The discovery of a new class of large-conductance Ca2+-activated K+ channel opener targeted for overactive bladder: Synthesis and structure-activity relationships of 2-amino-4-azaindoles

Article abstract of DOI:10.1016/S0960-894X(03)00324-X

2-Amino-4-azaindoles have been identified as a structurally novel class of BK_Ca channel openers. Their synthesis from 2-chloro-3-nitropyridine is described together with their in vitro properties assessed by ⁸⁶Rb⁺ efflux and whole-cell patch-clamp assays using HEK293 cells stably transfected with the BK_Ca α subunit. In vitro functional characterization of BK_Ca channel opening activity was also assessed by measurement of relaxation of smooth muscle tissue strips obtained from Landrace pig bladders. The preliminary SAR data indicate the importance of steric bulk around the 2-amino substituent.

Full text of DOI:10.1016/S0960-894X(03)00324-X

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2003–2007
The Discovery of a New Class of Large-Conductance Ca2+-
activated K+ Channel Opener Targeted for Overactive Bladder:
Synthesis and Structure–Activity Relationships of
2-Amino-4-azaindoles
Sean C. Turner,* William A. Carroll, Tammie K. White, Murali Gopalakrishnan,
Michael J. Coghlan, Char-Chang Shieh, Xu-Feng Zhang, Ashutosh S. Parihar,
Steven A. Buckner, Ivan Milicic and James P. Sullivan
Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road,
Abbott Park, IL 60064, USA
Received 24 January 2003; revised 20 March 2003; accepted 21 March 2003
Abstract—2-Amino-4-azaindoles have been identified as a structurally novel class of BKCa channel openers. Their synthesis from
2-chloro-3-nitropyridine is described together with their in vitro properties assessed by 86Rb+ efflux and whole-cell patch-clamp
assays using HEK293 cells stably transfected with the BKCa a subunit. In vitro functional characterization of BKCa channel
opening activity was also assessed by measurement of relaxation of smooth muscle tissue strips obtained from Landrace pig blad-
ders. The preliminary SAR data indicate the importance of steric bulk around the 2-amino substituent.
# 2003 Elsevier Science Ltd. All rights reserved.
Overactive bladder (OAB) is characterized by symptoms
of increased urinary urgency, frequency, and/or incon-
tinent episodes.1 Until recently, urge urinary incon-
tinence (UUI) has been the most noticeable
manifestation of this condition although it represents
only a subset of the potential patient population. In the
US alone, estimates for patients who suffer from OAB
range from 13–17 million.2 Fewer than 50% of indivi-
duals consult their health care provider, and only about
20% of sufferers are on pharmacological therapy. Con-
sequently, OAB is thought to be underestimated and
insufficiently diagnosed due to lack of physician and
patient awareness, limited practical and effective treat-
ments, and patient embarrassment.
Detrusor instability may also have myogenic origins
that are idiopathic, or they may be caused by other
conditions including urethral obstruction. Treatments
employed to reduce bladder overactivity include oral
drug therapy, instillation drug therapy and para-
sympathetic nerve ablation.
Existing pharmacological therapies for OAB include
antimuscarinics that, although widely prescribed, are
limited by the high incidence of side effects such as dry
mouth.3 The ATP-sensitive potassium channel (KATP
)
openers cromakalim4 and ZD-61695 (see Fig. 1) have
been shown to exert potent relaxant activity on bladder
detrusor smooth muscle. However, the clinical utility of
KATP openers has been limited as a result of hemo-
dynamic side effects.
OAB is associated with the urodynamic finding of
involuntary bladder contractions, referred to as detru-
sor instability. Detrusor instability may be of neurologic
origin (e.g., Alzheimer’s disease, Parkinson’s disease or
stroke) which is referred to as bladder hyperreflexia.
Recently, there has been growing interest in the thera-
peutic potential of modulators of large conductance
calcium-activated potassium channels (BKCa).6 These
channels are regulated by both intracellular Ca2+ levels
and membrane potential, and are present in many exci-
table cell types localized in the CNS and smooth mus-
cle.7 The role of BKCa channels in several disease states
*Corresponding author. Tel.:+1-847-935-0404; fax:+1-847-935-5466;
e-mail: sean.turner@abbott.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00324-X

2004
S. C. Turner et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2003–2007
Figure 1.
has been investigated, including stroke, overactive
bladder and sexual dysfunction.8
treated with CO2 (g) to precipitate the pure 2-
hydroxy azaindole 11. A number of different chlor-
inating agents (SOCl2, PCl3 and PCl5) were examined
but the best conversion to the key 2-chloro inter-
mediate 12 was obtained with POCl3 at 105 ꢀC. Dis-
placement of the chloro moiety with primary and
secondary amines and anilines gave the 2-amino tar-
gets in moderate yields.
In vitro studies carried out with nonselective BKCa
openers such as NS-004 (3, Neurosearch) showed that
these compounds can relax bladder smooth muscle tis-
sues.9 Subsequently, NS-8 (4, Nippon Shinyaku), a
pyrrole BKCa opener, has been shown to be efficacious
in vivo in rat cystometry and in the rat hyper-reflexia
model where it abolished isovolumetric contractions
following intravenous and intravesicular administra-
tion.10 Recently, a series of disubstituted triazolones
have been reported with relaxant activity on isolated rat
bladder strips.11
Surprisingly, when unrecrystallized 8 was catalytically
reduced using the same conditions already described
(Pd-C/EtOH), this afforded the N-hydroxy derivative 13
through partial nitro reduction and subsequent cycli-
zation via addition to the nitrile. Cleavage of the N–O
bond using Ra–Ni, in the presence of 3% NaOH solu-
tion, gave the 2-amino 3-ester analogue 14. Both 13 and
14 were found to be inactive at BKCa channels.
Using NS-8 as a starting point, we synthesized several
different classes of fused pyrrole derivatives as potential
BKCa openers. The 2-amino-4-azaindoles 5 were found
to be active in 86Rb+ efflux, whole-cell patch-clamp
technique and pig bladder smooth muscle relaxation
assays.
Compounds were evaluated for BKCa channel opening
activity using a 86Rb+ efflux assay in which HEK293
cells stably transfected with BKCa a subunits were trea-
ted with the test compound (10 mM).13 The EC50 value
was determined for compounds that showed increases in
86Rb+ efflux>40% relative to the positive control
ionomycin (10 mM).
The synthesis of the 2-amino-4-azaindoles was
accomplished by the route depicted in Scheme 1. The
potassium enolate of ethyl cyanoacetate 6 was heated
with 2-chloro-3-nitropyridine 7 to give the 2-adduct 8
which was recrystallized from methanol.12 The 1H
NMR (CDCl3) showed this to be completely in the
enol tautomeric form 9 at room temperature. Cataly-
tic hydrogenation of the nitro group using Pd-C gave
the 3-amino product without any competitive reduc-
tion of the pyridyl ring. Thermal cyclization of 10 gave
a crude mixture that was dissolved in NaOH and then
Further electrophysiological characterization of active
compounds was performed using a whole-cell patch
clamp technique. The HEK293 cells stably transfected
with BKCa a subunits were voltage-clamped at a hold-
ing potential of À80 mV. The changes in ionic currents
in the presence of compounds were measured at the
testing potential of +40 mV for 400 ms.
Scheme 1. Reagents and reaction conditions: (i) KOtBu, i-PrOH, rt, 30 min then 82 ꢀC, 6 h (74%); (ii) Pd/C, H2, EtOH, 3 h, 50 psi (99%); (iii)
xylene, reflux, 20 h; (iv) NaOH then CO2 (g) (46% over 2 steps); (v) POCl3, 105 ꢀC, 2 h (45%); (vi) R1R2NH, 110 ꢀC, 18 h; (vii) Pd/C, H2, EtOH, 3 h,
50 psi (48%); (viii) Ra/Ni, KOH, water (45%).

S. C. Turner et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2003–2007
2005
Table 1. Effect of 2-substitution of 5 on 86Rb+ efflux in HEK293 cells stably transfected with BKCa a subunits
Compd
2-Substituent
86Rb+ Efflux BK-alpha (% response)
86Rb+ Efflux BK-alpha EC50 (mM)
11
12
15
16
17
18
19
20
OH
Cl
N(CH3)2
65
0
60
57
63
0
13.4
NA
5.13
6.94
6.75
NA
NA
NA
N(CH2CH3)2
N(CH3)(CH2CH2N(CH3))2
N((CH2)2CH3)CH2CH2OH
NHPh
0
0
NHCH2Ph
21
22
58
0
8.12
NA
23
24
25
0
0
0
NA
NA
NA
26
27
0
NA
65
3.02
28
65
5.07
29
30
31
32
33
34
35
36
28
55
61
0
15.1
17.0
3.34
NA
4.43
NA
5.86
74
0
62
62
3.86
(continued on next page)

2006
S. C. Turner et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2003–2007
Table 1 (continued)
Compd
2-Substituent
86Rb+ Efflux BK-alpha (% response)
0
86Rb+ Efflux BK-alpha EC50 (mM)
37
38
NA
0
NA
Values are the mean of two or three experiments—each performed in duplicate.
NA=Not active.
A selection of the more potent compounds was also
evaluated for in vitro functional activity using tissue
strips obtained from Landrace pig bladders.14 Tissues
were stimulated by a low-frequency (0.05 Hz) current
that produced a stable twitch response. The potent
KATP channel opener P107515 completely eliminated the
stimulated twitch response in a dose-dependant fashion.
The maximal efficacy of each compound was expressed
relative to P1075.
greater responses than that observed with the standard
BKCa channel opener NS-8.
The most potent compounds from 86Rb+ efflux studies
were also evaluated for their functional effects in iso-
lated pig bladder detrusor strips (Table 3). Four com-
pounds were effective in suppressing electrically
stimulated contractions, with compound 28 displaying
potency 7-fold greater than that of NS-8.
SAR studies were conducted by varying the R1/R2 sub-
stitution of 5 as outlined in Table 1. For acyclic alipha-
tic groups, smaller substituents were generally favored
(15>16>18). For cyclic aliphatics, the 6- and 7-mem-
bered rings were preferred over the 5-membered. The
bridged analogue 33 was potent but the fused bicycle 34
was inactive. Replacement of the piperidine group with
morpholine retained BKCa opening activity but the
N-methyl piperazine and homopiperazines were inac-
tive. All examples bearing an NH group at the 2-posi-
tion (for example the aniline and benzylamine
substitutions) were inactive.
In summary, we have identified 2-amino-4-azaindoles as
a novel structural class of BKCa channel openers. Their
BKCa channel activity has been established using in
vitro by evaluation in 86Rb efflux and whole-cell patch-
clamp assays. Their potential to act as relaxants of
bladder smooth muscle has been demonstrated.
References and Notes
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Compd
Outward current in the presence of
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NS-8
15
28
21.7Æ7.0%
75.5Æ8.6%
107.8Æ24.5%
104.8Æ11.4%
31
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Compd
Efficacy (% P1075)
EC50 (mM)
(À) Cromakalim
95
93
58
67
31
63
0.257
63.1
23.5
24.8
9.26
25.9
NS-8
15
27
28
31
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