Design and synthesis of 4-substituted benzamides as potent, selective, and orally bioavailable IKs blockers

Article abstract of DOI:10.1021/jm015505u

Multiple delayed rectifier potassium currents, including I_Ks, are responsible for the repolarization and termination of the cardiac action potential, and blockers of these currents may be useful as antiarrhythmic agents. Modification of compound 5 produced 19(S) that is the most potent I_Ks blocker reported to date with > 5000-fold selectivity over other cardiac ion channels. Further modification produced 24A with 23% oral bioavailability.

Full text of DOI:10.1021/jm015505u

3764
J . Med. Chem. 2001, 44, 3764-3767
Design a n d Syn th esis of 4-Su bstitu ted
Ben za m id es a s P oten t, Selective, a n d
Or a lly Bioa va ila ble IKs Block er s
J ohn Lloyd,* J oan B. Schmidt, George Rovnyak,
Saleem Ahmad, Karnail S. Atwal, Sharon N. Bisaha,
Lidia M. Doweyko, Philip D. Stein, Sarah C. Traeger,
Arvind Mathur, Mary Lee Conder, J ohn DiMarco,
Timothy W. Harper, Tonya J enkins-West,
Paul C. Levesque, Diane E. Normandin,
F igu r e 1. Known IKs blockers and the screening hit 3.
Anita D. Russell, Randolph P. Serafino,
Mark A. Smith, and Nicholas J . Lodge
5 µM). Although a substantial effort to optimize the
activity of these analogues led to compounds with much
greater potency against IKs, they also showed significant
block of IKr as well as affinity for other receptors.
Further directed screening using the tetralone 3 as a
template identified the phenyl ether 4 and the ketone
6, both of which had moderate activity against IKs (Table
1). In our initial studies we found that the basic amine
was not necessary, as the amide 5 was equipotent to
the amine 4. We also found that we could replace the
ketone in 6 with an amide. Benzamides such as 7 then
became our primary target.
Bristol-Myers Squibb Pharmaceutical Research Institute,
P.O. Box 4000, Princeton, New J ersey 08543-4000
Received J anuary 18, 2001
Ab st r a ct : Multiple delayed rectifier potassium currents,
including IKs, are responsible for the repolarization and
termination of the cardiac action potential, and blockers of
these currents may be useful as antiarrhythmic agents.
Modification of compound 5 produced 19(S) that is the most
potent IKs blocker reported to date with >5000-fold selectivity
over other cardiac ion channels. Further modification produced
24A with 23% oral bioavailability.
Using high throughput automated synthetic proce-
dures, we initially varied the amine component of the
amide while keeping the 4-hexyloxybenzoic acid portion
of the molecule constant. After the synthesis and testing
of over 100 benzamide analogues from amines chosen
for structural diversity, it became clear that amides
derived from small, branched alkylamines were the
most potent. The amide derived from 3,3-dimethylbu-
tylamine (8) showed a >10-fold enhancement in potency
over the amide 7 (Table 2). It was interesting to note
the sharply defined structural requirements of the alkyl
chain as changing the length of the alkyl chain or
deletion of one or both of the terminal methyl groups
from compound 8 resulted in significantly decreased
potency (see Supporting Information). We were also
encouraged since compound 8 showed only weak (<50%)
block of IKr at 10 µM.
Constraint of a conformationally mobile alkyl group
in some cases can increase potency. Therefore, we
replaced the 3,3-dimethylbutylamine with cyclized
amines (Table 2). Bridging from the nitrogen to the
â-carbon with a two-carbon link as in compound 9
resulted in a large loss of activity. Constraint from the
R-carbon to the terminal methyl with a one-carbon link
resulted in compound 10 of equal potency to compound
8. Constraint of the â-carbon to the terminal methyl
group with a two-carbon link as in compound 11
resulted in a slight improvement in potency for the more
active enantiomer. Decreasing the distance from the
nitrogen to the cyclopentane in compound 12 resulted
in substantial loss of potency, as did increasing the ring
size from five to six carbons in compound 13. However,
moving the methyl groups to the adjacent carbon (14)
resulted in recovery of some IKs blocking potency.
While studying the constrained amines, we simulta-
neously investigated the replacement of the 4-hexyloxy-
phenyl group. Automated synthesis was used to produce
amides of 3,3-dimethylbutylamine with a diverse set of
Multiple delayed rectifier potassium currents are
responsible for the repolarization and termination of the
cardiac action potential. Blockade of these potassium
currents delays repolarization, prolongs action potential
duration, and thus produces an antiarrhythmic (class
III) effect.1 The delayed rectifier potassium current is
carried by at least two different potassium channels, IKr
2
and IKs
.
The pore forming R-subunits of IKr and IKs
channels are encoded by HERG3 and KvLQT14 potas-
sium channel genes, respectively. IKr is characterized
by rapid activation and deactivation kinetics whereas
IKs exhibits slow activation and deactivation kinetics.
Blockade of IKr and action potential prolongation at
resting heart rates can cause severe arrhythmias.5
Because of the slow deactivation kinetics of IKs, this
current may accumulate at fast heart rates and thus
selective block may lengthen action potential duration
more at fast heart rates than at slow rates. Conse-
quently, blockers of IKs may not show the negative rate
6
dependence that is exhibited by agents that block IKr
.
In this Letter we report the discovery of orally bioavail-
7
able benzamides as potent and selective blockers of IKs
.
Directed screening for inhibitors of the IKs current was
carried out using voltage clamp techniques. Initially,
compounds were chosen for testing based on similarity
to the known IKs blockers azimilide (1) and clofilium (2)
(Figure 1). In most cases, compounds were first screened
in Xenopus oocytes expressing the cloned IKs â-subunit,
minK.8 MinK associates with an endogenous IKs R-sub-
unit in Xenopus oocytes to form the IKs current.9
Compounds showing greater than 30% inhibition of this
current at 10 µM were further tested for inhibition of
the native IKs current in guinea pig ventricular myo-
cytes. Using this protocol, we discovered a tetralone ana-
logue 3 that blocked IKs with moderate potency (IC50
)
* To whom correspondence should be addressed: 609-252-5327
(voice), 609-252-6804 (fax), john.lloyd@bms.com (e-mail).
10.1021/jm015505u CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/17/2001

Letters
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23 3765
Ta ble 3. Acid Modifications of Benzamides
Ta ble 1. Screening Hits and Initial Modifications
a
a
All compounds gave satisfactory spectral and analytical data
Compounds gave satisfactory spectral and analytical data (C,
b
b
(C, H, N; (0.4% of theoretical values). Molar concentration
H, N; (0.4% of theoretical values). Molar concentration required
required to inhibit 50% of the IKs current in isolated guinea pig
ventricular myocytes n ) 2-5 at two to four concentrations (see
Supporting Information for protocol).
to inhibit 50% of the IKs current in isolated guinea pig ventricular
myocytes n ) 2-5 at two to four concentrations (see Supporting
Information for protocol).
Ta ble 4. Heterocycle Substituted Benzamides
Ta ble 2. Inhibition of IKs with 4-Hexyloxybenzamides
a
All compounds gave satisfactory spectral and analytical data
b
(C, H, N; (0.4% of theoretical values). Molar concentration
required to inhibit 50% of the IKs current in isolated guinea pig
ventricular myocytes n ) 2-5 at two to four concentrations (see
Supporting Information for protocol). c Enantiomers separated by
chiral HPLC; the absolute stereochemistry was not determined.
a
All compounds gave satisfactory spectral and analytical data
b
(C, H, N; (0.4% of theoretical values). Molar concentration
acids. Again, the requirements for the substituent on
the aromatic ring were quite specific (see Supporting
Information). Shortening of the alkyl chain or substitu-
tion of a carbon for the oxygen of the hexyloxy substitu-
ent in compound 8 resulted in >10-fold decreases in po-
tency. Ortho substitution or ring fusion also was not tol-
erated. However, we found that the biphenyl analo-
gue 15 was nearly as potent as compound 8 (Table 3).
We also found that the distal phenyl ring in the biphenyl
compound 15 could be replaced by a heterocycle such
as indole (16) and maintain equal potency to compound
8.
Substitution of a heterocycle for the distal phenyl ring
of the biphenyl compound 15 was interesting in light of
the highly lipophilic character of these compounds and
their anticipated low aqueous solubility. This led us to
synthesize the phenyl oxadiazole 17 that had similar
potency to the indole 16 (Table 4). Based on our earlier
observations that an alkyl substituent of the proper
length enhanced the activity of the 4-alkoxybenzamides,
required to inhibit 50% of the IKs current in isolated guinea pig
ventricular myocytes n ) 2-5 at two to four concentrations (see
Supporting Information for protocol). c Enantiomers separated by
chiral HPLC.
we synthesized the butyloxadiazole analogue 18 that
was 10-fold more potent than the indole 16. When this
modification was combined with the constrained amine
used in compound 11, the resulting compound 19(S)
showed 10-fold enhancement in potency over the 3,3-di-
methylbutyl analogue 18 and was 4-fold more potent
than its enantiomer 19(R). In our assay system, these
compounds were both more potent than the benzodiaz-
epine 20 that is one of the most potent compounds pre-
viously reported.7 Both 19(S) and 19(R) had little block
of IKr (IC50 >30 µM), sodium, or calcium currents (IC50
>10 µM). Thus we had achieved our objective of iden-
tifying a potent and selective blocker of the IKs current.
Our next goal was to identify an orally active agent.
The oral bioavailability of compound 19(S) was <1% in
rats. Analysis by LC/MS of the bile of the orally dosed

3766 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23
Ta ble 5. Side Chain and Heterocycle Replacements
Letters
Sch em e 1
Sch em e 2
a
All compounds gave satisfactory spectral and analytical data
b
(C, H, N; (0.4% of theoretical values). Molar concentration
required to inhibit 50% of the IKs current in isolated guinea pig
ventricular myocytes n ) 2-5 at two to four concentrations (see
Supporting Information for protocol). c Enantiomers separated by
chiral HPLC; the absolute stereochemistry was not determined.
d
C, H, N; (0.5% of theoretical values.
member of this series, 19(S), has an IC50 of 2 nM with
no significant block of IKr, INa, or ICa up to 10 µM.
Compound 19(S) is the most potent and selective IKs
blocker reported to date. We have identified compound
24A with 23% oral bioavailability. Future studies will
include further optimization of the heterocycle and the
trifluoropropyl chain of compound 24A to improve
potency while preserving this level of oral activity.
Ch em istr y. Most of the benzamides were synthesized
by either reaction of 4-hexyloxybenzoyl chloride with
various amines or the reaction of an amine with various
acids using 1-(3-dimethylaminopropyl)-3-ethylcarbodi-
imide hydrochloride (EDC) as the coupling agent (Scheme
1).
Ta ble 6. Oral Bioavailability and iv Half-Life in Rats
an ) 4-6 rats both iv and po.
animals showed that the amide and cyclopentane re-
mained intact; however, the butyl chain was extensively
metabolized. These studies suggested the need to block
degradation of the butyl group, and we achieved this
by modification of the butyl chain and the heterocycle
(Table 5). Substantially reducing the size of the butyl
group was not a useful option since the methyl analogue
21 was 100-fold less potent than 19(S). The propyl
analogue 22 and cyclopropylmethyl analogue 23 were
both slightly less potent than compound 19(S). We also
synthesized the isomeric oxadiazole 24 which contained
a trifluoromethyl substituent on the alkyl chain to block
metabolism.
Several of the more potent compounds were screened
for maximal plasma levels after oral dosing in rats. Oral
bioavailability was determined for compounds that
showed the highest plasma levels. The compound with
the greatest oral bioavailability was compound 24A
which was 23% orally bioavailable and had a half-life
after iv administration of 3.2 h in rats (Table 6).
Although 24A had lower potency (IC50 ) 40 nM)
compared to compound 19(S), it showed little block
(<30%) of IKr, INa, or ICa at concentrations up to 10 µM.
We have shown that amides of benzoic acids are
potent and selective blockers of IKs. The most potent
A variety of methods were used to synthesize the
cycloalkylamines (Scheme 2). The 3-tert-butylpyrrolidine
(29) was synthesized from 3-tert-butylpyrrole (28).10
Dimethylcyclopentylmethylamine (33) was synthesized
from 2,2-dimethylcyclopentanone (32) with tosylmeth-
ylisocyanide11 followed by reduction of the nitrile. The
2,2-dimethylcyclopentylamine (35) was synthesized from
2,2-dimethylcyclopentanone (34) by reaction with am-
monium formate followed by hydrolysis.12
The individual isomers of compound 19 were initially
separated by chiral preparative HPLC (Chiracel OD,
15% 2-propanol, water). The absolute stereochemistry
was later determined by synthesis of the individual
isomers from intermediates of known stereochemistry.
2,2-Dimethylcycopentane carboxylic acid (37)13 was
coupled with (S)-R-methylbenzylamine to form a mix-
ture of diastereomeric amides (Scheme 3). After separa-
tion of the amides, compound 38 was crystallized and
the absolute configuration (S,S) was determined by
X-ray analysis.14 The individual amides were reduced
to the amines which were coupled with the benzoic acid
to give compounds 19(S) and 19(R). The benzoic acid
was synthesized from monomethylteraphthalate and
N-hydroxypentamidine15 followed by hydrolysis of the
methyl ester.

Letters
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23 3767
Sch em e 3
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