Discovery and structure-activity relationships of a novel series of benzopyran-based KATP openers for urge urinary incontinence

Article abstract of DOI:10.1016/j.bmc.2008.11.055

A novel series of benzopyran derivatives were synthesized and evaluated as K_ATP channel openers. Structure-activity relationships were investigated around 4-position of the benzopyran nucleus. Optimization of 4-substituent with some heterocyclic rings led to compound 13b bearing a benzo[d]isoxazol-3-one moiety as a potent and selective K_ATP channel opener in vitro. In two anesthetized rat models of myogenic bladder overactivity, compound 13b was found to inhibit spontaneous bladder contractions.

Full text of DOI:10.1016/j.bmc.2008.11.055

Bioorganic & Medicinal Chemistry 17 (2009) 855–866
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery and structure–activity relationships of a novel series of
benzopyran-based KATP openers for urge urinary incontinence
*
Xuqing Zhang , Yuhong Qiu, Xiaojie Li, Sheela Bhattacharjee, Morgan Woods, Patricia Kraft,
Scott G. Lundeen, Zhihua Sui
Drug Discovery, Johnson & Johnson Pharmaceutical Research and Development, LLC, 665 Stockton Drive, Exton, PA 19341, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of benzopyran derivatives were synthesized and evaluated as KATP channel openers. Struc-
ture–activity relationships were investigated around 4-position of the benzopyran nucleus. Optimization
of 4-substituent with some heterocyclic rings led to compound 13b bearing a benzo[d]isoxazol-3-one
moiety as a potent and selective KATP channel opener in vitro. In two anesthetized rat models of myogenic
bladder overactivity, compound 13b was found to inhibit spontaneous bladder contractions.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 8 January 2008
Revised 11 November 2008
Accepted 13 November 2008
Available online 27 November 2008
Keywords:
Urge urinary incontinence
KATP channel openers
Benzopyran
1. Introduction
prevalent form of UI, with reported prevalence rate from 40% to
70% of all diagnosed UI cases.5 OAB is characterized by the symp-
Ion channels play a fundamental role in the hormeostasis of cell
function through the regulation of the transmembrane movement
of ions. Cellular activity can be affected by modifications of the
activities of the ion channels. This leads to changes in membrane
potential difference. Potassium channels are a diverse and ubiqui-
tous group of ion channels.1 They principally regulate the resting
membrane potential of the cell and attenuate the level of excita-
tion of cells. A functional KATP channel is a hetero-octamer assem-
bled from four inward rectifying potassium channel subunits
(Kir6.2) and four sulfonylurea receptor (SUR) subunits.2 There are
two SUR genes, SUR1 and SUR2. SUR1/Kir6.2 channels are found
in the pancreas and brain. Two major splice variants arise from
the SUR2 gene, SUR2A and SUR2B, that differ only at the C-terminal
42 amino acids. SUR2A/Kir6.2 channels are found in cardiac and
skeletal tissues whereas SUR2B/Kir6.2 channels are found in
smooth muscles of many tissues including bladder.3 A number of
diseases or conditions may be treated with potassium channel
openers.4 This includes overactive bladder, urinary incontinence,
male erectile dysfunction, female sexual disorders, premature la-
bor, benign prostate hyperplasia (BPH), dysmenorrhea, neurode-
generation, stroke, pain, coronary artery disease, angina,
ischemia, eating disorders, irritable bowl syndrome, alopecia.
Urinary incontinence (UI) is a disease that can affect the overall
quality of life of a patient. Overactive bladder (OAB) is the most
toms of increased urinary frequency, urgency, and involuntary loss
of urine. A primary cause of OAB is an oversensitive bladder that
contracts unexpectedly and involuntarily. The ideal pharmaceuti-
cal agent should suppress the involuntary contraction while leav-
ing the normal voiding contractions intact. ATP-sensitive
potassium channel openers (KCO) could serve as such agents. The
ATP-sensitive potassium channels (KATP) are expressed in bladder
smooth muscle and function as key regulators of the resting mem-
brane potential in these cells. Compounds that selectively open
these channels hyperpolarize the cell and decrease cellular excit-
ability, resulting in suppression of involuntary bladder contrac-
tions, while leaving the normal micturition circuitry intact.6
A number of structurally distinct series of KATP channel openers
have been described. Among them, N-cyanoguanidine, benzopy-
ran, tertiary carbinol, dihydropyridine and squarate scaffolds have
been reported to activate bladder KATP channel in vitro and to inhi-
bit bladder function in vivo.7 Clinical studies have been conducted
on newer generation of KATP channel openers such as ZD-6169 and
ZD-0947 for treatment of OAB.
The present work aimed at developing novel benzopyrans
structurally related to cromakalim (1, Fig. 1) by varying nature of
4-substitution at the core structure. Particular attention was paid
to identification of structural requirements leading to improve-
ment of potency and selectivity on bladder. Benzopyrans represent
the most thoroughly studied KCOs. Previous structure-activity
studies indicated that 2, 4 and 6-positions of the nucleus are highly
susceptible to modulate biological activity. Initial screening of
* Corresponding author. Tel.: +1 610 458 4137.
E-mail address: xzhang5@prdus.jnj.com (X. Zhang).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.055

856
X. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 855–866
sired products (8g–j) in good yields with easy purification. When
Cl
heterocyclic nucleophiles 11b were 1H-indazol-3-ylamines or
benzo[d]isoxazol-3-ylamines, opening of epoxides 4 only yielded
corresponding 12 with NH as the linker between benzopyran core
and the heterocycles. However, benzo[d]isoxazol-3-ones 11b as
the nucleophiles reacted with epoxides 4 in the presence of
K2CO3 to afford a pair of regio-isomers in almost 1:1 ratio as O-
alkylated derivatives 12 and N-alkyated products 13. 13C NMR
spectra were used for differentiation of these two isomers. For
example, 12a and 13b were isolated from the epoxide-opening
reaction. Compound 12a presented a characteristic C signal next
to the linker O at 72.6 ppm, which was 26 ppm downfield than that
of the N-linked isomer 13b.
O
N
O
O
O
O
NC
OH
Me
NC
OH
Me
Me
Me
Cromakalim, 1
2
Figure 1.
3. Results and discussion
in-house compounds with benzopyran core resulted in compound
2 (Fig. 1) bearing a 3-chloro-benzoate group as a potent KATP chan-
nel opener (EC50 = 0.31 lM). We envisaged that modification on
Compounds were assayed for their activity as KATP channel
openers using primary TE671 human medulloblastoma cells.12
The effect of test compounds on KATP channels were evaluated on
a fluorometric imaging plate reader (FLIPR, Molecular Devices) at
room temperature. Following stabilization, increasing concentra-
tions of test compounds were superfused and isometic force was
measured. Glyburide, a KATP channel blocker, was added to a final
concentration to check the specificity of the test compound as a
KATP channel openers. Hyperpolarization resulting from KATP chan-
nel opening was observed as a decrease in fluorescent intensity.
All of the benzopyrans below are presented as KATP channel
openers. The KATP channel activation activity for all of these com-
pounds is reversed by glyburide. The initial effort was directed at
expanding SAR on structure 2 (6a in Table 1). Replacement of 3-
chloro-benzoate group of 6a with 3-chloro-benzamide group at
4-position of the benzopyran core resulted in slight increase in
activity, as shown for compound ( )-6b. Shifting of 3-Cl to 4-Cl
substitution on the phenyl ring resulted in a slight loss of activity
(6c). Replacement of Cl with CN group diminished the potency
(6d). Partial loss in activity was observed for the compounds with
bis-substitutions, as illustrated in 6e and 6f. Replacement of the
phenyl group with a pyridinyl group seemed to be tolerated for
KATP activity (6g). While partial loss in activity was observed for
6h with sulfonamide group as the linker, phosphinic amide group
(6i) as the linker was detrimental to activity. Resolution of enanti-
omers (+)-6b and (ꢀ)-6b showed that absolute stereochemistry
was critical for KATP activity as activity resided predominantly with
(3S,4R) enantiomer (ꢀ)-6b. Based on this information, all com-
pounds in Tables 2–4 were prepared as single enantiomer with
(3S,4R) configuration.
this template should generate our starting point to search structur-
ally novel, more potent and selective KATP channel openers. Our
strategy was to replace the benzoate group of compound 2 with
heterocycles or heterocycles attached via a heteroatom linker. In
this paper, we describe synthesis, extensive SAR investigations
and discovery of a potent and selective benzopyran bearing
benzo[d]isoxazol-3-one moiety as KATP channel opener targeting
UI.
2. Chemistry
The preparations of all compounds in this paper are shown in
Schemes 1–3. Epoxides 4 were prepared from asymmetric epoxi-
dation of chromenes 3 by Jocobsen’s catalyst (Scheme 1).8 Opening
of epoxides 4 with ammonia afforded amino-alcohols 5.9 Ana-
logues 6a–6i reported in this paper were prepared by following
the literature known methods (Scheme 2, condition a).10 Com-
pounds 10a and 10b were prepared by reacting amino-alcohols 5
with corresponding 2-methoxy-1H-quinazolin-4-ones 9 in reflux-
ing toluene (Scheme 2, condition b). Treatment of amino-alcohols
5 with acid anhydrides 14 in neat condition at 120 °C generated
corresponding phthalimide derivatives 13o and 13p in moderate
yields (Scheme 2, condition c). Scheme 3 showed the general pro-
cedure for preparation of compounds 8a–j, 10c–g, 12a–i and 13a–
n. Opening of epoxides 4 with appropriately substituted heterocy-
clic nucleophiles in the presence of a base such as K2CO3 in DMF
gave corresponding hydroxyl chromans. The required nucleophiles
7, 11a and 11b were commercially available or prepared by the lit-
erature known methods.11 Yield of epoxide-opening reaction is
critically dependent on nature of nucleophiles. Generally the reac-
tion proceeds in good yield when a S-containing nucleophile is
used. Using N or O containing nucleophiles results in longer reac-
tion time, higher temperature and decreased chemical yield. While
K2CO3-induced opening of epoxides 4 with heterocyclic substrates
was successful for preparation of a variety of analogues, it gave
very low yield for preparation of analogues 8g and 8h. Using pyr-
idine in refluxing EtOH provided an alternative way to yield the de-
Structurally modifying the carboxyl group of 2 to a five-mem-
bered heterocyclic ring such as pyrazole or imidazole resulted in
less potent compounds (Table 2). For 6-CN substituted benzopyran
analogues, introduction of a Cl group on the side chain phenyl ring
improved activity comparing with non-substituted phenyl group,
as illustrated in 8a–c versus 8d–e. Pyrazole derivative 8a seemed
to be the most potent analogue in this series with EC50 1.50
lM,
while imidazole analogues 8b and 8c were less active with EC50
2.75 and 7.45
lM correspondingly. Introduction of a methyl group
NH2
OH
O
a
b
R1
R1
R1
Me
Me
Me
O
5
O
O
4
Me
Me
Me
3
Scheme 1. Reagents and conditions: (a) Jacobsen’s catalyst, bleach in DCM; (b) NH3 in MeOH, rt.

X. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 855–866
857
R2
A
NH
O
NH2
O
OH
Me
OH
Me
Me
a
R1
R1
Me
5
A = -C(O)-, -SO2- or -P(O)Me-
6a-6i
O
N
NH2
O
R2
b
OH
Me
Me
R1
N
H
NH
O
O
OH
Me
R1
N
R2
5
Me
OMe
N
H
10a,10b
R2
9
NH2
O
c
OH
Me
Me
R1
R2
O
O
N
O
OH
Me
Me
R1
5
O
O
O
14
13o,13p
Scheme 2. Reagents and conditions: (a) Ar-A–Cl, TEA in DCM, 0 oC–rt; (b) toluene, refluxing; (c) neat, 120 oC.
on the imidazole ring was detrimental to activity (8d). For the
benzopyran core, replacement of 6-CN with 6-Cl group abolished
activity (8f). 6-Aza-chroman analogues 8h–i were tolerated for
activity comparing with their corresponding 6-CN substituted
chroman analogues. Among 6-aza-chroman analogues, pyrazole
8i seemed to be more active than imidazoles 8h and 8j.
13d bearing no substitution on the phenyl ring showed weak activ-
ities (EC50 6.94 lM for 13a and EC50 14.1 lM for 13d), substitution
on the phenyl ring with a Cl group at either 50 or 60-position of 13a
and a NO2 group at 50-position of 13d resulted in great improve-
ment of activity, as shown in 13b (EC50 0.67
lM), 13c (EC50
2.44 M) and 13e (EC50 4.10
l
l
M). 50-Cl substituted derivative
Extensive SAR was also conducted by replacing the benzoate
group of compound 2 with heterocycles attached via a heteroatom
linker, as shown in Table 3. Quinazolinone derivatives 10a (EC50
13b was about 4-fold more potent than its 6’-Cl substituted isomer
13c, 5-fold more potent than cromakalim, 10-fold more potent
than pinacidil and almost equipotent as P1075. Brief survey of 6-
substitution of the benzopyran core revealed that a variety of sub-
stitutions were well tolerated for KCO activity. As illustrated in
13f-i, these compounds containing 6-benzosulfone or 6-benzosulf-
onamide substitution still maintained potent activity with EC50
3.71
the open chain analogue 6b. Benzoxazole derivative 10c was sub-
stantially less active with EC50 16.5 M. Introduction of benzoimi-
dazole sulfanyl side chain resulted in a series of compounds 10d–g
with decreased activity (EC50 from 2.60 to 14.7 M) comparing
with compound 6b. Benzoisoxazoleoxy (12a, EC50 7.85 M), ben-
zo[d]-isothiazoleoxy (12b, EC50 16.1 M), benzo-isoxazolylamino
(12c, EC50 6.18 M) and indazolylamino (12d, EC50 6.85 M) were
lM) and 10b (EC50 2.29
lM) were ꢁ10-fold less potent than
l
l
ꢁ1
l
M. Other substitutions shown in Table 4 including 6,7-di-Cl
l
(13j, EC50 10.3
l
M) and 6-benzophenone (13k, EC50 6.45 M)
l
l
groups resulted in substantial loss of activity. Additional substitu-
tion such as F, Cl or methoxy group on the benzosulfone group of
13f was detrimental to activity, as shown for 13l–n. Finally, more
than 10-fold drop of activity was observed by replacement of the
benzo[d]isoxazol-3-one core with phthalimide moiety (13o, EC50
l
l
also not well tolerated for sub-micro molar activity comparing
with compound 6b. Substitution at 6-position of the benzopyran
core was also investigated. While 6-benzosulfone analogue 12e
showed good activity with EC50 1.37
meta-F group on the phenyl group resulted in tremendous loss in
activity (12f, EC50 7.77 M). Replacement of 6-CN group with 6-
l
M, further substitution of a
3.16
A selective binding of KCOs to different types of KATP channels in
different organs is one of the most demanding challenges in KATP
lM and 13p, EC50 6.04 lM).
l
-
sulfonamide (12g), 6,7-dichloro (12h) or 6-benzophenone (12i)
group led to nearly inactive compounds.
channel research. On the basis of successful cloning of KIR and of
SUR, it is widely accepted that SUR subtypes SUR1, SUR2A and
SUR2B are responsible for differential effects of KCOs on each type
of KATP channels. The small molecules prepared here targeted
SUR2B/Kir6.2 channel in smooth muscles such as bladder. Thus, se-
lected potent KCOs from screening assay were further evaluated
Further elucidation of structural and electronic properties of the
pharmacophore was accomplished by exploring
a series of
benzo[d]isoxazol-3-ones and their close analogues shown in Table
4. While benzo[d]isoxazol-3-one 13a and benzo[d]isothiazol-3-one

858
X. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 855–866
R2
R2
Y
Y
X
X
N
N
H
O
OH
Me
7
R1
R1
Me
Me
O
O
a or b
Me
X or Y = N
8a-j
4
R2
N
Y
O
X
N
a
OH
R1
R2
HX
R1
Me
Me
+
Y
Me
O
O
Me
X, Y = N , O or S
11a
10c-g
4
R2
Y
N
R2
Y
O
X
O
N
OH
Me
Y
OH
Me
a
R1
R1
R2
+ R1
N
Me
Me
+
O
O
O
Me
HX
Me
12
4
X, Y = N, O or S
11b
13a-n
Scheme 3. Reagents and conditions: (a) K2CO3, DMF rt–100 oC; (b) pyridine, EtOH, reflux for 8g–8j.
Table 1
SAR study of compounds 6 for KATP channel openers on TE671 cell
Ar
X
NC
OH
Me
O
Me
6
Compounds
–X–Ar
Ar
Stereochemistry
EC50 (l
M)b
Pinacidila
3.54
0.23
1.25
0.31
0.12
2.07
0.07
0.78
21.1
0.81
7.06
0.56
1.54
18.6
P1075a
Cromakalima
6a
( )-6b
(+)-6b
(ꢀ)-6b
6c
6d
6e
6f
6g
–OC(O)–Ar
3-Cl–Phenyl
3-Cl–Phenyl
3-Cl–Phenyl
3-Cl–Phenyl
4-Cl–Phenyl
4-CN–Phenyl
3,4-Methylenedioxy-phenyl
3,4-Di-Cl-phenyl
4-Cl-3-Pyridinyl
3-Cl-Phenyl
( )
( )
(3R,4S)
(3S,4R)
( )
–NHC(O)–Ar
–NHC(O)–Ar
–NHC(O)–Ar
–NHC(O)–Ar
–NHC(O)–Ar
–NHC(O)–Ar
–NHC(O)–Ar
–NHC(O)–Ar
–NHSO2–Ar
–NHP(O)(Me)–Ar
( )
(3S,4R)
(3S,4R)
(3S,4R)
(3S,4R)
(3S,4R)
6h
6i
Phenyl
a
Available from Sigma.
b
Average value for pinacidil, P1075 and cromakalim on 14 tests and average value for other compounds on 3 tests.
for SUR2B selectivity over SUR1 and SUR2A. In these studies, b-TC-
6 cell assay was used as the indicator of SUR2/Kir6.2 activity while
COS-7 assay was used as the indicator of SUR2A/Kir6.2 activity.
Table 5 summarized the selectivity of the most potent compounds
from screening. Benzo[d]isoxazol-3-one based benzopyrans (13b,
13f, 13g, 13i) showed good SUR2B/Kir selectivity over SUR1/Kir.
All compounds in Table 5 showed better selectivity than pinicidil
and cromakalim. The most selective compound 13b presented
comparable SUR2B/Kir6.2 against SUR1/Kir6.2 selectivity as
P1075. On the other hand, the benzopyran analogues were found

X. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 855–866
859
Table 2
Table 3
SAR study of compounds 8 for KATP channel openers on TE671 cell
SAR study of compounds 10 and 12 for KATP channel openers on TE671 cell
5'
Ar
Het
5
6'
Z
6
R
OH
N
R2
R1
S
Me
Y
7'
X
8'
O
7
R
5
8
Me
OH
6
R1
S
8
Me
O
7
Me
8
5
6
10 and 12
Compounds
-Het-Ar
EC50 (l
M)a
R1
Compounds
R1
R2
X
Y
Z
EC50 (l
M)a
7
8
Pinacidil
P1075
Cromakalim
10a
10b
10c
10d
10e
10f
10g
12a
12b
12c
12d
12e
12f
12g
12h
5.82
0.38
3.48
3.71
2.29
16.5
2.60
8.44
3.09
14.7
7.85
16.1
6.18
6.85
1.37
7.77
> 30
24.8
20.4
Pinacidil
P1075
Cromakalim
5.82
0.38
3.48
6-CN
6-CN
6-CN
6-CN
6-CN
6-CN
6-CN
6-CN
6-CN
6-CN
6-CN
6-SO2Ph
—
NH
NH
NH
O
NH
NH
NH
NH
—
—
—
—
—
C(O)
C(O)
—
—
—
—
—
O
S
O
NH
O
O
O
60-Cl
60-Cl
60-OEt
60-F
NH
NH
S
S
S
S
O
O
NH
NH
NH
NH
NH
NH
NH
6
NC
NC
NC
NC
NC
Cl
Cl
8a
8b
8c
8d
8e
8f
1.50
2.75
7.45
>30
N
N
60-NO2
60-Cl
60-Cl
—
60-Cl
70-Cl
70-Cl
70-Cl
70-Cl
70-Cl
70-Cl
N
N
6
6
6
6
Cl
6-SO2-m-F-Ph
6-SO2NEt2
6,7-Di-Cl
—
—
—
N
N
Cl
O
O
12i
6-C(O)Ph
a
Average value on P3 tests.
Me
N
N
Compound 13b was also evaluated in vitro using rat bladder
strip assay (organ bath)12 as shown in Fig. 2. Compound 13b was
found to be more active than other two KCOs, pinacidil and ZD-
N
N
13.0
>30
0947 with EC50 2.04
had EC50 0.14 M.
lM. For comparison, P1075 in the same test
l
According to all in vitro data, compound 13b was identified as a
potent and selective KATP channel openers in vitro. It was then
evaluated in two in vivo models of overactive bladder. The detailed
procedures are described in the experimental section. Compound
13b was first evaluated in a rat acetic acid model (Fig. 3). Cystom-
etry was performed using anesthetized female Sprague-Dawley
rats with exhibiting unstable bladder contractions due to saline
infusion. Administration of 13b (lower panel) at 0.3 mg/kg/1mL
via iv using 10% of PEG 300 and 20% of HPBCD as vehicle caused
a clear reduction in the number of unstable contractions compara-
ble to P1075 (middle panel), which demonstrates that 13b is capa-
ble of suppressing spontaneous bladder contractions at the testing
concentration. Compound 13b is further evaluated in a rat partial
outlet obstruction model. Cystometry was performed using anes-
thetized female Sprague-Dawley rats with bladder hypertrophy
and exhibiting unstable bladder contractions due to intravesical
outflow obstruction. Rats hypertrophied bladder cystometry plots
before and after treatment with and P1075 and 13b are presented
in Fig. 4. The pretreatment plot (upper panel) shows spontaneous
spikes which develop during the bladder filling phase. The lower
panel shows the cystometry after oral administration of compound
13b at 3 mg/kg. The spontaneous bladder contractions observed
during the filling phase have been significantly reduced. For com-
parison, P1075 in the same model shows the similar pharmacolog-
ical effect on the rat hyperophied bladder as shown in the middle
panel. Thus, 13b demonstrated its ability to prevent the spontane-
ous contractions in the hypertrophied rat bladder during filling.
N
N
6
N
N
6
N
8g
8h
12.9
4.33
N
N
6
N
Cl
6
Cl
Cl
N
8i
1.64
13.8
N
N
6
N
N
N
8j
a
Average value on P3 tests.
to be ꢁ6-fold SUR2B/Kir selective against SUR2A/Kir6.2, which
indicated that discrimination between cardiac (SUR2A/Kir6.2)
and smooth muscle (SUR2B/Kir6.2) was feasible.

860
X. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 855–866
Table 4
P1075 EC50 = 0.14 µM
13b EC50 = 2.04 µM
SAR study of compounds 13 for KATP channel openers on TE671 cell
R2
100
85
70
55
40
25
10
-5
PINACIDIL EC50 = 9.82 µM
ZD0947 EC50 = 17.7 µM
5'
6'
7'
4'
Y
O
N
O
5
R
6
OH
R1
S
Me
7
Me
8
13
Compounds
R1
R2
Y
EC50 (l
M)a
Pinacidil
P1075
Cromakalim
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
5.82
0.38
3.48
6.94
0.67
2.44
14.1
4.10
0.75
1.56
1.08
0.82
10.3
6.45
7.33
7.99
1.71
3.16
10-4 10-3 10-2 10-1 100 101 102 103
µM
6-CN
6-CN
6-CN
6-CN
6-CN
6-SO2Ph
6-SO2Ph
—
O
O
O
S
50-Cl
60-Cl
—
Figure 2. Organ bath study on rat bladder strips.
50-NO2
50-Cl
60-Cl
50-Cl
50-Cl
50-Cl
50-Cl
50-Cl
50-Cl
50-Cl
50-Cl
S
O
O
O
O
O
O
O
O
O
C(O)
AVANCE400 spectrometer. Chemical shifts are reported in ppm
downfield from TMS as an internal standard. Thin-layer chroma-
tography was carried out using 2.5 ꢂ 7.5 cm Silica Gel 60
6-SO2-Piperidinyl
6-SO2NEt2
6,7-Di-Cl
6-C(O)Ph
(250 lM layer) plates with UV detection. Anhydrous sodium sul-
6-SO2-p-MeO-Ph
6-SO2-p-Cl-Ph
6-SO2-m-F-Ph
6-CN
fate was employed to dry organic extracts prior to concentration
by rotary evaporation. Flash chromatography was usually done
using a CombiFlash companion system with pre-packed silica gel
cartridges purchased from AnaLogix. Solvents from J.T. Baker or Al-
drich and all other commercially available reagents were used
without further purification. Melting points were taken using a
Thomas-Hoover MelTemp apparatus without any correction.
Microanalysis was done by Quantitative Technologies Inc., White-
house, NJ. Mass spectra were obtained on a Hewlett–Packard
5989A quadrupole mass spectrometer. HPLC analysis was carried
on Agilent 1100 Series LC/MSD equipment. High resolution mass
spectra were obtained on M-Scan’s VG Analytical ZAB 2SE high
field mass spectrometer.
13o
5'
N
13p
6-CN
C(O)
6.04
a
Average value on P3 tests.
Table 5
Selectivity of SUR2B/Kir6.2 against SUR1/Kir6.2 and SUR2A/Kir6.2
Compounds
SUR2B TE-671 SUR1 b-TC- 6 SUR2A COS-7 SUR1/
SUR2A/
Compounds 6a–6h were prepared according to the literatures.
The spectroscopic data were the same as the reported data.10
EC50
(l
M)
EC50
(
lM)
EC50 (lM)
SUR2B SUR2B
Pinacidil
Cromakalim 1.25
3.54
60.8
33.7
60.5
99.8
44.3
31.2
53.3
21.6
4.89
0.62
1.67
2.44
8.15
3.19
17
27
263
149
59
6.1
3.9
2.7
6.4
3.3
5.2
3.8
5.1.1. (3S,4R)-N-(6-Cyano-3-hydroxy-2,2-dimethyl-chroman-4-
yl)-methylphenylphosphinic amide (6i)
P1075
13b
13f
13g
13i
0.23
0.67
0.75
1.56
0.82
(3S,4R)-4-Amino-3-hydroxy-2,2-dimethyl-chroman-6-carboni-
trile 5 (325 mg, 1.50 mmol) in DCM (10 mL) at 0 oC was treated
20
65
with TEA (316 lL, 2.25 mmol) followed by methylphenylphosphi-
nic chloride (261 mg, 1.50 mmol). The reaction was then slowly
warmed to room temperature over 2 h. Saturated NaHCO3 was
added and the aqueous phase was extracted 2ꢂ with DCM. The
combined organic layers were washed with brine, dried over anhy-
drous Na2SO4, filtered and concentrated as a yellow solid, which
was then purified by silica gel column chromatography to as yield
the title compound as a pale yellow solid (400 mg, 75%).
4. Conclusion
We have identified a novel series of benzopyrans as KATP chan-
nel openers. Extensive SAR studies revealed that benzo[d]isoxazol-
3-one as 4-subsutition of the benzopyran core was effective phar-
macophore for potent activity. The best compound 13b was dem-
onstrated to be potent in cell and rat bladder strip functional
assays with smooth muscle selectivity. It was also efficacious in
two rat models of bladder overactivity.
1H NMR: (CDCl3) d 8.15–7.52 (m, 5H), 7.10 (d, J = 7.5 Hz, 1H),
6.62 (d, J = 7.5 Hz, 1H), 6.08 (s, 1H), 4.23 (d, J = 9.5 Hz, 1H), 3.15
(d, J = 9.5 Hz, 1H), 1.55–1.42 (m, 9H). MS (m/z): MH+ 357.
5.1.2. (3S,4R)-4-[5-(4-Chloro-phenyl)-pyrazol-1-yl]-3-hydroxy-
2,2-dimethyl-chroman-6-carbonitrile (8a)
5. Experimental
(S,S)-2,2-Dimethyl-1a,7b-dihydro-2H-1,3-dioxa-cyclopropa[a]-
naphthalene-6-carbonitrile 4 (500 mg, 2.49 mmol) and 5-(4-chloro-
phenyl)-1H-pyrazole 7 (445 mg, 2.50 mmol) in DMF (5 mL) were
treated with K2CO3 (520 mg, 3.75 mmol) at 70 oC for 4 h. The
reaction was cooled down and the solid was filtered off. The filtrate
5.1. Chemistry. General procedures
1H NMR spectra were recorded at 400 MHz or 300 MHz with d-
chloroform or MeOD-d4 as solvent on a Brucker AVANCE300 or

X. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 855–866
861
Dosing
20
15
10
5
0
7500
8000
8500
9000
11500
10500
9500
10000
10500
20
10
0
-10
20
10500
11000
12000
12500
10
0
-10
8500
9000
9500
10000
11000
11500
12000
12500
Figure 3. Cystometry on acetic acid treated rats.
was then partitioned between EtOAc and water. The organic layer
was further washed with sat. NH4Cl solution, water and brine, dried
over anhydrous Na2SO4, filtered and concentrated as a yellow solid,
which was then purified by silica gel column chromatography to as
yield the title compound as a yellow solid (420 mg, 45%).
1H NMR: (CDCl3) d 7.72 (m, 1H), 7.35 (m, 5H), 6.82 (d, J = 7.5 Hz,
1H), 6.72 (s, 1H), 6.31 (s, 1H), 5.35 (d, J = 7.8 Hz, 1H), 4.05 (d,
J = 7.8 Hz, 1H), 2.16 (s, 3H), 1.65 (s, 3H), 1.28 (s, 3H). MS (m/z):
MH+ 360.
1H NMR (CDCl3) d 8.00 (s, 1H), 7.70 (m, 2H), 7.55 (m, 1H), 7.50
(s, 1H), 7.30 (m, 2H), 7.10 (s, 1H), 7.00 (m, 1H), 5.30 (m, 1H), 4.00
(m, 1H), 1.50 (s, 3H), 1.30 (s, 3H). MS (m/z): MH+ 380.
5.1.6. (3S,4R)-3-Hydroxy-2,2-dimethyl-4-(2-phenyl-imidazol-1-
yl)-chroman-6-carbonitrile (8e)
The same procedure for 8a was followed to give the title com-
pound as a white solid. Yield 48%.
5.1.3. (3S,4R)-4-[2-(4-Chloro-phenyl)-imidazol-1-yl]-3-
hydroxy-2,2-dimethyl-chroman-6-carbonitrile (8b)
The same procedure for 8a was followed to give the title com-
pound as a white solid. Yield 41%.
1H NMR: (CDCl3) d 7.82 (d, J = 3.5 Hz, 2H), 7.45 (m, 3H), 7.31 (d,
J = 6.5 Hz, 1H), 6.82 (d, J = 7.5 Hz, 1H), 6.60 (s, 1H), 6.32 (s, 1H), 5.32
(d, J = 7.5 Hz, 1H), 3.88 (d, J = 7.5 Hz, 1H), 1.55 (s, 3H), 1.25 (s, 3H).
MS (m/z): MH+ 346.
1H NMR: (CDCl3) d 7.72 (m, 3H), 7.45–7.35 (m, 4H), 6.85 (m,
2H), 5.90 (br, 1H), 5.30 (m, 1H), 4.02 (m, 1H), 1.65 (s, 3H), 1.28
(s, 3H). MS (m/z): MH+ 380.
5.1.7. (3S,4R)-6-Chloro-2,2-dimethyl-4-(2-phenyl-imidazol-1-
yl)-chroman-3-ol (8f)
The same procedure for 8a was followed to give the title com-
pound as a white solid. Yield 37%.
5.1.4. (3S,4R)-4-[5-(4-Chloro-phenyl)-imidazol-1-yl]-3-
hydroxy-2,2-dimethyl-chroman-6-carbonitrile (8c)
The same procedure for 8a was followed to give the title com-
pound as a yellow solid. Yield 55%.
1H NMR: (CDCl3) d 7.82 (d, J = 7.5 Hz, 1H), 7.55 (d, J = 7.4 Hz,
1H), 7.38 (m, 4H), 7.02 (s, 1H), 6.78 (d, J = 8.5 Hz, 1H), 6.71 (s,
1H), 6.40 (s, 1H), 5.42 (d, J = 8.0 Hz, 1H), 4.12 (d, J = 7.1 Hz, 1H),
1.58 (s, 3H), 1.22 (s, 3H). MS (m/z): MH+ 355.
1H NMR: (CDCl3) d 7.70 (m, 2H), 7.52 (s, 1H), 7.49 (m, 1H), 7.35
(m, 2H), 7.10 (s, 1H), 6.95 (m, 1H), 6.65 (s, 1H), 5.28 (d, J = 3.6 Hz,
1H), 4.30 (m, 1H), 2.75 (m, 1H), 1.60 (s, 3H), 1.35 (s, 3H). MS (m/
z): MH+ 380.
5.1.8. (3S,4R)-2,2-Dimethyl-4-(2-phenyl-imidazol-1-yl)-3,4-
dihydro-2H-pyrano[3,2-c]pyridin-3-ol (8g)
(S,S)-2,2-Dimethyl-1a,7b-dihydro-2H-1,3-dioxa-6-aza-cyclo-
propa[a]naphthalene 4 (450 mg, 2.54 mmol) and 2-phenyl-1H-
imidazole 7 (366 mg, 2.55 mmol) in EtOH (10 mL) were treated
with pyridine (1 mL). The reaction was refluxed for 10 hrs. The sol-
vent was removed in vacuo and the residue was purified by silica
5.1.5. (3S,4R)-3-Hydroxy-2,2-dimethyl-4-(4-methyl-2-phenyl-
imidazol-1-yl)-chroman-6-carbonitrile (8d)
The same procedure for 8a was followed to give the title com-
pound as a white solid. Yield, 50%.

862
X. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 855–866
Pre-dosing
Post-dosing
60
40
20
0
60
40
20
0
4:53:20 4:55:00 4:56:40 4:58:20 5:00:00 5:01:40 5:03:20 5:05:00
1:05:00 1:06:40 1:08:20 1:10:00 1:11:40 1:13:20 1:15:00 1:16:40
40
20
0
40
20
0
:40
1:00:00 1:03:20 1:06:40 1:10:00 1:13:20 1:16:40 1:20:00
4:00:00 4:03:20 4:06:40 4:10:00 4:13:20 4:16:40 4:20:00
40
30
20
10
0
30
20
10
0
53:20 55:00 56:40 58:20 1:00:00 1:01:40 1:03:20 1:05:00 1:06:40
4:20:00 4:23:20 4:26:40 4:30:00 4:33:20 4:36:40 4:40:00 4:43:20
Figure 4. Cystometry on partially obstructed rats.
gel chromatography to give the title compound as a brown solid
(340 mg, 42%).
5.1.12. (3S,4R)-4-(6-Chloro-4-oxo-1,4-dihydro-quinazolin-2-
ylamino)-3-hydroxy-2,2-dimethyl-chroman-6-carbonitrile
(10b)
1H NMR: (CDCl3) d 8.20 (m, 1H), 7.80 (m, 2H), 7.50 (s, 1H), 7.40
(m, 3H), 6.75 (m, 2H), 6.40 (s, 1H), 6.55 (s, 1H), 5.45 (m, 1H), 3.75
(m, 1H), 1.55 (s, 3H), 1.22 (s, 3H). MS (m/z): MH+ 322.
4-Amino-3-hydroxy-2,2-dimethyl-chroman-6-carbonitrile
5
(1.09 g, 5 mmol) and 6-chloro-2-methoxy-1H-quinazolin-4-one
(1.05 g, 5 mmol) in toluene (15 mL) in a seal tube was heated
at 150 oC for 4 h. The solvent was removed and the residue
was purified by silica gel chromatography with hexanes and
ethyl acetate to yield the title compound as white solid
(812 mg, 41%).
5.1.9. (3S,4R)-4-[2-(4-Chloro-phenyl)-imidazol-1-yl]-2,2-
dimethyl-3,4-dihydro-2H-pyrano[3,2-c] pyridin-3-ol (8h)
The same procedure for 8g was followed to give the title com-
pound as a yellow solid. Yield 32%.
1H NMR: (CDCl3) d 8.20 (m, 1H), 7.70 (m, 2H), 7.55 (s, 1H), 7.30
(m, 2H), 6.80 (s, 1H), 6.70 (m, 1H), 6.65 (s, 1H), 6.55 (s, 1H), 5.45 (m,
1H), 4.00 (m, 1H), 1.60(s, 3H), 1.25 (s, 3H). MS (m/z): MH+ 356.
1H NMR: (CDCl3) d 7.95 (s, 1H), 7.65 (m, 1H), 7.60 (d, J = 7.5 Hz,
1H), 7.51 (s, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H),
5.31 (d, J = 4.0 Hz, 1H), 3.72 (d, J = 7.5 Hz, 1H), 1.58 (s, 3H), 1.38
(s, 3H). MS (m/z): MH+ 398.
5.1.10. (3S,4R)-4-[5-(4-Chloro-phenyl)-pyrazol-1-yl]-2,2-
dimethyl-3,4-dihydro-2H-pyrano[3,2-c]pyridin-3-ol (8i)
The same procedure for 8g was followed to give the title com-
pound as a white solid. Yield 30%.
5.1.13. (3S,4R)-3-Hydroxy-2,2-dimethyl-4-(4-oxo-1,4-dihydro-
quinazolin-2-ylamino)-chroman-6-carbonitrile (10a)
The same procedure for 10b was followed to give the title com-
pound as a white solid. Yield 45%.
1H NMR: (CDCl3) d d 8.20 (m, 1H), 7.70 (s, 2H), 7.60 (s, 1H), 7.32
(m, 2H), 7.22 (m, 2H), 6.70 (m, 2H), 5.00 (d, J = 3.6 Hz, 1H), 3.72 (d,
J = 3.6 Hz, 1H), 1.60 (s, 3H), 1.30 (s, 3H). MS (m/z): MH+ 356.
1H NMR: (CDCl3) d 6.95–7.85 (m, 7H), 5.20 (s, br, 1H), 5.05 (d,
J = 8.5 Hz, 1H), 4.10 (d, J = 8.5 Hz, 1H), 1.55 (s, 3H), 1.41 (s, 3H).
MS (m/z): MH+ 363.
5.1.11. (3S,4R) 4-[5-(4-Chloro-phenyl)-imidazol-1-yl]-2,2-
dimethyl-3,4-dihydro-2H-pyrano[3,2-c]pyridin-3-ol (8j)
The same procedure for 8g was followed to give the title com-
pound as a white solid. Yield 25%.
5.1.14. (3S,4R)-4-(5-Chloro-benzooxazol-2-ylamino)-3-
hydroxy-2,2-dimethyl-chroman-6-carbonitrile (10c)
The same procedure for 8a was followed to give the title com-
pound as a white solid. Yield 60%.
1H NMR: (CD3OD) d 8.20 (d, J = 1.5 Hz, 1H), 7.93 (s, 1H),
7.72 (d, J = 3.0 Hz, 2H), 7.70 (s, 1H), 7.30 (d, J = 3.0 Hz, 2H),
6.90 (d, J = 1.5 Hz, 1H), 6.72 (s, 1H), 4.45 (d, J = 3.3 Hz, 1H),
4.30 (d, J = 3.3 Hz, 1H), 1.57 (s, 3H), 1.32 (s, 3H). MS (m/z):
MH+ 356.
1H NMR: (CDCl3) d 7.65 (s, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.12 (d,
J = 8.0 Hz, 2H), 7.05 (d, J = 6.5 Hz, 1H), 6.92 (d, J = 7.5 Hz, 1H), 6.42
(s, 1H), 5.05 (d, J = 7.1 Hz, 1H), 3.82 (d, J = 7.1 Hz, 1H), 1.55 (s, 3H),
1.35 (s, 3H). MS (m/z): MH+ 372.

X. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 855–866
863
5.1.15. (3S,4R)-4-(5-Ethoxy-benzothiazol-2-ylamino)-3-
hydroxy-2,2-dimethyl-chroman-6-carbonitrile (10d)
The same procedure for 8a was followed to give the title com-
pound as a white solid. Yield, 61%.
Compound 13d: 1H NMR: (CDCl3) d 7.90 (m, 1H), 7.80 (m, 1H),
7.70 (s, 1H), 7.60–7.40 (m, 3H), 6.95 (m, 1H), 6.10 (m, 1H), 4.80 (m,
1H), 4.10 (m, 1H), 1.55 (s, 3H), 1.40 (s, 3H). MS (m/z): MH+ 353.
1H NMR: (CDCl3) d 7.70 (s, 1H), 7.50–7.40 (m, 2H), 7.10 (s, 1H),
6.90 (m, 2H), 5.05 (m, 1H), 4.10 (m, 2H), 3.80 (m, 1H), 1.55 (s, 3H),
1.40 (m, 3H), 1.30 (s, 3H). MS (m/z): MH+ 396.
5.1.21. (3S,4R)-4-(6-Chloro-benzo[d]isoxazol-3-ylamino)-3-
hydroxy-2,2-dimethyl-chroman-6-carbonitrile (12c)
The same procedure for 8a was followed to give the title com-
pounds as a white solid. Yield, 55%.
5.1.16. (3S,4R)-4-(5-Fluoro-benzothiazol-2-ylamino)-3-
hydroxy-2,2-dimethyl-chroman-6-carbonitrile (10e)
The same procedure for 8a was followed to give the title com-
pound as a white solid. Yield, 48%.
1H NMR: (DMSO) d 8.00 (s, 1H), 7.70 (s, 1H), 7.60–7.50 (m, 3H),
6.95 (m, 1H), 5.80 (m, 1H), 4.65 (m, 1H), 3.88 (m, 1H), 1.45 (s, 3H),
1.20 (s, 3H). MS (m/z): MH+ 370.
1H NMR: (CDCl3) d 7.70 (s, 1H), 7.45–7.40 (m, 2H), 7.25 (m, 1H),
7.00 (m, 1H), 6.90 (m, 1H), 5.95 (br, 1H), 5.05 (d, J = 5.1 Hz, 1H),
3.80 (d, J = 5.1 Hz, 1H), 1.50 (s, 3H), 1.32 (s, 3H). MS (m/z): MH+ 370.
5.1.22. (3S,4R)-4-(5-Chloro-1H-indazol-3-ylamino)-3-hydroxy-
2,2-dimethyl-chroman-6-carbonitrile (12d)
The same procedure for 8a was followed to give the title com-
pounds as a white solid. Yield, 61%.
1H NMR: (CDCl3) d 7.50 (m, 1H), 7.15 (m, 2H), 7.05 (m, 2H), 6.85
(m, 1H), 5.85 (m, 1H), 5.35 (m, 1H), 1.62 (s, 3H), 1.38 (s, 3H). MS
(m/z): MH+ 351.
5.1.17. (3S,4R)-3-Hydroxy-2,2-dimethyl-4-(5-nitro-
benzothiazol-2-ylamino)-chroman-6-carbonitrile (10f)
The same procedure for 8a was followed to give the title com-
pound as a white solid. Yield 60%.
5.1.23. (3S,4R)-6-Benzenesulfonyl-4-(5-chloro-benzo[d]-
isoxazol-3-ylamino)-chroman-3-ol (12e)
1H NMR: (CDCl3) d 8.42 (s, 1H), 8.20 (m, 1H), 7.70 (s, 1H), 7.50
(m, 2H), 6.90 (m, 1H), 5.15 (m, 1H), 3.80 (m, 1H), 1.55 (s, 3H),
1.35 (s, 3H). MS (m/z): MH+ 397.
The same procedure for 8a was followed to give the title com-
pounds as a white solid. Yield, 40%.
1H NMR: (CDCl3) d 7.90 (s, 1H), 7.75 (d, J = 6.5 Hz, 2H), 7.60 (s,
1H), 7.55–7.30 (m, 5H), 7.20 (d, J = 7.0 Hz, 1H), 6.70 (d, J = 7.0 Hz,
1H), 5.70 (d, J = 4.5 Hz, 1H), 4.90 (m, 1H), 4.15 (m, 1H), 3.90 (d,
J = 4.5 Hz, 1H), 1.45 (s, 3H), 1.25 (s, 3H). MS (m/z): MH+ 485.
5.1.18. (3S,4R)-4-(5-Chloro-1H-benzoimidazol-2-ylsulfanyl)-3-
hydroxy-2,2-dimethyl-chroman-6-carbonitrile (10g)
The same procedure for 8a was followed to give the title com-
pound as a white solid. Yield 55%.
1H NMR: (CDCl3) d 10.50 (br, 1H), 8.00 (s, 1H), 7.50–7.40 (m,
4H), 7.20 (m, 1H), 6.90 (m, 1H), 4.78 (m, 1H), 4.10 (m, 1H), 1.60
(s, 6H). MS (m/z): MH+ 396.
5.1.24. (3S,4R)-4-(5-Chloro-benzo[d]isoxazol-3-ylamino)-6-(3-
fluoro-benzenesulfonyl)-2,2-dimethyl-chroman-3-ol (12f)
The same procedure for 8a was followed to give the title com-
pounds as a white solid. Yield, 35%.
5.1.19. (3S,4R)-4-(6-Chloro-benzo[d]isoxazol-3-yloxy)-3-hydro-
xy-2,2-dimethyl-chroman-6-carbonitrile (12a) and (3S,4R)-4-(6-
chloro-3-oxo-3H-benzo[d]isoxazol-2-yl)-3-hydroxy-2,2-dime-
thyl-chroman-6-carbonitrile (13b)
1H NMR: (CDCl3) d 7.90 (s, 1H), 7.60–7.20 (m, 9H), 6.80 (d,
J = 5.5 Hz, 1H), 5.60 (d, J = 5.5 Hz, 1H), 4.90 (m, 1H), 4.20 (br, 1H),
3.90 (d, J = 5.5 Hz, 1H), 1.50 (s, 3H), 1.35 (s, 3H). MS (m/z): MH+
503.
The same procedure for 8a was followed to give the title com-
pounds as white solids in ꢁ1:1 ratio. Total yield, 66%.
Compound 12a: 1H NMR: (CDCl3) d 7.70 (s, 1H), 7.60–7.50 (m,
2H), 7.35–7.30 (m, 2H), 6.95 (d, J = 8.2 Hz, 1H), 5.90 (d, J = 6.0 Hz,
1H), 4.15 (m, 1H), 3.60 (d, J = 2.0 Hz, 1H), 1.55 (s, 3H), 1.40 (s,
3H). 13C NMR: (CDCl3) d 164.5, 163.2, 147.1, 135.9, 132.2, 129.8,
125.8, 125.1, 123.4, 120.3, 117.0, 114.6, 110.3, 103.9, 92.1, 75.9,
72.6, 21.5, 21.3. MS (m/z): MH+ 371.
5.1.25. (3S,4R)-4-(5-Chloro-benzo[d]isoxazol-3-ylamino)-3-
hydroxy-2,2-dimethyl-chroman-6-sulfonic acid diethylamide
(12g)
The same procedure for 8a was followed to give the title com-
pound as a white solid. Yield, 51%.
1H NMR: (CDCl3) d 7.80 (s, 1H), 7.55 (d, J = 4.5 Hz, 1H), 7.45 (m,
2H), 7.20(m, 1H), 6.90 (d, J = 5.0 Hz, 1H), 5.00 (s, 1H), 4.90 (s, 1H),
3.90 (m, 1H), 3.60 (m, 1H), 3.10 (m, 4H), 1.50 (s, 3H), 1.30 (s,
3H), 1.00 (t, J = 4.5 Hz, 6H). MS (m/z): MH+ 480.
Compound 13b:
[
a]
+86.6o (c = 6.2, MeOH); 1H NMR:
D
(CDCl3) d 7.70 (d, J = 8.5 Hz, 1H), 7.50 (d, J = 8.5 Hz, 2H), 7.30–
7.25 (m, 3H), 6.95 (d, J = 8.2 Hz, 1H), 5.62 (d, J = 8.5 Hz, 1H),
4.20 (d, J = 8.5 Hz, 1H), 3.20 (br, 1H), 1.55 (s, 3H), 1.35 (s,
3H). 13C NMR: (CDCl3)
d
162.9, 161.2, 157.5, 138.6, 132.4,
5.1.26. (3S,4R)-6,7-Dichloro-4-(5-chloro-benzo[d]isoxazol-3-
ylamino)-2,2-dimethyl-chroman-3-ol (12h)
130.1, 129.8, 125.8, 121.6, 118.8, 116.5, 116.2, 114.7, 103.9,
87.4, 77.0, 46.6, 22.2, 21.5. MS (m/z): MH+ 371. Anal. Calcd
for C19H15N2O4Cl: C, 61.55; H, 4.08; N, 7.56. Found: C, 61.38;
H, 3.98; N, 7.75.
The same procedure for 8a was followed to give the title com-
pound as a white solid. Yield, 37%.
1H NMR: (CDCl3) d 7.55 (s, 1H), 7.45 (d, J = 4.5 Hz, 1H), 7.30 (d,
J = 4.5 Hz, 2H), 6.90 (d, J = 4.5 Hz, 1H), 6.85 (m, 1H), 5.00 (m, 1H),
4.85 (m, 1H), 4.25 (m, 1H), 1.50 (s, 3H), 1.35 (s, 3H). MS (m/z):
MNa+ 435.
5.1.20. (3S,4R)-4-(Benzo[d]isothiazol-3-yloxy)-3-hydroxy-2,2-
dimethyl-chroman-6-carbonitrile (12b) and 2-(6-chloro-3-
hydroxy-2,2-dimethyl-chroman-4-yl)-benzo[d]isothiazol-3-one
(13d)
The same procedure for 8a was followed to give the title com-
pounds as white solids in ꢁ1:1 ratio. Total yield, 60%.
Compound 12b: 1H NMR: (CDCl3) d 7.98 (d, J = 8.1 Hz, 1H), 7.82
(d, J = 8.0 Hz, 1H), 7.72 (s, 1H), 7.61 (t, J = 7.5 Hz, 1H), 7.53 (d,
J = 8.1 Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H), 6.98 (d, J = 8.5 Hz, 1H), 6.12
(d, J = 7.5 Hz, 1H), 4.82 (d, J = 2.1 Hz, 1H), 4.12 (d, J = 7.5 Hz, 1H),
1.59 (s, 3H), 1.46 s, 3H). MS (m/z): MH+ 353.
5.1.27. (3S,4R)-4-(5-Chloro-benzo[d]isoxazol-3-ylamino)-3S-
hydroxy-2,2-dimethyl-chroman-6-yl-phenyl-methanone (12i)
The same procedure for 8a was followed to give the title com-
pound as a white solid. Yield, 31%.
1H NMR: (CDCl3) d 7.90 (s, 1H), 7.70 (s, 1H), 7.50 (m, 4H), 7.30
(m, 3H), 7.25 (m, 1H), 6.75 (d, J = 4.5 Hz, 1H), 5.65 (d, J = 4.5 Hz,
1H), 5.00 (m, 1H), 4.20 (br, 1H), 3.70 (d, J = 4.5 Hz, 1H), 1.45 (s,
3H), 1.30 (s, 3H). MS (m/z): MH+ 449.

864
X. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 855–866
5.1.28. (3S,4R)-3-Hydroxy-2,2-dimethyl-4-(3-oxo-3H-benzo[d]-
isoxazol-2-yl)-chroman-6-carbonitrile (13a)
O-linked regio-isomer: 1H NMR: (CDCl3) d 7.80 (s, 1H), 7.70 (d,
J = 5.5 Hz, 2H), 7.50 (m, 2H), 7.40 (m, 1H), 7.00 (d, J = 5.0 Hz, 1H),
5.90 (d, J = 4.5 Hz, 1H), 4.15 (m, 1H), 3.80 (d, J = 2.0 Hz, 1H), 3.20
(m, 4H), 1.50 (s, 3H), 1.35 (s, 3H), 1.10 (t, J = 4.5 Hz, 6H). MS (m/
z): MH+ 481.
1H NMR: (CDCl3) d 7.71 (s, 1H), 7.65–7.48 (m, 4H), 7.30 (m, 1H),
6.95 (d, J = 5.1 Hz, 1H), 5.90 (d, J = 5.1Hz, 1H), 4.25 (m, 1H), 3.80 (d,
J = 2.0 Hz, 1H), 1.55 (s, 3H), 1.40 (s, 3H). MS (m/z): MH+ 337.
5.1.29 (3S,4R)-4-(6-Chloro-3-oxo-3H-benzo[d]isoxazol-2-yl)-3-
hydroxy-2,2-dimethyl-chroman-6-carbonitrile (13c)
The same procedure for 8a was followed to give the title com-
pound as a white solid. Yield, 32%.
5.1.35. (3S,4R)-6-Chloro-2-(6,7-dichloro-3-hydroxy-2,2-
dimethyl-chroman-4-yl)-benzo[d]isoxazol-3-one (13j)
The same procedure for 8a was followed to give the title com-
pounds as white solids in ꢁ1:1 ratio. Total yield, 72%.
Compound 13j: 1H NMR: (CDCl3) d 7.50 (d, J = 4.0 Hz, 1H), 7.45
(s, 1H), 7.40 (d, J = 4.0 Hz, 1H), 7.25 (m, 1H), 6.80 (d, J = 5.0 Hz, 1H),
5.85 (d, J = 1.0 Hz, 1H), 4.30 (m, 1H), 3.70 (d, J = 2.0 Hz, 1H), 1.55 (s,
6H). MS (m/z): MH+ 414.
1H NMR: (CDCl3) d 7.65 (m, 1H), 7.55 (m, 1H), 7.35 (s, 1H), 7.20
(m, 2H), 6.95 (m, 1H), 5.62 (m, 1H), 4.40 (m, 1H), 4.20 (m, 1H), 1.60
(s, 3H), 1.35 (s, 3H). MS (m/z m/z): MNa+ 393.
5.1.30. (3S,4R)-3-Hydroxy-2,2-dimethyl-4-(6-nitro-3-oxo-3H-
benzo[d]isothiazol-2-yl)-chroman-6-carbonitrile (13e)
The same procedure for 8a was followed to give the title com-
pound as a white solid. Yield, 45%.
O-linked regio-isomer: 1H NMR: (CDCl3) d 7.50 (d, J = 5.0 Hz, 1H),
7.35 (d, J = 5.0 Hz, 1H), 7.15 (m, 2H), 6.80 (d, J = 5.0 Hz, 1H), 5.65 (d,
J = 4.5 Hz, 1H), 4.80 (br, 1H), 4.20 (d, J = 4.5 Hz, 1H), 1.55 (s, 3H),
1.35 (s, 3H). MS (m/z): MH+ 414.
1H NMR: (CDCl3) d 9.95 (br, s, 1H), 9.05 (s, 1H), 8.58 (d,
J = 8.5 Hz, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.5 Hz, 1H),
7.61 (s, 1H), 7.08 (d, J = 8.8 Hz, 1H), 4.56 (m, 1H), 3.02 (m, 1H),
1.65 (s, 3H), 1.45 (s, 3H). MS (m/z): MH+ 398.
5.1.36. (3S,4R)-2-(6-Benzoyl-3-hydroxy-2,2-dimethyl-chroman-
4-yl)-6-chloro-4-benzo[d]isoxazol-3-one (13k)
The same procedure for 8a was followed to give the title com-
pounds as white solids in ꢁ1:1 ratio. Total yield, 70%.
Compound 13k: 1H NMR: (CDCl3) d 7.75 (m, 1H), 7.60 (m, 3H),
7.45 (m, 2H), 7.25 (m, 2H), 7.20 (m, 2H), 6.95 (d, J = 4.5 Hz, 1H),
5.70 (m, 1H), 4.20 (d, J = 5.0 Hz, 1H), 3.80 (br, 1H), 1.60 (s, 3H),
1.40 (s, 3H). MS (m/z): MH+ 450.
5.1.31. (3S,4R)-2-(6-Benzenesulfonyl-3-hydroxy-2,2-dimethyl-
chroman-4-yl)-6-chloro-benzo[d]isoxazol-3-one (13f)
The same procedure for 8a was followed to give the title com-
pound as a white solid. Yield, 34%.
1H NMR: (CDCl3) d 7.75 (m, 3H), 7.60 (d, J = 6.5Hz, 1H), 7.55–
7.35 (m, 4H), 7.20 (d, J = 7.0Hz, 1H), 7.10 (s, 1H), 6.90 (d,
J = 7.0 Hz, 1H), 5.60 (d, J = 6.5 Hz, 1H), 4.15 (m, 1H), 4.00 (d,
J = 4.5 Hz, 1H), 1.55 (s, 3H), 1.30 (s, 3H). MS (m/z): MH+ 486.
O-linked regio-isomer: 1H NMR: (CDCl3) d 8.05 (s, 1H), 7.75 (m,
3H), 7.60–7.40 (m, 5H), 7.30 (d, J = 4.5 Hz, 1H), 6.95 (d, J = 4.5 Hz,
1H), 6.15 (m, 1H), 4.45 (m, 1H), 2.30 (m, 1H), 1.60 (s, 3H), 1.40
(s, 3H). MS (m/z): MH+ 450.
5.1.32. (3S,4R)-2-(6-Benzenesulfonyl-3-hydroxy-2,2-dimethyl-
chroman-4-yl)-5-chloro-4-benzo[d] isoxazol-3-one (13g)
The same procedure for 8a was followed to give the title com-
pound as a white solid. Yield, 41%.
5.1.37. (3S,4R)-6-Chloro-[2-hydroxy-6-(4-methoxy-benzene-
sulfonyl)-2,2-dimethyl-chroman-4-yl]-benzo[d]isoxazol-3-one
(13l)
The same procedure for 8a was followed to give the title com-
pounds as white solids in ꢁ1:1 ratio. Total yield, 58%.
Compound 13l: 1H NMR: (CDCl3) d 7.70 (m, 4H), 7.50 (s, 1H),
7.20 (d, J = 5.0 Hz, 1H), 7.10 (s, 1H), 6.90 (d, J = 5.0 Hz, 1H), 6.85
(d, J = 5.5 Hz, 2H), 5.60 (d, J = 6.5 Hz, 1H), 4.15 (m, 1H), 3.80 (s,
3H), 1.55 (s, 3H), 1.30 (s, 3H). MS (m/z): MH+ 516.
O-linked regio-isomer: 1H NMR: (CDCl3) d 7.90 (s, 1H), 7.80 (m,
3H), 7.50 (m, 2H), 7.30 (m, 1H), 6.90 (m, 3H), 5.85 (d, J = 4.5 Hz,
1H), 4.15 (m, 1H), 3.85 (m, 1H), 3.80 (s, 3H), 1.50 (s, 3H), 1.35 (s,
3H). MS (m/z): MH+ 516.
1H NMR: (CDCl3) d 7.95 (s, 1H), 7.85 (d, J = 5.5 Hz, 2H), 7.80 (d,
J = 6.0 Hz, 1H), 7.55–7.40 (m, 6H), 6.96 (d, J = 6.0 Hz, 1H), 5.85 (d,
J = 4.5 Hz, 1H), 4.10 (m, 1H), 3.90 (m, 1H), 1.50 (s, 3H), 1.40 (s,
3H). MS (m/z): MH+ 486.
5.1.33. (3S,4R)-6-Chloro-2-[3-hydroxy-2,2-dimethyl-6-(piper-
idine-1-sulfonyl)-chroman-4-yl]-benzo[d] isoxazol-3-one (13h)
The same procedure for 8a was followed to give the title com-
pounds as white solids (ꢁ1:1). Total yield, 67%.
Compound 13h: 1H NMR: (CDCl3) d 7.65 (d, J = 6.0 Hz, 1H), 7.55
(d, J = 6.5 Hz, 2H), 7.30 (s, 1H), 7.20 (d, J = 6.5 Hz, 1H), 7.15 (s, 1H),
7.00 (d, J = 6.5 Hz, 1H), 5.70 (d, J = 7.0 Hz, 1H), 4.20 (m, 1H), 3.90 (d,
J = 4.5 Hz, 1H), 2.75 (t, J = 1.5 Hz, 4H), 1.60 (s, 3H), 1.50 (m, 4H),
1.35 (m, 2H), 1.35 (s, 3H). MS (m/z): MNa+ 515.
5.1.38. (3S,4R)-6-Chloro-2-[6-(4-chloro-benzenesulfonyl)-3-
hydroxy-2,2-dimethyl-chroman-4-yl]-benzo[d]isoxazol-3-one
(13m)
The same procedure for 8a was followed to give the title com-
pounds as white solids in ꢁ1:1 ratio. Total yield, 60%.
Compound 13m: 1H NMR: (CDCl3) d 7.70 (m, 4H), 7.55 (s, 1H),
7.35 (d, J = 5.0 Hz, 2H), 7.20 (d, J = 5.0 Hz, 1H), 7.10 (s, 1H), 7.00
(d, J = 5.5 Hz, 1H), 5.60 (d, J = 6.5 Hz, 1H), 4.15 (m, 1H), 4.00 (m,
1H), 1.55 (s, 3H), 1.30 (s, 3H). MS (m/z): MH+ 504.
O-linked regio-isomer: 1H NMR: (CDCl3) d 7.75 (s, 1H), 7.60 (d,
J = 5.5 Hz, 2H), 7.50 (m, 2H), 7.25 (d, J = 5.5 Hz, 1H), 7.00 (d,
J = 6.0 Hz, 1H), 5.90 (d, J = 4.5 Hz, 1H), 4.20 (m, 1H), 3.90 (s, 1H),
2.90 (t, J = 2.5 Hz, 4H), 1.60 (m, 4H), 1.50 (s, 3H), 1.40 (m, 2H),
1.35 (s, 3H). MS (m/z): MNa+ 515.
O-linked regio-isomer: 1H NMR: (CDCl3) d 8.00 (s, 1H), 7.80 (m,
3H), 7.50 (m, 4H), 7.30 (m, 1H), 7.00 (d, J = 5.5 Hz, 1H), 5.90 (d,
J = 4.5 Hz, 1H), 4.15 (m, 1H), 3.80 (d, J = 1.0 Hz, 1H), 1.50 (s, 3H),
1.35 (s, 3H). MS (m/z): MH+ 521.
5.1.34. (3S,4R)-(5-Chloro-3-oxo-3H-benzo[d]isoxazol-2-yl)-3-
hydroxy-2,2-dimethyl-chroman-6-sulfonic acid diethylamide
(13i)
The same procedure for 8a was followed to give the title com-
pounds as white solids (ꢁ1:1). Total yield, 56%.
Compound 13i: 1H NMR: (CDCl3) d 7.65 (m, 2H), 7.55 (d,
J = 6.5 Hz, 1H), 7.40 (s, 1H), 7.15 (d, J = 6.5 Hz, 1H), 6.95 (d,
J = 6.5 Hz, 1H), 5.70 (d, J = 7.0 Hz, 1H), 4.20 (m, 1H), 3.90 (m, 1H),
3.00 (m, 4H), 1.60 (s, 3H), 1.35 (s, 3H), 1.00 (t, J = 4.5 Hz, 6H). MS
(m/z): MH+ 481.
5.1.39. (3S,4R)-6-Chloro-2-[6-(3-fluoro-benzenesulfonyl)-3-
hydroxy-2,2-dimethyl-chroman-4-yl]-benzo[d]isoxazol-3-one
(13n)
The same procedure for 8a was followed to give the title com-
pounds as white solids in ꢁ1:1 ratio. Total yield, 56%.

X. Zhang et al. / Bioorg. Med. Chem. 17 (2009) 855–866
865
Compound 13n: 1H NMR: (CDCl3) d 7.75 (d, J = 5.5 Hz, 1H), 7.65
(d, J = 5.0 Hz, 1H), 7.5 (m, 2H), 7.45 (m, 2H), 7.20 (m, 2H), 7.10 (s,
1H), 7.00 (d, J = 5.5 Hz, 1H), 5.60 (d, J = 6.5 Hz, 1H), 4.15 (m, 1H),
1.50 (s, 3H), 1.35 (s, 3H). MS (m/z): MH+ 504.
according to manufacturer’s instructions. The amount of DNA used
per well was 240 ng (SUR2A: Kir6.2 = 210 ng:30 ng). The plates
were incubated at 37 oC with 5% CO2 for 48 h. FLIPR assay was car-
ried out as described above.
O-linked regio-isomer: 1H NMR: (CDCl3) d 8.00 (s, 1H), 7.80 (d,
J = 5.5 Hz, 1H), 7.70 (d, J = 5.0 Hz, 1H), 7.55 (d, J = 5.0 Hz, 1H),
7.50 (m, 2H), 7.25 (m, 3H), 7.00 (d, J = 5.5 Hz, 1H), 5.90 (d,
J = 4.5 Hz, 1H), 4.15 (m, 1H), 3.90 (m, 1H), 1.50 (s, 3H), 1.35 (s,
3H). MS (m/z): MH+ 504.
6.1.2. Rat bladder strips organ bath assay
Longitudinal strips (2 mm ꢂ 1 cm) of rat bladder were dissected
and hung in organ chambers containing Krebs buffer (NaCl,
118.2
lM; KCl, 4.7
l
M; MgSO4, 1.2 M; CaCl2,
l
M; KH2PO4, 1.2
l
2.5 M, NaHCO2, 25
l
l
M; glucose, 11.1 lM). The solution was
maintained at 37 oC and gassed with 95% O2 and 5% CO2. After
the optimal tension was reached through repeated stretching and
5.1.40. (3S,4R)-4-(1,3-Dioxo-1,3-dihydro-pyrrolo[3,4-c]pyridin-
2-yl)-3-hydroxy-2,2-dimethyl-chroman-6-carbonitrile 13p
(3S,4R)-4-Amino-3-hydroxy-2,2-dimethyl-chroman-6-carboni-
carbachol (1
M carbachol. When the contraction was stabilized, an accumu-
lative dosage curve was constructed for each compound. The stock
solutions were prepared at 100 M in DMSO and further diluted in
saline. The maximum tension induced by carbachol minus baseline
was defined as 100% tension. The relaxation was expressed as per-
cent tension.
lM) challenge, the strips were pre-contracted with
1
l
trile
5
(220 mg, 1 mmol) and furo[3,4-c]pyridine-1,3-dio-
ne(150 mg, 1 mmol) in toluene (5 mL) in sealed tube was heated
at 120 oC for 10 h. The reaction was cooled down and the solvent
was removed. The residue was purified by silica gel chromatogra-
phy with hexanes: ethyl acetate 1:1 to yield the title compound as
a yellow solid (200 mg, 55%).
l
1H NMR: (CDCl3) d 9.05 (d, J = 6.0 Hz, 1H), 9.00–9.10 (br s, 1H),
7.45 (d, J = 7.5 Hz, 1H), 7.15 (s, 1H), 6.95 (d, J = 7.5 Hz, 1H), 5.35 (d,
J = 8.2 Hz, 1H), 4.52 (dd, J = 8.0, 5.8 Hz, 1H), 3.32 (s, 1H), 1.61 (s,
3H), 1.38 (s, 3H). MS (m/z): MH+ 350.
6.2. In vivo studies
6.2.1. Rats acetic acid model
Female Sprague-Dawley rats 180–220 g (Charles River, Wil-
mington, MA) were used. The anesthesia was induced by exposure
to isoflurane (induction at 3–4% isoflurane + oxygen) briefly (about
10 min). The anesthesia is maintained by s.c. or i.p. injection of 1.0–
1.2 g/kg urethane. Body temperature is maintained at 36–38 °C
using a warming blanket. The urinary bladder is exposed through
a midline incision of the abdomen and urine is emptied by applica-
tion of light pressure. A PE 50 catheter is inserted through the apex
of the bladder dome and secured by a purse-string silk suture (5-
O). The tubing was attached (using a ‘T’ connector) to a Statham
pressure transducer (Model P23Db) and to a Harvard infusion
pump. The right jugular vein was cannulated for compound admin-
istration. After the preparation was rested for I h, cystometric eval-
uations were initiated by infusing saline containing 0.25% acetic
acid into the bladders at 2.4 mL/h. The infusion continued for
60–90 min until consistent micturation pattern was obtained.
Vehicle or test compounds dissolved in 10% PEG300/20% HPBCD
was delivered intravenously. Bladder infusion was continued
for another 60 min. Micturition parameters were continuously
monitored and recorded on a on a GOULD Ponemah Physiology
Platform.
5.1.41. (3S,4R)-4-(5-Chloro-1,3-dioxo-1,3-dihydro-isoindol-2-
yl)-3-hydroxy-2,2-dimethyl-chroman-6-carbonitrile 13o
The same procedure for 13p was followed to give the title com-
pound as a white solid. Yield, 66%.
1H NMR: (CDCl3) d 7.80–7.70 (m, 3H), 7.40 (m, 1H), 7.15 (s, 1H),
6.90 (m, 1H), 5.30 (m, 1H), 4.45 (m, 1H), 4.35 (m, 1H), 1.55 (s, 3H),
1.30 (s, 3H). MS (m/z): MH+ 351.
6. Biological evaluations
All animal studies were approved by the Johnson & Johnson
Pharmaceutical Research & Development 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.
6.1. In vitro studies
6.1.1. FLIPR assay
TE671 and b-TC cells were obtained from ATCC and grew in de-
sired media. The cells were seeded in black 96-well plates at 50–
60 k per well the day before. On the day of assaying, media was dis-
6.2.2. Rats partial outlet obstruction model
Female Sprague-Dawley rats (Charles River, Wilmington, MA;
190–210 g) were anesthetized with isoflurane and through a mid-
line incision the bladder and urethra were exposed. A 4–0 silk lig-
ature was tied around the proximal urethra in the presence of a
stainless steel rod (1 mm diameter). The rod was then removed
resulting in a partial occlusion. The abdominal musculature was
closed using 3–0 silk and the skin was closed with surgical staples.
Each rat received 150,000 U of bicilin C-R, i.m. (Wyeth Laborato-
ries, Philadelphia, PA). After six 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 tunneled under the skin and exteriorized
through an opening in the back of the neck. The abdominal incision
was sutured and the free end of the catheter was sealed. Two days
after catheter implantation, the animals were used for cystometric
evaluations. The night before testing the animals were placed in
the metabolic cages, the catheter was connected to a Harvard infu-
sion pump, and bladders were perfused overnight with saline at
2 mL/h. The next morning the bladder catheter was attached (using
a ‘T’ connector) to a Statham pressure transducer (Model P23Db)
carded and 100
lL of FLIPR buffer (20
l
M HEPES, pH 7.4, 120
lM
NaCl, 2 M KCl, 2
l
l
M CaCl2, 1 M mg Cl2, and 5 l
l
M Glucose) was
added to each well. Equal volume of membrane potential dye
(Molecular Devices) prepared in FLIPR buffer was also added to
each well. The plate was incubated at room temperature for 15–
30 min before placed in FLIPR. The fluorescence was monitored be-
fore and after the addition of various concentrations of standards
and test compounds. The maximal decrease in fluorescence inten-
sity obtained by pinacidil and P-1075 after subtracting buffer con-
trol was designated as 100%. EC50 values were determined by the
concentration of compounds required to produce 50% of the max-
imal response induced by pinacidil and P-1075. Glyburide, a KATP
channel blocker, was added 3 min after the compound addition
to a final concentration of 5 lM.
SUR2A was expressed in COS-7 cells transient transfection. The
day before the transfection, COS-7 cells were plated into 96-well
black well plates at 20 K/well in DMED media without antibiotics.
Transfection was carried out with Lipofectamine 2000 (Invitrogen)

866
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through a force displacement transducer (Grass FTO3). The cysto-
metric evaluation of bladder function was started by infusing sal-
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