N-acyl-1,2,3,4a,5,10b-hexahydro-[1]benzopyrano-[3,4-b][1,4]oxazine-9- carbonitriles as bladder-selective potassium channel openers

Article abstract of DOI:10.1016/S0968-0896(00)00260-1

Optically active N-acyl-5,5-dimethyl-1,2,3,4a,5,10b-hexahydro-[1]benzopyrano[3,4-b][1,4] oxazine-9-carbonitriles 2-22 were synthesized as rigid analogues of cromakalim. The (4aR,10bR)-N-benzoyl derivative (-)-11 was identified as a bladder-selective KCO (IC_{50, bladder}=8.2 μM, IC_{50, portal vein}=34.5 μM). Among the analogues of 11 with substitution on the benzoyl moiety, the 3-methyl analogue (-)-14 showed highly potent and selective activity at portal vein (IC_{50, bladder}=279 μM, IC_{50, portal vein}=0.54 μM). The 4-bromo analogue (-)-19 (IC_{50, bladder}=2.0 μM, IC_{50, portal vein}=8.1 μM) and the 4-hydroxy analogue (-)-21 (IC_{50, bladder}=3.8 μM, IC_{50, portal vein}=75 μM) showed enhanced activity at the bladder, while maintaining unprecedented bladder selectivity in vitro. The N-benzenesulfonyl analogue (-)-22, a bioisoster of (-)-11, showed similar activity at the bladder with enhanced selectivity (IC_{50, bladder}=11.6 μM, IC_{50, portal vein}=120 μM).

Full text of DOI:10.1016/S0968-0896(00)00260-1

Bioorganic & Medicinal Chemistry 9 (2001) 383±393
N-Acyl-1,2,3,4a,5,10b-hexahydro-[1]benzopyrano-
[3,4-b][1,4]oxazine-9-carbonitriles as Bladder-Selective
Potassium Channel Openers
Hsin-I. Chiu,a Yen-Chung Lin,a Chen-Yu Cheng,a,* Ming-Cheng Tsaib
and Hon-Cheng Yuc
aInstitute of Pharmaceutical Sciences, College of Medicine, National Taiwan University, Taipei, Taiwan 10018
bDepartment of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan 10018
cDepartment of Urology, College of Medicine, National Taiwan University, Taipei, Taiwan 10018
Received 10 July 2000;accepted 20 September 2000
AbstractÐOptically active N-acyl-5,5-dimethyl-1,2,3,4a,5,10b-hexahydro-[1]benzopyrano[3,4-b][1,4]oxazine-9-carbonitriles 2±22 were
synthesized as rigid analogues of cromakalim. The (4aR,10bR)-N-benzoyl derivative (À)-11 was identi®ed as a bladder-selective KCO
(IC50, bladder=8.2 mM, IC50, portal vein=34.5 mM). Among the analogues of 11 with substitution on the benzoyl moiety, the 3-methyl
analogue (À)-14 showed highly potent and selective activity at portal vein (IC50, bladder=279mM, IC50, portal vein=0.54 mM). The 4-bromo
analogue (À)-19 (IC50, bladder=2.0 mM, IC50, portal vein=8.1 mM) and the 4-hydroxy analogue (À)-21 (IC50, bladder=3.8mM, IC50, portal
vein=75 mM) showed enhanced activity at the bladder, while maintaining unprecedented bladder selectivity in vitro. The N-benzene-
sulfonyl analogue (À)-22, a bioisoster of (À)-11, showed similar activity at the bladder with enhanced selectivity (IC50,
blad-
der=11.6 mM, IC50, portal vein=120 mM). # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
blood vessels, cardiac muscle, and other smooth muscles.2
These agents may ®nd use in the treatment of a variety
of diseases such as hypertension, asthma, myocardial
ischemia, and urinary incontinence. The relaxant e€ects
of cromakalim and pinacidil on bladder detrusor muscle
have also been reported.3À5 However, their clinical uti-
lity to treat UI is limited due to their pronounced
hemodynamic e€ects. A series of aniline tertiary carbi-
nols represented by ZD6169 are the ®rst compounds
claimed as bladder-selective KCOs.6 More recent exam-
ples of KCOs with bladder-selectivity include a 4-
phenyl-1,4-dihydropyridine derivative ZM2440857 and
a series of diaminocyclobutenedione derivatives repre-
sented by Way 133537.8 In this article, we would like to
report the discovery of novel bladder-selective KCOs
among a series of optically active N-acyl-1,2,3,4a,5,10b-
hexahydro-[1]benzopyrano[3,4-b][1,4]oxazine-9-carboni-
triles (2±22) (Fig. 1). As described in our preliminary
account of this study,9 we designed these compounds on
the basis of rigidization of the amide function and 3-
hydroxyl group in cromakalim. The synthesis of a rela-
ted series of racemic 1-benzopyrano[3,4-b][1,4]oxazines
((Æ)-I) as rigid analogues of cromakalim was ®rst
reported by Fleischhacker et al.10 Although the resem-
blance of I to cromakalim based on conformational
Urinary incontinence (UI), a widespread and distressing
condition with the elderly population, is classi®ed into
four types: urge, stress, re¯ex and over¯ow incon-
tinence.1 Urinary urge incontinence (UUI), the largest
category among the four types of incontinence, has been
demonstrated to be caused by bladder detrusor muscle
instability. Current treatments for UUI include anti-
muscarinics, antispasmodics and mixed antimuscarinic/
antispasmodic agents, which have met with limited suc-
cess due to their low ecacy and/or high incidence of
side e€ects. Therefore, the search for more speci®c and
ecacious pharmacotherapy against UUI remains a
worthwhile endeavor from both a scienti®c and a com-
mercial point of view.
Typical ATP-sensitive potassium channel openers
(KCOs), such as cromakalim ((Æ)-1) and pinacidil, have
been demonstrated to possess potent relaxant activity on
*Corresponding author. Tel.: +886-2-23964202;fax: +886-2-23512086;
e-mail: cyc@ha.mc.ntu.edu.tw
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00260-1

384
H.-I. Chiu et al. / Bioorg. Med. Chem. 9 (2001) 383±393
analysis was described, the supporting pharmacological
data were not given, while in our hands, enantiomers of
I only showed weak KCO activity.9
Scheme 2. Thus, epoxide (À)-23 was treated with aqu-
eous ammonia to give aminoalcohol (+)-26, which was
converted to acetamide (+)-27. Inversion of the 3-OH
group in (+)-27 was accomplished by treatment with
(diethylamino)sulfur tri¯uoride (DAST), followed by
hydrolysis of the oxazoline intermediate.14 The resulting
cis-aminoalcohol (À)-28 was treated with 2-nitrobenz-
enesulfonyl chloride15 to give sulfonamide (À)-29, which
then underwent the desired double alkylation with 1-
bromo-2-chloroethane, followed by deprotection of the
amine function to form the tricyclic cis-hexahydro-
[1]benzopyrano[3,4-b][1,4]oxazine intermediate (À)-31.
Acylation of 31 as described then provided the target
compounds 7±11. As an isostere of benzamide (À)-11,
benzenesulfonamide 22 was prepared from (À)-31 via
treatment with benzenesulfonyl chloride.
Chemistry
All the target compounds were prepared in optically
active form11 from chiral starting materials. Compounds
2±6, which maintain the stereochemistry of levcromaka-
lim ((À)-1), the active enantiomer of cromakalim, were
synthesized according to Scheme 1. The starting epoxide
(À)-23, prepared from 6-cyano-2,2-dimethyl-2H-1-benzo-
pyran via (S,S)-Mn(salen)-catalyzed asymmetric epoxida-
tion,12 was subjected to ring-cleavage by 2-aminoethanol
to give the diol intermediate (+)-24. Treatment of 24 with
Ph3P/DEAD13 resulted in the desired cyclization to form
the tricyclic 1,2,3,4a,5,10b-hexahydro-[1]benzopyrano-
[3,4-b][1,4]oxazine intermediate (+)-25. Acylation of 25
under appropriate conditions as shown then provided
the target compounds 2±6.
In Scheme 3 is shown a shorter route to cis-hexahydro-
[1]benzopyrano[3,4-b][1,4]oxazines, as exempli®ed by
the preparation of compounds 12±21. This improved
synthesis was based on our accidental observation that
compound (+)-3 was converted to its more stable cis-
isomer or compound (+)-8 upon treatment with a
strong base such as NaH. The energy di€erence between
The cis-isomers of 2±6, namely compounds 7±11, were
also prepared in a stereospeci®c fashion as shown in
Figure 1.

H.-I. Chiu et al. / Bioorg. Med. Chem. 9 (2001) 383±393
385
Scheme 1. Reagents: (a) H2NCH2CH2OH, THF, re¯ux;(b) Ph P, DEAD, THF, rt;(c) HCO COCH3, pyridine, THF, À70 ꢀC to rt;(d) (RCO) O,
3
2
2
pyridine, rt;(e) RCOCl, Et 3N, THF, rt.
Scheme 2. Reagents: (a) NH3 (30%), THF:EtOH=1:1;(b) CH COCl, Et3N, THF;(c) i. DAST, CH 2Cl2;ii. 6 N HCl, CH 3CN, re¯ux;(d) 2-nitro-
3
benzenesulfonyl chloride, Et3N, THF;(e) BrCH 2CH2Cl, K2CO3, DMF, 70 ꢀC;(f) PhSH, K 2CO3, DMF;(g) HCO 2COCH3, pyridine, THF, À70 ꢀC;
(h) (RCO)2O, pyridine, rt;(i) PhCOCl, Et 3N, THF, rt;(j) benzenesulfonyl chloride, Et 3N, 100 ꢀC.

386
H.-I. Chiu et al. / Bioorg. Med. Chem. 9 (2001) 383±393
the cis-isomers and their corresponding trans-isomers
was later con®rmed by a molecular mechanics calcula-
tion (Sybyl, version 6.3), which gave values of 14.12 and
13.74 kcal/mol for benzamide 6 and its cis-isomer 11
respectively. Thus, intermediate (À)-25 was prepared
from epoxide (+)-23 as described for (+)-25. Acylation
of (À)-25 with substituted benzoyl chlorides as shown
provided the trans-benzamides 32±40. Compounds 32±
40 were then subjected to treatment with an excess of
NaH, which resulted in epimerization at C-10b, and
provided the desired cis-benzamides 12±20. Compound
(À)-21 was obtained via O-demethylation of (À)-16
with boron tribromide. The structure assignment of the
target compounds is supported by X-ray crystal analy-
sis, as shown in Figure 2 for compounds 18 and 19. The
X-ray structures of compounds 3 and 8 have been
reported in our previous communication.9 Signi®cant
di€erences between the X-ray structures of the cis ana-
logues, such as 18 and 19, and that of cromakalim16 can
be visualized. The carbonyl groups in the cis analogues
point to the opposite direction from the carbonyl group
in cromakalim, and the phenyl rings in cis analogues
11±21 extend to an area in space not accessible by the
pyrrolidone ring in cromakalim.
Results and Discussion
The KCO activities of target compounds were evaluated
in vitro with preparations of spontaneously containing
rat portal vein and KCl-stimulated rat detrusor strip
based on the literature procedure17 (Table 1). Most
compounds tested, with the exception of 2 and 6,
demonstrated signi®cant relaxant activity on both rat
portal vein and detrusor strip. As compared to levcro-
makalim, these rigid analogues are less potent. How-
ever, the observed relaxant activity was antagonized by
the potassium channel blocker glibenclamide, indicating
the direct involvement of potassium channels. Com-
pounds 2 and 6 showed weak enhancement activity on
Scheme 3. Reagents: (a) H2NCH2CH2OH, THF, re¯ux;(b) Ph 3P, DEAD, THF;(c) R-C 6H4COCl, Et3N, THF;(d) NaH, DMF, 0 ꢀC;(e) BBr
S(CH3)2, ClCH2CH2Cl, re¯ux.
-
3
Figure 2. X-ray structures of compounds 18 and 19.

H.-I. Chiu et al. / Bioorg. Med. Chem. 9 (2001) 383±393
387
Table 1. Mechanoinhibitory activity of N-acyl-1,2,3,4a,5,10b-hexahydro-[1]benzopyrano[3,4-b][1,4]oxazine-9-carbonitriles on rat portal vein and
rat detrusor stripsa
IC50 (mM)
IC50 (mM) in the presence
of glibenclamideb
Compound
Portal veinc
Detrusord
IC50 ratioe
Portal vein
Detrusor
f
(+)-2
(+)-3
(+)-4
(+)-5
+
152Æ28.4
70.1Æ24.2
47.2Æ18.1
104Æ8.7
66Æ20
Ð
0.08
1.38
1.22
Ð
1.21
0.31
0.39
0.33
4.23
1.1
NDg
79.3Æ10
92.6Æ5
140Æ3
158Æ0.6
153Æ15
318Æ19
244Æ11
123Æ10
279Æ22
53.2Æ3
5.9Æ2.1
64.9Æ10.6
127Æ12.3
+
(+)-6
(À)-7
ND
62.8Æ18
51.8Æ17.6
8.8Æ3.1
117Æ7
(À)-8
2.7Æ1.0
60.9Æ2
52.6Æ5
58.9Æ5
45.5Æ3
201Æ18.1
121Æ14
58.3Æ4.1
36.8Æ2.9
57.1Æ7.9
58.9Æ6.5
61.4Æ6.1
12.9Æ0.8
15.7Æ1.7
97Æ6.8
242Æ7
(À)-9
20.1Æ8.9
18.4Æ2.5
34.5Æ0.71
98.3Æ12.8
71Æ7.8
51.8Æ3.06
56.4Æ5.2
8.15Æ1.9
88.7Æ7.9
29Æ4.1
141Æ7
(À)-10
(À)-11
(À)-12
(À)-13
(À)-14
(À)-15
(À)-16
(À)-17
(À)-18
(À)-19
(À)-20
(À)-21
(À)-22
(Æ)-ZD6169
Levcromakalim
268Æ21
32.8Æ2
592Æ23.7
85.9Æ6
2.4
0.54Æ0.02
24.1Æ3.1
37.4Æ5.6
6.6Æ0.5
279Æ7.9
12.5Æ1.3
28.8Æ1.7
48.6Æ0.88
25.4Æ2.3
2.05Æ0.1
5.4Æ0.04
3.8Æ0.6
0.002
1.9
1.3
0.13
0.1
>300
39.9Æ3.6
117Æ4.7
80.7Æ7.3
92.3Æ7.4
26.1Æ3.9
50.9Æ2
2.6Æ0.2
8.1Æ0.9
4
10.6Æ1.4
74.6Æ9.7
120Æ12
1.96
19.6
10.3
0.12
0.16
61.4Æ2.8
27.4Æ4.4
2.7Æ0.3
8.7Æ0.9
11.6Æ0.8
0.97Æ0.2
0.82Æ0.2
0.12Æ0.01
7.7Æ0.5
4.9Æ0.2
0.13Æ0.08
aData represent the mean of three experiments each performed in duplicate.
b1 mM.
cSpontaneously contracting rat portal vein.
dIsolated rat detrusor strips exposed to extracellular KCl (20 mmol/L).
eIC50,portal vein/IC50,bladder
.
fThe ``+'' sign indicates enhancement of the spontaneous contraction.
gND: Not determined.
Table 2. Structures, optical rotations, IR, mass, and elemental analyses or chromatographic purities for 2±22
Compound
R
[a]d (w/v %, CH3OH)
IR, nmax (cmÀ1
)
EI±MS
EAa or HPLC area %b
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
HCO
CH3CO
C2H5CO
(CH3)2CHCO
C6H5CO
HCO
CH3CO
C2H5CO
(CH3)2CHCO
C6H5CO
2-CH3C6H4CO
2-CF3C6H4CO
3-CH3C6H4CO
3-CF3C6H4CO
3-NO2C6H4CO
4-CH3C6H4CO
4-CF3C6H4CO
4-BrC6H4CO
4-OCH3C6H4CO
4-OHC6H4CO
C6H5SO2
+79.8 (1)
+33.3 (0.13)
+24.6 (0.51)
+56.9 (0.1)
+53.9 (0.26)
À125 (0.12)
À244 (0.1)
À310 (0.1)
À155 (0.2)
À114 (0.07)
À70 (0.2)
2980, 2220, 1670
2978, 2223, 1657
2970, 2240, 1660
2977, 2224, 1651
2981, 2224, 1642
2935, 2225, 1665
2980, 2223, 1650
2981, 2223, 1650
2973, 2223, 1646
2980, 2224, 1636
2983, 2224, 1639
3062, 2226, 1705
2979, 2225, 1607
2220, 1648, 1124
2990, 2224, 1640
2975, 2222, 1635
2926, 2220, 1627
1941, 2221, 1617
2972, 2224, 1698
2964, 2226, 1609
2979, 2225, 1654
272 (M+, base)
286 (M+, base)
C, H, N
C, H, N
C, H, N
98.4
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
98.7
100
97.5
97.4
100
97.2
300 (M+, base)
314 (M+), 154 (base)
348 (M+), 105 (base)
272 (M+, base)
286 (M+), 86 (base)
300 (M+, base)
314 (M+, base)
348 (M+), 105 (base)
362 (M+), 119 (base)
416 (M+), 116 (base)
362 (M+), 119 (base)
416 (M+), 116 (base)
393 (M+), 150 (base)
362 (M+), 119 (base)
416 (M+), 116 (base)
426 (M+), 184 (base)
378 (M+), 135 (base)
364 (M+), 121 (base)
384 (M+), 173 (base)
À69 (1)
À85.5 (1.5)
À86 (2)
À38.3 (0.6)
À52.5 (1)
À82.7 (4)
C, H, N
C, H, N
C, H, N
97.6
À77.8 (3)
À106 (1)
À9.6 (0.2)
À32.4 (1)
97.7
aAnalytical results were within Æ0.4% of the theoretical values.
bHPLC conditions: CHIRALCEL OD, n-hexane:IPA=1:1, UV 254 nm, 0.5 ml/min.

388
H.-I. Chiu et al. / Bioorg. Med. Chem. 9 (2001) 383±393
the portal vein, while maintaining weak relaxant activity
on the detrusor. The cis-isomers 7±11 were found to be
more potent than their corresponding trans-isomers 2±6,
in contrast to the earlier report, that the (3R,4R)-cis-
isomer of levcromakalim is 50 times weaker than
levcromakalim itself.18
showed relaxant activity at the detrusor approaching
that of (Æ)-ZD6169 and levcromakalim, while maintain-
ing unprecedented in vitro bladder-selectivity. Further
SAR study and in vivo assays on selected compounds
from the series are in progress.
During the course of this study, it was noted with great
interest that the cis-benzamide 11 demonstrated potent
and selective relaxant activity at the bladder detrusor
Experimental
General procedures
(IC50, bladder=8.2 mM;IC
ratio=4.2). Subsequently
50
compounds 12±21 were synthesized in order to investi-
gate the e€ects of substitution on the benzene ring. As
indicated by the reduced activities of 12 and 13, ortho-
substituents, irrespective of their electronic properties,
are detrimental. It was surprising to see that introduc-
tion of an ortho-methyl substituent resulted in 63-fold
increase in the potency at the portal vein and 34-fold
decrease in the potency at the bladder, compound 14
being a highly potent and selective KCO at the blood
Melting points were taken in a capillary tube with a
MEL-TEMP II apparatus by Laboratory Devices. IR
spectra were determined with a Perkin±Elmer 1760-X
FT-IR spectrometer. NMR spectra were recorded on
Bruker AM-200 and AMX-400 NMR spectrometers;
chemical shifts were recorded in parts per million down-
®eld from Me4Si. Mass spectra were recorded on a Jeol
JMS-D300 mass spectrometer;HRMS were obtained
with a Jeol JMS-HX110 spectrometer. Elemental ana-
lysis was performed with a Perkin±Elmer 2400-CHN
instrument. Optical rotation was determined with a
Jasco DIP-370 digital polarimeter. TLC was performed
on Merck (art. 5715) silica gel plates and visualized under
UV light (254 nm). Flash column chromatography was
performed with Merck (art. 9385) 40±63 mm silica gel 60.
Anhydrous tetrahydrofuran was distilled from sodium±
benzophenone prior to use.
vessel (IC50, portal vein=0.54 mM;IC
ratio=0.002). In
50
contrast, the polar 3-tri¯uoromethyl and 3-nitro groups
(15, 16) had little e€ect on the activity at the portal vein
but they caused moderate decrease in activity at the
detrusor. As for the para-substituents, 4-methyl and 4-
tri¯uoromethyl groups had similar but lesser e€ects
than the 3-methyl group, and resulted in compounds 17
and 18 being potent and selective KCOs at the portal
vein. However, when substituents with lone-pair elec-
trons were introduced at the para position, activity at
the bladder was selectively enhanced. The 4-bromo
analogue (19) was found to be the most potent at the
detrusor in our series, with its IC50, bladder of 2.0 mM
(IC50 ratio=4), amounting to that of levcromakalim
and (Æ)-ZD6169, while the 4-hydroxy analogue (21)
demonstrated the highest bladder-selectivity (IC50, blad-
der=3.8 mM;IC 50 ratio=19.6). The benzenesulfonamide
(3S,4R)-Dihydro-2,2-dimethyl-4-(2-hydroxyethylamino)-
3-hydroxy-1-benzopyran-6-carbonitrile ((+)-24). A solu-
tion of epoxide 23 (2 g, 9.9 mmol), 2-aminoethanol
(4.8 mL, 79.2 mmol) and dry THF (20 mL) was re¯uxed
under N2 overnight. The reaction mixture was evapo-
rated, and the residue was chromatographed (silica gel;
CH2Cl2:CH3OH=19:1) to a€ord (+)-24 (1.9 g, 95%) as
a yellow oil: Rf=0.28 (CH2Cl2:CH3OH=19:1); 1H
NMR (200 MHz, CDCl3) d 1.2 (s, 3H), 1.4 (s, 3H), 2.7
(dt, J=13, 5 Hz, 1H), 2.9 (dt, J=13, 5 Hz, 1H), 3.6 (d,
J=10 Hz, 1H), 3.7 (d, J=10 Hz, 1H), 3.8 (m, 2H), 6.8
(d, J=9 Hz, 1H), 7.4 (d, J=9 Hz, 1H), 7.7 (s, 1H); 13C
NMR (50 MHz, CDCl3) d 18.8, 26.9, 46.1, 56.4, 62.3,
71.4, 79.7, 103.5, 118.3, 119.5, 124.6, 132.2, 132.5, 157.3;
IR (KBr) 3363, 2977, 2225, 1609 cmÀ1;HRMS (EI,
70 eV) m/z calcd for C14H18O3N2 262.1317, found
262.1313;262 (M +), 190 (base).
(À)-22 (IC50, bladder=11.6 mM;IC
ratio=10.3) was
50
found to essentially maintain both the potency and
selectivity of compound 11 at the bladder, indicating
their bio-isosteric relationship. It is noteworthy that
bladder-selectivity was not observed with (Æ)-ZD6169
in our in vitro assay, although (À)-ZD6169 was docu-
mented as a bladder-selective KCO.6 However, this is
not inconsistent with the results obtained by Zeneca's
researchers,19 who have suggested pharmacokinetic
reasons for the in vivo selectivity demonstrated by
ZD6169.
(4aS,10bR)-5,5-Dimethyl-1,2,3,4a,5,10b-hexahydro-[1]-
benzopyrano[3,4-b][1,4]oxazine-9-carbonitrile ((+)-25).
To a stirred solution of (+)-24 (400 mg, 1.53 mmol) and
triphenylphosphine (480 mg, 1.83 mmol) in dry THF
(10 mL) at room temperature under N2 was added drop-
wise diethyl azodicarboxylate (0.285 mL, 1.83 mmol).
The resulting mixture was stirred at 25 ꢀC for 24 h and
evaporated. The residue was partitioned between ether
and water. The aqueous layer was separated, acidi®ed
with 6 N HCl until the pH reached 2.0, and then
extracted with CH2Cl2 (100 mL). The CH2Cl2 layer was
dried (MgSO4) and evaporated. The residue was chro-
matographed (silica gel;CH 2Cl2:CH3OH=19:1) to
a€ord (+)-25 (168 mg, 45%) as a white solid: Rf=0.49
(CH2Cl2:CH3OH=19:1);mp: 132±133 ꢀC; 1H NMR
(200 MHz, CDCl3) d 1.3 (s, 3H), 1.4 (s, 3H), 3.0 (dd,
Conclusion
Ecient syntheses of optically active N-acyl-1,2,3,4a,
5,10b-hexahydro-[1]benzopyrano[3,4-b][1,4]oxazine-9-
carbonitriles (2±22) as rigid analogues of levcromakalim
have been accomplished. In in vitro assays, the N-benzoyl
analogue (À)-11 revealed itself as a bladder-selective
KCO. Pronounced substituent e€ects were observed with
analogues of 11 (12±21). The 3-methyl analogue ((À)-14)
emerged as a highly potent and selective KCO at the
portal vein, while para-substituents possessing lone-pair
electrons enhanced the potency at the bladder. The 4-
bromo and 4-hydroxy analogues ((À)-19 and (À)-21)

H.-I. Chiu et al. / Bioorg. Med. Chem. 9 (2001) 383±393
389
J=13, 3 Hz, 1H), 3.1 (d, J=10 Hz, 1H), 3.1 (td, J=12,
4 Hz, 1H), 3.6 (td, J=12, 3 Hz, 1H), 3.7 (d, J=10 Hz,
1H), 3.9 (dd, J=12, 3 Hz, 1H), 6.8 (d, J=9 Hz, 1H), 7.4
(d, J=9 Hz, 1H), 7.7 (s, 1H); 13C NMR (50 MHz,
CDCl3) d 21.1, 27.1, 46.2, 52.3, 69.3, 79.1, 81.7, 103.9,
118.4, 119.7, 123.9, 130.8, 133.1, 156.7;HRMS (EI,
70 eV) m/z calcd for C14H16O2N2 244.1212, found
244.1218;244.2 (M +, base).
(4aS,10bR)-5,5-Dimethyl-1,2,3,4a,5,10b-hexahydro-N-(2-
methylpropionyl) - [1]benzopyrano[3,4 - b][1,4]oxazine - 9 -
carbonitrile ((+)-5). To a solution of (+)-25 (58.5 mg,
0.25 mmol) and triethylamine (0.052 mL, 0.375 mmol)
in dry THF (5 mL) was added isobutyryl chloride
(0.04 mL, 0.375 mmol) at 0 ꢀC under N2. The reac-
tion mixture was let warm to room temperature and
®ltered. The ®ltrate was evaporated, and the residue was
chromatographed (silica gel;EtOAc: n-hexane=1: 2)
to a€ord (+)-5 (55.3 mg, 73%): Rf=0.3 (EtOAc:n-
hexane=1: 2);mp: 158±160 ꢀC; 1H NMR (200
MHz, CDCl3) d 1.1 (m, 8H), 1.2 (s, 3H) 1.5 (s, 3H),
2.8 (m, 1H), 3.7 (d, J=4 Hz, 1H), 3.7 (d, J=5 Hz, 1H),
4.1(m, 1H), 6.8 (d, J=4 Hz, 1H), 7.2 (s, 1H), 7.4 (d,
J=1 Hz, 1H); 13C NMR (50 MHz, CDCl3) d 1.4, 14.6,
19.7, 19.9, 20.1, 20.6, 27.3, 68.3, 76.8, 77.5, 78.1, 79.4,
104.0, 118.7, 123.1, 131.1, 132.6, 179.4;HRMS (EI,
70 eV) m/z calcd for C18H22O3N2 314.1631, found
315.1634.
(4aS,10bR)-5,5-Dimethyl-N-formyl-1,2,3,4a,5,10b-hexa-
hydro - [1]benzopyrano[3,4 - b][1,4]oxazine - 9 - carbonitrile
((+)-2). A mixture of formic acid (0.14 mL, 3.69
mmol), triethylamine (0.51 mL, 3.69 mmol), and acetyl
chloride (0.26 mL, 3.69 mmol) in THF (10 mL) was stir-
red at À70 ꢀC for 15 min. The mixture was then trans-
ferred to a solution of (+)-25 (300 mg, 1.23 mmol) in
THF (5 mL). The reaction mixture was stirred for 3 h
while the temperature was raised form À70 ꢀC to room
temperature. The mixture was then ®ltered, and the ®l-
trate was evaporated in vacuo. The oily residue was
taken up in ethyl acetate, washed with water (25 mLÂ2),
dried (MgSO4), and evaporated to give (+)-2 (251.6 mg,
22%) as a white solid, which was crystallized from
ether. (+)-2: Rf=0.34 (CH2Cl2:CH3OH=19:1);mp:
(4aS,10bR)-N-Benzoyl-5,5-dimethyl-1,2,3,4a,5,10b-hexa-
hydro - [1]benzopyrano[3,4 - b][1,4]oxazine - 9 - carbonitrile
((+)-6). Rf=0.83 (CH2Cl2:CH3OH=19:1);mp: 195±
197 ꢀC; H NMR (400 MHz, CDCl3) d 1.3 (s, 3H), 1.5
1
197 ꢀC; H NMR (200 MHz, CDCl3) d 1.8 (s, 3H), 1.4
(s, 3H), 3.6±3.7 (m, 2H), 3.7±3.8 (m, 2H), 3.9 (d,
J=10 Hz, 1H), 4.6 (d, J=10 Hz, 1H), 6.9 (d, J=8 Hz,
1H), 7.4 (d, J=8 Hz, 1H), 7.4±7.5 (m, 4H); 13C NMR
(50 MHz, CDCl3) d 19.5, 26.3, 55.9, 67.6, 75.3, 78.4,
103.7, 118.5, 119.2, 121.7, 128.1, 128.7, 130.3, 131.3,
131.9, 134.7, 155.3, 173.2.
1
(s, 3H), 3.1 (td, J=4, 3 Hz, 1H), 3.3 (d, J=10 Hz, 1H),
3.6 (td, J=9, 9. 12 Hz, 1H), 3.9 (dd, J=3, 3 Hz, 1H), 4.4
(d, J=9 Hz, 1H), 4.5 (d, J=15 Hz, 1H), 6.9 (d, J=9 Hz,
1H), 7.5 (d, J=9 Hz, 1H), 7.5 (d, J=9 Hz, 1H), 7.6 (s,
1H), 8.2 (s, 1H); 13C NMR (50 MHz, CDCl3) d 19.6,
26.4, 44.4, 56.8, 67.7, 76.9, 82.5, 104.9, 116.9, 118.8,
120.6, 132.8, 134.0, 157.6, 160;HRMS (EI, 70 eV) m/z
calcd for C15H16N2O3 272.1161, found 272.1161.
(30S,40R)-N-(6-Cyano-3,4-dihydro-2,2-dimethyl-3-hydroxy-
2H-[1]benzopyran-4-yl)-2-acetamide ((+)-27). Rf=0.23
(CH2Cl2:CH3OH=19:1);mp: 188±190 ꢀC; 1H NMR
(400 MHz, CDCl3) d 1.2 (s, 3H), 1.5 (s, 3H), 2.1 (s, 3H),
3.6 (d, J=9 Hz, 1H), 4.3 (s, 1H), 5.0 (dd, J=9, 8 Hz,
1H), 6.2 (d, J=8 Hz, 1H), 6.8 (d, J=9 Hz, 1H), 7.39 (d,
J=9 Hz, 1H), 7.5 (s, 1H); 13C NMR (100 MHz, CD3
OD) d 18.5, 23.1, 26.3, 50.3, 75.4, 80.3, 103.7, 118.6,
119.0, 122.5, 132.4, 133.2, 157.0, 173.4;IR (KBr) 3400,
(4aS,10bR)-N-Acetyl-5,5-dimethyl-1,2,3,4a,5,10b-hexa-
hydro - [1]benzopyrano[3,4 - b][1,4]oxazine - 9 - carbonitrile
((+)-3). A solution of (+)-25 (500 mg, 2.05 mmol) and
acetic anhydride (5 mL) in pyridine (5 mL) was stirred at
room temperature overnight. The mixture was then
treated with 10% NaOH and extracted with CH2Cl2
(10 mLÂ2). The organic layer was washed with 2 N HCl
(10 mL) and then evaporated. The residue was chroma-
2900, 2220, 1640 cmÀ1;HRMS (EI, 70 eV) m/z calcd for
+
C14H16O3N2 260.1161, found 260.1160;261 (MH
base).
,
tographed (silica gel;CH Cl2:CH3OH=19:1) to a€ord
2
3 (350 mg, 90%) as a white solid, which was crystallized
from EtOAc/n-hexꢀane: Rf=0.62 (CH2Cl2:CH3OH=
(3R,4R)-Dihydro-2,2-dimethyl-4-amino-3-hydroxy-1-benzo-
pyran-6-carbonitrile ((À)-28). A solution of compound 27
(400mg, 1.54mmol), diethylaminosulfur tri¯uoride (0.223
mL, 1.09mmol), and dry CH2Cl2 (5 mL) was stirred at
room temperature under N2 for 30 min. The mixture
was evaporated, re-dissolved in 6 N HCl:CH3CN
(5 mL:5 mL), and re¯uxed overnight. The resulting
solution was basi®ed with 10% NaOH to a pH of 12
and extracted with CH2Cl2. The CH2Cl2 layer was dried
and evaporated to give (À)-28 (276 mg, 82%): Rf=0.12
(CH2Cl2:CH3OH=19:1);mp: 140±141 ꢀC; 1H NMR
(400 MHz, CD3OD) d 1.3 (s, 3H), 1.5 (s, 3H), 1.9 (br,
1H), 3.6 (d, J=4 Hz, 1H), 4.1 (d, J=4 Hz, 1H), 6.8 (d,
J=9 Hz, 1H), 7.4 (d, J=9 Hz, 1H), 7.7 (s, 1H); 13C
NMR (100 MHz, CD3OD) d 23.6, 24.5, 47.1, 71.1, 79.5,
103.2, 117.9, 119.5, 125.8, 132.3, 133.0, 157.5;IR (KBr)
3600, 2900, 2200, 1600 cmÀ1;HRMS (EI, 70 eV) m/z
calcd for C12H14O2N2 218.1055, found 218.1041;218
(M+, base).
1
19:1);mp: 137±139 C; H NMR (200 MHz, CDCl3) d
1.2 (s, 3H), 1.4 (s, 3H), 3.6±3.7 (m, 3H), 3.7±3.8 (m, 1H),
3.9±4.0 (m, 1H), 4 (br, s, 1H), 6.7 (d, J=8 Hz, 1H), 7.2
(s, 1H), 7.3 (d, J=8 Hz, 1H); 13C NMR (50 MHz,
CDCl3) d 20.7, 23.1, 27.3, 54.8, 55.1, 67.6, 75.3, 79.5,
103.8, 118.6, 119.8, 123.1, 131.7, 132.6, 156.1, 172.6.
(4aS,10bR)-N-Propionyl-5,5-dimethyl-1,2,3,4a,5,10b-hexa-
hydro - [1]benzopyrano[3,4 - b][1,4]oxazine - 9 - carbonitrile
((+)-4). Rf=0.37 (CH2Cl2:CH3OH=19:1);mp:
170 ꢀC; H NMR (200 MHz, CDCl3) d 1.0 (t, 2H), 1.2
1
(s, 3H), 1.4 (s, 3H), 2.4 (d, J=7 Hz, 2H), 3.6±3.8 (m,
4H), 3.9±4.1 (m, 1H), 4.8 (s, 1H), 6.8 (d, J=9 Hz, 1H),
7.2 (s, 1H), 7.3 (d, J=9 Hz, 1H); 13C NMR (50 MHz,
CDCl3) d 1.4, 9.7, 20.7, 27.3, 28.2, 67.8, 75.6, 79.5,
103.9, 118.6, 119.8, 123.2, 1331.6, 132.6, 156.0, 175.9;
HRMS (EI, 70 eV) m/z calcd for C17H20O3N2 300.1469,
found 300.1474.

390
H.-I. Chiu et al. / Bioorg. Med. Chem. 9 (2001) 383±393
(30R,40R)-N-(6-Cyano-3,4-dihydro-2,2-dimethyl-3-hydroxy-
2H-[1]benzopyran-4-yl)-2-nitrobenzenesulfonamide ((À)-
29). To a stirred solution of aminoalcohol 28 (2.4 g,
11 mmol) and triethylamine (2.3 mL, 10.5 mmol) in dry
THF (60 mL) was added dropwise 2-nitrobenezesulfo-
nyl chloride (2.9 g, 13.2 mmol). The resulting mixture
was stirred at 40 ꢀC under N2 overnight. The mixture
was then ®ltered, and the ®ltrate was evaporated. The
(À)-8. Rf=0.48 (CH2Cl2:CH3OH=19:1);mp: 176±
177 ꢀC; H NMR (200 MHz, CDCl3) d 1.3 (s, 3H), 1.5
1
(s, 3H), 2.3 (s, 3H), 3.0 (td, J=3, 12 Hz, 1H), 3.4±3.6
(m, 3H), 3.9 (dd, J=3, 12 Hz, 1H), 5.8 (d, J=3 Hz, 1H),
6.9 (d, J=8 Hz, 1H), 7.3 (d, J=2 Hz, 1H), 7.4 (dd, J=2,
8 Hz, 1H); 13C NMR (50 MHz, CDCl3) d 22.1, 23.9,
24.9, 42.2, 45.4, 66.9, 74.2, 76.7, 104.4, 118.8, 119.2,
119.7, 132.4, 133.2, 158.1, 170.8.
residue was chromatographed (silica gel;CH Cl2:CH3
2
OH=19:1) to give (À)-29 (4.1 g, 93%) as a yellow
(À)-9. Rf=0.53 (CH2Cl2:CH3OH=19:1);mp: 141±
142 ꢀC; H NMR (200 MHz, CDCl3) d 1.1 (t, J=7 Hz,
1
solid: Rf=0.35 (CH2Cl2:CH3OH=19:1);mp: 238±
239.5 ꢀC; H NMR (400 MHz, DMSO-d6) d 1.3 (s, 3H),
3H), 1.2 (s, 3H), 1.4 (s, 3H), 2.4 (q, J=7 Hz, 2H), 2.9
(td, J=3, 12 Hz, 1H), 3.3 (d, J=3 Hz, 1H), 3.4±3.5 (m,
2H), 3.8 (dd, J=3, 11 Hz, 1H), 13C NMR (50 MHz,
CDCl3) d 9.8, 23.9, 24.9, 27.1, 41.3, 45.4, 66.9, 74.2,
78.7, 104.3, 118.8, 119.7, 119.8, 132.3, 133.1, 158.1,
174.1;HRMS (EI, 70 eV) m/z calcd for C17H20N2O3
300.1476, found 300.1473.
1
1.5 (s, 3H), 2.2 (d, J=8 Hz, 1H), 3.5 (dd, J=7, 4 Hz,
1H), 4.9 (s, 1H), 6.2 (s, 1H), 6.9 (d, J=9 Hz, 1H), 7.5
(d, J=7 Hz, 1H), 7.6 (s, 1H), 7.7±7.8 (m, 2H), 7.9 (m,
1H), 8.2 (m, 1H); 13C NMR (100 MHz, DMSO-d6)
d 24.5, 25.3, 50.9, 69.1, 80.3, 102.5, 118.2, 119.6, 122.2,
124.8, 130.6, 133.1, 133.2, 133.9, 135.0, 147.8, 157.7;
IR (neat) 1600, 2200, 3000, 3200 cmÀ1;MS (EI, 70 eV)
403 (M+, base). Anal. calcd for C18H17N3O6S: C,
53.59;H, 4.25;N, 10.42;found: C, 53.45;H, 4.34;N,
10.31.
(À)-10. Rf=0.62 (CH2Cl2:CH3OH=19:1);mp: 192±
193 ꢀC; H NMR (200 MHz, CDCl3) d 1.2 (d, J=6 Hz,
1
3H), 1.2 (d, J=6 Hz, 3H), 1.3 (s, 3H), 1.5 (s, 3H), 2.9
(m, J=6 Hz, 1H), 3.0 (t, J=11 Hz, 1H), 3.4 (d, J=3 Hz,
1H), 3.5±3.6 (m, 2H), 3.9 (dd, J=3, 11 Hz, 1H), 5.8 (d,
J=3 Hz, 1H), 6.9 (d, J=8 Hz, 1H), 7.2 (s, 1H), 7.4 (dd,
J=2, 8 Hz, 1H); 13C NMR (50 MHz, CDCl3) d 19.7,
19.9, 23.9, 24.9, 30.9, 41.4, 45.3, 67.1, 74.3, 78.7, 104.4,
118.8, 119.6, 119.8, 132.2, 133.2, 158.1, 177.4.
(4aR,10bR)-5,5-Dimethyl-1,2,3,4a,5,10b-hexahydro-[1]-
benzopyrano[3,4-b][1,4]oxazine-9-carbonitrile ((À)-31). A
mixture of (À)-29 (4 g, 9.92 mmol), K2CO3 (6.4 g, 49.6
mmol), 1-bromo-2-chloroethane (1.65mL, 19.8mmol),
NaI (0.3 g, 1.98 mmol), and dry DMF (10 mL) was stir-
red at 70 ꢀC under N2 for 3 days. The mixture was cooled
to room temperature and ®ltered. The ®ltrate was eva-
porated to give a residue, which was chromatographed
(silica gel;EtOAc: n-hexane=1: 1) to give (À)-30.
(À)-11. Rf=0.73 (CH2Cl2:CH3OH=19:1);mp: 195±
196 ꢀC; H NMR (200 MHz, CDCl3) d 1.4 (s, 3H), 1.5
1
(s,3H), 3.0 (td, J=3, 12 Hz, 1H), 3.4±3.5 (m, 2H), 3.6 (d,
J=3 Hz, 1H), 3.8 (dd, J=3, 12 Hz, 1H), 6.0 (d, J=3 Hz,
1H), 7.0 (d, J=8 Hz, 1H), 7.3±7.5 (m, 7H); 13C NMR
(50 MHz, CDCl3) d 14.1, 23.4, 24.5, 43.2, 45.4, 60.3,
66.7, 73.6, 78.2, 104.1, 109.4, 118.5, 119.1, 126.9, 128.7,
130.4, 131.7, 134.4, 157.6, 171.6.
A mixture of sulfonamide 30 (690 mg, 1.61 mmol), thio-
phenol (0.198 mL, 1.93 mmol), K2CO3 (412 mg, 3.22
mmol), and dry THF (5 mL) was stirred under N2 at
room temperature for 30 min. The mixture was evapo-
rated, and the residue was chromatographed (silica gel;
EtOAc:n-hexane=1:1) to a€ord (À)-31 (273 mg, 70%)
as a white solid: Rf=0.39 (CH2Cl2:CH3OH=19:1);mp:
(4aR,10bR)-N-Benzenesulfonyl-5,5-dimethyl-1,2,3,4a,5,
10b-hexahydro-[1]benzopyrano[3,4-b][1,4]oxazine-9-carbo-
nitrile ((À)-22). Mp: 135 ꢀC; 1H NMR (200 MHz,
CDCl3) d 1.2 (s, 3H), 1.3 (s, 3H), 2.8±2.9 (dt, J=14 Hz,
1H), 3.1 (d, J=3 Hz, 1H), 3.2±3.4 (dt, J=10, 12 Hz,
1H), 5.0 (d, J=2 Hz, 1H), 6.84 (d, J=9 Hz, 1H), 7.4 (d,
J=6 Hz, 2H), 7.5±7.7 (m, 3H), 7.9 (d, J=9 Hz, 2H); 13C
NMR (50 MHz, CDCl3) d 23.8, 24.8, 40.7, 49.8, 66.2,
73.3, 78.5, 104.7, 118.8, 118.9, 119.4, 127.1, 130.2, 133.0,
133.6,133.8, 141.2, 157.9;HRMS (EI, 70 eV) m/z calcd
for C20H20N2O4S 384.1144, found 384.1144.
154.5±155.5 ꢀC; H NMR (400 MHz, CDCl3) d 1.4 (s,
1
3H), 1.7 (s, 3H), 2.0 (br, 1H), 2.5 (dd, J=3, 26 Hz, 1H),
2.6 (td, J=3, 11 Hz, 1H), 3.5 (d, J=3 Hz, 1H), 3.5 (td,
J=3, 11 Hz, 1H), 3.7 (dd, J=3, 11 Hz, 1H), 4.0 (m, 1H),
6.8 (d, J=8 Hz, 1H), 7.4 (d, J=8 Hz, 1H), 7.8 (s, 1H);
13C NMR (100 MHz, CDCl3) d 23.4, 24.4, 39.1, 47.3,
68.6, 74.7, 78.1, 103.4, 118.1, 119.6, 121.9, 132.2, 132.9,
157.8;IR (neat) 2935, 2225, 1611, 1489 cm À1;MS (EI,
70 eV) 244 (M+, base). Anal. calcd for C14H16N2O2: C,
68.83;H, 6.60;N, 11.47;found: C, 68.52;H, 6.57;N,
11.49.
(4aS,10bR)-5,5-Dimethyl-1,2,3,4a,5,10b-hexahydro-N-(2-
methylbenzoyl)-[1]benzopyrano[3,4-b][1,4]oxazine-9-car-
bonitrile ((À)-32). By the same procedure for the synth-
esis of (+)-5, this compound was obtained from (À)-25
(115 mg, 0.47 mmol) and 2-methylbenzoyl chloride
(90 mg, 0.56 mmol) in 73% yield (124 mg);mp: 164±
(4aR,10bR)-5,5-Dimethyl-N-formyl-1,2,3,4a,5,10b-hexa-
hydro - [1]benzopyrano[3,4 - b][1,4]oxazine - 9 - carbonitrile
((À)-7). Rf=0.38 (CH2Cl2:CH3OH=19:1);mp: 212±
213 ꢀC; H NMR (200 MHz, CDCl3) d 1.3 (s, 3H), 1.5
1
165 ꢀC; H NMR (200 MHz, CDCl3) d 1.3 (s, 3H), 1.5
1
(s, 3H), 3.1 (td, J=3, 12 Hz, 1H), 3.4 (d, J=11 Hz, 1H),
3.5 (d, J=3 Hz, 1H), 3.6 (td, J=3, 11 Hz, 1H), 4 (dd,
J=3, 12 Hz, 1H), 5.6 (d, J=3 Hz, 1H), 6.9 (d, J=8 Hz,
1H), 7.3 (s, 1H), 7.5 (d, J=8 Hz, 1H), 8.4 (s, 1H); 13C
NMR (50 MHz, CDCl3) d 22.3, 23.5, 40.4, 43.6, 66.0,
72.5, 77.1, 103.1, 117.3, 117.6, 118.0, 131.1, 132.1, 156.6,
161.4.
(s, 3H), 2.4 (s, 3H), 3.6±3.7 (m, 3H), 4.7 (d, J=10 Hz,
1H), 6.9 (d, J=8 Hz, 1H), 7.2±7.5 (m, 6H); 13C NMR
(50 MHz, CDCl3) d 14.8, 20.0, 20.5, 27.2, 68.4, 76.0,
79.3, 104.2, 118.9, 122.4, 126.7, 130.4, 131.5, 132.7,
135.6, 155.9, 173.7;IR (KBr) 2973, 2651, 2223, 1682,
1650 cmÀ1;HRMS (EI, 70 eV) m/z calcd for C22H22O3N2

H.-I. Chiu et al. / Bioorg. Med. Chem. 9 (2001) 383±393
391
362.1630, found 362.1610;362.2 (M +), 119 (base).
NMR (50 MHz, CDCl3) d 20.0, 26.8, 56.7, 7.17, 76.1,
79.0, 104.3, 119.2, 119.7,.121.8, 126.4, 129.0, 130.8,
132.7, 133.2, 133.8, 138.7, 155.8, 172.3;HRMS (EI,
70 eV) m/z calcd for C22H19N2O3F3 416.1348, found
416.1354;416.2 (M +), 173 (base).
(À)-33. Mp: 164±165 ꢀC; 1H NMR (200 MHz, CDCl3) d
1.3 (s, 3H), 1.5 (s, 3H), 3.5±3.5 (m, 2H), 3.8±3.9 (br,
3H), 4.8 (br, 1H), 6.9 (d, J=8 Hz, 1H), 7.4 (d, J=8 Hz,
1H), 7.6±7.7 (m, 4H), 7.8 (d, J=8 Hz, 1H); 13C NMR
(50 MHz, CDCl3) d 20.5, 27.2, 56.7, 67.5, 76.1, 79.2,
104.4, 109.9, 119.7,.127.4, 128.3, 129.0, 130.5, 132.9,
133.2,133.8, 134.9, 155.9, 170.5;IR (KBr) 2967, 2222,
1656, 1490, 1312 cmÀ1;HRMS (EI, 70 eV) m/z calcd for
C22H19N2O3F3 416.1366, found 416.1372;416.2 (M +),
173 (base).
(À)-39. Mp: 199±201 ꢀC; 1H NMR (200 MHz, CDCl3) d
1.2 (s, 3H), 1.5 (s, 3H), 3.6±3.7 (m, 2H) 3.7±3.8 (m, 1H),
3.9 (m, 2H), 4.3 (d, J=9 Hz, 1H ), 6.9 (d, J=9 Hz, 1H),
7.4 (d, J=4 Hz, 2H), 7.5 (d, J=8 Hz, 2H), 7.6 (d,
J=8 Hz, 2H); 13C NMR (50 MHz, CDCl3) d 20.0, 26.8,
56.7, 68.1, 75.9, 79.9, 104.3, 119.2, 119.7,.121.9, 126.4,
130.3, 130.7, 132.6, 134.0, 155.8, 172.7;IR (KBr) 3243,
2986, 2225, 1751, 1690 cmÀ1;HRMS (EI, 70 eV) m/z
calcd for C21H19N2O3Br 427.0579, found: 426.0574;
427.9 (M+), 184 (base).
(À)-34. Mp: 175±176 ꢀC; 1H NMR (200 MHz, CDCl3) d
1.3 (s, 3H), 1.5 (s, 3H), 3.6±3.7 (m, 2H), 3.8±3.9 (m, 2H),
4.6 (d, J=10 Hz, 1H), 6.9 (d, J=8 Hz, 1H), 7.2±7.5 (m,
6H); 13C NMR (50 MHz, CDCl3) d 20.1, 21.8, 26.9,
56.4, 68.2, 75.9, 78.9, 104.2, 119.0, 122.3, 125.7, 129.1,
129.2, 130.9, 132.5, 139.2, 155.8, 173.9;IR (KBr) 3247,
2979, 2861, 2222, 1695 cmÀ1;HRMS (EI, 70 eV) m/z
calcd for C22H22O3N2 362.1631, found 362.1646;362.2
(M+), 119 (base).
(À)-40. Mp: 148±150 ꢀC; 1H NMR (200 MHz, CDCl3) d
1.2 (s, 3H), 1.5 (s, 3H), 3.6 (dt, J=9, 8 Hz, 2H), 3.8 (s,
3H), 3.9 (m, 3H), 4.7 (d, J=10 Hz, 1H), 6.9 (d, J=9 Hz,
3H), 7.3 (d, J=8 Hz, 2H), 7.5 (d, J=9 Hz, 2H); 13C
NMR (50 MHz, CDCl3) d 19.9, 26.8, 55.9, 56.6, 68.1,
75.9, 78.9, 104.2, 114.5, 119.8, 122.35, 127.3, 130.7,
130.9, 132.4, 155.8, 162.6, 173.4;IR (KBr): 3243, 2986,
2225, 1751, 1690 cmÀ1;HRMS (EI, 70 eV) m/z calcd for
C22H22O4N2 378.1580, found 378.1575;378.1 (M +), 135
(base).
(À)-35. A white solid; 1HNMR (200 MHz, CDCl3) d 1.3
(s, 3H), 1.5 (s, 3H), 3.6±3.7 (m, 2H), 3.7±3.8 (dd, J=4,
8 Hz, 1H), 3.8±3.9 (d, J=10 Hz, 2H), 4.5 (d, J=10 Hz,
1H), 6.9 (d, J=9 Hz, 1H), 7.4 (d, J=8 Hz, 2H), 7.5±7.6
(t, J=8, 9 Hz, 1H), 7.8±7.9 (m, 3H); 13C NMR (50 MHz,
CDCl3) d 1.4, 19.9, 26.8, 56.9, 68.1, 76.2, 76.2, 78.9,
104.4, 119.2, 119.6, 121.8, 125.7, 129.9, 130.8, 131.9,
132.7, 136.1, 155.8, 172.1;IR (KBr) 3243, 2220, 1656,
1490, 1325, 1127 cmÀ1;HRMS (EI, 70 eV) m/z calc'd for
C22H19N2O3F3 416.1343, found 416.1340;416.1 (M +),
173 (base).
(4aR,10bR)-5,5-Dimethyl-1,2,3,4a,5,10b-hexahydro-N-(2-
methylbenzoyl)-[1]benzopyrano[3,4-b][1,4]oxazine-9-car-
bonitrile ((À)-12). A mixture of compound (À)-32
(124 mg, 0.34 mmol), NaH (60%, 80 mg, 2.04 mmol),
and DMF (4 mL) was stirred under N2 at 0 ꢀC for 3 h.
The mixture was then quenched with 2 drops of acetic
acid. DMF was removed via Kugel-Rhor distillation
(5 mm Hg, 75 ꢀC). The residue was partitioned between
CH2Cl2 and H2O. The organic layer was dried with
MgSO4 and evaporated. The residue was chromato-
graphed (silica gel;EtOAc: n-hexane=1:2) to a€ord (À)-
12 (66 mg, 53%) as a white solid: Rf=0.27 (EtOAc:
1
(À)-36. A white solid; H NMR (200 MHz, CDCl3) d
1.3 (s, 3H), 1.5 (s, 3H), 3.7 (d, J=8 Hz, 1H), 3.8 (d,
J=9 Hz, 1H), 3.9 (d, J=10 Hz, 2H), 4.5 (d, J=10 Hz,
1H), 6.9 (d, J=9 Hz, 1H), 7.4 (d, J=7 Hz, 2H), 7.6±7.7
(t, J=8, 8 Hz, 1H), 7.9 (d, J=8 Hz, 1H), 8.3 (d,
J=8 Hz, 1H), 8.5 (s, 1H ); 13C NMR (50 MHz, CDCl3)
d 14.8, 19.9, 26.8, 51.2, 57.0, 68.2, 76.2, 78.9, 104.4,
119.3, 119.6,.121.5, 123.8, 126.5, 130.6, 130.8, 132.7,
134.6, 136.8, 148.8, 170.1;IR (KBr) 3246, 2921, 2225,
1668, 1534 cmÀ1;HRMS (EI, 70 eV) m/z calcd for
C21H19N3O5 393.1325 (M+), found 393.1340;393.2
(M+), 150 (base).
1
n-hexane=1:2); H NMR (200 MHz, CDCl3) d 1.3 (s,
3H), 1.5 (s, 3H), 2.1 (s, 3H), 2.8±2.9 (dt, 1H), 3.4±3.5
(m, 3H), 3.6±3.7 (d, J=11 Hz, 1H), 5.9 (s, 1H), 6.8 (d,
J=8 Hz, 1H), 7.2 (s, 5H), 7.4 (d, J=8 Hz, 1H); 13C
NMR (50 MHz, CDCl3) d 14.5, 19.8, 21.4, 23.8, 24.9,
60.7, 74.3, 78.7, 104.6, 119.0, 119.5, 126.1, 126.6, 129.9,
131.2, 131.5, 132.2, 134.6, 135.3, 158.1, 172.2;HRMS
(EI, 70 eV) m/z calcd for C22H22N2O3 362.1631, found
362.1637.
1
(À)-37. H NMR (200 MHz, CDCl3) d 1.3 (s, 3H), 1.5
(s, 3H), 2.4 (s, 2H), 3.7 (m, 2H), 3.9 (m, 3H), 4.5 (d,
J=10 Hz, 1H), 6.9 (d,J=8 Hz, 1H), 7.2 (d, J=7 Hz,
2H), 7.4 (d, J=8 Hz, 2H), 7.5 (d, J=8 Hz, 2H); 13C
NMR (50 MHz, CDCl3) d 1.4, 14.8, 20.0, 21.9, 26.8,
56.5, 62.6, 68.1, 75.9, 78.9, 104.2, 119.0, 122.3, 128.8,
129.9, 130.8, 132.3, 132.5, 142.3, 155.8, 173.9;IR (KBr)
3224, 3041, 2989, 2222, 1752 cmÀ1;HRMS (EI, 70 eV)
m/z calcd for C22H22O3N2 362.1638, found 362.1626;
362.2 (M+), 119 (base).
1
(À)-13. A white solid; H NMR (200 MHz, CDCl3) d
1.3 (s, 3H), 1.5 (s, 3H), 3.0±3.1 (t, J=12, 10 Hz, 1H), 3.3
(d, J=14 Hz, 1H), 3.5±3.6 (m, 2H), 3.8 (d, J=12 Hz,
1H), 5.9 (s, 1H), 6.9 (d, J=9 Hz, 1H), 7.4 (d, J=7 Hz,
2H), 7.5±7.6 (m, 2H), 7.7 (d, J=7 Hz, 2H); 13C NMR
(50 MHz, CDCl3) d 1.3, 14.5, 21.3, 23.8, 24.9, 43.7, 46.2,
67.0, 74.3, 78.7, 104.8, 119.1, 121.2, 124.7, 127.6, 129.8,
130.6, 131.7, 132.1, 133.5, 135.8, 158.1, 170.5;HRMS
(EI, 70 eV) m/z calcd for C22H19N2O3F3 416.1366,
found 416.1372.
1
(À)-38. A yellow oil; H NMR (200 MHz, CDCl3) d 1.3
(s, 3H), 1.5 (s, 3H), 3.6±3.7 (m, 2H), 3.8 (d, J=9 Hz,
2H), 3.9 (d, J=10 Hz, 2H), 4.6 (d, J=10 Hz, 1H), 6.9 (d,
J=9 Hz, 1H), 7.4 (d, J=7 Hz, 2H), 7.7 (s, 4H); 13C
(À)-14. A white solid; 1H NMR (200MHz, CDCl3) d 1.3
(s, 3H), 1.4 (s, 3H), 2.3 (s, 3H), 2.9±3.0 (dt, J=12 Hz, 1H),

392
H.-I. Chiu et al. / Bioorg. Med. Chem. 9 (2001) 383±393
3.4±3.5 (m, 3H), 3.7 (d, J=9 Hz, 1H), 5.9 (s, 1H), 6.8 (d,
J=9 Hz, 1H), 7.2±7.5 (m, 6H); 13C NMR (50 MHz,
CDCl3) d 14.5, 21.8, 23.9, 43.8, 45.9, 60.7, 62.5, 67.2,
74.1, 78.7, 104.6, 119.0, 119.7, 127.8, 129.8, 131.9, 132.3,
133.3, 141.2, 158.1, 172.2;HRMS (EI, 70 eV) m/z calcd
for C22H22N2O3 362.1631, found 362.1625.
(4aR,10bR)-5,5-Dimethyl-1,2,3,4a,5,10b-hexahydro-N-(4-
hydroxylbenzoyl) - [1]benzopyrano[3,4 - b][1,4]oxazine - 9 -
carbonitrile ((À)-21). A mixture of (À)-16 (0.0759 g,
0.2 mmol), BBr3-S(CH3)2 (375mg, 1.2 mmol), and ClCH2
CH2Cl (8 mL) was re¯uxed overnight, and then cooled
to room temperature. The mixture was treated with
aqueous NaHCO3 solution, and extracted with CH2Cl2
(50 mLÂ2). The organic layers was combined and eva-
porated to give (À)-21 (59.2 mg, 81.3%) as a white solid:
1
(À)-15. A white solid; H NMR (200 MHz, CDCl3) d
1.3 (s, 3H), 1.5 (s, 3H), 2.9±3.1 (m, 2H), 3.4±3.7 (m, 2H),
3.9±4.1 (m, 1H), 5.9 (d, J=2 Hz, 1H), 6.9 (m, J=8 Hz,
1H), 7.4±7.5 (m, 2H), 7.5±7.6 (m, 2H), 7.7±7.8 (m, 2H);
13C NMR (50 MHz, CDCl3) d 23.8, 24.9, 43.2, 46.5,
66.3, 66.9, 73.9, 74.6, 78.6, 78.8, 104.7, 118.8, 119.5,
119.4, 127.4, 127.6, 130.3, 132.1, 133.1, 157.9, 169.4;
HRMS (EI, 70 eV) m/z calcd for C22H19N2O3F3
416.1350, found 416.1349.
1
Rf=0.43 (EtOAc:n-hexane=1:2); H NMR (200 MHz,
CDCl3) d 1.3 (s, 3H), 1.5 (s, 3H), 3.0 (m, 1H), 3.5 (s,
3H), 3.7 (m, 1H), 5.9 (s, 1H), 6.8 (d, J=8 Hz, 3H), 7.2
(d, J=8 Hz, 2H), 7.4 (d, J=7 Hz, 2H), 7.9 (br, 1H); 13C
NMR (50 MHz, CDCl3) d 1.4, 14.5, 23.9, 24.9, 46.2,
60.9, 67.1, 74.1, 78.7, 104.4, 116.2, 119.1, 119.6, 125.4,
129.8, 132.2, 133.4, 158.2, 159.49, 172.8;HRMS (EI,
70 eV) m/z calcd for C21H20N2O4 364.1425, found
364.1423.
1
(À)-16. H NMR (200 MHz, CDCl3) d 1.3 (s, 3H), 1.5
(s, 3H), 3.0±3.1 (dt, J=12, 13 Hz, 1H), 3.3 (d, J=13 Hz,
2H), 3.5±3.6 (m, 2H), 3.8 (d, J=12 Hz, 1H), 5.9 (s, 1H),
6.9 (d, J=8 Hz, 1H), 7.4 (d, J=8 Hz, 2H), 7.7 (d,
J=8 Hz, 1H), 7.8 (d, J=7 Hz, 1H), 8.3 (d, J=8 Hz, 1H);
13C NMR (50 MHz, CDCl3) d 23.8, 24.9, 30.1, 43.8, 46.4,
67.0, 74.2, 78.7, 104.8, 119.0, 119.2, 122.9, 125.6, 130.6,
132.0, 133.4, 133.6, 136.5,.148.7, 158.1, 169.5;HRMS
(EI, 70 eV) m/z calcd for C21H19N3O5 393.1325, found
393.1337.
In vitro assay for PCO activity
The assay was performed with preparations of portal
vein strips and urinary bladder detrusor from adult
male Wistar rats. Literature procedures using the same
tissues from guinea pigs were adapted.15 The portal vein
and the bladder detrusor strips were placed respectively
in 1 mL and 5 mL organ baths containing modi®ed
Krebs±Henselent bu€er of the following composition
(in mmol/L): NaCl 118, KCl 4.7, CaCl2 2.52, MgSO4
1.19, KHPO4 1.19, chlortrimeton maleate 0.5 mg/L,
NaHCO3 25, glucose 11.48. A 0.5 gram preload tension
was applied to the portal vein to induce spontaneous
contraction, while the urinary bladder detrusor was
allowed to equilibrate under a 1 gram preload tension.
(À)-17. A white solid; 1H NMR (200 MHz, CDCl3) d 1.2
(s, 3H), 1.5 (s, 3H), 2.3 (s, 3H), 2.9±3.0 (t, 1H), 3.4±3.5
(m, 1H), 3.7±3.8 (m, 1H), 5.9 (s, 1H), 6.9 (d, J=9 Hz,
1H), 7.3 (m, 4H), 7.4±7.6 (m, 1H); 13C NMR (50 MHz,
CDCl3) d 14.6, 21.8, 25.0, 67.2, 74.2, 78.7, 104.6, 119.0,
119.7, 124.3, 128.1, 129.3, 131.6, 132.3, 133.3, 134.9,
139.3, 158.1;HRMS (EI, 70 eV) m/z calcd for C22H22
N2O3 362.1631, found 362.1624.
Acknowledgements
1
(À)-18. A white solid; H NMR (200 MHz, CDCl3) d
1.3 (s, 3H), 1.5 (s, 3H), 2.9±3.1(dt, J=3, 7, 13 Hz, 1H),
3.3 (d, J=13 Hz, 1H), 3.4 (d, 1H), 3.5±3.6 (m, 1H), 3.7±
3.9 (d, J=9 Hz, 1H), 5.9 (s, 1H), 6.9 (d, J=8 Hz, 1H),
7.4 (d, J=8 Hz, 1H), 7.6 (d, J=8 Hz, 2H), 7.7 (d,
J=8 Hz, 2H); 13C NMR (50 MHz, CDCl3) d 23.8, 24.9,
39.0, 43.6, 46.1, 67.0, 74.2, 78.7, 104.7, 119.1, 119.5, 126.3,
127.9, 132.0, 133.2, 133.5, 138.4,.158.1, 166.8, 170.7.
This research was supported by the National Science
Council of the R.O.C. under Grant No. NSC 87-2314-
B002-156-M38.
References and Notes
1. For recent reviews on urinary incontinence, see: (a)
Andersson, K.-E.;Appell, R.;Cardozo, L. D.;Chapple, C.;
Drutz, H. P.;Finkbeiner, A. E.;Haab, F.;Navarrete, R. V.
BJU Int. 1999, 84, 923. (b) Thuro€, J. W.;Chartier-Kastler,
E.;Corcus, J.;Humke, J.;Jonas, U.;Palmtag, H.;Tanagho,
E. A.; World J. Urol. 1998, 16 (suppl. 1), S48. (c) Owens, R.
G.;Karram, M. M. Drug Saf. 1998, 19, 123. (d) Butera, J. A.;
Argentieri, T. M. Expert. Opin. Ther. Pat. 1998, 8, 1017. (e)
Newgreen, D. T. Emerging Drugs 1998, 3, 209. (f) Hattori, T.
Drugs Today 1998, 34, 125.
(À)-19. A white solid; 1H NMR (200 MHz, CDCl3) d 1.3
(s, 3H), 1.5 (s, 3H), 3.0±3.9 (dt, 1H), 3.4 (d, J=14 Hz,
2H), 3.5±3.6 (m, 2H), 3.8 (d, J=14 Hz, 1H), 5.9 (s, 1H),
6.9 (d, J=8 Hz, 1H), 7.3 (d, J=8 Hz, 2H), 7.4 (d,
J=8 Hz, 2H), 7.5 (d, J=8 Hz, 2H); 13C NMR (50 MHz,
CDCl3) d 23.9, 24.9, 30.1, 43.7, 46.1, 67.1, 74.2, 78.7,
104.7, 119.1, 119.4, 125.4, 129.2, 132.1, 132.5, 133.4,
133.7, 158.1, 171.1.
2. For recent reviews on potassium channel openers, see: (a)
Poyser, R. H.;Hamilton, T. C. Drugs of the Future 1994, 19,
39. (b) Emp®eld, J. R.;Russell, K. Annu. Rep. Med. Chem.
1996, 30, 81. (c) Pirotte, B.;Fontaine, J.;Lebrun, P. Curr.
Med. Chem. 1995, 2, 573. (d) Atwal, K. S. Curr. Med. Chem.
1996, 3, 227. (e) Potassium Channels and their Modulators:
From Synthesis to Clinical Experience;Evans, J. M.;Hamilton,
T. C.;Longman, S. D.;Stemp, G., Eds;Taylor & Francis,
1996.
(À)-20. Mp: 169±170 ꢀC; 1H NMR (200 MHz, CDCl3) d
1.2 (s, 3H), 1.4 (s, 3H), 3.0 (t, 1H), 3.5 (s, 3H), 3.7 (s,
4H), 5.9 (s, 1H), 6.8±6.9 (m, 3H), 7.4 (d, J=7 Hz, 5H);
13C NMR (50 MHz, CDCl3) d 14.8, 21.8, 23.9, 24.9,
55.8, 62.5, 67.2, 74.2, 78.7, 104.6, 114.5, 119.0, 119.8,
128.6, 129.4, 129.7, 132.3, 133.3, 158.1, 171.9;HRMS
(EI, 70 eV) m/z calcd for C22H22N2O4 378.1579, found
378.1564.

H.-I. Chiu et al. / Bioorg. Med. Chem. 9 (2001) 383±393
393
3. Bonev, A. D.;Nelson, M. T. Am. J. Physiol. 1993, 264
(Cell Physiol. 33), C1190.
10. Wolf, C.;Weiss-Greiler, P.;Wolschann, P.;Ecker, G.;
Fleischhacker, W. Pharmazie 1996, 51, 836.
4. Nurse, D. E.;Restorick, J. M.;Mundy, A. R. Br. J. Urol.
1991, 68, 27.
5. De Moura, R. S.;De Mello, R. F.;D'Aguinaga, S. J. Urol.
1993, 149, 1174.
11. The high optical purity of target compounds was indicated
by the fact that only single isomers were observed when the
chromatographic purity of target compounds was determined
using a chiral column (cf. Table 2).
6. (a) Grant, T.;Frank, C. A.;Kau, S. T.;McLaren, F. M.;
Ohnmacht, C. J.;Russell, K.;Shapiro, H. S.;Trivedi, S.
Bioorg. Med. Chem. Lett. 1993, 12, 2723. (b) Grant, T. L.;
Ohnmacht, C. J.;Howe, B. B. TIPS 1994, 15, 402. (c) Emp-
®eld, J. R.;Mayhugh, D.;Ohnmacht, C. J.;Frank, C. A.;
Grant, T.;Li, J. Bioorg. Med. Chem. Lett. 1997, 7, 775.
7. Li, J. H.;Yasay, G. D.;Kau, S. T.;Ohnmacht, C. J.;Trai-
nor, D. A.;Bonev, A. D.;Heppner, T. J.;Nelson, M. T. Arz-
neim.-Forst./Drug Res. 1996, 46, 525.
8. (a) Butera, J. A.;Antane, M. M.;Antane, S. A.;Argentieri,
T. M.;Freeden, C.;Grace€a, R. F.;Hirth, B. H.;Jenkine, D.;
Lennox, J. R.;Mzltelan, E.;Norton, N. W.;Quagliato, D.;
Sheldon, J. H.;Spinelli, W.;Warga, D.;Wojdan, A.;Woods,
M. J. Med. Chem. 2000, 43, 1187. (b) Wojdan, A.;Freeden,
C.;Woods, M.;Oshiro, G.;Spinelli, W.;Colatsky, T. J.;
Sheldon, J. H.;Norton, N. W.;Warga, D.;Antane, M. M.;
Antane, S. A.;Butera, J. A.;Argentieri, T. M. J. Pharmacol.
Exp. Ther. 1999, 289, 1410.
12. Lee, N. H.;Muci, A. R.;Jacobson, E. C. Tetrahedron
Lett. 1991, 32, 5055.
13. Mitsunobu, O. Synthesis 1981, 1.
14. Houge-Frydrych, C. S. V.;Pinto, I. L. Tetrahedron Lett.
1989, 30, 3349.
15. Fukuyama, T.;Jow, C.-K.;Cheung, M. Tetrahedron Lett.
1995, 36, 6373.
16. Cassidy, F.;Evans, J. M.;Smith, D. M.;Stemp, G.;Edge,
C.;Williams, D. J. Chem. Commun. 1989, 377.
17. Zografos, P.;Li, J. H.;Kau, S. T. Pharmacol. 1992, 45,
216.
18. Quast, U.;Villhauer, E. B. Eur. J. Pharmacol. 1993, 245, 165.
19. Ohnmach, C. J.;Russel, K.;Emp®eld, J. R.;Frank, C. A.;
Gibson, K. H.;Mayhugh, D. R.;McLaren, F. M.;Shapiro, H.
S.;Brown, F. J.;Trainor, D. A.;Ceccarelli, C.;Lin, M. M.;
Masek, B. B.;Forst, J. M.;Harris, R. J.;Hulsizer, J. M.;
Lewis, J. J.;Silverman, S. M.;Smith, R. W.;Waraick, P. J.;
Kau, S. T.;Chun, A. L.;Grant, T. L.;Howe, B. B.;Li, J. H.;
Trivedi, S.;Halterman, T. J.;Yochim, C.;Dyro€, M. C.;
Kirkland, M.;Neilson, K. L. J. Med. Chem. 1996, 39, 4592.
9. Cheng, C. Y.;Chiu, H. I.;Chang, M. J.;Lin, Y. C.;Tsai,
M. C.;Yu, H. C. Bioorg. Med. Chem. Lett. 1998, 8, 463.