A potent and selective indole N-type calcium channel (Cav2.2) blocker for the treatment of pain

Article abstract of DOI:10.1016/j.bmcl.2010.11.067

N-type calcium channels (Ca_v2.2) have been shown to play a critical role in pain. A series of low molecular weight 2-aryl indoles were identified as potent Ca_v2.2 blockers with good in vitro and in vivo potency.

Full text of DOI:10.1016/j.bmcl.2010.11.067

Bioorganic & Medicinal Chemistry Letters 21 (2011) 869–873
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A potent and selective indole N-type calcium channel (Cav2.2) blocker
for the treatment of pain
Sriram Tyagarajan a, , Prasun K. Chakravarty a, Min Park a, Bishan Zhou a, James B. Herrington b,
⇑
Kevin Ratliff b, Randall M. Bugianesi b, Brande Williams b, Rodolfo J. Haedo b, Andrew M. Swensen b,
Vivien A. Warren b, McHardy Smith b, Maria Garcia b, Gregory J. Kaczorowski b, Owen B. McManus b,
Kathryn A. Lyons c, Xiaohua Li c, Maria Madeira c, Bindhu Karanam c, Mitchell Green c, Michael J. Forrest d,
Catherine Abbadie d, Erin McGowan d, Shruti Mistry d, Nina Jochnowitz d, Joseph L. Duffy a
a Departments of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
b Drug Metabolism and Pharmacokinetics, Merck Research Laboratories, Rahway, NJ 07065, USA
c Ion Channels, Merck Research Laboratories, Rahway, NJ 07065, USA
d Pharmacology, Merck Research Laboratories, Rahway, NJ 07065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
N-type calcium channels (Cav2.2) have been shown to play a critical role in pain. A series of low molecular
weight 2-aryl indoles were identified as potent Cav2.2 blockers with good in vitro and in vivo potency.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 6 August 2010
Revised 9 November 2010
Accepted 16 November 2010
Available online 21 November 2010
Keywords:
Pain
N-type calcium channel
Cav2.2 blockers
Indoles
Chung model
Several lines of evidence point to the involvement of N-type
calcium channels (Cav2.2 channels) in pain.1 These channels are
present in presynaptic terminals of primary afferent nociceptors.
Cav2.2 channels are highly expressed in the dorsal horn of the
spinal cord, which is the final common path for pain signal trans-
mission. These channels are up regulated in pain conditions.2
Cav2.2 knockout mice exhibit reduced sensitivity to pain.3 The
peptide ziconitide (PrialtÒ, Elan) is a potent and selective blocker
of Cav2.2 channels and is a marketed drug for treating severe
chronic pain.4
ability and thus be effective in treating pain without the side ef-
fects of ziconotide.7
From our high throughput screening efforts (Table 1) we identi-
fied compound 1 as a Cav2.2 blocker with modest potency in the
in vitro Cav2.2 fluorescent calcium-influx assay (FLIPR), and found
it to be selective over cardiac ion channels with weak block of
Table 1
Screening hit to lead
The use of ziconotide is limited by the need for intrathecal
administration and the narrow therapeutic window for CNS side
effects (sedation, confusion and dizziness).5 These effects may arise
from the state-independent block of Cav2.2 by ziconitide. Further-
more, systemic administration of the peptide is associated with
profound hemodynamic effects.6 It is believed that selective
state-dependent block of Cav2.2 channels with small molecules
will affect neuronal signaling only under conditions of hyper excit-
OH
N
NH
O
N
O
N
O
N
O
N
HO
O
H
O
1
2
3
Screening Hit
Lead
Ion channel targets
Cav2.2 (IC50 M)
1
2
3
,
l
0.88
62%
44%
0.6
40%
25%
0.87
52%
ND
Cav1.2 (inh @ 10
Nav1.5 (inh @ 10
lM)
⇑
Corresponding author.
E-mail address: sriram_tyagarajan@merck.com (S. Tyagarajan).
lM)
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.067

870
S. Tyagarajan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 869–873
Table 2
Cav1.2 and Nav1.5. Utilizing this indole scaffold, modifications of
substituents led to compound 2, a somewhat simplified indole ana-
log. Building upon the hypothesis that a C-3 substituent may not
be necessary, a more simplified indole analog, compound 3, was
identified as the lead for this series. Using this simplified indole
scaffold, we set out to explore a series of novel C-2 substituted
indoles as small molecule Cav2.2 blockers.
SAR at C-5 position of the indole
R
5
2
N
HO
Entry
R
Cav2.2 FLIPR
Cav1.2 FLIPR
(% inh @ 10
89% (0.87)
l
M) (IC50
,
l
M) (% inh @ 10
lM)
The synthesis of these indoles is outlined in Scheme 1.
Alkylation of the commercially available 4-nitrophenyl acetic acid
4 afforded the dimethyl ester 5. Reduction of the nitro group and
subsequent diazotization generated the hydrazine 6. Condensation
of the hydrazine 6 with aryl acetophenones afforded the 2-aryl
indoles 7. Hydrolysis of the ester to the acid and subsequent ami-
dation yielded the corresponding amides 8. Finally N-1 substituted
analogs were generated via treatment with the corresponding alkyl
halides and sodium hydride.
The synthesized compounds were evaluated for their ability
to block voltage gated Cav2.2 using an in vitro fluorescent
calcium-influx FLIPR assay.8 Selected compounds were screened
for activity against ancillary targets including hERG (MK-0499
binding assay measures the displacement of 35S-labeled MK-
0499, a known hERG blocker), CYP inhibition and Cav1.2 (diltiazem,
DLZ) binding.9 Compounds selective in these ancillary assays were
examined in various rat in vivo pain models and their pharmacoki-
netic profiles were evaluated.
We first explored the C-5 position of the indoles as shown in
Table 2. The dimethyl moiety is critical for Cav2.2 in vitro potency.
Several des-methyl analogs or truncated analogs such as com-
pound 10 were inactive in the in vitro Cav2.2 FLIPR assay. The
dimethyl ester 11 and the alcohol derivative 12 had significantly
reduced potency in the Cav2.2 FLIPR assay. Amides were well toler-
ated at C-5 position of the indole. The cyclopropyl amide 13 and
the trifluromethyl amide 14 had weaker block than the dimethyl
amide 3. The more hindered lipophilic t-butyl analog 15 had
improved Cav2.2 block in the FLIPR assay. These compounds were
also tested for the block of L-type Cav1.2. As illustrated in Table 2,
the compounds exhibited a range of activity against Cav1.2.
With an optimized t-butyl analog installed at C-5 position of the
indole, we turned our attention to the N-1 position of the indole
(Table 3). We focused our efforts on finding a suitable metaboli-
cally stable substituent at this position. Although the Boc substitu-
ent in 2 was active, both the Boc derivative 16 and the free indole
17 were less potent. Substituting this position with a liphophilic
t-butyl ester, analog 18 improved the Cav2.2 potency. Introduction
of a t-butyl acetamide, 19 afforded significantly improved Cav2.2
potency. Replacing the amide with an ether, analog 20 or the
amide isostere dimethyl isoxazole group, analog 21 reduced
N
3
52%
O
F
F
O
10
36%
43%
F
O
11
12
70%
44%
64%
32%
O
HO
H
N
13
14
15
89% (1.15)
91% (1.17)
97% (0.68)
ND
ND
90%
O
H
F3C
N
O
O
N
H
Table 3
SAR at N-1 position of the indole
H
N
O
N
R
Entry R
15
Cav2.2 FLIPR
Cav1.2 FLIPR
MK-0499
(% inh @ 10
l
M) (IC50
,
l
M) (% inh@ 10
l
M) (% inh@ 10 lM)
97% (0.68)
90%
64%
HO
16
17
59%
78%
ND
ND
ND
ND
O
O
O
H
O
18
19
91% (0.66)
98% (0.19)
48%
78%
60%
33%
b
a
HO
O
O
NO2
O
O
NO2
4
5
NH
R3
O
O
c
O
NH2
O
R2
20
21
99% (0.52)
99% (0.54)
70%
ND
81%
65%
N
N
H
H
7
6
O
R3
H
R3
N
H
N
R4
e
d
R4
R2
O
N
R1
N
O
R2
N
H
O
9
8
Scheme 1. Reagents and conditions: (a) (i) EtOH, H2SO4; (ii) NaH, DMF, EtI; (b) 10%
Pd/C, EtOH; (ii) NaNO2, HCl, SnCL2Á2H2O; (c) ArCOMe, EtOH, ZnCl2, HOAc; (d) (i)
KOH, MeOH; (ii) 1-chloro-N,N-trimethylpropenylamine, R4NH2, TEA, DCM; (e) NaH,
R1CH2Br, DMF.
22
36%
ND
ND
Cl

S. Tyagarajan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 869–873
871
Table 4
Table 6
SAR at C-2 position of the indole
In vivo activity of compound 30
H
Entry
% Reversal (1 h)
% Reversal (3 h)
% Reversal (8 h)
N
CFA (3 mpk, po)
CFA (10 mpk, po)
CFA (30 mpk, po)
SNL (10 mpk, po)
SNL (30 mpk, po)
36
57
66
45
68
37
52
68
47
50
17
22
32
14
27
R
O
N
O
HN
Cav1.2 FLIPR
Entry
R
Cav2.2 FLIPR
(IC50
PXR EC50
M)
,
lM)
(% inh@ 10
lM)
(l
analog 24, maintains the Cav2.2 potency and decreased the PXR
potency. Replacing the methyl groups with a more liphophilic
trifluoromethyl groups, analog 25, afforded modest in vitro block
on Cav2.2, but greater potency as a PXR activator. Replacing the
trifluromethyl groups in 25 with fluoro substituents, analog 27 im-
proved Cav2.2 block but the compound remained a PXR activator.
Introduction of a trifluoromethoxy aryl group at the C-2 position
improves the in vitro Cav2.2 potency and selectivity against PXR.
The 2-trifluormethoxy aryl, 3-trifluomethoxy and the 4-trifluoro-
methoxy aryl groups were all well tolerated at this position.
Replacement with a methoxyphenyl substituent maintains the
in vitro block but the compound was selective towards the PXR
receptor.
19
23
24
25
26
27
28
29
30
31
32
33
34
35
3,5-MePh
Ph
4-FPh
0.18
0.47
0.50
0.76
0.40
0.27
0.07
0.06
0.15
0.28
0.14
0.24
0.24
0.57
79%
79%
82%
61%
88%
93%
79%
93%
85%a
60%
47%
24%
40%
64%
>30
1.81
>30
3,5-CF3Ph
3-CF3,-5-FPh
2,4,-FPh
2-OCF3Ph
3-OCF3Ph
4-OCF3Ph
2-OCH3Ph
t-butyl
10.76
> 30
0.94
> 30
> 30
> 30
0.74
0.57
6.64
2.46
0.60
CONHt-Bu
Thiazole
2-Pyridyl
a
IC50 = 0.91 lM.
We observed that alkyl groups at C-2, as in 32, tend to be PXR
activators while aryl groups at the same position can be more
selective against PXR. Conversely, aryl groups at C-2 (23–31) gen-
erally exhibited more pronounced Cav1.2 block in comparison to
alkyl groups at C-2 position.
Cav2.2 potency, while a benzyl analog 22 was not potent. These
analogs exhibited modest selectivity in favor of Cav2.2 over
Cav1.2. Although 19 was potent and selective for block of Cav2.2
channels, the compound exhibited poor in vitro metabolic stability
in incubations with rat and human liver microsomes (<5% parent
remaining after 1 h).
We hypothesized that the 3,5-dimethyl phenyl substituent was
a metabolic liability, and we set out to find a suitable replacement
for this substituent (Table 4). The resulting compounds were
distinguished on basis of Cav2.2 FLIPR potency. Having optimized
the N-1 amide substituent, MK-0499 activity was minimal in all
subsequent compounds. The compounds were counter screened
for Cav1.2 activity and for PXR activation as a measure of the
potential for CYP induction.10 The unsubstituted phenyl analog
23 at C-2 position afforded good potency but also had moderate
activity in the PXR induction assay. Introduction of a fluoro group,
Table
5 shows the pharmacokinetic profile of selected
compounds that exhibited excellent Cav2.2 block and good
selectivity against PXR). Compounds 28 and 29 maintained a good
pharmacokinetic profile but very poor stability in human liver
microsomal incubation. The 4-trifluoromethoxy phenyl analog 30
has modest pharmacokinetic profile in rat but a better metabolic
profile than indoles 28 or 29. Compound 30 exhibited similar met-
abolic stability in liver preparations from human and rat, as well as
dog and monkey.
Compound 30 was further evaluated for pharmacokinetic
properties in rat and dog. The compound afforded a moderate oral
exposure following
a 3 mpk dose in rats (Cmax = 0.29 lM,
AUCN = 0.34 M h kg/mg) and a high clearance and distribution
l
(CL = 64 mL/min/kg, Vdss = 5.1 L/kg) for an overall oral bioavailabil-
Table 5
Pharmacokinetic data for selected compounds
H
N
R
N
O
O
NH
Dose(iv/po) (mg/Kg)
c
Entry
28
R
PK species
Rat
HLMa (%)
0.04
RLMb (%)
2.5
AUCN
0.57
Clpd
21
T1/2 (h)
F (%)
F3CO
1.0/3.0
1.0/3.0
2.68
38
OCF3
29
Rat
0.82
20
22
20
0.83
11
2.27
26
30
30
OCF3
OCF3
Rat
1.0/3.0
1.0/3.0
0.34
1.02
64
1.4
4.6
68
Dog
5.6
18%
a
b
c
Percent of parent compound remaining in human liver microsomes following incubation after 60 min.
Percent of parent compound remaining in rat liver microsomes following incubation after 60 min.
lM h kg/mg.
d
mL/min/kg.

872
S. Tyagarajan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 869–873
Baseline
30 min
15 min
2:52
2:24
1:55
1:26
0:57
0:28
0:00
-1*9*%
-37%
***
Vehicle
3 mg/kg IV
10 mg/kg IV
Figure 1. Effect of compound 30 at 3 and 10 mg/kg IV on motor coordination.
ity of 68% based on plasma analysis. However, in dog, the PK profile
showed low clearance (4.6 mL/min/kg), good plasma half life (4.6 h)
adjuvant (CFA)-induced inflammatory pain model, compound 30
reverses mechanical allodynia in a dose dependant manner.11
When dosed at 30 mg/kg, compound 30 afforded robust maximal
and modest oral exposure moderate oral exposure (AUCN
1.0 M h kg/mg) for an overall modest bioavailability of 18%.
Compound 30 was a weak inhibitor of common isoforms of
cytochrome P450 (IC50 > 10 M vs CYP3A4, CYP2D6 and CYP2C9).
=
l
68% reversal of paw pressure analgesia (Cplasma = 0.75
brain = 0.17 M, t = 3 h). In the rat spinal nerve ligation (SNL) mod-
el of neuropathic pain, the compound afforded robust maximal 68%
reversal of paw pressure analgesia(Cplasma = 0.75 M, Cbrain = 0.17
M, t = 3 h). In the rat spinal nerve ligation (SNL) model of neuro-
pathic pain, the compound afforded robust maximal 68% reversal
lM,
C
l
l
From these collective results, compound 30 was identified for
additional characterization in the in vivo rat pain efficacy models.
This potent Cav 2.2 channel blocker was efficacious in various
preclinical pain models (Table 6). In the rat complete Freund’s
l
l
of allodynia at 30 mpk PO (Cplasma = 0.41 lM, Cbrain = 0.26 lM,
t = 1 h).12,13 Compound 30 affords excellent in vivo efficacy in all
the rodent pain models relatively at low plasma and brain
concentration.
50%PEG200_50%DMSO N=4
Compound 30 N=2
140
120
100
80
The ancillary pharmacology of 30 was also evaluated. In the rat
Rotarod model of motor coordination,14 compound 30 was studied
at doses of 3 and 10 mg/kg (IV) (Fig 1). Due to poor solubility,
compound 30 could not be evaluated at higher doses. Significant
impairment of motor coordination caused by compound 30 was ob-
served at higher plasma (8.0
no impairment of motor coordination was observed at 3 mg/kg IV
(plasma and brain exposure 1.9 and 1.4 M, respectively). Signifi-
cant efficacy (EC50) in the rat SNL model was obtained at a plasma
exposure of 0.44 M, which leads to a four fold window for efficacy
lM) and brain level (6.0 lM). However,
l
l
60
over the no effect level for ancillary CNS effects.
In order to study the cardiovascular properties of indole 30, the
compound was administered at rising doses of 1, 3, and 10 mg/kg
IV in barbiturate anesthetized and ventilated dogs (Fig 2 and Fig 3).
The compound afforded an exposure-dependent decrease in mean
1 mg/kg/30min
2 mg/kg/30min
7 mg/kg/30min
40
0
30
60
90
arterial pressure (MAP) above 2
l
M plasma concentration, reach-
Minutes After Treatment
ing maximal À40% reduction at maximal exposure (32
lM). A con-
Figure 2. Effect of compound 30 on MAP in anesthetized dogs.
comitant decrease in HR was also observed, to a maximal À32%
reduction at maximal exposure. These effects may be related to
L-type Cav1.2 calcium channel activity (IC50 = 908 nM).
In summary, a novel aryl indole 30, was discovered as a po-
tent, selective and state-dependent blocker of Cav2.2 channels
has been identified from a screening lead. Compound 30 displays
excellent in vitro block in rat Cav2.2 FLIPR assay and has robust
in vivo efficacy in the rat pain models. It has good pharmacoki-
netic profile in rat and dogs. Compound 30 is clean in CYP3A4
& PXR. In cardiovascular dog studies, compound 30 caused signif-
icant dose dependent reduction in blood pressure and heart rate
50%PEG200_50%DMSO N=4
Compound 30
140
120
100
80
starting at a relatively modest plasma concentration (2–3 lM).
The observed CV effects are attributed to blocking of Cav1.2chan-
nels (L-type). Lack of selectivity between Cav2.2 and Cav1.2 block
is an issue which is prevalent in this indole series and structural
modifications that improve calcium channel subtype selectivity
will be reported in due course.
60
1 mg/kg/30min
7 mg/kg/30min
2 mg/kg/30min
40
Acknowledgments
0
30
60
90
Minutes After Treatment
The authors thank Joseph Leone and Amanda Makarewicz for
their outstanding technical contribution to this work.
Figure 3. Effect of compound 30 on HR in anesthetized dogs.

S. Tyagarajan et al. / Bioorg. Med. Chem. Lett. 21 (2011) 869–873
873
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