Design and SAR of novel potassium channel openers targeted for urge urinary incontinence. 1. N-cyanoguanidine bioisosteres possessing in vive bladder selectivity

Article abstract of DOI:10.1021/jm9905099

A structurally novel series of adenosine 5'-triphosphate-sensitive potassium (K(ATP)) channel openers is described. As part of our efforts directed toward identifying novel, bladder-selective potassium channel openers (KCOs) targeted for urge urinary incontinence (UUI), we found that bioisosteric replacement of the N-cyanoguanidine moiety of pinacidil (1, Figure 1) with a diaminocyclobutenedione template afforded squaric acid analogue 2, the prototype of a novel series of K(ATP) channel openers with unique selectivity for bladder smooth muscle in vive. Further modification of the heterocyclic ring to give substituted aryl derivatives (3) afforded potent KCOs that possessed the desired detrusor selectivity when administered orally. The effects of these potassium channel agonists on bladder contractile function was studied in vitro using isolated rat detrusor strips. Potent relaxants were evaluated in vive in a rat model of bladder instability. Lead compounds were evaluated concomitantly in normotensive rats for their effects on mean arterial blood pressure (MAP) and heart rate as a measure of in vive bladder selectivity. (R)-4-[3,4-Dioxo-2-(1,2,2-trimethyl- propylamino)-cyclobut-1-enyαmino]-3-ethyl-benzonitrile (79) met our potency and selectivity criteria and represents an attractive development candidate for the treatment of UUI. Electrophysiological studies using isolated rat bladder detrusor myocytes have demonstrated that compound 79 produces significant hyperpolarization which is glyburide-reversed, thus consistent with the activation of K(ATP). The design, synthesis, structure-activity relationships (SAR), and pharmacological activity associated with this series of novel KCOs will be discussed.

Full text of DOI:10.1021/jm9905099

J . Med. Chem. 2000, 43, 1187-1202
1187
Design a n d SAR of Novel P ota ssiu m Ch a n n el Op en er s Ta r geted for Ur ge
Ur in a r y In con tin en ce. 1. N-Cya n ogu a n id in e Bioisoster es P ossessin g in Vivo
Bla d d er Selectivity
J ohn A. Butera,*,† Madelene M. Antane,† Schuyler A. Antane,† Thomas M. Argentieri,‡ Chris Freeden,#
Russell F. Graceffa,† Bradford H. Hirth,† Douglas J enkins,† J oseph R. Lennox,† Edward Matelan,†
N. Wesley Norton,‡ Dominick Quagliato,† J effrey H. Sheldon,‡ Walter Spinelli,# Dawn Warga,‡
Alexandra Wojdan,# and Morgan Woods‡
Chemical Sciences, Cardiac Diseases, Urologic Diseases, Woman’s Health Discovery Research, Wyeth-Ayerst Research,
CN 8000, Princeton, New J ersey 08543-8000
Received October 8, 1999
A structurally novel series of adenosine 5′-triphosphate-sensitive potassium (KATP) channel
openers is described. As part of our efforts directed toward identifying novel, bladder-selective
potassium channel openers (KCOs) targeted for urge urinary incontinence (UUI), we found
that bioisosteric replacement of the N-cyanoguanidine moiety of pinacidil (1, Figure 1) with a
diaminocyclobutenedione template afforded squaric acid analogue 2, the prototype of a novel
series of KATP channel openers with unique selectivity for bladder smooth muscle in vivo. Further
modification of the heterocyclic ring to give substituted aryl derivatives (3) afforded potent
KCOs that possessed the desired detrusor selectivity when administered orally. The effects of
these potassium channel agonists on bladder contractile function was studied in vitro using
isolated rat detrusor strips. Potent relaxants were evaluated in vivo in a rat model of bladder
instability. Lead compounds were evaluated concomitantly in normotensive rats for their effects
on mean arterial blood pressure (MAP) and heart rate as a measure of in vivo bladder selectivity.
(R)-4-[3,4-Dioxo-2-(1,2,2-trimethyl-propylamino)-cyclobut-1-enylamino]-3-ethyl-benzonitrile (79)
met our potency and selectivity criteria and represents an attractive development candidate
for the treatment of UUI. Electrophysiological studies using isolated rat bladder detrusor
myocytes have demonstrated that compound 79 produces significant hyperpolarization which
is glyburide-reversed, thus consistent with the activation of KATP. The design, synthesis,
structure-activity relationships (SAR), and pharmacological activity associated with this series
of novel KCOs will be discussed.
In tr od u ction
persistent instability causes chronic sensory urgency
and involuntary urine loss. The etiology of urge UI is
multivariant and can be associated with spinal chord
injury, bladder hypertrophy secondary to outlet obstruc-
tion, or urinary tract infections. Antimuscarinic agents
are widely prescribed for the treatment of urge UI
although their usefulness is limited by antimuscarinic
side effects resulting in low patient compliance (<30%).
Stress UI is characterized by involuntary urine loss
during physical exertion and occurs when intravesical
pressure exceeds the maximum urethral pressure in the
absence of detrusor overactivity. Sympathomimetic
agents are often prescribed for the treatment of stress
UI because the bladder sphincter is mostly under
sympathetic control. Postmenopausal women may be
successfully treated with hormone replacement therapy
because bladder and urethral tone is largely under
estrogenic control. Overflow incontinence is defined as
involuntary urine loss associated with overdistension
of the bladder. Other than agents used to treat the
symptoms of urge or stress UI, few treatments are
available specifically for this type of incontinence.
Urinary incontinence (UI), officially defined as invol-
untary loss of urine, can become a devastating disorder
to the patient due to its significant social and hygienic
impact and negative effect on quality of life.1 UI often
proceeds undiagnosed and untreated in up to 50% of
patients due to embarrassment or their unwillingness
to discuss the symptoms with their primary care physi-
cian. UI affects over 13 million people in the United
States alone and about 10-20% of the world popula-
tion.2 In the United States, this amounts to over $16
billion in health care costs.
The pharmacology of incontinence and current treat-
ment strategies have been thoroughly reviewed.1,3-10
Briefly, UI can be categorized into three major types:
urge incontinence (bladder instability), stress inconti-
nence, and overflow incontinence. Urge UI, also known
as hyperreflexive bladder, is characterized by abnormal
spontaneous detrusor contractions which are unrelated
to bladder urine volume and accounts for 35-65% of
all UI cases depending on the age of the patient.2,11 The
Potassium channels play a crucial role in controlling
physiological function of excitable cells; thus modulation
of these channels provides novel mechanistic approaches
to treat cell dysfunction. The structure and function of
* To whom correspondence should be addressed. Tel: 732-274-4289.
Fax: 732-274-4129. E-mail: buterj@war.wyeth.com.
† Chemical Sciences.
#
Cardiac Diseases.
‡ Urologic Diseases.
10.1021/jm9905099 CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/23/2000

1188 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Butera et al.
F igu r e 1. Bioisosteric replacement of the pinacidil (1) N-cyanoguanidine moiety with diaminocyclobutenedione affords a novel
series of bladder selective KCOs (3).
Sch em e 1. Synthesis of
N-Aryl-1,2-diaminocyclobutenedionesa
F igu r e 2. Bladder-selective KCOs reported by AstraZeneca.
physiologically important potassium channels and rel-
evant SAR have been thoroughly reviewed.12-25 By
virtue of their ability to hyperpolarize cell membranes
a
(a) THF or Et2O, 3,4-diethoxy-3-cyclobutene-1,2-dione, reflux;
and prevent the influx of Ca2+ ions through voltage-
(b) NaH, DMF, 3,4-diethoxy-3-cyclobutene-1,2-dione, 80 °C; (c)
gated Ca2+ channels, KCOs relax smooth muscle cells.
Several potent KATP channel openers such as pinacidil
(Chart 1) and cromakalim have been studied clinically
as antihypertensive agents. It has been shown that the
ATP-dependent potassium channel does exist in the
bladder and that it can be activated by numerous
antihypertensive KCOs.26-29 The results of a small
clinical study with cromakalim suggest that KCOs may
show promise for the treatment of detrusor instability
and hyperreflexia although full utility will only be
realized with compounds that have minimal hemody-
namic side effects (hypotension and tachycardia).30,31
Workers at Zeneca Pharmaceuticals have recently de-
scribed a structurally novel series of anilide tertiary
carbinol KCOs (4)32,33 and a series of dihydropyridine
KCOs (5)34 which are claimed to be bladder-selective
smooth muscle relaxants in vivo. When administered
orally, ZD6169 (4) (Figure 2) has been shown to activate
the bladder KATP channel and to selectively increase
bladder compliance in the rat and dog without signifi-
cant hemodynamic effects.35,36 This compound is cur-
rently undergoing Phase II clinical trials for the treat-
ment of urge UI.
In this article, we report the results of our investiga-
tion of a novel series of diaminocyclobutenedione de-
rivatives that were demonstrated to be potent and
bladder-selective agonists of the KATP channel. A pre-
liminary account of these efforts has been presented.37
Our strategy was to utilize the 1,2-diaminocyclobutene-
3,4-dione template as a putative bioisostere for the
N-cyanoguanidine moiety seen in the pinacidil class of
antihypertensive KCOs to generate a structurally novel
series of KCOs. The compounds were evaluated for their
ability to relax KCl precontracted isolated rat bladder
smooth muscle strips in vitro. Electrophysiological stud-
ies on isolated bladder myocytes demonstrated that
relaxation within this class of agents was mediated
through activation of the ATP-sensitive potassium
channel. In vivo performance of the active compounds
was assessed in a rodent model of unstable bladder.
Upon oral administration, lead compounds produced
significant bladder relaxant effects at doses that caused
minimal effects on hemodynamics, thus demonstrating
R3NH2, EtOH, reflux.
a clear distinction between these selective agents and
the antihypertensive KCOs.
Ch em istr y
A series of 1,2-diaminocyclobutene-3,4-diones was
generated using the synthetic route shown in Scheme
1. Treatment of 3,4-diethoxy-3-cyclobutene-1,2-dione
with 1-1.3 equiv of the requisite substituted aniline or
heteroarylamine in refluxing THF, ethanol, or diethyl
ether (method A) afforded the intermediate amido-esters
7 in modest to excellent yields. For some deactivated
anilines possessing bulky or electron-withdrawing groups,
it was necessary to carry out the reaction in the presence
of sodium hydride in DMF or THF to preform the more
reactive sodium salt of the aniline (method B).
The di-n-butoxy and diisopropoxy analogues of 3,4-
diethoxy-3-cyclobutene-1,2-dione could also be used as
starting material. The amido-esters 7 were converted
to 1,2-diaminocyclobutene-3,4-diones 8 by treatment
with 1-5 equiv of the desired alkylamine in ethanol.
Recrystallization from ethanol or acetonitrile typically
afforded analytically pure compounds. Optically active
(R)- and (S)-2-amino-3,3-dimethylbutane were prepared
by the method of Manley and Quast from pinacolone
using R-methylbenzylamine as a chiral auxiliary.38
Chiral purities of enantiomers 42 and 43 were deter-
mined to be 99% ee and 97% ee, respectively (see
Experimental Section). As it is unlikely for epimeriza-
tion of the chiral center to occur during the displacement
reactions, any derivative possessing the (R)-2-amino-
3,3-dimethylbutane moiety is assumed to be ≈99% ee.
Optically active (R) and (S) starting materials for the
syntheses of compounds 60, 61, 63, 64, 67-69 were
obtained commercially. Percent yields and physical
properties for test compounds 2 and 9-87 are shown
in Tables 1-3 and 5-6.
P h a r m a cology
Compounds were first evaluated in vitro for their
ability to relax KCl precontracted rat detrusor muscle
strips. Following stabilization, increasing concentrations

Potassium Channel Openers Targeted for UUI. 1
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1189
Ta ble 1. In Vitro Effects of Pyridyl Analogues on Precontracted Rat Bladder Smooth Muscle Strips (IC50, µM)a
a
IC50: drug concentration (µM) that relaxed KCl-induced contractions in rat detrusor strips by 50%. b Structures of compounds confirmed
1
d
by H NMR, IR, and MS. c Analytical results are within(0.4% of the theoretical value unless otherwise noted. Number of experiments.
e See compound 2. f See compound 10. C: calcd, 61.08; found, 60.37. C: calcd, 62.27; found, 61.40. C: calcd, 62.32; found, 62.86.
g
h
i
of test compounds were superfused and isometric force
was measured. Glyburide, a specific blocker of the KATP
channel, was added at the end of each experiment and
percent recovery of contractile activity was recorded. As
a measure of intrinsic selectivity, some compounds were
screened in similar tissue baths using rat aortic rings.
Selected compounds were evaluated for in vivo ef-
ficacy in a rat pathophysiological model of bladder
instability previously described by Malmgren and co-
workers.39,40 Compounds that were efficacious in reduc-
ing the frequency of spontaneous bladder contractions
in vivo were selected for hemodynamic assessment in a
separate group of animals. Effects on mean arterial
pressure and heart rate were recorded after oral ad-
ministration of drug.
R-configuration of the 3,3-dimethyl-2-butyl alkyl moiety
retains the activity of the racemate in both the 4- (9 vs
2) and 3-pyridyl (11 vs 10) series. It has been shown in
a series of pinacidil-type KCOs that when the alkyl-
amine moiety contains an R-methyl group in addition
to a C(CH3) group, only the R-configuration can be
readily accommodated at the receptor.38
Due to the similar potencies of 4- and 3-pyridyl
analogues 9 and 11 and to the consistently higher yields
of the arylamine condensation reaction in the 3-pyridyl
series over the 4-pyridyl series, the SAR of the alkyl-
amine moiety (R2) and pyridyl ring substituents were
explored within the 3-pyridyl series. Exchange of a tert-
butyl group for the 3,3-dimethyl-2-butyl moiety results
in slightly lower activity (13 vs 10), while reducing the
steric bulk further to i-propyl (18) or H (19) results in
complete loss of activity. This requirement for a bulky
lipophilic group on the alkylamine side is consistent
with the SAR reported for the pinacidil-type N-cyano-
guanidine antihypertensives.41 Substitution is tolerated
at the 2-position of the pyridine ring as seen with
compounds 14 (2-Cl) and 15 (2-OCH3). Interestingly,
while a 2-OCH3 group is tolerated, a 2-OH substituent
(16) results in complete loss of activity most likely due
to existence of the pyridinone tautomer. Substitution
of a Br atom onto the 5-position of the 3-pyridyl ring
results in a 6-fold loss in activity (17 vs 10).
Finally, to confirm the mechanism of action of our
biological lead compounds, we studied their effects on
membrane currents in isolated rat detrusor smooth
muscle cells using voltage clamp and current clamp
recording techniques.
Resu lt a n d Discu ssion
The in vitro bladder relaxant effects of pinacidil (1)
and pyridyl cyclobutenedione analogues 2 and 9-19 are
shown in Table 1. Compound 2, the direct cyclobutene-
dione analogue of (()pinacidil, was found to be about
2-fold less potent than pinacidil as a bladder smooth
muscle relaxant (IC50s ) 1.36 µΜ and 0.63 µΜ, respec-
tively). The corresponding 3-pyridyl analogue (10) was
only slightly less potent than 2, while the 2-pyridyl
derivative (12) was found to possess bladder smooth
muscle contracting properties. This SAR is consistent
with that reported for the pinacidil-type N-cyanoguani-
dine antihypertensive series where the order of potency
was reported as 4-pyridyl = 3-pyridyl . 2-pyridyl.41 The
In an effort to expand the SAR to bicyclic heteroaryl
derivatives, compounds 20 -27 were prepared and the
results are shown in Table 2. Replacement of the
aminopyridine ring with 3-, 6-, or 8-amino-quinoline
resulted in loss of activity. The 5-amino-isoquinoline
derivative 24 was slightly more potent than the corre-
sponding pyridyl analogue 13. Interestingly, replacing
the 3-pyridyl group of compound 11 with 5-amino-

1190 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Butera et al.
Ta ble 2. In Vitro Effects of Bicyclic Heteroaryl Analogues on Precontracted Rat Bladder Smooth Muscle Strips (IC50, µM)a
a
IC50: drug concentration (µM) that relaxed KCl-induced contractions in rat detrusor strips by 50%. b Structures of compounds confirmed
by 1H NMR, IR, and MS. The presence of hydrates and partial solvent adducts was confirmed by KF analysis and 1H NMR. c Analytical
results are within (0.4% of the theoretical value unless otherwise noted. Number of experiments. e See compound 20.
d
Ch a r t 1
indazolyl (27) resulted in a 2.5-fold increase in bladder
relaxant activity while the corresponding 6-amino-
indazolyl analogue (26) was 2.4-fold less active.
We next sought to examine N-(4-substituted)-aryl
analogues as potential surrogates for the 4-pyridyl
moiety. Keeping R2 constant as 3,3-dimethyl-2-butyl
(either the R-configuration or racemic), N-aryl ana-
logues 28-41 were prepared, and the in vitro bladder
relaxant effects are shown in Table 3. The unsubstituted
phenyl analogue 28 was found to be 3.2-fold less potent
than the corresponding 4-pyridyl analogue 9. As seen
with examples 29-40, bladder relaxant activity is
increased 10-fold when a cyano group is installed at the
4-position (34). A bromine atom (29) or a methoxy group
(30) at position-4 is tolerated; however, replacement
with any of the other groups shown in Table 3 caused a
precipitous loss of bladder relaxant activity. Homolo-
gation of the cyano group in 34 with a methylene group
to give phenyl-acetonitrile analogue 41 resulted in
retention of potent bladder relaxant activity. The ob-
served SAR for the 4-position is consistent with that
reported for a series of aryl-N-cyanoguanidine KCOs .42
Compound 89 (BMS-182264), a putative hybrid of
pinacidil (1) and cromakalim (88), was shown to be the
most potent aortic smooth muscle relaxant in a series
of structurally novel KCOs (Chart 1).42 In our study, it
(IC50 ) 0.52 µM), we chose to evaluate its performance
in a pathophysiological model of bladder instability. The
in vivo effects of 34 and N-cyanoguanidine analogue 89
in the rat model of bladder instability and effects on
MAP are shown in Table 4. When administered orally
at 10 mg/kg, compound 34 reduced the frequency of
spontaneous bladder contractions by 83% while, in a
separate set of animals, no significant changes in MAP
relaxed bladder smooth muscle strips with an IC50
0.19 µM.
)
Due to the potent bladder relaxant effects associated
with cyanophenyl-aminocyclobutenedione compound 34

Potassium Channel Openers Targeted for UUI. 1
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1191
Ta ble 3. In Vitro Effects of Aryl Analogues on Precontracted Rat Bladder Smooth Muscle StripssSAR at Position-4. (IC50, µM)a
p
a
IC50: drug concentration (µM) that relaxed KCl-induced contractions in rat detrusor strips by 50%. b Structures of compounds confirmed
by 1H NMR, IR, and MS. The presence of hydrates and partial solvent adducts was confirmed by KF analysis and 1H NMR. c Analytical
results are within (0.4% of the theoretical value unless otherwise noted. Number of experiments. e Prepared by oxidation of sulfide 31;
d
see Experimental Section. f C: calcd, 54.71; found, 54.08. H: calcd, 6.51; found, 6.03. C: calcd, 68.77; found, 68.20. C: calcd, 69.43;
g
h
i
found, 68.88
Ta ble 4. In Vivo Effects of Compounds 34 and 89 on
Frequency of Spontaneous Blader Contractions in the Rat
Hypertrophic Bladder Model and Effects on MAP in
Normotensive Rats (Percent Change from Pre-Drug Value,
suitable surrogate for the chiral 3,3-dimethyl-2-butyl
group. Bladder relaxant activity was found to be re-
markably sensitive to steric modifications on the lipo-
philic alkylamine side chain. The requirement of
R-branching is demonstrated by the total loss of activity
observed by simply removing the R-methyl group (49,
IC50 > 30 µM). Modulation of branching and steric bulk
at the position â to the nitrogen also reduces activity,
but the effects are much less dramatic (47, IC50 ) 1.9
µM; 48, IC50 ) 3.34 µM; and 50; IC50 ) 2.1 µM). Further
reducing the overall lipophilicity and steric bulk to
2-propyl (51, IC50 ) 13.7 µM) or methyl (52, IC50 > 30
X ( SE)
spontaneous bladder contractions
blood pressurec
dose
dose
(mg/kg)
po
frequency
(mg/kg)
po
maximum
comp
na
(% change)b
na % change
34
89
10
10
4
8
-83 ( 12
-27 ( 10
30
10
4
7
+8 ( 3
-19 ( 6
a
b
Number of experiments. Vehicle (PEG-200) effects: -2 ( 11.
c Initial BP values ranged from 105 ( 2 to 116 ( 5 mmHg.
µM), or installing a linear lipophilic group (53, IC50
>
were observed at a dose of 30 mg/kg. In contrast, the
direct N-cyanoguanidine analogue 89 only marginally
reduced the frequency of bladder contractions at 10 mg/
kg, while causing a 19% drop in MAP. Having demon-
strated this remarkable in vivo bladder smooth muscle
selectivity relative to hemodynamic effects associated
with compound 34, our SAR efforts turned to optimiza-
tion of in vitro potency while maintaining in vivo
selectivity.
We chose to vary the position of the nitrile on the
arylamine and the structure of the alkylamine moiety.
Compounds 42-74 were prepared, and the results are
shown in Table 5. Consistent with prior trends, detrusor
relaxant activity resides mostly in the (R)- antipode as
demonstrated by enantiomers 42 (IC50 ) 0.37 µM) and
43 (IC50 ) 24.3 µM). Transposing the nitrile group to
the meta (44, IC50 ) 3.05 µM) and ortho (45, IC50 > 30
µM) positions results in progressive loss of activity. The
tert-butyl group (46, IC50 ) 0.56 µM) was found to be a
30 µM), abolished activity. In a similar way, cyclic alkyl
groups (i.e., 55, 56, 59) are not tolerated. Interestingly,
the endo-norbornyl analogue 57 retains some bladder
relaxant activity, while the corresponding exo-analogue
58 is completely devoid of activity. This observation is
consistent with SAR reported by workers at Green Cross
for a series of related N-pyridyl-N′-bicycloalkyl-N′′-
cyanoguanidine derivatives shown to be potent antihy-
pertensive KCOs.43
Compared to compound 34, a remarkable 10-fold
enhancement of in vitro potency is observed with the
(R)-R-methylbenzylamine analogue, 60 (IC50 ) 0.056
µM). Consistent with previously observed trends, the
corresponding (S)-enantiomer (61, IC50 > 30 µM) was
much less potent. In an attempt to further optimize in
vitro activity associated with 60, modifications in the
R-methylbenzylamine moiety were studied with com-
pounds 62-74, and surprisingly, we found a very

1192 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Butera et al.
Ta ble 5. In Vitro Effects of Cyanophenyl Analogues on Precontracted Rat Bladder Smooth Muscle StripssAlkyl SAR (IC50, µM)a
rat bladder strip
a
IC50: drug concentration (µM) that relaxed KCl-induced contractions in rat detrusor strips by 50%. b Structures of compounds confirmed
by 1H NMR, IR, and MS. The presence of hydrates and partial solvent adducts was confirmed by KF analysis and 1H NMR. c Analytical
results are within (0.4% of the theoretical value unless otherwise noted. Number of experiments. e See compound 34. f N: calcd, 13.85;
d
g
h
i
found, 12.89. C: calcd, 62.98; found, 62.38. N: calcd, 15.46; found, 14.95. The compound caused a further contraction of the detrusor
j
k
strip. No effect. C: calcd, 69.14; found 68.57.
narrow window of activity in this subset of compounds.
Consistent with the (R)-3,3-dimethyl-2-butyl SAR, re-
moval of the R-methyl group (62, IC50 > 30 µM) or
introduction of a second methyl group (65, IC50 > 30
µM) is detrimental to activity. Exchange of the R-methyl
group for R-trifluoromethyl (66), R-hydroxymethyl (67),
or R-ethyl (68, 69) all resulted in attenuation of activity.
Likewise, smooth muscle relaxant properties were
diminished with a variety of substituents at position-4
of the benzene ring (70-74). Replacement of the ben-
zene ring with a cyclohexane ring (63, IC50 ) 2.6 µM)
resulted in a 50-fold drop in activity.
Our criteria for lead progression required that an in
vivo bladder inhibitory ED50 dose in the rat should not
cause greater than a 10% drop in mean arterial pres-
sure. Potent in vitro lead 60 was evaluated in vivo (data

Potassium Channel Openers Targeted for UUI. 1
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1193
Ta ble 6. In Vitro Effects of N-4-Cyanophenyl-N′-(R)-3,3-dimethyl-2-butyl Analogues on Precontracted Rat Bladder Smooth Muscle
Strips (IC50, µM)a
rat bladder strip
a
b
IC50 drug concentration (µM) that relaxed KCl-induced contractions in rat detrusor strips by 50%. Structures of compounds confirmed
by 1H NMR, IR, and MS. The presence of hydrates and partial solvent adducts has been confirmed by KF analysis and 1H NMR. c Analytical
results are within (0.4% of the theoretical value unless otherwise noted. Number of experiments. e Racemic compound. f N: calcd, 11.06;
d
g
found, 10.37. No effect.
Ta ble 7. Comparison of In Vitro and In Vivo Effects of
not shown) and was found to not meet that criteria and
Compounds 79, ZD-6169 (4), and Celikalim (90)
thus was dropped from further consideration as a
biological lead.
hemodynamic
bladder effects
effects
selectivity
Revisiting N-4-cyanophenyl-N′-(R)-3,3-dimethyl-2-bu-
tyl analogue 34 as a starting point for a second iteration
of SAR, we chose to focus on the introduction of
additional substituents onto the aromatic ring, and the
results are shown in Table 6. The 3-chloro analogue (75,
IC50 ) 4.3 µM) was somewhat less potent than 34. The
corresponding 3-methyl analogue (76, IC50 ) 0.63 µM)
retained most of the activity of the parent compound
in vitro in vivo
in vivo
ED20
mg/kg
ratio
(MAP ED20/
bladder ED50)
a
b
c
IC50
ED50
comp
(µM)
mg/kg
79
0.09
0.93
0.03
0.13
2.4
0.3
2.3
6.96
0.2
17.7
2.9
0.7
ZD-67169 (4)
Celikalim (90)
a
IC50 drug concentration (µM) that relaxed KCl-induced con-
tractions in rat detrusor strips by 50%. ED50: drug dose (po) that
b
while homologation to the 3-ethyl species (77, IC50
)
caused a 50% reduction in the frequency of spontaneous bladder
contractions in the rat hypertrophied model of bladder instability.
Vehicle (PEG-200) effects: -2 ( 11. c ED20: drug dose (po) that
caused a 20% drop in MAP in normotensive rats. Initial BP values
ranged from 105 ( 2 to 116 ( 5 mmHg.
17.6 µM) resulted in a 34-fold reduction in activity. The
3-hydroxy analogue (83) was totally devoid of activity.
Introduction of a 2-methyl substituent (78, IC50 ) 1.08
µM) had little effect on potency. In contrast to the trend
at position-3, homologation of methyl to ethyl (79,
IC50 ) 0.09 µM) at position-2 results in a 6-fold increase
in bladder relaxant potency. However, further increase
in lipophilic steric bulk from 2-ethyl to 2-propyl (80,
IC50 ) 23.1 µM) abolishes most of the bladder relaxant
activity. Interestingly, some bladder relaxant activity
of abnormal spontaneous contractions in the rat are
shown in Table 7. Comparative data are presented for
ZD-6169 (4), a compound which has been shown to
activate the bladder KATP channel and to selectively
increase bladder compliance in the rat and dog without
significant hemodynamic effects, and for the antihyper-
tensive KATP channel opener celikalim (90). A thorough
pharmacological comparison of these three KCOs has
been reported elsewhere.44
Oral administration of 79 at doses between 0.03 and
10 mg/kg produced a dose-dependent decrease in the
frequency of spontaneous bladder contractions. The
calculated ED50 value for the compound is 0.13 mg/kg
(po). A representative cystometric recording before and
after administration of 79 is shown in Figure 3. The
upper portion shows the characteristic bladder instabil-
ity and spontaneous spikes in bladder pressure that
develop during the filling phase of the experiment. It is
is maintained with a 2-benzoyl substituent (86, IC50
)
4.78 µM), albeit at a level 9-fold lower than 34. Other
substituents at position-2 which were found to improve
in vitro bladder relaxant activity include a methoxy
group (81, IC50 ) 0.29 µM), a chlorine atom (84, IC50
)
0.041 µM), and a bromine atom (85, IC50 ) 0.075 µM).
As a result of the potent in vitro bladder relaxant
properties associated with (R)-4-[3,4-dioxo-2-(1,2,2-tri-
methyl-propylamino)-cyclobut-1-enylamino]-3-ethyl-ben-
zonitrile (79, WAY-133537), we chose to fully evaluate
the compound in our in vivo models for efficacy and
bladder selectivity. The in vivo effects of 79 on inhibition

1194 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Butera et al.
F igu r e 3. Rat hypertrophied bladder model: a representative
cystometric tracing. Spontaneous spikes in bladder pressure
are observed during the filling phase, indicating an increasing
amount of instability prior to the micturition pressure (upper
panel). After treatment with 1.0 mg/kg 79, the frequency of
spontaneous bladder contractions is significantly reduced and
micturition pressure is unchanged (lower panel).
believed that the sense of urinary urgency that develops
in patients suffering from this type of incontinence
results from similar spontaneous bladder contractions.
A sustained micturition contraction at the end of the
cycle results in urine output. The lower portion of the
chart shows bladder pressure and void volume after
exposure to 1.0 mg/kg (po) 79. Spontaneous bladder
contractions are significantly reduced and, importantly,
micturition pressure and void volume remain relatively
unchanged. The maximal effect on reduction in the
frequency of bladder contractions was observed at 1.0
h post-dose. As shown in Table 7, celikalim (ED50 ) 0.3
mg/kg) possessed similar in vivo potency to 79; however,
in this model, ZD-6169 (ED50 ) 2.4 mg/kg) was ap-
proximately 18-fold less potent than 79.
F igu r e 4. Comparison of in vivo bladder relaxant and
hemodynamic effects of 79 and celikalim (90).
As a measure of relative in vivo selectivity, we chose
to compare the blood pressure lowering properties of 79,
ZD-6169, and celikalim, and the data are shown in
Table 7. All three compounds dose-dependently lowered
MAP in normotensive rats following oral administra-
tion. Celikalim (ED20 ) 0.2 mg/kg), a KCO developed
as an antihypertensive, was the most potent blood
pressure lowering agent, followed 79 (ED20 ) 2.3 mg/
kg) and ZD-6169 (ED20 ) 6.96 mg/kg). Although all
three agents were effective at inhibiting spontaneous
bladder contractions, compound 79, with a selectivity
ratio of 17.7, demonstrated bladder efficacy at doses
which produced minimal hemodynamic changes. In our
studies, ZD-6169 also exhibited some degree of bladder
selectivity in vivo with a ratio of 2.9 in favor of bladder
effects. The antihypertensive agent celikalim, with a
ratio of 0.7, showed no selectivity toward the bladder
over cardiovascular effects. A graphical representation
of the observed bladder and hemodynamic effects of 79
and celikalim is shown in Figure 4. The top panel
illustrates the remarkable in vivo bladder selectivity of
79 in contrast to the nonselective effects of celikalim
shown in the lower panel.
F igu r e 5. Representative current clamp tracing from a single
rat detrusor myocyte. Control RMP was approximately -30
mV; after exposure to 79 (WAY-133537, 1 µM), the cell
hyperpolarized by approximately 14 mV. This hyperpolariza-
tion was reversed with the addition of glyburide (5 µM) to the
tissue bath.
taken. Contractile inhibition with all test compounds
in the isolated bladder strip assay was reversed upon
exposure to 6 µM glyburide; thus suggesting that the
relaxant mechanism involves activation of the ATP-
dependent potassium channel in the rat bladder. A full
electrophysiological profile of 79 has been reported.44
For the purpose of this manuscript, some cell electro-
physiological data are shown in Figures 5 and 6. In
current clamp studies on isolated rat detrusor myocytes,
exposure to 1 µM 79 caused a hyperpolarization of
approximately 14 mV which was reversed with the
addition of 5 µM glyburide (Figure 5). In whole-cell
To elucidate the underlying mechanism of action of
this novel series of KCOs, several studies were under-

Potassium Channel Openers Targeted for UUI. 1
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1195
muscle over isolated aortic rings (data not shown). In
general, in vitro IC50 values for relaxation of precon-
tracted aortic tissues were 2-3 times lower than values
observed for detrusor muscle. Favorable pharmacody-
namics and/or pharmacokinetics may play a role in the
observed in vivo selectivity associated with cyclobutene-
dione 79 and its analogues. Alternatively, it has been
proposed45 that bladder selectivity with agents such as
ZD-6169 may be explained by a biphasic concentration-
response for activation of the ATP-dependent potassium
channel (i.e., observed activation of the ATP-dependent
potassium channel at lower concentrations of ZD-6169,
but inhibition of the channel at concentrations > 20 µM
ZD-6169) as well as by possible effects on other potas-
sium channels. Studies to further understand the
observed in vivo bladder selectivity of 79 and its
analogues are currently in progress.
Con clu sion s
We have demonstrated that structural modifications
to prototypical KCOs in the N-cyanoguanidine class of
agents originally developed as antihypertensives have
provided novel compounds with potential utility in
noncardiovascular therapeutic areas. Replacement of
the N-cyanoguanidine template in pinacidil with a 1,2-
diaminocyclobutene-3,4-dione moiety afforded a novel
series of KATP channel openers possessing potent in vitro
bladder relaxant activity. A systematic SAR study on
both the aryl and alkylamine groups produced definitive
trends which are summarized in Figure 7. Several
agents were evaluated in vivo in a pathophysiological
model of bladder instability in the rat and also for their
effects on MAP and heart rate in normotensive rats.
These studies culminated with the identification of (R)-
4-[3,4-dioxo-2-(1,2,2-trimethyl-propylamino)-cyclobut-1-
enylamino]-3-ethyl-benzonitrile (79) as an agent which
selectively inhibits spontaneous detrusor contractions
when administered orally at doses that caused no
significant changes in hemodynamic parameters. Mecha-
nistic studies have demonstrated that the underlying
mechanism of action for the compound involves activa-
tion of the KATP channel in the bladder.
Cyclobutenedione 79 was evaluated in a full battery
of safety assessment and ancillary studies. Its oral
bioavailability in the rat (calculated from drug plasma
levels after oral vs iv administration) was determined
to be 70%. It was found to be clean in a NOVA Screen
receptor binding profile and was found to be AMES
negative at all concentrations tested. A two week pilot
toxicological study in the rat at doses up to 200 mg/kg
resulted in no observed drug-related abnormalities. In
our studies, compound 79 compared favorably in overall
profile to ZD-6169, a compound currently undergoing
Phase II clinical evaluation for the treatment of urge
UI. Due to its potent bladder relaxant effects and its
unique in vivo selectivity profile, compound 79 repre-
sents a true “second-generation” bladder-selective KCO
with potential for development as a novel agent to treat
urge urinary incontinence.
F igu r e 6. Whole-cell voltage clamp tracings from an isolated
rat detrusor myocyte. Cells were held at -50 mV and stepped
in +10 mV increments for 1000 ms (figure shows currents
elicited at -50, -20, 10, and 40 mV). Compound 79 (WAY-
133537) increased outward current at test potentials over the
voltage range associated with activation of the ATP-dependent
potassium channel. The increases in outward current were
reversed in the presence of 79 (WAY-133537) with the subse-
quent addition of glyburide (5 µM) to the tissue bath.
voltage clamp studies on isolated rat detrusor myocytes,
79 increased outward current at all test potentials over
the voltage range associated with activation of the ATP-
dependent potassium channel (Figure 6). The increase
in outward current was also reversed by exposure to
glyburide. Results from both of these cell electrophysi-
ological studies are consistent with activation of the
KATP channel.
Although the mechanism of action studies are con-
clusive for activation of the KATP channel, the underlying
mechanism of action for the in vivo bladder selectivity
of this series of compounds and their differentiation
from the vascular-selective antihypertensive KCOs is
less clear. Intrinsically, neither compound 79 or ZD-
6169 demonstrated in vitro selectivity for detrusor
Exp er im en ta l Section
Melting points were determined on a Thomas-Hoover capil-
lary melting point apparatus or on an Electrothermal IA9300
and are uncorrected. 1H NMR spectra were recorded on either
a Varian XL-300 or on a Varian Unity Plus-400 spectrometer

1196 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Butera et al.
α
α
F igu r e 7. 1,2-Diaminocyclobutene-3,4-diones: trends in in vitro SAR.
using tetramethylsilane (TMS) as an internal standard. The
chemical shifts are reported in parts per million (δ) downfield
from TMS. Mass spectra were recorded on either a Finnigan
MAT 8230 or a Finnigan MAT SSQ710C spectrometer. The
chromatography (4:1 ethyl acetate/hexane) afforded 3.15 g
(49%) of monoadduct as a white solid: mp 140-145 °C. To
the compound (0.328 g, 1.50 mmol) in acetonitrile (30 mL) was
added (()-2-amino-3,3-dimethylbutane (0.200 mL, 1.52 mmol).
A precipitate formed while stirring overnight. The crude
reaction mixture was vacuum filtered, and the precipitate was
dried in vacuo to yield 0.27 g (66%) of title compound as a white
solid: mp 243-245 °C; 1H NMR (DMSO-d6) δ 9.67 (s, 1H), 8.58
(d, 1H),7.98 (d, 1H), 7.66 (d, 1H), 7.38 (m, 1H), 3.96-4.00 (m,
1H), 1.18 (d, 3H), 0.95 (s, 9H); IR (KBr) 3200, 1800, 1665, 1600
cm-1; MS (m/z) 273 (M+).
3-ter t-Bu t yla m in o-4-(p yr id in -3-yla m in o)-cyclob u t -3-
en e-1,2-d ion e (13). Prepared in the manner described for
compound 10: mp 250-252 °C; 1H NMR (DMSO-d6) δ 8.57 (s,
1H), 8.23 (d, 1H), 7.96 (d, 1H), 7.37 (m, 1H) 1.43 (s, 9H); IR
(KBr) 1790, 1685, 1600 cm-1; MS (m/z) 245 (M+).
3-(Isop r op yla m in o)-4-(p yr id in -3-yla m in o)-cyclobu t-3-
en e-1,2-d ion e (18). Prepared in the manner described for
compound 10: mp 258-260 °C; 1H NMR (DMSO-d6) δ 9.62
(br s, 1H), 8.55 (s, 1H), 8.22 (d, 1H), 7.94 (br d, 1H), 7.73 (br
s, 1H), 7.36 (dd, 1H), 4.20 (br m, 1H), 1.25 (d, 6H); IR (KBr)
3200, 1800, 1660, 1610 cm-1; MS (m/z) 231(M+).
3-Am in o-4-(p yr id in -3-yla m in o)-cyclob u t -3-en e-1,2-d i-
on e (19). Prepared in the manner described for compound
10: mp 297 °C (dec); 1H NMR (DMSO-d6) δ 8.56 (s, 1H), 8.22
(d, 1H), 7.92 (d, 1H), 7.37 (m, 1H); IR (KBr) 3200, 1800, 1670,
1625 cm-1; MS (m/z) 189 (M+).
infrared spectra were recorded on
a Nicolet Avatar 360
spectrophotometer. C, H, N combustion analyses were deter-
mined on a Perkin-Elmer 2400 analyzer, and all compounds
are within (0.4% of the theoretical value unless otherwise
indicated. Organic extracts were dried over magnesium sulfate
or sodium sulfate and were concentrated in vacuo with rotary
evaporators. When necessary, purification of compounds was
accomplished by flash column chromatography using 230-400
mesh silica gel, by radial chromatography using a Chroma-
totron 7924 (Harrison Research) with precast silica gel rotors
obtained from Analtech, Inc., or by HPLC using a Waters Prep
500 instrument with silica Prep-Pak cartridges. Thin-layer
chromatography was performed on silica gel 60 F-254 (0.25
mm thickness) plates. Visualization was accomplished with
UV light, I2 vapor, or 10% phosphomolybdic acid in ethanol.
Structures of all tested compounds have been confirmed by
1H NMR, IR, MS, and combustion analysis. Yields, melting
points, and analytical results are tabulated in Tables 1-3 and
5-6. The syntheses and spectral data for representative
compounds from each of the Tables 1-3 and 5-6 are shown
below.
Rep r esen ta tive P yr id yl An a logu es in Ta ble 1: (()-3-
(P yr id in -4-yla m in o)-4-(1,2,2-tr im eth yl-p r op yla m in o)-cy-
clobu t-3-en e-1,2-d ion e (2). To a solution of 3,4-diethoxy-3-
cyclobutene-1,2-dione (5.00 g, 29.4 mmol) in absolute ethanol
(100 mL) was added a suspension of 4-aminopyridine (2.77 g,
29.4 mmol) in ethanol (50 mL). The reaction mixture was
refluxed for 4 h and was then concentrated to give crude
product. Flash chromatography (ethyl acetate) afforded 0.68
g (13%) of monoadduct as a white solid: mp 120-125 °C. To
the compound (0.332 g, 1.52 mmol) in acetonitrile (30 mL) was
added (()-2-amino-3,3-dimethylbutane (0.200 mL, 1.52 mmol).
A precipitate formed while stirring overnight. The crude
reaction mixture was vacuum filtered, and the precipitate was
dried in vacuo to yield 0.328 g (79%) of title compound as a
pale yellow solid: mp 255-257 °C; 1H NMR (DMSO-d6) δ 9.80
(s, 1H), 8.42 (dd, 2H), 7.72 (d, 1H), 7.45 (dd, 2H), 3.96-4.00
(m, 1H), 1.18 (d, 3H), 0.91 (s, 9H); IR (KBr) 3200, 1800, 1675,
1600 cm-1; MS (m/z) 274 (MH+).
Repr esen tative Bicyclic Heter oar yl An alogu es In Table
2: (()-3-(Qu in olin -3-yla m in o)-4-(1,2,2-tr im eth yl-p r op yl-
a m in o)-cyclobu t-3-en e-1,2-d ion e (20). To a solution of
3-aminoquinoline (2.12 g, 14.69 mmol) in ethanol (60 mL) was
added 3,4-diethoxy-3-cyclobutene-1,2-dione (2.50 g, 14.69 mmol),
and the resulting mixture was heated to reflux for 24 h. The
mixture was cooled, and the precipitate was filtered, washed
with diethyl ether, and dried in vacuo to give 3.14 g (80%) of
monoadduct as a yellow solid which was used without puri-
fication: 1H NMR (DMSO-d6) δ 11.14 (s, 1H), 8.92 (dd, 1H),
8.20 (dd, 1H), 7.90 (m, 2H), 7.63 (m, 2H). To the compound
(0.30 g, 1.12 mmol) in ethanol (8 mL) was added (()-2-amino-
3,3-dimethylbutane (0.18 mL, 1.34 mmol), and the resulting
mixture was heated at 50 °C overnight. The precipitate was
filtered, washed with diethyl ether, and dried in vacuo to afford
0.30 g (83%) of title compound as a light tan solid: mp 242-
1
(()-3-(P yr idin -3-ylam in o)-4-(1,2,2-tr im eth yl-pr opylam i-
n o)-cyclobu t-3-en e-1,2-d ion e (10). To a solution of 3,4-
diethoxy-3-cyclobutene-1,2-dione (5.00 g, 29.4 mmol) in abso-
lute ethanol (100 mL) was added a suspension of 3-amino-
pyridine (2.77 g, 29.4 mmol) in ethanol (50 mL). The mixture
was heated at reflux for 18 h, then concentrated. Flash
246 °C; H NMR (DMSO-d6) δ 9.93 (s, 1H), 8.90 (d, 1H), 8.38
(br s, 1H), 7.97 (d, 1H), 7.84 (dd, 1H), 7.75-7.55 (m, 2H), 4.00
(m, 1H), 1.20 (d, 3H), 0.94 (s, 9H); IR (KBr) 3160, 2960, 1795,
1650 cm-1; MS (m/z) 324 (MH+).
3-ter t-Bu tyla m in o-4-(qu in olin -3-yla m in o)-cyclobu t-3-
en e-1,2-d ion e (21). Prepared in the manner described for

Potassium Channel Openers Targeted for UUI. 1
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1197
1
compound 20: mp 257-260 °C; H NMR (DMSO-d6) δ 10.00
at room temperature, diluted with diethyl ether, and filtered
to give 1.22 g (80%) of monoadduct of sufficient purity for the
second step. The compound (0.50 g, 1.90 mmol) was added to
a solution of (()-2-amino-3,3-dimethylbutane (0.51 mL, 3.80
mmol) in ethanol (10 mL) and was stirred at room temperature
overnight. Filtration afforded 0.536 g (89%) of title compound
as a yellow solid: mp 285-286 °C; 1H NMR (DMSO-d6) δ 9.55
(s, 1H), 7.57 (d, 1H), 7.41 (d, 2H), 7.25 (d, 2H), 3.95 (m, 1H),
2.44 (s, 3H), 1.17 (d, 3H), 0.91 (s, 9H); IR (KBr) 3400, 3200,
2920, 1800, 1660, 1575, 1525, 1450 cm-1; MS (m/z) 318 (M+).
(br d, 1H), 8.9 (d, 1H), 8.4 (m, 1H), 8.02 (d, 1H), 7.96 (br d,
1H), 7.86 (dd, 1H), 7.66-7.54 (m, 2H), 1.45 (s, 9H); IR (KBr)
3400, 3200, 2980, 1785, 1680 cm-1; MS (m/z) 296 (MH+).
3-ter t-Bu tyla m in o-4-(qu in olin -6-yla m in o)-cyclobu t-3-
en e-1,2-dion e (22). To a solution of 3,4-diethoxy-3-cyclobutene-
1,2-dione (5.00 g, 29.4 mmol) in absolute ethanol (100 mL) was
added a suspension of 6-aminoquinoline (4.24 g, 29.4 mmol)
in ethanol (50 mL). The mixture was heated at reflux for 18
h, cooled, and vacuum filtered to afford 6.64 g (84%) of
monoadduct (mp: 185-187, dec) which was used without
further purification. An aliquot (3.00 g, 11.2 mmol) was
dissolved in tert-butylamine (50 mL). The solution was refluxed
for 3 h, cooled, and concentrated. The residue was recrystal-
lized twice from ethanol and then triturated with diethyl ether
to give 1.05 g (31%) of the title compound as a pale yellow
solid: mp 234-236 °C; 1H NMR (DMSO-d6) δ 9.92 (s, 1H), 8.76
(d, 1H), 8.24 (d, 1H), 8.01 (d, 1H), 7.98 (s, 1H), 7.97 (d, 1H),
7.88 (d, 1H), 7.48 (m, 1H), 1.45 (s, 9H). IR (KBr) 1780, 1665,
1610 cm-1; MS (m/z) 295 (M+).
(()-3-(4-Tr iflu or om et h oxy-p h en yla m in o)-4-(1,2,2-t r i-
m eth yl-p r op yla m in o)-cyclobu t-3-en e-1,2-d ion e (32). Pre-
pared in the manner described for compound 34: mp 282-
1
285 °C; H NMR (DMSO-d6) δ 9.66 (s, 1H), 7.60 (d, 1H), 7.53
(d, 2H), 7.33 (d, 2H), 3.99 (m, 1H), 1.17 (d, 3H), 0.91 (s, 9H);
MS (m/z) 357 (M + H)+.
(()-4-[3,4-Dioxo-2-(1,2,2-t r im et h yl-p r op yla m in o)-cy-
clobu t-1-en yla m in o]-ben zon itr ile (34). 4-Aminobenzoni-
trile (17.58 g, 149 mmol) was added to a solution of 3,4-
diethoxy-3-cyclobutene-1,2-dione (25.31 g, 149 mmol) in absolute
ethanol (450 mL). The mixture was heated at reflux overnight
and the resulting suspension was filtered hot to remove a small
amount of bis-adduct. The filtrate was gradually concentrated
to afford several crops of 4-(3,4-dioxo-2-ethoxy-cyclobut-1-
enylamino)-benzonitrile, as a bright yellow solid, which were
(()-3-(Qu in olin -8-yla m in o)-4-(1,2,2-t r im et h yl-p r op yl-
a m in o)-cyclobu t-3-en e-1,2-d ion e (23). To a solution of
8-aminoquinoline (1.00 g, 6.94 mmol) in ethanol (20 mL) was
added 3,4-diethoxy-3-cyclobutene-1,2-dione (1.03 mL, 6.94
mmol), and the resulting mixture was heated to reflux for 24
h. The mixture was cooled, diluted with ethanol, and filtered.
The crude product was triturated with chloroform/hexanes and
then purified by flash chromatography (ethyl acetate/hexane)
to give 1.31 g (70%) of monoadduct: 1H NMR (DMSO-d6) δ
9.75 (br m, 1 H), 8.86 (dd, 1 H), 8.26 (br m, 1H), 8.20 (dd, 1H),
7.57 (m, 2H), 7.53 (dd, 1H), 4.95 (q, 2H), 1.59 (t, 3H). To the
compound (0.300 g, 1.12 mmol) in ethanol (5 mL) was added
(()-2-amino-3,3-dimethylbutane (0.18 mL, 1.34 mmol), and the
resulting mixture was heated at 45 °C overnight, diluted with
hexanes, and filtered to give 0.294 g (81%) of title compound
1
collected by filtration and combined: yield 29.11 g (81%); H
NMR (DMSO-d6) δ 11.07 (s, 1H), 7.81 (d, 2H), 7.56 (d, 2H),
4.79 (q, 2H), 1.46 (t, 3H). To this product (13.00 g, 53.7 mmol)
in ethanol (360 mL) was added (()-2-amino-3,3-dimethylbu-
tane (7.2 mL, 54 mmol). The mixture was heated at reflux
overnight. Gradual concentration of the reaction solution
afforded two crops of 4-[3,4-dioxo-2-(1,2,2-trimethyl-propyl-
amino)-cyclobut-1-enylamino]-benzonitrile, as a yellow pre-
cipitate, which were collected by filtration and combined: yield
11.34 g (71%); mp 241-243 °C; 1H NMR (DMSO-d6) δ 9.89 (s,
1H), 7.78 (d, 2H), 7.72 (d, 1H), 7.60 (d, 2H), 3.96 (m, 1H), 1.18
(d, 3H), 0.91 (s, 9H).
1
as a yellow solid: mp 244-245; H NMR (DMSO-d6) δ 10.45
(s, 1H), 8.97 (dd, 1H), 8.62 (d, 1H), 8.41 (dd, 1H), 8.29 (dd,
1H), 7.6 (m, 3H), 4.10 (m, 1H), 1.21 (d, 3H), 0.94 (s, 9H); IR
(KBr) 3280, 2960, 1790, 1670 cm-1; MS (m/z) 323 (MH+).
(+)-(R)-4-[3,4-Dioxo-2-(1,2,2-t r im et h yl-p r op yla m in o)-
cyclobu t-1-en yla m in o]-ben zoic Acid Meth yl Ester (36).
3-ter t-Bu tyla m in o-4-(isoqu in olin -5-yla m in o)-cyclobu t-
3-en e-1,2-d ion e (24). To a solution of 3,4-diethoxy-3-cy-
clobutene-1,2-dione (5.00 g, 29.4 mmol) in absolute ethanol
(100 mL) was added a suspension of 5-aminoisoquinoline (4.24
g, 29.4 mmol) in ethanol (50 mL). The mixture was heated to
reflux overnight and then filtered to yield 2.30 g (29%) of
monoadduct as a solid: mp 182 (dec) °C. The compound (0.300
g, 1.12 mmol) was dissolved in tert-butylamine (50 mL) and
refluxed for 3 h. The mixture was cooled, concentrated, and
triturated with diethyl ether to afford 0.120 g (39%) of the title
compound as a white solid, one-eighth hydrate: mp 268-270
°C (dec); 1H NMR (DMSO-d6) δ 9.75 (s, 1H), 9.35 (s, 1H), 8.62
(d, 1H), 8.19 (s, 1H), 8.01 (d, 1H), 7.88 (d, 1H), 7.80 (d, 1H),
Prepared in the manner described for compound 34: mp 279-
1
280 °C; [R]25 ) +7.54° (DMSO); H NMR (DMSO-d6) δ 9.87
D
(s, 1H), 7.93 (d, 2H), 7.68 (d, 1H), 7.56 (d, 2H), 4.99 (m, 1H),
3.82 (s, 3H), 1.18 (d, 3H), 0.92 (s, 9H); MS (m/z) 331 (M + H)+.
(()-3-(4-Met h a n esu lfon yl-p h en yla m in o)-4-(1,2,2-t r i-
m eth yl-p r op yla m in o)-cyclobu t-3-en e-1,2-d ion e (37). To a
suspension of 3-(4-methylsulfanyl-phenylamino)-4-(1,2,2-tri-
methyl-propylamino)-cyclobut-3-ene-1,2-dione (31, 0.100 g,
0.314 mmol) in ethanol (3 mL) was added potassium peroxy-
monosulfate (OXONE, 0.77 g, 1.26 mmol) as a solution in water
(2 mL). The mixture was stirred overnight at room tempera-
ture and filtered, and the solid was washed with water and
dried in vacuo. The solid was triturated with methanol/ethyl
acetate to afford 0.05 g (45%) of sulfone as an off-white solid:
7.68 (t, 1H), 1.47 (s, 9H); IR (KBr) 3200, 1785, 1670, 1600 cm-1
;
MS (m/z) 295 (M+).
1
mp 307-310 °C; H NMR (DMSO-d6) δ 9.91 (s, 1H), 7.85 (d,
(()-3-(6-Meth oxy-qu in olin -8-ylam in o)-4-(1,2,2-tr im eth yl-
p r op yla m in o)-cyclobu t-3-en e-1,2-d ion e (25). Prepared in
the manner described for compound 23 utilizing 8-amino-6-
methoxyquinoline: mp 243-245 °C; 1H NMR (DMSO-d6) δ
10.43 (br s, 1H), 8.78 (dd, 1H), 8.64 (d, 1H), 8.28 (dd, 1H), 8.13
(d, 1H), 7.56 (dd, 1H), 7.02 (d, 1H), 4.11 (m, 1H), 3.88 (s, 3H),
2H), 7.71 (d, 1H), 7.65 (d, 2H), 3.99 (m, 1H), 3.17, (s, 3H), 1.20
(d, 3H), 0.91 (s, 9H); IR (KBr) 3400, 3150, 2925, 1800, 1680,
1575, 1530, 1450 cm-1; MS (m/z) 351 (M + H)+.
(+)-(R)-3-(4-Tr iflu or om eth yl-p h en yla m in o)-4-(1,2,2-tr i-
m eth yl-p r op yla m in o)-cyclobu t-3-en e-1,2-d ion e (40). Pre-
pared in the manner described for compound 34: mp 296-
1.20 (d, 3H), 0.94 (s, 9H); IR (KBr) 3400, 3220, 2950, 1780 cm-1
;
1
299 °C; [R]25 ) +4.20° (DMSO); H NMR (DMSO-d6) δ 9.81
D
MS (m/z) 353 (M+).
(s, 1H), 7.63 (m, 5H), 4.00 (m, 1H), 1.17 (d, 3H), 0.91 (s, 9H);
Rep r esen ta tive Ar yl An a logu es in Ta ble 3: (+)-(R)-3-
P h en yla m in o-4-(1,2,2-tr im eth yl-p r op yla m in o)-cyclobu t-
3-en e-1,2-d ion e (28). Prepared in the manner described for
MS (m/z) 341 (M + H)+.
Rep r esen ta tive Ben zon itr ile An a logu es in Ta ble 5:
(+)-(R)-4-[3,4-Dioxo-2-(1,2,2-tr im eth yl-p r op yla m in o)-cy-
clobu t-1-en yla m in o]-ben zon itr ile (42). Prepared in the
compound 34: mp 279-280 °C; [R]25 ) +6.96; 1H NMR
D
manner described for compound 34: mp 273-274 °C; [R]25
)
(DMSO-d6) δ 9.55 (s, 1H), 7.57 (d, 1H), 7.40 (d, 2H), 7.33 (m,
2H), 7.01 (m, 1H), 3.99 (m, 1H), 1.17 (d, 3H), 0.91 (s, 9H); MS
(m/z) 273 (M + H)+.
D
1
+5.88° (DMSO); H NMR (DMSO-d6) δ 9.88 (s, 1H), 7.79 (d,
2H), 7.71 (d, 1H), 7.61 (d, 2H), 3.99 (m, 1H), 1.17 (d, 3H), 0.91
(s, 9H); MS (m/z) 298 (M + H)+. The chiral purity of 42 was
determined using a Chiralpak AS column with an 80:20
isocratic hexane:ethanol mobile phase at a flow rate of 1.0 mL/
min. Under these conditions, compound 42 possessed a reten-
tion time of 9.40 min (>99% ee).
(()-3-(4-Meth ylsu lfan yl-ph en ylam in o)-4-(1,2,2-tr im eth yl-
pr opylam in o)-cyclobu t-3-en e-1,2-dion e (31). 4-(Methylthio)-
aniline (0.722 mL, 5.80 mmol) was added to a stirring solution
of 3,4-diethoxy-3-cyclobutene-1,2-dione (1.29 mL, 8.70 mmol)
in absolute ethanol (29 mL). The mixture was stirred overnight

1198 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Butera et al.
(-)-(S)-4-[3,4-Dioxo-2-(1,2,2-t r im et h yl-p r op yla m in o)-
cyclobu t-1-en yla m in o]-ben zon itr ile (43). To a solution of
(S)(-)-2-amino-3,3-dimethyl-butane (0.50 g, 4.90 mmol) in
ethanol (100 mL) was added 4-(3,4-dioxo-2-ethoxy-cyclobut-1-
enylamino)-benzonitrile (1.00 g, 4.13 mmol). The mixture was
stirred for 2 days at room temperature and vacuum filtered.
The solid was dissolved in methanol and filtered. The filtrate
was concentrated in vacuo and the residue was triturated with
acetone to afford 350 mg (29%) of title compound as a pale
yellow solid: mp 241-243 °C; [R]25D ) -7.7° (DMSO); 1H NMR
(DMSO-d6) δ 9.89 (s, 1H), 7.78 (d, 2H), 7.72 (d, 1H), 7.60 (d,
2H), 3.96-3.98 (m, 1H), 1.17 (d, 3H), 0.91 (s, 9H); IR (KBr)
3210, 2250, 1800, 1685 cm-1; MS (m/z) 297 (M+). The chiral
purity of 43 was determined using a Chiralpak AS column with
an 80:20 isocratic hexane: ethanol mobile phase at a flow rate
of 1.0 mL/min. Under these conditions, compound 43 possessed
a retention time of 7.52 min (97% ee).
4-[3,4-Dioxo-2-(1-m eth yl-1-ph en yl-eth ylam in o)-cyclobu t-
1-en yla m in o]-ben zon itr ile (65). Prepared in the manner
1
described for compound 34: mp >300 °C; H NMR (DMSO-
d6) δ 10.08 (s, 1H), 8.38 (s, 1H), 7.79 (d, 2H), 7.61 (d, 2H), 7.48-
7.27 (m, 5H), 1.78 (s, 6H); IR (KBr) 3200, 2230, 1790, 1675,
1600 cm-1; MS (m/z) 331 (M+).
(()-4-[3,4-Dioxo-2-(2,2,2-tr iflu or o-1-ph en yl-eth ylam in o)-
cyclobu t-1-en yla m in o]-ben zon itr ile (66). To a solution of
N-2,2,2-trifluoro-1-phenylethyl-N-1′-(phenyl)ethylamine46 (1.65
g, 6.22 mmol) and ammonium formate (1.17 g, 18.6 mmol) in
methanol (150 mL) was added 10% palladium on activated
carbon. The suspension was refluxed for 4 h, filtered through
Celite, and concentrated to a volume of approximately 10 mL.
4-(3,4-Dioxo-2-ethoxy-cyclobut-1-enylamino)-benzonitrile (1.00
g, 4.13 mmol) and ethanol (20 mL) were added, and the
mixture was heated at reflux for 18 h, cooled slightly, and
vacuum filtered to remove a small amount of solid. The filtrate
was chromatographed (methanol/dichloromethane), and the
resulting yellow solid was crystallized from chloroform and
ether to afford 0.72 g (47%) of product as a pale yellow solid:
(()-3-[3,4-Dioxo-2-(1,2,2-t r im et h yl-p r op yla m in o)-cy-
clobu t-1-en yla m in o]-ben zon itr ile (44). Prepared in the
manner described for compound 34: mp 296-298 °C (dec); 1H
NMR (DMSO-d6) δ 9.79 (s, 1H), 7.94 (m, 1H), 7.63-7.69 (m,
2H), 7.51-7.55 (m, 1H), 7.44-7.46 (m, 1H), 3.93-4.03 (m, 1H),
1
mp 206-207 °C; H NMR (DMSO-d6) δ 9.99 (s, 1H), 8.88 (d,
1H), 7.82 (d, 2H), 7.60-7.46 (m, 9H), 5.98 (m, 1H), 3.73 (s,
3H); IR (KBr) 3200, 2200, 1800, 1690, 1570 cm-1; MS (m/z)
371 (M+).
1.17 (d, 3H), 0.91 (s, 9H); IR (KBr) 3200, 2200, 1800, 1675 cm-1
;
MS (m/z) 297 (M+).
(+)-(R)-2-[3,4-Dioxo-2-(1,2,2-t r im et h yl-p r op yla m in o)-
(-)-4-[3,4-Dioxo-2-((R)-1-ph en yl-pr opylam in o)-cyclobu t-
1-en yla m in o]-ben zon itr ile (68). Prepared in the manner
cyclobu t-1-en yla m in o]-ben zon itr ile (45). Prepared in the
manner described for compound 34: mp 258-259 °C; [R]25
)
D
described for compound 34: mp 242-243 °C; [R]25 ) -52.73
D
+45.29° (DMSO); 1H NMR (DMSO-d6) δ 9.69 (s, 1H), 8.02 (br
d, 1H), 7.79 (dd, 1H), 7.62 (m, 1H), 7.57 (d, 1H), 7.22 (m, 1H),
4.03 (m, 1H), 1.19 (d, 3H), 0.92 (s, 9H); MS (m/z) 298 (M +
H)+.
1
(DMSO); H NMR (DMSO-d6) δ 9.84 (s, 1H), 8.12 (br d, 1H),
7.76 (d, 2H), 7.56 (d, 2H), 7.42-7.27 (m, 5H), 5.06 (m, 1H),
1.94 (m, 2H), 0.90 (t, 3H); IR (KBr) 3200, 2220, 1790, 1670,
1600 cm-1; MS (m/z) 331 (M+).
4-(2-ter t-Bu tyla m in o-3,4-d ioxo-cyclobu t-1-en yla m in o)-
ben zon itr ile (46). Prepared in the manner described for
compound 34: mp 257-260 °C (dec); H NMR (DMSO-d6) δ
9.97 (s, 1H), 8.02 (s, 1H), 7.78 (d, 2H), 7.61 (d, 2H), 1.42 (s,
9H); IR (KBr) 3220, 2240, 1800, 1680, 1620 cm-1; MS (m/z)
269 (M+).
(+)-4-[3,4-Dioxo-2-((S)-1-ph en yl-pr opylam in o)-cyclobu t-
1-en yla m in o]-ben zon itr ile (69). Prepared in the manner
1
described for compound 34: mp 241-243 °C; [R]25 ) +52.33
D
1
(DMSO); H NMR (DMSO-d6) δ 9.84 (s, 1H), 8.21 (br d, 1H),
7.76 (d, 2H), 7.56 (d, 2H), 7.42-7.27 (m, 5H), 5.06 (m, 1H),
1.94 (m, 2H), 0.90 (t, 3H); IR (KBr) 3200, 2220, 1790, 1670,
1600 cm-1; MS (m/z) 331 (M+).
4-(2-Isop r op yla m in o-3,4-d ioxo-cyclobu t-1-en yla m in o)-
ben zon itr ile (51). Prepared in the manner described for
(-)-(R)-4-{2-[1-(4-Meth yl-ph en yl)-eth ylam in o]-3,4-dioxo-
cyclobu t-1-en yla m in o}-ben zon itr ile (70). Prepared in the
1
compound 34: mp 290-292 °C (dec); H NMR (DMSO-d6) δ
manner described for compound 34: mp >300 °C; [R]25
)
9.89 (s, 1H), 7.79 (d, 1H), 7.76 (d, 2H), 7.57 (d, 2H), 4.19-4.20
D
(m, 1H), 1.25 (d, 6H); IR (KBr) 3220, 2230, 1800, 1620 cm-1
;
1
-56.01 (DMSO); H NMR (DMSO-d6) δ 9.81 (s, 1H), 8.10 (m,
1H), 7.76 (d, 2H), 7.55 (d, 2H), 7.29 (d, 2H), 7.19 (d, 2H), 5.26
(m, 1H), 1.57 (d, 3H); IR (KBr) 3200, 2220, 1790, 1670, 1600
cm-1; MS (m/z) 331 (M+).
MS (m/z) 255 (M+).
(()-(Exo)-4-[2-(bicyclo[2.2.1]h ep t-2-yla m in o)-3,4-d ioxo-
cyclobu t-1-en yla m in o]-ben zon itr ile (58). Prepared in the
manner described for compound 34: mp 288-290 °C; 1H NMR
(DMSO-d6) δ 9.79 (s, 1H), 7.78 (d, 2H), 7.76 (m, 1H), 7.58 (d,
2H), 3.90-3.98 (m, 1H), 2.22-2.34 (m, 2H), 1.76-1.86 (m, 1H),
1.08-1.56 (m, 7H); IR (KBr) 2220, 1790, 1650, 1600 cm-1; MS
(m/z) 307 (M+).
(-)-(R)-4-{2-[1-(4-Meth oxy-p h en yl)-eth yla m in o]-3,4-d i-
oxo-cyclobu t-1-en yla m in o}-ben zon itr ile (71). To a solution
of (1R, 1′R)-N-(1′-phenylethyl)-1-(4′′-methoxyphenyl)ethyl-
amine (1.37 g, 5.36 mmol; prepared from 4′-methoxyacetophe-
none utilizing the procedure described by Manley and Quast38
)
4-(2-Cycloh eptylam in o-3,4-dioxo-cyclobu t-1-en ylam in o)-
ben zon itr ile (59). Prepared in the manner described for
and ammonium formate (1.01 g, 16.0 mmol) in methanol (125
mL) was added 10% palladium on activated carbon. The
suspension was refluxed for 2 h, filtered through Celite, and
concentrated. 4-(3,4-Dioxo-2-ethoxy-cyclobut-1-enylamino)-
benzonitrile (1.00 g, 4.13 mmol) was added to a solution of the
resulting residue in ethanol (30 mL). The mixture was heated
at reflux for 18 h, cooled slightly, and vacuum filtered. The
precipitate was chromatographed (methanol/dichloromethane)
and recrystallized (methanol/dichloromethane) to afford 0.21
1
compound 34: mp 273-275 °C (dec); H NMR (DMSO-d6) δ
9.89 (s, 1H), 7.86 (d, 1H), 7.77 (d, 2H), 7.58 (d, 2H), 4.07-4.11
(m, 1H), 1.42-1.98 (m, 12H); IR (KBr) 3250, 2200, 1800, 1675
cm-1; MS (m/z) 309 (M+).
(-)-4-[3,4-Dioxo-2-((R)-1-p h en yl-eth yla m in o)-cyclobu t-
1-en yla m in o]-ben zon itr ile (60). Prepared in the manner
described for compound 34: mp 273-274 °C; [R]25 ) -53.20
D
(DMSO); 1H NMR (DMSO-d6) δ 9.91 (s, 1H), 8.21 (d, 1H), 7.72
(d, 1H), 7.79-7.31 (m, 9H), 5.29 (m, 1H), 1.59 (d, 3H); IR (KBr)
3200, 2230, 1790, 1670, 1600 cm-1; MS (m/z) 317 (M+).
(+)-4-[3,4-Dioxo-2-((S)-1-p h en yl-eth yla m in o)-cyclobu t-
1-en yla m in o]-ben zon itr ile (61). Prepared in the manner
g (15%) of product as a yellow solid: mp >300 °C; [R]25
)
D
1
-46.95 (DMSO); H NMR (DMSO-d6) δ 9.89 (s, 1H), 8.13 (d,
1H), 7.77 (d, 2H), 7.56 (d, 2H), 7.34 (d, 2H), 6.95 (d, 2H), 5.23
(m, 1H), 3.73 (s, 3H), 1.57 (d, 3H); IR (KBr) 3200, 2200, 1800,
1670, 1575 cm-1; MS (m/z) 347 (M+).
described for compound 34: mp 269-270 °C; [R]25 ) +46.47
D
(-)-(R)-4-{3,4-Dioxo-2-[1-(4-tr iflu or om eth oxy-p h en yl)-
eth yla m in o]-cyclobu t-1-en yla m in o}-ben zon itr ile (72). To
a solution of (1R,1′R)-N-(1′-phenylethyl)-1-(4′′-trifluorometh-
oxyphenyl)-ethylamine (1.92 g, 6.21 mmol; prepared from 4′-
trifluoromethoxyacetophenone utilizing the procedure de-
scribed by Manley and Quast38) and ammonium formate (1.17
g, 18.6 mmol) in methanol (150 mL) was added 10% palladium
on activated carbon. The suspension was refluxed for 2 h,
filtered through Celite, and concentrated. 4-(3,4-Dioxo-2-
ethoxy-cyclobut-1-enylamino)-benzonitrile (1.00 g, 4.13 mmol)
(DMSO); 1H NMR (DMSO-d6) δ 9.91 (s, 1H), 8.21 (d, 1H), 7.72
(d, 1H), 7.79-7.31 (m, 9H), 5.29 (m, 1H), 1.59 (d, 3H); IR (KBr)
3200, 2230, 1790, 1670, 1600 cm-1; MS (m/z) 317 (M+).
4-[3,4-Dioxo-2-(b en zyla m in o)-cyclob u t -1-en yla m in o]-
ben zon itr ile (62). Prepared in the manner described for
1
compound 34: mp 288-290 °C (dec); H NMR (DMSO-d6) δ
9.91 (s, 1H), 8.10 (m, 1H), 7.79 (d, 2H), 7.75 (d, 2H), 7.55 (d,
2H), 7.91-7.78 (m, 5H), 4.82 (d, 2H); IR (KBr) 3190, 2220,
1790, 1660, 1575 cm-1; MS (m/z) 303 (M+).

Potassium Channel Openers Targeted for UUI. 1
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1199
was added to a solution of the resulting residue in ethanol (35
mL). The mixture was heated at reflux for 18 h, cooled slightly,
and vacuum filtered. The precipitate was combined with a
second crop of solid obtained from the cooled filtrate, chro-
matographed (methanol/dichloromethane), and recrystallized
(methanol/dichloromethane) to afford 0.74 g (45%) of product
chiral purity of 79 was determined using a Pirkle covalent
(S,S) Whelk O1 column with a mobile phase consisting of
either 100% ethanol or 1:1 hexane:ethanol at a flow rate of
0.5 mL/min. Under these conditions, the (R)-(+)-enantiomer
(eutomer, 98.2%) possessed a retention time of 9.51 min and
the (S)-(-)-enantiomer (distomer, 1.6%) possessed a retention
time of 11.49 min. Chiral purity was determined to be 96.6%
ee.
as a white solid: mp 281-284 °C (dec); [R]25 ) -55.94
D
(DMSO); 1H NMR (DMSO-d6) δ 9.94 (s, 1H), 8.22 (d, 1H), 7.78
(d, 2H), 7.60-7.51 (m, 4H), 7.40 (d, 2H), 5.33 (m, 1H), 1.60 (d,
3H); IR (KBr) 3200, 2200, 1800, 1670, 1560 cm-1; MS (m/z)
401 (M+).
(+)-(R)-4-[3,4-Dioxo-2-(1,2,2-tr im eth yl-pr opylam in o)-cy-
clobu t-1-en ylam in o]-3-m eth oxy-ben zon itr ile (81). Meth od
A. To a cooled solution of 3-methoxybenzonitrile (10.00 g, 75
mmol) in acetic anhydride (150 mL) was added 70% nitric acid
(60 mL) at a rate that kept the reaction mixture below 50 °C.
After 1 h the reaction mixture was poured onto ice (400 g) and
extracted with ethyl acetate. The ethyl acetate layer was
washed with water and dried (Na2SO4). Concentration under
reduced pressure and flash chromatography with ethyl acetate/
hexanes gave 2.69 g (20%) of 3-methoxy-4-nitrobenzonitrile:
1H NMR (DMSO-d6) δ 8.04 (d, 1H), 7.93 (d, 1H), 7.61 (dd, 1H),
3.97 (s, 3H). 3-Methoxy-4-nitrobenzonitrile (2.6 g, 14.6 mmol)
was suspended in ethanol (100 mL) and added to a mixture of
5% platinum on carbon (250 mg) in ethanol (150 mL). The
reaction mixture was stirred under a hydrogen gas atmo-
sphere. After 24 h the reaction was filtered, concentrated, and
chromatographed (ethyl acetate/hexanes, 25/75) to yield 1.3 g
(60%) of 4-amino-3-methoxybenzonitrile: 1H NMR (DMSO-d6)
δ 7.1 (m, 2H), 6.65 (d, 1H), 5.77 (br s, 2H), 3.79 (s, 3H).
4-Amino-3-methoxybenzonitrile (1.3 g, 8.77 mmol) and 3,4-
diethoxy-3-cyclobutene-1,2-dione (1.5 g, 8.82 mmol) in ethanol
(50 mL) were heated in an oil bath at 110-115 °C for 3 days.
The reaction mixture was filtered hot, and the filtrate was
reduced to half its volume. Filtration gave 0.58 g (24%) of
monoadduct as a yellow solid: mp 159-161 °C; 1H NMR
(DMSO-d6) δ 10.42 (br s, 1H), 7.54 (d, 1H), 7.44 (dd, 1H), 7.39
(d, 1H), 4.70 (q, 2H), 3.86 (s, 3H), 1.36 (t, 3H); IR (KBr) 3413,
3026, 2650, 2576, 2439, 1719, 1630, 1548 cm-1; MS (m/z) 272
(M+). 4-(2-Ethoxy-3,4-dioxo-cyclobut-1-enylamino)-3-methoxy-
benzonitrile (0.58 g, 2.1 mmol) and a solution of (R)-2-amino-
3,3-dimethylbutane (4.2 mmol) in ethanol (21 mL) were stirred
at room temperature for 24 h. The reaction mixture was
filtered, and the filtrate was concentrated to a foam, which
was dissolved in acetonitrile (10 mL). Upon standing at room
temperature fluffy pale yellow needles formed. This was
filtered and rinsed sparingly with acetonitrile to yield 0.46 g
(67%) of title compound as pale yellow needles: mp 280-282
(-)-(R)-4-{2-[1-(4-Nitr o-p h en yl)-eth yla m in o]-3,4-d ioxo-
cyclobu t-1-en yla m in o}ben zon itr ile (73). To 4-(3,4-dioxo-
2-ethoxy-cyclobut-1-enylamino)-benzonitrile (0.598 g, 2.47 mmol)
in ethanol (50 mL) were added (R)-R-methyl-4-nitrobenzyl-
amine hydrochloride (0.50 g, 2.5 mmol) and N,N-diisopropyl-
ethylamine (0.43 g, 2.5 mmol). The mixture was heated at
reflux for 16 h. After cooling, the precipitate was filtered off
to afford 0.70 g (78%) of product as an orange solid: mp 290-
295 °C; [R]25 ) -100.52 (DMSO); 1H NMR (DMSO-d6) δ 9.98
D
(s, 1H), 8.32 (d, 1H), 8.25 (d, 2H), 7.79 (d, 2H), 7.68 (d, 2H),
7.57 (d, 2H), 5.42 (m, 1H), 1.61 (d, 3H); IR (KBr) 3200, 2220,
1790, 1670, 1600 cm-1; MS (m/z) 362 (M+).
Rep r esen ta tive N-4-Cya n op h en yl-N′-(R)-3,3-d im eth yl-
2-bu tyl An a logu es in Ta ble 6: (+)-(R)-4-[3,4-Dioxo-2-
(1,2,2-tr im eth yl-p r op yla m in o)-cyclobu t-1-en yla m in o]-3-
eth yl-ben zon itr ile (79). Meth od A. 4-Amino-3-ethylbenzo-
nitrile (0.86 g, 5.88 mmol) and 3,4-diethoxy-3-cyclobutene-1,2-
dione (1.0 g, 5.88 mmol) were refluxed in acetonitrile (2 mL)
for 21 h. Additional 3,4-diethoxy-3-cyclobutene-1,2-dione (0.5
g, 2.9 mmol) was added to the reaction mixture. After 48 h
the reaction mixture was cooled to room temperature and then
diluted with ethyl acetate, and a solid was filtered. The filtrate
was concentrated, and the resulting solid was taken up in ethyl
acetate (5 mL) and sonicated. Filtration gave 0.54 g (34%) of
monoadduct as a light brown solid: 1H NMR (DMSO-d6) δ
10.56 (br s, 1H), 7.70 (br s overlapping a doublet at d 7.69,
2H), 7.30 (d, 1H), 4.71 (q, 2H), 2.74 (q, 2H), 1.37 (t, 3H), 1.15
(t, 3H). 4-(2-Ethoxy-3,4-dioxo-cyclobut-1-enylamino)-3-ethyl-
benzonitrile (1.26 g, 4.66 mmol) and a solution of (R)-2-amino-
3,3-dimethylbutane (9.3 mmol) in ethanol (58 mL) were stirred
at room temperature for 24 h. The resulting yellow solution
was concentrated to a yellow oil, which was dissolved in
acetonitrile (20 mL) and stirred at room temperature. The
resulting yellow solid was filtered and rinsed with ethyl acetate
°C (dec); [R]25 ) +62.86 (DMSO); 1H NMR (DMSO-d6) δ 9.49
D
to yield 0.79 g (52%) of title compound as an off-white solid:
1
mp 236-237 °C; [R]25 ) +68.20 (DMSO); H NMR (DMSO-
(br s, 1H), 8.31 (d, 1H), 7.99 (d, 1H), 7.50 (d, 1H), 7.43 (dd,
1H), 4.03 (m, 1H), 3.97 (s, 3H), 1.18 (d, 3H), 0.90 (s, 9H); MS
(m/z) 327 (M+).
D
d6) δ 8.04 (br s, 1H), 8.04 (d, 1H), 7.69-7.57 (m, 3H), 4.05 (m,
1H), 2.71 (q, 2H), 1.27-1.16 (overlapping doublet and triplet,
6H), 0.91 (s, 9H); IR (KBr) 3278, 2959, 2222, 1793, 1674, 1598,
1576, 1522 cm-1; MS (m/z) 325 (M+).
(+)-(R)-4-[3,4-Dioxo-2-(1,2,2-tr im eth yl-pr opylam in o)-cy-
clobu t-1-en ylam in o]-3-m eth oxy-ben zon itr ile (81). Meth od
B. 4-Amino-3-methoxybenzonitrile (1.32 g, 8.90 mmol) was
dissolved in DMF (20 mL) and purged with nitrogen. NaH
(0.263 g, 80%, 8.9 mmol) was added, and the resulting mixture
was stirred for 30 min at 70 °C. The mixture was cooled to
room temperature and added via syringe to a solution of 3,4-
diethoxy-3-cyclobutene-1,2-dione (2.27 g, 13.35 mmol) in DMF
(10 mL). The mixture was stirred for 48 h at room temperature
and diluted with brine (50 mL) and extracted with dichlo-
romethane. The organic phase was washed with 1.0 N HCl
and brine. It was dried and concentrated under heated vacuum
to afford an oil. Trituration with diethyl ether/hexanes afforded
0.97 g (41%) of 4-(2-ethoxy-3,4-dioxo-cyclobut-1-enylamino)-3-
methoxy-benzonitrile as a light yellow solid. This was con-
verted to the title compound by treatment with (R)-2-amino-
3,3-dimethylbutane as described above in method A. The
compound was identical in all respects with the sample
obtained from method A.
(+)-(R)-4-[3,4-Dioxo-2-(1,2,2-t r im et h yl-p r op yla m in o)-
cyclobu t-1-en yla m in o]-3-eth yl-ben zon itr ile (79). Meth od
B. To a solution of 4-amino-3-ethylbenzonitrile (10 g, 68.5
mmol) in DMF (150 mL) at 0 °C was added, portion-wise, NaH
(2.26, 80%, 75.3 mmol). The mixture was heated to 60 °C for
1 h and was then cooled to room temperature. The 3,4-
diethoxy-3-cyclobutene-1,2-dione (13.17 mL, 89.04 mmol) was
added, and the resulting mixture was heated to 80 °C for 4 h.
The reaction was cooled, diluted with brine (150 mL), and
extracted with dichloromethane. The organic phase was
washed with 1.0 N HCl and brine. It was dried and concen-
trated under heated vacuum to afford an oil. Trituration with
diethyl ether/hexanes afforded 10.93 g (59%) of 4-(2-ethoxy-
3,4-dioxo-cyclobut-1-enylamino)-3-ethyl-benzonitrile as an off-
white solid. The intermediate (7.8 g, 28.88 mmol) was added
to a solution of (R)-2-amino-3,3-dimethylbutane in ethanol (209
mL of a 0.166 N solution; 34.7 mmol) and was stirred for 48 h
at room temperature. The reaction mixture was concentrated,
and the residue was triturated with 5:1 petroleum ether/
diethyl ether. Filtration afforded 4.86 g (52%) of analytically
pure title compound. An additional crop of 1.95 g (21%) was
obtained from the mother liquor. The compound was identical
in all respects with the sample obtained from method A. The
(+)-(R)-3-Ch lor o-4-[3,4-d ioxo-2-(1,2,2-tr im eth yl-p r op yl-
a m in o)-cyclobu t-1-en yla m in o]-ben zon itr ile (84). To 3,4-
diethoxy-3-cyclobutene-1,2-dione (2.0 g, 11.75 mmol) and
4-amino-3-chlorobenzonitrile (1.8 g, 11.80 mmol) in DMF (10
mL) was added 60% NaH (0.94 g, 23.5 mmol). The mixture
was stirred at room temperature overnight and then parti-

1200 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Butera et al.
tioned between dichloromethane and 10% citric acid. The
organic phase was washed with brine, dried, and concentrated.
The residue was purified by flash column chromatography
(hexanes/ethyl acetate) to afford 0.70 g (21%) of 4-(2-ethoxy-
3,4-dioxo-cyclobut-1-enylamino)-3-chloro-benzonitrile. The com-
pound (0.3 g, 1.08 mmol) was added to an ethanolic solution
of (R)-2-amino-3,3-dimethylbutane (5.4 mL of 0.2 N solution;
1.08 mmol). The reaction was stirred overnight at room
temperature and then concentrated. The residue was tritu-
rated with acetonitrile, sonicated, and filtered to afford 0.22 g
with KOH. Signals were acquired (3 kHz high-frequency cutoff,
12 bit resolution) and analyzed using a 586-based personal
computer.
Cell resting membrane potential (RMP) was assessed by
adding nystatin to the pipet solution (100 µg/mL) and utilizing
the perforated patch technique.48 After stable intracellular
access was achieved, RMP was recorded for a 5 min control
period. Next, compound (0.3 and 1.0 µM) was superfused until
changes in voltage amplitude reached steady state (ap-
proximately 3-5 min). After this time, the effects of glyburide
(5 µM) on RMP was evaluated.
Whole-cell voltage clamp recordings were made without
nystatin using broken patch access. Currents were evoked
using the voltage steps described in the figure legends. After
stability was achieved, control currents were recorded. Next,
compound (0.3 and 1 µM) was added to the superfusate.
Currents were recorded for 5 to 10 min or until compound
effects reached steady state. This was followed either by
washout or addition of glyburide (5 µM) to the superfusate.
C. Isola ted Bla d d er Str ip Con tr a ction Stu d ies. Female
Sprague-Dawley rats (Charles River, Wilmington, MA; 250
to 350 g) were rendered unconscious via inhalation of CO2 and
exsanguinated. The entire bladder was removed and placed
into room temperature PSS of the following composition
(mM): NaCl (118.4), KCl (4.7), CaCl2 (2.5), MgSO4 (1.2), KH2-
PO4 (1.2), NaHCO3 (24.9), and D-glucose (11.1) gassed with
O2-CO2, 95%/5%, to achieve a pH of 7.4. The dome of the
bladder was isolated from the trigone region, and the mucosa
was removed. The detrusor was then cut into strips 4-5 mm
wide by 10 mm long. One end was secured to the bottom of a
10 mL tissue bath and the other to a Grass isometric force
transducer (Grass Instruments, Quincy, MA). Tissues were
pretensioned (0.25 to 0.5 g) and allowed to equilibrate for 30
min. Strips were then contracted with an additional 15 mM
KCl and again allowed to equilibrate for approximately 90 min.
Compounds were administered directly into the tissue baths
as cumulative concentrations, and responses were allowed to
reach steady-state. Signals were digitized (486 based personal
computer, 12 bit resolution, 1 s sampling interval, custom
software) for online analysis. Since isolated bladder strips
contract with irregular frequency and amplitude, a 5 min area-
under-the-contraction curve was used to assess contractility
after achieving steady state for each concentration.
In Vivo Stu dies: A. Rat Hyper tr oph ied Bladder Model.
The method for producing hypertrophied, unstable bladders
was modified from that reported by Malmgren et al.39 Briefly,
female Sprague-Dawley rats (Charles River, Wilmington, MA;
190-210 g) were anesthetized with isoflurane, and the bladder
and urethra were exposed through a 2 cm midline incision. A
4-0 silk ligature was tied around the proximal urethra in the
presence of a stainless steel rod (1 mm diameter). The rod was
then removed thus resulting in a partial premeasured occlu-
sion. The abdominal musculature was closed using 3-0 silk,
and the skin was closed with surgical staples. Each rat
received 150 000 units (im) of bicilin C-R (Wyeth Laboratories,
Philadelphia, PA). During the following 6 week period, the
increased urethral outlet resistance from the partial occlusion
caused the bladders to hypertrophy and become unstable.
(61%) of title compound: 220-222 °C; [R]25 ) + 61.39
D
(DMSO); 1H NMR (DMSO-d6) δ 9.42 (s, 1H), 8.31 (d, 1H), 8.06
(d, 1H), 7.81 (m, 2H), 4.05 (m, 1H), 1.19 (d, 3H), 0.91 (s, 9H);
MS (m/z) 332 (M + H)+.
(+)-(R)-3-Br om o-4-[3,4-d ioxo-2-(1,2,2-tr im eth yl-p r op yl-
a m in o)-cyclobu t-1-en yla m in o]-ben zon itr ile (85). To 60%
NaH (0.5 g, 12.5 mmol) in THF (50 mL) was added a solution
of 4-amino-3-bromobenzonitrile (1.2 g, 6.09 mmol) and 3,4-
diethoxy-3-cyclobutene-1,2-dione (1.40 mL, 9.5 mmol) in THF
(10 mL). The mixture was heated for 2 h under argon at 80
°C. The reaction was cooled, quenched with 2 N HCl, and
stirred for 30 min at room temperature. The water layer was
removed by pipet, and the organic layer was diluted with
dichloromethane, dried, and concentrated to afford crude
monoadduct which was purified by flash column (dichlo-
romethane) to give 1.09 g (56%) of 4-(2-ethoxy-3,4-dioxo-
cyclobut-1-enylamino)-3-bromo-benzonitrile. The compound
(0.4 g, 1.25 mmol) was added to an ethanolic solution of (R)-
2-amino-3,3-dimethylbutane (8.1 mL of 0.2 N solution; 1.62
mmol). The reaction was stirred overnight at room tempera-
ture and then concentrated. The residue was triturated with
acetonitrile, sonicated, and filtered to afford 0.28 g (59%) of
title compound: 229-231 °C; [R]25 ) +51.85 (DMSO); 1H
D
NMR (DMSO-d6) δ 9.30 (s, 1H), 8.29 (d, 1H), 8.21 (d, 1H), 7.87
(dd, 1H), 7.65 (d, 1H), 4.05 (m, 1H), 1.20 (d, 3H), 0.93 (s, 9H);
MS (m/z) 376/378 (M + H)+.
P h a r m a cologica l Meth od s. All animal studies were ap-
proved by the Wyeth-Ayerst Institutional Animal Care and
Use Committee and were performed in accordance with the
guidelines of the Animal Welfare Act and the American
Association for Accreditation of Laboratory Animal Care.
In Vitr o Stu d ies: A. Isola tion of Ra t Detr u sor Cells.
Rat detrusor cells were isolated in a manner previously
described for guinea-pig detrusor.47 Male Sprague-Dawley
rats (Charles River, Wilmington, MA; 200-400 g) were eu-
thanized by CO2 inhalation and exsanguination. The urinary
bladder was rapidly removed and placed in 37 °C physiological
salt solution (PSS) with the following composition (mM): Na
glutamate (80.0), NaCl (54.7), KCl (5.0), NaHCO3 (25.0),
MgCl2‚2H2O (2.5), D-glucose (11.8), and CaCl2 (0.2) gassed with
O2-CO2, 95%/5%, for a final pH of 7.4. The dome of the bladder
was isolated from the trigone region, and the mucosa was
removed. The detrusor was then cut into 2-3 mm wide strips
and placed into fresh buffer for 1 h. Tissues were then
transferred into 10 mL of PSS isolation buffer plus collagenase
type VIII (1.0 mg/mL) and pronase (0.25 mg/mL). After 10 min
the isolation buffer was replaced with fresh buffer for an
additional 10 min. The tissue was then washed 3 times in fresh
collagenase and pronase free PSS and stored at room temper-
ature until studied. Cells for study were prepared by triturat-
ing one to two pieces of detrusor tissue in 5 mL of fresh
isolation buffer for 5 min with a polished Pasteur pipet (tip
diameter ∼1.5 mm) attached to a modified Harvard Respirator
pump (Harvard Apparatus, Southnatic, MA) at a rate of 20×/
min with an approximate volume of 5 mL. Cells were studied
After 6 weeks, the ligature was removed under isoflurane
anesthesia, and a flared catheter (PE60) was placed in the
dome of the bladder and secured with a purse-string suture.
The catheter was exteriorized under the skin and through an
opening in the back of the neck. The abdominal incision was
sutured and the free end of the catheter sealed. Following
surgery, animals were given bicilin C-R (150 000 units/rat, im).
Two days after catheter implantation, the animals were
used for cystometric evaluation. The night before testing, the
animals were placed into metabolic cages. The catheter was
connected to a Harvard infusion pump, and bladders were
perfused overnight with saline at a rate of 2 mL/h. The next
morning a Statham pressure transducer (model P23Db) was
positioned in line with the Harvard infusion pump (using a
“T” connector) to record bladder pressure. A plastic beaker
attached to a force displacement transducer (Grass FTO3) was
in
a temperature regulated tissue bath at 32.5° C and
continually superfused with PSS.
B. Cell Electr op h ysiology. Single-cell recordings were
performed with a List-Medical EPC-7 patch clamp amplifier
(Adams & List Assoc., Westbury, NY). Pipet electrodes had
tip resistances of 2-4 MΩ and were filled with the following
composition (mM): KCl (126.0), MgCl2‚6H2O (4.5), ATP Mg
salt (4.0), GTP tris salt (0.3), creatine PO4 (14.0), D-glucose
(9.0), EGTA (9.0), HEPES (9.0). The pH was adjusted to 7.4

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