Synthesis and structure-activity relationships of 3-aryloxindoles: A new class of calcium-dependent, large conductance potassium (maxi-K) channel openers with neuroprotective properties

Article abstract of DOI:10.1021/jm0101850

A series of 3-aryloxindole derivatives were synthesized and evaluated as activators of the cloned maxi-K channel mSlo expressed in Xenopus laevis oocytes using electrophysiological methods. The most promising maxi-K openers to emerge from this study were

Full text of DOI:10.1021/jm0101850

J . Med. Chem. 2002, 45, 1487-1499
1487
Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip s of 3-Ar yloxin d oles: A New
Cla ss of Ca lciu m -Dep en d en t, La r ge Con d u cta n ce P ota ssiu m (Ma xi-K) Ch a n n el
Op en er s w ith Neu r op r otective P r op er ties
Piyasena Hewawasam,*,† Matthew Erway,† Sandra L. Moon,‡ J ay Knipe,§ Harvey Weiner,‡
Christopher G. Boissard,‡ Debra J . Post-Munson,‡ Qi Gao,| Stella Huang, Valentin K. Gribkoff,‡ and
Nicholas A. Meanwell†
Departments of Chemistry, Neuroscience, Metabolism and Pharmacokinetics, Analytical Research and Development, and
Discovery Analytical Sciences, The Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway,
Wallingford, Connecticut 06492
Received April 25, 2001
A series of 3-aryloxindole derivatives were synthesized and evaluated as activators of the cloned
maxi-K channel mSlo expressed in Xenopus laevis oocytes using electrophysiological methods.
The most promising maxi-K openers to emerge from this study were (()-3-(5-chloro-2-
hydroxyphenyl)-1,3-dihydro-3-hydroxy-6-(trifluoromethyl)-2H-indol-2-one ((()-8c) and its 3-des-
hydroxy analogue (()-11b. The individual enantiomers of (()-8c were synthesized, and the
maxi-K channel-opening properties were shown to depend on the absolute configuration of the
single stereogenic center with the efficacy of (-)-8c superior to that of both (+)-8c and the
racemic mixture when evaluated at a concentration of 20 µM. Racemic 11b exhibited greater
efficacy than either the racemic 8c or the more active enantiomer in the electrophysiological
evaluation. In vitro metabolic stability studies conducted with (()-8c and (()-11b in rat liver
S9 microsomal fractions revealed significant oxidative degradation with two hydroxylated
metabolites observed by liquid chromatography/mass spectrometry for each compound in
addition to the production of 8c from 11b. The pharmacokinetic properties of (()-8c and (()-
11b were determined in rats as a prelude to evaluation in a rat model of stroke that involved
permanent occlusion of the middle cerebral artery (MCAO model). In the MCAO model,
conducted in the spontaneously hypertensive rat, the more polar 3-hydroxy derivative (()-8c
did not demonstrate a significant reduction in cortical infarct volume when administered
intravenously at doses ranging from 0.1 to 10 mg/kg as a single bolus 2 h after middle cerebral
artery occlusion when compared to vehicle-treated controls. In contrast, intravenous admin-
istration of (()-11b at a dose of 0.03 mg/kg was found to reduce the measured cortical infarct
volume by approximately 18% when compared to vehicle-treated control animals. Intraperi-
toneal administration of (()-11b at a dose of 10 mg/kg 2 h following artery occlusion was shown
to reduce infarct volume by 26% when compared to vehicle-treated controls. To further probe
the effects of compounds (()-8c and (()-11b on neurotransmitter release in vitro, both
compounds were examined for their ability to reduce electrically stimulated [3H]-glutamate
release from rat hippocampal slices that had been preloaded with [3H]-glutamate. Only (()-
11b was able to demonstrate a significant inhibition [3H]-glutamate release in this assay at a
concentration of 20 µM, providing concordance with the profile of these compounds in the MCAO
model. Although (()-11b showed some promise as a potential developmental candidate for the
treatment of the sequelae of stroke based on its efficacy in the rat MCAO model, the
pharmacokinetic profile of this compound was considered to be less than optimal and was not
pursued in favor of derivatives with enhanced metabolic stability.
In tr od u ction
of the consequences of this devastating and debilitating
event represents one of the most prominent unmet
medical needs in the clinical arena. Strokes are classi-
fied into two types: ischemic stroke, which results from
vessel occlusion, and hemorrhagic stroke, in which there
is intracerebral hemorrhage resulting in neuronal dam-
age. Hemmorrhagic strokes represent approximately
20% of all such events but are very difficult to classify,
leading to some caution when treating stroke victims
with the thrombolytic agents that have been developed
to facilitate dissolution of the occluding thrombus. These
agents are utilized to restore blood flow to ischemic
neuronal tissue, and tissue plasminogen activator has
recently been approved for use in a limited patient
Stroke is presently recognized as the third leading
cause of adult disability and death in the United States
and Europe with an incidence in excess of 700 000 per
year in the United States and over 2 million per annum
worldwide.1 Providing clinicians with effective drugs
that provide stroke victims with protection against some
* To whom correspondence should be addressed. Tel.: (203)677-
7815. Fax: (203)677-7702. E-mail: hewawasp@bms.com.
† Department of Chemistry.
‡ Department of Neuroscience.
§ Department of Metabolism and Pharmacokinetics.
| Department of Analytical Research and Development.
Department of Discovery Analytical Sciences.
10.1021/jm0101850 CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/03/2002

1488 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Hewawasam et al.
Ch a r t 1. Structures of NS-004 (1), NS-1619 (2),
N-Benzylated Benzimidazol-2-one Derivatives (3), and
3-Aryl-3-hydroxyoxindoles (4)
population.2 In an effort to provide clinicians with
effective neuroprotective therapies to be administered
poststroke, numerous mechanistic approaches have
been examined in the past decade including antagonists
of AMPA/kainate3-5 and N-methyl-D-aspartate (NMDA)
excitatory amino acid receptors6 and inhibitors of neu-
ronal adenosine reuptake.7,8 However, to date, none of
these potential neuroprotective agents have succeeded
due to either a lack of demonstrated clinical efficacy or
poor side effect profiles,9 resulting in the current situ-
ation in which there is no approved neuroprotective
therapy. We have approached the problem of poststroke
neuroprotection by developing potent and specific open-
ers of large conductance, Ca2+-activated (maxi-K) potas-
sium channels. This strategy is designed to protect
neurons by enhancing an intrinsic mechanism of cell
regulation that should ultimately reduce abnormally
high levels of Ca2+ entry,10,11 thereby reducing the
release of potentially pathogenic neurotransmitters12
and controlling neuronal hyperexcitability.13
mally high Ca2+ entry. Because intracellular levels of
Ca2+ are low in nonischemic tissue, a maxi-K opener
would not be expected to significantly affect channels
under these circumstances, thereby reducing the po-
tential for undesirable side effects.
The range of synthetic and naturally occurring com-
pounds that demonstrate maxi-K channel-opening ac-
tivity and their associated electrophysiological and
biochemical pharmacological properties has recently
been reviewed.22,23 The benzimidazolone derivatives NS
004 (1) and NS 1619 (2) are prominent prototypes
among the small molecule openers of maxi-K chan-
nels24-26 that have been studied in some detail, both in
vitro and in vivo.27-30 A recent study of the structurally
homologous N-benzylated benzimidazol-2-one deriva-
tives (3) has illuminated fundamental aspects of this
maxi-K-opening pharmacophore and provided the first
insights into structure-activity relationships (Chart
1).31
As part of a broad-based effort directed toward the
identification of potent and selective activators of neu-
ronal maxi-K channels that would be useful as neuro-
protective agents, we recently disclosed a structurally
novel class of maxi-K channel openers based upon
3-aryl-3-hydroxyoxindoles (4).32 More specifically, the
maxi-K channel-opening activity of 3-(5-chloro-2-hy-
droxyphenyl)-1,3-dihydro-3-hydroxy-6-(trifluoromethyl)-
2H-indol-2-one (8c) was described and shown to be
dependent upon the absolute configuration at the ste-
reogenic center. In this paper, a more detailed analysis
of structure-activity relationships associated with the
BK-opening pharmacophore inherent to 8c is presented.
In addition, the potential of 8c and its analogues to
function as neuroprotective agents, as predicted by
demonstration of neuroprotective effects in the middle
cerebral artery occlusion (MCAO) rat model of stroke,
is described.
Ba ck gr ou n d a n d Ra tion a le
Potassium channels have emerged as interesting
targets for drug discovery based on their structural
diversity, tissue distribution, and regulatory role in cell
function, thereby providing an opportunity to design
drugs that target specific K+ channels that may selec-
tively intervene in a wide range of physiological pro-
cesses and disease states. Because they are subject to
regulation by a range of physiological factors including
voltage, the metabolic state of a cell, intracellular Ca2+
concentration, and receptor-activated processes,14,15 they
play a critical role in regulating cell membrane potential
and neuronal excitability.16-19 The Ca2+-activated po-
tassium (KCa) channel subfamily is characterized by
their dependence on the intracellular concentration of
Ca2+ ions for activity but are also regulated by trans-
membrane voltage and phosphorylation state.20 KCa
channels have been further subdivided based on channel
conductance into three distinct categories: the large
conductance maxi-K or BK channels are those that
exhibit a single channel conductance of greater than 150
picoSiemens (pS) while intermediate conductance and
low conductance channels are categorized as 50-150 pS
and less than 50 pS, respectively. Maxi-K channels are
of particular interest because of their large channel
conductance and their expression in a range of excitable
cell types, including neurons and smooth muscle cells.20,21
In view of this and as a consequence of their central
role in regulating cell activity, modulators of maxi-K
channels are potentially useful agents for the treatment
of a variety of disease states associated with both the
central nervous system and smooth muscle function.22,23
Ch em istr y
The racemic 3-aryl-3-hydroxyindol-2-ones (7 or 8)
were prepared in a straightforward fashion from the
corresponding isatins 5,33 as depicted in Scheme 1. The
addition of an aryl Grignard reagent (6) to the sodium
salt of an isatin 5 in tetrahydrofuran (THF) afforded
the corresponding 3-aryl-3-hydroxyindol-2-ones 7 in 80-
95% yields. For derivatives of 7 substituted with a
methoxy moiety, demethylation was accomplished using
BBr3 in CH2Cl2 to provide the corresponding phenols
8.
The opening of maxi-K channels is stimulated by an
increase in intracellular Ca2+; openers of these channels
augment this opening of the channel by Ca2+. As a
consequence, the pharmacological modulation of channel
function is largely dependent on the presence of high
levels of intracellular Ca2+, a circumstance encountered
during neuronal ischemia.10-11,23 Maxi-K channel open-
ing stimulated by Ca2+ in the presence of a pharmaco-
logical opener would function to limit further Ca2+
entry, thus interrupting the cascade of events leading
to ischemic neuronal death that is initiated by abnor-
A more direct approach to phenol derivatives was
developed that relied upon the reaction of magnesium
phenolates (9), prepared by treating phenols with eth-
ylmagnesium bromide, with isatins 5 in either CH2Cl2

New Class of Potassium Channel Openers
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1489
Sch em e 1
Sch em e 2
or toluene,34 as summarized in Scheme 1. Although it
was anticipated that only electron-rich magnesium
phenolates would react efficiently with isatins 5, this
reaction proved to be quite general and a variety of
electron-deficient magnesium phenolates coupled ef-
fectively to afford adducts 8. Moreover, meta-substituted
magnesium phenolates were found to react with isatins
5 with excellent regioselectivity, affording only those
adducts in which the phenol was substituted at the
least-hindered of the two ortho positions. However, the
steric interactions that underlie this selectivity pre-
vented the reaction of 5 with meta-disubstituted mag-
nesium phenolates, which reacted only poorly. As a
consequence, tractable products could not be isolated.
Several of the 3-aryl-3-hydroxyoxindoles 7 were re-
duced to the corresponding 3-aryloxindoles 10, as shown
in Scheme 1. Dehydroxylation was accomplished by
heating 7 in a sealed tube with an excess of Et3SiH in
CF3CO2H at 110-120 °C. As might be anticipated, the
rate of this dehydroxylation reaction was dependent
upon the electronic nature of the substituents present
on the oxindole aromatic ring of 7. Thus, unsubstituted
and electron-rich oxindoles were deoxygenated much
more rapidly than oxindoles substituted with electron-
withdrawing moieties. Demethylation of the methyl
ether moiety of 10 with BBr3 in CH2Cl2 provided the
corresponding phenols 11 (Scheme 2).
The dehydroxylated oxindole derivatives were viewed
as potential precursors to homochiral 3-aryl-3-hydroxy-
oxindoles by way of the asymmetric hydroxylation
reaction developed by Davis,35 as depicted in Scheme
2.32 Treating the racemic indolone 10c with potassium
bis(trimethylsilyl)amide in THF at -78 °C under an
atmosphere of argon followed by the addition of the
chiral oxaziridine, (1R)-(-)-9 afforded only racemic 7c,
as determined by 1H nuclear magnetic resonance (NMR)
in the chiral solvent (L)-trifluoromethylphenyl carbinol.36
In this solvent, the CH3O protons of the two enanti-
omers of racemic 7c resonated at δ 3.49 and 3.47 ppm.
It was subsequently determined that the potassium salt
of 10c was extremely sensitive to oxidation by oxygen
dissolved in THF. Consequently, the reaction was
repeated using dry degassed THF. Under these circum-
stances, oxidation of the potassium salt of 10c with (1S)-

1490 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Hewawasam et al.
of the oxindole derivatives to be compared with a
prototypical maxi-K channel opener.
The structure-activity relationships described in
Table 1 provide a basic understanding of the oxindole
maxi-K channel-opening pharmacophore, extending the
findings of the earlier study, which was restricted to
3-hydroxy oxindole derivatives. In addition to identify-
ing the optimal substitution patterns associated with
the heterocycle and 3-phenyl moieties, the data in Table
1 further illuminate the effect of asymmetry on maxi-
K-opening efficacy and reveal some subtle differences
between this chemotype and the benzylated benzimid-
azolones reported earlier.31 From the limited structure-
activity data associated with the 3-des-hydroxy series,
compounds 10b and 11a ,b, it appears that an electron-
withdrawing substituent is not an essential structural
element since the prototype 11a demonstrates efficacy
comparable to NS-004 (1). However, as observed with
the benzimidazolone chemotype,31 the introduction of
the 6-CF3 group (11b) markedly enhances channel-
opening efficacy, results that taken together are indica-
tive of greater intrinsic activity in this series. Compound
11b also provided an opportunity to evaluate one aspect
of the contribution of the phenol moiety by methylation,
which eliminates the potential to function as a hydrogen
bond donor. While the significantly attenuated efficacy
of ether 10b as compared to 11b may be compatible with
this suggestion, a deleterious effect associated with
increased steric bulk cannot be disregarded.
F igu r e 1. X-ray structure of (3R)-(-)-5-bromo-3-(5-chloro-2-
methoxyphenyl)-1,3-dihydro-3-hydroxy-2H-indol-2-one ((-)-7e)
showing the atomic labeling scheme.
(+)-9 at -78 °C followed by gradual warming to 0 °C
and quenching with glacial acetic acid gave the desired
3-hydroxyindolone (-)-7c. The enantiomeric purity of
(-)-7c was determined to be >95% after examination
of the 1H NMR spectrum in the chiral solvent (L)-
trifluoromethylphenyl carbinol.36 When the reaction was
repeated with (1R)-(-)-9, the opposite enantiomer, (+)-
7c, was obtained, again in >95% ee. Because suitable
single crystals of either enantiomer (+)-7c or (-)-7c
could not be obtained, (3R)-(-)-5-bromo-3-(5-chloro-2-
methoxyphenyl)-1,3-dihydro-3-hydroxy-2H-indol-2-one
((-)-7e) was prepared in an analogous fashion (Scheme
2), using (1S)-(+)-9 as the oxidant, and the absolute
configuration was determined by single crystal X-ray
analysis. The solid state structure of this compound is
shown in Figure 1. In analogy with this stereochemical
assignment, the absolute configurations of (+)-7c and
(-)-7c are tentatively assigned as depicted in Scheme
2. Finally, the ether moieties of (+)-7c and (-)-7c were
demethylated by exposure to BBr3 in CH2Cl2 to afford
the corresponding phenols (-)-8c and (+)-8c, respec-
tively. The compounds prepared by these methods are
compiled in Table 1 along with relevant physicochemical
data.
The 3-hydroxy oxindoles were the major focus of this
study and subject to a more extensive structure-activity
survey that included an evaluation of the effects of
chirality on channel-opening properties. The latter was
examined in the context of prototypes methyl ether 7c
and the derived phenol 8c, a pair of compounds that
provided contrasting results. Ether 7c exhibited modest
efficacy that was not dependent on the absolute config-
uration at C-3 while the racemic phenol 8c more
effectively enhanced channel conductance, extending the
trend seen between 10b and 11b, but in a fashion
sensitive to the identity of the single stereogenic center.
Thus, the channel-opening activity of (-)-8c was supe-
rior to that of both (+)-8c and the racemic mixture. The
aqueous solubility properties of (()-8c were such that
evaluation at the higher concentration of 30 µM was
permitted. In this experiment, a dose-related increase
in the efficacy of channel opening was observed with
an increase in outward current in oocytes expressing
mSlo to 200.8 ( 17.8% of control. Unfortunately, at-
tempts to evaluate the individual enantiomers of 8c at
a concentration of 30 µM were unsuccessful due to
precipitation of the test compound under the assay
conditions.
Resu lts a n d Discu ssion
The effect of the target compounds on outward K+
current was determined by using a two electrode voltage
clamp recording from Xenopus laevis oocytes expressing
cloned mSlo37 (or hSlo38) maxi-K channels, as described
previously.39 The voltage clamp protocols consisted of
500-750 ms voltage steps in +20 mV increments from
a holding potential of -60 to +140 mV, which were
conducted in the absence and presence of test compound.
Compounds under evaluation were maintained at a
concentration of 20 µM in the recording chamber, and
all experiments were concluded by incubation of the
oocytes for 10 min with 50 nM iberiotoxin (IbTx).
Voltage clamp protocols in the presence of this selective
blocker of the maxi-K channel allowed quantification
of mSlo or hSlo current expression. In all cases, a
minimum of five different oocytes was used to evaluate
the effect of a single drug concentration on channel
current, and for each compound tested, the average
percentage change in mSlo (or hSlo) IbTx sensitive
current relative to drug-free control (100%) was deter-
mined. The results obtained are presented in Table 1
along with data for NS-004 (1), which allows the efficacy
A more detailed electrophysiological evaluation of (()-
8c was conducted using an inside-out excised patch from
a COS cell expressing mSlo. In this assay, (()-8c
significantly increased the mean open time and reduced
periods of closure of mSlo maxi-K channels at a con-
centration as low as 100 nM (Figure 2A). The effects of
(()-8c on the human maxi-K channel hSlo expressed
in HEK cells were also examined (Figure 2B). In both
systems, (()-8c was found to increase the probability
of the open state of the channels at a concentration of 1
µM.

New Class of Potassium Channel Openers
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1491
Ta ble 1. Structure and Physical Properties of 3-Substituted 1,3-Dihydro-indol-2-one Derivatives and Effect on Maxi-K-Mediated
Outward Current in Xenopus laevis Oocytes Expressing Cloned Maxi-K Channels
% increase in
compd
no.
method of
mSlo current
at 20 µM
R1
R2
R3
R4
R5
R6 preparation mp (°C)a
molecular formula
1 (NS-004)
7a
7b
(()-7c
(+)-7c
(-)-7c
7d
(-)-7e
8a
8b
(()-8c
(+)-8c
(-)-8c
8d
8e
8f
8g
8h
8i
8j
132 ( 13b
87 ( 4
H
CF3
CF3
CF3
CF3
CF3
H
H
H
H
H
H
C6H5
3-Cl-C6H4
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
A
A
A
247-249
268-270
207-210
243-245
244-245
218-220
257-259
216-218
209-211
210-213
200-201
198-200
156-158
205-207
239-242
215-217
225-227
158-161
232-235
191-193
184-186
194-196
192-194
226-228
180-185
159-160
276-280
231-233
210-215
C15H10F3NO2
H
H
H
H
H
H
H
H
H
H
H
H
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
97 ( 6b
120 ( 7b
117 ( 3b
116 ( 4b
109 ( 3
NT
C15H9ClF3NO2
C16H11ClF3NO3
C16H11ClF3NO3
C16H11ClF3NO3
C16H11ClF3NO3
C15H11BrClNO3
C14H10ClNO3‚0.4H2O
C15H10F3NO3
C15H9ClF3NO3
C15H9ClF3NO3
C15H9ClF3NO3
C15H9ClF3NO3
C15H9ClF3NO3
C15H9ClF3NO3
C14H9ClN2O5‚0.5H2O
C15H12ClNO3
C14H9ClFNO3
C14H8Cl3NO3
C16H8ClF6NO3
C15H9F4NO3
C15H9BrF3NO3‚0.25H2O
C15H9F3INO3
C16H12F3NO3
C21H14F3NO3
2-OCH3, 5-Cl-C6H3
2-OCH3, 5-Cl-C6H3
2-OCH3, 5-Cl-C6H3
2-OCH3, 5-Cl-C6H3
2-OCH3, 5-Cl-C6H3
2-OH, 5-Cl-C6H3
2-OH-C6H4
2-OH, 5-Cl-C6H3
2-OH, 5-Cl-C6H3
2-OH, 5-Cl-C6H3
2-OH, 5-Cl-C6H3
2-OH, 5-Cl-C6H3
scheme 2
scheme 2
A
scheme 2
C
C
A and B
scheme 2
scheme 2
A and B
A and B
A and B
C
C
H
CF3
Br
H
H
H
H
H
CF3
H
H
NO2
CH3
F
H
H
H
H
H
H
H
H
95 ( 3
90 ( 3
CF3
CF3
CF3
CF3
H
H
H
H
H
131 ( 7
123 ( 11
141 ( 9
108 ( 8
122 ( 7
127 ( 13
78 ( 5
CF3 2-OH, 5-Cl-C6H3
H
H
H
Cl
2-OH, 5-Cl-C6H3
2-OH, 5-Cl-C6H3
2-OH, 5-Cl-C6H3
2-OH, 5-Cl-C6H3
100 ( 3
106 ( 6
117 ( 8
127 ( 9
97 ( 7
H
Cl
C
C
8k
8l
8m
8n
8o
8p
8q
8r
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3 2-OH, 5-Cl-C6H3
A and B
H
H
H
H
H
H
H
H
H
2-OH, 5-F-C6H3
2-OH, 5-Br-C6H3
2-OH, 5-I-C6H3
2-OH, 5-CH3-C6H3
2-OH, 5-C6H5-C6H3
2-OH-C10H6
2-OH, 4-NH2-C6H3
2-OH, 5-OCH3-C6H3
2-OH, 5-(NCH3-
piperazinyl)C6H3
2-OH, 5-CF3-C6H3
2-OH, 4-CF3-C6H3
2-OH, 3-Cl-C6H3
2-OH, 4-Cl-C6H3
2-OH, 4,5-diCl-C6H2
2-OH, 3,5-diCl-C6H2
C
C
C
C
C
C
C
C
C
89 ( 3
107 ( 7
106 ( 4
106 ( 3
116 ( 4
92 ( 2
H
H
H
H
C19H12F3NO3‚0.85H2O
C15H11F3N2O3
C16H12F3NO4
8s
8t
81 ( 7
91 ( 2
H
C20H20F3N3O3
8u
8v
8w
8x
8y
H
H
H
H
H
H
H
H
H
H
H
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
OH
OH
OH
C
C
C
C
C
C
C
175-177
198-201
202-205
188-190
152-154
210-212
198-200
210-212
210-213
256-258
266-268
130 ( 11
108 ( 7
116 ( 5
93 ( 6
107 ( 5
108 ( 6
94 ( 4
124 ( 4
110 ( 0
139 ( 6
164 ( 8
C16H9F6NO3
C16H9F6NO3
C15H9ClF3NO3
C15H9ClF3NO3
C15H8Cl2F3NO3‚0.1H2O
C15H8Cl2F3NO3
C15H10ClF3N2O3
C15H8ClF3INO3
C16H11ClF3NO2
C14H10ClNO2
8z
8za
8zb
10b
11a
11b
2-OH, 4-NH2-5-Cl-C6H2 OH
2-OH, 3-I, 5-Cl-C6H2
2-OCH3, 5-Cl-C6H3
2-OH, 5-Cl-C6H3
2-OH, 5-Cl-C6H3
OH
H
H
see exptl
D
D and B
D and B
CF3
H
C15H9ClF3NO2
a
b
All new compounds exhibited spectroscopic and combustion data in accord with the designated structure. Compounds 1 (NS-004),
7b, (()-7c, (+)-7c, and (-)-7c were shown to have identical effects on mSlo- and hSlo-mediated BK currents.39
The optimal regiochemistry for the heterocyclic CF3
substituent was determined by comparing the activity
of 8c with its isomers 8d -f. It is clear from the data in
Table 1 that a 6-CF3 moiety is superior to the 5-CF3
isomer, in the context of both a methyl ether (7d ) and
a phenol (8d ) as the 3-aryl element. However, efficacy
is largely restored with the 7- and 4-substituted CF3
isomers 8e and 8f, respectively, and maintained al-
though not enhanced with the C-6, C-4 disubstitution
pattern found in 8k . None of the remaining aromatic
substituents examined (8g-j) that complete this survey
afforded active compounds.
an inactive compound. Somewhat remarkably, activity
was not restored by substitution at the 5-position by
either F, Br, or I, since compounds 8l-n are ineffective
openers of the mSlo channel. The chloro-substituted
regioisomers 8w and 8x were also inactive, but a 5-CF3
moiety, compound 8u , was found to be compatible with
good efficacy although, again, the 5-position is optimal
in this limited series, as demonstrated by the lack of
channel-opening activity associated with 8v. Further
underscoring the uniqueness of the 5-Cl substituent,
introduction of additional halogens provides inactive
derivatives 8y and 8z. In addition, a 2-hydroxyl or
2-methoxyl moiety also appears to be a critical aspect
of the pharmacophore since 7b and 7a are also ineffec-
tive channel openers.
The maxi-K channel-opening properties of this chemo-
type also exhibited sensitivity to the identity and
pattern of substitution of the 3-aryl element. Of par-
ticular note and in contrast to the benzylated benzim-
idazolone series, removal of the 5-Cl atom (8b) afforded
Several additional substituents appended to the 3-
phenyl ring were introduced in an effort not only to

1492 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Hewawasam et al.
F igu r e 2. (A) Example of an inside-out excised patch from a COS cell expressing mSlo. The channel open probability was very
low in control medium, and application of (()-8c (0.1 uM; 2 min) produced a rapid and reversible large increase in channel
opening. The holding potential was +20 mV, and the calcium concentration at the intracellular patch face was 3 mM. (B) Whole
cell recording of the effects of (()-8c on the human maxi-K channel hSlo expressed in HEK cells. The compound produced a
concentration-dependent increase in maxi-K channel-mediated outward current elicited by +30 mV voltage steps from a holding
potential of -60 mV. The pipet calcium concentration was 1.0 mM.
Ta ble 2. Whole Brain and Plasma Concentrations of (()-8c and (()-11b after IV Administration (4.6 mg/kg) to Male Rats
plasma levela
brain levelb
mean brain/plasma ratio
compd
0.25 hr
2 hr
0.25 hr
2 hr
0.25 hr
2 hr
(()-8c
(()-11b
1.2 ( 0.2
3.3 ( 0.3
0.231 ( 0.004
0.28 ( 0.01
1.34 ( 0.06
2.4 ( 0.2
0.87 ( 0.05
0.23 ( 0.01
1.1
0.73
3.8
0.82
a
b
Concentration as µg/mL; mean of three animals ( standard deviation. Concentration as µg/g wet weight; mean of three animals (
standard deviation.
Ta ble 3. Mean Whole Brain and Plasma Pharmacokinetic
Parameters of (()-8c after IV Administration (4.6 mg/kg) to
Ratsa
probe structure-activity relationships but also to en-
hance aqueous solubility. However, none of the substit-
uents examined provided compounds (8r , 8t, and 8za )
with significant channel-opening activity, reinforcing
the sensitivity of this region of the pharmacophore to
the nature and pattern of substitution. The structure-
activity pattern that has emerged for the 3-aryloxindole
class of maxi-K channel openers indicates a pharma-
cophore highly sensitive to structural variation with
optimal activity restricted to a pattern of substitution
largely identified with two benzimidazolone prototypes
NS-004 (1) and NS-1619 (2).
param
plasma
brain
brain/plasma ratioc
Cmax (ng/g)
Tmax (hr)
AUCb
1.4
0.25
4.8
1.9
2.5
2.5
T1/2 (hr)
3.4
a
Male rats (24) were dosed, and three animals were killed at
b
each of eight time points after dosing. Expressed as µg hr/mL
for plasma and µg hr/g for brain calculated by the trapezoidal rule
over 0-8 h after dosing. c On the basis of ratio of AUC values.
suggesting a prolonged elimination of (()-8c from brain
tissue. To more fully explore the relationship between
brain and plasma levels of (()-8c over time, a larger
group of rats was administered (()-8c intravenously
and whole brain and plasma concentrations were de-
termined out to 8 h postdosing. The results from this
expanded study (Table 3) verified that (()-8c rapidly
entered rat brain after IV administration and attained
concentrations there that exceeded those of plasma (a
total brain/plasma AUC ratio of 2.5, evaluated over 8
h). Furthermore, (()-8c was eliminated somewhat more
slowly from rat brain than from plasma (elimination
half-lives of 3.4 and 2.5 h, respectively).
Meta bolism a n d P h a r m a cok in etics
As a prelude to evaluating compounds in animal
models of stroke, the pharmacokinetic properties of the
most promising maxi-K openers (()-8c and (()-11b
were examined in rats, with particular attention focused
on determining the extent of brain penetration following
intravenous administration. Table 2 shows the plasma
and whole brain concentrations of each compound at 15
min and 2 h after IV dosing. Brain levels of (()-11b
exceeded those of (()-8c at 15 min, but by 2 h, (()-8c
concentrations were greater than those of (()-11b ,

New Class of Potassium Channel Openers
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1493
Ta ble 4. Percent Reduction of Neocortical Infarct Volume in Rats (IV Administration 2 h Post-MCAO)a
dose (mg/kg)
study
compd
(()-8c
(group)
vehicle
0.005
0.01
0.03
0.1
0.3
1.0
5.0
10.0
A
percent
mean
(SE)
n
4%
161.8
(8)
10%
152.2
(6.4)
13
10%
151.0
(8)
4%
168.5
(8.3)
13
161.0
(5.3)
12
9
13
(()-11b
A
percent
mean
(SE)
n
18%b
133.1
(9.1)
8
9%
147.7
(8.7)
13
0%
163.1
6.4
6%
152.6
(6.2)
9
toxic
162.3
(6.7)
13
10
B
percent
mean
(SE)
n
4%
148.8
(8.8)
9
-7% (increase)
12%b
135.4
(6.5)
9
154.3
(6.3)
9
165.4
(7.7)
9
lubeluzole
A
percent
mean
(SE)
n
13%c
157.5
(5.3)
9
22%b
141.1
(8.6)
9
18%b
148.3
(5.6)
9
180.9
(8.3)
9
a
Neocortical infarct volume in mm3 expressed as group mean (with standard error). Percent reduction as compared to vehicle-treated
controls from the same study. p e 0.01, Student’s t test or ANOVA and Dunnett’s t test. c p e 0.02, Student’s t test or ANOVA and
b
Dunnett’s t test.
Ta ble 5. Neocortical Infarct Volume in Rats and Percent
When incubated with a rat liver S9 preparation, both
Reductiona after Administration of (()-11b (Intraperitoneal
(()-8c and (()-11b were metabolized. Over 1 h, 39% of
(()-8c was metabolized to two monohydroxylated me-
tabolites, detected by liquid chromatography (LC)/mass
spectrometry (MS) profiling, while 76% of (()-11b was
metabolized to at least three monohydroxylated me-
tabolites, one of which was identified as (()-8c.48
Administration 2 h Post-MCAO)
dose of (()-11b (mg/kg)
study
vehicle
5
10
20
A
percent
mean
(SE)
n
6%
161.8
(6.5)
9
26%b
127.3
(5.0)
9
171.4
(7.0)
9
Effects of (()-8c a n d (()-11b on Mod els of
P er m a n en t F oca l In fa r ct in th e Ra t
B
percent
mean
(SE)
n
24%b
113.2
(8.3)
8
9%
135.5
(4.7)
7
Because both (()-8c and (()-11b demonstrated ad-
equate brain penetration in rats, they were evaluated
in a model of focal stroke that involved a permanent
occlusion of the middle cerebral artery (MCAO model)
conducted in the spontaneously hypertensive rat (SHR).
This procedure results in a reliably large neocortical
infarct volume that is measured by means of vital dye
exclusion in serial slices of the brain harvested 24 h
after initiating arterial occlusion. In this protocol,
compounds were administered intravenously or intra-
peritoneally 2 h postocclusion in order to mimic a
clinical situation in which patients present to the
hospital with significant delay after experiencing a
stroke. This protocol also provides a stringent test for
the ability of drug candidates to confer protection to
neuronal tissue.
In this model, the more polar 3-hydroxy derivative
(()-8c did not show a significant reduction in cortical
infarct volume when administered intravenously at
doses ranging from 0.1 to 10 mg/kg as a single bolus 2
h after MCAO when compared to vehicle-treated (2%
dimethyl sulfoxide (DMSO), 98% propylene glycol)
controls (Table 4). However, perhaps reflecting its
greater brain-penetrating ability, (()-11b demonstrated
activity in this model of cerebral ischemia, as shown in
Tables 4 and 5. Intravenous administration of (()-11b
at a dose of 0.03 mg/kg reduced the measured cortical
infarct volume by approximately 18% as compared to
control (Table 4). Intraperitoneal administration of (()-
11b at a dose of 10 mg/kg 2 h following artery occlusion
149.0
(6.2)
11
a
Neocortical infarct volume in mm3 expressed as group mean
(with standard error). Percent reduction as compared to vehicle-
b
treated controls from the same study. p e 0.01, Student’s t test
or ANOVA and Dunnett’s t test.
reduced infarct volume by 26% when compared to
vehicle-treated control (Table 5). Under these conditions,
the extent of neuroprotection demonstrated by (()-11b
was either equivalent or superior to a range of experi-
mental neuroprotectants that possess diverse mecha-
nisms of action. For comparison, the noncompetitive
NMDA receptor antagonist Cerestat40 (CN1102, Cam-
bridge Neuroscience) was inactive in this model, a result
consistent with the poor performance of this compound
in clinical trials. However, lubeluzole,41 which is thought
to affect the glutamate/nitric oxide synthase pathway,
showed efficacy comparable to (()-11b, with a 22%
reduction in infarct volume (Table 4).
One possible explanation for the inactivity of the
hydroxyl analogue (()-8c may be that this compound
did not achieve adequate brain levels at early time
points when compared to (()-11b. As a general observa-
tion but for reasons not clearly apparent, we have found
that maxi-K openers with a brain concentration of
g2000 ng/g at 15 min following administration of a dose
of 5 mg/kg show significant activity in MCAO stroke
model.42 However, given the complex profile of (()-11b
in the MCAO model of stroke, the narrow dosing

1494 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Hewawasam et al.
Ta ble 6. Effect of Maxi-K Channel Openers (()-8c and
(()-11b on Electrically Stimulated [3H]-Glutamate Release in
Hippocampal Slices In Vitro
Con clu sion
In summary, we have identified a novel class of
maxi-K channel openers and demonstrated that chan-
nel-opening activity is sensitive to both the nature and
the pattern of substitution of both aromatic elements
as well as the absolute configuration of an asymmetric
center. The preliminary structure-activity data for this
series indicate the importance of both an electron-
withdrawing substituent on the oxindole nucleus and
the presence of a phenolic hydroxyl for effective expres-
sion of BK channel-opening properties. The 3-aryl
oxindole (()-11b showed promise as a potential candi-
date for further development based on its in vitro and
in vivo profiling. However, because of its metabolic
instability, particularly hydroxylation at the 3-position
of the heterocycle, further development of (()-11b as a
neuroprotective agent for the treatment of stroke was
not pursued in favor of derivatives with a more favor-
able pharmacokinetic profile.43,49
electrically stimulated
[3H]-glutamate release
compd
concn (µM)
(% control)a
(()-8c
1
20
20
89 ( 5 (4)
93 ( 7 (5)
71 ( 8 (5)
(()-11b
a
The results are the mean ( SEM with the number of
experiments given in parentheses.
window for efficacy, and the observation that both
compounds enter brain tissue, alternate explanations
were sought.
As part of an effort to illuminate further reasons for
the differences in performane of (()-8c and (()-11b, the
effects of the compounds on neurotransmitter release
in vitro were examined. While the experiments with
either mSlo or hSlo channels expressed in oocytes
described above provide a clear understanding of the
effects of (()-8c and (()-11b on a specifically configured
K+ channel, the biochemical pharmacological effects
under the more complex circumstances of neurotrans-
mitter release in vitro proved to be instructive. This
setting provides a more realistic evaluation of the
performance of the compounds under conditions in
which the maxi-K channels are configured in a more
natural fashion. The amino acid glutamate mediates
much of the excitatory synaptic transmission in the
brain and is thought to be a key mediator in the process
of learning. However, excess glutamate release has also
been implicated in disease states such as epilepsy and
ischemic brain damage, damaging neurons in the latter
by initiating a cascade of events that lead to cell death.12
Because ischemia-related damage maybe reduced by
limiting glutamate release, compounds (()-8c and (()-
11b were evaluated for their ability to interfere with
electrically stimulated [3H]-glutamate release in an in
vitro assay. Briefly, this assay is performed using rat
hippocampal slices, which are preloaded with [3H]-
glutamate. Electrical stimulation evokes the release of
[3H]-glutamate, and the synaptic pool of this neuro-
transmitter is approximately 50% of the total released
during stimulation, as estimated by the effects of total
pharmacological synaptic blockade. As shown in Table
6, only (()-11b was able to demonstrate a significant
inhibition of electrically stimulated [3H]-glutamate re-
lease in vitro at a concentration of 20 µM. One possible
explanation for the differences between compounds (()-
11b and (()-8c is that the latter compound, as the
hydroxylated derivative of (()-11b, is more hydrophilic,
which may limit its penetration into the tissue wedges.
This possibility is consistent with the results of brain
penetration studies, which demonstrated that (()-8c
reached lower initial levels in the brain in vivo when
compared to (()-11b at 0.25 h (Table 2). Alternatively,
the maxi-K channels expressed naturally in rat hippo-
campal slices may be configured such that the biochemi-
cal pharmacological effects of (()-8c do not reflect the
channel-opening effects observed with the cloned chan-
nels in oocytes. Finally, the possibility that the ad-
ditional hydroxyl present in (()-8c leads to additional
pharmacological activities that override the effects of
maxi-K channel opening cannot be excluded.
Exp er im en ta l Section
Melting points were recorded on a Thomas-Hoover capillary
apparatus and are uncorrected. Magnetic resonance (NMR)
spectra were recorded on a Bruker AM FT instrument operat-
ing at 300 MHz for proton (1H) and 75 MHz for carbon (13C)
or a Varian Gemini 300 operating at 300 MHz for proton (1H).
All spectra were recorded using tetramethylsilane (TMS) as
an internal standard, and signal multiplicity was designated
according to the following abbreviations: s ) singlet, d )
doublet, t ) triplet, q ) quartet, m ) multiplet, brd s ) broad
singlet. Infrared (IR) spectra were obtained using a Nicolet
MX1 FT spectrometer, scanning from 4000 to 400 cm-1, and
calibrated to the 1601 cm-1 absorption of a polystyrene film.
Optical rotations were determined on a Perkin-Elmer 41
polarimeter in the solvents indicated. Low-resolution mass
spectra and the apparent molecular ion (MH+) or (M - H)-
were determined on a Finnigan TSQ 7000. Analytical samples
were dried in vacuo at 78 °C or in the presence of P2O5 at room
temperature for at least 12 h. Elemental analyses were
provided by Bristol-Myers Squibb’s Analytical Chemistry
Department. For those compounds whose elemental analyses
were not performed, approximate purity was determined to
be >95% by NMR.
Meth od A. Gr ign a r d Ad d ition to Isa tin s (Sch em e 1).
Gen er a l P r oced u r e. A solution of aryl Grignard reagent (6,
1-2 equiv) in THF was added to a stirred cold (-20 °C)
solution of the Na salt of the isatin in THF under an
atmosphere of N2. The mixture was allowed to warm to room
temperature and stirred until the isatin was consumed,
typically 1-2 h. The reaction mixture was diluted with ether,
cooled in an ice-bath, and then quenched with 1 N HCl. The
organic layer was separated, washed with 0.5 N NaOH, water,
and brine, and then dried (Na2SO4). The crude solid isolated
after evaporation of the solvents was triturated with CH2Cl2
to afford pure 1,3-dihydro-3-hydroxy-3-aryl-2H-indol-2-one
derivatives (7) in 80-95% yield.
Met h od B. Dem et h yla t ion of Ar ylm et h yl E t h er
(Sch em e 1). Gen er a l P r oced u r e. Demethylation of the
methyl ether moiety of 7 or 10 was carried out using BBr3 in
CH2Cl2. To a cold (-78 °C), stirred solution of 7 (R ) OMe) or
10 (R ) OMe) in anhydrous CH2Cl2, BBr3 (3 equiv, 1 M
solution in CH2Cl2) was added under an atmosphere of N2. The
mixture was subsequently stirred in an ice bath until starting
material disappeared by thin-layer chromatography (TLC;
typically 1-2 h). The reaction mixture was quenched with
saturated NaHCO3 and acidified with 1 N HCl. Where the
product was soluble in CH2Cl2, the organic layer was sepa-
rated, washed with brine, and then dried (MgSO4). For
products insoluble in CH2Cl2, the organic layer was evaporated
at room temperature and the aqueous residue was extracted
with EtOAc. The extract was washed with brine, dried over

New Class of Potassium Channel Openers
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1495
Na2SO4, and concentrated in vacuo to give 8 or 11, respectively.
The crude products were purified either by trituration with
CH2Cl2 or recrystallization from EtOAc/hexanes to afford pure
product in 80-95% yield.
230 mg of pure title compound. Concentration of the mother
liquor followed by retrituration with CH2Cl2 gave an additional
72 mg of product bringing the total to 302 mg, 84% yield; mp
244-245 °C; [R]D -132.6° (MeOH). IR (KBr, cm-1): 3300-
3100, 1722, 1320, 1250, 1125. 1H NMR (DMSO-d6): δ 3.42 (3H,
s), 6.90 (1H, s), 6.93 (1H, d, J ) 8.7 Hz), 7.04 (1H, d, J ) 7.7
Hz), 7.05 (1H, s), 7.19 (1H, d, J ) 7.7 Hz), 7.35 (1H, dd, J )
8.7 Hz, J ′ ) 2.7 Hz), 7.79 (1H, d, J ) 2.7 Hz), 10.67 (1H, brd
s). MS: m/e 358 (MH+). Anal. (C16H11ClF3NO3) C, H, N.
(+)-3-(5-Ch lor o-2-m et h oxyp h en yl)-1,3-d ih yd r o-3-h y-
d r oxy-6-(tr iflu or om eth yl)-2H-in d ol-2-on e (7c). mp 243-
245 °C; [R]D +129.9° (MeOH). IR (KBr, cm-1): 3400, 1720,
1320, 1240, 1125. 1H NMR (DMSO-d6): δ 3.42 (3H, s), 6.89
(1H, s), 6.94 (1H, d, J ) 8.7 Hz), 7.04 (1H, d, J ) 7.7 Hz), 7.06
(1H, s), 7.20 (1H, d, J ) 7.7 Hz), 7.36 (1H, dd, J ) 8.6 Hz,
J ′ ) 2.7 Hz), 7.81 (1H, d, J ) 2.7 Hz), 10.66 (1H, s). MS: m/e
358 (MH+). Anal. (C16H11ClF3NO3) C, H, N.
(()-3-(5-Ch lor o-2-m et h oxyp h en yl)-1,3-d ih yd r o-3-h y-
d r oxy-5-(tr iflu or om eth yl)-2H-in d ol-2-on e (7d ). mp 218-
220 °C. IR (KBr, cm-1): 3350, 1730, 1325, 1260, 1150, 1120.
1H NMR (DMSO-d6): δ 3.41 (3H, s), 6.88 (1H, s), 6.93 (1H, d,
J ) 8.8 Hz), 7.01 (1H, d, J ) 8.1 Hz), 7.09 (1H, d, J ) 1.6 Hz),
7.35 (1H, dd, J ) 8.7 Hz, J ′ ) 2.7 Hz), 7.56 (1H, dd, J ) 8.1
Hz, J ′ ) 1.1 Hz), 7.80 (1H, d, J ) 2.7 Hz), 10.77 (1H, s). 13C
NMR (DMSO-d6): δ 55.97, 74.26, 109.51, 113.61, 120.06,
120.11, 121.8 (m, CF3), 124.50, 126.37, 126.94, 128.72, 131.38,
133.10, 146.70, 154.31, 177.55. MS: m/e 358 (MH+). Anal.
(C16H11ClF3NO3) C, H, N.
Meth od C. Ad d ition of Ma gn esiu m P h en ola tes to
Isa tin s (Sch em e 1). Gen er a l P r oced u r e. A solution of
EtMgBr (1.1 equiv) in THF was added dropwise to a cold (0
°C) solution of the phenol (1.2 equiv) in anhydrous THF. The
resultant white suspension of bromomagnesium phenolate was
concentrated to dryness using a rotary evaporator, the residue
was dissolved in anhydrous CH2Cl2, and the isatin (1 equiv)
was added as a solid. The reaction was performed at room
temperature with progress monitored by TLC. In those cases
where no reaction was observed at ambient temperature, the
reaction mixture was heated at reflux until the isatin was
consumed before it was cooled to room temperature. The
reaction mixtures were quenched with 1 N HCl, and the
organic layer was separated and washed with water and brine
before it was dried over MgSO4. The crude products remaining
after concentration were purified by either flash column
chromatography or recrystallization to afford pure 1,3-dihydro-
3-hydroxy-3-(2-hydroxyaryl)-2H-indol-2-ones (8).
Meth od D. Deh yd r oxyla tion of 3-Ar yl-3-h yd r oxyoxin -
d oles 7 (Sch em e 1). Gen er a l P r oced u r e. A mixture of 7 (1
equiv), Et3SiH (3 equiv), and CF3CO2H (3 equiv) was heated
at 110-120 °C in a sealed tube for 1-3 days until deoxygen-
ation was complete. Excess Et3SiH and CF3CO2H were evapo-
rated, and the residue was subjected to flash chromatography
(silica gel/3% MeOH in CH2Cl2) to afford the desired deoxy-
genated product 10 in yields ranging from 80 to 90%.
(()-3-(5-Ch lor o-2-h ydr oxyph en yl)-1,3-dih ydr o-3-h ydr ox-
y-2H-in d ol-2-on e (8a ). mp 216-218 °C. IR (KBr, cm-1): 3520,
1
3328, 3204, 1700, 776, 820. H NMR (DMSO-d6): δ 3.39 (1H,
(()-1,3-Dih ydr o-3-h ydr oxy-3-ph en yl-6-(tr iflu or om eth yl)-
1
brd s), 6.60 (1H, d, J ) 8.5 Hz), 6.82 (3H, m), 7.14 (2H, m),
7.69 (1H, d, J ) 2.7 Hz), 9.59 (1H, s), 10.27 (1H, s). 13C NMR
(DMSO-d6): δ 74.87, 109.13, 116.46. 121.18, 122.16, 123.71,
126.83, 127.91, 128.82, 130.19, 132.41, 143.21, 152.52, 177.67.
MS: m/e 276 (MH+). Anal. (C14H10ClNO3‚0.4H2O) C, H, N.
(()-1,3-Dih yd r o-3-h yd r oxy-3-(2-h yd r oxyp h en yl)-6-(tr i-
flu or om eth yl)-2H-in d ol-2-on e (8b). mp 209-211 °C (dec).
1H NMR (DMSO-d6): δ 6.59 (1H, d, J ) 7.9 Hz), 6.62 (1H, s),
6.87 (1H, t, J ) 7.3 Hz), 6.98 (1H, d, J ) 7.9 Hz), 7.00 (1H, s),
7.10 (1H, m), 7.17 (1H, d, J ) 7.5 Hz), 7.73 (1H, dd, J ) 7.7
Hz, J ′ ) 1.5 Hz), 9.36 (1H, s), 10.53 (1H, s). MS: m/e 310
(MH+).
2H-in d ol-2-on e (7a ). mp 247-249 °C. H NMR (DMSO-d6):
δ 6.85 (1H, s), 7.12 (1H, s), 7.24-7.34 (7H, m), 10.70 (1H, s).
MS: m/e 294 (MH+). Anal. (C15H10F3NO2) H, N; C: calcd,
61.44; found, 60.96.
(()-3-(3-Ch lor op h en yl)-1,3-d ih yd r o-3-h yd r oxy-6-(t r i-
flu or om eth yl)-2H-in d ol-2-on e (7b). mp 268-270 °C. 1H
NMR (DMSO-d6): δ 7.05 (1H, s), 7.06-7.09 (1H, m) 7.14 (1H,
s), 7.31-7.41 (4H, m), 7.42 (1H, s), 10.80 (1H, s). MS: m/e 328
(MH+). Anal. (C15H9ClF3NO2) H, N; C: calcd, 54.98; found,
55.41.
(()-3-(5-Ch lor o-2-m et h oxyp h en yl)-1,3-d ih yd r o-3-h y-
d r oxy-6-(tr iflu or om eth yl)-2H-in d ol-2-on e (7c). mp 207-
210 °C. IR (KBr, cm-1): 3400, 1730, 1320, 1250, 1125, 1170.
1H NMR (DMSO-d6): δ 3.41 (3H, s), 6.91 (1H, s), 6.94 (1H, d,
J ) 8.7 Hz), 7.05 (2H, m), 7.19 (1H, d, J ) 8.2 Hz), 7.35 (1H,
dd, J ) 8.6 Hz, J ′ ) 2.7 Hz), 7.80 (1H, d, J ) 2.7 Hz), 10.67
(1H, s). 13C NMR (DMSO-d6): δ 55.95, 74.25, 105.21. 113.52,
118.45, 122.28, 124.27, 124.46, 126.83, 128.70, 129.47 (q),
131.31, 136.60, 143.88, 154.30, 177.29. MS: m/e 358 (MH+).
Anal. (C16H11ClF3NO3) C, H, N.
(-)-3-(5-Ch lor o-2-m et h oxyp h en yl)-1,3-d ih yd r o-3-h y-
d r oxy-6-(tr iflu or om eth yl)-2H-in d ol-2-on e (7c). Potassium
bis(trimethylsilyl)amide solution (2.2 mL, 1.1 mmol, 0.5 M in
toluene) was added dropwise under an atmosphere of argon
to a cold (-78 °C) stirred solution of (()-3-(5-chloro-2-meth-
oxyphenyl)-1,3-dihydro-6-(trifluoromethyl)-2H-indol-2-one (342
mg, 1 mmol) in dry THF (3 mL) that had been degassed by
heating at reflux for 1 h while bubbling through a stream of
argon. The resultant light yellow solution of the potassium
enolate was stirred at -78 °C for 30 min before adding a
solution of (1S)-(+)-(10-camphorsulfonyl)oxaziridine (252 mg,
1.1 mmol) in dry degassed THF (2 mL) dropwise over 5 min.
The mixture was stirred at -78 °C for 1 h and then allowed
to warm in an ice-bath (0-5 °C). The reaction mixture was
quenched with glacial AcOH (0.1 mL), diluted with ether (25
mL) followed by addition of a saturated NH4Cl (10 mL)
solution. The organic layer was separated, washed with
saturated NaHCO3, water, and brine, and then dried (Na2SO4).
Filtration and evaporation of the solvents gave 0.54 g of crude
product, which was triturated with ether to precipitate the
insoluble (camphorsulfonyl)imine byproduct, which was re-
moved by filtration. Concentration of the filtrate gave 0.39 g
of product, which was triturated with boiling CH2Cl2 to afford
(()-3-(5-Ch lor o-2-h ydr oxyph en yl)-1,3-dih ydr o-3-h ydr ox-
y-6-(tr iflu or om eth yl)-2H-in d ol-2-on e (8c). mp 210-213 °C.
1
IR (KBr, cm-1): 3300, 1725, 1320, 1250, 1170, 1140. H NMR
(DMSO-d6): δ 6.61 (1H, dd, J ) 8.5 Hz, J ′ ) 2.6 Hz), 6.81
(1H, s), 7.02, (1H, s), 7.05 (1H, d, J ) 7.7 Hz), 7.17 (2H, m),
7.71 (1H, d, J ) 2.7 Hz), 9.72 (1H, s), 10.60 (1H, s). 13C NMR
(DMSO-d6): δ 74.37, 105.17, 116.48. 118.30, 122.34, 124.32,
125.92, 126.84, 128.33, 129.14, 129.26 (q), 136.87, 144.11,
152.37, 177.30. MS: m/e 344 (MH+). Anal. (C15H9ClF3NO3) C,
H, N.
(+)-3-(5-Ch lor o-2-h ydr oxyph en yl)-1,3-dih ydr o-3-h ydr ox-
y-6-(tr iflu or om eth yl)-2H-in d ol-2-on e (8c). Demethylation
of (-)-7c (170 mg, 0.475 mmol) was carried out following the
general procedure described in method B to provide pure titled
compound (+)-8c (164 mg, 100%) as a white solid; mp 200-
201 °C; [R]D + 119.8° (MeOH). IR (KBr, cm-1): 3540, 3350,
1
1725, 1320, 1160, 1130. H NMR (DMSO-d6): δ 6.60 (1H, d,
J ) 8.5 Hz), 6.82 (1H, brd s), 7.02, (1H, s), 7.05 (1H, d, J ) 7.6
Hz), 7.17 (2H, m), 7.71 (1H, d, J ) 2.7 Hz), 9.75 (1H, brd s),
10.61 (1H, s). MS: m/e 344 (MH+). Anal. (C15H9ClF3NO3) C,
H, N.
(-)-3-(5-Ch lor o-2-h ydr oxyph en yl)-1,3-dih ydr o-3-h ydr ox-
y-6-(tr iflu or om eth yl)-2H-in d ol-2-on e (8c). mp 198-200 °C;
[R]D - 121.5° (MeOH). IR (KBr, cm-1): 3540, 3300, 1725, 1320,
1125. 1H NMR (DMSO-d6): δ 6.60 (1H, d, J ) 8.6 Hz), 6.82
(1H, brd s), 7.02, (1H, s), 7.05 (1H, d, J ) 7.6 Hz), 7.17 (2H,
m), 7.72 (1H, d, J ) 2.7 Hz), 9.75 (1H, brd s), 10.61 (1H, s).
MS: m/e 344 (MH+). Anal. (C15H9ClF3NO3) C, H, N.
(+)-3-(5-Ch lor o-2-h ydr oxyph en yl)-1,3-dih ydr o-3-h ydr ox-
y-5-(tr iflu or om eth yl)-2H-in d ol-2-on e (8d ). mp 156-158 °C.
1
IR (KBr, cm-1): 3350, 1740, 1325, 1260, 1160, 1120. H NMR

1496 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Hewawasam et al.
(DMSO-d6): δ 6.62 (1H, d, J ) 8.5 Hz), 6.80 (1H, s), 6.98 (1H,
d, J ) 8.1 Hz), 7.09 (1H, d, J ) 1.2 Hz), 7.17 (1H, dd, J ) 8.5
Hz, J ′ ) 2.7 Hz), 7.56 (1H, dd, J ) 8.1 Hz, J ′ ) 1.2 Hz), 7.73
(1H, d, J ) 2.7 Hz), 9.73 (1H, s), 10.72 (1H, s). 13C NMR
(DMSO-d6): δ 74.37, 109.44, 116.52, 120.16, 121.75 (q), 122.37,
126.42, 126.75, 126.93, 128.37, 129.28, 133.33, 146.94, 152.36,
177.56. MS: m/e 344 (MH+). Anal. (C15H9ClF3NO3) C, H, N.
(()-3-(5-Br om o-2-h ydr oxyph en yl)-1,3-dih ydr o-3-h ydr ox-
y-6-(tr iflu or om eth yl)-2H-in d ol-2-on e (8m ). mp 194-196
°C. 1H NMR (DMSO-d6): δ 6.56 (1H, d, J ) 8.5 Hz), 6.82 (1H,
brd s), 7.02 (1H, s), 7.05 (1H, d, J ) 7.7 Hz), 7.19 (1H, dd, J )
7.7 Hz, J ′ ) 0.6 Hz), 7.28 (1H, dd, J ) 8.5 Hz, J ′ ) 2.6 Hz),
7.84 (1H, d, J ) 2.6 Hz), 9.77 (1H, s), 10.62 (1H, s). MS: m/e
390 (MH+).
(()-1,3-Dih ydr o-3-h ydr oxy-3-(5-iodo-2-h ydr oxyph en yl)-
6-(tr iflu or om eth yl)-2H-in d ol-2-on e (8n ). mp 192-194 °C.
1H NMR (DMSO-d6): δ 6.45 (1H, d, J ) 8.4 Hz), 6.78 (1H, s),
7.01 (1H, s), 7.04 (1H, d, J ) 7.6 Hz), 7.18 (1H, d, J ) 7.6 Hz),
7.43 (1H, dd, J ) 8.4 Hz, J ′ ) 2.3 Hz), 8.01 (1H, d, J ) 2.3
Hz), 9.74 (1H, s), 10.59 (1H, s). MS: m/e 436 (MH+). Anal.
(C15H9F3INO3) C, H, N.
(()-3-(5-Ch lor o-2-h ydr oxyph en yl)-1,3-dih ydr o-3-h ydr ox-
y-7-(tr iflu or om eth yl)-2H-in d ol-2-on e (8e). mp 205-207 °C.
1
IR (KBr, cm-1): 3250, 1745, 1340, 1240, 1175, 1120. H NMR
(DMSO-d6): δ 6.59 (1H, d, J ) 8.5 Hz), 6.99 (1H, t, J ) 7.7
Hz), 7.10 (1H, d, J ) 7.1 Hz), 7.16 (1H, dd, J ) 8.5 Hz, J ′ )
2.7 Hz), 7.44 (1H, d, J ) 7.8 Hz), 7.72 (1H, d, J ) 2.7 Hz),
9.79 (1H, s), 10.79 (1H, s). 13C NMR (DMSO-d6): δ 73.41,
110.10 (q), 116.37, 121.43, 122.27, 125.16, 125.61, 126.83,
127.53, 128.30, 129.37, 134.40, 140.72, 152.38, 178.02. MS: m/e
344 (MH+). Anal. (C15H9ClF3NO3‚0.2H2O) C, H, N.
(()-1,3-Dih yd r o-3-h yd r oxy-3-(2-h yd r oxy-5-m eth ylp h en -
yl)-6-(tr iflu or om eth yl)-2H-in d ol-2-on e (8o). mp 226-228
1
°C (dec). H NMR (DMSO-d6): δ 2.26 (3H, s), 6.47 (1H, s, J )
8.1 Hz), 6.59 (1H, s), 6.90 (1H, dd, J ) 8.1 Hz, J ′ ) 2.0 Hz),
6.98-7.01 (2H, m), 7.17 (1H, d, J ) 7.7 Hz), 7.55 (1H, d, J )
2.0 Hz), 9.11 (1H, s), 10.51 (1H, s). MS: m/e 324 (MH+). Anal.
(C16H12F3NO3) C, H, N.
(()-1,3-Dih yd r o-3-h yd r oxy-3-(4-h yd r oxy-1,1′-bip h en yl-
3-yl)-6-(tr iflu or om eth yl)-2H-in d ol-2-on e (8p ). mp 180-185
°C (dec). 1H NMR (DMSO-d6): δ 6.67 (1H, d, J ) 8.3 Hz), 7.03
(1H, s), 7.08 (1H, d, J ) 7.7 Hz), 7.19 (1H, d, J ) 7.7 Hz), 7.29
(2H, t, J ) 7.3 Hz), 7.40-7.48 (3H, m), 7.62 (2H, d, J ) 7.3
Hz), 8.04 (1H, d, J ) 1.9 Hz), 9.60 (1H, brd s), 10.58 (1H, s).
MS: m/e 386 (MH+). Anal. (C21H14F3NO3) C, H, N.
(()-1,3-Dih yd r o-3-h yd r oxy-3-(2-h yd r oxy-1-n a p h th a len -
yl)-6-(tr iflu or om eth yl)-2H-in d ol-2-on e (8q). mp 159-160
°C. 1H NMR (DMSO-d6): δ 6.96 (1H, d, J ) 8.4 Hz), 7.06 (1H,
s), 7.16 (1H, d, J ) 7.8 Hz), 7.22-7.31 (3H, m), 7.42 (1H, brd
s), 7.71 (1H, d, J ) 8.7 Hz), 7.78 (1H, d, J ) 8.0 Hz), 9.35 (1H,
brd s), 9.99 (1H, brd s), 10.48 (1H, brd s). MS: m/e 358 (M -
H)-. Anal. (C19H12F3NO3‚0.85H2O) C, H, N.
(()-3-(4-Am in o-2-h ydr oxyph en yl)-1,3-dih ydr o-3-h ydr ox-
y-6-(tr iflu or om eth yl)-2H-in d ol-2-on e (8r ). mp 276-280 °C
(dec). 1H NMR (DMSO-d6): δ 4.92 (2H, s), 5.88 (1 H, d, J )
1.9 Hz), 6.06 (1H, dd, J ) 8.3, J ′ ) 1.9 Hz), 6.35 (1H, s), 6.98
(1H, s), 7.01 (1H, d, J ) 7.7 Hz), 7.17 (1H, d, J ) 7.7 Hz), 7.27
(1H, d, J ) 8.3 Hz), 8.86 (1H, s), 10.42 (1H, s). MS: m/e 325
(MH+).
(()-3-(5-Ch lor o-2-h ydr oxyph en yl)-1,3-dih ydr o-3-h ydr ox-
y-4-(tr iflu or om eth yl)-2H-in d ol-2-on e (8f). mp 239-242 °C.
1
IR (KBr, cm-1): 3300, 1725, 1330, 1250, 1170, 1140. H NMR
(DMSO-d6): δ 6.56 (1H, d, J ) 8.5 Hz), 6.76 (1H, s), 7.07-
7.13 (3H, m), 7.39 (1H, t, J ) 7.9 Hz), 7.66 (1H, s), 9.57 (1H,
s), 10.71 (1H, s). 13C NMR (DMSO-d6): δ 74.93, 113.23, 116.01,
118.26, 121.60, 121.84,125.50 (q), 127.77, 127.95, 128.76,
129.57, 129.73, 145.00, 152.40, 176.89. MS: m/e 344 (MH+).
Anal. (C15H9ClF3NO3) C, H, N.
(()-3-(5-Ch lor o-2-h ydr oxyph en yl)-1,3-dih ydr o-3-h ydr ox-
y-5-n itr o-2H-in d ol-2-on e (8g). mp 215-217 °C (dec). 1H
NMR (DMSO-d6): δ 6.67 (1H, d, J ) 8.5 Hz), 6.97 (1H, s), 7.02
(1H, d, J ) 8.6 Hz), 7.18 (1H, dd, J ) 8.5 Hz, J ′ ) 2.7 Hz),
7.62 (1H, d, J ) 2.3 Hz), 7.73 (1H, d, J ) 2.6 Hz), 8.17 (1H,
dd, J ) 8.6 Hz, J ′ ) 2.4 Hz), 9.88 (1H, s), 11.12 (1H, s). MS:
m/e 321 (MH+).
(()-3-(5-Ch lor o-2-h ydr oxyph en yl)-1,3-dih ydr o-3-h ydr ox-
y-5-m eth yl-2H-in d ol-2-on e (8h ). mp 225-227 °C (dec). IR
(KBr, cm-1): 3350, 1710, 1240, 820. 1H NMR (DMSO-d6): δ
2.13 (3H, s), 6.49 (1H, s), 6.60 (1H, d, J ) 9.0 Hz), 6.63 (1H, d,
J ) 1.0 Hz), 6.67 (1H, d, J ) 7.8 Hz), 6.94 (1H, dd, J ) 7.75
Hz, J ′ ) 1.0 Hz), 7.12 (1H, dd, J ) 8.5 Hz, J ′ ) 2.8 Hz), 7.68
(1H, d, J ) 2.7 Hz), 9.57 (1H, s), 10.17 (1H, s). MS: m/e 290
(MH+). Anal. (C15H12ClNO3) C, H, N.
(()-3-(5-Ch lor o-2-h ydr oxyph en yl)-1,3-dih ydr o-5-flu or o-
3-h yd r oxy-2H-in d ol-2-on e (8i). mp 158-161 °C (dec). IR
(KBr, cm-1): 3300, 1710, 1250. 1H NMR (DMSO-d6): δ 3.36
(1H, brd s), 6.61 (1 H, d, J ) 8.5 Hz), 6.68 (1H, dd, J ) 8.0 Hz,
J ′ ) 2.6 Hz), 6.77 (1H, dd, J ) 8.5 Hz, J ′ ) 4.3 Hz), 6.98 (1H,
ddd, J ) 8.5 Hz, J ′ ) 8.0 Hz, J ′′ ) 2.6 Hz), 7.15 (1H, dd, J )
8.5 Hz, J ′ ) 2.7 Hz), 7.68 (1H, d, J ) 2.7 Hz), 9.65 (1H, s),
10.32 (1H, s). MS: m/e 294 (MH+).
(()-1,3-Dih yd r o-3-h yd r oxy-3-(2-h yd r oxy-5-m et h oxy-
p h en yl)-6-(tr iflu or om eth yl)-2H-in d ol-2-on e (8s). mp 231-
1
233 °C. H NMR (DMSO-d6): δ 3.72 (3H, s), 6.50 (1H, d, J )
8.6 Hz), 6.65 (1H, s), 6.69 (1H, dd, J ) 8.6 Hz, J ′ ) 3.1 Hz),
7.02 (1H, d, J ) 7.5 Hz), 7.00 (1H, s), 7.17 (1H, d, J ) 7.5 Hz),
7.33 (1H, d, J ) 3.1 Hz), 8.90 (1H, s), 10.53 (1H, s). MS: m/e
340 (MH+). Anal. (C16H12F3NO4) C, H, N.
(()-1,3-Dih ydr o-3-h ydr oxy-3-[2-h ydr oxy-5-(4-m eth ylpip-
er a zin -1-yl)p h en yl]-6-(t r iflu or om et h yl)-2H -in d ol-2-on e
(8t). mp 210-215 °C (dec). IR (KBr, cm-1): 3350, 1735, 1320,
1250, 1170, 1125. 1H NMR (DMSO-d6): δ 2.26 (3H, s), 2.52
(4H, m), 3.04 (4H, m), 3.32 (3H, s), 6.47 (1H, d, J ) 8.6 Hz),
6.58 (1H, s), 6.71 (1H, dd, J ) 8.6 Hz, J ′ ) 2.9 Hz), 6.99 (1H,
s), 7.01 (1H, d, J ) 8.3 Hz), 7.16 (1H, d, J ) 8.3 Hz), 7.38 (1H,
d, J ) 2.9 Hz), 8.81 (1H, s), 10.49 (1H, s). MS: m/e 408 (MH+).
(()-1,3-Dih yd r o-3-h yd r oxy-3-[2-h yd r oxy-5-(t r iflu or o-
m eth yl)p h en yl]-6-(tr iflu or om eth yl)-2H-in d ol-2-on e (8u ).
(()-3-(5-Ch lor o-2-h yd r oxyp h en yl)-4,6-d ich lor o-1,3-d i-
h yd r o-3-h yd r oxy-2-H-in d ol-2-on e (8j). mp 232-235 °C
1
(dec). IR (KBr, cm-1): 3400, 1730, 1275. H NMR (DMSO-d6):
δ 6.61 (1H, d, J ) 8.5 Hz), 6.79 (1H, d, J ) 1.7 Hz), 6.81 (1H,
s), 6.94 (1H, d, J ) 1.7 Hz), 7.14 (1H, dd, J ) 8.5 Hz, J ′ ) 2.7
Hz), 7.71 (1H, d, J ) 2.7 Hz), 9.71 (1H, s), 10.71 (1H, s). 13C
NMR (DMSO-d6): δ 74.77, 108.31, 116.32, 121.22, 122.07,
127.55, 128.20, 128.39, 130.40, 134.05, 146.26, 152.27, 176.89.
MS: m/e 344 (MH+). Anal. (C14H8Cl3NO3) C, H, N.
(()-3-(5-Ch lor o-2-h ydr oxyph en yl)-1,3-dih ydr o-3-h ydr ox-
y-4,6-bis(tr iflu or om eth yl)-2H-in d ol-2-on e (8k ). mp 191-
193 °C. IR (KBr, cm-1): 3700-2500, 1740, 1280, 1170, 1130.
1H NMR (DMSO-d6): δ 6.59 (1H, d, J ) 8.5 Hz), 7.06 (1H, s),
7.15 (1 H, dd, J ) 8.5 Hz, J ′ ) 2.6 Hz), 7.34 (1H, s), 7.45 (1H,
s), 7.68 (1H, d, J ) 2.6 Hz), 9.74 (1H, s), 11.07 (1H, s). MS:
m/e 412 (MH+). Anal. (C16H8ClF6NO3‚0.2H2O) C, H, N.
(()-1,3-Dih ydr o-3-(5-flu or o-2-h ydr oxyph en yl)-3-h ydr ox-
y-6-(tr iflu or om eth yl)-2H-in d ol-2-on e (8l). mp 184-186 °C.
1H NMR (DMSO-d6): δ 6.57 (1H, dd, J ) 8.8, J ′ ) 4.7 Hz),
6.78 (1H, brd s), 6.94 (1H, ddd, J ) 8.5 Hz, J ′ ) 8.5 Hz, J ” )
3.2 Hz), 7.01 (1H, s), 7.03 (1H, d, J ) 7.7 Hz), 7.18 (1H, d,
J ) 7.7 Hz), 7.49 (1H, dd, J ) 10.0 Hz, J ′ ) 3.2 Hz), 9.41 (1H,
s), 10.59 (1H, s). MS: m/e 328 (MH+). Anal. (C15H9F4NO3) H,
N; C: calcd, 55.06; found, 54.69.
1
mp 175-177 °C. H NMR (DMSO-d6): δ 6.77 (1H, d, J ) 8.3
Hz), 7.02-7.09 (3H, m), 7.19 (1H, d, J ) 7.7 Hz), 7.51 (1H, d,
J ) 8.3 Hz), 8.08 (1H, s), 10.40 (1H, s), 10.67 (1H, s). MS: m/e
378 (MH+). Anal. (C16H9F6NO3) C, H, N.
(()-1,3-Dih yd r o-3-h yd r oxy-3-[2-h yd r oxy-4-(t r iflu or o-
m eth yl)p h en yl]-6-(tr iflu or om eth yl)-2H-in d ol-2-on e (8v).
mp 198-201 °C. 1H NMR (DMSO-d6): δ 6.97 (1H, s), 7.01-
7.06 (3H, m), 7.11 (1H, dd, J ) 8.7 Hz, J ′ ) 2.5 Hz), 7.23 (1H,
d, J ) 7.7 Hz), 7.99 (1H, d, J ) 8.7 Hz), 10.18 (1H, s), 10.67
(1H, s). MS: m/e 378 (MH+). Anal. (C16H9F6NO3‚0.7H2O) C,
H, N.
(()-3-(3-Ch lor o-2-h ydr oxyph en yl)-1,3-dih ydr o-3-h ydr ox-
y-6-(tr iflu or om eth yl)-2H-in d ol-2-on e (8w ). mp 202-205
1
°C. H NMR (DMSO-d6): δ 6.77 (1H, s), 6.90 (1H, t, J ) 7.8
Hz), 7.04 (1H, s), 7.02 (1H, d, J ) 7.8 Hz), 7.21 (1H, d, J ) 7.8

New Class of Potassium Channel Openers
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1497
Hz), 7.30 (1H, dd, J ) 8.0 Hz, J ′ ) 1.6 Hz), 7.69 (1 H, dd, J )
8.0 Hz, J ′ ) 1.0 Hz), 9.46 (1H, s), 10.68 (1H, s). MS: m/e 344
(MH+).
124.20, 125.61, 126.06, 128.05, 128.41, 130.76, 134.72, 143.69,
154.26, 176.92. MS: m/e 328 (MH+). Anal. (C15H9ClF3NO2) C,
H, N.
(()-3-(4-Ch lor o-2-h ydr oxyph en yl)-1,3-dih ydr o-3-h ydr ox-
y-6-(tr iflu or om eth yl)-2H-in d ol-2-on e (8x). mp 188-190 °C
(dec). 1H NMR (DMSO-d6): δ 6.64 (1H, d, J ) 2.0 Hz), 6.75
(1H, s), 6.95 (1H, dd, J ) 8.3 Hz, J ′ ) 2.0 Hz), 7.00 (1H, d,
J ) 8.3 Hz), 7.02 (1H, s), 7.19 (1H, d, J ) 8.3 Hz), 7.75 (1H, d,
J ) 8.3 Hz), 9.96 (1H, s), 10.59 (1H, s). MS: m/e 344 (MH+).
In Vitr o a n d In Vivo Meth od s. Recor d in g of Wh ole
Cell Ma xi-K (m Slo or h Slo)-Med ia ted Ou tw a r d Cu r r en ts
Exp r essed in Xen opu s Oocytes. The ability of compounds
listed in Table 1 to open maxi-K channels and increase whole
cell outward (K+) maxi-K-mediated currents was assessed
under voltage clamp conditions by determining their ability
to increase cloned mammalian (mSlo or hSlo) maxi-K-medi-
ated outward current heterologously expressed in Xenopus
oocytes.37,38 The two maxi-K constructs employed represent
nearly structurally identical homologous proteins and have
proven to be pharmacologically identical in our tests. To isolate
maxi-K current from native (background, nonmaxi-K) current,
the specific and potent maxi-K channel-blocking toxin, IbTX,44
was employed at a supramaximal concentration (50 nM). The
relative contribution of maxi-K channels current to total
outward current was determined by subtraction of the current
remaining in the presence of IbTX (nonmaxi-K current) from
the current profiles obtained in all other experimental condi-
tions (control, drug, and wash).39 It was determined that at
the tested concentration the compounds profiled did not affect
nonmaxi-K native currents in the oocytes.39 All compounds
were tested in at least five oocytes and are reported at the
single concentration of 20 µM; the effect of compounds on
maxi-K current was expressed as the percent of control IbTX-
sensitive current, and it is listed in Table 1. Recordings were
accomplished using standard two electrode voltage clamp
techniques;45 voltage clamp protocols consisted of 500-750 ms
duration step depolarizations from a holding potential of -60
to +140 mV in 20 mV steps. The experimental media (modified
Barth’s solution) consisted of (in mM) NaCl (88), NaHCO3 (2.4),
KCl (1.0), HEPES (10), MgSO4 (0.82), Ca(NO3)2 (0.33), and
CaCl2 (0.41); pH 7.5.
P h a r m a cok in etic Stu d ies. For evaluation of brain up-
take, (()-8c and (()-11b were each dissolved (10% DMSO/
65% poly(ethylene glycol)/25% water; 2.5 mg/mL), and 2 mL/
kg of each solution was administered as a bolus via the tail
vein to male Sprague-Dawley rats (ca. 225 g). Three animals
from each group were killed (CO2 inhalation) at 0.25 and 2 h,
the whole brain was removed, and a blood sample was
obtained. Aqueous whole brain homogenates and plasma
samples were extracted with CH3CN and analyzed by a high-
performance liquid chromatography assay with UV (290 nm)
detection. For the pharmacokinetic evaluation of (()-8c in rats,
24 male rats were dosed as above, with three animals killed
at intervals out to 8 h after dosing. Brains and blood samples
were removed and analyzed for (()-8c. For metabolite iden-
tification, (()-8c and (()-11b were separately incubated at a
final concentration of 25 µg/mL in a freshly prepared rat liver
9000 g supernatant fraction (S9) fortified with cofactors
(NADPH, MgCl2, glucose-6-phosphate, and glucose-6-phos-
phate dehydrogenase) for 1 h.50 Over time, aliquots of each
incubation mixture were removed, extracted with CH3CN, and
analyzed for (()-8c or (()-11b. Metabolite identification (LC/
MS/MS: Sciex API III tandem quadrupole) was conducted on
samples following the 1 h incubation.
(()-3-(4,5-Dich lor o-2-h yd r oxyp h en yl)-1,3-d ih yd r o-3-
h ydr oxy-6-(tr iflu or om eth yl)-2H-in dol-2-on e (8y). mp 152-
1
154 °C. H NMR (CDC3/DMSO-d6): δ 5.22 (1H, s), 6.83 (1H,
s) 7.04 (1H, s), 7.14 (1H, d, J ) 7.8 Hz), 7.20 (1H, d, J ) 7.8
Hz), 7.32 (1H, s), 9.50 (1H, brd s), 9.96 (1H, brd s). MS: m/e
378 (MH+).
(()-3-(3,5-Dich lor o-2-h yd r oxyp h en yl)-1,3-d ih yd r o-3-
h ydr oxy-6-(tr iflu or om eth yl)-2H-in dol-2-on e (8z). mp 210-
1
212 °C. H NMR (DMSO-d6): δ 6.19 (1H, brd s), 6.91 (1H, s),
6.97 (1H, s), 7.21(1H, d, J ) 7.8 Hz), 7.43 (1H, d, J ) 7.7 Hz),
9.25 (1H, s), 10.38 (1H, s). MS: m/e 376 (M - H).
(()-3-(4-Am in o-5-ch lor o-2-h ydr oxyph en yl)-1,3-dih ydr o-
3-h yd r oxy-6-(tr iflu or om eth yl)-2H-in d ol-2-on e (8za ). mp
198-200 °C. 1H NMR (DMSO-d6): δ 5.18 (2H, s), 6.09 (1H, s),
6.50 (1H, s), 6.98 (1H, s), 7.05 (1H, d, J ) 7.6 Hz), 7.18 (1H, d,
J ) 7.2 Hz), 7.44 (1H, s), 9.15 (1H, s), 10.45 (1H, s). MS: m/e
359 (MH+). Anal. (C15H10ClF3N2O3) C, H; N: calcd, 7.81; found,
7.33.
(()-3-(5-Ch lor o-3-iod o-2-h yd r oxyp h en yl)-1,3-d ih yd r o-
3-h yd r oxy-6-(t r iflu or om et h yl)-2H -in d ol-2-on e (8zb). A
solution of iodine monochloride (104 mg, 0.64 mmol) in glacial
AcOH (10 mL) was added to a cold (5-10 °C) stirred solution
of 8c (200 mg, 0.58 mmol) in AcOH (20 mL) maintained under
an atmosphere of nitrogen. The mixture was allowed to warm
to room temperature and stirred overnight before it was
poured into cold water and extracted with ether. The ether
extract was washed with 10% NaHSO3 solution, water, and
brine and then dried over Na2SO4. Filtration followed by
concentration in a vacuum gave a brown solid (0.25 g), which
was triturated with warm CH2Cl2 to afford pure iodinated
phenol 8zb; mp 210-212 °C (dec). IR (KBr, cm-1): 3600-2500,
1
1730, 1320, 1170, 1140. H NMR (DMSO-d6): δ 7.06 (1H, s),
7.09 (1H, s), 7.12 (1H, d, J ) 7.8 Hz), 7.25 (1H, d, J ) 7.8 Hz),
7.69 (1H, d, J ) 2.6 Hz), 7.75 (1H, d, J ) 2.6 Hz), 9.25 (1H, s),
10.70 (1H, s). MS: m/e 470 (MH+). Anal. (C15H8ClF3INO3) C,
H, N.
(()-3-(5-Ch lor o-2-m et h oxyp h en yl)-1,3-d ih yd r o-6-(t r i-
flu or om et h yl)-2H -in d ol-2-on e (10b ). mp 210-213 °C. IR
(KBr, cm-1): 3200, 1710, 1320, 1250, 1170, 1120, 1050. 1H
NMR (DMSO-d6): δ 3.57 (3H, s), 4.91 (1H, s) 7.02 (1H, d, J )
8.8 Hz), 7.06 (2H, m), 7.21, (1H, dd, J ) 7.7 Hz, J ′ ) 0.6 Hz),
7.35 (2H, m), 10.77 (1H, s). 13C NMR (DMSO-d6): δ 48.25,
56.07, 105.05. 113.58, 118.26, 122.42, 124.18, 126.03, 127.38,
128.60 (q), 128.70, 130.65, 134.37, 143.58, 156.09, 176.73.
MS: m/e 342 (MH+). Anal. (C16H11ClF3NO2) C, H, N.
(()-3-(5-Ch lor o-2-h ydr oxyph en yl)-1,3-dih ydr o-2H-in dol-
2-on e (11a ). mp 256-258 °C. IR (KBr, cm-1): 3300, 3200,
1680, 820, 750. 1H NMR (DMSO-d6): δ 4.75 (1H, s), 6.78 (1H,
d, J ) 8.6 Hz), 6.85 (2H, t, J ) 8.1 Hz), 6.92 (1H, d, J ) 7.2
Hz), 7.08 (1H, d, J ) 2.5 Hz), 7.11-7.17 (2H, m), 8.78 (1H, s),
10.46 (1H, s). 13C NMR (DMSO-d6): δ 48.04, 109.09, 116.87,
121.31, 122.20, 123.72, 126.61,127.64, 127.99, 130.01, 130.05,
MCAO P r otocol. Male SHRs (from Taconic Farms, Inc. or
Harlan), weighing 240-350 gm, were anesthetized with a
combination of ketamine (40 mg/kg, i.m.) and xylazine (4.5 mg/
kg). According to the method described by Tamura et al.,46
a
142.78, 154.40, 177.17. MS: m/e 260 (MH+). Anal. (C14H10
-
subtemporal approach was used to occlude the left MCA. Prior
to making the initial incision, 0.2 mg of xylocaine was injected
into the scalp. Muscle tissue covering the rostral end of the
zygoma at its fusion to the squamosal bone was excised. With
a no. 6 dental burr, a craniotomy was performed to locate the
MCA. A 2 mm segment of the MCA, containing the origins of
the lenticulostriate arteries, was electrocauterized with bipolar
forceps, and then, the cauterized artery was severed to ensure
permanent occlusion. Sterile Gelfoam was placed in the wound
that was then sutured. Colonic and temporalis temperatures
were recorded immediately prior to surgery, at the time of
occlusion, and 1 h postocclusion. Body temperature was
ClNO2) C, H, N.
(()-3-(5-Ch lor o-2-h yd r oxyp h en yl)-1,3-d ih yd r o-6-(t r i-
flu or om eth yl)-2H-in d ol-2-on e (11b). Demethylation of 10b
(1.37 g, 4 mmol) was carried out following the general
procedure described in method B to afford 11b (1.21 g, 93%)
as an off-white solid; mp 266-268 °C. IR (KBr, cm-1): 3320,
1690, 1310, 1250, 1160, 1125. 1H NMR (DMSO-d6): δ 4.85 (1H,
s), 6.75 (1H, d, J ) 8.6 Hz), 7.04 (1H, s), 7.11, (1H, d, J ) 7.7
Hz), 7.17 (1H, dd, J ) 8.6 Hz, J ′ ) 2.6 Hz), 7.22 (1H, d, J )
8.0 Hz), 7.26 (1H, d, J ) 2.4 Hz), 9.82 (1H, s), 10.73 (1H, s).
13C NMR (DMSO-d6): δ 48.47, 104.94, 116.84. 118.16, 122.27,

1498 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
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