Discovery of 1-aryloxyethyl piperazine derivatives as Kv1.5 potassium channel inhibitors (part I)

Article abstract of DOI:10.1016/j.ejmech.2014.03.075

Kv1.5 potassium channel is an efficacious and safe therapeutic target for the treatment of atrial fibrillation (AF), the most common arrhythmia that threatens human. Herein, by modifying the hit compound 7k from an in-house database, 48 derivatives were synthesized for the assay of their Kv1.5 inhibitory effects by whole cell patch clamp technique. Six compounds which showed better potency than the positive compound dronedarone were selected for the next evaluation of their drug-like properties. Compound 8 exhibited balanced solubility and permeability. It also showed acceptable pharmacodynamics profile with very low acute toxicity. Taking all these data into account, compound 8 can serve as a promising lead for the development of novel therapeutic agent for the treatment of AF.

Full text of DOI:10.1016/j.ejmech.2014.03.075

Accepted Manuscript
Discovery of 1-Aryloxyethyl Piperazine Derivatives as Kv1.5 Potassium Channel
Inhibitors (Part I)
Xiaoke Guo, Xianglei Ma, Qian Yang, Jing Xu, Lu Huang, Jianmin Jia, Jiaojiao Shan,
Li Liu, Weilin Chen, Hongxi Chu, Jinlian Wei, Xiaojin Zhang, Haopeng Sun, Yiqun
Tang, Qidong You
PII:
S0223-5234(14)00289-X
10.1016/j.ejmech.2014.03.075
EJMECH 6859
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 December 2013
Revised Date: 22 March 2014
Accepted Date: 27 March 2014
Please cite this article as: X. Guo, X. Ma, Q. Yang, J. Xu, L. Huang, J. Jia, J. Shan, L. Liu, W. Chen,
H. Chu, J. Wei, X. Zhang, H. Sun, Y. Tang, Q. You, Discovery of 1-Aryloxyethyl Piperazine Derivatives
as Kv1.5 Potassium Channel Inhibitors (Part I), European Journal of Medicinal Chemistry (2014), doi:
10.1016/j.ejmech.2014.03.075.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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ACCEPTED MANUSCRIPT

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Highlights
1.
2.
3.
4.
Lead compound 7k was identified by screening as a moderate Kv1.5 inhibitor.
48 derivatives were designed, synthesized and bio-evaluated.
Physicochemical, pharmacodynamics and acute toxicity studies were undertaken.
Compound 8 deserves further evaluations as a promising lead for treatment of AF.

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Discovery of 1-Aryloxyethyl Piperazine Derivatives as Kv1.5 Potassium Channel
Inhibitors (Part I)
a, b
d
c
d
d
a, c
d
Xiaoke Guo , Xianglei Ma , Qian Yang , Jing Xu , Lu Huang , Jianmin Jia , Jiaojiao Shan , Li
e
a, b
a, b
a, b
a
a, b, c, *
Liu , Weilin Chen , Hongxi Chu , Jinlian Wei , Xiaojin Zhang , Haopeng Sun
, Yiqun
d, *
a, b, *
Tang , Qidong You
a
Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University,
Nanjing 210009, China
b
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009,
China
c
Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China
d
Research Division of Pharmacology, China Pharmaceutical University, Nanjing 210009, China
e
Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing
2
10009, China
*
Corresponding author. Tel. /fax: +86-25-8327-1351; Tel. /fax: +86-25-8327-1070.
E-mail addresses: sunhaopeng@163.com (Haopeng Sun); youqidong@gmail.com (Qidong You);
tyq@cpu.edu.cn (Yiqun Tang)

ACCEPTED MANUSCRIPT
Abstract: Kv1.5 potassium channel is an efficacious and safe therapeutic target for the treatment of
atrial fibrillation (AF), the most common arrhythmia that threatens human. Herein, by modifying the
hit compound 7k from an in-house database, 48 derivatives were synthesized for the assay of their
Kv1.5 inhibitory effects by whole cell patch clamp technique. Six compounds which showed better
potency than the positive compound dronedarone were selected for the next evaluation of their
drug-like properties. Compound 8 exhibited balanced solubility and permeability. It also showed
acceptable pharmacodynamics profile with very low acute toxicity. Taking all these data into account,
compound 8 can serve as a promising lead for the development of novel therapeutic agent for the
treatment of AF.
Keywords: Atrial fibrillation; Kv1.5 potassium channel; structure−activity relationship

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1
.
Introduction
Atrial fibrillation (AF) is the most common arrhythmia that threatens human beings [1-3]. It can
obviously increase the onset risk of a series of severe diseases, including stroke (2~5 -fold), heart
failure (3-fold) and dementia (2-fold) [4-5]. It is estimated that the morbidity of AF will at least double
in the next 5 decades [6]. As a result, it is critical to discover efficacious and safe therapeutic agents.
Antiarrhythmic drugs have been used for nearly 100 years and remain the preferred choice for the
treatment of AF in the clinical trials till now [7]. However, these drugs such as dofetilide [8, 9] and
sotalol [10], are limited in clinical use because of the production of negative feedback on the
ventricular repolarizations, leading to QT interval prolongation and/or the potentially life threatening
arrhythmia torsades de pointes (Tdp) [10-12]. Therefore, novel drugs with improved safety are urgently
required [1, 14-15].
The ultra-rapid potassium current (IKur), encoded by Kv1.5 gene, plays a significant role in the
repolarization of the atrial action potential [16-18]. Kv1.5 potassium channel is recognized as an ideal
target because it is only recorded in the human atrial myocytes while is absent in the ventricular
myocytes [19-21].With the aim to discover novel Kv1.5 potassium channel inhibitors, an in-house
database was screened using whole cell patch clamp technique. The hit compound 7k was identified
with moderate inhibitory activity against human Kv1.5 potassium channel (hKv1.5 IC50 = 48.36 ꢀM).
Considering the alkoxyl chain including two carbon is a common segment in the drugs now on the
market (amiodarone, dromedarone and vernakalant), we introduced this fragment into the structure of
7
k (Figure 1). A series of resulted compounds were designed and synthesized.
Herein we present the optimization, structure−activity relationship (SAR) studies, in vitro and in
vivo characterization of the designed compounds. Although some agents with the similar core to our

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series II compounds had earlier been reported in other paper [22] as class III antiarrhythmic drugs, we
performed a comprehensive SAR study with more detailed information about the design of this scaffold.
Among these derivatives, compound 8, possessing potent Kv1.5 inhibitory activity and satisfactory
physicochemical properties, was selected for further evaluation. The acceptable pharmacodynamics
profile and favorable safety results promoted 8 as a promising lead for treatment of AF.
2
.
Chemistry
To improve the Kv1.5 inhibitory activity, an alkoxy chain was introduced into the structure of the
hit 7k. Treatment of thiophene-2-carboxylic acid with SOCl gave the thiophene-2-carbonyl chloride
2
(
1a). The substituted phenols were treated with BrCH CH Br in the presence of K CO to yield the
2 2 2 3
3
a-3r. The 3a-3r was reacted with piperazine hydrobromide (1:1) to give the 4a-4r in 35% - 70%
yields. Series I compounds 1~ 11 (Scheme 1) were obtained after reacting 4a-4r with 1a in DMF.
In order to investigate the SAR, several structural modifications were conducted on the aromatic
ring and the linker. Thiophene ring (Ring A) was replaced by benzene or thiazole and the linker was
changed from carbonyl to acylamino (Figure 2). The synthesis of these compounds was outlined in
Scheme 2. Substituent benzoyl chlorides (1b-1e) were prepared as the same procedure of 1a.
Commercially available aromatic amines were acylated with chloroacetyl chloride, and compounds
2
4
3
3
a-2e were obtained in 75% - 95% yields. Series II-IV compounds were (Scheme 2) synthesized from
a-4r treated with 1b-1e or 2a-2e in DMF.
.
Results and Discussion
.1. IKur blockade Assays
The synthesized derivatives were evaluated for their inhibitory activities against Kv1.5 (IKur
)
potassium channel using whole cell patch clamp technique in a representative HEK 293 cell stably

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expressing hKv1.5 channels [23, 24]. The results for series I compounds were shown in Table 1.
On the basis of the structure of 7k, an alkoxy chain (-OCH CH -) was introduced to increase the
2
2
inhibitory activity against Kv1.5 potassium channel. The resulting compound 5 exhibited a higher
Kv1.5 potency (~8 fold) compared to 7k. Then a series of substituents with varying electronic and
steric properties were introduced into the phenyl ring to modulate the Kv1.5 inhibitory activity (1~ 11).
The results (Table 1) showed that the molecule without substitution at phenyl exhibited decreased
activity compared to compounds with steric substituents (e.g. unsubstituted 1 vs methoxyl substituted 4
or vs chlorine substituted 8, 2- to 50-fold potency decrease), revealing that bulky groups at phenyl ring
was preferred for the optimization of the compounds. The biological activities of compounds with
substitution at para position were better than those ones substituted at ortho position (2 > 3; 8 > 10). In
this series, the most potent compound 8 (IC50 = 0.72 ꢀM) displayed ~3 fold improved potency than the
positive control dronedarone (IC50 = 2.37 ꢀM).
With the aim to investigate the SARs, thiophene ring (Ring A) was replaced by benzene and the
linker was changed from carbonyl to acylamino, leading to series II and III compounds (Table 2 and
Table 3). No significant variation was observed when ring A was changed from thiophene to benzene
(
IC50 = 37.50 ꢀM, 12 IC50 = 38.78 ꢀM, 21 IC50 = 28.57 ꢀM), demonstrating a structural tolerance at
this position. The derivatives with 4-methylphenyl at ring A (18, 29) instead of phenyl (12, 21) resulted
in improved activities against Kv1.5. Meanwhile, the compounds with 2-chlorphenyl (20, 32) at ring A
exhibited decreased Kv1.5 potency. Compounds with either the electron-withdrawing group, such as
chlorine group (13, 24) and cyano group (14, 25), or electron-donating group, such as methyl group (15,
2
2), at the R obviously showed an increased activity against Kv1.5 compared to 12 (or 21) without
2
substituent. Replacing the linker from carbonyl (II series) to acylamino (III series) remained or slightly

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increased the activity (21 vs 12; 26 vs 16; 28 vs 17; 32 vs 20), suggesting that the linker could bear
different steric modifications.
We further synthesized a series of thiazole derivatives (series IV compounds) to expand the SAR
analysis. The results (Table 4) showed that replacing the phenyl with thiazolyl maintained (21 vs 34; 24
vs 46) or slightly increased (23 vs 42) the activities. Compounds with small substituents improved
activity, which was same with series I-III, but with bulky substituents, such as dimethyl group (37, 38
and 39), 4-C(Me) (40), 2-OCH CH (43), 4-COOC H (47) and naphthyl (48) led to significant loss of
3
2
3
2
5
activity, indicating that it was not suitable to introduce large groups at phenyl.
3
.2. Physicochemical properties Studies
After the IKur blockade assay, six compounds that possessed excellent activities against Kv1.5
potassium channel (IC50 < positive control 2.37 ꢀM) were picked out to determine their
physicochemical properties (pKa, log D7.4 and permeability), in order to identify potential druglike
compounds. The pKa and partition coefficient (log D, pH 7.4) were determined according to the
methods of Avdeef and Tsinman [25, 26] on a Gemini Profiler instrument (pION) by the
“
goldstandard” Avdeef−Bucher potentiometric titration method [26, 27]. Permeability coefficients
were determined using MDCK (Madin-Darby canine kidney) and MDR1-MDCK (stably expressing
multidrug resistance gene 1) cells.
As shown in Table 5, six compounds exhibited acceptable physicochemical properties, especially
compounds 8, 36 and 45, which showed superior permeability and LogD (lipid-water distribution
coefficient), the key factors influencing drug absorption [28].
In the permeability experiment, we also investigated whether these compounds were substrates of
P-glycoprotein (Pgp) using MDR1-MDCK (stably expressing multidrug resistance gene 1) cell line.

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Efflux ratio was equal to Papp (apparent permeability coefficient) (B-A)/ Papp (A-B), R is efflux ratio in
T
MDR1-MDCK cell while RW is efflux ratio in MDCK cell. According to the results, all of our
compounds were not the substrates of Pgp (R < 1.50) [29, 30].
Based on the above evaluation, compound 8 with was potent in Kv1.5 inhibitory effect, and was
balanced in solubility and permeability. As a result, it was chosen for further in vivo evaluation
.
3
.3. Effect of 8 on AF model induced by CaCl -ACh
2
An AF model induced by CaCl -ACh (calcium chloride and acetylcholine) in SD rats was applied
2
to evaluate the anti-AF effect of compound 8 as described previously [24], and the results were
characterized by the changes of duration of AF and AERP (atrial effective refractory period) before
and after treatment with 8. Figure 2A showed that there was no significant difference between 5
groups (8 treatment groups, model and two positive control groups) during the model-creation
(
(
Day1-Day3). Then duration of AF was significantly reduced when the animals were treated with 8
Day4-Day7). The effect of 8 (6.45 ꢀM, 2.50 mg/kg) was close to that of sotalol (8.09 ꢀM, 2.50
mg/kg). In addition, the effect of 8 (3.23 ꢀM, 1.25 mg/kg) was better than that of dronedarone (2.25
ꢀ
M, 1.25 mg/kg). The AERP of 8-treated group was increased to normal level (Figure 2B), the effect
was close to sotalol and better than dronedarone. These data showed that compound 8 could reverse
the AF induced by CaCl -ACh in vivo.
2
3
.4. Acute toxicity studies of 8
Acute toxicity of compound 8 was observed by tail intravenous injection (IOCV) on mice (Table
). LD50 of injection was 73.65 mg/kg, far higher than the effective dose (1.25 mg/kg), indicating that
6
compound 8 possessed a high security in the treatment of AF.
4
.
Conclusion

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We have designed and synthesized 48 novel compounds modified from the compound 7k which
was obtained through the screening of an in-house database by whole cell patch clamp technique.
Structural modifications mainly addressing the aromatic ring, coupling chain and substituents at phenyl
enabled modulation of Kv1.5 inhibitory potency. Most of the derivatives endowed improved potency
compared to 7k and some of them possessed more potent inhibitory activity than dronedarone (positive
control). The physicochemical properties of some of the potent compounds were evaluated to consider
the drug-like property. Among them, compound 8, with outstanding activity, balanced LogD and
permeability, was selected for further in vivo evaluations. This compound also showed comparable in
vivo potency with sotalol and dronedarone and remarkable safety in rats. Based on these results, it can
serve as a promising lead for further development of novel agents treating AF.
5
.
Experimental Section
5
.1. Chemistry
Starting materials, reagents, and analytical grade solvents were purchased from commercial
sources and, unless otherwise noted, were used without further purification. Column and thin-layer
chromatography (CC and TLC, resp.) were performed on silica gel (200-300 mesh) and silica gel
GF254 resp., supplied by Qingdao Marine Chemical Factory. Melting points were determined with a
Melt-Temp II apparatus and were reported without correction. IR spectra were recorded on a Nicolet
1
13
iS10 Avatar FT-IR spectrometer using KBr film. The H-NMR and C-NMR spectra were measured on
a Bruker AV-300 instrument using deuterated solvents with tetramethylsilane (TMS) as internal
standard. Chemical shifts were reported as d values (parts per million) relative to TMS peak. Coupling
constants were reported in hertz. The multiplicity was defined by an s (singlet), d (doublet), t (triplet),
or m (multiplet). EI-mass spectra were recorded with a Shimadzu GCMS-2010 mass spectrometer.

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High-resolution mass spectra (HR-MS) were recorded on a Water Q-Tof micro mass spectrometer.
Purity for final compounds was greater than 95% and was measured by HPLC with Agilent
Technologies 1260 infinity C18 4.60 mm × 150 mm column using a mixture of solvent methanol/water
at the flow rate of 0.5 mL/min and peak detection at 245 nm under UV.
For experimental procedures and spectral characterization data see the Supporting Information.
5
.2. Whole cell patch clamp assay
The HEK 293 cell line that stably expressed hKv1.5 potassium channel was a kindly gift from Dr.
Gui-Rong Li (Department of Medicine and Department of Physiology, Li Ka Shing Faculty of
Medicine, The University of Hong Kong, Pokfulam, Hong Kong, SAR, China) [23]. Transfected HEK
2
93 cells were maintained at 37 °C in Minimal Eagle Medium (MEM) or Dulbecco's Modified Eagle
Medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 2 mmol/L
L-glutamine, 0.1 mmol/L nonessential amino acids, 1 mmol/L sodium pyruvate, and 0.2 mg/mL
geneticin (Invitrogen Corporation, Carlsbad, CA). Cells were passaged weekly and used at ≤ 80%
confluence. For electrophysiological recordings, the cells were harvested from the culture dish by
trypsinization, and then washed twice with standard MEM or DMEM and maintained in culture
medium at room temperature for later use on the same day.
The whole cell membrane currents were recorded by the patch clamp technique, using an EPC-10
double patch clamp amplifier (HEKA, Pfalz, Germany). Recording pipettes, made from borosilicate
glass (1.2 mm, o.d.), pulled with a pipette puller (PIP5, HEKA, Germany), had resistances of between
4
and 6MΩ when filled with the pipette solution. Pulse software (HEKA, Pfalz, Germany) was used to
generate voltage pulse protocols and to record and analyse data. Standard voltage protocols for IKur
:
5
00-ms depolarizing pulse steps to +60 mV from a holding potential of -80 mV at 0.1 Hz [23]

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Compounds were applied at least 5 min after current stabilization. IC50 value was determined by using
cell-by-cell fits with the Hill equation for three cells at least for all concentrations. Data were presented
as the mean and standard deviation (x ± SD), n = 3. All experiments were performed at 25 °C.
5
.3. CaCl -Ach induced AF Model
2
SD rats (250 ± 20 g) were anesthetized with 10% chloral hydrate (intraperitoneal injection, 0.3
mL/kg), followed by iv administration of CaCl (10 mg/mL) and acetylcholine (ACh; 66 mg/mL)
2
through the caudal vein (1 mL/kg) once a day for 7 days. The ECG was recorded for the complete
experiment procedure from rat anesthetization to the ECG being restored to normal, approximately 50
min for each rat each day. On Day 4, rats were randomly divided into four groups (n = 10/group): (1)
AF model group: repeated prior procedure until day 7, CaCl (10 mg/mL, i.v.) and ACh (66 mg/mL,
2
i.v.); (2) and (3) 8 treatment groups (1.25 mg/kg and 2.50 mg/kg, ip) combined with CaCl (10 mg/mL)
2
and ACh (66 mg/mL) (i.v.) from Day 4 to Day 7; (4) positive control: sotalol hydrochloride (2.50
mg/kg, ip) or dronedarone hydrochloride (1.25 mg/kg, ip) combined with CaCl (10 mg/mL, i.v.) and
2
ACh (66 mg/mL, i.v.) from Day 4 to Day 7. Controls were anesthetized with 10% chloral hydrate (3
mL/kg, ip), followed by physiological saline i.v. for 7 days. The disappearance of the P wave and
appearance of the f wave was determined as the beginning of AF while the end was designated by
disappearance of the f wave and the appearance of the regular P wave. After 7 days of administration,
SD rats were anesthetized and the hearts were rapidly excised and placed in ice-cold Krebs-Henseleit
buffe. The atrial appendage was separated and pinned to the bottom of a recording chamber perfused
with oxygenated Krebs-Henseleit buffer maintained at 37 ºC. Atrial muscles were equilibrated for 40
min and contractile responses recorded for a series of continuous S1–S2 couplings at 10 Hz and 150%
threshold voltage. Atrial ERP was measured (BL-420 system of Tai Meng, Cheng Du) using an

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incremental technique, with 1-ms steps at basic drive cycle lengths of 50 ms [23].
Data were analyzed using Student’s t test and presented as the mean (x ± SD), n = 6.
5
.4. Acute toxicity
7
0 SPF mice were divided into 7 groups (1 for control and 6 for treatment groups) and
administrated with distilled water (30 mL/kg) or compound 8 (84.6, 79.8, 75.3, 71, 67 and 63.2 mg/kg).
Response of mice (eg. behavior, mental state, excrement and death) was observed in 0-0.5 h
(
continuously), 0.5-6 h (0.5 h interval), 1-15 day (1 day interval). Data were analyzed using Student’s t
test, LD50 was calculated by Bliss-Finney method [31].
Acknowledgments
The authors thank Dr. Gui-Rong Li (Department of Medicine and Department of Physiology, Li Ka
Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, SAR, China) for
providing the HEK293 cells in which Kv1.5 channels are stably expressed.
This work was supported by the Project Program of Jiang Su Key Laboratory of Drug Design and
Optimization and State Key Laboratory of Natural Medicine, the innovation Program for the
Postgraduates in Jiangsu in 2012, National Major Science and Technology Project of China
(
Innovation and Development of New Drugs. No. 2012ZX09103101-002 and No. 2012ZX09401-005).
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[
[
26]. Zhang, X. J.; Li, X.; Sun, H. P.; Wang, X. J.; Zhao, L.; Gao, Y.; Liu, X. R.; Zhang, S. L.; Wang, Y.
Y.; Yang, Y. R.; Zeng, S.; Guo, Q. L.; You, Q. D. Garcinia Xanthones as Orally Active Antitumor
Agents. J. Med. Chem. 2013, 56, 276-292.
27]. Avdeef, A.; Bucher, J. J. Accurate measurements of the concentration of hydrogen ions with a
glass electrode: calibrations using the Prideaux and other universal buffer solutions and a
computer-controlled automatic titrator. Anal. Chem. 1978, 50, 2137-2142.

ACCEPTED MANUSCRIPT
[
[
[
28]. Wohnsland, F.; Faller, B. High-Throughput Permeability pH Profile and High-Throughput
Alkane/Water log P with Artificial Membranes. J. Med. Chem. 2001, 44, 923-930.
29]. Liu, Y.; Zeng, S. Advances in the MDCK-MDR1 cell model and its applications to screen drug
permeability. Acta Pharmacol. Sin. 2008, 43, 559-564.
30]. Doan, K. M. M.; Humphreys, J. E.; Webster, L. O.; Wring, S. A.; Shampine, L. J.; Serabjit-Singh,
C. J.; Adkison, K. K.; Polli, J. W. Passive Permeability and P-Glycoprotein-Mediated Efflux
Differentiate Central Nervous System (CNS) and Non-CNS Marketed Drugs. J. Pharmacol. Exp.
Ther. 2002, 303, 1029-1037.
[
31]. Wang, A. P., Wu, H., Zhang, S. Y. Acute toxicity test. In Preclinical safety evaluation and
practice of drugs, 1st, ed.; Yuan, B. J., Wang, Z. J.; Military Medical Science Press: Beijing, 1997,
pp 23-42.

ACCEPTED MANUSCRIPT
Figure Captions
Figure 1. Design of target compounds by comparison of the hit 7k with market drugs.
Figure 2. A: Effect of compound 8 on duration of AF; B: Effect of compound 8 on AERP. (AERP =
Atrial Effective Refractory Period; Data are expressed as mean ± SD, n = 6; * P < 0.05, ** P < 0.01 vs
control)

ACCEPTED MANUSCRIPT
Figure 1.
Figure 2.

ACCEPTED MANUSCRIPT
Table 1. Kv1.5 inhibition profile of series I compounds
a
a
Comp.
R
-
IC50 (hKv1.5) ꢀM
48.36 ± 7.42
37.50 ± 4.38
1.52 ± 0.29
Comp.
R
4-CN
4-F
IC50 (hKv1.5) ꢀM
10.17 ± 1.05
12.71 ± 2.14
0.72 ± 0.13
7
k
6
7
1
H
2
3
4
5
4-Me
2-Me
4-OMe
8
4-Cl
13.53 ± 1.61
21.92 ± 3.53
6.44 ± 0.77
9
4-Br
12.18 ± 1.56
9.74 ± 1.02
10
11
2-Cl
4-OCF
2,4-diCl
1.48 ± 0.13
3
Dronedarone
2.37 ± 0.13
a
Data are expressed as mean ± SD, n = 3.
Table 2. Kv1.5 inhibition profile of series II compounds
a
a
Comp.
R1
R2
H
IC50 (hKv1.5) ꢀM
38.78 ± 5.37
3.97 ± 0.58
Comp.
17
R1
R2
IC50 (hKv1.5) ꢀM
5.50 ± 1.08
1
1
1
1
1
2
3
4
5
6
H
H
4-OMe 4-Cl
4-Cl
4-CN
4-Me
H
18
4-Me
H
7.14 ± 1.35
H
4.23 ± 0.79
19
4-Me 4-Cl
4.51 ± 0.52
H
25.27 ± 4.21
16.07 ± 3.93
20
4-Cl
H
56.75 ± 9.81
4-OMe
a
Data are expressed as mean ± SD, n = 3.

ACCEPTED MANUSCRIPT
Table 3. Kv1.5 inhibition profile of series III compounds
a
a
Comp.
R1
R2
H
IC50 (hKv1.5) ꢀM
28.57 ± 4.29
11.82 ± 1.76
7.33 ± 1.14
Comp.
28
R1
R2
IC50 (hKv1.5) ꢀM
2.62 ± 0.31
2
2
2
2
2
2
2
1
2
3
4
5
6
7
H
H
4-OMe 4-Cl
2-Me
4-OCF
4-Cl
4-CN
H
29
4-Me
H
16.11 ± 1.97
4.49 ± 0.75
H
30
4-Me 4-CN
3
H
4.38 ± 0.72
31
4-Me
4-Cl
4-Cl
4-Cl
H
5.02 ± 0.69
H
9.10 ± 1.30
32
29.42 ± 5.11
3.59 ± 0.47
4-OMe
4-OMe
10.13 ± 1.19
14.46 ± 2.04
33
4-Cl
4-CN
a
Data are expressed as mean ± SD, n = 3.

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Table 4. Kv1.5 inhibition profile of series IV compounds
a
a
Comp.
R
IC50 (hKv1.5) ꢀM
29.34 ± 6.32
8.93 ± 1.18
Comp.
41
R
IC50 (hKv1.5) ꢀM
9.13 ± 1.34
3
3
3
3
3
3
4
4
4
5
6
7
8
9
0
8
H
4-OMe
4-Me
42
4-OCF
2.29 ± 0.26
3
b
2-Me
1.24 ± 0.16
43
2-OCH
CH
11.9%
2
3
2,4-diMe
3,4-diMe
2,6-diMe
17.16 ± 1.94
50.24 ± 8.23
85.93 ± 10.07
63.18 ± 10.48
44
2-Cl
3.39 ± 0.51
0.97 ± 0.08
5.18 ± 0.72
45
4-CN
4-Cl
46
b
4-C(Me)
47
4-COOC
H
32.1%
3
2
5
68.03 ± 10.29
a
b
Data are expressed as mean ± SD, n = 3; Percent inhibition of current at 11 ꢀM.

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Table 5. Physicochemical properties of compounds
MDCK
MDR1-MDCK
b
b
Comp. pKa Log D7.4
Papp (nm/sec)
Papp (nm/sec)
T W
R = R /R
b
c
R
W
R
T
B-A
A-B
B-A
A-B
2
8
6.40
6.35
5.23
7.11
6.74
6.37
3.24
1.94
3.73
2.45
3.94
1.72
311 ± 50 200 ± 14 0.64
328 ± 6
256 ± 7
161 ± 6
0.78
0.84
1.22
0.87
1.39
0.84
0.95
1.00
219 ± 44 213 ± 11 0.97 193 ± 48
11
191 ± 33
170 ± 6
0.89 315 ± 54 391 ± 42 1.24
3
4
4
6
2
5
342 ± 42 322 ± 35 1.09 323 ± 32 289 ± 21 0.92
169 ± 18
167 ± 2
122 ± 8
1.50 196 ± 21
163 ± 6
189 ± 2
1.42
1.14
148 ± 21 1.14 198 ± 16
a
b
c
ND for Not Determined; Data are expressed as mean ± SD, n = 3;
W T
R (or R ) = Papp (A-B)/ Papp (B-A).

ACCEPTED MANUSCRIPT
Table 6. Determination of LD50 of compound 8 on mice by IOCV
Compound 8
LD50
IOCV (mg/kg)
73.65
9
5% confidence interval
70.17-77.50

ACCEPTED MANUSCRIPT
Scheme 1. Reagents and conditions: i) BrCH CH Br, K CO , reflux; ii) (1) piperazine, EtOH, HBr
2
2
2
3
(
40% aqueous solution), (2) HCl, (3) NaOH; iii) SOCl , CH Cl , reflux; iv) K CO , DMF, 80 °C.
2 2 2 2 3
Scheme 2. Reagents and conditions: i) SOCl , CH Cl , reflux; ii) ClCH COCl, TEA, DMF, 5 °C; iii)
2
2
2
2
K CO , DMF, 80 °C.
2
3

Supporting Information
ACCEPTED MANUSCRIPT
Discovery of 1-Aryloxyethyl Piperazine Derivatives as Kv1.5 Potassium Channel
Inhibitors (Part I)
a, b
d
c
d
d
a, c
d
Xiaoke Guo , Xianglei Ma , Qian Yang , Jing Xu , Lu Huang , Jianmin Jia , Jiaojiao Shan , Li
e
a, b
a, b
a, b
a
a, b, c, *
Liu , Weilin Chen , Hongxi Chu , Jinlian Wei , Xiaojin Zhang , Haopeng Sun
, Yiqun
d, *
a, b, *
Tang , Qidong You
a
Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University,
Nanjing 210009, China
b
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009,
China
c
Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China
d
Research Division of Pharmacology, China Pharmaceutical University, Nanjing 210009, China
e
Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing
2
10009, China
ꢀ
Experimental section
1
.
General Procedure for the Synthesis of Intermediates 1a-1e
SOCl (0.30 mol) was added to a solution of aromatic acid (0.06 mol) in CH Cl (80 mL), the
2
2
2
mixture was stirred and refluxed for 4 h, then evaporated until dryness to give compounds 1a-1e with
colorless oily liquid which was immediately applied for further reaction as a crude product after quick
weighing.
2
.
General Procedure for the Synthesis of Intermediates 2a-2e

Supporting Information
ACCEPTED MANUSCRIPT
To a solution of commercially available aromatic amines (0.02 mol) and TEA (0.02 mol) in DMF
40 mL) was added chloroacetyl chloride (0.024 mol) in DMF (10 mL). The mixture was stirred at r.t.
(
for 2 h, taken up in water (300 mL) and filtered to obtain the compounds 2a-2e in 75.91-95.37% yields.
3
.
General Procedure for the Synthesis of Intermediates 3a-3r
K CO (0.02 mol) was suspended in BrCH CH Br (30 mL, 0.06 mol), and dropwise added different
2
3
2
2
substituted phenols (0.015 mol) in CH CN (10 mL) at 50 °C, then the mixture was refluxed for 24 h
3
and filtered, the filtrate was concentrated and purified by CC (column chromatography) over silica gel
(
eluent, PE:EA = 10:1) to give the white solid 3a-3r in 40.28-74.32 % yields.
4
.
General Procedure for the Synthesis of Intermediates 4a-4r
HBr (aq. 40%, 0.02 mol and 2.60 mL) was dropwise added to a solution of piperazidine (0.02 mol)
in EtOH (50 mL) at r.t., and then 3a-3r (0.01 mol) in EtOH (50 mL) was added. The mixture was
refluxed for 48 h, filtered and concentrated. The residue was dissolved in water, acidified with 10%
HCl aq. to pH = 5-6 and washed with CH Cl , water layer was basified by 10-50 % NaOH till pH > 9
2
2
and extracted with CH Cl , dried over NaSO and evaporated until dryness to obtain the compounds
2
2
4
4
5
a-4r with colorless oil in 35.24-70.98 % yields.
.
Thiophen-2-yl(4-(2-phenoxyethyl)piperazin-1-yl)methanone hydrochloride (1)
Compound 1a (0.015 mol) was dropwise added to a solution of 4a (0.01 mol) and TEA (0.02 mol)
in dry CH Cl under ice-salt bath, the mixture was stirred at r.t. for 8 h and washed with water (× 3),
2
2
saturation sodium bicarbonate solution (× 2) and saturation sodium chloride solution (× 2), the organic
layer was concentrated and recrystallized in PE: EA = 3 : 2, and then the residue was acidulated with
HCl in EtOH to obtain the title compound 1 as white solid (28.71%, m.p. = 258-259 °C)
-1 1
IR (KBr): 2577, 2511, 2425, 1606, 1464, 1426, 1243, 847, 758, 734 cm ; H-NMR (300 MHz, D O)
2

Supporting Information
ACCEPTED MANUSCRIPT
δ 7.39 (d, 1H, J = 4.86 Hz, Ar-H), 7.14 (d, 1H, J = 3.48 Hz, Ar-H), 7.06 (t, 2H, J = 7.89 Hz, Ar-H),
6
.84 (t, 1H, J = 4.20 Hz, Ar-H), 6.79-6.70 (m, 3H, Ar-H), 4.10 (t, 2H, J = 4.56 Hz, -CH CH O-), 3.75
2 2
1
3
(
(
brs, 4H, -N(CH CH ) N-), 3.36 (t, J = 4.68, -CH CH O-), 3.22 (brs, 4H, -N(CH CH ) N-); C-NMR
2 2 2 2 2 2 2 2
75MHz, CDCl , basic group) δ 163.0, 156.7, 136.5, 128.9, 128.3, 128.1, 126.2, 125.4, 115.4, 65.7,
3
+
5
6
8
6
6.5, 53.1, 44.9; MS (EI) m/z 316 [M ]; Anal. (C H ClN O S.1/2H O) C, H, N. Calcd for: 56.42,
17
21
2
2
2
.13, 7.74; Found: 56.72, 5.89, 7.59; HPLC purity: 99.44%, retention time: 4.69 min (methanol/water
0:20, v/v).
.
Thiophen-2-yl(4-(2-(p-tolyloxy)ethyl)piperazin-1-yl)methanone (2)
4
b (0.01 mol) and TEA (0.02 mol) was dissolved in dry CH Cl and cooled to 0 °C and 1a (0.015
2
2
mol) was added dropwise, then the solution was warmed to r.t. After being stirred for 8 h, the mixture
was washed with water (× 3), saturation sodium bicarbonate solution (× 2) and saturation sodium
chloride solution (× 2), the organic layer was concentrated and recrystallized in PE: EA = 3 : 2 to yield
compound 2 as white solid (52.58 %, m.p. = 84-86 °C).
-1
1
IR(KBr): 3850, 2943, 2832, 1601, 1521, 1513, 1455, 1427, 1248, 996, 803, 737 cm ; H-NMR
(
300 MHz, CDCl ) δ 7.33-7.30 (m, 1H, Ar-H), 7.27-7.23 (m, 1H, Ar-H), 7.09-7.02 (m, 3H, Ar-H),
3
6
2
.82-6.78 (m, 2H, Ar-H), 4.10 (t, 2H, J = 5.73 Hz, -CH CH O-), 3.77 (t, 4H, J = 5.10, -N(CH CH ) N-),
2 2 2 2 2
.83 (t, 2H, J = 5.79 Hz, -CH CH O-), 2.62 (t, 4H, J = 5.13, -N(CH CH ) N-), 2.28 (s, 3H, -CH ); MS
2
2
2
2 2
3
+
(
EI) m/z 330 [M ]; Anal. (C H N O S) C, H, N. Calcd for: 65.43, 6.71, 8.48; Found: 65.39, 6.95, 8.42;
18 22 2 2
HPLC purity: 99.08%, retention time: 6.41 min (methanol/water 80:20, v/v).
7
.
Thiophen-2-yl(4-(2-(o-tolyloxy)ethyl)piperazin-1-yl)methanone hydrochloride (3)
Compound 3 was synthesized from 4c and 1a with the procedure described for compound 1 (yellow
solid, 45.51%). m.p. = 207-209 °C.

Supporting Information
ACCEPTED MANUSCRIPT
-1
1
IR(KBr): 3438, 3053, 2918, 2411, 2389, 1607, 1464, 1429, 1265, 988, 734 cm ; H-NMR (300
MHz, D O) δ 7.68 (d, 1H, J = 4.80 Hz, Ar-H), 7.44 (d, 1H, J = 3.33 Hz, Ar-H), 7.24-7.13 (m, 2H,
2
Ar-H), 6.92-6.86 (m, 3H, Ar-H), 4.37 (t, 2H, J = 4.59 Hz, -CH CH O-), 4.05 (s, 4H, -N(CH CH ) N-),
2
2
2
2 2
3
3
8
.60 (t, 2H, J = 4.53 Hz, -CH CH O-), 3.47 (s, 4H, -N(CH CH ) N-), 2.26 (s, 3H, -CH ); MS (EI) m/z
2 2 2 2 2 3
+
30 [M ]; Anal. (C H ClN O S) C, H, N. Calcd for: 58.94, 6.28, 7.65; Found: 59.02, 6.33, 8.09.
1
8
23
2
2
.
Thiophen-2-yl(4-(2-(4-methoxyphenoxy)ethyl)piperazin-1-yl)methanone (4)
Compound 4 was synthesized from 4d and 1a with the procedure described for compound 2
(
yellow solid, 59.72%). m.p. = 82-84 °C.
-1
1
IR(KBr): 3438, 3089, 2940, 2832, 1600, 1514, 1434, 1232, 1051, 823, 737 cm ; H-NMR (300
MHz, CDCl ) δ 7.45 (d, 1H, J = 5.16 Hz, Ar-H), 7.29-7.26 (m, 1H, Ar-H), 7.06-7.02 (m, 1H, Ar-H),
3
6
.84-6.79 (m, 4H, Ar-H), 4.08 (t, 2H, J = 5.43 Hz, -CH CH O-), 3.79-3.72 (m, 7H, -OCH ,
2 2 3
-
N(CH CH ) N-), 2.83 (t, 2H, J = 5.49 Hz, -CH CH O-), 2.63 (t, 4H, J = 4.80 Hz, -N(CH CH ) N-);
2 2 2 2 2 2 2 2
1
3
C-NMR (75MHz, CDCl ) δ 162.9, 153.5, 152.3, 136.6, 128.3, 128.0, 126.1, 115.1, 114.2, 66.1, 56.7,
3
+
5
5.2, 53.0, 45.0; MS (EI) m/z 346 [M ]; Anal. (C H N O S) C, H, N. Calcd for: 62.40, 6.40, 8.09;
18
22
2
3
Found: 62.29, 6.59, 7.90; HPLC purity: 98.10%, retention time: 4.87 min (methanol/water 80:20, v/v).
9
.
Thiophen-2-yl(4-(2-(4-(trifluoromethoxy)phenoxy)ethyl)piperazin-1-yl)methanone
hydrochloride (5)
Compound 5 was synthesized from 4e and 1a with the procedure described for compound 1 (white
solid, 73.39%). m.p. = 208-210 °C.
IR(KBr): 3050, 2932, 2500, 2386, 1608, 1521, 1508, 1463, 1427, 1260, 1200, 1160, 987, 846, 728
-1 1
cm ; H-NMR (300 MHz, D O) δ 7.45 (d, 1H, J = 5.25 Hz, Ar-H), 7.25-7.21 (m, 1H, Ar-H), 7.10-7.06
2
(
m, 2H, Ar-H), 6.92-6.89 (m, 1H, Ar-H), 6.82-6.78 (m, 2H, Ar-H), 4.17 (t, 2H, J = 4.83 Hz,