Amidine derived inhibitors of acid-sensing ion channel-3 (ASIC3)

Article abstract of DOI:10.1016/j.bmcl.2009.06.021

A series of indole amidines modified at the 2-position of the indole ring were evaluated as inhibitors of Acid-Sensing Ion Channel-3 (ASIC3), a novel target for the treatment of chronic pain.

Full text of DOI:10.1016/j.bmcl.2009.06.021

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4059–4063
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Amidine derived inhibitors of acid-sensing ion channel-3 (ASIC3)
a
a
a
a
Scott D. Kuduk a, , Ronald K. Chang , Jenny M.-C. Wai , Christina N. Di Marco , Victoria Cofre ,
Robert M. DiPardo a, Sean P. Cook b, Matthew J. Cato b, Aneta Jovanovska b, Mark O. Urban b, Michael Leitl b,
Robert H. Spencer b, Stefanie A. Kane b, George D. Hartman a, Mark T. Bilodeau a
*
a Department of Medicinal Chemistry, Sumneytown Pike, PO Box 4, West Point, PA 19486, USA
b Department Pain Research Merck Research Laboratories, Sumneytown Pike, PO Box 4, West Point, PA 19486, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of indole amidines modified at the 2-position of the indole ring were evaluated as inhibitors of
Acid-Sensing Ion Channel-3 (ASIC3), a novel target for the treatment of chronic pain.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 13 May 2009
Revised 1 June 2009
Accepted 3 June 2009
Available online 13 June 2009
Keywords:
ASIC3
Pain
Ion channel
Amidine
Despite the presence of a multitude of analgesic medications on
the market, chronic pain is a condition that continues to afflict a
significant portion of the population. Chronic pain can result from
different pathways including inflammation associated with osteo-
arthritis and direct damage to the nervous system in the form of
neuropathic pain. Because of this, it is imperative that the search
for new analgesics to treat chronic pain continues.1
The neurons in the central nervous system contain a number of
ion channels and receptors that are sensitive to acidosis.2 Pro-
longed illness or trauma often leads to a drop in pH, which activate
these signaling pathways.3 It is widely accepted that one of these
classes of ion channels is the vanilloid receptor subtype-1 (VR1),
which is sensitive to a variety of stimuli,3 and is activated under
tissue acidification to pH of less than 6.0.4 Consequently, a number
of VR1 antagonists are currently being examined in clinic trials for
the treatment of pain.5,6
A second class of pH sensitive ion channels is the acid-sensing
ion channels (ASICs), which are members of the proton-gated epi-
thelial Na+/degenerin superfamily.7 There are currently seven
known ASICs subunits (ASIC1a/b, ASIC1b2, ASIC2a/b, ASIC3, and
ASIC4) that are susceptible to activation by very modest changes
in pH. In some cases, the ASICs can even detect subtle decreases
in extracellular pH from 7.4 to 7.0.8 Studies have demonstrated
that increased ASIC activity and expression can result from a num-
ber of inflammatory mediators.9 Thus, similar to the VR1 class of
ion channels, the ASICs are also believed to act as messengers of
pain resulting from acidosis.10,11 The ASIC3 channel, in particular,
is highly expressed in nociceptors12,13 and in vivo studies in ASIC3
knock-out mice14 have suggested that the ASIC3 channel may be a
viable target for the blocking of chronic inflammatory pain.
Few small molecule inhibitors of ASIC channels have been re-
ported, hindering the further understanding of the physiological
role of ASIC3. The potassium sparing diuretic agent amiloride 1
has been shown to be a non-selective inhibitor of ASIC channels15
and exhibits a modest effect in rat pain models at very high plasma
concentrations.16 We recently reported an SAR study for a series of
modified amiloride derivatives that exhibited enhanced activity for
ASIC3, but suffered from poor pharmacokinetic properties.17
group from Abbott has previously shown that amidine A-317567
A
2
is a much more potent blocker of ASIC3 than amiloride
(Fig. 1).18 In addition, A-317567 showed full efficacy in the rat
Complete Freud’s Adjuvant (CFA)-induced inflammatory thermal
H2N
Cl
N
N
NH2
N
N
NH2
NH2
NH2
NH
O
1: Amiloride
2: A-317567
Figure 1. Known ASIC channel blockers.
* Corresponding author.
E-mail address: scott_d_kuduk@merck.com (S.D. Kuduk).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.021

4060
S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4059–4063
H2N
HN
Boc
Boc
N
HO
HO
N
a
NH2
NH
N
I
B
H
CN
CN
16
4b
3: MW = 277
ASIC3 Inhibition: 33% @ 1 µM
b
Figure 2. Lead compound 3.
H
H
N
N
c
R1
I
NH2
NH2
NH
hyperalgesia model and a skin incision model of post-operative
pain.
NH
Identifying new small molecule leads as inhibitors of ASIC3 has
been impaired by the lack of an effective high-throughput assay to
screen for hits. All biochemical evaluation of ASIC3 inhibition has
been carried out using labor intensive electrophysiology studies,
thus precluding a large HTS campaign as a means of identifying
lead structures. Upon further analysis of compound 2, we proposed
the amidine moiety as a critical requisite for ASIC3 activity.
Accordingly, the MRL sample repository was mined for aryl ami-
dines that were subsequently evaluated in the ASIC3 electrophys-
iology assay. Indole bis-amidine 3 was identified and proved to be
a relatively potent, low MW lead compared to compound 2, and
this communication will describe the preliminary SAR for this
compound class (Fig. 2).
The synthesis of 3 and related analogs is shown in Schemes 1
and 2. Commercially available indole boronic acids 4a and 4b
served as the starting points. Suzuki cross-coupling of 4a, b with
4-cyanophenyl iodide affords 5a, b. These may be directly con-
verted to the corresponding bis amidines 3 and 7 via generation
of the O-ethyl imidates under acidic conditions followed by reac-
tion with ammonia. The mono amidine products 6 and 10 can be
accessed via the same route by using iodobenzene in the Suzuki
step with subsequent amidine formation. The Boc group in indole
10b-n
17
Scheme 2. Reagents and conditions: (a) NIS, CH3CN. (b) (i) HCl, EtOH, 80 °C; (ii)
NH3–EtOH, rt to 45 °C. (c) Pd(dppf)Cl2, K2CO3, DMF, R1B(OH)2, 80 °C.
5b can be removed and then undergo a base mediated methylation
to provide 8, which can then proceed to bis amidine 9. Finally, the
benzimidazole bis-amidine 13 is prepared via condensation of dia-
mine 11 with acid chloride 12 followed by amidine formation un-
der standard conditions.
In order to facilitate a higher throughput synthesis of substi-
tuted analogs of 10, an alternative approach was taken as shown
in Scheme 2. Iodide 16 was sought as a useful intermediate, but at-
tempts to prepare it via direct metallation of the 2-position of tert-
butyl-5-cyano-indole-1-carboxylate followed by quenching with
an iodide source were largely unsuccessful. However, conversion
of indole boronic acid 4b directly to iodide 16 could be effected
using N-iodosuccinimide (NIS) in CH3CN in near quantitative
yield.19 Amidine formation with concomitant removal of the Boc
group afforded iodide 17. Subsequent Suzuki couplings with the
appropriate boronic acids afforded analogs 10b–n in a single step.
H2N
HO
B
HO
b
a
NC
NH2
N
HN
N
N
CN
H
CN
Boc
NH
Boc
4a
c, b
5a
3
Me
Me
N
H2N
HN
N
b
b
NC
NH2
NH
N
NH2
NH
CN
H
8
9
6
d, e
Boc
H
N
Boc
H2N
N
HO
HO
N
a
NC
B
NH2
NH
HN
CN
CN
4b
c, b
5b
7
H
H
N
N
H2N
HN
NC
NH2
NH2
ClOC
f, b
+
NH2
NH
NH2
NH
N
CN
10
11
12
13
Scheme 1. Reagents and conditions: (a) Pd(dppf)Cl2, K2CO3, DMF, 4-iodobenzonitrile, 80 °C; (b) (i) HCl, EtOH, 80 °C; (ii) NH3–EtOH, rt to 45 °C; (c) Pd(dppf)Cl2, K2CO3, DMF,
iodobenzene, 80 °C; (d) HCl(g), EtOAc, 0 °C; (e) NaHMDS, MeI, DMF; (f) BF3–OEt2, dioxane, 0–120 °C.

S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4059–4063
4061
H
H2N
HN
N
NH2
N
H
Me
H2N
HN
N
H2N
HN
NH
13: ASIC3 EP = 53% @ 10 µM
N
NH2
NH
NH2
NH
7: ASIC3 EP = 39% @ 1 µM
9: ASIC3 EP = 14% @ 1 µM
H2N
HN
NH2
NH
N
H
3: ASIC3 EP = 83% @ 10 µM
EP = 33% @ 1 µM
H
N
NH2
NH
NH2
H
N
H2N
HN
N
H
NH
10: ASIC3 EP = 82% @ 10 µM
(IC50 = 2.7 µM)
6: ASIC3 EP = 18% @ 1 µM
15: ASIC3 EP = 46% @ 10 µM
Figure 3. SAR of amidines.
The initial SAR exploration for select amidine structures is
shown in Figure 3. For this initial analysis, compounds were
tified with an ASIC3 IC50 = 2.7 lM. As a result, all further SAR inves-
tigation was carried out using 10 as the core scaffold for
exploration. It should also be noted that the primary amidine
was a requirement. Attempts to add substituents onto the amidine
moiety present in compound 10 via alkylation, acylation, and
hydroxylation all had deleterious effects on ASIC3 inhibition (data
not shown).
A library approach was employed using the chemistry described
in Scheme 2 to investigate SAR of the aryl group at the 2-position of
the indole. It was quickly found that a phenyl ring was required
(data not shown) with a strong preference for 3,5-substituted aryl
rings. The SAR analysis with this substitution pattern is shown in
Table 1. Compounds were screened initially in the electrophysiol-
screened in the electrophysiology assay at 10 and/or 1 lM concen-
trations.20 The indole isomer of lead 3 that has the amidine at the
6-position of the indole ring, bis-amidine 7, gave slightly greater
channel block at 1 lM. The N-methyl analog 9 and benzimidazole
13 were both markedly less potent compared to 7. The evaluation
of having a single amidine group present was also examined. Re-
moval of the amidine on the indole (15) significantly reduced
channel block inhibition relative to the amidine on the phenyl ring
(6 and 10). In these latter cases, having the amidine at the 6-posi-
tion of the indole (10) gave the compound with better potency,
consistent with the preference observed with bis amidine 7. In fact,
10 was the most potent mono-amidine ASIC channel blocker iden-
ogy assay at 10 and/or 1 lM, followed by a five point titration.
Table 1
Inhibition of ASIC3 by select amidine derivatives 10a–k
H
N
R
NH2
NH
10a-n
Compd
R
% Inh. @ 1
nd
lM
% Inh. @ 10
88
lM
IC50 (nM)a
2765
a
Cl
Cl
b
92
98
133
(continued on next page)

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S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4059–4063
Table 1 (continued)
Compd
R
% Inh. @ 1
l
M
% Inh. @ 10
lM
IC50 (nM)a
Cl
c
d
e
f
91
nd
135
Cl
Cl
Cl
Cl
83
84
82
93
92
77
78
83
91
87
94
nd
nd
99
96
92
98
nd
nd
nd
nd
nd
272
291
278
59
NC
Cl
N
Cl
Cl
F
Br
g
h
i
Cl
Br
59
Br
MeO
374
95
F
H2N
j
Cl
PhHNH2C
k
114
77
Cl
BnHN
l
Cl
Me
m
51
Cl
Ph
n
nd
Cl
a
Electrophysiology recording, inhibition of current was expressed as percent inhibition of peak current vs. baseline peak current. N = 3.

S. D. Kuduk et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4059–4063
4063
450
400
350
300
250
200
150
100
50
100
vehicle
20 mg/kg naproxen
30 mg/kg 10b
75
50
25
0
*
*
0
BL
CFA
30 min
Figure 4. Effect of 10b in the rat CFA inflammatory pain model (left); data represented as % reversal of mechanical hypersensitivity (right); p <0.05 vs vehicle.
Highlighting the preference for meta-substitution, the 3,5-di-
chloro analog 10b was ꢀ20-fold more potent (ASIC3 IC50 = 133 nM)
than the phenyl comparator 10a. Placement of additional groups
on 10b at the ortho (10c) or para-positions (10d) had no effect or
led to a decrease in channel inhibition. Similarly, insertion of a pyr-
idine (10e) led to a modest twofold drop in activity. The lipophilic-
ity of the substituents also appears to have effects on activity. For
example, exchanging one of the chlorines with a fluorine (10f), led
to a decrease in activity, while replacement with a bromine (10g)
led to one of the most potent compounds observed (IC50 = 59 nM).
Addition of a second bromine (10h) did not provide any further
improvements. Interestingly, as shown with compounds 10i–n,
the meta position is remarkably tolerant of substitution so long
as the other meta group has a halogen (ideally chlorine) in this
position.
In order to more fully characterize this indole amidine class of
ASIC3 channel blockers, 3,5-dichloro analog 10b was examined
in vivo in the rat Complete Freud’s Adjuvant (CFA) model of inflam-
matory pain.21 In this study, 10b showed a robust reversal of
mechanical hypersensitivity 30 min post-dosing in male Spra-
gue–Dawley rats at 30 mg/kg ip that was comparable to the NSAID
naproxen (dosed at 20 mg/kg po). Interestingly, when plasma22
and brain were analyzed for compound levels of 10b at 30 min,
all results were below the level of quantification (Fig. 4).
any oral bioavailability in rat and also exhibits high partitioning
into red blood cells. Accordingly, efforts have been initiated to re-
place the amidine moiety to improve upon these latter two issues,
and results will be reported in due course.
References and notes
1. Childers, W. E.; Gilbert, A. M.; Kennedy, J. D.; Whiteside, G. T. Expert Opin. Ther.
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Biochem. Biophys. Res. Comm. 2001, 281, 1183.
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6. Kyle, D. J.; Tafesse, L. Expert Opin. Ther. Patents 2006, 16, 977.
7. Wemmie, J. A.; Price, M. P.; Welsh, M. J. Trends Neurosci. 2006, 29, 578.
8. Waldmann, R. Adv. Exp. Med. Biol. 2001, 502, 293.
9. Mamet, J.; Baron, A.; Lazdunski, M.; Voilley, N. J. Neurosci. 2002, 22, 10662.
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13. Yiangou, Y. Eur. J. Gastroenerol. Hepatol. 2001, 13, 891.
14. Sluka, K. A.; Price, M. P.; Breese, N. M.; Stucky, C. L.; Wemmie, J. A.; Welsh, M. J.
Pain 2003, 106, 229.
15. Ugawa, S.; Ueda, T.; Ishida, Y.; Nishigaki, M.; Shibata, Y.; Shimada, S. J. Clin.
Invest. 2002, 110, 185.
16. Ferreira, J.; Santos, A. R. S.; Calixto, J. B. Life Sci. 1999, 65, 1059.
17. Kuduk, S. D.; Di Marco, C. N.; Chang, R. K.; DiPardo, R. M.; Cook, S. P.; Cato, M. J.;
Jovanaskova, A.; Urban, M. O.; Leitl, M.; Spencer, R. H.; Kane, S. A.; Bilodeau, M.
T.; Hartman, G. D.; Bock, M. G. Bioorg. Med. Chem. Lett. 2009, 19, 2514.
18. Dube, G. R.; Lehto, S. G.; Breese, N. M.; Baker, S. J.; Wang, X.; Matulenko, M. A.;
Honore, P.; Stewart, A. O.; Moreland, R. B.; Brioni, J. D. Pain 2005, 117, 88.
19. Thiebes, C.; Prakash, G. K. S.; Petasis, N. A.; Olah, G. A. Synlett 1998, 141.
20. Acid-evoked (pH5.5 for 3 s) current was recorded at À60 mV using an
automated patch clamp instrument (PatchXpress, MDS, Inc.). The
intracellular solution contained (mM): 119 K-gluconate, 15 KCl, 3.2 MgCl2, 5
EGTA, 5 HEPES, 5 K2ATP, pH 7.3. The extracellular solution contained (mM):
150 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 12 Dextrose, and 10 HEPES (pH 7.4) or 10
MES (pH5.5). Compounds were applied 120 s prior to acid application. Peak
current following compound incubation was expressed as a fraction of the
control (vehicle) peak current. IC50 values were determined by fitting data to
the Hill equation.
To follow up on this result, a rat pharmacokinetic study was car-
ried out. Compound 10b was not orally bioavailable, which was not
necessarily unexpected due to the presence of the amidine moi-
ety.23 The iv plasma pharmacokinetics showed very high cleare-
ance (Cl = 52 ml/min/kg) with
a short half-life (t1/2 = 2.0 h).
However, analysis of the blood samples showed lower clearance
(Cl = 8.5 ml/min/kg) with a more moderate half-life (t1/2 = 4.4 h)
indicating that 10b was partitioning into red blood cells.
In summary, screening a sub-set of the MRL sample repository
for aryl amidines led to the discovery of indole bis-amidine 3 as
a low MW, modest ASIC channel blocker. SAR analysis quickly
21. Stein, C.; Millan, M. J.; Herz, A. Pharm. Biochem. Behav. 1988, 31, 445.
22. Compound 10b did not show any binding to rat plasma proteins.
23. Kamanka, T.; Park, Y.-J.; Lin, L. S.; de Laszlo, S.; McCauley, E. D.; Van Riper, G.;
Egger, L.; Kidambi, U.; Mumford, R. A.; Tong, S.; Tang, W.; Colletti, A.; Teffera,
Y.; Stearns, R.; MacCoss, M.; Schmidt, J. A.; Hagmann, W. K. Bioorg. Med. Chem.
Lett. 2004, 14, 2323.
led to the identification of mono-amidine 10b as
a potent
(IC50 = 133 nM) ASIC3 channel blocker that exhibited efficacy sim-
ilar to that of naproxen in the rat CFA model of mechanical hyper-
algesia. This indole amidine class of compounds does not possess