trans-N,N′ꢀ
dimethylcyclohexaneꢀ1,2ꢀdiamine (0.036 mL, 0.227 mmol) and toluene (5 mL). The pressure tube was
sealed and the mixture was warmed to 110 ºC and was allowed to stir for 18 h. The material was
allowed to cool to ambient temperature then was filtered through Celite with ethyl acetate. The filtrate
was concentrated under reduced pressure and purified via column chromatography (SiO , 1% ethyl
2
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