Discovery of Indole- and Indazole-acylsulfonamides as Potent and Selective NaV1.7 Inhibitors for the Treatment of Pain

Article abstract of DOI:10.1021/acs.jmedchem.8b01550

3-Aryl-indole and 3-aryl-indazole derivatives were identified as potent and selective Na_v1.7 inhibitors. Compound 29 was shown to be efficacious in the mouse formalin assay and also reduced complete Freund's adjuvant (CFA)-induced thermal hyper

Full text of DOI:10.1021/acs.jmedchem.8b01550

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Article
Discovery of Indole- and Indazole-Acylsulfonamides as Potent
and Selective NaV1.7 Inhibitors for the Treatment of Pain
Guanglin Luo, Ling Chen, Amy Easton, Amy Newton, Clotilde Bourin, Eric Shields, Kathy
Mosure, Matthew G. Soars, Ronald J Knox, Michele Matchett, Rick L Pieschl, Debra Post-
Munson, Shuya Wang, James Herrington, John Graef, Kimberly Newberry, Digavalli V Sivarao,
Arun Senapati, Linda Bristow, Nicholas A. Meanwell, Lorin A Thompson, and Carolyn D. Dzierba
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01550 • Publication Date (Web): 21 Dec 2018
Downloaded from http://pubs.acs.org on December 22, 2018
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Journal of Medicinal Chemistry
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Discovery of Indole- and Indazole-Acylsulfonamides as Potent and Selective Na 1.7 Inhibitors for the
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Treatment of Pain
Guanglin Luo*, Ling Chen, Amy Easton, Amy Newton, Clotilde Bourin, Eric Shields, Kathy Mosure, Matthew G.
Soars, Ronald J. Knox, Michele Matchett, Rick L. Pieschl, Debra J. Post-Munson, Shuya Wang, James Herrington,
John Graef, Kimberly Newberry, Digavalli V. Sivarao, Arun Senapati, Linda J. Bristow, Nicholas A. Meanwell, Lorin
A. Thompson, and Carolyn Dzierba
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Bristol-Myers Squibb Research and Development, PO Box 4000, Princeton, New Jersey 08543-4000, United
States.
ABSTRACT: 3-Aryl-indole and 3-aryl-indazole derivatives were identified as potent and selective Na 1.7 inhibitors.
v
Compound 29 was shown to be efficacious in the mouse formalin assay and also reduced complete Freund’s
adjuvant (CFA)-induced thermal hyperalgesia and chronic constriction injury (CCI) induced cold allodynia, models
of inflammatory and neuropathic pain, respectively, following intraperitoneal (IP) doses of 30 mg/kg. The
observed efficacy could be correlated with the mouse dorsal root ganglion exposure and Na 1.7 potency
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associated with 29.
INTRODUCTION
Chronic pain is an immense unmet medical need that affects about 20% of the adult population in the
United States and incurs costs of approximately $600 billion annually.1 Current pain therapeutics, including
opioids and general analgesics, have many issues such as intolerable side effects, limited efficacy, and the
2
potential for drug abuse and diversion. New safe and efficacious analgesic agents are greatly needed and one
attractive strategy is to develop drugs with novel mechanisms of action that would avoid some of the problems
3
observed with existing drugs. The voltage-gated sodium channel Na 1.7 has emerged as a promising target for
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a new class of analgesics based on genetic studies.4-7 Na 1.7, which is one of nine human Na channel subtypes
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(
Na 1.1-1.9), controls the passage of sodium ions into sensory neurons and plays a key role in the perception of
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8
pain in humans. Na 1.7 is highly expressed in nociceptive sensory neurons within the dorsal root ganglia (DRG)
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and mutations in SCN9A, the gene encoding for Na 1.7, have been linked to a variety of pain disorders.9,10 Loss-
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of-function mutations in SCN9A are associated with congenital insensitivity to pain,11,12 while gain-of-function
mutations result in painful conditions such as inherited erythromelalgia and paroxysmal extreme pain disorder.13
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Other than anosmia, patients with loss-of-function mutations in the Na 1.7 gene appear to be normal. In
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addition, knockout studies in mice have recapitulated the human phenotype and show the absence of pain-
related behaviors following application of nociceptive stimuli.15,16 These results suggest that the selective
inhibition of Na 1.7 will not incur on-target safety issues. However, one challenge with targeting Na 1.7 for the
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treatment of pain is obtaining selectivity for this channel over other Na family members. Selectivity over Na 1.5,
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which is expressed in atrial and ventricular myocytes, is particularly important in order to reduce the potential for
adverse cardiovascular events such as arrhythmia.17
Given the high level of target validation, there has been significant documented effort to identify selective
1
8-25
Na 1.7 inhibitors for the treatment of pain, with representative hNa 1.7 inhibitors shown in Figure 1.
One
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class of arylsulfonamide, represented by PF-05089711 (1), was advanced into phase II clinical trials, while 2 was
2
3
reported to be orally efficacious in animal models of pain, a result that was reproducible in our hands. Another
major class of hNa 1.7 inhibitors are based on acylsulfonamides, with 3 and 4 representative, although detailed
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structure-activity relationships (SARs) for these compound have not been described.26,27 In previous work, we
2
8-30
have disclosed a series of potent and selective sulfonamide-based Na 1.7 blockers.
In other studies, we have
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been interested in retaining the acylsulfonamide functionality associated with Na 1.7 inhibitors while replacing
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the middle linker region with saturated or unsaturated monocyclic or bicyclic scaffolds. While several prototype
molecules were prepared, potent hNa 1.7 inhibition was discovered in a series of 3-aryl indoles and 3-aryl
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indazoles related to 4 but which were specifically configured to provide an opportunity to more broadly study
substitution patterns around the core heterocycle, notably at the 2-position where substituents would be
anticipated to affect the conformation of the 3-aryl moiety (Figure 1). Herein we disclose SAR studies around
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these cores and profile 29 as one of the more promising compounds that demonstrates activity in multiple models
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5
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5
5
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of pain.
Chart 1. Structures of representative Na 1.7 inhibitors.
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F
O
O
F
S
O
O
Cl
S
N
N
S
H
N
N
H
O
N
H
Cl
N
H N
2
Cl
N NH
1 (PF-05089771)
2
F
O
O O
S
O
O O
S
N
N
H
N
H
O
O
N
Cl
Cl
N
N
Cl
O
3
4 (PF-05241328)
Figure 1. Design principles leading to 3-arylindole and 3-arylindazole acylsulfonamide-based Na 1.7 inhibitors.
v
O
O
O
O
O O
S
O O
S
O O
S
O O
S
R1
N
R1
N
N
N
N
N
N
N
H
R2
R3
H
H
R2 N
R3
H
Linker
R3
R3
X
X
X
X
RESULTS AND DISCUSSION
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The inhibitory activity of target compounds towards hNa 1.7 and hNa 1.5 channels was assessed using
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1
1
1
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1
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2
2
2
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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
an automated 384-well IonWorks Barracuda® patch-clamp assay designed to measure the potency of test
compounds on the inactivated channel. The data for compounds 5-27, compiled in Table 1, reveals good
inhibitory selectivity for hNa 1.7 compared to hNa 1.5. In the methyl sulfonamide series, the simple
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unsubstituted phenyl analog 5 inhibited hNa 1.7 activity with an IC value of 740 nM with no significant activity
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50
0
1
2
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4
5
6
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0
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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
towards hNa 1.5 (>30 µM), data that meets targeted selectivity. Substitution of the pendent phenyl ring at the
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meta- or para-position with Me or Et groups (6-9) showed comparable or improved hNa 1.7 potency.
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Interestingly, the introduction of a 4-CF phenyl substituent (10) greatly increased hNa 1.7 potency (Na 1.7 IC =
3
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50
8
8 nM), while 3-CF substitution (11) was associated with significantly lower potency (Na 1.7 IC = 1420 nM). Bis-
3
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substitution, especially at the ortho-position (12 and 13), generally led to a reduction in potency. Larger aliphatic
substitutions at the para-position of the phenyl ring had only a modest effect on the potency, as exemplified by
14 and 15. While a 4-OCF substituent (16) showed inhibitory activity comparable to 10, the removal of a single
3
fluorine atom from 10 to afford 17 led to a 3-fold reduction in potency relative to prototype 10. Further SARs
around the methylsulfonamide group were explored in the context of 18-23. Replacing the methyl group in 10
with a cyclopropane moiety retained potency but, interestingly, in this series the 4-CHF analog 18 exhibited
2
modestly more potent Na 1.7 inhibition than the 4-CF analog 19 that was accompanied by better selectivity over
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3
3
hNa 1.5. Other groups in the R position, compounds 20-23, showed poorer potency relative to the methyl or
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2
cyclopropyl substituents. Fluorine substitution in the R position next to the amide group was also explored in
2
4-27 with the result that this pattern generally improved the potency, particularly when combined with a
cyclopropyl sulfonamide moiety.
a
Table 1. Structures and hNa 1.7 and hNa 1.5 inhibition IC data for compounds 5-27.
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50
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O
O
O
1
2
3
4
5
6
7
8
9
S
N
R3
N
H
R2
R1
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
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8
9
0
1
2
3
4
5
6
7
8
9
0
hNa 1.7
hNa 1.5
V
V
R1
R2
R3
Compd
b
c
IC (nM)
IC (µM)
50
5
0
5
6
H
4-Me
3-Me
4-Et
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
740 ± 540
740 ± 270
380 ± 300
170 ± 100
220 ± 75
>30
>30
>30
>30
>30
20
7
8
9
3-Et
10
4-CF3
3-CF3
2,3-diF
2-Cl-4-CF3
88 ± 31
1
1
1420 ± 190
NT
12
2900 ± 1000
600 ± 310
363 ± 19
530 ± 30
80 ± 27
>30
8.6
>30
16
1
1
1
1
1
1
3
4
5
6
7
8
i
4- Bu
i
4-O Bu
4-OCF3
4-CHF2
4-CF3
18
262 ± 90
104 ± 51
>30
8.4
1
2
9
0
4-CHF2
4-CF3
H
H
69 ± 4
>30
7.5
CF3
810 ± 390
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2
1
4-CF3
H
N
1700 ± 320
19
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
2
2
3
4-CF3
4-CF3
H
H
420 ± 83
>3000
3.9
18
S
F
F
24
4-CF3
4-CF3
F
F
Me
Me
49 ± 34
16 ± 8
NT
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
5
3.8
2
2
6
7
4-CHF2
4-CHF2
F
F
91 ± 43
38 ± 5
>30
NT
a
b
Values are the means of at least two separate experiments. The hNa 1.7 inhibition data were collected using
an IonWorks Barracuda automated electrophysiology platform using a protocol where cells were held at −60 mV.
The hNa 1.5 data were collected on the same platform using a protocol where cells were held at −50 mV. NT =
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c
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not tested.
The SARs surrounding the 3-pyridinyl analogs are documented in Table 2. While the unsubstituted
pyridine analog 28 was inactive, substitution at the 5- and/or 6-positions of the pyridine generally led to improved
activity. Introduction of the substituent groups found in 3 and 4 afforded analog 29 which exhibited potent
hNa 1.7 inhibition (IC = 108 nM) and good selectivity (hNa 1.5 IC =19 µM). Removal of the Cl group modestly
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50
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50
compromised potency (30) while replacement of the iso-butyl group with the cyclopropylmethyl group found in
31 had little effect on activity. However, replacement of the Cl group with CN gave 32, which exhibited enhanced
potency while maintaining selectivity, while substitution with a OMe group (33) substantially diminished the
potency. Replacing the iso-butyl group in 29 with a difluoroethyl moiety (34) substantially decreased the potency
but, interestingly, trifluoroethyl substitution provided an analog 35 with very good inhibitory potency. To improve
the physicochemical properties of the series, a hydroxyl group was added to the iso-butyl substituent to give 36;
however, this change abrogated potency, indicating that hydrophilic polar groups are not tolerated in this region.
SARs around the methyl sulfonamide group were explored in the context of 37-44 where, as observed in the
phenyl series, the cyclopropyl derivatives 37-39 maintained or improved potency compared to the methyl
homologue. The hNa 1.7 inhibition potency gradually decreased as the substituent size increased, as exemplified
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by 40-42. In addition, replacing the methyl sulfonamide in this pyridinyl series with a sulfamoyl group, probed by
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
3 and 44, also led to a decrease in inhibitory activity. 5-Fluoro-substituted indole analogs with a cyclopropyl in
5
the R position, compounds 45-48, were more potent than the corresponding methyl analogs while analogs with
2
a chlorine at R were less potent than the des-Cl analogs. Compound 48 expressed potent hNa 17 inhibition with
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an IC value of 59 nM. Finally, the larger N-substituents explored in the context of 49-51 substantially lowered
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
the hNa 1.7 inhibitory potency.
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a
Table 2. Structures and hNa 1.7 and hNa 1.5 IC inhibition data for compounds 28-51.
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V
50
R3
N
O
O
O
R5
S
N
H
R4
N
R2
R1
hNa 1.7
hNa 1.5
V
V
Compd
28
R1
H
R2
R3
R4
R5
b
b
IC (nM)
IC (nM)
50
5
0
H
Me
Me
H
H
Me
Me
>3000 d
NT
19
2
3
3
9
0
1
O
O
O
O
O
Cl
108 ± 71
164 ± 27
123d
H
Cl
Me
Me
Me
Me
Me
H
H
H
H
H
Me
Me
Me
Me
Me
>30
NT
32
CN
OMe
Cl
70 ± 32
21
3
3
3
4
510 ± 120
850 ± 850
>30
>30
F
O
O
F
F
F
3
3
5
6
Cl
Cl
Me
Me
H
H
Me
Me
63 ± 47
12
F
>30000 d
>30
O
HO
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3
3
3
4
7
8
9
0
O
O
O
Cl
Cl
Me
Me
Me
Me
Me
H
H
H
H
H
290 ± 220
82 ± 5
12
NT
>30
5.7
14
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
OMe
Cl
85 ± 18
O
O
154 ± 74
460 ± 260
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
41
42
Cl
Cl
Cl
Me
Me
H
H
1300 d
NT
O
O
1300 d
4
4
3
4
N
6.3
N
4200 d
O
O
O
Cl
Cl
Cl
H
Me
Me
H
F
NT
7.2
5.1
16
45
46
Me
Me
520 ± 440
230 ± 170
89 ± 60
Me
F
4
4
4
5
5
7
8
9
0
1
O
O
Me
F
H
Me
F
59 ± 35
3.7
1.7
24
O
O
O
Cl
Cl
Cl
i-Pr
H
H
H
Me
Me
700 ± 530
3330 ± 1200
8100 ± 6700
i-Butyl
i-Butyl
11
a
b
Values are the means of at least two separate experiments. The hNa 1.7 inhibition data were collected using
an IonWorks Barracuda automated electrophysiology platform using a protocol where cells were held at −60 mV.
The hNa 1.5 data were collected on the same platform using a protocol where cells were held at −50 mV. Tested
only once. NT = not tested.
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c
d
V
The SARs around the indazole core were also studied and the results of this phase of the survey are
documented in Table 3. When compared to the indole core, the indazole derivatives were equally potent, with
good levels of selectivity. In the trifluoromethylphenyl series 52-54 the potency was increased with addition of a
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Journal of Medicinal Chemistry
5
-F on the indazole core incorporating a cyclopropyl sulfonamide moiety, leading to 54 with an IC value of 13
5
0
1
2
nM for hNa 1.7 inhibition. In the substituted pyridine series 55-59, methyl and cyclopropylsulfonamides were
V
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
both well tolerated. Removal of the chlorine from the pyridyl led to improved selectivity over Na 1.5 (compare
V
2
55 and 57). The addition of fluorine in the R position of the indazole (58) led to increased potency and further
modification of the sulfonamide to a cyclopropyl further increased potency, with the Na 1.7 IC value for 59 = 45
V
50
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
nM.
a
Table 3. Structures and hNa 1.7 and hNa 1.5 inhibition IC data for compounds 52-59.
V
V
50
O
O
O
R3
S
N
N
N
H
R2
R1
hNa 1.7
hNa 1.5
V
V
R1
R2
R3
Compd
b
c
IC (nM)
IC (µM)
50
5
0
5
5
5
2
3
4
H
F
Me
Me
194 ± 64
52 ± 20
13 ± 2
>30
>30
6.4
6.5
F3C
F3C
F3C
F
Cl
55
H
Me
67 ± 48
O
N
N
N
N
N
Cl
5
5
5
5
6
7
8
9
H
H
F
104 ± 57
314 ± 90
170 ± 25
45 ± 30
5.9
>30
>30
11
O
O
O
O
Me
Me
F
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a
b
Values are the means of at least two separate experiments. The hNa 1.7 inhibition data were collected using
V
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
an IonWorks Barracuda automated electrophysiology platform using a protocol where cells were held at −60 mV.
c
The hNa 1.5 data were collected on the same platform using a protocol where cells were held at −50 mV.
V
The additional indazole derivatives shown in Figure 2 were prepared and tested in order to evaluate the
effect of the N-methyl substituent and its site of deployment on inhibitory potency. The N-unsubstituted indazole
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
6
6
0 exhibited lowered potency compared to the methylated prototype 55 as did the isomeric N-2 methyl indazoles
1 and 62 when compared to their N-1 methyl matched pairs 55 and 56. The latter SAR reflects observations
made with the indole series where a bromo or chloro substituent attached to the 2-position of the indole
heterocycle resulted in a 3-7-fold reduction potency (data not shown). However, indole 2-substitution was more
readily tolerated in a related series of Na 1.7 inhibitors that was spawned by observations made with this study
v
and this aspect of the SARs was pursued in that chemotype.32
Figure 2. Structures and Na 1.7 inhibition data for indazole derivatives 60-62.
v
O
O
O
O
O
O
O
O
O
H
S
S
S
N
N
N
N
N
N
N
H
N
H
N
H
N
N
N
Cl
Cl
Cl
O
O
O
60
Na 1.7 IC = 280 nM
61
Na 1.7 IC = 790 nM
62
Na 1.7 IC = 190 nM
v
50
v
50
v
50
The series of azaindole derivatives depicted in Figure 3 was prepared and evaluated in an effort to further
probe aspects of electronic properties of the core heterocycle and its relationship with inhibitory activity. Both
the 7-azaindole derivatives 63-65 and the 4-azaindole derivatives 66 and 67 exhibited lowered but measurable
hNa 1.7 inhibitory activity when compared to the corresponding indazole core derivatives while the activity of
V
the 5-azaindole analog 66 was poor, with an IC value of >30 µM.
50
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Journal of Medicinal Chemistry
1
2
Figure 3. Structures and Na 1.7 inhibition data for the series of azaindole derivatives 63-68.
v
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
O
O
O
O
O
O
O
N
S
N
S
N
S
N
N
N
N
N
N
H
H
H
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
N
Cl
O
F3C
F3C
63
64
Na 1.7 IC = 3020 nM
65
Na 1.7 IC = 642 nM
Na 1.7 IC = 1400 nM
v
50
v
50
v
50
O
O
O
O
O
O
O
O
O
S
S
S
N
N
N
N
N
N
H
H
H
N
N
N
N
N
Cl
Cl
F3C
O
O
66
Na 1.7 IC = 7820 nM
67
Na 1.7 IC = 2000 nM
68
Na 1.7 IC > 30,000 nM
v
50
v
50
v
50
Those compounds demonstrating potent activity in the Barracuda assay were further profiled in a series of in vitro
developability assays, with the results compiled in Table 4. These compounds were tested in a manual patch
clamp electrophysiology (EP) assay, with the results confirming the potency measured in the Barracuda assay. In
general, this set of compounds exhibited good metabolic stability in human, rat and mouse liver microsomes but
was associated with high plasma protein binding, with free fractions of <1.5% in both human and mouse plasma.
2
8
Unlike the aryl sulfonamides, all of the acyl sulfonamide derivatives described here did not significantly inhibit
CYP 3A4 and there was no evidence of time-dependent inhibition (data not shown). For example, both 29 and 47
exhibited acceptable CYP profiles (HLM-CYP IC s for compound 29 = 9.7 µM (2B6), 1.0 µM (2C8), 9.9 µM (2C9),
5
0
2
8 µM (2C19), >40 µM (2D6), >40 µM (3A4) and for 47 = >40 µM (2B6), 1.0 µM (2C8), 27 µM (2C9), 4.0 µM (2C19),
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Journal of Medicinal Chemistry
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>
40 µM (2D6), 15 µM (3A4) with no evidence of time-dependent inhibition. Moreover, both 29 and 47 showed
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
PXR IC values of >50 µM. Although the permeability of many of these analogs in a Caco-2 cell assay appeared
5
0
to be high, several compounds showed high efflux ratios. Notably, the addition of a 5-F substituent adjacent to
the acylsulfonamide moiety, compounds 24, 25, 27, 45, 47, and 48, reduced the efflux ratios in Caco-2 cells and
also plasma protein binding.31 However, this effect did not extend to the 5-fluoro indazole analog 54 which
exhibited a Caco-2 cell efflux ratio of >21.
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
Table 4. Further profiling of potent compounds.
PAMPA
permeability
c
Met. stab. In
liver
microsomes
Fu
Caco2
A-B/B-A
(nm/sec)
hNa 1.7
V
Caco2
b
Compd
H/M
EP IC5
0
pH = 5.5/7.4
efflux ratio
(nM)
(
%free)
(H/R/M)
(nm/sec)
1
1
1
2
2
2
2
3
4
4
4
4
5
5
5
0
4
8
4
5
7
9
8
5
6
7
8
4
5
9
96
23
8
100/74/54
100/94/100
87/92/87
<0.10/<0.10
0.10/0.30
0.20/0.40
0.20/0.50
0.25/0.35
0.30/0.70
<0.10/0.20
<0.10/0.20
0.10/0.20
<0.10/0.20
0.80/1.40
2.4/0.60
972/171
NT
<15/76
<15/78
NT
>5.1
>6.2
NT
525/135
712/140
1660/323
325/60
13
5
97/99/95
~20/125
46/86
6.1
1.9
2.4
>5.5
>7.6
3.4
3.0
3.1
1.6
>21
NT
100/100/-
93/82/86
12
8
81/195
<15/168
<15/114
20/67
95/66/100
97/77/93
443/216
-/391
27
1.1
3.3
25
19
21
11
43
100/85/100
100/37/100
98/98/99
495/205
335/310
802/164
851/254
592/102
1160/417
762/100
34/102
61/186
120/189
<15/312
NT
100/78/98
100/100/100
100/100/100
91/88/92
NT/0.80
NT/0.20
NT/1.1
NT
NT
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Journal of Medicinal Chemistry
a
b
Data are from previous tables. Values are percentage remaining after 10 minutes with 0.5 µM compounds
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
c
incubated in liver microsomes. Unbound percentage free fraction in human (H) and mouse (M) plasma. NT =
not tested.
Selected compounds were next examined in the manual EP assay using the mouse Na 1.7 channel isoform
V
and the mouse PK profiles determined as a prelude to selecting compounds for evaluation in the mouse formalin
model of pain. The results are compiled in Table 5 with the PK profiles of 29 and 47 following oral and IP dosing
captured in Figure 4. The mouse formalin model of pain measures spontaneous, nociceptive-like paw
flinching/licking responses after peripheral nociceptor activation and was chosen for in vivo efficacy screening
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
6
since formalin-induced nociceptive behaviors were abolished in global Na 1.7 knockout mice. The injection of
V
formalin elicits a biphasic behavioral flinching response; phase I occurs during the first 5 minutes post-injection
and results from direct nociceptor activation, whereas phase II occurs 15-45 minutes after the injection and is
thought to reflect a tonic state of pain similar to the relatively long-lasting stimuli in human disease, such as
neuropathic pain.33 Treatment of mice with compound 2 (30 mg/kg, PO), used as a positive control, 60 minutes
prior to the injection of formalin had no effect on the phase I responses but abolished phase II nociceptive
behaviors (Figure 5). When compound 29 was dosed IP at 3 or 10 mg/kg, there was no significant effect on either
phase I or phase II responses in mice (Figure 5). However, phase II nociceptive behaviors were completely
abolished when 29 was dosed at 30 mg/kg, IP but with no effect on the phase I symptoms. The percent reversal
of nociceptive behaviors in the second phase is plotted in Figure 6. All other compounds shown in Table 5 were
also tested at 30 mg/kg, PO, but failed to show significant efficacy in this model. Interestingly, when the indazole
5
5 was assessed in the mouse formalin model, dosing of the drug resulted in prostration, possibly due to sedation,
as well as hypothermia (see note in Table 5). In contrast, none of the mice dosed with indole derivatives showed
any detrimental side effects during the course of the in vivo pain model studies, indicating that small structural
changes in this series can lead to significantly different profiles.34 Compound 55 is structurally analogous to 4 but,
unfortunately, details on the in vivo pharmacology of 4 that might place this observation in perspective have not
been disclosed although this compound was advanced into clinical trials as part of a micro-dosing study.35
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Table 5. Mouse data for potent compounds.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Free
Plasma
Fu
Plasma
Conc.b
(M)
DRG
Conc.
mNa 1.7
mNa 1.7
Dosea
Active in
Formalin
V
V
Com
pd.
mDRG
(%free)
EP IC50
mDRG IC50
PO/IP
(mg/kg)
Conc.
(nM)
(nM)
Test
Total/Free (nM)b
(nM)
5
3.9
27
1.8
5
100
-
30
30
30
30
30
30
30
3c
1.4
340
490
1211
118
116
616
792
0.56
340/340
Yes
No
No
Yes
No
No
No
No
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
2
4
4
4
5
5
7
9
6
7
8
5
140 ± 7.8
173 ± 3.5
59 ± 5
-
231
35
10
115
37
-
12
11
-
-
2720/95
11000/-
990/84
-
3.5
-
8.5
-
58 ± 20
14
24
12
44 ± 11
132 ± 15
0.28 ± 0.18
-
-
a
Compound dosed PO to male mice in 0.5% Methocel/0.1% Tween/99.4% water (10 mL/kg) and measured at 75
minutes, with the exception of 29 which was dosed IP. Results are presented as the mean, n = 3–9 per group.
Compound 55 at 30 mg/kg caused prostration or sedation and hypothermia in three mice and was tested at a
lower 3 mg/kg dose.
b
C
Figure 4. Mouse PK profiles of compounds 29 and 47 dosed at 30 mg/kg IP and PO.
Figure 5. Effect of 2 (30 mg/kg, PO) or 29 (3, 10, 30 mg/kg, IP) on phase I/II formalin-induced nociceptive
behaviors. Results are presented as the mean ± SEM response (n = 12-15 per group) and were analyzed by ANOVA
followed by Dunnett’s post-hoc tests; ***P<0.001 compared to vehicle/formalin group.
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Journal of Medicinal Chemistry
Phase I & II
1
2
3
4
5
6
7
8
9
1
000
control (n=12)
800
vehicle (n=14)
Cmpd 2 30 mg/kg (n=12)
Cmpd 29 3 mg/kg (n=14)
Cmpd 29 10 mg/kg (n=14)
Cmpd 29 30 mg/kg (n=15)
6
00
00
4
***
***
***
200
0
***
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
1
2
phase
Figure 6. Effect of 2 (30 mg/kg, PO) or 29 (3, 10, 30 mg/kg, IP), or 47 (3, 10, 30 mg/kg, PO) on percentage reversal
of phase II formalin-induced nociceptive behaviors. Results are presented as the mean ± SEM response (n = 12-
5 per group) and were analyzed by ANOVA followed by Dunnett’s post-hoc tests; ***P<0.001 compared to
1
vehicle/formalin group.
% Reversal in phase II
%
Reversal in phase II
150
100
50
1
50
00
***
***
***
***
1
**
5
0
0
0
control veh Cmpd 2
3
10
30
control veh Cmpd 2
3
10
30
Cmpd 29 mg/kg
Cmpd 47 mg/kg
Why compound 29 demonstrated complete reversal of nociceptive behavior while 47, a close analog with
similar in vitro properties, was not efficacious is enigmatic. Analogous discrepancies between PK and efficacy the
3
6
formalin test model have been reported with aminotriazine-based Na 1.7 inhibitors and arylsulfonamide-based
V
2
8
Na 1.7 inhibitors.
In an effort to further investigate this anomaly, we studied additional aspects of the
V
biochemical pharmacology of the compounds. Kinetic studies with 29 and 47 on hNa 1.7 also showed that both
V
compounds exhibited similar on- and off-rates (onset at -70 mV: tau on for 29 and 47 were 9.6 and 12.1 seconds,
respectively; recovery times at -120 mV: tau off for 29 and 47 were 45 and 99 seconds, respectively. The tissue
binding of both 29 and 47 in dorsal root ganglia (DRG) and trigeminal ganglia (TGG) tissue from both mouse and
rat were examined since these are believed to be key sites of action of Na 1.7 inhibitors. It was found that 29 had
v
similar unbound free fractions in the DRG and TGG tissues of mouse (3.5%) and rat (3.4%). The unbound free
fractions for 47 were more variable; in the mouse these were 8.4% (DRG) and 6.9% (TGG), whereas lower free
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Page 16 of 71
fractions were seen in the rat DRG (3.9%) and TGG (4.5%). It was our belief that compounds able to achieve free
tissue concentrations in the DRG above the Na 1.7 manual EP IC would show efficacy in the formalin-induced
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
V
50
pain model. As can be seen from Table 5, using the total mouse DRG exposure at 30 mg/kg (2724 nM for 29 and
910 nM for 47), mouse DRG binding (3.5% free for 29 and 8.5% free for 47), and mouse DRG Na 1.7 manual EP
V
IC (11 nM for 29 and 14 nM for 47), we can calculate the free[DRG]/IC values for 29 and 47 as 8.7 and 5.5,
5
0
50
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
respectively. This small difference in values fails to adequately explain why 29 is efficacious in the formalin-
induced pain model but 47 is not. However, the ability to accurately measure the in vitro properties of very
lipophilic compounds can be challenging and precise determination of the free fraction in plasma is often less
accurate for compounds that are highly protein bound. Determining the free fraction in DRG tissue is also
technically challenging. Assuming the free drug concentration in the DRG needs to cover the mNaV1.7 IC by 10-
5
0
fold, a 2-fold decrease in the calculated DRG free fraction may be enough to predict that compound 47 would be
inactive. With regard to activity at other Na subtypes, both compounds exhibit similarly advantageous selectivity
V
(
for 29: Barracuda assay results were IC values of 16, >30, 4.8, 18.5 µM for hNa 1.2/1.3/1.4/1.5, respectively).
5
0
V
PK studies with 47 showed that plasma exposures in CD1 mice were similar following either oral or IP
administration at 30 mg/kg, so the route of administration does not appear to explain the cause of the lack of
efficacy with this analog (see Figure 4). No off-target activities were seen in an in-house, CEREP-like, safety panel
assay for either 29 or 47 that could explain their different profiles (data not shown). It is worth noting that in
some of the efficacy models, including the formalin mouse model, there is a steep dose response curve which
may suggest that a somewhat modest increase or decrease in the dose may have a dramatic effect on the
behavioral response based on tissue exposure. The doses tested in the formalin mouse model were limited to 30
mg/kg in an effort to avoid potential off-target activities, particularly since the plasma exposures at this dose were
high (Figure 4).
Compounds 29 and 47 were also tested in the mouse complete Freund’s adjuvant (CFA) model of
inflammatory pain.37 It has been shown that Na 1.7 knockout mice do not develop CFA-induced thermal
V
hyperalgesia.16 Treatment with compound 29 (30 mg/kg, IP) significantly reduced CFA-induced thermal
hyperalgesia without affecting thermal response latency of the untreated, contralateral (left) paw. Compound 29
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Journal of Medicinal Chemistry
was efficacious in reversing thermal hyperalgesia at 60 minutes post-treatment but not at 180 minutes after
treatment (Figure 7). Similar to previous results, 47 (30 mg/kg, PO) was not effective in the CFA thermal
hyperalgesia model. Plasma exposures at these doses/routes, determined in satellite groups of mice, were 63 ±
4.8 µM and 38 ± 10 µM for 29 and 47, respectively. This corresponded to a mouse free plasma concentration of
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
26 nM for 29 (0.2% plasma free fraction) and 532 nM for 47 (1.4% plasma free fraction), corresponding to ~4-5-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
fold coverage of the mouse cellular IC values that were determined by electrophysiology (mNa 1.7 IC = 35 nM
5
0
v
50
for 29, mNa 1.7 IC = 115 nM for 47).
v
50
Figure 7. Effect of compound 2 (60 mg/kg, PO) or 29 (3, 10, 30 mg/kg, IP) on latency of thermal hyperalgesia in
CFA mice. Results are presented as the mean ± SEM response (n = 12-14/group) and were analyzed by ANOVA
followed by Dunnett’s post-hoc tests; ***P<0.001 compared to vehicle/control group.
CFA Injected paw (180 min)
CFA injected paw (60 min)
1
2
8
4
0
12
***
***
***
***
8
***
4
0
Saline Veh Cmpd 2
Inj
3
10
30
Saline Veh Cmpd 2
Inj
3
10
30
Cmpd 29 mg/kg
Cmpd 29 mg/kg
Next, 29 was tested in the mouse chronic constriction injury (CCI) model in which three ligatures are loosely
tied around the left sciatic nerve to simulate the conditions associated with nerve compression injury.38 In
contrast to mice receiving sham surgery, CCI mice showed increased sensitivity to cold and a faster latency to
0
exhibit nociceptive-like behaviors when placed on a cold plate maintained at 5 C. CD1 mice receiving CCI or sham
surgery were tested 5 weeks later and were treated IP with either vehicle or 29 30 minutes prior to testing. As
can be seen from Figure 8 (left), an IP dose of 30 mpk 29 produced a significant increase in the latency in the
response to cold in CCI mice. Figure 8 (right) shows that the cold allodynia response in CCI mice was reversed by
a modest 25% following treatment with a dose of 30 mpk of 29. The total plasma exposures at 60 minutes post-
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Page 18 of 71
dose were 3.3 ± 1.0, 27 ± 6.8, and 68 ± 31 µM following administration of 3, 10, and 30 mg/kg, respectively, which
corresponds to a free plasma concentrations of 6.6, 54, and 136 nM, respectively.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 8. Effect of compound 2 (60 mg/kg, PO) or 29 (3, 10, 30 mg/kg, IP) on latency of cold allodynia in CCI mice.
Results are presented as the mean ± SEM response (n = 12-14 per group) and were analyzed by ANOVA followed
by Dunnett’s post-hoc tests; ***P<0.001, *p<0.05 compared to vehicle/control group.
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
Cold Plate (Cmpd 29, ip, 60 min)
Cold Plate (Cmpd 29, ip, 60 min)
1
25
00
125
100
75
50
25
0
***
***
***
***
1
7
5
0
5
*
*
25
0
Sham Veh Cmpd 2
3
10
30
Sham Veh Cmpd 2
3
10
30
Cmpd 29 mg/kg
Cmpd 29 mg/kg
To further explore the differences between 29 and 47, sural fascicle recording studies were performed in
order to determine if the compounds would differentially affect mechanoreceptor-mediated afferent
transmission in wild-type mice, a modified form of the model described by Koltzenburg et al.39 The assay was
validated with systemic mexiletine (IP; 10 mg/kg) and local tetrodotoxin (300 nM applied to the nerve)
applications (data not shown). Systemic IP dosing of either 29 or 47 at doses of 30 mg/kg was followed by
recording of the sural nerve mechano-sensitivity at 5, 30, 60, 90, and 120 minutes post-dose. While brush and
low-threshold pressure (1.6 mN) did not show any difference between treated and non-treated animals,
differences were noted at the higher pressures of 98 mN and 980 mN, as shown in Figure 9. At the longer time
points of 90–120 minutes, both 29 and 47 showed significant reductions in mechanically-elicited afferent firing,
with the systemic exposures measured 2 hours after IP dosing determined to be 89 µM and 57 µM, respectively.
In a second study, 29 or 47 were applied directly onto the nerve fascicle, with the effects summarized in Figure
10. Intriguingly, in this study, only compound 29 and not compound 47 showed a significant effect.
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Journal of Medicinal Chemistry
Figure 9. Effects of 29 and 47 (30 mg/kg) on mechano-sensitivity of the mouse sural nerve when dosed IP.
indicates significant difference from vehicle (2-way ANOVA followed by Dunnett’s test).
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
*
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 10. Effects of 29 and 47 on mechano-sensitivity of the mouse sural nerve when applied directly to the
nerve. *indicates significant difference from vehicle (2-way ANOVA followed by Dunnett’s test).
CHEMISTRY
Scheme 1 describes the general synthesis of compounds 5-15. Bromination of methyl indole-6-
carboxylate (69) using NBS afforded bromide 70 which, after methylation of the indole nitrogen atom, provided
71. Following hydrolysis of 71, the resultant acid 72 was converted to the acylsulfonamide 73 using the
40
Mukaiyama reagent for the acid activation. Finally, a Suzuki coupling of bromide 73 with a phenylboronic acid
derivative afforded the aryl indoles 5-15.
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
.
Scheme 1 Synthetic approaches to indoles 5-15.
O
O
O
H
a
b
c
H
N
N
O
O
N
O
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
Br
Br
69
70
71
O
O
O
O
O
S
O
O
N
N
d
S
e
N
N
H
OH
N
H
Br
Br
R
72
73
5-15
a
Reagents and conditions: (a) NBS, DMF, rt, 2 h, 93%; (b) MeI, K CO , DMF, rt, 18 h, 90%; (c) LiOH, THF-MeOH,
2
3
H O, rt, 18 h, 92%; (d) Methansulfonamide, 2-chloro-1-methylpyridin-1-ium iodide, Et N, DMAP, CH Cl , rt, 1.5 h,
2
3
2
2
9
3%; (e) R-ArB(OH) , PdCl (dppf), Na CO , dioxane, 100 ºC, 2 h.
2
2
2
3
Scheme 2 describes the synthesis routes employed to prepare 16-23. Indole 69 was converted to the N-
methylated iodoindole 75 through the intermediacy of 74.41 The stable boronate 76, prepared from iodide 75,
was purified and characterized.42 Boronate 76 reacted with bromide 80, which was prepared from 79 under
standard Mitsunobu conditions, to generate 77a. After hydrolysis, the acid 78a was converted to 16 as described
above. Alternatively, the boronate formation and Suzuki coupling from 75 could be carried out in a single pot42
to afford intermediates 77b/c, which led to compounds 17-23.
Scheme 2. Synthetic approaches to the preparation of 16-23.
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Journal of Medicinal Chemistry
O
Me
1
2
O
O
N
Me
O
O
H
N
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
H
N
a
b
N
c
d
O
O
O
B
O
O
I
I
69
74
75
76
O
O
O
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
Me
Me
Me
O
O
S
N
N
N
Br
Br
O
OH
N
H
e
f
g
O
OH
7
7a
78a
79
80
O
O
O
16
O
O
O
Me
Me
Me
O
O
O
S
Me
N
N
N
R1
O
OH
N
H
h
e
f
N
O
I
R
R
R
75
77b: R = 4-CHF2
7c: R = 4-CF3
78b: R = 4-CHF2
78c: R = 4-CF3
17-23
7
a
Reagents and conditions: (a) I , KOH, DMF, rt, 18 h, 91%; (b) MeI, K CO , DMF, rt, 18 h, 89%; (c) Pd(PPh ) , Et N,
2
2
3
3 4
3
4
,4,5,5-tetramethyl-1,3,2-dioxaborolane, dioxane, 80 ºC, 2 h, 81%; (d) 4-isobutoxyphenyl bromide, PdCl (dppf),
2
Na CO , dioxane, 80 ºC, 12 h; (e) LiOH, THF-MeOH, H O, rt, 18 h, 88%; (f) DMAP, 2-chloro-1-methylpyridin-1-ium
2
3
2
1
iodide, Et N, R SO NH , CH Cl , rt, 1 h, 28%; (g) PPh , DIAD, THF, rt, 2 h, 40%; (h) Pd(PPh ) , Et N, 4,4,5,5-
3
2
2
2
2
3
3 4
3
tetramethyl-1,3,2-dioxaborolane, dioxane, 80 ºC, 2 h; then MeOH, R-Ar-Br, Cs CO , 80 ºC, 4 h, 60-63%.
2
3
Scheme 3 describes the synthesis of the 5-fluoroindole derivatives 24-27. The 5-fluoro-6-indole ester 84
was prepared from intermediate 83 which, in turn, was generated by using the Leimgruber-Batcho reaction in
4
3
which 81 is exposed to 82. Iodination and methylation of 84 took place in one pot to generate intermediate 85,
which could be converted into 24-27 as described previously.
Scheme 3. Synthetic approaches to the preparation of 24-27.
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
F
O N
CO Me
2
2
O N
CO Me
2
2
MeO
MeO
a
b
H
N
N
O
N
F
F
8
1
82
83
Me
84
O
O
F
N
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
O
e
c
d
N
I
O
F
B
O
O
8
5
86
O
O
O
F
Me
Me
Me
O
O
S
N
N
N
R1
O
OH
N
f
g
H
F
F
87a/b
88a/b
a: R = 4-CF3
b: R = 4-CHF2
24-27
R
R
R
a: R = 4-CF3
b: R = 4-CHF2
a
Reagents and conditions: (a) DMF, 100 ºC, 1 h, 86%; (b) Pd/C, H , EtOAc, rt, 2 h, 70%; (c) I , KOH, DMF, rt, 16 h;
2
2
then MeI, rt, 5 h, 85%; (d) Pd(PPh ) , Et N, 4,4,5,5-tetramethyl-1,3,2-dioxaborolane, dioxane, 80 ºC, 2 h; (e) MeOH,
3 4
3
R-Ar-Br, Cs CO , dioxane, 80 ºC, 4 h; (f) LiOH, THF, H O, rt, 18 h; (g) DMAP, 2-chloro-1-methylpyridin-1-ium iodide,
2
3
2
1
Et N, R SO NH , CH Cl , rt, 1 h.
3
2
2
2
2
Scheme 4 describes the synthetic approaches to 3-(3-pyrindinyl)indole derivatives 28-48. Boronate 76 or
iodide 75 were converted into 28-31, 33, and 36-44 as described above. Intermediate bromide 92 was
conveniently prepared from the 2-fluoropyridine 91, while bromide 94 was prepared by the reaction of
hydroxypyridine 93 with 2,2-dimethyloxirane. All other bromides used were commercially available. Cyano
4
4
derivative 32 was directly prepared from the chloride 29, while 34 and 35 were prepared from bromide 73 and
boronates 97a/b, respectively, which, in turn, were prepared from the pyridine intermediates 96a/b following a
literature procedure.27,45 Intermediates 96a/b were prepared from 2-fluoro-3-chloro-pyridine (95) while the 5-
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Journal of Medicinal Chemistry
fluoro-indole derivatives 45-48 were prepared from the boronate 86 and the corresponding bromides following
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the established conditions.
Scheme 4. Synthetic approaches to the preparation of 28-48.
O
Me
O
O
O
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
Me
Me
Me
O
O
N
S
O
N
N
N
R1
O
OH
N
a
c
d
H
B
O
O
76
89a-f
90a-f
28-31,33,36-44
N
N
N
R
R
R
b
O
Me
O
O
S
N
N
H
O
Me
f
29
N
O
N
I
75
CN 32
O
Cl
Br
Cl
Br
Cl
Br
e
Cl
Br j
O
N
O
N
F
N
HO
N
HO
91
92
93
94
O
Me
O
O
S
N
O
B
N
Cl
Cl
g
h
i
H
Cl
O
+ 73
O
F
N
R2
O
N
R2
N
9
5
N
9
6a/b
97a/b
34,35
2
2
R2
a: R = CHF
a: R = CHF
Cl
2
2
O
2
2
b: R = CF3
b: R = CF
3
O
O
F
O
O
F
Me
N
Me
N
Me
O
O
Me
S
N
R1
N
O
OH
N
O
H
a
c
d
F
F
B
O
O
N
O
N
N
86
98a/b
a: R = Cl
b: R = H
99a/b
a: R = Cl
b: R = H
45-48
R
R
R
O
O
a
Reagents and conditions: (a) R-pyridyl bromide, PdCl (dppf), Na CO , dioxane, 80 ºC, 12 h; (b) Pd(PPh ) , Et N,
2
2
3
3 4
3
4
,4,5,5-tetramethyl-1,3,2-dioxaborolane, dioxane, 80 ºC, 2 h, then MeOH, R-pyridyl bromide, Cs CO , 80 ºC, 4 h;
2
3
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1
(
c) LiOH, THF-MeOH, H O, rt, 18 h; (d) R SO NH , DMAP, 2-chloro-1-methylpyridin-1-ium iodide, Et N, CH Cl , rt,
2
2
2
3
2
2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
h; (e) Cs CO , 2-methylpropanol, DMSO, 90 ºC, 10 h, 91%; (f) (1,1'-binaphthalen)-2-yl di-tert-butylphosphine,
2
3
2
Zn, Pd(O CCF ) , Zn(CN) , DMAC, 95 ºC, 3 h, 56%; (g) R CH OH, Cs CO , DMSO, 80 ºC, 18 h; (h) (1,5-
2
3 2
2
2
2
3
Cyclooctadiene)(methoxy)iridium(i) dimer, bis(pinacolato)diboron, 4,4'-di-tert-butyl-2,2'-bipyridine, tBuOMe, 80
ºC, 30 min; (i) PdCl (dppf), Na CO , dioxane, 100 ºC, 2 h; (j) K CO , 2,2-dimethyloxirane, 2-butanone, 80 ºC, 18 h,
2
2
3
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
9%.
Scheme 5 describes the synthesis of the N-alkylated indole derivatives 49-51. Alkylation of the 3-
bromoindole 70 with either 2-iodoprapane or 1-iodo-2-methylpropane led to formation of intermediates 100a/b,
respectively. Suzuki coupling to install the pyridyl side chain followed by hydrolysis of the acid and coupling with
a sulfonamide led to acylsulfonamides 49-51.
Scheme 5. Synthetic approaches to the N-alkylated indole derivatives 49-51.
R1
O
O
N
O
R1
O
H
a
b
c
N
O
N
O
Br
N
Br
Cl
O
70
1
00a/b
101a/b
1
1
a: R = iPr
a: R = iPr
1
1
b: R = iBu
b: R = iBu
R1
N
O
R1
N
O
O
O
S
R2
OH
d
N
H
N
N
Cl
Cl
49-51
O
O
1
02a/b
1
a: R = iPr
1
b: R = iBu
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Journal of Medicinal Chemistry
a
1
Reagents and conditions: (a) R -I, K CO , DMF, rt, 18 h, 44-46%; (b) 5-bromo-3-chloro-2-isobutoxypyridine,
2
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
PdCl (dppf), Na CO , dioxane, 80 ºC, 12 h; (c) LiOH, THF-MeOH, H O, rt, 18 h; (d) R SO NH , DMAP, 2-chloro-1-
2
2
3
2
2
2
methylpyridin-1-ium iodide, Et N, CH Cl , rt, 1 h.
3
2
2
Scheme 6 describes the synthetic routes used to procure the indazole derivatives 52, 55-57, and 60-62.
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
4
6
Methylation of indazole 103 led to two separable isomers 104 and 105, with the major isomer 105 subjected to
a selective Suzuki coupling at the iodo substituent to afford bromide 106 which, in turn, was transformed into 52
following literature conditions.47 Methylation of the indazole 107 led to the formation of the two separable
isomers 108 and 109, whose structures were identified by NOE studies. Isomer 109 was converted to the N-1
methyl indazole analogs 55 and 56, while isomer 108 was converted to the N-2 methyl indazole derivatives 61
and 62 following the established conditions. Derivatives 57 and 60 were prepared from the bromides 114a/b
which were obtained from iodides 105 and 103, respectively, following the previously outlined conditions.
Scheme 6. Synthetic approaches to the preparation of indazole derivatives 52, 55-57, and 60-62.
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O
O
O
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Br
N
S
N
N
H
N
N
Br
N
H
Br
N
I
Br
N
I
a
b
c
N
N
+
N
I
1
03
104
105
F3C
106
52
F3C
O
O
O
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
H
N
d
N
N
OMe
OMe
OMe
N
N
N
+
Br
Br
Br
1
07
108
109
O
O
O
O
O
S
N
N
OH
g
N
H
R
N
OMe
N
N
N
e
f
109
N
N
N
Cl
55,56
Cl
Cl
O
O
O
110
111
O
O
O
O
O
S
N
N
N
O
OH
N
R
N
N
N
H
f
g
e
108
N
N
N
Cl
Cl
Cl
O
O
O
112
113
61,62
R1
N
R1
N
O
O
O
Br
S
R1
N
N
N
H
Br
N
b
c
N
N
N
I
R2
R2
O
1
O
1
2
1
2
1
05: R = Me
114a: R = Me/R = H
57: R = Me/R = H
1
1
2
1
2
1
03: R = H
114b: R = H/R = Cl
60: R = H/R = Cl
a
Reagents and conditions: (a) K CO , DMF, MeI, rt, 72 h, 104 (14%), 105 (48%); (b) 4-trifluoromethylphenyl
2
3
bromide, PdCl (dppf), Na CO , dioxane, 80 ºC, 1 h; (c) Methanesulfonamide, Mo(CO) , DBU, tri-tert-
2
2
3
6
butylphosphonium
tetrafluoroborate,
trans-di-mu-acetatobis[2-[bis(2-
methylphenyl)phosphino]benzyl]dipalladium, dioxane, 140 ºC, 10 min, 61%; (d) NaH, THF, MeI, rt, 18 h, 108 (18%),
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Journal of Medicinal Chemistry
1
09 (34%); (e) 5-bromo-3-chloro-2-isobutoxypyridine, PdCl (dppf), Na CO , dioxane, 100 ºC, 2 h; (f) LiOH, THF-
2
2
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
MeOH, H O, rt, 18 h; (g) RSO N , DMAP, 2-chloro-1-methylpyridin-1-ium iodide, Et N, CH Cl , rt, 1 h.
2
2
2
3
2
2
Scheme 7 describes the synthesis route leading to 5-fluoroindazole derivatives 53, 54, 58, and 59.
Nitration of 115 in concentrated H SO /HNO afforded nitro derivative 116. After ester formation and nitro
2
4
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
reduction, the indazole intermediate 119 was formed from 2-methylaniline precursor 118. Iodination and
methylation of 119 in one pot led to intermediate 120 along with small amounts of the minor isomer. The isomers
were separable and identified by NOE studies. Iodide 120 was transformed into the final products, 53, 54, 58,
and 59, through the established routes and conditions.
Scheme 7. Synthetic approaches to the 5-fluoroindazole derivatives 53, 54, 58, and 59.
CO H
O N
2
CO H
O N
2
CO Me
H N
2
CO Me
2
2
2
2
a
b
c
F
F
F
F
115
116
117
118
O
F
O
H
d
e
N
N
O
OMe
R =
R =
N
N
N
F
F3C
O
I
119
major
1
21a/122a
121b/122b
120
O
O
O
O
O
S
f
N
g
N
h
N
R2
OMe
OH
N
N
N
N
H
F
F
F
R
R
R
121a/b
122a/b
53, 54, 58, 59
a
Reagents and conditions: (a) H SO , HNO , 0 ºC, 3 h; (b) H SO , MeOH, 65 ºC, 20 h, 92% for 2 steps; (c) Pd/C, H ,
2
4
3
2
4
2
EtOAc, rt, 2 h, 95%; (d) KOAc, AcOH, isoamyl nitrite, CHCl3, rt, 3 h, 13%; (e) I , KOH, DMF, rt, 18 h, then MeI, 20 h,
2
59%; (f) PdCl (dppf), Na CO , dioxane, 80 ºC, 18 h; (g) LiOH, THF-MeOH, H O, rt, 18 h; (h) Sulfonamides, DMAP, 2-
2
2
3
2
chloro-1-methylpyridin-1-ium iodide, Et N, CH Cl , rt, 1 h.
3
2
2
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Scheme 8 describes routes to access the azaindole derivatives 63-68. The synthesis of the 7-azaindole
derivatives 63-65 originated with 123. Ester formation, iodination, and indole methylation led to intermediate
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
26 which was converted into analogs 63-65 following previously outlined conditions. The 4-azaindole derivatives
66 and 67 were prepared from bromide 131 which, in turn, was generated from 129 after iodination and
methylation, following the established conditions. Finally the 5-azaindazole 68 originated from 132, which was
prepared using literature conditions.48 Iodination of 132 provided iodide 133 which reacted with 4-
trifluoromethylphenyl boronic acid to afford 134. After hydrolysis of 134 to acid intermediate 135, analog 68 was
obtained after acylsulfonamide formation.
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
Scheme 8. Synthetic approaches to the azaindole derivatives 63-68.
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1
Journal of Medicinal Chemistry
O
O
O
O
O
O
H
N
N
H
a
H
N
b
N
c
N
I
d or e
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N
1
N
OMe
OMe
N
OH
OMe
OH
I
23
124
125
126
O
O
O
N
N
N
S
N
g
N
R =
N
R =
N
OMe f
N
H
R
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
Cl
O
F3C
127/128b
R
R
R
1
27a/b
128a/b
63-65
1
27/128a
O
O
O
Br
N
S
N
N
R
H
N
H
Br
H
Br
N
b
c
N
h
N
N
N
I
N
N
Cl
131
Cl
O
O
1
29
130
66,67
O
O
b
N
I
e
OMe
N
OMe
N
N
32
1
133
O
O
O
O
O
N
S
OMe
N
N
N
OH
g
H
N
N
N
f
F3C
F3C
F3C
1
34
135
68
a
Reagents and conditions: (a) H SO , MeOH, 70 ºC, 4 h; (b) I , KOH, DMF, rt, 18 h; (c) MeI, K CO , DMF, rt, 18 h; (d)
2
4
2
2
3
127a: Pd(PPh ) , Et N, 4,4,5,5-tetramethyl-1,3,2-dioxaborolane, dioxane, 80 ºC, 2 h, then MeOH, R-Ar-Br, Cs CO ,
3
4
3
2
3
8
0 ºC, 4 h; (e) 127b: AR-B(OH) , PdCl (dppf), Na CO , dioxane, 80 ºC, 18 h; (f) LiOH, THF-MeOH, H O, rt, 18 h; (g)
2
2
2
3
2
Sulfonamides, DMAP, 2-chloro-1-methylpyridin-1-ium iodide, Et N, CH Cl , rt, 1 h; (h) RSO NH , Mo(CO) , DBU,
3
2
2
2
2
6
tri-tert-butylphosphonium
tetrafluoroborate,
trans-di-mu-acetatobis[2-[bis(2-
methylphenyl)phosphino]benzyl]dipalladium, dioxane, 140 ºC, 10 min.
CONCLUSION
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