Design and SAR of novel potassium channel openers targeted for urge urinary incontinence. 2. Selective and potent benzylamino cyclobutenediones

Article abstract of DOI:10.1021/jm9905108

A novel series of benzylamine, potassium channel openers (KCOs) is presented as part of our program toward designing new, bladder-selective compounds for the treatment of urge urinary incontinence (UUI). We have found that the in vitro potency of (R)-4-[3,4-dioxo-2-(1,2,2-trimethyl- propylamino)-cyclobut-1-enylamino]-3-ethyl-benzonitrile 1 in the relaxation of precontracted rat detrusor strips can also be obtained with cyanobenzylamine derivative 4 (IC₅₀ = 0.29 μM) (Figure 3). Addition of a 2-Cl substituted benzylamine moiety and changing the alkylamino substituent of 4 to a t-Bu amine gives 31 (IC₅₀ = 0.14 μM) - a compound with similar in vitro potency as 4 as well as relaxant activity on bladder smooth muscle in vivo when administered orally (31, ED₅₀ = 3 mg/kg) in a rodent model of bladder instability. Further modifications, particularly the replacement of the t-Bu amino substituent with a tert-amylamine, gave a similarly active compound 60 (IC₅₀ = 0.10 μM) which shows excellent in vivo efficacy (ED₅₀ = 0.6 mg/kg). Moreover, 60, 3-(2,4-dichloro-6-methyl-benzylamino)-4- (1,1-dimethylpropylamino)-cyclobut-3-ene-1,2-dione (WAY-151616), shows excellent tissue selectivity for bladder K channels over arterial tissue (60, MAP ED₂₀ = 100 mg/kg; selectivity: MAP ED₂₀/bladder ED₅₀ = 166). Other manipulations of the benzylamino cyclobutenediones, acylation of the benzylamine, conversion of the benzylamine substituent to a benzamide, homologation of the benzylamine to a phenethylamine, and incorporation of a methyl group at the benzyl carbon, all led to substantial loss of in vitro activity, although some in vivo activity was maintained in the acylated analogues. Compound 60 represents an attractive candidate for development in the treatment of UUI.

Full text of DOI:10.1021/jm9905108

J . Med. Chem. 2000, 43, 1203-1214
1203
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. 2. Selective a n d P oten t Ben zyla m in o Cyclobu ten ed ion es
Adam M. Gilbert,*,# Madelene M. Antane,† Thomas M. Argentieri,‡ J ohn A. Butera,† Gerardo D. Francisco,#
Chris Freeden,§ Eric G. Gundersen,† Russell F. Graceffa,† David Herbst,† Bradford H. Hirth,† J oseph R. Lennox,†
Geraldine McFarlane,† N. Wesley Norton,‡ Dominick Quagliato,† J effrey H. Sheldon,‡ Dawn Warga,‡
Alexandra Wojdan,§ and Morgan Woods§
Chemical Sciences, Wyeth-Ayerst Research, 401 North Middletown Road, Pearl River, New York 10965-1299,
Chemical Sciences, Cardiac Diseases, Urologic Diseases, Women’s Health Discovery Research, Wyeth-Ayerst Research,
CN 8000, Princeton, New J ersey 08543-8000
Received October 8, 1999
A novel series of benzylamine, potassium channel openers (KCOs) is presented as part of our
program toward designing new, bladder-selective compounds for the treatment of urge urinary
incontinence (UUI). We have found that the in vitro potency of (R)-4-[3,4-dioxo-2-(1,2,2-
trimethyl-propylamino)-cyclobut-1-enylamino]-3-ethyl-benzonitrile 1 in the relaxation of pre-
contracted rat detrusor strips can also be obtained with cyanobenzylamine derivative 4
(IC50 ) 0.29 µM) (Figure 3). Addition of a 2-Cl substituted benzylamine moiety and changing
the alkylamino substituent of 4 to a t-Bu amine gives 31 (IC50 ) 0.14 µM)sa compound with
similar in vitro potency as 4 as well as relaxant activity on bladder smooth muscle in vivo
when administered orally (31, ED50 ) 3 mg/kg) in a rodent model of bladder instability. Further
modifications, particularly the replacement of the t-Bu amino substituent with a tert-amylamine,
gave a similarly active compound 60 (IC50 ) 0.10 µM) which shows excellent in vivo efficacy
(ED50 ) 0.6 mg/kg). Moreover, 60, 3-(2,4-dichloro-6-methyl-benzylamino)-4-(1,1-dimethyl-
propylamino)-cyclobut-3-ene-1,2-dione (WAY-151616), shows excellent tissue selectivity for
bladder K channels over arterial tissue (60, MAP ED20 ) 100 mg/kg; selectivity: MAP ED20/
bladder ED50 ) 166). Other manipulations of the benzylamino cyclobutenediones, acylation of
the benzylamine, conversion of the benzylamine substituent to a benzamide, homologation of
the benzylamine to a phenethylamine, and incorporation of a methyl group at the benzyl carbon,
all led to substantial loss of in vitro activity, although some in vivo activity was maintained in
the acylated analogues. Compound 60 represents an attractive candidate for development in
the treatment of UUI.
In tr od u ction
The previous paper in this issue from our group
describes (R)-4-[3,4-dioxo-2-(1,2,2-trimethyl-propylamino)-
cyclobut-1-enylamino]-3-ethyl-benzonitrile 1, a potent
and orally active KATP channel opener (KCO) that
effectively relaxes isolated rat detrusor strips in vitro
(IC50 ) 0.09 µM), is effective in a rat hypertrophied
model of bladder instability (ED50 ) 0.13 mg/kg) without
significant effects on mean arterial blood pressure
(MAP) and heart rate, and represents an attractive
developmental candidate for the treatment of urge
urinary incontinence (UUI).1,2
Metabolism studies of 1 using preparations of rat or
human liver microsomes produces phenylamine 2 as the
primary metabolite. At 10 µM concentrations of sub-
strate and 1 mg/mL concentrations of microsomes, the
apparent rate of metabolism (from 0 to 10 min) of 1 was
0.52 nmol/min/mg and 0.64 nmol/min/mg in rat and
human liver microsomes, respectively.
F igu r e 1. Rat liver microsome metabolism of 1 produces
aniline 2 as the major metabolite.
binding profile, concern over long-term patient exposure
to 2 prompted us to search for biological leads with
different metabolic profiles. Various manipulations were
performed on 1: (i) introduction of bulky flanking
groups on the phenyl ring of 1 failed to block or slow
metabolic cleavage,3 (ii) modification of the electronics
of the phenyl ring (primarily by changing the p-
substituent) resulted in the loss of in vitro activity,1 (iii)
acylation of the phenylamino nitrogen in 1 failed to
block production of 2 (compounds deacylate and then
release 2 upon exposure to rat liver microsomes),3 and
(iv) a large number of cyclobutenedione mimetics were
synthesized (heterocyclic, carbocyclic and acyclic tem-
plates); however, no suitable replacement could be found
which possessed the potency of the cyclobutenedione
molecules in vitro nor their in vivo efficacy (Figure 2).4
Although 1 and primary metabolite 2 were found to
be Ames negative and clean in Nova Screen receptor
* To whom correspondence should be addressed. Tel: 914-732-4865.
Fax: 914-732-5561. E-mail: gilbera@war.wyeth.com.
#
Chemical Sciences, Wyeth-Ayerst Research, New York.
† Chemical Sciences, Wyeth-Ayerst Research, New J ersey.
§ Cardiac Diseases.
‡ Urologic Diseases, Women’s Health Discovery Research.
10.1021/jm9905108 CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/03/2000

1204 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Gilbert et al.
F igu r e 2. Attempts to block metabolism of cyclobutenedione 1 to prevent liberation of phenylamine 2.
F igu r e 3. Progression from aryl cyclobutenedione 1 to benzylamino cyclobutenedione 4.
Sch em e 1a
A potentially more promising idea would be to ho-
mologate the phenylamine moiety in 1 to a benzylamines
thereby eliminating the possible concern of long-term
exposure to a metabolically liberated aniline. Direct
homologation of aniline 1 to benzylamine 3 results in a
substantial loss of in vitro potency. Removal of the o-Et
group produces 4, a compound with a similar in vitro
potency to 3 but much weaker in vivo efficacy (Figure
3). Structure-activity relationship efforts were under-
taken toward improving the in vivo profile of benzyl-
amino analogue 4 with the view of maintaining the
potency and selectivity of 1.
In this article, we report our study of a series of novel
benzylamino cyclobutenedione derivatives. Extensive
manipulation of the substituents on both the benzyl-
a
(a) 3,4-Diethoxy-3-cyclobutene-1,2-dione or 3,4-di-n-butoxy-3-
cyclobutene-1,2-dione, THF or EtOH, 23 °C; (b) R4R5NH or
(R1R2)PhCH(R3)NH2, THF or EtOH, 23, 66, or 78 °C; (c) (1) NaH,
THF/DMF, 23 °C, (2) (R6CO)2O, 23 °C.
amino and alkyl amino groups produces selective ben-
zylamino cyclobutenedione bladder agonists of the KATP
channel which upon oral administration in rats causes
significant bladder effects and minimal effects on MAP
that are similar to biological lead 1.
deprotonating 7 with NaH in DMF and then reacting
the resulting anion with an acid anhydride to give 8.
Ch em istr y
P h a r m a cology
Benzylamino cyclobutenediones were synthesized us-
ing the procedure shown in Scheme 1. The reaction of
a benzylamine ((R1R2)PhCH(R3)NH2) or an alkylamine
(R4R5NH) with 1 to 1.2 equiv of 3,4-diethoxy-3-cy-
clobutene-1,2-dione or 3,4-di-n-butoxy-3-cyclobutene-1,2-
dione in THF or EtOH at 23 °C gives monosubstituted
benzylamino cyclobutenediones 5 or monosubstituted
alkylamine cyclobutenediones 6. Performing these reac-
tions in refluxing THF or EtOH gives symmetrically
disubstituted amino cyclobutenediones in addition to the
desired monosubstituted compounds. Reacting 5 or 6
with the other amine component at 25 °C or in refluxing
THF or EtOH gives diamino cyclobutenediones 7. In
contrast to the synthesis of the phenylamino cyclobutene-
diones, the order of addition of the amines to the 3,4-
dialkoxy-3-cyclobutene-1,2-diones is not important. Both
monosubstituted cyclobutenediones 5 and 6 are suf-
ficiently reactive to combine with benzylamines and
alkylamines to produce 7. Selective acylation of the
benzylamine component can be accomplished by first
Compounds were first evaluated in vitro for their
ability to relax KCl precontracted rat detrusor muscle
strips. Following stabilization, increasing concentrations
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 to
look for the recovery of contractile activity.
Selected compounds were evaluated for in vivo ef-
ficacy in rat hypertrophied model of bladder instability
previously described by Malmgren and co-workers.5,6 In
vivo active compounds were selected for hemodynamic
assessment in a separate group of animals. Effect on
mean arterial pressure (MAP) was recorded after oral
administration of drug.
Resu lts/Discu ssion
All of the benzylamino cyclobutenediones below are
presented as KATP channel openers. The bladder relax-
ant activity for all of these compounds is reversed by
glyburide (specific data not presented). In addition, the

Potassium Channel Openers Targeted for UUI. 2
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1205
Ta ble 1. In Vitro Effects of Alkylamine Variation in 4 on Precontracted Rat Bladder Smooth Muscle Strips (IC50, µM)a
cmpd
R1
R2
% yield
mp (°C)
formulab
anal.c
IC50 (µM)a
nd
4
9
(R)-3,3-dimethyl-2-butyl
H
H
H
Me
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
93
64
89
68
68
86
79
95
58
64
64
83
88
81
44
86
31
4
288-291 (dec)
C18H21N3O2
C, H, N
C, H, Ne
C, H, Nf
C, H, N
C, H, N
C, H, Ng
C, H, N
C, H, N
C, H, Ni
C, H, N
C, H, N
C, H, Nj
C, H, N
C, H, Nk
C, H, N
C, H, Nl
C, H, N
C, H, Nn
0.29 ( 0.04
2
3
4
2
3
7
4
4
4
4
2
3
2
3
4
4
1
4
H
>260
C
C
C
C
C
C
C
12H9N3O2
13H11N3O2
14H13N3O2
15H15N3O2
15H15N3O2
16H17N3O2
16H17N3O2
>30
10
11
12
13
14
15
16h
17
18
19
20
21
22
23
24
25m
Me
Me
n-Pr
i-Pr
n-Bu
t-Bu
t-Bu
302-306 (dec)
244-246
>30
>30
21.9 ( 7.2
14.0 ( 4.7
>30
0.27 ( 0.04
18.3 ( 2.8
4.0 ( 1.1
2.2 ( 0.5
10.2 ( 2.2
1.3 ( 0.2
1.1 ( 0.4
21.3 ( 7.2
9.4 ( 2.7
14.4
241-245
276-278
250-252
283-287 (dec)
224-226 (dec)
254-257
C17H19N3O2
(R)-2-butyl
(S)-2-butyl
2-i-Bu
3-pentyl
tert-amyl
2-hydroxy-1,1-dimethyl-ethyl
cyclopentyl
2,2,3,3,3-pentafluoropropyl
4-CNC6H4CH2
C
C
C
C
C
C
C
C
16H17N3O2
16H17N3O2
16H17N3O2
17H19N3O2
17H19N3O2
16H17N3O3
17H17N3O2
15H10F5N3O2
253-256
255-257
265-268 (dec)
257-260
253-257
283-286 (dec)
252-256
>260
C20H14N4O2
>30
a
IC50: drug concentration that relaxed KCl-induced contractions in rat detrusor strips by 50%. The bladder relaxant activity could be
reversed by the addition of glyburide. Structures of compounds confirmed by 1H NMR, IR, and MS. c Analytical results are within (0.4%
b
of the theoretical value unless otherwise noted. Number of experiments. e C: calcd, 63.43; found, 62.93. f C: calcd, 64.72; found, 64.19.
d
g
h
C: calcd, 66.90; found, 66.24. Compound 16 was synthesized from 3-methoxy-4-(N-tert-butyl-N-methylamino)-cyclobut-3-ene-1,2-dione
i
j
k
and 4-cyanobenzylamine. C: calcd, 68.67; N: 14.13; found, C: 67.17; N: 13.69. C: calcd, 67.83; found, 67.32. C: calcd, 68.67; found,
68.10. l C: calcd, 69.14; found, 68.61. Compound 25 was isolated as a minor impurity in the synthesis of 3-ethoxy-4-(4-cyano-benzylamino)-
m
n
cyclobut-3-ene-1,2-dione. C: calcd, 70.17; found, 69.57.
closely related phenylamino analogues such as 1 have
been established as KATP channel openers by cellular
electrophysilogy.1,2
Although potent in the KCl-contracted rat detrusor
muscle strip assay, compound 4 lacked comparable in
vivo efficacy compared to 1. Initial SAR investigations
focused on manipulation of the alkylamine portion of 4
(Table 1). Primary amino (9), monomethylamino (10),
F igu r e 4. Comparison of benzylamino cyclobutenedione 4
with its corresponding vinylogous imide 26.
and dimethylamino (11) analogues have extremely weak
in vitro activity. Short-chained alkyl substituents (n-
Pr, i-Pr, and n-Bu) in 12-14 also show weak inhibitory
activity although some modest activity is observed with
branched i-Pr compound 13. The t-Bu compound 15
(with a highly branched alkyl group) shows activity
(IC50 ) 0.27 µM) which is comparable to 4. Methylation
of the t-Bu amine in 15 gives 16, a compound with
reduced activity. A similar trend was observed for the
phenylamino compounds.1
Both the (R) 17 and (S) 18 enantiomers of the 2-butyl
alkyl compounds showed similar activity in the rat
detrusor muscle strip assay. A compound that lacks the
R branching, the 2-i-Bu compound 19, shows compara-
tively weaker inhibition, thus following similar trends
seen in the phenyl series.1 The 3-pentyl and 1,2-
dimethyl-propyl compounds 20 and 21 have inhibitory
activity that is comparable to 17 and 18, but they are
not as potent as 4 or 15. That the area of space probed
by the alkyl amino substituent must be hydrophobic is
suggested by the reduced activity of 22, the hydroxyl-
ated analogue of 15. Cycloalkyl groups appear to be poor
choices as 23 is only moderately active. A pentafluoro-
n-propyl analogue 24 is slightly more potent than the
corresponding n-propyl analogue 12 but not as potent
as the more highly branched alkyls. Finally a p-CN
benzylamino substituent was incorporated, but the
resulting cyclobutenedione 25 had an IC50 > 30 µM.
Overall, highly branched alkyl chains were optimal
((R)-3,3-dimethyl-2-butyl 4 and t-Bu 15) with good
activity seen in compounds with branching in the carbon
R to the amine (4, 15, 17, 18, 20, and 21). In addition,
the nitrogen bearing the alkyl group must have a
hydrogen as shown by the N(H)t-Bu compound 15
(IC50 ) 0.27 µM) which is almost 68 times more potent
than its N(Me)t-Bu analogue 16 (IC50 ) 18.3 µM).
Structurally modifying the benzylamine moiety of 4
to the corresponding racemic benzamide 26 resulted in
a loss of activity (26, IC50 ) 11.5 µM) (Figure 4). It is
not clear whether this loss of activity is due to a
conformational change and/or modulation in pKa of the
benzylamine proton. It is believed that this effect is not
due to 4 containing the enantiomerically pure (R)-3,3-
dimethyl-2-butylamino moiety while 26 contains the
racemic 3,3-dimethyl-2-butylamino group.7
Attempts were also made to improve activity by
homologating the benzylamine substituent to a phen-
ethylamino amino group. However, this transformation
also led to a marked loss in activity. Potent (4-CN)
benzylamine 4 loses most of its in vitro activity when
homologated to the phenethylamino (4-CN) analogue 27
(IC50 > 30 µM). Similarly the (4-CN, t-Bu) derivative
15 also undergoes a loss of in vitro activity when
homologated to 28 (IC50 > 30 µM) (Figure 5).
The effect of adding a methyl group to the benzylic
carbon was also studied. The (4-CNPhC(Me)H) cy-

1206 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Gilbert et al.
similar activity to 15 although the SEM value of this
compound is large. Changing the 2-Cl group in 31 to a
Et group (32) also gives a compound with good potency.
One should note that this 4-CN, 2-Et substitution
pattern is identical to the phenyl substitution pattern
in 1.1 The 2,4 relationship of the Cl and Et substitution
in 31 and 32 appears to be optimal; movement of the
Cl and Et groups from the 2-position to the 3-position
(33 and 34) results in a loss of activity.
F igu r e 5. Comparison of benzylamino cyclobutenediones 4
and 15 with their homobenzylic analogues 27 and 28.
It is possible to substitute a pyridine ring for the
phenyl group as shown for 4-pyridyl analogue 35,
although a 10-fold loss in activity is seen compared to
15. The 3-pyridyl compound 36 is equipotent to 35
although the 2-pyridyl 37 is less potent. It appears that,
for the t-Bu benzylamino cyclobutenediones, the pyridyl
ring does not give an increase in activity as shown for
the phenylamino compounds;1 35 and 36 have compa-
rable potency to the unsubstituted benzyl compound 30.
The 4-OMe analogue 38 also has similar potency to 30,
but the 4-F analogue 39 appears to be slightly better.
Ortho substituted analogues of 39, (2,4-F2) 40, and (2-
Cl, 4-F) 41 fail to give increases in activity. The 2-Cl
moiety in 41 fails to give a similar increase in activity
as seen going from the 4-CN compound 15 to the (2-Cl,
4-CN) derivative. Movement of the F to the 3-position
(42) or the 2-position (43) gives compounds of similar
potency. The (2,5-F2) analogue 43 has similar activity
compared to the (2,4-F2) analogue 40 while the (2,6-F2)
compound 45 shows a slightly higher IC50 value.
F igu r e 6. Comparison of benzylamino cyclobutenedione 4
with its R-methyl benzyl analogue 29.
clobutenedione 29 was prepared, and a noticeable loss
in activity is seen compared to benzylamine 4 (29,
IC50 ) 6.5 µM; 4, IC50 ) 0.29 µM) (Figure 6).
Compound 15 was next used for an SAR optimization
study where substituents on the benzylamine were
varied and the t-Bu amino group was held constant
(Table 2). The electron-withdrawing 4-CN group in 15
is important for the molecule’s potency as seen in the
unsubstituted 30salthough this molecule still possesses
inhibitory activity. Introduction of a 2-Cl substituent in
15 produces 31 (IC50 ) 0.14 µM), a compound with
Ta ble 2. In Vitro Effects of Alkylamine Variation in 15 on Precontracted Rat Bladder Smooth Muscle Strips (IC50, µM)a
% yield
step 1
% yield
step 2
cmpd
R1
R2
R3
R4
(methodb)
mp (°C)
formulac
anal.d
IC50 (µM)a
ne
15
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
CN
H
CN
CN
CN
CN
H
H
H
H
Cl
Et
H
H
Cl
Et
H
H
H
H
H
H
H
H
95 (B)
87 (A)
87 (A)
87 (B)
81 (B)
45 (B)
87 (A)
87 (A)
87 (A)
87 (A)
87 (A)
87 (A)
87 (A)
87 (A)
87 (A)
87 (A)
87 (A)
87 (A)
87 (A)
87 (A)
87 (A)
44 (B)
87 (A)
87 (A)
95
84
50
37
30
52
76
66
94
74
86
18
59
75
68
73
56
69
74
74
38
37
62
47
283-287 (dec)
306-307 (dec)
243-245
C16H17N3O2
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, Nf
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, Ng
C, H, N
C, H, N
C, H, N
C, H, N
0.27 ( 0.04
3.4 ( 0.6
0.14 ( 0.34
0.24 ( 0.03
1.1 ( 0.1
6.1 ( 3.8
3.0 ( 0.2
2.6 ( 0.2
11.5 ( 4.3
4.8 ( 2.1
1.5 ( 0.3
2.1 ( 0.9
1.4 ( 0.4
2.4 ( 0.4
3.9 ( 1.1
2.1 ( 0.3
7.5 ( 4.5
1.7 ( 1.0
1.3 ( 0.6
0.22 ( 0.08
1.1 ( 0.4
0.27 ( 0.08
0.29 ( 0.02
0.63 ( 0.15
4
6
4
4
4
4
6
4
4
4
4
4
4
3
4
4
4
2
4
2
4
3
2
4
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
15H18N2O2
16H16ClN3O2
18H21N3O2
16H16ClN3O2
18H21N3O2
14H17N3O2
14H17N3O2
14H17N3O2
16H20N2O3
15H17FN2O2
15H16F2N2O2
15H16ClFN2O2
15H17FN2O2
15H16F2N2O2
15H17FN2O2
15H16F2N2O2
15H17ClN2O2
15H16Cl2N2O2
224-228
276-278 (dec)
233-235
271 (dec)
296 (dec)
236 (dec)
4-pyridyl
3-pyridyl
2-pyridyl
H
H
F
Cl
H
H
F
F
H
Cl
Cl
Cl
Et
Me
Me
OMe
F
F
F
H
H
H
H
Cl
Cl
Cl
H
Br
Br
Me
H
H
H
H
F
H
H
H
H
H
F
H
F
278-279 (dec)
296-298 (dec)
227-228 (dec)
292-294
295-297 (dec)
273-274 (dec)
260-262 (dec)
269 (dec)
308-309 (dec)
229-230 (dec)
246-250 (dec)
265-266 (dec)
228-232
267-271 (dec)
228-229
F
H
H
H
H
H
H
H
H
H
H
H
Me
Cl
H
Me
H
C18H23BrN2O2
C
C
C
C
15H16N2O2
17H21BrN2O2
17H21BrN2O2
17H22N2O2
a
IC50: drug concentration that relaxed KCl-induced contractions in rat detrusor strips by 50%. The bladder relaxant activity could be
reversed by the addition of glyburide. Method A: (1) 3,4-alkoxy-3-cyclobutene-1,2-dione, t-BuNH2; (2) R1R2R3R4PhNH2, Method B: (1)
b
3,4-alkoxy-3-cyclobutene-1,2-dione, R1R2R3R4PhNH2; (2) t-BuNH2. c Structures of compounds confirmed by 1H NMR, IR, and MS.
Analytical results are within (0.4% of the theoretical value unless otherwise noted. e Number of experiments. f C: calcd, 69.43; found,
d
g
68.64. C: calcd, 57.00; found, 56.55.

Potassium Channel Openers Targeted for UUI. 2
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1207
Ta ble 3. In Vitro Effects of Alkylamine/Benzylamine Variation in 27 on Precontracted Rat Bladder Smooth Muscle Strips (IC50, µM)a
% yield
step 1
% yield
cmpd R1 R2 R3
R4
(methodb) step 2
mp (°C)
formulac
anal.d
IC50 (µM)a ne
31
53
54
55
CN Cl
CN Cl
H
H
H
t-Bu
87 (A)
95 (B)
95 (A)
10 (B)
50
52
29
67
243-245
298-300
235-239
C16H16ClN3O2
C, H, N
C, H, N
0.14 ( 0.34
0.18 ( 0.33
4
4
3
4
(R)-3,3-dimethyl-2-butyl
(R)-3,3-dimethyl-2-butyl
C
C
C
18H20ClN3O2
Cl
Cl
17H20Cl2N2O3 C, H, Nf 0.34 ( 0.23
CN Cl Me (R)-3,3-dimethyl-2-butyl
>300
19H22ClN3O2
0.04 CH2Cl2
C, H, N
1.9 ( 1.1
9.8 ( 5.1
56
57
58
59
60
61
62
63
Cl
CN Cl
CN Cl Me tert-amyl
Cl Me (R)-3,3-dimethyl-2-butyl
95 (A)
86 (A)
10 (B)
86 (A)
86 (A)
78 (B)
86 (A)
86 (A)
63
65
62
40
92
59
44
12
>300
216-220
C
C
C
C
C
C
C
C
18H22Cl2N2O2 C, H, N
12H18ClN3O2
18H20ClN3O2
16H18Cl2N2O2 C, H, N
17H20Cl2N2O2 C, H, N
17H21ClN2O2
16H18Cl2N2O2 C, H, N
19H23N3O2 C, H, N
4
6
2
4
4
4
3
2
H
tert-amyl
C, H, Ng 0.13 ( 0.03
C, H, N
258-262 (dec)
196-197
0.16 ( 0.01
0.21 ( 0.04
0.10 ( 0.03
4.4 ( 4.1
0.28 ( 0.03
0.11 ( 0.01
Cl
Cl
H
Cl
H
tert-amyl
Cl Me tert-amyl
Cl Me tert-amyl
247-248
224-226
C, H, N
H
Cl Cl
tert-amyl
tert-amyl
239-241 (dec)
183-186
CN Et
H
a
IC50: drug concentration that relaxed KCl-induced contractions in rat detrusor strips by 50%. The bladder relaxant activity could be
reversed by the addition of glyburide. Method A: (1) 3,4-alkoxy-3-cyclobutene-1,2-dione, R4NH2; (2) R1R2R3PhNH2, Method B: (1) 3,4-
b
alkoxy-3-cyclobutene-1,2-dione, R1R2R3PhNH2; (2) R4NH2 c Structures of compounds confirmed by 1H NMR, IR, and MS. Analytical
d
results are within (0.4% of the theoretical value unless otherwise noted. e Number of experiments. f C: calcd, 57.48; found, 57.96. C:
g
calcd, 61.54; found, 60.81.
The 4-Cl compound 46 has a similar IC50 in the rat
detrusor muscle strip assay as the 4-F derivative 39.
The addition of a 2-Cl substituent (47) fails to increase
activity, but the corresponding (2,4-Cl2, 6-Me) analogue
48 is noticeably more potent (IC50 ) 0.22 µM). The (2,6-
Cl2) derivative 49 loses this extra potencysshowing that
the 4-substituent is important for submicromolar activ-
ity in this series. 4-Bromine substituents give potent
compounds when combined with 2 or 2,6 substitution:
(4-Br, 2-Et) analogue 50 (IC50 ) 0.27 µM), (4-Br, 2,6-
Me2) analogue 51 (IC50 ) 0.29 µM). Sub micromolar
activity is also produced with a 2,4-methyl substitution
pattern on the benzylamine 52 (IC50 ) 0.63 µM).
Two of the t-Bu benzylamino cyclobutenediones were
tested in the rat bladder instability model (Table 5).
Rats were dosed orally, and the reduction in the
frequency of spontaneous bladder contractions was
measured. Compound 15 causes a 52% reduction in the
frequency of bladder contractions at 3 mg/kg, results
similar to that seen with the equipotent analogue 4 (15,
ED50 ) 3 mg/kg; 4 frequency, -69% reduction at 3 mg/
kg). The (4-CN, 2-Cl) benzylamine cyclobutenedione 31
showed in vivo results similar to those of 4 and 15 (31,
ED50 of 3.0 mg/kg).
It was next decided to vary both the alkyl and the
benzylamine moieties in compounds similar to 31 in
hopes of increasing in vitro activity and in vivo efficacy.
It is known from the SAR of compounds in Tables 1 and
2 that excellent in vitro potency can be obtained in
benzylamine cyclobutenediones with 2,4 disubstituted
or 2,4,6 trisubstituted benzylamines. In addition, the
nature the alkylamine moiety has a large effect. This
led to another phase of our SAR study with the view of
combining previously studied (see above) benzylamines/
alkylamines that would lead to increased potency.
As stated above, these new elaborations began with
31 (Table 3). Replacement of the t-Bu substituent with
a (R)-3,3-dimethyl-2-butyl group gives equipotent ana-
logue 53. Changing the 4-CN group in 31 to a 4-Cl group
produces the (2,4-Cl) derivative 54, a compound with
similar potency to 31. Addition of a 6-Me group to 53 to
give 55 causes a loss in activity. The (2,4-Cl2, 6-Me)
pattern (56) slightly reduces the activity of 55. This is
quite surprising since the addition of a 6-Me group to
47 in the t-Bu series (Table 2) gives a noticeable increase
in activity.
Introduction of a tert-amylamine group within this
subset of leads proved important as will be shown below.
The tert-amyl analogue of 31 and 53 was prepared (57),
and this gave an equipotent compound (IC50 ) 0.13 µM).
This result was surprising since in the 4-CN benzyl
series (Table 1) the tert-amyl derivative 21 has reduced
activity compared to the (R)-3,3-dimethyl-2-butyl 4 and
t-Bu 15 analogues. In general the addition of ortho
substituents to the para substituted benzyl ring in the
tert-amyl series gives potent compounds as previously
documented in the t-Bu series. The (2-Cl, 4-CN, 6-Me)
analogue 58 (IC50 ) 0.16 µM) as well as the (2,4-Cl)
derivative 59 (IC50 ) 0.21 µM) possess good relaxant
activity. From the t-Bu series (Table 2), it was known
that adding a 6-Me group to the (2,4-Cl) derivative
increased potency; this was also confirmed for the tert-
amyl series as the (2,4-Cl, 6-Me) analogue 60 (WAY-
151616) has an IC50 as potent as any of the best
benzylamino cyclobutenediones (60, IC50 ) 0.10 µM).
The importance of the 4-Cl substituent in 60 can be seen
in the (2-Cl, 6-Me) analogue 61 which loses activity.
However, changing the 6-Me group in 61 to a 6-Cl gives
a potent compound (62, IC50 ) 0.28 µM). Finally, (2-Et,
4-CN) derivative 63 is a potent compoundsnote again
that the (2-Et, 4-CN) was the same substitution pattern
used in the potent and efficacious phenylamino cy-
clobutenedione 1.
Additional tert-amyl cyclobutenedione analogues are
presented in Table 4. Overall, the 2,4 disubstitution and
2,4,6 trisubstitution patterns on the phenyl ring gener-
ally produces the most potent compounds. While these
new tert-amyl compounds were very potent, no com-

1208 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Gilbert et al.
Ta ble 4. In Vitro Effects of Benzylamine Variation for tert-Amyl Cyclobutenediones on Precontracted Rat Bladder Smooth Muscle
Strips (IC50, µM)a
cmpd
R1
Cl
F
F
F
F
H
F
CN
Me
Me
Me
H
H
H
Me
MeS
Br
R2
R3
R4
R5
% yield
mp (°C)
formulab
anal.c
IC50 (µM)a
nd
60
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
H
H
H
H
F
F
Cl
Cl
H
H
H
H
H
Me
Me
H
Cl
H
F
Cl
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
Me
H
H
H
Me
H
H
H
H
H
H
H
H
Me
Cl
Cl
Me
H
H
H
Me
92
72
80
60
62
80
65
32
45
42
26
59
45
54
30
23
71
247-248
C17H20Cl2N2O2
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, Ne
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, Nf
0.10 ( 0.03
0.36 ( 0.14
0.22 ( 0.04
1.5 ( 1.0
4
4
2
3
2
2
2
2
3
3
2
4
2
2
2
1
3
255-257
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
16H19FN2O2
16H18F2N2O2
16H18ClFN2O2
16H18F2N2O2
16H18F2N2O2
16H18ClFN2O2
17H18ClN3O2
18H24N2O2
242-244
201-203
273-275
1.6 ( 0.8
242-244
0.75 ( 0.45
0.87 ( 0.42
0.26 ( 0.02
0.53 ( 0.23
0.31 ( 0.08
0.20 ( 0.08
4.4 ( 4.1
H
H
242-244
267-271
Me
Me
Me
Me
Me
H
H
H
Me
177-178
284-285 (dec)
265-271 (dec)
224-226
19H26N2O2
18H23ClN2O2
17H21ClN2O2
18H24N2O2
18H24N2O2
18H24N2O2
227-231
0.83 ( 0.66
222-224
>30
>30
5.6
1.2 ( 1.1
248
280-281 (dec)
246-250 (dec)
17H22N2O2S
18H23BrN2O2
H
a
IC50: drug concentration that relaxed KCl-induced contractions in rat detrusor strips by 50%. The bladder relaxant activity could be
reversed by the addition of glyburide. Structures of compounds confirmed by 1H NMR, IR, and MS. c Analytical results are within (0.4%
b
of the theoretical value unless otherwise noted. Number of experiments. e C: calcd, 64.57; found, 63.81. f C: calcd, 57.00; found, 56.55.
d
Ta ble 5. Comparison of in Vivo Effects of Benzylamine
Cyclobutenediones 4, 15, 31, and 60 on Frequency of
Spontaneous Bladder Contractions in the Rat Hypertrophic
Bladder Model (percent change from pre-drug value, X ( SE)
spontaneous
bladder contractions
blood pressurea
dose
dose
(mg/kg)
po
frequency
ED50
(mg/kg)
po
maximum
% change
cmpd
nb (% change)c (mg/kg)d
n
4
15
31
60
3
3
3
3
5
4
6
4
-69 ( 16
-52 ( 8
-49 ( 8
-82 ( 5
NT
3.0
3.0
0.6
10
4
+7 ( 3
NT
NT
10
6
-7 ( 3
a
Initial BP values ranged from 105 ( 2 to 116 ( 5 mmHg.
Number of experiments. c Vehicle (PEG-200) effects: -2 ( 11.
ED50
b
d
F igu r e 7. Rat hypertrophied bladder model: cystometry plot
of 60 (WAY-151616). Spontaneous spikes during the bladder
filling phase are observed, indicating the presence of bladder
instability prior to the micturition contraction (upper panel).
After treatment with 3 mg/kg (po) of 60, the frequency of
spontaneous bladder contractions is reduced and a normal
micturition response is observed (lower panel).
: drug dose (po) that caused a 50% reduction in the
frequency of spontaneous bladder contractions in a rat hypertro-
phied model of bladder instability. NT ) not tested.
pound from this series is more potent than compounds
such as 31 and 60.
Compound 60, one of the most potent benzylamines
in the in vitro rat detrusor muscle strip assay, was
tested in the rat hypertrophied model of bladder insta-
bility (Table 5). Gratifyingly, 60 shows an excellent 82%
reduction in the frequency of spontaneous bladder
contractions at 3 mg/kg (po). A dose-response study
demonstrates that 60 possesses a reduced ED50 versus
compounds 15 and 31 (60, ED50 ) 0.6 mg/kg). The effect
of 60 on MAP in normotensive rats was also tested. At
10 mg/kg, compound 60 only decreased blood pressure
7%. A dose-response study with 60 shows that the ED20
for blood pressure lowering was greater than 100 mg/
kg.
(upper panel) shows spontaneous spikes which develop
during the bladder filling phase. These spontaneous
contractions lead to concomitant urine leakage as
demonstrated by increases in urine volume output. At
the end of bladder filling (∼10 min), a micturition
contraction is observed, and the bladder empties. The
lower panel shows the cystometry after oral administra-
tion of compound 60 at 3 mg/kg. The spontaneous
bladder contractions observed during the filling phase
have been significantly reduced. In addition, no urine
leakage is observed as urine output remains at zero
during the bladder filling phase. After bladder filling
(∼10 min), a normal micturition response is observed,
and urine output increases. The onset of action for 60
at this dose is 1 h, and the duration of action is 3.7 h.
A rat hypertrophied bladder cystometry plot for one
bladder filling/emptying cycle before and after treatment
with 60 is presented in Figure 7. The pretreatment plot

Potassium Channel Openers Targeted for UUI. 2
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1209
Ta ble 6. Comparison of Benzylamine Cyclobutenediones 4, 80, 60, and 81 on in Vitro Precontracted Rat Bladder Smooth Muscle
Strips and the in Vivo Frequency of Spontaneous Bladder Contractions in the Rat Hypertrophic Bladder Model (in vitro data: IC50
,
d
µM);a in vivo data: reported as ED50
)
IC50
ED50
cmpd R1 R2 R3
R4
R5
mp (°C)
formulab
anal.c
(µM)a
(mg/kg; po)d
4
CN
H
H
H
(R)-3,3-dimethyl-2-butyl 288-291 (dec) C18H21N3O2
C, H, N 0.27 ( 0.04
F: -69% at
3 mg/kg, poe
8.0 ( 4.4
0.6
80
60
81
CN
Cl
Cl
H
H
n-PrCO (R)-3,3-dimethyl-2-butyl 101-103
H
C22H27N3O3
C, H, N 12.2 ( 10.2
Cl Me
Cl Me n-PrCO tert-amyl
tert-amyl
247-248
117-118
C
C
17H20Cl2N2O2 C, H, N 0.10 ( 0.10
21H26Cl2N2O3 C, H, N C ) 245 ( 15%f 3.9 ( 1.4
a
IC50: Drug concentration that relaxed KCl-induced contractions in rat detrusor strips by 50%. The bladder relaxant activity could be
reversed by the addition of glyburide. Structures of compounds confirmed by 1H NMR, IR, and MS. c Analytical results are within (0.4%
b
d
of the theoretical value unless otherwise noted. ED50: Drug dose (po) that 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. e F: decrease in frequency
of bladder contractions in the rat hypertrophied model of bladder instability. A dose-response study was not performed on this compound
to determine an ED50
.
f C ) 245 ( 15%: a 245% additional contraction of a KCl precontracted rat bladder strip at 30 µM.
Ta ble 7. Comparison of in Vitro and in Vivo Effects of
Celikalim 82, ZD-6169 83, Compound 1, and Compound 60
Ch a r t 1
bladder effects
hemodynamic effects
selectivity
in vitro in vivo
in vivo
ED20
ratio
(MAP ED20/
bladder ED50
IC50
ED50
compd (µM)a (mg/kg)b
(mg/kg)c
82d
83d
1d
0.03
0.93
0.09
0.10
0.3
2.4
0.13
0.6
0.2
6.96
2.3
0.7
2.9
17.7
166
60
100
a
IC50: drug concentration that relaxed KCl-induced contrac-
Thus 60 prevents the spontaneous contractions in the
hypertrophied rat bladder during filling and prevents
urine leakage while still allowing normal urination after
bladder filling is complete.
b
tions in rat detrusor strips by 50%. ED50: drug dose (po) that
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
In an attempt to further modify solubility and absorp-
tion properties, putative prodrugs of compounds 4 and
60 were prepared by deprotonating benzylamine nitro-
gen with NaH and trapping the resulting anion with
n-PrCOCl (Scheme 1). Acylation of the aniline nitrogen
in the previously described phenylamino cyclobutene-
diones was shown to have small effects on in vitro
potency and in vivo efficacy,3 but in the benzylamine
series, acylation attenuates in vitro potency while the
compounds maintain some in vivo activity (Table 6). The
(4-CNbenzyl, (R)-3,3-dimethyl-2-butyl) compound 4 is
moderately active in vivo (frequency, -69% at 3 mg/
kg) despite its potent IC50 (IC50 ) 0.27 µM). The
corresponding n-butylamide 80 is 42 times less potent
in vitro but maintains some in vivo efficacy when
administered po (ED50 ) 8 mg/kg). The (2,4-Cl, 6-Me,
tert-amyl) analogue 60 has good in vitro (IC50 ) 0.10
µM) and very good in vivo activity (ED50 ) 0.6 mg/kg).
The corresponding n-butylamide 81 is a bladder smooth
muscle contractor in vitro8 and also maintains some in
vivo relaxant activity (ED50 ) 3.9 mg/kg; po). These data
suggest that n-butylamide analogues 80 and 81 may be
acting as prodrugs, either by liberating parent benzyl-
amines or a comparably active metabolite in vivo.
d
ranged from 105 ( 2 to 116 ( 5 mmHg. Reference 2.
as a bladder relaxant agent (ratio MAP ED20/bladder
ED50 ) 0.7) (Table 7).2 ZD-6169 83 exhibits weaker
activity in vitro than 82 as well as being weaker in vivo
as a bladder relaxant in our models. However, 83 shows
selectivity in the rat hypertrophied model of bladder
instability over MAP (ratio MAP ED20/bladder ED50
)
2.9).2 Diamino cyclobutenedione 1 shows excellent in
vitro potency in the precontracted rat bladder strip
model as well as potent in vivo bladder activity. Impor-
tantly, the hemodynamic effects of 1 are much less
potent than its observed bladder effects (ratio MAP
ED20/bladder ED50 ) 17.7).2 Compound 60 shows even
greater promise as a selective agent for UUI. While not
as active in the rat hypertrophied bladder model as 1
(60, ED50 ) 0.6 mg/kg; 1, ED50 ) 0.13 mg/kg), its ED20
for blood pressure lowering is 100 mg/kg. Thus 60 is
166-fold more selective for bladder effects versus he-
modynamic effects and shows great promise as a selec-
tive agent.
Con clu sion s
We have shown that the novel phenylamino cyclobu-
tendione KCO 1 can be successfully modified into a
series of related benzylamino cyclobutenediones. Con-
version of the anline portion of 1 to a benzylamine in
conjunction with changing the alkylamine portion of 1
from a (R)-3,3-dimethyl-2-butyl moiety to a t-Bu group
gives a series of compounds with potent in vitro potency
in the KCl-contracted rat bladder strip assay. Ortho and
An assessment of the efficacy and bladder selectivity
of novel KCOs 1 and 60 was performed and compared
to celikalim 82, an antihypertensive KCO, and to ZD-
6169 83, an anilide tertiary carbinol KCO currently in
Phase II clinical trials for the treatment of UUI (Chart
1). Celikalim 82 shows both bladder and hemodynamic
effects in our models and could not be used selectively

1210 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Gilbert et al.
4-{[3,4-Dioxo-2-(1,2,2-tr im eth yl-pr opylam in o)-cyclobu t-
1-en yla m in o]-m eth yl}-3-eth yl-ben zon itr ile (3). 4-Cyano-
2-ethylbenzaldehyde oxime10 (2.9 g, 17 mmol) and 142 mL of
2 N HCl were stirred at room temperature. Acetone (100 mL)
was added to produce a homogeneous solution. After 5 days
the reaction mixture was diluted with 500 mL of EtOAc, and
solid NaCl was added until the aqueous layer was saturated.
After the mixture was stirred at room temperature for 1 h,
the EtOAc layer was separated and dried over Na2SO4.
Concentration under reduced pressure yielded 7.0 g of a solid
which was chromatographed (SiO2 gel) with hexanes and then
hexanes:EtOAc (8:1) to give 2.0 g (12.6 mmol, a 75% yield) of
4-cyano-2-ethylbenzaldehyde, which was used without further
purification.
para substituents on the benzyl moiety improve activity.
Electron-withdrawing groups (CN, F, Cl, Br) in the para
position appear to be optimal, although in some cases
activity is maintained with electron-donating groups.
Ortho substituents appear to improve in vitro activity.
Unfortunately, the N-benzyl-N′-t-Bu cyclobutenediones
have weaker in vivo activity than their N-phenyl-N′-
alkyl counterparts.
Replacement of the t-Bu group with a tert-amyl
moiety increases the in vitro potency of the benzylamino
cyclobutenedionessmany of the tert-amyl compounds
have in vitro IC50s below 1.0 µM. As in the case of the
t-Bu compounds, para substituents on the phenyl ring
give optimal activity; ortho substituents can further
improve activity. The potent tert-amyl compound 60 was
tested in vivo and showed excellent efficacy (60,
ED50 ) 0.6 mg/kg, po). Most importantly, 60 represents
a series of novel KCOs with in vivo selectivity for
bladder relaxant effects over hemodynamic effects (ratio
MAP ED20/bladder ED50 ) 166). Stability assessment
studies using rat and human liver microsomes show
that 60 remians intactsno benzylamine is produced as
a metabolite. Compound 60 was also found to be clean
in a NOVA Screen receptor binding profile and AMES
negative at all concentrations tested (up to 5000 µg/
plate). In addition, a 10 day pilot toxicological study in
the rat at doses up to 200 mg/kg produced no drug
related abnormalities. 3-(2,4-Dichloro-6-methyl-benzy-
lamino)-4-(1,1-dimethyl-propylamino)-cyclobut-3-ene-
1,2-dione 60 represents an attractive developmental
candidate for the treatment of UUI.
4-Cyano-2-ethylbenzaldehyde (2.0 g, 13 mmol) in 63 mL of
MeOH was cooled to 0 °C. Solid NaBH4 (480 mg, 13 mmol)
was added. The reaction mixture was stirred at 0 °C for 1 h.
A second portion of NaBH4 (0.48 g, 13 mmol) was added, and
stirring was continued at 0 °C for 40 min. A third portion of
NaBH4 (0.48 g, 13 mmol) was added, and the reaction mixture
was stirred at 0 °C for 20 min. The ice bath was then removed,
and stirring was continued at room temperature for 1 h. Water
(28 mL) was then added, and the reaction mixture was stirred
at room temperature for 24 h. The solution was then diluted
with EtOAc (100 mL) and divided into two portions, which
were each extracted with EtOAc (300 mL) and brine (40 mL).
The combined EtOAc extracts were concentrated under re-
duced pressure, and the resulting residue was chromato-
graphed (SiO2 gel) with hexanes:EtOAc (7:1) and then hexane:
EtOAc (3:1) to yield 1.53 g (9.5 mmol, a 75% yield) of 4-cyano-
2-ethylbenzyl alcohol a white solid: 1H NMR (DMSO-d6) δ
7.70-7.50 (m, 3H), 5.39 (br m, 1H), 4.59 (s, 2H), 2.60 (q, 2H),
1.18 (t, 3H).
4-Cyano-2-ethylbenzyl alcohol (950 mg, 5.9 mmol), 1.05 g
(7.1 mmol) of phthalimide, 1.85 g (7.1 mmol) of Ph3P, and 39
mL of THF were mixed and cooled to 0 °C in an ice bath.
Diethylazodicarboxylate (1.09 mL, 6.9 mmol) was added drop-
wise. The reaction mixture was allowed to warm to room
temperature as the ice bath melted. After 24 h the reaction
mixture was concentrated under reduced pressure, and the
resulting residue was chromatographed (SiO2 gel) with hex-
anes/EtOAc (5/1), hexanes/EtOAc (3/1), and then hexanes/
EtOAc (2/1) to yield 1.85 g (6.37 mmol, a 108% yield) of a white
solid: 1H NMR (DMSO-d6) δ 8.00-7.80 (m, 4H), 7.70 (s, 1H),
7.59 (d, 1H), 7.30 (d, 1H), 4.86 (s, 2H), 2.81 (q, 2H), 1.22 (t,
3H).
Exp er im en ta l Section
Melting points were determined on a Thomas-Hoover Mel-
temp apparatus and are uncorrected. The proton nuclear
magnetic resonance (1H NMR) spectra were recorded at 300
MHz on a Bruker DPX-300 spectrometer using tetramethyl-
silane (δ 0.0) as an internal standard. Infrared (IR) spectra
were obtained as KBr pellets. Combustion analyses were
obtained using a Perkin-Elmer Series II 2400 CHNS/O ana-
lyzer. Mass spectra were obtained using a Micromass Platform
Electrospray Ionization Quadrapole mass spectrometer. Flash
chromatography was performed using EM Science 230-400
mesh silica gel. Thin-layer chromatography (TLC) was per-
formed on Analtech silica gel GHLF 250 µM prescored plates.
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 1-4 and
6. The syntheses and spectral data for representative com-
pounds from these tables are shown below. The term “rota-
mers” which appears as a comment in some of the 1H NMR
data is defined as conformational isomers due to restricted
rotation about the vinylogous amide bond.
(R)-3-Eth oxy-4-(1,2,2-tr im eth yl-pr opylam in o)-cyclobu t-
3-en e-1,2-d ion e. Tetrahydrofuran (15 mL), 50 mL (10 mmol)
of a 0.2 M solution of (R)-2-amino-3,3-dimethylbutane9 in
absolute EtOH, and 2.26 g (10 mmol) of 3,4-dibutoxy-3-
cyclobutene-1,2-dione were stirred together for approximately
65 h at room temperature. The waxy solid remaining after
removal of solvent was dissolved in chloroform (15 mL) and
flash chromatographed on SiO2 gel (hexanes/EtOAc). The
appropriate fractions were freed of solvent to yield 2.41 g (9.53
mmol, a 95% yield) of a cream-colored waxy solid: mp 90-99
°C. Two recrystallizations of 1.1 g of this material from
hexanes provided 833 mg of the title compound as a white
solid: mp 90-93 °C; 1H NMR (DMSO-d6) δ 8.73 and 8.50 (two
br d, 1H, rotamers), 4.64 (m, 2H), 3.92 and 3.41 (two m, 1H,
rotamers), 1.71 (m, 2H), 1.38 (m, 2H), 1.11 (m, 3H), 0.91 (t,
3H), 0.84 (m, 9H); IR (KBr) 3135, 1800, 1690 cm-1; MS 253
(M+). Anal. C14H23NO3: C, H, N.
N-(4-Cyano-2-ethylbenzyl)phthalimide (1.73 g, 6.0 mmol),
1.07 mL of 35% N2H4 (12 mmol), and 105 mL of absolute EtOH
were heated at 65 °C under argon for 3 h and then at 85 °C
for 5 h. The reaction mixture was concentrated under reduced
pressure, resuspended in 35 mL of absolute EtOH, filtered,
and rinsed with absolute EtOH (2 × 30 mL). Concentration
under reduced pressure yielded a solid, which was suspended
in 100 mL of EtOAc and filtered. The filtrate was concentrated
under reduced pressure to give 770 mg (4.81 mmol, an 81%
yield) of 4-cyano-2-ethylbenzylamine as a moist solid: 1H NMR
(CDCl3) δ 7.50 (m, 3H), 3.94 (s, 2H), 2.70 (m, 2H), 1.43 (br m,
2H), 1.21 (t, 3H).
4-Cyano-2-ethylbenzylamine (0.21 g, 1.3 mmol) was placed
in 5.5 mL of absolute EtOH. (R)-3-Ethoxy-4-(1,2,2-trimethyl-
propylamino)-cyclobut-3-ene-1,2-dione (300 mg, 1.3 mmol) was
added, followed by 5 mL of CH2Cl2. The clear solution was
stirred at room temperature for 5 days. The reaction mixture
was concentrated under reduced pressure, and the resulting
solid was chromatographed (SiO2 gel) with 1% MeOH in CH2-
Cl2, then 3% MeOH in CH2Cl2 to give 190 mg (0.56 mmol, a
43% yield) of the (R) isomer of the 3 as a light tan solid: mp
208-212 °C; [R]25 13.4° (c 0.0086 g/mL, DMSO); 1H NMR
D
(DMSO-d6) δ 7.71 (m, 2H), 7.60 (br m, 1H), 7.48 (br d, 1H),
7.27 (br d, 1H), 4.85 (m, 2H), 3.90 (m, 1H), 2.69 (q, 2H), 1.17
(t, 3H), 1.10 (d, 3H), 0.86 (s, 9H); IR (KBr) 3200, 2970, 2230,
1800, 1650 cm-1; MS 339 (M+). Anal. C20H25N3O2 Calcd: C,
70.77; H, 7.42; N, 12.38. Found: C, 70.05; H, 7.29; N, 12.13.

Potassium Channel Openers Targeted for UUI. 2
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1211
3-Eth oxy-4-(4-cya n o-ben zyla m in o)-cyclobu t-3-en e-1,2-
d ion e. To 1.2 g of 4-cyanobenzylamine (9.1 mmol) in 40 mL
of absolute EtOH was added 1.6 g (9.4 mmol) of 3,4-diethoxy-
3-cyclobutene-1,2-dione. The reaction mixture was stirred at
room temperature for 5 days. The resulting white suspension
was filtered and dried under vacuum at 0.4 mm and 65 °C to
give 1.07 g (4.16 mmol, a 46% yield) of the title compound as
a white solid: 1H NMR (DMSO-d6) δ 9.29 and 9.08 (two br m,
1H, rotamers), 7.83 (d, 2H), 7.49 (d, 2H), 4.80-4.50 (m, 4H),
1.36 and 1.28 (two t, 3H, rotamers); MS 257 (MH+).
4-[(2-iso-P r opylam in o-3,4-dioxo-cyclobu t-1-en ylam in o)-
m eth yl]-ben zon itr ile (13). The title compound was prepared
according to the procedure of compound 10 using 1.0 g (3.9
mmol) of 3-ethoxy-4-(4-cyano-benzylamino)-cyclobut-3-ene-1,2-
dione and 1.2 mL (828 mg, 14 mmol) of i-PrNH2: yield 900
mg (3.35 mmol, an 86% yield); mp 276-278 °C; 1H NMR
(DMSO-d6) δ 7.84 (d, 2H), 7.70 (br m, 1H), 7.51 (d, 2H), 7.40
(br m, 1H), 4.79 (d, 2H), 4.09 (br m, 1H), 1.18 (d, 6H); IR (KBr)
3150, 2980, 2250, 1800, 1660 cm-1; MS 270 (MH+). Anal.
C
15H15N3O2 Calcd: C, 66.90; H, 5.61, N, 15.60. Found: C,
66.24; H, 5.45, N, 15.39.
(R)-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]-m eth yl}-ben zon itr ile (4). 3-Ethoxy-
4-(4-cyano-benzylamino)-cyclobut-3-ene-1,2-dione (400 mg, 1.56
mmol) in 15 mL of absolute EtOH and 11.7 mL of a 0.2 M
solution of (R)-2-amino-3,3-dimethylbutane in absolute EtOH
(2.3 mmol) were heated at reflux for 17 h. The reaction mixture
was cooled to room temperature, and the resulting white
suspension was filtered, rinsed with absolute EtOH (2 × 10
mL), and dried under vacuum (0.4 mm, 85 °C) to give 450 mg
(1.45 mmol, a 93% yield) of 4 as a white solid: mp 288-291
°C (dec); [R]25D ) +28.2° (9.7 mg/mL, DMSO); 1H NMR (DMSO-
d6) δ 7.85 (d, 2H), 7.70 (br m, 1H), 7.52 (d, 2H), 7.29 (br d,
1H), 4.84 (m, 2H), 3.91 (br m, 1H), 1.10 (d, 3H), 0.85 (s, 9H);
IR (KBr) 3200, 2960, 2250, 1800, 1650 cm-1; MS 312 (MH+).
Anal. C18H21N3O3: C, H, N.
4-[(2-n -Bu t yla m in o-3,4-d ioxo-cyclob u t -1-en yla m in o)-
m eth yl]-ben zon itr ile (14). The title compound was prepared
according to the procedure of compound 10 using 250 mg (0.98
mmol) of 3-ethoxy-4-(4-cyano-benzylamino)-cyclobut-3-ene-1,2-
dione and 0.096 mL (72 mg, 0.98 mmol) of n-BuNH2: yield
1
220 mg (0.78 mmol, a 79% yield); mp 250-252 °C; H NMR
(DMSO-d6) δ 7.84 (d, 2H), 7.80 (br m, 1H), 7.50 (d, 2H), 7.40
(br m, 1H), 4.78 (d, 2H), 3.49 (br m, 2H), 1.47 (m, 2H), 1.29
(m, 2H), 0.87 (t, 3H); IR (KBr) 3160, 2950, 2250, 1810, 1640
cm-1; MS 283 (M+). Anal. C16H17N3O2: C, H, N.
4-[(2-ter t-Bu tyla m in o-3,4-d ioxo-cyclobu t-1-en yla m in o)-
m eth yl]-ben zon itr ile (15). The title compound was prepared
according to the procedure of compound 10 using 1.5 g (5.9
mmol) of 3-ethoxy-4-(4-cyano-benzylamino)-cyclobut-3-ene-1,2-
dione and 0.62 mL (432 mg, 5.9 mmol) of t-BuNH2: yield 1.59
g (5.62 mmol, a 95% yield); mp 283-287 °C (dec); 1H NMR
(DMSO-d6) δ 7.85 (d, 2H), 7.84 (br t, 1H), 7.56 (br s, 1H), 7.52
(d, 2H), 4.81 (d, 2H), 1.35 (s, 9H); IR (KBr) 3150, 2980, 2250,
1800, 1660 cm-1; MS 284 (MH+). Anal. C16H17N3O2: C, H, N.
4-Cya n o-N-[3,4-d ioxo-2-(1,2,2-tr im eth yl-p r op yla m in o)-
cyclobu t-1-en yl]-ben za m id e (26). To a -10 °C solution of
3,4-diethoxy-3-cyclobutene-1,2-dione (0.51 mL, 582 mg, 3.42
mmol), 500 mg (3.42 mmol) of 4-cyanobenzamide, and 35 mL
of DMF was added 205 mg (6.84 mmol) of an 80% NaH/mineral
oil dispersion. The mixture was allowed to stir at 0 °C for 2 h.
After pouring into 100 mL of brine/5 mL of 1 N HCl(aq), the
resulting mixture was extracted with EtOAc (3 × 50 mL). The
combined organics were dried over MgSO4, filtered, and
evaporated with 10 g of SiO2 gel. The resulting powder was
placed on top of a SiO2 gel flash column and eluted with
hexanes/EtOAc (1.5/1 to 1/1) to produce 260 mg (0.96 mmol, a
28% yield) of 4-cyano-N-[3,4-dioxo-2-ethoxy-cyclobut-1-enyl]-
benzamide.
4-[(2-Am in o-3,4-d ioxo-cyclobu t-1-en yla m in o)-m eth yl]-
ben zon itr ile (9). 3-Ethoxy-4-(4-cyano-benzylamino)-cyclobut-
3-ene-1,2-dione (250 mg, 0.98 mmol) and 4.8 mL of NH3(g)
saturated CH3CN were mixed in 4.8 mL absolute ethanol at
23 °C. After standing for several hours, the resulting white
precipitate was filtered, rinsed with EtOAc, and dried under
vacuum to give 140 mg (0.62 mmol, a 64% yield) of 9 as a white
1
solid: mp > 260 °C; H NMR (DMSO-d6) δ 8.00-7.20 (br s,
1H), 7.83 (d, 2H), 7.50 (d, 2H), one N-H proton not seen; IR
(KBr) 3420, 3100, 1800, 1650, cm-1; MS 227 (M+). Anal.
C12H9N3O2: C, H, N.
4-[(2-Met h yla m in o-3,4-d ioxo-cyclob u t -1-en yla m in o)-
m eth yl]-ben zon itr ile (10). 3-Ethoxy-4-(4-cyano-benzylamino)-
cyclobut-3-ene-1,2-dione (250 mg, 0.98 mmol), 0.12 mL (0.98
mmol) of an 8 M solution of MeNH2 in EtOH, and 19.5 mL of
absolute ethanol were allowed to stand at room temperature
for 3 days. The resulting white precipitate was filtered, rinsed
with EtOAc, and dried under vacuum to give 210 mg (0.87
mmol, an 89% yield) of 10 as a white solid: mp 302-306 °C
(dec); 1H NMR (DMSO-d6) δ 7.90 (br m, 1H), 7.84 (d, 2H), 7.50
(d, 2H), 7.30 (br m, 1H), 4.77 (d, 2H), 3.11 (br s, 3H); IR (KBr)
3180, 2980, 2250, 1800, 1650 cm-1; MS 241 (M+). Anal.
C13H11N3O2 Calcd: C, 64.72; H, 4.60; N, 17.42. Found: C,
64.19; H, 4.44; 17.10.
To a -10 °C solution of 260 mg (0.96 mmol) of 4-cyano-N-
[3,4-dioxo-2-ethoxy-cyclobut-1-enyl]-benzamide and 10 mL of
MeCN was added 0.195 mL (147 mg, 1.44 mmol) of 2-amino-
3,3-dimethylbutane. After the mixture was stirred at -10 °C
for 2 h, the solvent was evaporated, and the resulting residue
was triturated with CH2Cl2/petroleum ether to give 300 mg
(0.92 mmol, a 96% yield) of 26 as a white solid: mp 265-266
4-[(2,2-Dim eth ylam in o-3,4-dioxo-cyclobu t-1-en ylam in o)-
m eth yl]-ben zon itr ile (11). 3-Ethoxy-4-(4-cyano-benzylamino)-
cyclobut-3-ene-1,2-dione (250 mg, 0.98 mmol) was dissolved
in 4.8 mL of CH2Cl2 and 4.8 mL of absolute EtOH to give a
clear solution. N,N-Diisopropylethylamine (0.17 mL, 126 mg,
0.98 mmol) was added followed by 79.6 mg (0.98 mmol) of N,N-
dimethylamine hydrochloride, and the resulting mixture was
stirred at 23 °C. The resulting white precipitate was filtered,
rinsed with EtOAc, and dried under vacuum to give 170 mg
(0.67 mmol, a 68% yield) of 11 as a white solid: mp 244-246
°C; 1H NMR (DMSO-d6) δ 8.21 (t, J ) 6.4 Hz, 1H), 7.82 (d,
J ) 8.4 Hz, 2H), 7.52 (d, J ) 8.4 Hz, 2H), 4.82 (d, J ) 6.4 Hz,
2H), 3.16 (s, 6H); IR (KBr) 3190, 2200, 1760, 1730 cm-1; MS
255 (M+). Anal. C14H13N3O2: C, H, N.
1
°C; H NMR (DMSO-d6) δ 8.15 (d, J ) 8.4 Hz, 2H), 8.01 (d,
J ) 8.4 Hz, 2H), 7.68 (br d, 1H), 4.05-4.09 (m, 1H), 1.17 (d,
J ) 6.8 Hz, 3H), 0.91 (s, 9H); MS 325 (M+). Anal. C18H19N3O3:
C, H, N.
4-{2-[3,4-Dioxo-2-(1,2,2-tr im eth yl-p r op yla m in o)-cyclo-
bu t-1-en yla m in o]-eth yl}-ben zon itr ile (27). To a solution
of 913 mg (5.0 mmol) of 2-(aminoethyl)benzonitirle hydrochlo-
ride,11 1.27 g (5.0 mmol) of 3-butoxy-4-(1,2,2-trimethyl-propyl-
amino)-cyclobut-3-ene-1,2-dione, and 10 mL of pyridine was
added 0.71 mL (506 mg, 5.0 mmol) of Et3N. After being stirred
at room temperature for 70 h, the mixture was evaporated to
an off-white solid, triturated with 3 × 25 mL of H2O, washed
with 3 × 25 mL of Et2O, and dried under vacuum to produce
1.39 g (4.27 mmol, an 85% yield) of 27 as a white solid: mp
4-[(2-n -P r op yla m in o-3,4-d ioxo-cyclobu t-1-en yla m in o)-
m eth yl]-ben zon itr ile (12). The title compound was prepared
according to the procedure of compound 10 using 250 mg (0.98
mmol) of 3-ethoxy-4-(4-cyano-benzylamino)-cyclobut-3-ene-1,2-
dione and 0.08 mL (58 mg, 0.98 mmol) of n-PrNH2: yield 180
mg (0.67 mmol, a 68% yield); mp 241-245 °C; 1H NMR
(DMSO-d6) δ 7.84 (d, 2H), 7.80 (br m, 1H), 7.50 (d, 2H), 7.45
(br m, 1H), 4.78 (d, 2H), 3.44 (m, 2H), 1.50 (m, 2H), 0.86 (t,
3H); IR (KBr) 3170, 2980, 2250, 1800, 1660 cm-1; MS 269 (M+).
Anal. C15H15N3O2: C, H, N.
1
273-275 °C (dec); H NMR (DMSO-d6) δ 7.76 (d, J ) 7.3 Hz,
2H), 7.44 (d, J ) 6.1 Hz, 2H), 7.30-7.22 (brm, 1H), 7.22-7.15
(brm, 1H), 3.92-3.84 (brm, 1H), 3.72-3.82 (m, 2H), 2.94 (t,
J ) 5.3 Hz, 2H), 1.08 (d, J ) 5.2 Hz, 3H), 0.83 (s, 9H); IR
(KBr) 3300, 3150, 2960, 1780, 1600 cm-1; MS 325 (M+). Anal.
C
19H23N3O2: C, H, N.
4-[2-(2-ter t-Bu tylam in o-3,4-dioxo-cyclobu t-1-en ylam in o)-
eth yl]-ben zon itr ile (28). To a solution of 913 mg (5.0 mmol)
of 2-(aminoethyl)benzonitrile hydrochloride,11 1.12 g (5.0 mmol)

1212 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Gilbert et al.
of 3-butoxy-4-(tert-butylamino)-cyclobut-3-ene-1,2-dione, and
10 mL of pyridine was added 0.71 mL (506 mg, 5.0 mmol) of
Et3N. After being stirred at room temperature for 70 h, the
mixture was evaporated to an off-white solid, triturated with
3 × 25 mL of H2O, washed with 3 × 25 mL of Et2O, and dried
under vacuum to produce 767 mg (2.58 mmol, a 52% yield) of
28 as an off-white solid: mp 307-308 °C (dec); 1H NMR
(DMSO-d6) δ 7.77 (d, J ) 7.3 Hz, 2H); 7.45 (d, J ) 6.1 Hz,
2H); 7.39 (m, 2H), 3.79 (q, J ) 4.9 Hz, 2H), 2.94 (t, J ) 5.3
Hz, 2H), 1.33 (s, 9H); IR (KBr) 3250, 3200, 2960, 2400, 1780,
1660 cm -1; MS 297 (M+). Anal. C17H19N3O2: C, H, N.
(88.68 mmol, a 59% yield) of 2-chloro-4-cyanobenzyl bromide
as a white solid: mp 81-84 °C; 1H NMR (DMSO-d6) δ 8.10 (d,
1H), 7.82 (m, 2H), 4.69 (s, 2H). IR (KBr) 2220 cm-1
.
A mixture of 2-chloro-4-cyanobenzyl bromide (20.29 g, 88.0
mmol) and 17.92 g (96.8 mmol) of potassium phthalimide in
200 mL of DMF was stirred at room temperature, and the
reaction temperature rose to approximately 36 °C after ap-
proximately 5 min with formation of a tan suspension. The
temperature then receded, and stirring was continued for 2 h
at room temperature. After removal of solvent, the residue was
triturated thoroughly with H2O and dried. The resulting buff
solid was treated with approximately 500 mL of boiling EtOAc,
gravity filtered to remove a small amount of white insoluble
material, heated to boiling, treated with charcoal, and filtered.
Concentration and cooling of the filtrate precipitates 20.26 g
(68.3 mmol, a 78% yield) of the N-(2-chloro-4-cyanobenzyl)-
4-{1-[3,4-Dioxo-2-(1,2,2-tr im eth yl-p r op yla m in o)-cyclo-
bu t-1-en yla m in o]-eth yl}-ben zon itr ile (29). To (R)-3-ethoxy-
4-(1,2,2-trimethyl-propylamino)-cyclobut-3-ene-1,2-dione (200
mg, 0.89 mmol) and 4.4 mL of absolute EtOH was added 130
mg (0.89 mmol) of 4-[(()-1-aminoethyl]benzonitrile, and the
resulting solution was stirred at 23 °C. After 15 h, the reaction
mixture was diluted with 6 mL of MeCN, and the resulting
precipitate was filtered and set aside (fraction #1). To the
filtrate of this reaction was added 130 mg (0.89 mmol) of 4-[(()-
1-aminoethyl]benzonitrile, and the resulting mixture was
heated to 85 °C. After 8 h, an additional portion of 260 mg
(1.78 mmol) of 4-[(()-1-aminoethyl]benzonitrile was added.
After a total of 32 h, the reaction mixture was cooled to 23 °C,
and the resulting precipitate was isolated by filtration and
washed with 2 × 5 mL of CH3CN. The resulting solid was set
aside (fraction #2). The filtrate was evaporated to one-third
of its volume which produces additional precipitate (fraction
#3). All three fractions are combined to give 150 mg (0.46
1
phthalimide as a white solid: mp 172.5-173.0 °C; H NMR
(DMSO-d6) δ 8.10 (d, 1H), 7.90 (m, 4H), 7.75 (dd, 1H), 7.52 (d,
1H), 4.88 (s, 2H); IR (KBr) 2220, 1770, 1715 cm-1
.
A mechanically stirred suspension of N-(2-chloro-4-cyanoben-
zyl)phthalimide (18.99 g, 64 mmol) in 150 mL of absolute
EtOH was treated with 6.41 g (128 mmol) of N2H4‚H2O, and
the resulting mixture was refluxed for 1 h and then allowed
to stand at room temperature for approximately 16.5 h. The
reaction mixture was stirred at room temperature, 90 mL of
2 N HCl was added slowly, and the mixture was stirred for 10
min. The mixture was filtered, and the insolubles were
triturated thoroughly with absolute EtOH and then with H2O.
The combined filtrates and triturates were evaporated, and
resulting solids were added to 250 mL of ice-H2O and basified
with 90 mL of 2.5 N NaOH. The resulting mass was extracted
thoroughly with CHCl3, and the extracts were washed with
H2O and brine and dried (anhydrous Na2SO4). Removal of
solvent gave a cream-colored solid which was recrystallized
from hexane to provide 6.85 g (41.11 mmol, a 64% yield) of
2-chloro-4-cyanobenzylamine as a white solid: mp 85.0-87.0
°C; 1H NMR (DMSO-d6) δ 7.96 (d, 1H), 7.82 (dd, 1H), 7.77 (m,
1H), 3.82 (s, 2H), 2.12 (br m, 2H); IR (KBr, cm-1) 3380, 3320,
2230.
1
mmol, 52% yield) of 29 as a white solid: mp 262-264 °C; H
NMR (DMSO-d6) δ 7.85 (d, J ) 8.4 Hz, 2H), 7.75 (br m, 1H),
7.55 (d, J ) 7.9 Hz, 2H), 7.21 (br m, 1H), 5.27-5.25 (br m,
1H), 3.96-3.94 (br m, 1H), 1.54 (d, J ) 7.0 Hz, 3), 1.11 (d,
J ) 6.8 Hz, 2H), 0.86 (s, 9H); IR (KBr) 3200, 2950, 1800, 1670
cm-1; MS 326 (M)+. Anal. C19H23N3O2 Calcd: C, 70.13; H, 7.13;
N, 12.91. Found: C, 69.46; H, 6.99; 12.96.
3-Bu toxy-4-ter t-bu tyla m in o-cyclobu t-3-en e-1,2-d ion e.
A solution of 3,4-dibutoxy-3-cyclobutene-1,2-dione (11.31 g, 50
mmol) and 3.66 g (50 mmol) of t-BuNH2 in 80 mL of THF was
stirred at room temperature for 71 h. The solvent was removed,
and a solution of the residue in chloroform was washed with
water and dried over Na2SO4. Removal of the solvent and
gravity chromatography on 350 g of neutral activity III SiO2
gel (eluting with hexanes/CHCl3) provided 9.83 g (43.69 mmol,
an 87% yield) of 3-butoxy-4-tert-butylamino-cyclobut-3-ene-1,2-
dione as white solid: mp 67.0-68.5 °C. Two recrystallizations
of 800 mg of this product afforded 551 mg of the title compound
as a white solid: mp 68-69 °C; 1H NMR (DMSO-d6) δ 8.75
and 8.59 (two br s, 1H, rotamers), 4.66 (br m, 2H), 1.72 (m,
2H), 1.40 (m, 2H), 1.31 (m, 9H), 0.91 (t, 3H); IR (KBr) 3140,
1780, 1700 cm-1; MS 225 (M+). Anal. C12H19NO3: C, H, N.
Tetrahydrofuran (50 mL), 6.76 g (30 mmol) of 3-butoxy-4-
tert-butylamino-cyclobut-3-ene-1,2-dione, and 5.0 g (30 mmol)
of 2-chloro-4-cyanobenzylamine were refluxed for 6 h and then
allowed to stand at room temperature for 16 h. Following
removal of solvent from the reaction mixture, the residue was
triturated thoroughly with Et2O and dried to give a buff solid.
This material in approximately 1.4 L of boiling acetone was
filtered to remove a small amount of white solid. The hot
filtrate was treated with charcoal, filtered, concentrated, and
cooled to afford 6.52 g (20.52 mmol, a 68% yield) of 31 as a
cream-colored solid. Two additional recrystallizations of this
material from acetone gave 4.78 g (15.03 mmol, a 50% yield)
1
of the title compound as a white solid: mp 243.5-245 °C; H
3-Ben zyla m in o-4-(ter t-bu tyla m in o)-cyclobu t-3-en e-1,2-
d ion e (30). The title compound was prepared according to the
procedure of compound 10 using 0.65 mL (638 mg, 6.0 mmol)
of benzylamine and 1.13 g (5.0 mmol) of 3-butoxy-4-tert-
butylamino-cyclobut-3-ene-1,2-dione: yield 1.08 g (4.18 mmol,
NMR (DMSO-d6) δ 8.10 (d, 1H), 7.88 (dd, 1H), 7.82 (m, 1H),
7.66 (br s, 1H), 7.61 (d, 1H), 4.88 (d, 2H), 1.34 (s, 9H); IR (KBr)
3320, 3230, 2240, 1780, 1665 cm-1; MS 317/319 (M+). Anal.
C
16H16ClN3O2: C, H, N, Cl.
1
an 84% yield); mp 306-307 °C (dec); H NMR (DMSO-d6) δ
3-ter t-Bu tyla m in o-4-[(p yr id in -4-ylm eth yl)-a m in o]-cy-
7.74 (br s, 1H), 7.49 (br s, 1H), 7.28-7.40 (complex m, 5H),
4.71 (d, J ) 6.2 Hz, 2H), 1.35 (s, 9H); IR (KBr) 3200, 1780,
1700 cm-1; MS 258 (M+). Anal. C15H18N2O2: C, H, N.
clobu t-3-en e-1,2-d ion e (35). To a solution of 1.13 g (5.0
mmol) of 3-butoxy-4-(4-cyano-benzylamino)-cyclobut-3-ene-1,2-
dione and 5 mL of pyridine was added 1.0 mL (1.08 g, 10 mmol)
of 4-(aminomethyl)pyridine, and the resulting solution was
stirred at 23 °C for 8 h. Evaporation of solvent leaves a white
solid. Ether (25 mL) is added, and the resulting slurry was
stirred at 23 °C for 1 h. Filtration and washing of the collected
solid with 3 × 10 mL of Et2O yields 990 mg (3.82 mmol, a
76% yield) of the title compound as a white solid: mp 271 °C
4-[(2-ter t-Bu tyla m in o-3,4-d ioxo-cyclobu t-1-en yla m in o)-
m eth yl]-3-ch lor o-ben zon itr ile (31). A mixture of 3-chloro-
4-methylbenzonitrile (22.74 g, 150 mmol), 32.04 g (180 mmol)
of NBS, and 2.46 g (15 mmol) of 2,2′-azobis-2-methylpropioni-
trile in 120 mL of CCl4 was carefully warmed to reflux
temperature whereupon a moderate exotherm occurred and
refluxing proceeded for approximately 10 min without external
heating. Heating was then resumed, and refluxing continued
for 26 h. The hot reaction mixture was suction filtered, and
the insolubles were rinsed with CCl4 (3 × 25 mL). The
combined CCl4 fractions were washed with H2O and dried over
Na2SO4. Removal of solvent gave a yellow mush which was
crystallized from hexane (using decolorizing charcoal). The
product again was recrystallized from hexane to yield 20.44 g
1
(dec); H NMR (DMSO-d6) δ 8.55 (dd, J ) -4.3, 1.1 Hz, 2H),
7.81 (brt, 1H), 7.58 (brs, 1H), 7.32 (dd, J ) 4.3, 1.1 Hz, 2H),
4.76 (d, J ) 4.8 Hz, 2H), 1.36 (s, 9H); IR (KBr) 3320, 3230,
1780, 1660 cm -1; MS 259 (M+). Anal. C14H17N3O2: C, H, N.
3-ter t-Bu tyla m in o-4-(2,4-d ich lor o-6-m eth yl-ben zyla m i-
n o)-cyclobu t-3-en e-1,2-d ion e (48). The title compound was
prepared according to the procedure for compound 10 using
3-ethoxy-4-tert-butylamino-cyclobut-3-ene-1,2-dione (0.22 g, 1.1

Potassium Channel Openers Targeted for UUI. 2
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6 1213
mmol) and 2,4-dichloro-6-methylbenzylamine (0.22 g, 1.2
mmol, containing approximately 5% of a compound which is
regioisomeric with respect to the substitution on the aryl
ring): yield 340 mg (0.99 mmol, an 89% yield) of a white solid
which contains approximately 5% of a compound which is
regioisomeric with respect to substitution on the aryl ring: mp
264-268 °C; 1H NMR (DMSO-d6) δ 7.54 (d, 1H), 7.46 (s, 1H),
7.43 (br t, 1H), 7.39 (d, 1H), 4.89 (d, 2H), 4.72 (d, minor isomer),
Anal. C12H18ClN3O2 Calcd: C, 61.54; H, 5.47; N, 12.66.
Found: 60.81; H, 5.40; N, 12.52.
3-Ch lor o-4-{[2-(1,1-d im et h yl-p r op yla m in o)-3,4-d ioxo-
cyclobu t-1-en ylam in o]-m eth yl}-5-m eth yl-ben zon itr ile (58).
To 3-(2-chloro-4-cyano-6-methyl-benzylamino)-4-ethoxy-cyclobut-
3-ene-1,2-dione (0.24 g, 0.79 mmol) prepared according to the
procedure for compound 55 were added 5 mL (3.7 g, 42.8 mmol)
of 1,1-dimethylpropylamine and 3 mL of CH2Cl2, and the
resulting mixture was heated at reflux for 8 h. The solvent
was removed under reduced pressure to give a solid, which
was triturated with EtOAc, filtered, rinsed with additional
EtOAc, and dried in vacuo (0.4 mm, 70 °C) to give 170 mg
(0.49 mmol, a 62% yield) of 58 as a white solid: mp 258-262
°C (dec); 1H NMR (DMSO-d6) δ 7.99 (s, 1H), 7.78 (s, 1H), 7.51
(br t, 1H), 7.35 (s, 1H), 4.98 (d, 2H), 2.46 (s, 3H), 1.66 (q, 2H),
1.29 (s, 6H), 0.80 (t, 3H); IR (KBr) 3200, 2980, 2200, 1800,
1650 cm-1; MS 345/347 (M+). Anal. C18H20ClN3O2: C, H, N.
2.40 (s, 3H), 2.31 (s, minor isomer), 1.34 (s, 9H); IR (KBr, cm-1
3200, 2950, 1800, 1650; MS 340/342/344 (M+). Anal. C16H18
Cl2N2O2: C, H, N.
)
-
3-Ch lor o-4-{[3,4-dioxo-2-(1,2,2-tr im eth yl-pr opylam in o)-
cyclobu t-1-en ylam in o]-m eth yl}-5-m eth yl-ben zon itr ile (55).
To 2-chloro-4-cyano-6-methylbenzaldehyde oxime (1.2 g, 6.2
mmol) in 12.3 mL of glacial HOAc was added 1.6 g (24 mmol)
of Zn powder. The slurry was heated to reflux. When the
bubbling subsided, a second portion of zinc powder (1.6 g, 24
mmol) was added, and the slurry was heated to boiling. When
the reaction had cooled to room temperature, it was diluted
with absolute EtOH, filtered through Celite, rinsed with
absolute EtOH, and concentrated under reduced pressure. The
resulting residue was mixed with 3,4-diethoxy-3-cyclobutene-
1,2-dione (0.91 mL, 6.2 mmol) and 5 mL of absolute EtOH and
then allowed to stand at room temperature for 18 h. The solid
precipitate which forms was filtered and rinsed with EtOAc
to give 510 mg of a white solid. This solid was dissolved in
CH2Cl2, filtered, and rinsed with CH2Cl2, and the filtrate was
concentrated under reduced pressure to give 190 mg (0.62
mmol, a 10% yield) of 3-(2-chloro-4-cyano-6-methyl-benzyl-
amino)-4-ethoxy-cyclobut-3-ene-1,2-dione as a white solid: 1H
NMR (DMSO-d6) δ 8.97 and 8.74 (br m, 1H, rotamers), 7.92
(s, 1H), 7.74 (s, 1H), 4.93 (br m, 1H), 4.78-4.60 (br m, 3H),
2.43 (s, 3H), 1.36 (br m, 3H); MS 304/306 (M+).
3-(2,4-Dich lor oben zylam in o)-4-(1,1-dim eth ylpr opylam i-
n o)-cyclobu t-3-en e-1,2-d ion e (60). The title compound was
prepared according to the procedure of compound 10 using
16.67 g (79.0 mmol) of 3-ethoxy-4-(1,1-dimethyl-propylamino)-
cyclobut-3-ene-1,2-dione and 15.02 g (79.0 mmol) of 2,4-
dichloro-6-methylbenzylamine: yield 25.7 g (72.3 mmol, a 92%
yield); mp 247.1-248.3 °C; 1H NMR (DMSO-d6) δ 7.54 (d, 1H),
7.44 (br t, 1H), 7.39 (d, 1H), 7.31 (s, 1H), 4.90 (d, 2H), 2.40 (s,
3H), 1.66 (q, 2H), 1.28 (s, 6H), 0.80 (t, 3H); IR (KBr) 3200,
2980, 1800, 1650 cm-1; MS 354/356/358 (M+). Anal. C17H20
Cl2N2O2: C, H, N.
-
N-(4-Cya n o-ben zyl)-N-[3,4-dioxo-2-(1,2,2-tr im eth yl-p r o-
p yla m in o)-cyclobu t-1-en yl]-bu tyr a m id e (80). Compound
4 (500 mg, 1.6 mmol) in 20 mL of THF was placed under Ar
at room temperature. Solid NaH (77 mg of a 60% dispersion
in mineral oil, 1.9 mmol) was added, and the reaction mixture
was stirred at room temperature for 20 min. Butyric acid
anhydride (0.79 mL, 4.8 mmol) was then added, and the
solution was stirred at room temperature for 3 h and then
refluxed for 8 h. The reaction mixture was loaded onto a plug
of silica gel and eluted with hexanes:EtOAc (1:1) to give a
residue, which was triturated repeatedly with hexanes to give
280 mg (0.73 mmol, a 45% yield) of 80 as a pale yellow solid:
3-(2-Chloro-4-cyano-6-methyl-benzylamino)-4-ethoxy-cyclobut-
3-ene-1,2-dione (0.19 g, 0.62 mmol) and 4.7 mL (0.94 mmol)
of a 0.2 M solution of (R)-2-amino-3,3-dimethylbutane in
absolute EtOH were stirred at room temperature for 2 d. The
slurry was filtered, rinsed with MeCN (3 × 3 mL), and dried
to give 150 mg (0.42 mmol, a 67% yield) of 55 as a white solid:
mp >300 °C; 1H NMR (DMSO-d6) δ 7.98 (s, 1H), 7.77 (s, 1H),
7.38 (br m, 1H), 7.18 (br d, 1H), 4.97 (m, 2H), 3.90 (m, 1H),
2.46 (s, 3H), 1.08 (d, 3H), 0.84 (s, 9H); IR (KBr) 3180, 2980,
mp 101-103 °C; [R]25 -101.9° (c 0.01, DMSO); 1H NMR
D
(DMSO-d6) δ 8.05 (br m, 1H), 7.83 (d, 2H), 7.45 (d, 2H), 5.22
(br m, 2H), 4.07 (m, 1H) 2.50-2.30 (m, 2H), 1.52 (m, 2H), 1.16
(d, 3H), 0.87 (s, 9H), 0.82 (t, 3H). IR (KBr) 3450, 3350, 2980,
2250, 1800, 1730 cm-1; MS 382 (MH)+. Anal. C22H27N3O3: C,
H, N.
2250, 1800, 1640 cm-1; MS 359/361 (M+). Anal. C19H22
-
ClN3O2‚0.04 CH2Cl2: C, H, N (the presence of 0.04 mol of CH2-
1
Cl2 confirmed by H NMR analysis).
3-Bu toxy-4-(1,1-dim eth yl-pr opylam in o)-cyclobu t-3-en e-
1,2-d ion e. A solution of 3,4-dibutoxy-3-cyclobutene-1,2-dione
(4.53 g, 20 mmol) and 1.74 g (20 mmol) of 1,1-dimethylpro-
pylamine in 20 mL of THF was stirred at room temperature
for approximately 19.5 h. The solvent was removed, and the
residue was chromatographed by gravity (CHCl3/hexane) on
neutral, activity III silica (150 g). The white solid isolated from
the appropriate eluates was recrystallized from hexane to give
4.105 g (17.18 mmol, an 86% yield) of the title compound as a
white solid: mp 56.5-57.5 °C. One gram (4.18 mmol) of this
material was recrystallized twice from hexane to provide 794
mg (3.32 mmol) of the title compound as a white solid: mp
56-57 °C; 1H NMR (DMSO-d6) δ 8.63 and 8.48 (two br s, 1H,
rotamers), 4.67 (m, br, 2H), 1.67 (m, br, 4H), 1.39 (m, 2H),
1.26 (m, br, 6H), 0.91 (t, 3H), 0.78 (t, 3H); IR (KBr) 3170, 1790,
1700 cm-1; MS: 239 (M+). Anal. C13H21NO3: C, H, N.
3-Ch lor o-4-{[2-(1,1-d im et h yl-p r op yla m in o)-3,4-d ioxo-
cyclobu t-1-en yla m in o]-m eth yl}-ben zon itr ile (57). 3-Bu-
toxy-4-(1,1-dimethylpropylamino)-cyclobut-3-ene-1,2-dione (957
mg, 4 mmol), 666 mg (4 mmol) of 2-chloro-4-cyanobenzylamine,
and 10 mL of THF were stirred together at room temperature
for 138 h. Removal of solvent, trituration of the residue with
Et2O, and drying provided a white solid. Recrystallization of
this solid from MeCN (charcoal) followed by a second recrys-
tallization from CH3CN gave 863 mg (2.60 mmol, a 65% yield)
of 57 as a white solid: mp 215.5-219.5 °C; 1H NMR (DMSO-
d6) δ 8.11 (d, 1H), 7.89 (m, 1H), 7.86 (m, 1H), 7.61 (d, 1H),
7.53 (s, 1H), 4.90 (d, 2H), 1.68 (q, 2H), 1.30 (s, 6H), 0.82 (t,
3H); IR (KBr) 3300, 2230, 1790, 1660 cm-1; MS 331/333 (M+).
N-(2,4-Dich lor o-6-m et h yl-b en zyl)-N-[2-(1,1-d im et h yl-
pr opylam in o)-3,4-dioxo-cyclobu t-1-en yl]-bu tyr am ide (81).
To 3-(2,4-dichloro-6-methyl-benzylamino)-4-(1,1-dimethyl-pro-
pylamino)-cyclobut-3-ene-1,2-dione (500 mg, 1.41 mmol) in 2
mL of DMF and 8 mL of THF was added NaH (62 mg of a
60% dispersion in mineral oil, 1.54 mmol) at 0 °C. The frothy
suspension was stirred for 1 h as the mixture was warmed to
23 °C. After cooling to 0 °C, 240 mg (1.54 mmol) of butyric
acid anhydride was added, and the reaction mixture was
stirred at 0 °C for 15 min and then allowed to warm to room
temperature. After being stirred for a total of 12 h, the reaction
mixture was poured into brine (50 mL) and extracted with
EtOAc (3 × 50 mL). The organic layer was dried over MgSO4
and decolorized (charcoal). The solvent was removed in vacuo,
and the remaining oil was triturated with Et2O/petroleum
ether to yield 310 mg (0.73 mmol, a 53% yield) of 77 as a white
1
solid: mp 117.2-118.4 °C; H NMR (DMSO-d6) δ 8.80 (br s,
1H), 7.39 (d, 1H), 7.28 (d, 1H), 5.06 (s, 2H), 2.34 (s, 3H), 2.29
(t, 2H), 1.67 (q, 2H), 1.51 (q, 2H), 1.30 (s, 6H), 0.82 (q, 6H); IR
(KBr) 3230, 2950, 1800, 1744, 1700, 1570 cm-1; MS 424 (M)+.
Anal. C21H26Cl2N2O3: C, H, N.
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 ted Bla d d er Str ip Con tr a c-
tion Stu d ies. Female Sprague-Dawley rats (Charles River,

1214 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 6
Gilbert et al.
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 follow-
ing composition (mM): NaCl (118.4), KCl (4.7), CaCl2 (2.5),
MgSO4 (1.2), KH2PO4 (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.5 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.
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
placed under the metabolic cage to collect and record urine
volume. The cystometric evaluation of bladder function was
started by infusing saline (20 mL/h), and after the first
micturition the infusion was maintained for 20 min. Two hours
after the first cystometry period, the rats were dosed orally
with the test compound, and a second cystometry was ap-
proximately 1 h after administration of test compound. Vehicle
(poly(ethylene glycol) 200) was similarly administered to
groups of rats that served as controls.
anesthetized with isofluorane. A femoral artery and vein were
cannulated with polyethylene tubing (PE50). The rats were
placed in Bollman cages, and the tail along with two cannulas
was extended through a hole at one end of the cage. The
animal was further immobilized by securely taping the tail to
the benchtop. Arterial blood pressure (BP) was obtained from
the cannulated femoral artery by means of a Statham pressure
transducer (model P23Db). Transducer signals were recorded
on a Grass (model 7) polygraph. Heart rate (HR) was calcu-
lated manually from the BP traces.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures for compounds 16-24, 32-34, 36-47, 50-54, 56, 59,
and 61-79. This material is available free of charge via the
Internet at http://pubs.acs.org.
Refer en ces
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Freeden, C.; Graceffa, R. F.; Hirth, B. H.; J enkins, D.; Lennox,
J . R.; Matelan, E.; Norton, N. W.; Quagliato, D.; Sheldon, J . H.;
Spinelli, W.; Warga, D.; Wojdan, A.; Woods, M. Design and SAR
of Novel Potassium Channel Openers Targeted for Urge Urinary
Incontinence. 1. N-Cyanoguanidine Bioisosteres Possessing in
Vivo Bladder Selectivity. J . Med. Chem. 2000, 43, 1187-1202.
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M. M.; Antane, S. A.; Butera, J . A.; Argentieri, T. M. Comparison
of the Potassium Channel Openers, WAY-133537, ZD6169 and
Celikalim on Isolated Bladder Tissue and In Vivo Bladder
Instability in the Rat. J . Pharmacol. Exp. Ther. 1999, 289, 1410-
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(3) Antane, S. A.; Hirth, B. H.; Antane, M. M.; Butera, J . A.; Ho, D.
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Freeden, C.; Wojdan, A. Substituted N-Aryl-1,2-diaminocy-
clobutene-3,4-diones II. Acylated Analogues as Novel KATP
Potassium Channel Openers Targeted For Urge Urinary Incon-
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American Chemical Society, 1997; American Chemical Society:
Washington, DC, 1997; MEDI 45.
(4) Unpublished results.
(5) Malmgren, A.; Sjogren, C.; Uvelius, B.; Andersson, K. E.;
Andersson, P. O. Cystometraical Evaluation of Bladder Instabil-
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(6) Malmgren, A.; Andersson, K. E.; Sjogren, C.; Andersson, P. O.
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(7) That the loss in the activity going from 4 to 26 is due to
conversion of the benzylamine of 4 to the benzamide of 26 and
not that 4 is a single enantiomer while 26 is a racemate is
suggested by analysis of chiral and racemic 3,3-dimethyl-2-
butylamino analogues presented in the phenylamino cyclobutene-
dione series (i.e., compare compounds 34 and 42 in ref 1).1
(8) C ) 245 ( 15% at 30 µM (administration of compound 77 to a
KCl-contracted rat bladder strip additionally contracts this
tissue 245 ( 15% at 30 µM).
(9) (R)-2-amino-3,3-methylbutane was prepared using a modification
of the procedure of Manley and Quast: Manley, P. W.; Quast,
U. Structure-Activity Studies of Potassium Channel Opening
in Pinacidil-Type Cyanoguanidines, Nitroethenediamines, Thio-
ureas, and Ureas. J . Med. Chem. 1992, 35, 2327.2340.
(10) J olad, S. D.; Rajagopal, S. 2-Bromo-4-Methylbenzaldehyde. Org.
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(11) Wiley: M. R.; Chirgadze, N. Y.; Clawson, D. K.; Craft, T. J .;
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B. Hem od yn a m ic Assessm en t. Male Sprague-Dawley
rats (Charles River, Wilmington, MA; 315-410 g) were
J M9905108