Molecular hybridization, synthesis, and biological evaluation of novel chroman IKr and IKs dual blockers

Article abstract of DOI:10.1016/j.bmcl.2009.01.022

The combination of I_Kr and I_Ks blockade could lead to synergistic and safe class III anti-arrhythmic effect with the enhanced efficacy and reduced risk. On the rationale of structural hybridization of azimilide and HMR-1556, a novel

Full text of DOI:10.1016/j.bmcl.2009.01.022

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1477–1480
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Molecular hybridization, synthesis, and biological evaluation of novel chroman
IKr and IKs dual blockers
a
Lupei Du a, Minyong Li a, Qian Yang a, Yiqun Tang b, Qidong You a, , Lin Xia
*
a Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Jiangsu 210009, China
b Department of Physiology, China Pharmaceutical University, 24 Tongjiaxiang, Jiangsu 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The combination of IKr and IKs blockade could lead to synergistic and safe class III anti-arrhythmic effect
with the enhanced efficacy and reduced risk. On the rationale of structural hybridization of azimilide and
HMR-1556, a novel series of IKr and IKs dual blockers were designed, synthesized and evaluated in vitro.
One compound, 3r (CPUY11018), deserves further evaluation for its potent anti-arrhythmic activity and
favorable cardiovascular profile.
Received 9 September 2008
Revised 11 December 2008
Accepted 9 January 2009
Available online 14 January 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Arrhythmia
Class III anti-arrhythmic agents
IKr and IKs dual blockers
Molecular hybridization
As one of the common but lethal manifestations affecting the
cardiovascular system, cardiac arrhythmia remains a major cause
of morbidity and mortality around the world.1 Sudden deaths, of-
ten induced by arrhythmias, are estimated to account for approxi-
mately half of all deaths from cardiovascular diseases.2 So far anti-
arrhythmic drugs are categorized, albeit imperfectly, by the Vaugh-
an Williams classification based upon their electrophysiological
mechanisms.3 Among these four classes of anti-arrhythmic drugs,
class III agents often prolong action potential duration (APD) and
increase the period of time so that the cardiac muscle is refractory
to a premature electrical stimulus by blocking one specific delayed
rectifier potassium current (IK).4
In the ventricular myocytes, the IK has two distinct components,
IKr (rapid delayed rectifier potassium current) and IKs (slow delayed
rectifier potassium current), with variable rates of activation and
rectification properties that sum to promote repolarization during
the plateau phase of the cardiac action potential.5 Biophysically, IKr
ion channel is clustered by four hERG subunits as a tetramer,6
while IKs channel is formed by co-assembly of KvLQT1 (or called
As a IKs selective blockers containing the chromene scaffold
from Hoechst,9 HMR-1556 (1) can increases the reverse rate-
dependence of refractoriness prolongation by dofetilide in vitro.10
Recent in vivo pharmacological evidences unveiled that this com-
pound can also decrease ventricular defibrillation threshold and
reverse the repolarization without rate-dependence in rabbits.11
Another potent class III anti-arrhythmic molecule, azimilide (2),
is a dual IKr and IKs blocker compared with existing class III anti-
arrhythmic agents.12 In animal and clinical studies, azimilide (2)
can prolong repolarization by increasing APD and effective refrac-
tory period, and terminate both atrial and ventricular arrhythmias
in vivo.13 Moreover, azimilide also demonstrated anti-fibrillatory
efficacy in both animal test and clinical trial.14 In addition, azimi-
lide is associated with low incidence of proarrhythmic events, such
as torsades de pointes (TdP) and its risk factor is similar to other IKr
blockers.15
So far there are numerous examples showing that the combined
blockade of IKr and IKs could prolong APD more than what would be
expected from simple summation of the two individual ef-
fects.10,11,16–18 Therefore, a dual IKr and IKs blocker could be pro-
posed to have a synergistic anti-arrhythmic effect on achieving
safer Class III activity. Along with this kind of streamline, we would
like to report the discovery of a novel series of chroman-based
blockers (Fig. 1) structurally associated from HMR-1556 (1) and
azimilide (2) by targeting IKr and IKs simultaneously.
KCNQ1)
a-subunits and minK (or called KCNE1) b-subunits but
mainly mediated by KvLQT1.7 IKr and IKs are distinguished by their
different kinetics, pharmacology, voltage dependence and rectifica-
tion properties. During long voltage-clamp depolarizing steps to
potentials approximating the plateau level of cardiac action poten-
tials, IKs is much larger than IKr, but during relatively brief pulses
that approximate APD both components are similar in magnitude.8
Our design began with the molecular hybridization knowledge
that two indispensable structural moieties19, including a chroman
ring in HMR-1556 (1) and imidazolidine-2,4-dione side chain in
azimilide (2), could be fused into single molecule (3a–u) with
* Corresponding author. Tel./fax: +86 25 83271351.
E-mail addresses: youqd@cpu.edu.cn, youqd@163.com (Q. You).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.022

1478
L. Du et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1477–1480
O
O
N
S
O
(CH2)n R2
F3C
O
OH
N
N
N
O
Molecular Hybridization
Cl
R1
O
O
1 HMR-1556
O
N
N
O
3a-u
N
N
N
O
2
azimilide
Figure 1. Hybridization design of dual blockers against IKr and IKs from azimilide (1) and HMR-1556 (2).
the goal of creating novel chemical entities more medically effec-
tive than individual components (Fig. 1). These hybrid molecules
are proposed to target the dual blockade of IKr and IKs potassium
channel. For example, in silico studies of interaction of 3r with
Figure 2. The schematic representation of 3r docking into the active site of hERG (A) and KCNQ1 (B) potassium channel.

L. Du et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1477–1480
1479
20
21
33,34
hERG (modulating IKr
)
and KCNQ1 (modulating IKs
)
homology
previously (Table 1).
It should be noted that most of com-
models show that 3r functionality is proposed to locate on the S6
transmembrane domain and the base of the pore helix (Figures
S1 and S2) after molecular mechanics (MM) and molecular dynam-
ics (MD) simulation following similar procedures we reported else-
where.22–24 In hERG homology model, simulation evidences
pounds, such as 3h, 3k, 3p, 3r and 3t, have potent blocking activi-
ties up to nanomolar scale against IKr and IKs. Therefore, these
interesting blocking activities trigger our ambition for further
pharmacological evaluation.
On the basis of in vitro blocking activity profile, one of the inter-
esting anti-arrhythmic compound, 3r was chosen for the evalua-
tion of its anti-arrhythmic activity in the isolated Langendorff
ischemic-reperfusion Sprague–Dawley (SD) rat heart and com-
pared with azimilide (2) and amiodarone.35 The arrhythmia scores
in repurfusion periods are collected in Figure 3 and AUCs are rep-
resented in Table 2. It should be noted that during the reperfusion
suggested possible hydrophobic, p-stacking and/or p-cation inter-
actions between the structural scaffold of 3r and Thr623, Ser649,
Tyr652, Ala653 and Phe656 (Fig. 2A). It needs to be emphasized
that among residues involved in the interaction of 3r and hERG,
two aromatic residues, Tyr652 and Phe656, are unique to hERG
and lead to the channel’s high affinity for blockers based on muta-
genesis studies.25,26 In the case of KCNQ1, 3r is still proposed to
period, 3r (1 and 3
rone (1 M), however, this AUC is significantly lower when com-
pared with azimilide (1 M). All evidences exhibit that anti-
arrhythmic activity of 3r anti-arrhythmic activity at 1 and 3
lM) gained a slightly higher AUC than amioda-
have strong hydrophobic and/or
p-stacking interaction with
l
Thr311, Ala336, Ile337, Ala340, Ala344 and Gly345 (Fig. 2B), by
having high accordance with molecular determinants by muta-
tion.27,28 3r also has good fit with common features derived from
IKr and IKs pharmacophore models we published before.21,29 Fur-
thermore, these compounds have been predicted to be high block-
ing activities by pharmacophore models of IKr and IKs blockers we
established elsewhere.21,29 Therefore, this series of compounds
are expected to have dual blockade against IKr and IKs. It should
be noted that such work is along the same pipeline of our research
interest in understanding factors contributing to potassium chan-
nels and their blockers.20,21,29–31
l
l
M
is slightly less than of amiodarone, but higher than of azimilide
(2). In brief, 3r has dose-dependent protection in arrhythmia.
To evaluate the electrophysiological profile of 3r, the compari-
son of its influence on ECG parameters with that elicited by the
The synthesis was performed, according to Scheme 1, via reac-
tion of 1-(benzylideneamino) imidazolidine-2,4-dione (4) with
haloalkane in basic condition to get compound 5a–b, followed by
reacting with N-methyl piperazine or piperidine in actonitrile
and cleavage of benzyl group in aqueous HCl to provide 7a–c. For
the synthesis of 3a–u, 7a-c was refluxed with 6-substitued-2,3-
dihydrochromen-4-one (8a–h) in EtOH/AcOH to provide 21 final
compounds. The absolute configuration of the final compound 3i
identified as E-configuration by 13C NMR.32 In order to enhance
water solubility and stability, most of final compounds were con-
verted into fumarates by reacting with ethanolic fumaric acid.
These molecules were evaluated for their blocking activities
against IKr and IKs using whole cell patch-clamp technique in the
fresh isolated ventricular myocytes from guinea pig as described
Figure 3. The arrhythmia scores during the reperfusion period.
O
O
O
(CH2)n R2
H2N
O
N
b
c
N
a
(CH2)nR2
N N
N
(CH2)nCl
N N
6a-c
N
N N
NH
O
O
O
O
7a-c
4
5a-b
7a R2=N-methylpiperazinyl, n=4
7b R2=piperidinyl, n=4
6a R2=N-methylpiperazinyl, n=4
6b R2=piperidinyl, n=4
5a n=4
5b n=3
7c R2=N-methylpiperazinyl, n=3
6c R2=N-methylpiperazinyl, n=3
O
(CH2)n R2
N
N
d
N
O
O
R1
O
R1
3a-u
3a R1=OCH3, R2=N-methylpiperazinyl, n=4; 3b R1=OC2H5, R2=N-methylpiperazinyl, n=4
3c R1=OC3H7, R2=N-methylpiperazinyl, n=4; 3d R1=OC4H9, R2=N-methylpiperazinyl, n=4
O
8a-h
3e R1=OH, R2=N-methylpiperazinyl, n=4;
3f R1=Cl, R2=N-methylpiperazinyl, n=4
8a R1=OCH3
8b R1=Cl
3g R1=CH3, R2=N-methylpiperazinyl, n=4; 3h R1=CONH2, R2=N-methylpiperazinyl, n=4
3i R1=OCH3, R2=piperidinyl, n=4;
3k R1=OC3H7, R2=piperidinyl, n=4;
3m R1=OH, R2=piperidinyl, n=4;
3o R1=CH3, R2=piperidinyl, n=4;
3j R1=OC2H5, R2=piperidinyl, n=4
3l R1=OC4H9, R2=piperidinyl, n=4
3n R1=Cl, R2=piperidinyl, n=4
8c R1=CH3
8d R1=CONH2
8e R1=OH
3p R1=OCH3, R2=N-methylpiperazinyl, n=3
8f R1=OC2H5
8g R1=OC3H7
8h R1=OC4H9
3q R1=OC2H5, R2=N-methylpiperazinyl, n=3; 3r R1=OC3H7, R2=N-methylpiperazinyl, n=3
3s R1=OC4H9, R2=N-methylpiperazinyl, n=3; 3t R1=CH3, R2=N-methylpiperazinyl, n=3
3u R1=Cl, R2=N-methylpiperazinyl, n=3
Scheme 1. The synthesis of chroman derivatives. Reagents and conditions: (a) DMF, Cl(CH2)nBr; (b) 1-methylpiperazine or piperidine, CH3CN; (c) HCl/H2O; (d) EtOH/AcOH,
reflux.

1480
L. Du et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1477–1480
Table 1
and left the QTc unchanged at 1 lM concentration. Moreover, ami-
IKs and IKr blocking activity of the various chromanone derived analogues
odarone at that concentration produced a 17% reduction of HR, 12%
of prolongation of QT and 13% reduction of QTc. Therefore, cardio-
vascular profile of compound 3r compared similarly with of azim-
ilide (2) and of amiodarone.
2
2
4
1
In summary, the compounds based on the chroman nucleus de-
scribed herein are potent IKr and IKs blocking class III anti-arrhyth-
mic agents. The combination of patch-clamp and Langendorff
assays highlight again that 3r (CPUY11018) is a potent anti-
arrhythmic agent with dose-dependent profile. This compound 3r
has been selected as a preclinical candidate for further evaluation.
More extensive pharmacological and toxicological assessment of
3r is ongoing and will be the subject of forthcoming publications.
Compound
R1
R2
n
IKr IC50
(l
M) IKs IC50 (lM)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
OMe
OEt
OPr
OBu
OH
N-Methylpiperazinyl
N-Methylpiperazinyl
N-Methylpiperazinyl
N-Methylpiperazinyl
N-Methylpiperazinyl
N-Methylpiperazinyl
N-Methylpiperazinyl
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
3
3
3
3
3
3
3
1.01
6.15
11.8
0.18
>100
4.7
0.65
>10
1.28
0.022
60
0.073
>100
0.33
22
1.04
0.0032
1.03
5.4
Cl
CH3
Acknowledgments
>10
CONH2 N-Methylpiperazinyl
0.45
7.47
0.45
0.4
0.0174
>10
OMe
OEt
OPr
OBu
OH
Piperidinyl
Piperidinyl
Piperidinyl
Piperidinyl
Piperidinyl
Piperidinyl
Piperidinyl
N-Methylpiperazinyl
N-Methylpiperazinyl
N-Methylpiperazinyl
N-Methylpiperazinyl
N-Methylpiperazinyl
N-Methylpiperazinyl
—
The authors gratefully acknowledged financial support from the
China National 863 Program (Grant No. 2007AA02Z307) and the
Innovation Program for the Postgraduates in Jiangsu in 2007.
Cl
0.0062
22
61
Supplementary data
CH3
OMe
OEt
OPr
OBu
CH3
Cl
18.9
0.0019
0.0269
0.0095
1.23
0.304
0.0058
0.24
0.54
4.34
0.106
39.7
0.619
3.87
0.18
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.01.022.
3q
3r
3s
3t
References and notes
3u
Azimilide (2)
—
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Table 2
The areas under curve during the reperfusion period
Compound
Concentration
—
AUCa
Animal control
Azimilide (2)
Amiodarone
3r
3r
3r
9695 622
1
1
l
l
M
M
2585 1280***
573.3 174.7***
4955 2028**
874 254***,#
850 286***,#
0.3
lM
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1
lM
3 M
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2005, 5, 75.
a
Each value corresponds to the mean SEM.
P < 0.01.
P < 0.001 versus IR (Ischemia-reperfusion).
P < 0.05 versus Azimilide.
**
***
#
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produced a 18% reduction of the heart rate, 13% prolongation of
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Table 3
Influence of 3r on ECG parameters evaluated by Langendorff in comparison with that
of positive controls azimilide and amiodaroned
Compound
Concentrations
Time
(min)
HRa
QTb
QTcc
Azimilide (2)
1
1
l
M
M
0
20
0
20
0
20
0
20
0
227 30
201 28
228 10
189 33
220 49
198 63
231 28
182 17
231 26
179 44
162.5 26.3
175 10
315.2 37.9
316.7 29.7
344.0 53.9
300.2 84.8
317.7 24.6
326.1 14.3
306.3 18.1
299.9 26.9
316.2 40.7
400.9 32.2
Amiodarone
l
173.3 28.7
193.3 24.9
166.7 20.8
186.7 35.1
158 8.4
178 20.5
178.3 29.2
248.0 66.9
3r
3r
3r
0.3
l
M
M
M
1
3
l
l
20
a
HR, heart rate calculated from the RR interval on the ECG signal.
QT, duration of the ventricular action potential (ms).
QTc, median Bazett rate corrected QT interval (ms/s1/2).
Each value corresponds to the mean SEM.
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b
c
d