Design, synthesis and evaluation of phenethylaminoheterocycles as K v1.5 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.05.035

Phenethylaminoheterocycles have been prepared and assayed for inhibition of the K_v1.5 potassium ion channel as a potential approach to the treatment of atrial fibrillation. A diverse set of heterocycles were identified as potent K_v1.

Full text of DOI:10.1016/j.bmcl.2014.05.035

Accepted Manuscript
Design, synthesis and evaluation of phenethylaminoheterocycles as Kv1.5 in-
hibitors
James A. Johnson, Ningning Xu, Yoon Jeon, Heather J. Finlay, Alexander
Kover, Mary L. Conder, Huabin Sun, Danshi Li, Paul Levesque, Mei-Mann
Hsueh, Timothy W. Harper, Ruth R. Wexler, John Lloyd
PII:
S0960-894X(14)00535-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.05.035
BMCL 21649
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Accepted Date:
17 April 2014
12 May 2014
Please cite this article as: Johnson, J.A., Xu, N., Jeon, Y., Finlay, H.J., Kover, A., Conder, M.L., Sun, H., Li, D.,
Levesque, P., Hsueh, M-M., Harper, T.W., Wexler, R.R., Lloyd, J., Design, synthesis and evaluation of
phenethylaminoheterocycles as Kv1.5 inhibitors, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://
dx.doi.org/10.1016/j.bmcl.2014.05.035
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Design, synthesis and evaluation of phenethylaminoheterocycles as Kv1.5
inhibitors
James A. Johnsona, , Ningning Xua, Yoon Jeona, Heather J. Finlaya, Alexander Kovera, Mary L. Conderb,
Huabin Sunb, Danshi Lib, Paul Levesqueb, Mei-Mann Hsuehc, Timothy W. Harperc, Ruth R. Wexlera, John
Lloyda
aDepartment of Discovery Chemistry, Bristol-Myers Squibb Research and Development, PO Box 5400, Princeton, NJ 08534-5400, USA
bDepartment of Discovery Biology, Bristol-Myers Squibb Research and Development, PO Box 5400, Princeton, NJ 08534-5400, USA
cDepartment of Preclinical Candidate Optimization, Bristol-Myers Squibb Research and Development, PO Box 5400, Princeton, NJ 08534-5400, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Phenethylaminoheterocycles have been prepared and assayed for inhibition of the Kv1.5
potassium ion channel as a potential approach to the treatment of atrial fibrillation. A diverse set
of heterocycles were identified as potent Kv1.5 inhibitors and were advanced to
pharmacodynamic evaluation based on selectivity and pharmacokinetic profile. Heterocycle
optimization and template modification lead to the identification of compound 24 which
demonstrated increased atrial effective refractory period in the rabbit pharmacodynamic model
with mild effects on blood pressure and heart rate.
Keywords:
Kv1.5
IKur
2009 Elsevier Ltd. All rights reserved.
Atrial fibrillation
Aminoheterocycle
AERP
selective inhibition of the Kv1.5 channel has emerged as a
potentially safer approach for treatment of AF and multiple IKur
Atrial fibrillation (AF) is a pervasive heart rhythm disorder1
characterized by non-synchronized atrial conduction. The
resulting rapid, irregular contractions of the atrium lead to a 5-
fold increased risk of stroke, exacerbation of heart failure and 2-
fold increase in mortality.2 AF is distinguished by a shortened
repolarization phase of the action potential of atrial myocytes and
shortened atrial effective refractory period (AERP). Current
pharmaceutical antiarrythmic therapies include non-selective ion
channel blockers that increase AERP to return the heart to normal
sinus rhythm.3 However, current therapies inhibit ion channels
that are expressed in the atrium and ventricle, which pre-disposes
patients to potentially lethal ventricular arrhythmias by delaying
ventricular conduction and repolarization.4 A potentially safer
approach to treat AF is to target atrial selective ion channels to
increase AERP without producing electrophysiologic effects in
the ventricle.
inhibitors have been disclosed in the past decade. 8,9
Phenethylbenzamides, especially o-anisoyl amides of the type
shown in Figure 1, have been previously reported as inhibitors of
the Kv1.x class of ion-channels.10 The enhanced activity for the o-
anisoyl amide may be due to the restricted rotation imparted by
the intramolecular hydrogen bond which favors a planar
conformation of the benzamide. Evidence of this hydrogen bond
is shown in the partial crystal structure of the anisoyl amide in
Figure 1, with an N-H…O hydrogen bond distance of 1.89 Å. We
sought to enforce this orientation by constructing constrained
heterocyclic analogs of the type shown in Figure 1. Replacement
of the hydrogen bond with
a covalent bond (Type A
heterocycles) resulted in reduced Kv1.5 potency. Constraining
bond rotation by fusing a heterocyclic ring, while maintaining the
hydrogen bond (Type B heterocycles), met with success and is
the subject of this manuscript.
The Kv1.5 ion channel conducts the ultrarapid delayed
rectifier potassium ion current (IKur
) that contributes to
repolarization of action potential in human atrial myocytes but is
5,6
not functionally expressed in the ventricle.
Inhibition of IKur
leads to increased AERP and restoration of arrhythmic tissue to
normal sinus rhythm in experimental models.7 For these reasons,
———
 Corresponding author. Tel.: +1-609-818-3086; fax: +1-609-818-3550; e-mail: james.a.johnson@bms.com

Figure 1. Crystal structure of anisoyl amide, reported Kv1.x inhibitor, and
design of phenethylaminoheterocycles
As a starting template, ,-dimethylphenethylamine 111 was
used to evaluate different heterocycles (Scheme 1).
Benzisoxazoles were synthesized starting from benzaldehydes
2a-f containing a leaving group in the ortho position (typically
fluorine, although nitro was also used). Oxime formation
followed by oxidation with NCS gave N-hydroxy imidoyl
chlorides 3a-f in high yields. Due to the propensity of N-hydroxy
imidoyl chlorides to self-condense, they were immediately
coupled to amine 1 to form the N-hydroxy benzamidines 4a-f.
Base induced intramolecular cyclization gave the desired
benzisoxazoles 5a-f.12 Benzisoxazole 5a was further modified by
demethylation using boron tribromide to give phenol 6. N-
methylation of the exocyclic amine gave 7, the site of alkylation
being determined by NOE spectroscopy.
Scheme 2. Synthesis of indazoles and benzisothiazole. Reagents and
conditions: (a) (1) CH3NHNH2, EtOH, reflux; (2) Br2, CCl4, -15 °C; (b) 1,
Et3N, dioxane (17%, 3 steps); (c) (1) CDI, DCM; (2) 1, Et3N (45%); (d)
Lawesson’s reagent, toluene, 85 °C (99%); (e) hydrazine, IPA, 100 °C (59%);
(f) K2CO3, 2-bromopropane, DMF, 100 °C (7%); (g) (1) n-BuLi, THF, -78
°C; (2) 3-chloro-4-methoxybenzisothiazole, -78 to 0 °C (19%).
Palladium catalyzed conditions15 were used to couple amine 1
to the 6,6-heterocycles quinazoline 14a16 and isoquinoline 14b17
which provided 15a and 15b in good yields (Scheme 3). The 2-
chloroquinazoline 17, prepared by arylation of 1 with 2,4-
dichloroquinazoline 16, could be hydrolyzed to give the
quinazolinone 18. The N-O bond of 5a was cleaved using
catalytic hydrogenation to give imidate 19 which was cyclized to
benzoxazinone 20 using CDI. To prepare the isoindolone, the
required primary carboxamide 21 was synthesized from 3-
methoxy anthranilic acid using Sandmeyer chemistry followed
by amide bond formation. Displacement of the bromide of 21
with cyanide followed by condensation with 1 resulted in
formation of isoindolone 22.
Scheme 1. Synthesis of benzisoxazoles. Reagents and conditions: (a) (1)
NH2OH, 50% NaOH(aq), EtOH, H2O; (2) NCS, DMF; (b) 1, Et3N, dioxane; (c)
t-BuOK, THF, reflux, overall yield for the sequence in parenthesis; (d) BBr3,
1,2-DCE, 190 °C (7%); (e) (1) NaH, DMF; (2) CH3I (48%).
The methodology used to make benzisoxazoles was modified
to obtain indazoles (Scheme 2). Formation of the hydrazone from
2-fluoro-6-methoxybenzaldehyde with methylhydrazine followed
by oxidation using bromine gave the hydrobromide salt 8.13
Coupling of 8 to amine 1 resulted in concomitant cyclization to
give the N-methyl indazole 9. To extend SAR at the N-1 position,
indazole 11 was first synthesized by cyclization of the thioamide
10 using hydrazine, then subsequently alkylated to give 12.
Regiochemistry of the alkylation was determined by observation
of coupling between the proton on the exocylic amine with the
adjacent methylene protons in the COSY NMR spectrum. The
benzisothiazole 13 was prepared by deprotonation of amine 1
using butyllithium at low temperature followed by addition of 3-
chloro-4-methoxybenzisothiazole.14

11
12
13
NH
NiPr
S
37% b
42% b
0.025
-
94
-
Scheme 3. Synthesis of quinazolines, isoquinoline, quinazolinone,
benzoxazinone, and isoindolone. Reagents and conditions: (a) Pd2(dba)3,
BINAP, t-BuONa, toluene, 75 °C, 14h (74% for 15a, 77% for 15b); (b) (1)
NaOCN, AcOH; (2) NaOH; (c) POCl3, N,N-dimethylaniline, reflux (35%, 2
steps); (d) 1, Et3N, THF (69%); (e) AcOH, 70 °C (96%), (f) 10% Pd/C, H2 (1
atm), MeOH (99%); (g) CDI, THF, 150 °C (59%), (h) (1) NaNO2, 10%
HBr(aq); (2) CuBr, 48% HBr(aq) (73%, 2 steps); (i) NH4Cl, EDC, HOBt,
Et3N, DCM (51%); (j) (1) CuCN, IPA, 180 °C; (2) 1, 75 °C (10%, 2 steps).
-
68
nd
a Inhibition is measured in duplicate at 3 concentrations and the mean values
used to calculate IC50 values.
b % Inhibition of current in L-929 cells at 0.3 M, 2-4 point determinations.
Both the quinazoline 15a and isoquinoline 15b had activity for
Kv1.5, with the isoquinoline 15b having superior potency (Table
3). However, both compounds also had potent inhibition of the
hERG potassium ion channel.19 The 2-chloroquinazoline 17 had
activity similar to the parent quinazoline 15a. For the carbonyl
containing heterocycles, the benzoxazinone 18 showed the best
potency against Kv1.5 with good selectivity against hERG. The
quinazolinone 20 lost potency relative to the benzoxazinone 18,
as did the isoindolone 22 to a lesser degree, but all carbonyl
containing heterocycles demonstrated improved permeability.
The compounds were assayed for inhibition of IKur current in
patch clamped mammalian L929 cells stably expressing
recombinant human Kv1.5.18 The 4-methoxy benzisoxazole 5a
showed good potency for Kv1.5 (Table 1) while demonstrating
selectivity against the hERG potassium ion channel (66%
inhibition at 10 M). Comparison of the C-4 des-methoxy
compound 5b to 5a highlights the significant role the methoxy
group plays at this position in terms of improving potency. A
substantial loss in activity was observed by moving the 4-
methoxy group to the 5 or 6 positions (5c and 5d respectively).
Replacing the C-4 methoxy group with a chloro, ethyl or hydroxy
group (5e, 5f, and 6 respectively) also resulted in a loss in
potency, establishing the methoxy group at the 4-position as the
optimal substituent. Removal of the intramolecular hydrogen
bond by N-methylation of the exocyclic amine (7) also resulted in
a loss in potency. The Caco-2 permeability could not be
determined (nd) for some compounds due to low recovery
(<50%) in the assay.
Table 3
Activity and permeability of 6,6-heterocycles
Compd
A
X
Kv1.5
IC50
hERG %
inhib. @
10 M
Caco-2 Pc PAMPA
a
A-B
Pc
Table 1
SAR of benzisoxazole
(M)
0.118
0.022
0.073
0.023
0.197
0.084
(nm/sec)
(nm/sec)
15a
15b
17
N
H
90
99
90
50
66
46
nd
nd
-
-
CH
N
H
-
Cl
-
Compd
5a
5b
5c
X
Kv1.5 IC50a (M)
0.043
Caco-2 Pc A-B (nm/sec)
18
O
-
816
695
790
20
NH
-
-
4-OMe
H
nd
116
-
22
270
0.784
a Inhibition is measured in duplicate at 3 concentrations and the mean values
used to calculate IC50 values.
5-OMe
6-OMe
4-Cl
37% b
5d
5e
21% b
36
nd
nd
-
Based on the in vitro potency for Kv1.5, compounds from each
series (5,6-, 6,6-, and carbonyl containing heterocycles) were
screened in a rat IV pharmacokinetic model to determine in vivo
exposure (Table 4).20 Benzisoxazole 5a, N-methylindazole 9 and
benzisothiazole 13 all had moderate half-life and clearance.
Isoquinoline 15b had high clearance. Both of the carbonyl
containing heterocycles 20 and isoindolone 22 had short half-life,
while parameters for benzoxazinone 18 could not be determined
due to intolerance in the rat model. Based on these PK profiles,
5a and 9 were considered suitable for further in vivo studies in
the rabbit pharmacodynamic model.21
33% b
5f
4-CH2CH3
4-OH
4-OMe
0.219
6
0.262
7
0.187
nd
a Inhibition is measured in duplicate at 3 concentrations and the mean values
used to calculate IC50 values.
b % Inhibition of current in L-929 cells at 0.3 M, 2-4 point determinations.
Comparison of the benzisoxazole 5a to the indazole 11
showed decreased activity against Kv1.5 but improved
permeability (Table 2). Potency could be increased by alkylating
the indazole with a methyl group (9), but the larger isopropyl
group of 12 proved detrimental to potency. The benzisothiazole
13 had an in vitro profile very similar to benzisoxazole 5a.
Table 4
Rat IV coarse PK profile for key compounds (n = 2)
Compd
Half-life (h)
Clearance
Vss (L/kg)
(ml/min/kg)
5a
9
2.4
2.1
1.8
1.8
0.8
0.8
22
12
28
124
10
35
3.7
1.7
3.9
14
Table 2
SAR of indazole and benzisothiazole
13
15b
20
22
0.7
1.1
a
Compd
A
Kv1.5 IC50
(M)
hERG % inhib.
@ 10 M
Caco-2 Pc A-B
(nm/sec)
9
NMe
0.198
69
23

In the anesthetized rabbit model, the compounds were dosed
at 0.3, 1, 3, and 10 mpk IV and electrophysiological effects on
AERP and VERP were measured. A non-significant effect on
AERP and VERP prolongation (<5%, n = 3) was found for
benzisoxazole 5a at plasma concentrations of 8.2 0.5 M when
dosed at 10 mpk (vehicle PEG400:EtOH:H2O, 1:1:1). N-
methylindazole 9 also had a non-significant effect on AERP
prolongation (6%, n = 1) at a 1 mpk dose (0.3 M plasma
concentration) and could not be dosed higher due to poor
solubility in the vehicle. Both compounds were highly protein
bound in rabbit plasma (>99.9%).
To further explore the pharmacodynamic response, analogs
with the N-methylindazole ring were prepared with
a
cyclohexane template which has previously been reported in
combination with anisoyl amides (Scheme 4).10 The cyclohexane
analog 23 had an activity and low permeability profile similar to
9 (Table 5). When dosed in the rabbit model, 23 also had a non-
significant effect on AERP (<5%) at the 10 mpk dose (20 M
plasma concentration, n = 4). To improve the physical properties
of the molecule, the methyl group on the indazole was removed
to give compound 24 which resulted in improved permeability
(Table 5, cf. 9 and 11).22 Despite having a similar IV
pharmacokinetic profile to compound 23 (Table 6), compound 24
demonstrated dose dependent prolongation of AERP in the rabbit
model (Figure 2). At a dose of 1 mpk (0.7 M plasma
concentration, n = 4), a 15% 0.9 increase in AERP was
observed, and a 22% 2.8 increase was achieved at 3 mpk (2.4
M). Prolongation of AERP by 10% in this rabbit model is
considered sufficient to produce a clinically relevant effect.9d
Dose dependent decreases in blood pressure (7% 4 at 1 mpk,
17% 3 at 3 mpk) and increase in heart rate (5% 2 at 1 mpk,
16%, 3 at 3 mpk) were also observed. There was no effect on
VERP, demonstrating the selectivity against hERG.
Figure 2. Effect on AERP and VERP in rabbit for compound 24
Compound 24 also had high protein binding in rabbit plasma
(99.7%) but was differentiated by its increased permeability
which may be a contributing factor for the observed PD response.
In summary, variations of heterocycles N-linked to
phenethylamines resulted in potent inhibitors of Kv1.5 and
allowed for optimization of hERG selectivity, permeability and
pharmacokinetic properties. Compounds containing 5,6-
heterocyclic rings (benzisoxazole, benzisothiazole and indazole)
had more favorable pharmacokinetic profiles and hERG
selectivity than the 6,6-heterocyles (quinazoline, isoquinoline).
Carbonyl containing heterocycles (isoindolone, quinazolinone)
showed improved permeability but had a short PK half-life.
Combination of the indazole with a cyclohexane-based template
gave compound 24 which demonstrated significant prolongation
of AERP in the rabbit pharmacodynamic model. Efforts to
mitigate hemodynamic effects while maintaining the PD
response will be the focus of future studies.
Acknowledgments
The authors wish to thank Dr. Jinping Gan and Lifei Wang for
biotransformation work as well as Robert Languish for high
resolution mass spectrometry support.
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body weight) were surgically implanted with jugular vein cannulas
and carotid artery cannulas. The rats were fasted overnight prior to
dosing and food provided at 8 hr after dosing. Dose preparation:
Compounds were dissolved in ethanol/PEG400/water (1:1:1) to
yield a final concentration of 2 M. Study Design: Each rat
received an infusion via the carotid artery of 0.5-mL/kg/min for 10
minutes to yield a total dose of 10 mol/kg. Serial blood samples
(0.25 mL) were collected at 0 (start of the infusion), 12 min, 1 hr,
2 hr and 4 hr after beginning the infusion. EDTA was used as the
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the trans template10 and found to have similar Kv1.5 activity (IC50
= 193 nM).

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Design, synthesis and evaluation of
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inhibitors
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James A. Johnson, Ningning Xu, Yoon Jeon, Heather J. Finlay, Alexander Kover, Mary L. Conder, Huabin Sun,
Danshi Li, Paul Levesque, Mei-Mann Hsueh, Timothy W. Harper, Ruth R. Wexler, John Lloyd