Atropisomeric 3-(β-hydroxyethyl)-4-arylquinolin-2-ones as maxi-K potassium channel openers

Article abstract of DOI:10.1021/jm061093j

The synthesis of a series of 3-β-hydroxyethyl-4-arylquinolin-2-ones is described. These compounds contain hydrophilic and hydrophobic substituents ortho to the phenolic OH in the C ring of the quinolinone. Electrophysiological evaluation of the panel of c

Full text of DOI:10.1021/jm061093j

1050
J. Med. Chem. 2007, 50, 1050-1057
Atropisomeric 3-(â-hydroxyethyl)-4-arylquinolin-2-ones as Maxi-K Potassium Channel Openers
Vivekananda M. Vrudhula,* Bireshwar Dasgupta, Jingfang Qian-Cutrone, Edward S. Kozlowski, Christopher G. Boissard,
Steven I. Dworetzky, Dedong Wu,† Qi Gao, Roy Kimura, Valentin K. Gribkoff,‡ and John E. Starrett, Jr.
Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, Connecticut 06492
ReceiVed September 18, 2006
The synthesis of a series of 3-â-hydroxyethyl-4-arylquinolin-2-ones is described. These compounds contain
hydrophilic and hydrophobic substituents ortho to the phenolic OH in the C ring of the quinolinone.
Electrophysiological evaluation of the panel of compounds revealed that 11 and 16 with an unbranched
ortho substituent retain activity as maxi-K ion channel openers. Members of this series of compounds can
exist as stable atropisomers. Calculated estimates of the energy barrier for rotation around the aryl-aryl
single bond in 3 is 31 kcal/mol. The atropisomers of (()-3, (()-4, and (()-11 were separated by chiral
HPLC and tested for their effect on maxi-K mediated outward current in hSlo injected X. laeVis oocytes.
The (-) isomer in each case was found to be more active than the corresponding (+) isomer, suggesting
that the ion channel exhibits stereoselective activation. X-ray crystallographic structures of (+)-3 and (+)-
11 were determined. Evaluation of the stability of (-)-3 at 80 °C in n-butanol indicated a 19.6% conversion
to (+)-3 over 72 h. In human serum at 37 °C (-)-3 did not racemize over the course of the 30 h study.
Introduction
incontinence (UI), and irritable bowel syndrome (IBS).3 Previ-
ously it was shown that the fluoroxindole class of compound
BMS-204352 (1, Chart 1) was neuroprotective and demonstrated
The transmembrane flux of inorganic ions such as potassium
(K+), sodium (Na+), and chloride (Cl-) across cell membranes,
and the resulting currents this produces, are critical for many
homeostatic cellular processes as well as specialized functions
such as muscle contraction, synaptic transmission, and secretion
of hormones. A plethora of transmembrane proteins mediate
the rapid transport of inorganic ions across cell membranes by
functioning as ion channels. Among these the K+ channels are
ubiquitously expressed in mammalian cells and represent the
largest and the most diverse group of ion channels from a
molecular perspective.1 These channels play a significant role
in the regulation of membrane potential, and in excitable cells
they regulate the frequency and form of the action potential,
the release of neurotransmitters, and contractility.2 The K+
channel superfamily is divided into a number of subfamilies
based on molecular structure and, to a degree, function. An
important subfamily is a group of K+ channels (KCa+) regulated
by the intracellular concentration of the divalent calcium ion
(Ca ++). On the basis of single-channel conductance, the Kca
channels are again subdivided into three types, BK (maxi-K),
IK, and SK channels referring respectively to big (100-300
picosiemens, (pS)), intermediate (25-100 pS), and small
conductance (2-25 pS) KCa channels. These channels vary in
pharmacology, distribution, function, and also in terms of
sensitivity to voltage and Ca2+ concentration. The BK channels,
the target of the molecules to be discussed in this article, are
regulated both by intracellular Ca2+ and voltage. Due to their
large single-channel conductance and their localization, they play
a key role in regulating voltage-dependent Ca2+ entry in
neuronal synapses and smooth muscle cells.1-3
efficacy in rodent models of acute focal stroke and TBI.4,5
A
pyrrole derivative NS-8 (2, Chart 1) which was also a maxi-K
channel opener was found to be effective in ViVo for the
treatment of UI in rodent models.6 Intracavernous injection of
DNA encoding the human BK channel (hSlo) to rats also
demonstrated that it was possible to alter nerve-stimulated penile
erection in this species. Effects lasting up to 2 months
postinjection were observed in this study, implicating utility for
maxi-K channel openers in male erectile dysfunction.7 Blockers/
inhibitors of maxi-K channel activity also have been described
and have potential utility in several therapeutic areas.3,8
Screening of several chemotypes in our laboratories has led
to the identification of 3-substituted-4-arylquinolin-2-ones as a
class of maxi-K channel openers.9 Among the number of
qunolinones tested, 3 turned out to be a potent maxi-K channel
opener (Chart 1). When examined in rats, 3 increased intrac-
avernous pressure, demonstrating its possible utility for the
treatment of male erectile dysfunction.10 Recently quinolinone
3 was also shown to decrease stress-induced colonic motility
and visceral nociception in rats, indicating potential utility in
mitigating irritable bowel syndrome.11 In an effort to further
identify novel maxi-K channel openers belonging to this class,
modifications done on ring B at positions 1 and 3 also led to
several derivatives with significant maxi-K channel opening
ability.12,13 Both 3 and its N-methyl derivative 4 turned out to
be potent leads with significant maxi-K channel opening
ability.9,10,12,13 With this series of compounds, the presence of
an underivatized phenolic hydroxyl group on ring C was also
identified as a general requirement for activity. In order to further
understand the maxi-K channel opening ability of this class of
compounds, modifications in the vicinity of the phenolic
hydroxyl group in the C ring were undertaken. A series of N1-
methylated analogues of 3 with hydrophilic and hydrophobic
substituents ortho to the phenolic OH were prepared and
evaluated for their activity as maxi-K channel openers. In this
article we describe the chemistry related to the modification of
the C ring in 3, and identification of stable atropisomeric
Activation of BK channels with small molecules can present
a number of opportunities for therapeutic intervention for
conditions such as stroke, traumatic brain injury (TBI), urinary
* Author to whom correspondence should be addressed. E-mail:
vivekananda.vrudhula@bms.com. Tel. 203-677-6697, Fax. 203-677-7702.
† Present address: Preformulation and Biopharmaceutics, AstraZeneca
Pharmaceuticals LP, 1800 Concord Pike, Wilmington, DE 19850.
‡ Present address: Scion Pharmaceuticals Inc., 200 Boston Ave., Suite
3600, Medford, MA 02155.
10.1021/jm061093j CCC: $37.00 © 2007 American Chemical Society
Published on Web 02/03/2007

Maxi-K Potassium Channel Openers
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 1051
Chart 1. Maxi-K Channel Openers
techniques for the preparation of oocytes expressing this
construct have been described in detail.18 The percent increase
in maxi-K channel-mediated outward current, a measure of the
comparative ability of these compounds to increase the open
probability of maxi-K channels, is shown in Table 1, as percent
iberiotoxin (IbTX)-sensitive current from the drug-free control.
Compounds displaying a value g130% for the increase in
outward control current at a maximum test concentration of 20
µM were considered significant openers of maxi-K channel. It
was found that compounds 11-16 were all less active than 3
or 4, indicating a lack of tolerance for steric bulk ortho to the
OH of the phenol. Olefin 11 and alcohol 16 with short
unbranched substituents displayed maxi-K channel opening
ability, yet their activity was less than that of 3 or 4 (Table 1).
However, compounds 12, 14, and 15 with branched substitutents
were inactive. When diol 15 showed poor activity as a maxi-K
channel opener, it was decided to separate and test the individual
isomers of 15. Chiral HPLC analysis (Figure 1) of 15, however,
led to the observation of four different isomers, suggesting that
the second element of asymmetry in 15 must be due to
atropisomerism caused by restricted rotation around the single
bond connecting rings B and C. Semipreparative HPLC separa-
tion of 15 on a chiral column led to the isolation of two of the
four diastereomers as pure compounds (isomers A and D, peaks
1 and 4, respectively) and another fraction containing a mixture
of two diastereomers (isomers B + C, peak 2 + peak 3). The
individual diastereomers as well as the mixture were found to
be inactive as maxi-K channel openers (Table 2). The next
attempt was to see if differential activity as maxi-K channel
openers could be established with a few examples of compounds
in this series. For this purpose, the marginally active olefin 11
and the more potent analogues 3 and 4 were chosen as
representative examples. Resolution of the individual atropiso-
mers of 11 was performed by a semipreparative chiral HPLC
method. The two atropisomers of 11 exhibited opposite rotation
and did not interconvert in ethanol even after 18 h at 40 °C.
Demonstration of differential biological activity by atropisomers
is known, for example, in the case of AMPA receptor
antagonists.19 Therefore, the two atropisomers of 11 were tested
as maxi-K channel openers. It was found that the maxi-K activity
resided with (-)-11, thus starting to demonstrate stereoselective
activation of the maxi-K channel.
quinolinone derivatives exhibiting differential activity as maxi-K
channel openers.
Chemistry. Scheme 1 illustrates the chemistry related to the
synthesis of the analogues for biological evaluation. Compounds
3 and 4 were prepared by methods previously described.9 The
primary hydroxyl group in 3 was protected as a TBDPS ether,
and the phenol was acetylated to give the differentially protected
silyl intermediate 5. Methylation of 5 gave 6, which upon
alkaline hydrolysis followed by allylation or methallylation, gave
the necessary ethers 7 or 8, respectively.
Several solvents were investigated for the Claisen rearrange-
ment of 7 to give 9. Refluxing dichlorobenzene14 was found to
be the best solvent, providing an 82% yield of the required
rearrangement product. These conditions were extended for the
conversion of 8 to 10. Fluoride-mediated desilylation of 9 and
10 provided the required olefinic analogues 11 and 12,
respectively. With this class of compounds, reduction of the
isolated double bond with Pt catalyst or Pd catalyst in MeOH
generally resulted in loss of Cl on the aromatic C ring. Sulfide-
poisoned Pt15 catalyst prevented the loss of Cl and gave the
required propyl and isobutyl analogues 13 and 14. Cis-
dihydroxylation using catalytic osmylation and hydroboration/
oxidation methodologies were employed in order to prepare the
more hydrophilic derivatives 15 and 16, respectively from the
olefin 9. Thus use of N-methylmorpholine N-oxide,16 along with
catalytic OsO4, followed by desilylation provided the necessary
diol 15 in 65% yield. Alcohol 16 was obtained in 80% yield
from 9 by hydroboration with borane-THF complex followed
by oxidation using H2O2. During this process, desilylation of
the silyl ether also occurred.
Results and Discussion
Given the observation of stable atropisomerism with (()-
11, we sought to investigate the propensity of the less sterically
encumbered analogues 3 and 4 to demonstrate similar effects.
In order to address the question of whether the atropisomers of
the more potent quinolinones 3 and 4 would exhibit larger
differential activity as maxi-K channel openers, chiral HPLC
resolution of 3 and 4 was accomplished on a semipreparative
scale. The individual atropisomers of 3 and 4 were tested as
maxi-K channel openers. Atropisomers (-)-3 and (-)-4 dem-
onstrated significantly higher activity than (+)-3 and (+)-4 as
openers of maxi-K channel respectively (Table 2). Thus, the
maxi-K channel does display stereoselective activation by one
atropisomer over the other. An enantiomeric 3-hydroxyoxindole
containing a chiral carbon exhibiting differential maxi-K channel
opening activity was described before.20 However, 3 and 4 are
the first examples of atropisomers displaying differential activity
as maxi-K openers.
At the time of initiation of this study with the 4-arylquino-
linones, it was felt that the steric bulk of small phenolic OH
and side chain â-hydroxyethyl at position 3 in quinolinone 1
would be insufficient to impose a strong rotational barrier
between the phenolic and quinolinone rings. In biphenyl
systems, the capacity of the ortho substituents to interfere with
passage through the planar transition state has been reported to
follow the order Br.CH3 > Cl > NO2 > CO2H . OCH3 >
F.17 It was also surmised by us that atropisomerism may not be
of any significant consequence especially under physiological
conditions. The phenols in this class of compounds are generally
important for their ability to function as openers of maxi-K
channels. Furthermore, in the absence of an X-ray crystal
structure of the mammalian maxi-K channel, a viable approach
of introduction of hydrophilic and hydrophobic substituents in
the vicinity of the phenolic OH may lead to further understand-
ing and profiling of this chemotype. Thus compounds 11-16
(Scheme 1) bearing substituents containing hydrophobic and
hydrophilic groups proximate to the phenolic OH of the C ring
in 4 were prepared and tested. This panel of compounds were
then evaluated for their effects on maxi-K channel-mediated
outward currents in hSlo- injected X. laeVis oocytes. The
Crystallization of (+)-3 from ethanol water afforded crystals
suitable for X-ray crystallographic analysis. The faster moving
isomer (+)-11 from chiral column chromatography (Figure 1b)
was also crystallized from ethyl acetate-hexane. X-ray crystal
structures of (+)-3 and (+)-11 were determined (Figure 2a and

1052 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
Vrudhula et al.
Scheme 1a
a Conditions: (a) TBDPSCl, imidazole, DMF; (b) Ac2O, Et3N, CH2Cl2; (c) MeI, acetone (d) LiOH·H2O, EtOH; (e) CH2dC(R)CH2Br, acetone, K2CO3;
(f) dichlorobenzene, reflux; (g) TBAF, THF; (h)Pt (sulfided)-C/H2, MeOH; (i) OsO4 (cat), NMMO, acetone, t-BuOH (j) BH3, THF 45 min, then H2O2,
MeOH.
Table 1. Effects of Target Quinolinone Derivatives on hSlo Maxi-K
ing with the side chain OH of a second molecule. The hydroxyl
group of the chlorophenol displayed a capability to participate
Currents Expressed in Xenopus Oocytesa
% IbTX-sensitive
current
concentration tested
in both intramolecular and intermolecular hydrogen bonding
with the side chain hydroxyl group. Comparison of the torsion
angles in (+)-11 and (+)-3 suggested that the side chain in ring
C of (+)-11 may have influenced the torsion angle. Attempts
to obtain single crystals of either (+)-4 or (-)-4 using a number
of solvents for crystallization were unsuccessful.
compd
[µM]
(()-3
(()-4
(()-11
(()-12
(()-13
(()-14
15c
253 ( 8.2
296 ( 13
140 ( 2.8
114 ( 8.7
127 ( 5.7
96.5 ( 7.0
112 ( 3.8
164 ( 6.4
20
20
20
10
10
5b
To understand the rotational barrier of atropisomers, it is
crucial to know how stable they are to elevation of temperature.
From the perspective of developing a therapeutic agent, it is
also important to understand the propensity to racemize under
physiological conditions, such as racemization in human plasma
at 37 °C. In the case of endothelin A antagonists, it is known
that serum proteins can influence the interconversion of atro-
pisomers.21 Computation of estimates of energy barrier for
rotation around the single bond joining rings B and C with 3
showed that the energy barrier was approximately 31 kcal/mol.
Generally, it requires a barrier of about 16-20 kcal/mol between
interconvertible isomers to permit their separation at room
temperature.22 At ambient temperature (∼20 °C) the calculated
energy barrier is high enough to preclude racemization of a
single atropisomer of 3. Studies were therefore undertaken to
investigate the thermal stability as well as the stability in human
serum at 37 °C with (-)-3 as an example.23 A supercritical fluid
chromatography method on a chiral column was developed for
studying the racemization of (-)-3. In n-butanol at 80 °C, 19.6%
of (-)-3 was converted to the opposite enantiomer after 72 h
(Figure 3). A stability study in human serum with (-)-3 at
37 °C showed that the compound was stable up to at least 30
20
20
(()-16
a Results are expressed as % IbTX-sensitive current (drug-free control
) 100%). Values obtained were the averages of at least four determinations.
b Solubility limitations prevented testing at higher concentrations. c Tested
as a diastereomerc mixture of four compounds.
Figure 2b respectively; data included in Supporting Information).
X-ray crystallographic analysis of orthorhombic crystal of (+)-3
as a hemi EtOH solvate displayed interesting characteristics
when compared with solvent free monoclinic crystals of (+)-
11. In (+)-11, the amide nitrogen was methylated, precluding
the possibility of hydrogen bonding, whereas in (+)-3, the NH
of the quinolinone clearly showed intermolecular hydrogen
bonding with the carbonyl oxygen of a second conformer.
Moreover, the chlorophenol ring C in (+)-3 was almost
perpendicular to ring B with a torsion angle of 91.3° and 87.1°,
respectively, for the two conformers in the crystal (Figure 2a).
The X-ray crystallographic analysis of (+)-11 revealed two
crystallogaphically independent conformers with torsion angles
of 78.2° and 75.9°, respectively for the two planes involving
rings B and C (Figure 2b). The carbonyl group of the
quinolinone participated only in intermolecular hydrogen bond-

Maxi-K Potassium Channel Openers
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 1053
Figure 1. Chiral analytical HPLC separation of atropisomers of (a) compound 15, (b) compound 11.
Table 2. Differential Effects of Atropoismeric Quinolinone Derivatives
energy barrier for interconversion revealed estimates of a high-
on hSlo Maxi-K Mediated Outward Current Expressed in Xenopus
energy barrier of 31 kcal/mol. The individual atropisomers of
3 were highly stable at physiologically relevant conditions and
exhibited differential activity as maxi-K channel openers,
suggesting that the maxi-K channel exhibits stereoselective
activation. This study with a quinolinone series emphasizes the
relevance of atropisomerism in the context of drug development.
Whenever atropisomerism may be involved, it should be
thoroughly investigated, and the distinct possibility of dealing
with the development of a chiral drug should be taken into
consideration.
Oocytesa
compd
% IbTX-sensitive
current
concentration tested [µM]
(()-3
(+)-3
259 ( 16
214 ( 21
292 ( 27
257 ( 16
184 ( 9.5
361 ( 30
140 ( 2.8
113 ( 3.6
135 ( 7.9
101( 3.4
100 ( 6.8
94.4 ( 5.6
10
10
10
10
10
10
20
20
20
20
20
20
(-)-3
(()-4
(+)-4
(-)-4
(()-11
(+)-11
(-)-11
15 isomer A
15 isomers B + C
15 isomer D
Experimental Section
h, which was the duration of the experiment. Under these
conditions no detectable racemization occurred.
General. Commercially available solvents and reagents of highest
available purity were used. Pooled human serum (catalog # BRH
72581) was purchased from Bioreclamation Inc. 1H and13 C NMR
spectra were run on a Bruker AC-300 or AC-500 instruments and
chemical shifts are reported in ppm (δ) with reference to (CH3)4-
Si. All evaporations were carried out under reduced pressure. LC-
MS analyses were carried out on a Shimadzu instrument on a YMC
Conclusion
In summary, 3-substituted-4-arylquinolin-2-ones with modi-
fications in ring C were prepared as maxi-K channel openers,
and their atropisomerism was investigated. Computation of the

1054 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
Vrudhula et al.
2-(3-(2-(tert-Butyldiphenylsilyloxy)ethyl)-1-methyl-2-oxo-6-
(trifluoromethyl)-1,2-dihydroquinolin-4-yl)-4-chlorophenyl Ac-
etate (6). A suspension of 5 (6.82 g, 10.3 mmol), methyl iodide
(4.37 g, 30.8 mmol), and K2CO3 (3.05 g, 30.8 mmol) in acetone
(200 mL) at ambient temperature was stirred for 18 h, after which,
the reaction mixture was diluted with ethyl acetate (200 mL),
washed with water (100 mL) and brine (100 mL), and dried
(MgSO4). After evaporation of the volatiles, purification of the
residue by chromatography (SiO2) with a step gradient elution using
hexane:ethyl acetate (9:1 to 7:3) afforded 6 as the major product
(5.52 g, 79%, LC-MS tR ) 2.3 min) as a white solid. MS [M + H]
1
) 678. H NMR (CD3OD) δ 7.82 (d, J ) 8.1 Hz, 1H), 7.73 (d, J
) 8.9 Hz, 1H), 7.52 (m, 1H), 7.48 (m, 4H), 7.34-7.21 (m, 9H),
3.91 (m, 1H), 3.76 (m, 4H), 2.88 (m, 1H), 2.70 (m, 1H), 1.71 (s,
3H), 0.94 (s, 9H).
4-(2-(Allyloxy)-5-chlorophenyl)-3-(2-(tert-butyldiphenylsilyl-
oxy)ethyl)-1-methyl-6-(trifluoromethyl)quinolin-2(1H)-one (7).
A solution of 6 (5.5 g, 8.1 mmol) in ethanol (80 mL) was stirred
at room temperature with LiOH·H2O (1.37 g, 32.5 mmol) for 4 h.
The volatiles were evaporated, and the residue was taken up in
ethyl acetate (300 mL) and washed with water (100 mL) and brine
(100 mL). Organic layer after drying (MgSO4) was evaporated. A
suspension of the residue in acetone (100 mL), allyl bromide (0.062
g, 0.51 mmol), and potassium carbonate (0.063 g, 0.64 mmol) was
stirred for 18 h at room temperature. The reaction mixture was
diluted with ethyl acetate (200 mL) and washed with water (100
mL), and brine (100 mL), dried (MgSO4), and evaporated. Purifica-
tion by column chromatography (SiO2) using a step gradient of
hexane:ethyl acetate (19:1 to 17:3) as eluant, afforded 7 (0.24 g,
85%, LC-MS tR ) 2.4 min) as a white solid. MS [M + H] ) 676.
1H NMR (CD3OD) δ 7.81 (d, J ) 8.9 Hz, 1H), 7.72 (d, J ) 8.9
Hz, 1H), 7.37 - 7.34 (m, 5H), 7.29 - 7.05 (m, 9H), 5.54 (m, 1H),
4.88 (m, 2H), 4.23 (m, 2H), 3.89 (m, 1H), 3.86 (s, 3H), 3.64 (m,
1H), 2.86 (t, J ) 7.2 Hz, 2H), 0.94 (s, 9H).
Figure 2. ORTEP drawings of (a) (+)-3 and (b) (+)-11 with thermal
ellipsoids at 30% probability for non-H atoms and open circles for
H-atoms. Carbon and hydrogen atoms are not labeled. See Supporting
Information for detailed interactions.
C18 column (4.6 × 50 mm) employing a 4 or 8 min linear gradient
of 0% to 100% solvent B in A (solvent A: 10% methanol, 90%
water, 0.1% TFA; solvent B: 90% methanol, 10% water, 0.1%
TFA) with UV detector set at 220 nm. A flow rate of 5 mL/min
was used. Preparative HPLC separations were carried out on a
Shimadzu instrument with the UV detector set at 220 nm on a 5
µm X-terra 30 × 100 reversed phase column. All runs employed
a linear gradient of B in A with a hold time of 2-5 min at the end.
A flow rate of 30 mL/min was used. Sovents A and B mentioned
above were used for elution. Chiral HPLC analyses were run on a
Shimadzu instrument equipped with photodiode array detection
(UVmax ) 234 nm) on a ChiralPak AD, 4.6 × 250 mm, 10 µm
particle size column. Mobile phase consisted of i-PrOH as the polar
component and hexane as the less polar component. Supercritical
fluid chromatographic (SFC) analyses at 35 °C were performed on
a Berger SFC instrument using a 4.6 × 250 mm chiralcel OJ-H 5
µm column at 150 bar with the UV detector set at 215 nm. An
isocratic elution using CO2:EtOH (9:1) at a flow rate of 2 mL/min
was employed for the analyses. Electrophysiological data reported
were the average of at least four determinations.
2-(3-(2-(tert-Butyldiphenylsilyloxy)ethyl)-2-oxo-6-(trifluorom-
ethyl)-1,2-dihydroquinolin-4-yl)-4-chlorophenyl Acetate (5). To
a solution of 3 (5.00 g, 13.0 mmol) in dry DMF (100 mL) were
added TBDPSCl (10.74 g, 39.1 mmol) and imidazole (2.66 g, 39.1
mmol) at ambient temperature under argon. The reaction mixture
was stirred for 3 h and diluted with ethyl acetate (300 mL). The
organic layer was washed with water (2 × 100 mL) and brine (100
mL) and dried (MgSO4). The solvent was evaporated to obtain the
oily silyl ether (6.61 g, 10.63 mmol, 81%). To the solution of the
silyl ether in dry dichloromethane (200 mL) were added Ac2O (5.43
g, 53.2 mmol) and Et3N (5.38 g, 53.2 mmol) at room temperature
under argon. The reaction mixture was stirred for 4 h, and the
mixture was diluted with ethyl acetate (200 mL). It was then washed
with water (2 × 100 mL) and brine (100 mL) and dried (MgSO4).
The solvent was evaporated, and the residue was purified by column
chromatography (SiO2) using hexane:ethyl acetate (19:1 to 4:1) as
eluant to give 5 (6.82 g, 96%, LC-MS tR ) 2.3 min) as a white
solid. MS [M + H] ) 664. 1H NMR (CD3OD) δ 7.74 (d, J ) 7.6
Hz, 1H), 7.60 (m, 1H), 7.52 (m, 5H), 7.37 (m, 2H), 7.31 (m, 5H),
7.23 (s, 1H), 7.17 (s, 1H), 3.94 (m, 1H), 3.80 (m, 1H), 2.89 (m,
1H), 2.72 (m, 1H), 1.76 (s, 3H), 0.97 (s, 9H).
3-(2-(tert-Butyldiphenylsilyloxy)ethyl)-4-(5-chloro-2-(2-methy-
lallyloxy)phenyl)-1-methyl-6-(trifluoromethyl)quinolin-2(1H)-
one (8). This compound was synthesized on a 0.75 mmol scale
from 6 in a similar manner as described in the synthesis of 7 using
methallyl bromide (1.2 mmol) as the alkylating agent. Purification
by column chromatography (SiO2) using hexane and ethyl acetate
(19:1 to 17:3) as eluant, afforded product 8 (0.31 g, 76%, LC-MS
1
tR ) 2.4 min) as a white solid. MS [M + H] ) 690. H NMR
(CD3OD) δ 7.78 (d, J ) 8.5 Hz, 1H), 7.71 (d, J ) 8.9 Hz, 1H),
7.49 (m, 5H), 7.35 (m, 2H), 7.29 (m, 4H), 7.18 (s, 1H), 7.11 (s,
1H), 7.04 (d, J ) 8.9 Hz, 1H), 4.63 (s, 1H), 4.57 (s, 1H), 4.11 (m,
2H), 3.86 (m, 1H), 3.77 (s, 3H), 3.65 (m, 1H), 2.82 (m, 2H), 1.30
(s, 3H), 0.93 (s, 9H).
4-(3-Allyl-5-chloro-2-hydroxyphenyl)-3-(2-(tert-butyldiphenyl-
silyloxy)ethyl)-1-methyl-6-(trifluoromethyl)quinolin-2(1H)-
one (9). A solution of 7 (0.24 g, 0.36 mmol) in 1,2-dichlorobenzene
was refluxed for 72 h, followed by evaporation of the volatiles.
Purification of the residue by column chromatography (SiO2) using
hexane:ethyl acetate (19:1 to 17:3) as eluant afforded 9 (0.20 g,
82%, LC-MS tR ) 2.4 min) as a white solid. MS [M + H] ) 676.
1H NMR (CD3OD) δ 7.81 (d, J ) 8.9 Hz, 1H), 7.74 (d, J ) 8.9
Hz, 1H), 7.52-7.50 (m, 5H), 7.36-7.24 (m, 8H), 6.01 (m, 1H),
5.09 (m, 2H), 3.86 (m, 1H), 3.79 (s, 3H), 3.72 (m, 1H), 2.76 (m,
1H), 0.94 (s, 9H).
3-(2-(tert-Butyldiphenylsilyloxy)ethyl)-4-(5-chloro-2-hydroxy-
3-(2-methylallyl)phenyl)-1-methyl-6-(trifluoromethyl)quinolin-
2(1H)-one (10). Claisen rearrangement of 8 on a 0.45 mmol scale,
including purification was carried out as described for the synthesis
of 9. After purification, 10 (0.22 g, 69%, LC-MS tR ) 2.41 min)
1
was obtained as a white solid. MS [M + H] ) 690. H NMR
(CDCl3) δ 7.73 (d, J ) 8.8 Hz, 1H), 7.54 - 7.18 (3 × m, 13H),
6.92 (d, J ) 2.5 Hz, 1H), 4.89 (s, 1H), 4.76 (s, 1H), 4.06 (m, 1H),
3.82 (m, 1H), 3.74 (s, 3H), 3.34 (s, 2H), 2.85 (m, 2H), 1.73 (s,
3H), 0.98 (s, 9H).
4-(3-Allyl-5-chloro-2-hydroxyphenyl)-3-(2-hydroxyethyl)-1-
methyl-6-(trifluoromethyl)quinolin-2(1H)-one (11). Silyl ether 9
(72 mg, 0.11 mmol) was treated with 1 M TBAF (1 mL, 10 equiv)

Maxi-K Potassium Channel Openers
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5 1055
Figure 3. Stability evaluation of (-)-3 at 80 °C in n-butanol by the SFC method.
in THF. The amber colored solution was stored at ambient
temperature overnight. The residue obtained after evaporating the
solvent was doissolved in DMF (1 mL) and purified by preparative
HPLC with 40-100% B in A (see General section) as eluant in a
20 min run. The major fraction thus obtained was evaporated, and
the residue was purified again by silica gel chromatography with
EtOAc:CH2Cl2 (1:1) as eluant to remove contaminating tetrabuty-
lammonium salt impurities. Fractions containing pure desilylated
olefin were combined to give 11 (38 mg, 81%, LC-MS tR ) 1.9
min). MS [M + H] ) 438. 1H NMR (CDCl3) δ 7.82 (dd, 1H, J )
8.9 and 1.9 Hz), 7.75 (d, 1H, J ) 8.9 Hz), 7.28 - 7.21 (m, 2H),
6.89 (d, 1H, J ) 2.6 Hz), 6.05 (m, 1H), 5.16-5.09 (m, 2H), 3.86
- 3.79 (m, 4H), 3.72-3.65 (m, 2H), 3.45 (m, 2H), 2.76-2.72 (m,
2H). 13C NMR (CDCl3) 163.9, 151.9, 145.8, 142.5, 137.1, 132.2,
131.6, 128.7, 127.4 (m), 126.3, 125.9, 125.5 (m), 125.4 (m), 122.4,
116.8, 116.6, 61.0, 35.2, 34.0, 30.8. Anal. (C22H19ClF3NO3) C, H,
N, Cl.
4-(5-Chloro-2-hydroxy-3-(2-methylallyl)phenyl)-3-(2-hydrox-
yethyl)-1-methy l-6-(trifluoromethyl)quinolin-2(1H)-one (12).
Desilylation of 10 (0.10 g, 0.15 mmol) in anhydrous THF (20 mL)
was carried out with 1 M of TBAF in THF (0.5 mL, 0.44 mmol)
at ambient temperature under argon as described above for 11.
Preparative HPLC using 40-100% B in A in a 20 min run followed
by evaporation of solvent from the fraction afforded product 12
(0.04 g, 61%, LC-MS tR ) 1.9 min) as a white solid. MS [M + H]
) 452. 1H NMR (CDCl3) δ 7.74 (d, J ) 9.4 Hz, 1H), 7.49 (d, J )
8.9 Hz, 1H), 7.29 (s, 1H), 7.22 (s, 1H), 6.91 (s, 1H), 4.89 (s, 1H),
4.75 (s, 1H), 3.94 (m, 1H), 3.80 (m, 1H), 3.78 (s, 3H), 3.45 (d, J
) 15.4 Hz, 1H), 3.38 (d, J ) 15.4 Hz, 1H), 2.86 (m, 1H), 2.66 (m,
1H), 1.75 (s, 3H). 13C NMR (CDCl3) 162.7, 150.4, 144.4, 143.9,
140.7, 131.4, 130.3, 127.4, 126.6 (m), 126.2, 125.1, 124.8 (m), 124.7
(m), 120.8, 114.8, 112.5, 61.0, 38.9, 32.4, 30.4, 25.9, 22.1, 19.5.
Anal. (C23H21ClF3NO3. O·1H2O) C, H, N, Cl.
4-(5-Chloro-2-hydroxy-3-isobutylphenyl)-3-(2-hydroxyethyl)-
1-methyl-6-(trifluoromethyl)quinolin-2(1H)-one (14). Reduction
of 10 (0.11 g, 0.16 mmol) in EtOH (20 mL) was carried out as
described above using sulfided Pt (0.08 g) for 4 h. The crude product
(0.10 g, 0.15 mmol) was desilylated with TBAF (1 M in THF, 0.5
mL, 0.44 mmol) as described in the synthesis of 11. The crude
product was purified by preparative HPLC using 30-100% B in
A in a 20 min run to afford product 14 (0.06 g, 89%, LC-MS tR )
2.0 min) as a white solid. MS [M + H] ) 454. 1H NMR (CDCl3)
δ 7.80 - 7.74 (m, 1H), 7.52 - 7.49 (m, 1H), 7.26 - 7.23 (m, 2H),
6.84 (d, J ) 2.6 Hz, 1H), 4.60 - 4.45 (m, 1H), 4.05 - 3.99 (m,
1H), 3.78 (s, 3H), 2.99 - 2.85 (m, 1H), 2.69 - 2.42 (m, 3H), 1.99
- 1.85 (m, 1H), 0.94 (s, 6H). 13C NMR (CDCl3) 162.7, 149.9,
144.7, 141.1, 133.2, 132.0, 131.6, 126.9, 126.8 (m), 126.5, 126.0,
124.9 (m), 124.8 (m), 120.9, 115.0, 114.9, 65.9, 60.8, 39.1, 32.1,
30.4, 29.2, 22.3. Anal. (C23H23ClF3NO3) C, H, N, Cl.
4-(5-Chloro-3-(2,3-dihydroxypropyl)-2-hydroxyphenyl)-3-(2-
hydroxyethyl)-1-methyl-6-(trifluoromethyl)quinolin-2(1H)-
one (15). To a solution of allyl derivative 9 (0.15 g; 0.22 mmol) in
acetone (9 mL) and water (3 mL) was added osmium tetroxide
(2.5 wt % in t-butanol; 0.3 mL) and 4-methylmorpholine N-oxide
(0.04 g; 0.40 mmol) under argon at ambient temperature. The
reaction mixture was stirred overnight, and excess osmium tetroxide
was destroyed by addition of sodium sulfite. Following dilution
with ethyl acetate (100 mL), washing with water and brine, and
drying (MgSO4), the EtOAc was evaporated and the crude product
was purified by preparative HPLC using 30-100% B in A in a 17
min run. Fractions containing the required silyl ether were combined
and evaporated in vacuo to provide a white solid (0.12 g, 77%). A
solution of the silyl ether in anhydrous THF (10 mL) was desilylated
as described above for the synthesis of 11. The crude product was
purified by reversed phase preparative HPLC as before to obtain
diol 15 (0.05 g, 65%, LC-MS tR ) 1.6 min) as a white solid. MS
1
[M + H] ) 472. H NMR (CD3OD) δ 7.84 (d, J ) 8.9 Hz, 1H),
4-(5-Chloro-2-hydroxy-3-propylphenyl)-3-(2-hydroxyethyl)-
1-methyl-6-(trifluoromethyl)quinolin-2(1H)-one (13). To a solu-
tion of olefinic silyl ether 9 (0.14 g, 0.20 mmol) in MeOH (30
mL) was added Pt (5%) on sulfided carbon. The mixture was stirred
under H2 at balloon pressure and ambient temperature for 18 h.
After filtration, MeOH was evaporated and the residue was
desilylated as described above for the preparation of 11. The crude
product was purified by silica gel column with EtOAc:CH2Cl2 (1:
1) as eluant to give 13 (0.06 g, 71%, LC- MS tR ) 1.9 min). MS
7.77 (d, J ) 9.0 Hz, 1H), 7.38 (d, J ) 2.6 Hz, 1H), 7.32 (m, 1H),
7.06 (m, 1H), 3.98 (m, 1H), 3.86 (s, 3H), 3.68 (m, 2H), 3.53 (m,
2H), 3.00 - 2.61 (m, 4H). Anal. (C22H21ClF3NO5) C, H, N, Cl.
4-(5-Chloro-2-hydroxy-3-(3-hydroxypropyl)phenyl)-3-(2-hy-
droxyethyl)-1-methyl-6-(trifluoromethyl)quinolin-2(1H)-one (16).
To a solution of olefin 9 (0.07 g, 0.10 mmol) in anhydrous THF
(20 mL) at 0 °C under argon was added borane-THF complex (2
mL, 1 M in THF). The reaction mixture was allowed to warm up
to ambient temperature and was stirred for 45 min. To the reaction
mixture was added hydrogen peroxide (30 wt % in H2O, 0.05 mmol)
and MeOH (2 mL). After 3 h, the reaction mixture was diluted
with ethyl acetate (200 mL). The organic layer was washed with
water and brine and dried (MgSO4). The silyl ether underwent
deprotection under the reaction conditions. Purification by reverse
phase preparative HPLC using the same conditions as in the case
1
[M + H] ) 440. H NMR (CD3OD) δ 7.81(dd, 1H, J ) 8.9 and
1.9 Hz), 7.75 (d, 1H, J ) 8.9 Hz), 7.26 (m, 2H), 6.96 (d, 1H, J )
2.6 Hz), 3.83 (s, 3H), 3.72-3.65 (m, 2H), 2.77-2.64 (m, 4H),
1.69-1.64 (m, 2H), 0.97 (t, 3H, J ) 7.4 Hz). 13C NMR (CD3OD)
164.0, 152.0, 146.0, 142.5, 134.6, 132.1, 131.7, 128.2, 127.4 (m),
126.1, 125.8, 125.5 (m), 125.4 (m), 122.4, 116.8, 61.0, 34.0, 33.3,
30.8, 24.1, 14.1. Anal. (C22H21ClF3NO3) C, H, N, Cl.

1056 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 5
Vrudhula et al.
of 15 afforded alcohol 16 (0.038 g, 80%, LC-MS tR ) 1.7 min) as
conformations of 3 were initially calculated using the Schrodinger
Ligprep utility and minimized for 500 steps using the Polak-Ribier
conjugate gradient method.25 Starting from this conformation, the
dihedral angle of interest was rotated from -180° to 180° in 5°
increments using the Macromodel Dihedral Drive utility. At each
of these angles, the structure was minimized in vacuum and the
total force-field self-energy of the minimized structure was recorded.
The barrier height was estimated as the difference in peak and
trough energies calculated during the dihedral rotation.
1
white solid. MS [M + H] ) 456. H NMR (CD3OD) δ 7.81 (d, J
) 8.8 Hz, 1H), 7.74 (d, J ) 8.9 Hz, 1H), 7.32 (s, 1H), 7.26 (s,
1H), 6.98 (d, J ) 2.4 Hz, 1H), 3.83 (s, 3H), 3.69 (m, 2H), 3.60 (t,
J ) 6.3 Hz, 2H), 2.82 - 2.69 (m, 4H), 1.87 (m, 2H). 13C
NMR (CD3OD) 163.9, 152.2, 146.0, 142.5, 134.0, 132.1, 132.0,
128.5, 127.4 (m), 126.2, 126.0, 125.6, 125.4 (m), 125.3 (m), 122.4,
116.8, 61.9, 61.1, 34.0, 33.5, 30.8, 27.6. Anal. (C22H21ClF3NO4)
C, H, N, Cl.
Electrophysiology Experiments.18 To evaluate the maxi-K
channel opening characteristics of the test compounds, experiments
were performed using standard two-electrode voltage clamp
techniques in Xenopus laeVis oocytes expressing the human maxi-K
hSlo channel. Oocytes were each injected with 50 ng of hSlo cRNA
and recorded from 2 to 5 days postinjection. From a holding
potential of -60 mV, outward potassium currents were evoked over
the voltage range of -40 to +140 mV using 20 mV depolarizing
steps. In all cases, a minimum of four to five different oocytes
were used to evaluate the effect of each compound at 5, 10, or 20
µM applied for 5 min. To determine the level of hSlo expression
and current in an oocyte, each experiment ended with a 10 min
application of the specific maxi-K channel blocker iberiotoxin
(IbTX; 100nM).26 Compound effects are presented in Tables 1 and
2 and are expressed as percent IbTX-sensitive currents relative to
compound-free control values measured at the peak effect.
Chiral Separation of 11. A solution of 11 (∼50 mg) in i-PrOH:
hexane (1:1, 2 mL) was applied in four injections on to a ChiralPak
AD, 21 × 250 mm, 10 µm particle size column. Elution with iPrOH:
hexane (1:19) was carried out for 50 min at a flow rate of 10 mL/
min. A detector with UVmax at 234 nm was employed. Fractions
containing the faster moving isomer, upon evaporation, gave 20.6
mg, [R]22 (EtOH) ) +20.7°, while the evaporation of fractions
D
containing the later peak gave 19.9 mg of the other isomer [R]22
D
(EtOH) ) -18.6° (98.7% enantiomeric purity). The two isomers
had identical proton NMR and LC-MS characteristics. Crystalliza-
tion of (+)-11 from ethyl acetate gave crystals suitable for X-ray
crystallographic analysis.
Chiral Separation of 15. Diol 15 was separated on the same
column as above by isocratic elution with 93:7 solvents B:A (A )
i-PrOH, B ) 0.05% TFA in hexane) for 130 min. Optical rotational
characterization of the diastereomers: Isomer A (peak 1), [R]22
D
(MeOH) ) +3.5°; Isomer B + C (peaks 2 + 3), [R]22 (MeOH)
) +2.7°; and Isomer D (peak 4), [R]22 (MeOH) ) -D5.3°.
D
Acknowledgment. The authors would like to thank Dr.
Stella Huang for conducting the NMR experiments in chiral
solvents.
Chiral Separation of 3. The conditions used were essentially
the same as those for 11 except that a longer run time (60 min)
was used. Characteristics of isomer eluting as first peak tR ) 46.1
min; NMR characteristics are the same as that of the racemate;
Supporting Information Available: Microanalyses data for
compounds 11-16. X-ray data information for (+)-3 and (+)-11.
Supporting information is available free of charge via the Internet
at http://pubs.acs.org.
[R]22 (MeOH) ) -8.8°. The later peak (tR ) 48.3 min) had the
D
same NMR spectral characteristics as the earlier peak with [R]22
D
(MeOH) ) +9.6°. Crystallization of (+)-3 from EtOH-H2O gave
single crystals suitable for X-ray crystallographic analysis.
Chiral Separation of 4. The racemate was separated as described
above for 15 except that a ratio of B:A ) 4:1 was used in a 25 min
isocratic run. Characteristics of the isomer eluting as first peak tR
) 8.5 min; NMR characteristics are the same as that of the
racemate;9 [R]22D (MeOH) ) -6.9°. The later peak (tR ) 19.0 min)
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had the same NMR spectral characteristics8 with [R]22 (MeOH)
D
) + 5.0°.
X-ray Crystallographic Data. Data obtained for (+)-3 and (+)-
11 are included in Supporting Information. X-ray crystallographic
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Stability of (-)-3 at 80 °C. A 1 mM solution of (-)-3 in
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Maxi-K Potassium Channel Openers
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JM061093J