Design and synthesis of novel cyanoguanidine ATP-sensitive potassium channel openers for the treatment of overactive bladder

Article abstract of DOI:10.1016/j.bmcl.2003.10.063

Thiourea derivatives were identified as glyburide-reversible potassium channel openers through high-throughput screening. Based on these findings, a number of novel cyanoguanidines were designed and synthesized, which hyperpolarized human bladder K_ATP channels. These agents are potent full agonists in relaxing electrically-stimulated pig bladder strips. The synthesis, SAR and biological properties of these agents are discussed.

Full text of DOI:10.1016/j.bmcl.2003.10.063

Bioorganic & Medicinal Chemistry Letters 14 (2004) 397–400
Design and synthesis of novel cyanoguanidine ATP-sensitive
potassium channel openers for the treatment of overactive bladder
Arturo Perez-Medrano,* Steven A. Buckner, Michael J. Coghlan, Robert J. Gregg,
Murali Gopalakrishnan, Michael E. Kort, John K. Lynch, Victoria E. Scott,
JamesP. Sullivan, Kristi L. Whiteaker and William A. Carroll
Abbott Laboratories, Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Park, IL 60064, USA
Received 29 August 2003; revised 16 October 2003; accepted 30 October 2003
Abstract—Thiourea derivatives were identified as glyburide-reversible potassium channel openers through high-throughput
screening. Based on these findings, a number of novel cyanoguanidines were designed and synthesized, which hyperpolarized
human bladder KATP channels. These agents are potent full agonists in relaxing electrically-stimulated pig bladder strips. The
synthesis, SAR and biological properties of these agents are discussed.
# 2003 Elsevier Ltd. All rights reserved.
Overactive bladder (OAB) isa prevalent urological dis-
order in humans that results in an involuntary loss of
urine. OAB has been hypothesized to arise primarily
from functional changes in detrusor smooth muscle
structure and function (myogenic etiology). It has been
suggested that supersensitivity to agonists, increase in
gap junctionsand enhanced electrical coupling between
smooth muscle cells could enable widespread dis-
However, this agent showed serious side effects on the
cardiovascular system. Clinically, no beneficial effects
were seen in the bladder at doses that were already
hypotensive.5 It isclear that new agentsare needed that
can be more selective for the bladder than for the car-
diovascular.
New KCOs6 like ZD-61697 2 and WAY1335378 3 have
recently appeared and reported to activate the bladder
KATP channelsin vitro and eslectively inhibit bladder
function in preclinical animal models.8,9
semination of depolarization signals leading to sponta-
1,2
neousnon-voiding contractio.ns
ATP-sensitive
potassium channels (KATPs), present in bladder smooth
muscle, play a critical role in controlling myogenic tone
and excitability. Compounds that selectively open these
channelshyperpolarize cell,s decreaes cellular (hyper)-
excitability and diminish smooth muscle activity result-
ing in suppression of unstable bladder contractions.3
Thus, KATP channel openers(KCOs) may be an attrac-
tive way of treating overactive bladder by inhibiting
overactivity during the filling phase without affecting
normal voiding contractions.
Clinical data, albeit limited, have provided support to
the concept of a role for KATP channelsfor the treat-
ment of overactive bladder.4 A pilot clinical trial with
the non-selective KCO, cromakalim 1, demonstrated an
improvement in the symptoms of bladder overactivity.4
Thiourea derivativeswere identified asglyburide-rever-
sible KCOs through high-throughput screening of our
library. Thiourea 4, an early analogue derived from a
HTS hit, showed potent activity in vitro as KATP channel
opener. Because of concerns about the potential toxicity
associated with the thiourea and the trichloromethyl
groups, chemistry efforts were first directed towards
* Corresponding author. Tel.:+1-847-937-9125; fax:+1-847-935-5466;
e-mail: arturo.perezmedrano@abbott.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.10.063

398
A. Perez-Medrano et al. / Bioorg. Med. Chem. Lett. 14 (2004) 397–400
finding suitable replacements for these two moieties. We
have synthesized a number of related aminals, which
hyperpolarize human bladder KATP channels. In this
study, the preparation, SAR and biological properties
of these agents will be discussed.
The aminal analogues were assayed for KATP activity in
cellsexipnrsges human bladder channels
K
ATP
(SUR2B17-/Kir6.2)18 (KATP-FMP). Membrane poten-
tial changeswere meausred with a Fluorometric Ima-
ging Plate Reader (FLIPR) using the fast membrane
potential (FMP) dye.19 These effects were reversed upon
addition of glyburide, a KATP channel inhibitor, con-
firming a KATP mechanism. The most potent com-
poundswere aols evaluated in vitro for functional
The synthesis of 4 issummarized in Scheme 1. Coupling
of aminal 510 with 3-pyridylisothiocyanate produced 4.
Trichloromethyl-aminal 6 wasprepared from 4, via
activation of the thiourea group with EDCI followed by
reaction with cyanamide in the presence of 2,6-lutidine
and titanium isopropoxide in methylene chloride.
Scheme 2 illustrates the synthesis of intermediates 8a–f
that contain replacementsfor the thiourea group. Thio-
methyl ethers 7a–c were prepared according to literature
procedures.11 Thiomethyl ethers 7d and 7e were
obtained from the treatment of 3-pyridylisothiocyanate
with the sodium salt of sulfamide or N,N-dimethylsulf-
amide followed by trapping of the sodium salt inter-
mediate with methyl iodide. Thiomethyls 7a–e were
transformed to 8a–e with ammonia in methanol.12 N-
Nitroguanidine 8f wasobtained by nitration of 3-pyr-
idylguanidine dihydrochloride.1
potassium channel opening activity using tissue strips
20
obtained from Landrace pig bladders(LPD).
The
SAR study reported herein is broken into three parts:
(1) Investigation of bioisosteres of the thiourea group;
(2) Replacement of the trichloromethyl; (3) Substitu-
tionsof the benzamide aromatic ring.
The SAR wasinitiated with the replacement of the tri-
chloromethyl group by tert-butyl and investigation of
bioisosteres of the thiourea group. The results are sum-
marized in Table 1. The t-Bu compound 11 showed
weaker activity than the parent analogue 4 suggesting
that the inductive effect wasimportant at the aminal
carbon for KATP activity. In general deviation from the
thiourea moiety resulted in a loss of activity in both cell
and tissue based assays. The only active isosteres were
cyanoguanidine and nitroguanidine. Nitroethylene and
sulfonylamides were practically inactive. Although the
cyanoguanidine 12a waosms ewhat weaker than
thiourea 11, it isworthwhile to note that the cyanogua-
nidine group can be viewed asa more acceptable group
than thiourea asa pharmaceutical entity. Therefore the
next step was to optimize the aminal R4 group having
the cyanoguanidine asthe preferred group. Table 1
illutrsatesattemptsin thisregard. Trichloromethyl
compound 6 was prepared for comparison and not sur-
prising was one of the most active compounds of this
series. However, it is interesting that this cyanoguani-
dine showed comparable activity with thiourea 4. Fur-
ther substitution of one of the chlorine atoms by a
methyl group resulted in dichloroethyl analogue 12g
and to a great extent KATP activity wasretained. Larger
groupssuch asdiethylalkyl or gem dimethylalkyl were
Preparation of 11 isoutlined in Scheme 3. Reaction of
4-chlorobenzamide with 2,2-dimethylpropionaldehyde
and benzotriazole afforded intermediate 9a14 (R4=t-Bu,
R2=Cl), which wastranfsormed to aminal
10 with
ammonia in methanol. Condensation of aminal 10 with
3-pyridylisothiocyanate furnished thiourea 11. Synthesis
of 12a–q was achieved by the two-step sequence depic-
ted in Scheme 3. Reaction of substituted amides with
the corresponding aldehyde15 and benzotriazole in the
presence of p-toluensulfonic acid13 produced benzo-
triazole intermediates 9a–m, which were coupled with
amines 8a–f to yield aminals 12a–q.16 Resolution of the
most potent compound 12g by preparative chiral HPLC
with a Chiralcel AS column provided the levorotatory
enantiomer 12g-(R)-(ꢀ)17 and the dextrorotatory enan-
tiomer 12g-(S)-(+). The absolute stereochemistry of the
enantiomer 12g-(S) wasdetermined by X-ray crytsal-
lography.
Scheme 1. Reagents and conditions: (a) 3-pyridylisothiocyanate, CH2Cl2, 60 ꢁC; (b) EDCI, CH2Cl2; (c) 2,6-lutidine, NH2CN, 3 A molecular sieves,
titanium isopropoxide, CH2Cl2, reflux.
Scheme 2. Reagents and conditions: (a) sulfamide, NaH, THF, MeI, 70%; (b) N,N-dimethylsulfamide, NaH, THF, MeI, 77%; (c) NH3, MeOH, 75–
85%; (d) HNO3, H2SO4, 66%.

A. Perez-Medrano et al. / Bioorg. Med. Chem. Lett. 14 (2004) 397–400
399
Scheme 3. Reagentsand conditions: (a) benzotriazole, R 2CHO, pTSOH, toluene, reflux; (b) NH3, MeOH, 74%; (c) 3-pyridylisothioyanate, CH2Cl2,
60 ꢁC, 33%; (d) 8a–f, K2CO3, DMF, 25–60%.
Table 1. In vitro activity of compounds 2, 4, 6, 11 and 12a–q
a
b
Compd
Z
R1
R2
R3
R4
KATP-FMP (pEC50
)
LPD (pEC50)
2
4
11
12a
12b
12c
12d
12e
12f
6
12g
ZD-6169
S
S
N–CN
6.4
6.1
5.6
5.2
5.6
5.7
5.1
4.7
5.1
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
CCl3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
CCl3
N–NO2
CH–NO2
N–SO2NH2
N–SO2N(CH3)
C(CN)2
N–CN
4.5
<4.0
<4.0
<4.0
Antagonist
6.0
5.7
5.6
5.3
4.7
N–CN
N–CN
N–CN
CCl2CH3
CCl2CH3
CCl2CH3
5.7
6.5
5.0
12g-(R)-(ꢀ)
12g-(S)-(+)
12h
12i
N–CN
N–CN
N–CN
N–CN
H
H
H
H
Cl
Cl
Cl
Cl
H
H
H
H
5.3
5.0
5.3
12j
4.7
12k
<4.0
12l
N–CN
N–CN
N–CN
N–CN
N–CN
N–CN
H
H
H
F
H
H
CH3
H
H
H
H
Br
H
Cl
F
F
CH3
H
CCl2CH3
CCl2CH3
CCl2CH3
CCl2CH3
CCl2CH3
CCl2CH3
5.6
5.5
5.4
5.4
5.3
5.0
12m
12n
12o
12p
12q
5.8
5.3
a Valuesare meansof two experiments.
b Valuesare meansof four experiments.
dramatically weaker, indicative of the importance of the
electronic effect of the halogen atomsover the tseric
effect. Examination of the enantiomersof 12g demon-
strated stereo differentiation in this series as the (R)-(ꢀ)-
enantiomer of 12g was15-fold more active than the ( S)-
(+)-enantiomer. Furthermore, this( R) enantiomer
showed in vitro KATP activity comparable to ZD-6169.
bon asthe preferred group. The preliminary SAR study
for the benzamide showed that substitution at the para
position seems to enhance KATP activity. Thistrend was
observed for the 4-chloro (12g) and 4-methyl (12l) ana-
loguesbut the data islimited and needsfurther
exploration.
In conclusion, a diverse series of novel cyanoguanidine
derivatives has been explored and several of which pos-
sess potent in vitro activity as KATP openers. These
Exploration of the substitution of the benzamide was
conducted with the 2,2-dichloroethyl at the aminal car-

400
A. Perez-Medrano et al. / Bioorg. Med. Chem. Lett. 14 (2004) 397–400
compounds are also potent full agonists in relaxing pig
bladder strips. Among these compounds, aminal 12g-
(R)-(ꢀ) wasaspotent asZD-6169 in human bladder
KATP channels. The steric and electronic requirements
for the aminal carbon and the applicability of the
cyanoguanidine group have been established for these
derivatives. Further SAR studies to determine the rele-
vance of the pyridine ring and in vivo studies as well as
in vivo selectivity comparison with literature KCO
standards are underway and will be presented in our full
paper.
Martin, S. W.; Radley, S. C.; Chess-Willams, R.; Kor-
stanje, C.; Chapple, C. R. Brit. J. Urol. 1997, 80, 405.
10. Weygand, F.; Steglich, W.; Lengyel, I.; Fraunberger, F.;
Maierhofer, A.; Oettmeier, W. Chem. Ber. 1966, 99, 1944.
11. (a)
Preparation
of
methyl
N0-cyano-N-(3-pyr-
idinyl)carbamimidothioate, 7a: Schou, C.; Ottosen, E. R.;
Petersen, H.; Bjoerkling, F.; Latini, S.; Hjarnaa, P. V.;
Bramn, E.; Binderup, L. Bioorg. Med. Chem. Lett. 1997,
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vinyl)-pyridin-3-yl-amine, 7b: Manley, P. W. Quast, U. J.
Med. Chem. 1992, 35, 2327. (c) 2-(3-Pyridinyl)-2-methyl-
mercapto-1-cyanoacrylnitrile, 7c, wasprepared according
to: Yokoyama, M.; Tohnishi, M.; Kurihara, A.; Ima-
moto, T. Chem. Lett. 1982, 1933.
12. Preparation of cyanoguanidine 8a: Peterson, H. J. J. Med.
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Acknowledgements
13. Dukat, M.; Abdel-Rahman, A. A.; Ismaiel, A. M.;
Ingher, S.; Teitler, M.; Gyermek, L.; Gleenon, R. J. Med.
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The authorswould like to thank Roger Henry for X-ray
crystal structure analysis.
14. Katritzky, A. R.; Drewniak, M. J. Chem. Soc., Perkin
Trans. 1 1988, 2339.
15. (a) 2,2-Dichloropropionaldehyde: De Buyck, L.; Verhe,
R.; De Kimpe, N.; Courtheyn, D.; Schamp, N. Bull. Soc.
Chim. Belg. 1980, 89, 441. (b) 3,3-Dimethyl-4-pentenal:
Buchi, G.; Vogel, D. E. J. Org. Chem. 1983, 48, 5406. (c)
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available 2,2-dimethylhexanol employing the Swern oxi-
dation: Swern, D.; Mancuso, A. J.; Huang, S. L. J. Org.
Chem. 1978, 43, 2480.
16. Z-Geometry was established for the double bond of
cyanoguanidines through X-ray crystal structure analysis
of 12g-(+). Although, derivatives 12b–e were obtained as
single isomers, the geometry of the double bond was not
determined.
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17. 12g waschromatographed over a Daicel Chiral Technol-
ogiesChiralcel AS chiral column (2.0 cm ꢂ25 cm) eluting
with 5% ethanol/hexanes(flow rate=10 mL/min) to pro-
vide
(R)-(ꢀ)-4-chloro-N-[2,2-dichloro-1-(N0-cyano-N’-
pyridin-3-ylguanidino)propyl]benzamide,
12g-(R)-(ꢀ);
[a]2D3 ꢀ22ꢁ (c 0.19, DMSO); mp 188–190 ꢁC; MS (ESI+)
m/z 425 (M+H)+; 1H NMR (300 MHz, DMSO-d6) d
ppm 9.93 (s, 1H), 8.75 (d, 1H, J=8.1 Hz), 8.52 (d, 1H,
J=2.0 Hz), 8.46 (d, 1H, J=4.4 Hz), 7.85 (d, 2H, J=8.5
Hz), 7.69 (m, 1H), 7.60 (d, 2H, J=8.5 Hz), 7.47 (dd, 1H,
J=8.0, 4.4 Hz), 7.16 (d, 1H, J=8.5 Hz), 6.55 (t, 1H,
J=8.5 Hz), 2.17 (s, 3H). Anal. calcd for C17H15Cl3N6O:
C, 47.96; H, 3.55; N, 19.74. Found: C, 48.14; H, 3.64; N,
19.47.
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