4-Aryl-3-(hydroxyalkyl)quinolin-2-ones: Novel maxi-K channel opening relaxants of corporal smooth muscle targeted for erectile dysfunction

Article abstract of DOI:10.1021/jm030005h

Novel 4-aryl-3-(hydroxyalkyl)quinoline-2-one derivatives were prepared and evaluated as openers of the cloned maxi-K channel hSlo expressed in Xenopus laevis oocytes by utilizing electrophysiological methods. The effect of these maxi-K openers on corporal

Full text of DOI:10.1021/jm030005h

J . Med. Chem. 2003, 46, 2819-2822
2819
Sch em e 1a
4-Ar yl-3-(h yd r oxya lk yl)qu in olin -2-on es:
Novel Ma xi-K Ch a n n el Op en in g
Rela xa n ts of Cor p or a l Sm ooth Mu scle
Ta r geted for Er ectile Dysfu n ction
Piyasena Hewawasam,*,† Wenhong Fan,† Min Ding,†
Kim Flint,‡ Deborah Cook,‡ Gregory D. Goggins,‡
Robert A. Myers,‡ Valentin K. Gribkoff,‡
Christopher G. Boissard,‡ Steven I. Dworetzky,‡
J ohn E. Starrett, J r.,† and Nicholas J . Lodge‡
Departments of Chemistry and Neuroscience/ Genitourinary
Drug Discovery, The Bristol-Myers Squibb Pharmaceutical
Research Institute, 5 Research Parkway,
Wallingford, Connecticut 06492
Received J anuary 7, 2003
Abstr a ct: Novel 4-aryl-3-(hydroxyalkyl)quinoline-2-one de-
rivatives were prepared and evaluated as openers of the cloned
maxi-K channel hSlo expressed in Xenopus laevis oocytes by
utilizing electrophysiological methods. The effect of these
maxi-K openers on corporal smooth muscle was studied in vitro
using isolated rabbit corpus cavernosum. From this study, a
potent maxi-K opener was identified as an effective relaxant
of rabbit corporal smooth muscle and shown to be active in an
in vivo animal model of male erectile function.
In tr od u ction . Erectile dysfunction (ED) has been
recognized as a common condition that affects ap-
proximately 30 million men in the U.S. and over 100
million worldwide.1 ED is clinically defined as the
persistent inability to achieve and maintain an erection
adequate for satisfactory sexual activity.1 The degree
of dysfunction may range from moderate to complete
ED depending on the age group and other medical
conditions such as diabetes, hypertension, and heart
disease.2 In the past 2 decades, a variety of topical and
oral drug therapies have been developed to treat male
ED.3,4 Sildenafil (Viagra) is a phosphodiesterase type 5
(PDE5) inhibitor that is a widely used treatment for
male ED. However, there have been reports of cardio-
vascular and retinal side effects associated with this
drug.5,6 There continues to be a need for new and
improved agents for the treatment of male ED.
Ra tion a le a n d Ba ck gr ou n d . It is widely believed
that impaired relaxation of the corpus cavernosum
smooth muscle is the primary cause of penile ED in a
vast majority of impotent men.7-9 As a consequence,
precise modulation of cavernosal (corporal) smooth
muscle tone is central to the development of effective
and improved treatments for ED. Relaxation of corporal
smooth muscle is accomplished by lowering of cytosolic
calcium (Ca2+), which is thought to be mediated by
several mechanistic pathways.10 One of the mechanisms
involves hyperpolarization of corporal smooth muscle
cells via activation of potassium channels.10 It has been
demonstrated that corporal smooth muscle cells express
several different K+ channels of which maxi-K channels
were found to be the most prominent subtype.11,12 Thus,
a
t
(a) (1) BuLi (2.2 equiv), THF, -78 to 40 °C; (2) add 4 at -40
°C then warm to 0 °C, 82%; (b) 3 N HCl, EtOH, reflux, 100%; (c)
BBr3, DCM, -78 to 0 °C, 98%; (d) MeO2C(CH2)nCOCl, pyridine,
DCM, 0-23 °C, 82-95%; (e) KN(SiMe3)2, THF, 23 °C, 1-16 h, 90-
100%; (f) 35% HBr in HOAc, 78-82%; (g) pyridine‚HCl, 200-210
°C, 15-20 min, 80-86%; (h) Me2S‚BH3, THF, 0-23 °C, 58-67%;
(i) MnO2, DCM, 23 °C, 54%; (j) EtO2CCH2P(O)(OEt)2, NaH, DMF,
i
86%; (k) Bu2AlH, THF/hexanes, -78 to 23 °C, 87%.
activation of maxi-K channels present in corporal
smooth muscle represents an important and attractive
mechanism for controlling corporal smooth muscle
function.11-13 Activation of maxi-K channels would lead
to membrane hyperpolarization, thereby closing the
voltage-gated Ca2+ channels with a consequent lowering
in cytosolic Ca2+ and smooth muscle relaxation. A
variety of KATP channel openers such as cromakalim,
pinacidil, and nicorandil have been shown to relax
precontracted corporal muscle tissue isolated from both
animals14,15 and humans.16
As part of a broad-based effort directed toward the
identification of orally bioavailable activators of maxi-K
channels that would be useful in smooth muscle relax-
ation, we synthesized a series of 4-aryl-3-(hydroxyalkyl)-
quinolin-2-one derivatives as shown in Schemes 1 and
2. 4-Aryl-3-hydroxyquinolin-2-ones (1) have been dis-
closed as maxi-K channel openers with antibacterial
activity.17 More recently, 4-aryl-3-aminoquinolin-2-ones
* To whom correspondence should be addressed. Phone: (203) 677-
7815. Fax: (203) 677-7702. E-mail: hewawasp@bms.com.
† Department of Chemistry.
‡ Department of Neuroscience/Genitourinary Drug Discovery.
10.1021/jm030005h CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/30/2003

2820 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Letters
Ta ble 1. Physical Properties of Compounds 10a -c, 11a -c,
Sch em e 2a
13b, and 17b: Effect on Maxi-K-Mediated Outward Current in
Xenopus laevis Oocytes Expressing the Cloned Maxi-K
Channels hSlo and Relaxation of Isolated Rabbit Corpus
Cavernosum Precontracted with Phenylephrine
% increase in
hSlo current
@20 µM
% inhibition of
compd
10a (n ) 1)c 232-235
mpa (°C)
force @ 10 µMb
114 ( 3
NT
10b (n ) 2)
10c (n ) 3)
11a (n ) 1)
11b (n ) 2)
11c (n ) 3)
13b
219-221
200-202
>250 (dec)
255-256
257-259
266-268
203-206
211 ( 20
258 ( 26
198 ( 8
378 ( 35
292 ( 30
223 ( 9
257 ( 18
132 ( 13
26.8 ( 5.2 (N ) 2)
35.7 ( 2.8 (N ) 2)
36.0 ( 9.6 (N ) 3)
83.7 ( 11.0 (N )3)
43.0 (N ) 1)
31.8 ( 2.1 (N ) 3)
46.0 ( 11.2 (N ) 7)
31.0 ( 5.9 (N ) 5)
17b
NS-004
a
All new compounds exhibited spectroscopic and combustion
data in accord with the designated structure.19
Percentage
b
inhibition of isometric force in response to a test compound in
isolated rabbit corpus cavernosum precontracted with phenyl-
ephrine (3 µM). Vehicle (DMSO) produced a 12.5 ( 6.7% inhibition
of force. c This n refers to that in Scheme 1.
a
(a) MeO2CCH2COCl, pyridine, DCM, 0-23 °C; (b) KOtBu,
i
THF, reflux, 83% for two steps; (c) Bu2AlH, THF, -78 °C, 78%;
(d) EtO2CCH2P(O)(OEt)2, NaH, DMF, 72%; (e) iBu2AlH, THF/
hexanes, -78 to 23 °C, 77%.
major product, which was reduced with iBu2AlH to
afford the alcohol 17b (Scheme 2). The 4-aryl-3-(hy-
droxyalkyl)quinolin-2-one derivatives synthesized by the
methods illustrated in Schemes 1 and 2 are listed in
Table 1 along with relevant physicochemical data.19
Resu lts a n d Discu ssion . The ability of the target
compounds to increase maxi-K-mediated whole-cell
outward K+ currents was determined by using two-
electrode voltage clamp recordings from Xenopus laevis
oocytes expressing cloned hSlo20 maxi-K channels, as
previously described.21 All compounds were tested in a
minimum of five different oocytes to evaluate the effect
of a single drug concentration (20 µM) on outward K+-
current sensitive to iberiotoxin (IbTx). The average
percentage change in hSlo current relative to drug free
control (100%) was determined for each compound
tested. The results obtained are presented in Table 1.
In this preliminary study, the optimal hydroxyalkyl
group at C-3 was examined while restricting the sub-
stitution pattern of the quinoline nucleus to 6-CF3 and
the 4-aryl moiety to either p-chlorophenol or p-chloro-
anisole, an element present in prototype maxi-K openers
1 and 2. As can be seen from the data presented in Table
1, all the compounds (except 10a ) were shown to be
potent openers of maxi-K channels. In all examples, the
p-chlorophenol derivatives (11a -c, 17b) were more
effective openers when compared to their methyl ether
analogues, 10a -c, and 13b. Surprisingly, all the methyl
ether derivatives (except 10a ) of this series exhibited
good efficacy when compared to the corresponding
methyl ether derivatives of quinolones, 1,17 and 2.18 The
hydroxyethyl derivative 11b was found to be the most
effective opener identified from this study.
Ch a r t 1. Structures of 4-Aryl-3-hydroxyquinolin-2-ones
(1) and 4-Aryl-3-aminoquinolin-2-ones (2)
(2) have been identified as brain penetrable maxi-K
channel openers with neuroprotective properties18 (Chart
1).
Ch em istr y. The requisite 2-aminobenzophenone de-
rivatives (5a -c) were prepared as described previ-
ously.18 Acylation of 5b with MeO2C(CH2)nCOCl (n )
1-3) gave the corresponding amides 6a -c, which upon
deprotonation with potassium bis(trimethylsilyl)amide
in THF undergoes cyclization to afford hydroxyquino-
lones 7a -c. Dehydration of 7a -c with HBr in AcOH
gave the quinolones 8a -c. Simultaneous deesterifica-
tion and demethylation of 8a -c with pyridine hydro-
chloride afforded 9a -c. Reduction of the carboxylic acid
moiety of 8a -c and 9a -c with a borane-methyl sulfide
complex provided the desired alcohols 10a -c and 11a -
c, respectively. Oxidation of 10a (n ) 1) with MnO2
provided the quinolone-3-carboxaldehyde 12. Olefina-
tion of 12 using triethyl phosphonoacetate and NaH in
i
DMF gave the ester 13a , which was reduced with Bu2-
AlH to the desired allylic alcohol 13b (Scheme 1).
Since demethylation of the methyl ether moiety of 13b
with a variety of demethylating agents led to the
formation of multiple products, the alcohol 17b was
prepared by the route shown in Scheme 2. Acylation of
2-amino-2′-hydroxybenzophenone derivative 5c with
methyl malonyl chloride gave the amide 14, which upon
treatment with KOtBu in THF afforded the lactone 15.
E ffect s of 4-Ar yl-3-(h yd r oxya lk yl)q u in olin -2-
on es on Isola t ed R a bb it Cor p u s Ca ver n osu m . A
rabbit corpus cavernosal tissue strip assay was used to
evaluate the functional effects of maxi-K openers on
smooth muscle relaxation.22 Corpus cavernosum strips
were isolated from the rabbit penis and suspended in
tissue baths containing warm (37 °C) physiological salt
solution. Isometric force was measured using standard
methods. Tissue strips were stimulated with the R-ago-
nist phenylephrine (3 µM) and allowed to reach a steady
level of force prior to the addition of test compounds.
i
Partial reduction of 15 with Bu2AlH in THF gave the
lactol 16. Alternatively, lactone 15 can be reduced to
i
the alcohol 11a (n ) 1, Scheme 1) with Bu2AlH by
simply changing the solvent to dichloromethane. Reac-
tion of the lactol 16 with triethyl phosphonoacetate in
the presence of NaH in DMF gave ester 17a as the

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 2821
The results are expressed as the percentage inhibition
of phenylephrine-induced force compared to that of the
vehicle control (Table 1).
As can be seen from the data presented in Table 1, a
majority of the compounds produced greater than a 30%
reduction of contractile force at 10 µM. In general,
relatively more efficacious maxi-K channel openers (i.e.,
p-chlorophenol analogues), 11a -c, and 17b were more
effective relaxants when compared to the corresponding
p-chloroanisole derivatives 10b, 10c, and 13b. Thus, a
weak correlation between maxi-K channel opening
activity and the ability to relax phenylephrine-induced
contractions in rabbit corporal tissue strip assay is
observed for this limited series. Compound 11b was
found to be particularly effective with an inhibition of
the contractile response by over 80% at 10 µM, which
is comparable to the maximum relaxation observed with
KATP openers Cromakalim (88%) and Pinacidil (87%) in
a similar assay.14 In a direct comparison, a prototype
maxi-K opener NS-004 showed 31% reduction at 10 µM.
Furthermore, 11b produced a concentration-dependent
relaxation (IC50 ) 5.0 µM) that developed slowly over a
period of 90 min. The ability of iberiotoxin (IbTx), a
selective maxi-K channel blocker, to reverse the relax-
ation produced by 11b was determined to assess the
contribution of maxi-K opening to relaxation. Interest-
ingly, the addition of IbTx (300 nM) to control tissues
resulted in an augmentation of the existing R-agonist
evoked force (30.6 ( 3.9% increase, n ) 7 strips). This
important observation provides direct evidence that
maxi-K channels play a physiologically relevant role in
the regulation of adrenergically mediated tone in the
corpus cavernosum. Addition of IbTx to the corporal
tissue strips treated with 11b (3 µM) almost fully
reversed the relaxation produced by this compound
(compound + IbTx ) 99.7 ( 5.8%, n ) 7 strips; vehicle
+ IbTx ) 104.1 ( 7.1, n ) 6 strips), supporting the
notion that 11b induced relaxation by opening maxi-K
channels.
F igu r e 1. Effect of 11b on intracavernous pressure (ICP)
response elicited by electrical stimulation of the cavernous
nerve at various time points following compound administra-
tion.
20, 35, 50 and 65 min after treatment (Figure 1).
Statistics were performed using the Student t-test, and
p < 0.05 was considered significant.
Administration of PEG 400 alone had no effect on
either baseline or electrically stimulated increases in
intracavernous pressure (data not shown). Intravenous
injection of compound 11b had no effect on basal
intracavernous pressure but produced a potentiation of
the electrically evoked increase in intracavernous pres-
sure. This potentiation was observed at time points g20
min following compound administration and reached
statistical significance 50 min postdose.
In summary, we have identified a novel series of
maxi-K channel openers and demonstrated that channel
opening activity is dependent on both the nature of the
3-(hydroxyalkyl) group and the 4-aryl moiety. Several
of these maxi-K openers are effective relaxants of
precontracted rabbit corpus cavernosal strips in vitro.
However, a weak correlation between this functional
effect and maxi-K channel opening activity was ob-
served, implicating a role for additional biochemical
mechanisms and tissue selectivity in the rabbit corporal
tissue strip assay. Nevertheless, we have identified a
potent maxi-K channel opener as an effective and maxi-
K-mediated relaxant of corpus cavernosum tissue in
vitro. Furthermore, we have demonstrated the in vivo
efficacy of this maxi-K opener in a rat model of erectile
function, indicating a potential utility in the treatment
of male ED. In conclusion, we have demonstrated the
efficacy of a maxi-K opener to elicit penile erection as a
novel mechanism for the treatment of ED.
Effects of 11b in a Ra t Mod el of Er ectile F u n c-
tion . The in vivo effects of 11b on erectile function were
evaluated by using a rat model that has been fully
described in the scientific literature.23 Male Fischer 344
rats (approximately 280-350 g) were anesthetized with
sodium pentobarbital. A carotid artery was cannulated
for the measurement of blood pressure, and both jugular
veins were cannulated for the administration of test
compound and the constant infusion of anesthetic
(sodium pentobarbital). The trachea was cannulated to
allow for artificial ventilation, and body temperature
was maintained at 37 ( 0.5 °C using a heating blanket
connected to a rectal probe thermistor. The right corpora
cavernosa was exposed and cannulated for the mea-
surement of intracavernous pressure. The cavernous
nerve was isolated, and a stainless steel bipolar elec-
trode was then placed around the nerve to allow for
electrical stimulation. Mean arterial blood pressure and
intracavernous pressure were continuously monitored.
The cavernous nerve was electrically stimulated for 30
s at a frequency of 20 Hz, typically using 0.3 mA pulses
of 0.22 ms duration. A second control stimulation was
performed 15 min after the first stimulation, and then
either vehicle (PEG 400) or compound was given intra-
venously. Repeated stimulations were performed at 5,
Su p p or tin g In for m a tion Ava ila ble: Experimental data
for various compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
Refer en ces
(1) NIH Consensus Conference. Impotence. NIH Consensus Devel-
opment Panel on Impotence. J . Am. Med. Assoc. 1993, 270, 83-
90.
(2) Feldman, H. A.; Goldstein, I.; Hatzichristou, D. G.; Krane, R.
J .; McKinlay, J . B. Impotence and its medical and psychosocial
correlates: results of the Massachusetts Male Aging Study. J .
Urol. 1994, 151, 54-61.
(3) Garcia-Reboll, L.; Mulhall, J . P.; Goldstein, I. Drugs for the
treatment of impotence. Drugs Aging 1997, 11, 140-151.
(4) Lue, T. F. Topical and oral agents for erectile dysfunction. J .
Formosan Med. Assoc. 1999, 98, 233-241.

2822 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Letters
(5) Kloner, R. A. Cardiovascular risk and sildenafil. Am. J . Cardiol.
2000, 86, 57-61.
(6) (a) Vobig, M. A.; Klotz, T.; Staak, M.; Bartz-Schmidt, K. U.;
Engelmann, U.; Walter, P. Retinal side-effects of sildenafil.
Lancet 1999, 353, 375. (b) Vobig, M. A. Retinal side-effects of
sildenafil. Lancet 1999, 353, 1442.
(7) Andersson, K. E.; Wagner, G. Physiology of penile erection.
Physiol. Rev. 1995, 75, 191-236.
(8) Carrier, S.; Brock, G.; Kour, N. W.; Lue, T. F. Pathophysiology
of erectile dysfunction. Urology 1993, 42, 468-481.
(9) Christ, G. J . The penis as a vascular organ. The importance of
corporal smooth muscle tone in the control of erection. Urol. Clin.
North Am. 1995, 22, 727-745.
(10) Saenz de Tejada, I. Molecular mechanisms for the regulation of
penile smooth muscle contractility. Int. J . Impot. Res. 2000, 12
(Suppl 4), 34-38.
(11) Christ, G. J .; Spray, D. C.; Brink, P. R. Characterization of K
currents in cultured human corporal smooth muscle cells. J .
Androl. 1993, 14, 319-328.
(18) Hewawasam, P.; Fan, W.; Knipe, J .; Moon, S. L.; Boissard, C.
G.; Gribkoff, V. K.; Starrett, J . E., J r. The Synthesis and
Structure-Activity Relationships of 4-Aryl-3-aminoquinolin-2-
ones: A New Class of Calcium-Dependent, Large Conductance,
Potassium (Maxi-K) Channel Openers Targeted for Post-Stroke
Neuroprotection. Bioorg. Med. Chem. Lett. 2002, 12, 1779-1783.
(19) All new compounds were characterized by 1H NMR, LRMS, and
elemental analysis.
(20) Dworetzky, S. I.; Trojnacki, J . T.; Gribkoff, V. K. Cloning and
Expression of a Human Large-Conductance Calcium-Activated
Potassium Channel. Mol. Brain Res. 1994, 27, 189-193.
(21) Gribkoff, V. K.; Lum-Ragan, J . T.; Boissard, C. G.; Post-Munson,
D. J .; Meanwell, N. A.; Starrett, J . E., J r.; Kozlowski, E. S.;
Romine, J . L.; Trojnacki, J . T.; McKay, M. C.; Zhong, J .;
Dworetzky, S. I. Effects of Channel Modulators on Cloned Large-
Conductance Calcium-Activated Potassium Channels. Mol. Phar-
macol. 1996, 50, 206-217.
(22) New Zealand white rabbits (3.4-3.7 kg) were euthanized by
sodium pentobarbital (100 mg/kg) overdose. The penis from each
animal was rapidly removed and immersed in a cold oxygenated
modified Krebs Henseleit bicarbonate buffer. The outer tunica
of the penis was removed, and the corpus cavernosum smooth
muscle was obtained and cut into strips approximately 3-5 mm
long. The strips were suspended in tissue baths containing
oxygenated modified Krebs Henseleit bicarbonate buffer (37 °C)
and allowed to equilibrate for at least 1 h under a resting tension
of 1.0 g. The development of tension was measured with a Grass
FT 0.3 force displacement transducer coupled to a BIOPAC data
acquisition system using AcqKnowledge software.
(12) Fan, S. F.; Brink, P. R.; Melman, A.; Christ, G. J . An analysis
of the Maxi-K+ (KCa) channel in cultured human corporal
smooth muscle cells. J . Urol. 1995, 153, 818-825.
(13) Christ, G. J .; Brink, P. R.; Melman, A.; Spray, D. C. The role of
gap junctions and ion channels in the modulation of electrical
and chemical signals in human corpus cavernosum smooth
muscle. Int. J . Impot. Res. 1993, 5, 77-96.
(14) Holmquist, F.; Andersson, K. E.; Fovaeus, M.; Hedlund, H. K(+)-
channel openers for relaxation of isolated penile erectile tissue
from rabbit. J . Urol. 1990, 144, 146-151.
(15) Moon, D. G.; Byun, H. S.; Kim, J . J . A KATP-channel opener as
a potential treatment modality for erectile dysfunction. Br. J .
Urol. Int. 1999, 83, 837-841.
(23) Rehman, J .; Chenven, E.; Brink, P.; Peterson, B.; Walcott, B.;
Wen, Y. P.; Melman, A.; Christ, G. Diminished neurogenic but
not pharmacological erections in the 2- to 3-month experimen-
tally diabetic F-344 rat. Am. J . Physiol. 1997, 272, H1960-H1971.
(16) Hedlund, P.; Holmquist, F.; Hedlund, H.; Andersson, K. E.
Effects of nicorandil on human isolated corpus cavernosum and
cavernous artery. J . Urol. 1994, 151, 1107-1113.
(17) Sit, S.-Y.; Meanwell, N. A. U.S. Patent 5,892,045, 1999.
J M030005H