Design and bio-evaluation of indole derivatives as potent Kv1.5 inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.08.041

Atrial fibrillation (AF) is one of the common arrhythmias that threaten human health. Kv1.5 potassium channel is reported as an efficacious and safe target for the treatment of AF. In this paper, we designed and synthesized three series of compounds through modifying the lead compound RH01617 that was screened out by the pharmacophore model we reported earlier. All of the compounds were evaluated by the whole-patch lamp technology and most of them possessed potent inhibitory activities against Kv1.5. Compounds IIIi and IIIl were evaluated for the target selectivity as well as the pharmacodynamic effects in an isolated rat model. Due to the promising pharmacological behavior, compound IIIl deserves further pharmacodynamic and pharmacokinetic evaluations.

Full text of DOI:10.1016/j.bmc.2013.08.041

Accepted Manuscript
Design and bio-evaluation of indole derivatives as potent Kv1.5 inhibitors
Xiaoke Guo, Qian Yang, Jing Xu, Li Zhang, Hongxi Chu, Peng Yu, Yingying
Zhu, Jinglian Wei, Weilin Chen, Yaozhong Zhang, Xiaojin Zhang, Haopeng
Sun, Yiqun Tang, Qidong You
PII:
S0968-0896(13)00735-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.08.041
BMC 11057
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
9 June 2013
21 August 2013
22 August 2013
Please cite this article as: Guo, X., Yang, Q., Xu, J., Zhang, L., Chu, H., Yu, P., Zhu, Y., Wei, J., Chen, W., Zhang,
Y., Zhang, X., Sun, H., Tang, Y., You, Q., Design and bio-evaluation of indole derivatives as potent Kv1.5 inhibitors,
Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.08.041
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*Graphical Abstract

Design and bio-evaluation of indole derivatives as potent Kv1.5 inhibitors
Xiaoke Guo a, b, 1, Qian Yang a, b, 1, Jing Xu d, Li Zhang d, Hongxi Chua, c, Peng Yu d, Yingying Zhu d, Jinglian Wei c,
Weilin Chen c, Yaozhong Zhang c, Xiaojin Zhang a, Haopeng Sun a, b, c, *, Yiqun Tang d, *, Qidong You a, b, *
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, School of Pharmacy, China Pharmaceutical University, Nanjing 210009,
China
d Research Division of Pharmacology, China Pharmaceutical University, Nanjing 210009, China
Abstract: Atrial fibrillation (AF) is one of the common arrhythmias that threaten human health. Kv1.5 potassium
channel is reported as an efficacious and safe target for the treatment of AF. In this paper, we designed and
synthesized 3 series of compounds through modifying the lead compound RH01617 that was screened out by the
pharmacophore model we reported earlier. All of the compounds were evaluated by the whole-patch lamp
technology and most of them possessed potent inhibitory activities against Kv1.5. Compounds IIIi and IIIl were
evaluated for the target selectivity as well as the pharmacodynamic effects in an isolated rat model. Due to the
promising pharmacological behavior, compound IIIl deserves further pharmacodynamic and pharmacokinetic
evaluations.
Keywords: Atrial fibrillation (AF); Kv1.5; Indole derivatives; Structure-activity relationship (SAR)
Corresponding author:
Haopeng Sun, Ph.D.
Lecturer of Medicinal Chemistry,
Tel. /fax: +86-25-8327-1351
E-mail: sunhaopeng@163.com
Qidong You, Ph.D.
Professor of Medicinal Chemistry
Tel. /fax: +86-25-8327-1351
E-mail: youqidong@gmail.com
Yiqun Tang, Ph.D.
Associate Professor of Pharmacology,
Tel. /fax: +86-25-8327-1070
E-mail: tyq@cpu.edu.cn

1.
Introduction
As the most common sustained form of arrhythmia, atrial fibrillation (AF) affects a large and growing
population, 1-4 some further projections 5 indicate that the morbidity of AF will at least double in the next 5 decades
with the ageing population and will lead to substantial mortality of stroke and heart failure. 6, 7
9
Antiarrhythmic medications remain a mainstay in the treatment of AF. 8 Currently the antiarrhythmic agents,
2, 10
such as dofetilide and sotalol, are limited in efficacy and considerable risks
for their unselective blockade of
11
the potassium currents in both atrial and ventricular myocytes. One interesting potential target is the ultra-rapid
potassium channel (IKur, encoded by Kv1.5 gene) which is functionally expressed in the atrium but not ventricle in
humans, 12-14 suggesting Kv1.5 potassium channel as a novel selective target for the treatment of AF. 15
16, 17
Our previous studies
reported a pharmacophore model of Kv1.5 (Figure 1) that was made up of one
hydrogen bond acceptor, one aromatic ring, and two hydrophobic groups. After structure-based virtual screening of
the compounds from the Maybridge database, in silico druglike property prediction and electrophysiological
evaluation were undertaken, 18, 19 leading to a lead compound RH01617 with outstanding Kv1.5 inhibitory activity
(Figure 1). Herein, with the aim of carefully investigating the structure-activity relationship (SAR), 3 series of
derivatives containing 30 compounds were designed, synthesized and bioevaluated. Among them, compounds IIIi
and IIIl showed potent activity and were chosen for further pharmacodynamic evaluations. The results showed that
compound IIIl possessed remarkable selectivity Kv1.5 potassium channel activity over hERG (human
ether-a-go-go gene) potassium channel and sodium channel, while the atrial selective profile was also proved in
the animal model in vitro.
Figure 1. A: Mapping of RH01617 to the pharmacophore model (Green: hydrogen bond acceptor; Orange:
aromatic ring; Blue: hydrophobic groups). B: Kv1.5 potassium channel inhibition of RH01617
2.
Result and discussion
2.1 Chemistry
Compounds Ia-Ii were prepared from methyl tryptophan ester hydrochloride and commercially available or
synthesized sulfonyl chloride derivatives (Scheme 1). Starting from 4-hydroxybenzenesulfonate and alkyl bromide,

compounds 2a-2e were obtained via substitution and acylation reaction. Compounds 3a-3b were obtained from
tryptophan or phenylalanine and CH3OH in the presence of SOCl2. Reaction of 2a-2e with 3a-3b in CH2Cl2
provided the target compounds Ia-Ii. Compound Ia was hydrolyzed by LiOH (1mol/L) to give the compound Ij.
With the purpose of investigating the SAR, we designed a compound Ik (Scheme 2) without the ester group.
1H-indole-3-carbaldehyde (4a) was synthesized using Vilsmeier Reaction from indole, and then be treated with
CH3NO2 and NH4OAc to give compound 5. 2-(1H-indol-3-yl)ethanamine (7) was obtained by deoxidized
compound 5 with NaBH4 and H2 (Pd/C) respectively, and then compound 7 reacted with 2a to get the compound
8a (Ik).
The synthetic routes of derivatives IIa-IIf and IIIa-IIIm were described in Schemes 3–4. Compound 7 reacted
with 2a, 2b or 2f respectively to form the compounds 8a-8c. Reaction of 8a-8c with 10a-10b that obtained from
substituted aniline (9a, 9b) provided the 2nd series of compounds. In the same way, treated compound 4 with
4-methylbenzenesulfonohydrazide (11) to get the compound 12. Reaction of compound 12 with 10a-10j provided
the 3rd series of compounds.
Scheme 1. Reagents and Conditions: i) DMF (or CH3OH), NaOH, 75 °C, 20 h; ii) CH2Cl2, SOCl2, reflux, 4 h; iii)
CH3OH, SOCl2, r.t., 30 min; iv) Et3N, CH2Cl2, r.t., 4 h; v) LiOH (1mol/L), THF, r.t., 10 h.
Scheme 2. Reagents and Conditions: i) POCl3, DMF, 5 °C for 20 min and 35 °C for 1 h; ii) CH3NO2, NH4OAc,
105 °C, 4 h; iii) NaBH4, THF, CH3OH, r.t., 30 min; iv) H2, Pd/C, CH3OH, 40 °C, 12 h; v) Et3N, CH2Cl2, r.t., 3 h.
Scheme 3. Reagents and Conditions: i) Et3N, CH2Cl2, r.t., 4 h; ii) ClCH2COCl, K2CO3, CH3CN, 0 °C and r.t., 2 h;

iii) NaH, DMF, N2 protection, 5 °C for 2 h and r.t. for 4 h.
Scheme 4. Reagents and Conditions: i) POCl3, DMF, 5 °C for 20 min and 35 °C for 1 h; ii) EtOH, reflux, 2 h; iii)
ClCH2COCl, K2CO3, DMF, r.t., 2 h; iv) NaH, DMF, N2 protection, 5 °C for 2 h and r.t. for 4 h.
2.2 Structure–activity relationship
The in vitro Kv1.5 inhibitory activity of the 30 target compounds were tested using whole cell patch-clamp
technique in a representative HEK 293 cell stably expressing hKv1.5 channels. (Table 1 and 2).
We started our investigation by examining stereo-hindrance effects of substituent groups on phenyl moiety of
the lead compound RH01617. Our results (Table 1) demonstrated that the steric hindrance at R2 (Ia-Ih) had no
apparent influence on the Kv1.5 activity, indicating a structural tolerance at this position. Removing the ester
group to avoid potential instability and the chirality led to compound Ik which maintained the potency of Kv1.5
close to Ia (Ik IC50 = 9.95, Ia IC50 = 8.37). However, a significant loss (~ 13 fold) in Kv1.5 inhibitory potency was
observed while hydrolyzing the ester group (Ia) to carboxyl group (Ij), presumably due to changed
physicochemical properties. Exchanging indole ring to phenyl group caused a dramatic drop of activity of Kv1.5
affinity (Ii vs Ia), inferring indole ring was a crucial group to the inhibitory activity.
Table 1. The activity of Kv1.5 inhibition of 1st series of compounds
HEK 293 hKv1.5 CHO hERG
Comp.
Ar
R2
R3
hERG / Kv1.5
IC50 (μM) a
8.37 ± 0.953
3.93 ± 0.428
3.56 ± 0.341
IC50 (μM) a, b
Ia
Ib
Ic
3-indolyl -OCH2CH2CH2CH3 -CH3
ND
ND
ND
3-indolyl
3-indolyl
3-indolyl
-OCH(CH3)2
-CH3
-CH3
-OCH2CH(CH3)2
Id
-CH3
2.38 ± 0.792
9.56 ± 1.207
4.02

Ie
If
3-indolyl
3-indolyl
3-indolyl
3-indolyl
-OCH3
-CH3
-Br
-CH3
-CH3
-CH3
-CH3
H
5.45 ± 0.688
4.72 ± 0.340
2.25 ± 0.191
6.45 ± 0.782
109.74 ± 9.359
65.42 ± 7.443
9.95 ± 0.941
ND
ND
Ig
Ih
Ii
4.66 ± 0.716
ND
2.07
0.32
-F
3-indolyl -OCH2CH2CH2CH3
ND
Ij
phenyl
-
-OCH2CH2CH2CH3 -CH3
-OCH2CH2CH2CH3
ND
Ik
-
3.18 ± 0.588
a Data are expressed as mean ± SD, n = 3. b ND for Not Determined
Lessening of hERG channel activity is supposed to mitigate the safety risks associated with current
antiarrhythmic agents. 20-26 With the aim to search a selective inhibitor of Kv1.5, 3 compounds (Id, Ig and Ik) that
possessed potent Kv1.5 activity were selected to test their in vitro hERG inhibitory activities using whole cell
27
patch-clamp technique in a representative CHO cell stably expressing hERG channels. The results showed that
this series of compounds were poor at Kv1.5 selectivity over hERG, especially the compound Ik which activity
ratio (hERG / Kv1.5) was 0.32. Recently, some researches inferred that discrete structural modification was a
28, 29
satisfactory approach to decrease hERG affinity.
Moreover, a comparison between the compound Ik with
some hERG inhibitors (dofetilide, sotalol and N-acetylprocainamide), showed (Figure 2) a similar chemical
structure – a long and flexibility chain. In order to reduce the hERG affinity, N-phenyl amide group was introduced
to compound Ik to change the molecular topological characteristics, resulting in 2nd series of compounds. To
further increase the rigidity and bulkiness of the molecular, the coupling chain was modified from ‘-CH2-CH2-’ to
‘-C=N-’, affording 3rd series of compounds.
Figure 2. The structure of hERG inhibitors and compound Ik
Compound IIe manifested similar Kv1.5 inhibitory effect to that of compound Ik (Table 2). Compounds with
methoxyl substitution at R2 (IIc and IId) showed more potent inhibitory activity than with methyl (IIa and IIb)
and with n-butoxyl (IIe and IIf). No remarkable difference in the activity was found using -Cl to replace -OCH3 at
R4 except IIf. The selectivity of the 2nd series of compounds was slightly ameliorated.
As shown in Table 2, the 3rd series of compounds changing the coupling chain from ‘-CH2-CH2-’ to ‘-C=N-’
increased the Kv1.5 activity (IIIa vs IIIe, IIIb vs IIIg). Electron-withdrawing groups such as trifluoromethoxy
group (IIIf) and 2,3-difluoro group (IIIi, IIIm) exhibited preferable activities than electron-donating groups such
as alkyl groups (IIIb, IIIc, IIId) and methoxy group (IIIe). The introduction of a methoxyl group at R5 (IIIk, IIIl,
IIIm) provided similar inhibitory potency to unsubstituted ones (IIIa, IIIg, IIIi). It is noteworthy that 3rd series of

compounds exhibited remarkably increased selectivity. Compound IIIl exhibited almost a 2600-fold selective
profile compared to compound Ik and 300-fold compared to compound IId, highlighting these compounds would
be safe inhibitors.
Table 2. The activity of Kv1.5 inhibition of 2nd and 3rd series of compounds
HEK 293 hKv1.5
IC50 (μM) a
CHO hERG
IC50 (μM) a, b
Comp.
R2
R4
R5
hERG / Kv1.5
IIa
IIb
-CH3
-OCH3
-Cl
-
38.73 ± 4.505
12.92 ± 1.968
1.72 ± 0.037
3.14 ± 0.822
6.21 ± 0.501
185.14 ± 12.293
0.81 ± 0.012
24.01 ± 1.529
1.77 ± 0.731
11.24 ± 0.694
16.91 ± 1.082
1.52 ± 0.079
1.90 ± 0.136
0.74 ± 0.057
0.25 ± 0.041
2.14 ± 0.758
3.73 ± 0.674
0.51 ± 0.035
2.18 ± 0.493
ND
-CH3
-
ND
IIc
-OCH3
-OCH3
-Cl
-
10.23 ± 1.230
5.95
3.66
IId
-OCH3
-
11.49 ± 1.931
IIe
-OCH2CH2CH2CH3
-OCH3
-Cl
-
ND
IIf
-OCH2CH2CH2CH3
-
ND
IIIa
IIIb
IIIc
IIId
IIIe
IIIf
IIIg
IIIh
IIIi
IIIj
IIIk
IIIl
IIIm
-
-
-
-
-
-
-
-
-
-
-
-
-
H
H
43.62 ± 5.725
53.85
2,4-diCH3
2,5-diCH3
3,4-diCH3
4-OCH3
4-OCF3
4-Cl
H
ND
H
ND
H
ND
H
ND
ND
H
H
ND
2-Cl
H
18.66 ± 2.018
42.97 ± 4.834
ND
25.22
2,4-diF
4-OCOCH2CH3
H
H
171.88
H
5-OCH3
5-OCH3
5-OCH3
ND
4-Cl
418.35 ± 33.219
ND
820.29
2,4-diF
a Data are expressed as mean ± SD, n = 3. b ND for Not Determined
Figure 3. Graphical depiction of the general SAR for Kv1.5 activity based on the IC50 results on HEK293 cell lines
Our newly synthesized 29 compounds exerted significant in vitro inhibitory effects on Kv1.5 potassium
channel and some of them had an obvious selective profile between Kv1.5 and hERG. Taken together (Figure 3),
these data indicated that the indole ring is an essential group for the activity against Kv1.5; ‘-C=N-’ double bond as

the coupling chain showed better Kv1.5 affinity than ‘-C–C-’ single bond and possessed improved selectivity; the
potency is not correlated with the size of substitution at R2 directly; electron-withdrawing group at R4 had a greater
contribution for activity than electron-donating group.
Compounds with potent Kv1.5 inhibitory effects and ideal selective profile were selected for further
pharmacodynamics studies.
2.3 Selective assay on sodium-channel
After investigating hERG potassium channel selectivity, a sodium channel inhibition assay was undertaken to
research the effects of 3 compounds on other ion-channels in heart. Compounds IIIa, IIIi and IIIl were chosen for
this test using whole cell patch-clamp technique in the fresh isolated ventricular myocytes from guinea pig (Table
3). Our compounds showed brilliant selectivity over sodium channel, especially compound IIIi (activity ratio =
459). This result proved once again our compounds could be safe and selective blockers of Kv1.5. Due to this
intriguing selective behavior, further pharmacological studies were taken on compounds IIIi and IIIl.
Table 3. The activity of sodium channel inhibition of the compounds
HEK 293 hKv1.5
Comp.
INa IC50 (μM) a
INa /Kv1.5
IC50 (μM) a
IIIa
IIIi
IIIl
77.97 ± 8.273
114.76 ± 12.314
52.60 ± 5.621
0.81 ± 0.012
96.26
459.06
103.14
0.25 ± 0.041
0.51 ± 0.035
a Data are expressed as mean ± SD, n = 3.
2.4 Pharmacodynamic studies of IIIi and IIIl
Compounds IIIi and IIIl had distinguished Kv1.5 activities and acceptable selective profiles and were further
evaluated for efficacy in the rat pharmacodynamics model. Effect on atrial effective refractory period (AERP) and
ventricular effective refractory period (VERP) of compounds IIIi and IIIl were tested respectively in isolated rat
hearts. Two compounds both demonstrated a dose dependent prolongation in AERP without increasing VERP
(Figure 4). Compared these two compounds, IIIl was more promising for its better selectivity between hERG and
Kv1.5 and the better linearity on rat model in vitro. These results indicated that compound IIIl could be a safe and
potent Kv1.5 inhibitor and it desiring further pharmacodynamic and pharmacokinetic evaluations.

Figure 4. Effect on ERP in rats for compounds IIIi (A) and IIIl (B). (AERP = Atrial Effective Refractory Period,
VERP = ventricular effective refractory period; Data are expressed as mean ± SEM, n = 3; * P < 0.05, ** P < 0.01)
3.
Conclusion
Drug targeted cardiac Kv1.5 channel is supposed to have a safety advantage over current market drugs. In this
study, we have designed and synthesized 29 novel compounds modified from the lead compound Ia which was
identified from virtual screening we previously reported, aiming at discovering potent and selective Kv1.5
inhibitors. Structural modifications mainly addressing the side chain of phenyl, ester group, coupling chain and
N-substituents at indolyl enabled modulation of Kv1.5 inhibitory potency, and most of the derivatives endowed
effective affinity, these compounds produced a concentration-dependent inhibition of hKv1.5 current. After
introducing the rigidity and bulkiness moieties, selectivity over hERG potassium channel and sodium channel of
our compounds was significantly improved, and the pharmacodynamic effect on VERP reflected the selectivity for
Kv1.5 over ventricular ion channels.
We discovered compound IIIl to be a very potent and selective blocker of Kv1.5. This compound also showed
a significant pharmacodynamic effect in rats. For these reasons it was chosen for further preclinical development.
4.
Experimental section
4.1. Chemistry
4.1.1. General methods, materials, and spectroscopic details
Melting points were measured with a Melt-Temp II instruments. IR spectra were recorded on a Nicolet Impact
410 spectrometer. EI-MS was recorded Shimadzu GC-MS 2050 apparatus; ESI-MS was recorded on Agilent 1100
LC/MSD (70 ev) spectrometers. 1H-NMR and 13C-NMR spectra were recorded on Bruker AV-300 or
AV-500MHz instruments in DMSO-d6 and CDCl3 using tetramethylsilane (TMS) as the internal standard.
Chemical shifts were reported as d values (parts per million) relative to solvent peak. Coupling constants were
reported in hertz. The multiplicity was defined by a s (singlet), d (doublet), t (triplet), or m (multiplet).

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.
4.1.2. Sodium 4-butoxybenzenesulfonate (1a)
To a stirred solution of sodium 4-hydroxybenzenesulfonate (1.96g, 0.010mol) and NaOH (0.44g, 0.011mol) in
DMF (15 mL) was added 1-bromobutane (2.20g, 0.016mol). The mixture was stirred at 75 °C for 20 h, and then
concentrated under reduced pressure to 1/3 of its original volume and filtrated to get the title compound 1a as
white solid (0.77g, 33.31%). Mp > 250 °C.
4.1.3. Sodium 4-isopropoxybenzenesulfonate (1b)
To a stirred solution of dried sodium 4-hydroxybenzenesulfonate (2.94g, 0.015mol) and NaOH (0.60g,
0.015mol) in CH3OH (100 mL) was added 2-bromopropane (2.77g, 0.023mol). The mixture was stirred at 80 °C
for 48 h, and then concentrated under reduced pressure. The residue was dissolved in acetone and filtrated to
remove the insoluble impurities and the filtrate was concentrated under reduced pressure to obtain the compound
Ib as yellow solid (1.98g, 55.41%). Mp > 250 °C.
4.1.4. Sodium 4-isobutoxybenzenesulfonate (1c)
Compound Ic was synthesized from 4-hydroxybenzenesulfonate and 1-bromo-2-methylpropane with the
procedure described for compound Ia (pink solid, 44.44%). Mp > 250 °C.
4.1.5. Sodium 4-(cyclopentyloxy)benzenesulfonate (1d)
Compound Id was synthesized from 4-hydroxybenzenesulfonate and bromocyclopentane with the procedure
described for compound Ia (pink solid, 41.62%). Mp > 250 °C.
4.1.6. 4-butoxybenzene-1-sulfonyl chloride (2a)
SOCl2 (1.3 mL, 0.018mol) and DMF (1~2 drops) were added to a stirring solution of compound 1a (0.77g,
0.003mol) in anhydrous CH2Cl2 (25 mL) under ice-water bath. After being refluxed for 4 h, the mixture was
filtrated to remove the insoluble impurities and the filtrate was washed with NaCl saturated solution (х 3), then
concentrated under reduced pressure to give the compound 2a as a yellow oil (0.46g, 61.33%).
4.1.7. 4-isopropoxybenzene-1-sulfonyl chloride (2b)
Compound 2b was synthesized from SOCl2 and sodium 4-isopropoxybenzenesulfonate (1b) with the procedure
described for compound 2a (colorless oil, 52.62%).
4.1.8. 4-isobutoxybenzene-1-sulfonyl chloride (2c)
Compound 2c was synthesized from SOCl2 and sodium 4-isobutoxybenzenesulfonate (1c) with the procedure

described for compound 2a (colorless oil, 40.40%).
4.1.9. 4-(cyclopentyloxy)benzene-1-sulfonyl chloride (2d)
Compound 2d was synthesized from SOCl2 and sodium 4-(cyclopentyloxy)benzenesulfonate (1d) with the
procedure described for compound 2a (colorless oil, 54.99%).
4.1.10. Methyl 2-amino-3-(1H-indol-3-yl)propanoate hydrochloride (3a)
To a solution of DL-tryptophane (8.16g, 0.040mol) in methanol (80 mL) was added dropwise SOCl2 (14.28g,
0.120mol), and the mixture was stirred at r.t. for 30 min. The solution was concentrated and filtered, then the
residue was washed with acetone and dried under infrared light (IR) to give the compound 3a as a white solid
(8.07g, 92.54%). Mp = 220 °C, Lit. 30 Mp = 221-222 °C.
4.1.11. Methyl 2-amino-3-phenylpropanoate hydrochloride (3b)
Compound 3b was synthesized from phenylalanine with the procedure described for compound 3a (white solid,
93.61%). Mp = 159 °C, Lit. 30 Mp = 156-157 °C.
4.1.12. 1H-indole-3-carbaldehyde (4a)
To a stirring solution of POCl3 (18.36g, 0.120mol) in DMF (25 mL), indole (11.7g, 0.100mol) in DMF (15 mL)
was added dropwise. The mixture was stirred at 35 °C for 1 h, and then be poured into ice water and neutralized
with 20% NaOH a.q. The solution was filtered and the residue was washed with water and dried under IR to give
the compound 4a as yellow needle (13.86g, 95.48%). Mp = 192-194 °C, Lit. 31 Mp = 196-197 °C.
4.1.13. 5-methoxy-1H-indole-3-carbaldehyde (4b)
Compound 4b was synthesized from 5-methoxy-1H-indole with the procedure described for compound 4a
(yellow solid, 89.4%). Mp = 57 °C, Lit. 31 Mp = 54-55 °C.
4.1.14. 3-(2-nitrovinyl)-1H-indole (5)
To a solution of 1H-indole-3-carbaldehyde (4a, 1.45g, 0.010mol) in nitromethane (15 mL) was added
ammonium acetate (0.77g, 0.010mol), the mixture was stirred at 105 °C for 4 h and evaporated until dryness to
give the compound 5 as yellow solid (1.09g, 57.98%). Mp = 168-170 °C, Lit. 32 Mp = 169-172 °C.
4.1.15. 3-(2-nitroethyl)-1H-indole (6)
NaBH4 was added slowly to a stirring solution of 3-(2-nitrovinyl)-1H-indole (5, 18.80g, 0.100mol) and
CH3OH (20 mL) in THF (100 mL), the mixture was stirred at r.t. for 30 min and concentrated to 1/5 of its original
volume and neutralized by HCl (1 mol/L). Then, the solution was extracted by EA (50 mL х 3) and the organic
layer was concentrated under reduced pressure. The crude material was purified by column chromatography (CC)
(petroleum ether/AcEt = 5:1) to get the compound 6 as yellow solid (9.12g, 47.95%). Mp = 56-58°C, Lit. 33 Mp =

55-57 °C.
4.1.16. 2-(1H-indol-3-yl)ethanamine (7)
To a solution of 3-(2-nitroethyl)-1H-indole (6, 9.12g, 0.048mol) in CH3OH (100 mL) was added Pd/C (10%,
1g), the mixture was stirred at 40 °C with continuous H2 for 12 h, and then was filtered and the filtrate was
concentrated under reduced pressure. The residue was dissolved in water (100 mL) and extracted by EA (40 mL х
3), the organic layer was evaporated until dryness to give the compound 7 as yellow solid (5.50g, 71.61%). Mp =
110-112 °C, Lit. 34 Mp = 112 °C.
4.1.17. N-(2-(1H-indol-3-yl)ethyl)-4-butoxybenzenesulfonamide (8a, Ik)
4-butoxybenzene-1-sulfonyl chloride (2a, 4.97g, 0.020mol) was added to
a stirring solution of
2-(1H-indol-3-yl)ethanamine (7, 3.20g, 0.020mol) and Et3N (4.04g, 0.040mol) in CH2Cl2 (100 mL). After being
stirred at r.t. for 3 h, the mixture was washed by water (30 mL х 3) and was concentrated under reduced pressure.
The crude material was purified by CC (petroleum ether/AcEt = 2:1) to get the compound 8a (Ik) as white solid
(4.64g, 85.91%). Mp = 84-87 °C.
1H-NMR(300 MHz, DMSO) δ 10.81 (1H, s), 7.73-7.70 (2H, m), 7.57 (1H, t, J = 5.70 Hz), 7.45-7.31 (2H, m),
7.12-6.92 (5H, m), 4.03 (2H, t, J = 6.30 Hz), 3.01-2.94 (2H, m), 2.81-2.76 (2H, m), 1.75-1.65 (2H, m), 1.49-1.36
(2H, m), 0.92 (3H, t, J = 7.50 Hz). MS (ESI) m/z 372 [M+]. HR-MS ([M + NH4]+) Calcd for C20H24N4O3S.NH4,
390.184; Found 390.1845.
4.1.18. N-(2-(1H-indol-3-yl)ethyl)-4-methoxybenzenesulfonamide (8b)
Compound
8b
was
synthesized
from
4-methoxybenzene-1-sulfonyl
chloride
(2e)
and
2-(1H-indol-3-yl)ethanamine (7, 3.20g, 0.020mol) with the procedure described for compound 8a (white solid,
12.64%). Mp = 94-96 °C.
4.1.19. N-(2-(1H-indol-3-yl)ethyl)-4-methylbenzenesulfonamide (8c)
Compound
8c
was
synthesized
from
4-methylbenzene-1-sulfonyl
chloride
(2f)
and
2-(1H-indol-3-yl)ethanamine (7, 3.20g, 0.020mol) with the procedure described for compound 8a (white solid,
73.95%). Mp = 107-109 °C.
4.1.20. 2-chloro-N-(4-methoxyphenyl)acetamide (10a)
To a solution of a4-methoxyaniline (6.16g, 0.050mol), Et3N (5.05g, 0.050mol) in DMF (40 mL) was added
chloroacely chloride (6.78g, 0.06mol) dissolved in DMF (15 mL) under ice bath. The mixture was stirred at r.t. for
2 h and poured into water and then filtered to get the compound compound 10a as yellow solid (9.61g, 96.25%).
Mp = 120-122 °C, Lit. 35 Mp = 124 °C.

4.1.21. 2-chloro-N-(4-chlorophenyl)acetamide (10b)
Compound 10b was synthesized from 4-chloroaniline with the procedure described for compound 10a (white
solid, 94.82%). Mp = 171-173 °C, Lit. 35 Mp = 170-171 °C.
4.1.22. 2-chloro-N-phenylacetamide (10c)
Compound 10b was synthesized from aniline with the procedure described for compound 10a (white solid,
80.33%). Mp = 133-135 °C. Lit. 35 Mp = 134-137 °C.
4.1.23. 2-chloro-N-(2,4-dimethylphenyl)acetamide (10d)
Compound 10d was synthesized from 2,4-dimethylaniline with the procedure described for compound 10a
(white solid, 93.76%). Mp = 152-153 °C.
4.1.24. 2-chloro-N-(2,5-dimethylphenyl)acetamide (10e)
Compound 10e was synthesized from 2,5-dimethylaniline with the procedure described for compound 10a
(white solid, 90.83%). Mp = 150 °C.
4.1.25. 2-chloro-N-(3,4-dimethylphenyl)acetamide (10f)
Compound 10f was synthesized from 3,4-dimethylaniline with the procedure described for compound 10a
(white solid, 97.82%). Mp = 106-107 °C.
4.1.26. 2-chloro-N-(4-(trifluoromethoxy)phenyl)acetamide (10g)
Compound 10g was synthesized from 4-(trifluoromethoxy)aniline with the procedure described for compound
10a (white solid, 97.79%). Mp = 131-133 °C.
4.1.27. 2-chloro-N-(2-chlorophenyl)acetamide (10h)
Compound 10h was synthesized from 2-chloroaniline with the procedure described for compound 10a (white
solid, 90.17%). Mp = 66-67 °C.
4.1.28. 2-chloro-N-(2,4-difluorophenyl)acetamide (10i)
Compound 10i was synthesized from 2,4-difluoroaniline with the procedure described for compound 10a
(white solid, 89.31%). Mp = 76-78 °C.
4.1.29. 4-(2-chloroacetamido)phenyl propionate (10j)
Compound 10j was synthesized from 4-(2-chloroacetamido)aniline with the procedure described for compound
10a (white solid, 83.66%). Mp = 109-110 °C.
4.1.30. N'-((1H-indol-3-yl)methylene)-4-methylbenzenesulfonohydrazide (12a)
A solution of 1H-indole-3-carbaldehyde (4a, 5.80g, 0.040mol) and 4-methylbenzenesulfonohydrazide (7.44g,
0.040mol) in EtOH (60 mL) was stirred at 80 °C for 2 h, then cooled to r.t. The mixture was concentrated and

purified by CC (CH2Cl2:CH3OH = 5:1) to obtain the compound 12a as white solid (7.78g, 62.06%). Mp =
142-144 °C (dec), Lit. 36 Mp = 145 °C.
4.1.31. N'-((5-methoxy-1H-indol-3-yl)methylene)-4-methylbenzenesulfonohydrazide (12b)
Compound 12b was synthesized from 5-methoxy-1H-indole-3-carbaldehyde (4b) with the procedure described
for compound 12a (white solid, 53.88%). Mp = 197-198 °C (dec).
4.1.32. Methyl 2-(4-butoxyphenylsulfonamido)-3-(1H-indol-3-yl)propanoate (Ia)
To a stirring solution of methyl 2-amino-3-(1H-indol-3-yl)propanoate hydrochloride (3a, 1.91g, 0.008mol) and
Et3N (3.03g, 0.030mol) in CH2Cl2 (40 mL) was added dropwise 4-butoxybenzene-1-sulfonyl chloride (2a, 1.86g,
0.008mol), the mixture was stirred at r.t. for 4 h and washed with water (х 3) and NaCl saturated solution (х 1).
Then, the organic layer was concentrated under reduced pressure to obtain the compound Ia as white solid (0.04g,
52.35%). Mp = 120-122 °C.
1
IR (KBr): 586, 1094, 1153, 1265, 1595, 1623, 1724, 3267, 3407, 3469 cm-1. H-NMR(300 MHz, DMSO) δ
8.10 (1H, s), 7.63 (2H, d, J = 2.10 Hz), 7.43 (1H, d, J = 7.80 Hz), 7.32 (1H, d, J = 8.10 Hz), 7.18-7.13 (1H, m),
7.09-7.01 (2H, m), 6.81 (2H, d, J = 8.70 Hz), 5.11 (1H, d, J = 9.00 Hz), 4.26-4.19 (1H, m), 3.96 (2H, t, J = 6.30
Hz), 3.45 (3H, s), 3.22 (2H, d, J = 5.70 Hz), 1.81-1.72 (2H, m), 1.55-1.43 (2H, m), 0.98 (3H, t, J = 7.20 Hz).
13C-NMR (75MHz, CDCl3) δ 171.3, 162.0, 135.6, 130.2, 128.7, 126.7, 123.0, 121.6, 119.1, 117.9, 113.9, 110.8,
108.5, 67.6, 55.5, 51.9, 30.5, 28.7, 18.6, 13.3. MS (ESI) m/z 430 [M+]. Anal. (C22H26N2O5S) C, H, N. Calcd for:
61.38, 6.09, 6.51; Found: 61.25, 6.35, 6.38.
4.1.33. Methyl 2-(4-isopropoxyphenylsulfonamido)-3-(1H-indol-3-yl) propanoate (Ib)
Compound Ib was synthesized from methyl 2-amino-3-(1H-indol-3-yl)propanoate hydrochloride (3a) and
4-isopropoxybenzene-1-sulfonyl chloride (2b) with the procedure described for compound Ia (white solid,
50.10%). Mp = 118-119 °C.
1
IR (KBr): 592, 745, 833, 1092, 1110, 1151, 1260, 1326, 1596, 1731, 2973, 3244, 3390 cm-1. H-NMR (300
MHz, CDCl3) δ 8.03 (1H, br s), 7.63 (2H, d, J = 8.10 Hz), 7.46-7.04 (5H, m), 6.82 (2H, d, J = 8.40 Hz), 5.05 (1H,
d, J = 8.70 Hz), 4.58 (1H, q, J = 6.30 Hz), 4.24 (1H, q, J = 3.30 Hz), 3.44 (3H, s), 3.24 (2H, d, J = 5.40 Hz), 1.34
(6H, d, J = 6.00 Hz). MS (EI) m/z 416 [M+]. Anal. (4C21H24N2O5S∙3H2O) C, H, N. Calcd for: 60.66, 5.98, 6.51;
Found: 58.66, 6.06, 6.42.
4.1.34. Methyl 2-(4-isobutoxyphenylsulfonamido)-3-(1H-indol-3-yl) propanoate (Ic)
Compound Ic was synthesized from methyl 2-amino-3-(1H-indol-3-yl)propanoate hydrochloride (3a) and
4-isobutoxybenzene-1-sulfonyl chloride (2c) with the procedure described for compound Ia (white solid, 11.53%).

Mp = 157-159 °C.
1
IR (KBr): 585, 739, 824, 1024, 1096, 1150, 1257, 1587, 1751, 2955, 3292, 3391cm-1. H-NMR (300 MHz,
CDCl3) δ 8.05 (1H, br s), 7.63 (2H, d, J = 8.70 Hz), 7.45-7.03 (5H, m), 6.82 (2H, d, J = 8.40 Hz), 5.07 (1H, d, J =
8.70 Hz), 4.23 (1H, q, J = 8.40 Hz), 3.72 (2H, d, J = 6.30 Hz), 3.46 (3H, s), 3.22 (2H, d, J = 5.3740 Hz), 2.08 (1H,
m, J = 6.30 Hz), 1.03 (6H, d, J = 6.60 Hz). MS (EI) m/z 430 [M+]. Anal. (C22H26N2O5S) C, H, N. Calcd for: 61.38,
6.09, 6.51; Found: 61.13, 6.08, 6.13.
4.1.35. Methyl 2-(4-(cyclopentyloxy)phenylsulfonamido)-3-(1H-indol-3-yl)propanoate (Id)
Compound Id was synthesized from methyl 2-amino-3-(1H-indol-3-yl)propanoate hydrochloride (3a) and
4-(cyclopentyloxy)benzene-1-sulfonyl chloride (2d) with the procedure described for compound Ia (white solid,
23.18%). Mp = 157-158 °C.
1
IR (KBr): 783, 824, 1092, 1152, 1258, 1325, 1384, 1593, 1724, 2359, 2949, 3274, 3386 cm-1. H-NMR (300
MHz, CDCl3) δ 8.04 (1H, br s), 7.44 (2H, d, J = 7.80 Hz), 7.34-7.03 (5H, m), 6.80 (2H, d, J = 8.70 Hz), 5.06 (1H,
d, J = 8.10 Hz), 4.76-4.74 (1H, m), 4.27-4.20 (1H, m), 3.44 (3H, s), 3.23 (2H, d, J = 5.70 Hz), 1.93-1.53 (8H, m).
MS (EI) m/z 442 [M+]. Anal. (2C23H26N2O5S∙H2O) C, H, N. Calcd for: 61.18, 6.03, 6.20; Found: 61.37, 6.20, 6.02.
4.1.36. Methyl 2-(4-methoxyphenylsulfonamido)-3-(1H-indol-3-yl)propanoate (Ie)
Compound Ie was synthesized from methyl 2-amino-3-(1H-indol-3-yl)propanoate hydrochloride (3a) and
4-methoxybenzene-1-sulfonyl chloride (2e) with the procedure described for compound Ia (white solid, 13.40%).
Mp = 136-137 °C.
IR (KBr): 566, 742, 1091, 1157, 1258, 1335, 1597, 1729, 3282, 3396 cm-1. 1H-NMR (300 MHz, CDCl3) δ 8.06
(1H, s), 7.62 (2H, d, J = 2.70 Hz), 7.44 (1H, d, J = 8.10 Hz), 7.33 (2H, d, J = 15.90 Hz), 7.19-7.19 (1H, m),
7.09-7.02 (1H, m), 6.85-6.81 (2H, m), 5.08 (1H, d, J = 9.00 Hz), 4.27-4.20 (1H, m), 3.82 (3H, s), 3.46 (3H, s), 3.23
(2H, d, J = 5.70 Hz). MS (EI) m/z 388 [M+]. Anal. (C19H20N2O5S) C, H, N. Calcd for: 58.75, 5.19, 7.21; Found:
58.38, 5.51, 7.12.
4.1.37. Methyl 2-(4-methylphenylsulfonamido)-3-(1H-indol-3-yl)propanoate (If)
Compound If was synthesized from methyl 2-amino-3-(1H-indol-3-yl)propanoate hydrochloride (3a) and
4-methylbenzene-1-sulfonyl chloride (2f) with the procedure described for compound Ia (white solid, 60.83%).
Mp = 108-109 °C.
IR(KBr): 518, 554, 586, 661, 755, 766, 818, 853, 950, 1091, 1159, 1213, 277, 1321, 1354, 1458, 1759, 2954,
3310, 3392 cm-1. 1H-NMR (300 MHz, CDCl3) δ 8.08 (1H, s), 7.61-7.31 (4H, m), 7.18-7.02 (5H, m), 5.12(1H, d, J
= 8.70 Hz), 4.28-4.21 (1H, m), 3.43(3H, s), 3.23 (2H, d, J = 5.40 Hz), 2.37 (3H, s). MS (EI) m/z 372 [M+]. Anal.

(C19H20N2O4S) C, H, N. Calcd for: 61.27, 5.41, 7.52; Found: 61.04, 5.28, 7.26.
4.1.38. Methyl 2-(4-bromophenylsulfonamido)-3-(1H-indol-3-yl)propanoate (Ig)
Compound Ig was synthesized from methyl 2-amino-3-(1H-indol-3-yl)propanoate hydrochloride (3a) and
4-bromobenzene-1-sulfonyl chloride (2g) with the procedure described for compound Ia (white solid, 49.27%).
Mp = 134-135 °C.
IR(KBr):555, 613, 737, 821, 949, 1008, 1068, 1089, 1110, 1158, 1226, 1275, 1334, 1176, 1458, 1573, 1732,
3215, 3379cm-1. 1H-NMR (300 MHz, CDCl3) δ 8.04 (1H, br s), 7.50-7.32 (6H, m), 7.22-6.99 (3H, m), 5.17(1H, d,
J = 8.70 Hz), 4.28-4.23 (1H, m), 3.53 (3H, s), 3.28-3.20 (2H, m). MS (EI) m/z 438 [M+]. Anal. (C18H17BrN2O4S) C,
H, N. Calcd for: 49.44, 3.92, 6.41; Found: 49.54, 3.85, 6.20.
4.1.39. Methyl 2-(4-fluorophenylsulfonamido)-3-(1H-indol-3-yl)propanoate (Ih)
Compound Ih was synthesized from methyl 2-amino-3-(1H-indol-3-yl)propanoate hydrochloride (3a) and
4-fluorobenzene-1-sulfonyl chloride (2h) with the procedure described for compound Ia (white solid, 42.30%).
Mp = 100-102 °C.
IR(KBr): 548, 661, 677, 745, 840, 951, 1008, 1091, 1153, 1168, 1244, 1291, 1338, 1432, 1458, 1494, 1591,
1
1731, 3269, 3353 cm-1. H-NMR (300 MHz, CDCl3) δ 8.05 (1H, br s), 7.70-7.32 (4H, m), 7.21-6.98 (5H, m),
5.13(1H, d, J = 8.70 Hz), 4.29-4.24 (1H, m), 3.51 (3H, s), 3.30-3.22 (2H, m). MS (EI) m/z 376 [M+]. Anal.
(C18H17FN2O4S) C, H, N. Calcd for: 57.44, 4.55, 7.44; Found: 57.19, 4.36, 7.25.
4.1.40. Methyl 2-(4-butoxyphenylsulfonamido)-3-phenylpropanoate (Ii)
Compound Ih was synthesized from methyl 2-amino-3-phenylpropanoate hydrochloride (3b) and
4-butoxybenzene-1-sulfonyl chloride (2a) with the procedure described for compound Ia (white solid, 44.26%).
Mp = 110-111 °C.
1
IR(KBr): 699, 805, 1018, 1093, 1157, 1260, 1344, 1597, 1729, 2958, 3286, 3414 cm-1. H-NMR (300 MHz,
DMSO) δ 7.66 (2H, d, J = 8.70 Hz), 7.26-7.23 (3H, m), 7.08-7.06 (2H, m), 6.87 (2H, d, J = 9.00 Hz), 4.98 (1H, d,
J = 9.00 Hz), 4.21-4.14 (1H, m), 3.99 (2H, t, J = 6.60 Hz), 3.51 (3H, s), 3.02 (2H, d, J = 6.00 Hz), 1.83-1.73 (2H,
m), 1.53-1.43 (2H, m), 0.98 (3H, t, J = 7.50 Hz). MS (ESI) m/z 391 [M+]. Anal. (C20H25NO5S) C, H, N. Calcd for:
61.36, 6.44, 3.58; Found: 61.07, 6.62, 3.38.
4.1.41. 2-(4-butoxyphenylsulfonamido)-3-(1H-indol-3-yl)propanoic acid (Ij)
LiOH
(1mol/L,
2.2
mL)
was
added
to
a
solution
of
methyl
2-(4-butoxyphenylsulfonamido)-3-(1H-indol-3-yl)propanoate (Ia, 0.43g, 0.001mol) in THF (10 mL). After being
stirred at r.t. for 10 h, the mixture was concentrated under reduced pressure and diluted acidified by HCl (0.1mol/L)

in water (20 mL). Then, the mixture was filtered to obtain the compound Ij as white solid (0.15g, 36.06%). Mp =
109-110 °C.
IR(KBr): 740, 830, 1096, 1150, 1726, 2395, 2954, 3400, 3664 cm-1. 1H-NMR (300 MHz, DMSO) δ 12.56 (1H,
s), 10.79 (1H, s), 8.04 (1H, d, J = 8.40 Hz), 7.48 (2H, d, J = 8.70 Hz), 7.30-7.26 (2H, m), 7.06-6.85 (5H, m), 3.98
(2H, d, J = 6.60 Hz), 3.88-3.80 (1H, m), 3.06-3.00 (1H, m), 2.86-2.80 (1H, m), 1.76-1.66 (2H, m), 1.51-1.38 (2H,
m), 0.98 (3H, t, J = 7.50 Hz). MS (ESI) m/z 416 [M+]. HR-MS ([M + NH4] +) Calcd for C21H28N3O5S, 434.1744;
Found: 434.1743. HPLC purity: 99.90%; retention time: 2.248 min (mobile phase: methanol/water 98:2, v/v).
4.1.42. N-(4-methoxyphenyl)-2-(3-(2-(4-methylphenylsulfonamido)ethyl)-1H-indol-1-yl)acetamide (IIa)
To a stirring solution of N-(2-(1H-indol-3-yl)ethyl)-4-methylbenzenesulfonamide (8c, 0.63g, 0.002mol) and
appropriate NaH in DMF (10 mL) was added 2-chloro-N-(4-methoxyphenyl)acetamide (10a, 0.80g, 0.004mol) at
5 °C under N2 flow. After being stirred at 5 °C for 2 h and at r.t. for another 4 h, the mixture was poured into water
and extracted with CHCl3 (20 mL х 3). The organic layer was concentrated and purified by CC (CH2Cl2/AcEt =
2:1) to obtain the compound IIa as white solid (0.42g, 43.75%). Mp = 160-161 °C.
1
IR(KBr): 698, 743, 1165, 1241, 1324, 1511, 1612, 1663, 3260, 3414 cm-1. H-NMR (300 MHz, DMSO) δ
10.15 (1H, s), 7.70-7.64 (3H, m), 7.48(2H, d, J = 9.00 Hz), 7.41-7.34 (4H, m), 7.14-7.08 (2H, m), 6.98-6.96 (1H,
m), 6.88 (2H, d, J = 9.00 Hz), 4.92 (2H, s), 3.71 (3H, s), 3.02-2.95 (2H, m), 2.82-2.72 (2H, m), 2.36 (3H, s).
13C-NMR (75MHz, CDCl3) δ 165.9, 142.7, 137.3, 131.7, 129.4, 128.1, 127.3, 126.8, 121.6, 121.2, 119.0, 118.5,
113.7, 111.8, 109.5, 54.7, 49.4, 43.5, 25.4, 20.4. MS (ESI) m/z 477 [M+]. Anal. (C26H27N3O4S) C, H, N. Calcd for:
65.39, 5.70, 8.80; Found: 65.39, 5.68, 8.48.
4.1.43. N-(4-chlorophenyl)-2-(3-(2-(4-methylphenylsulfonamido)ethyl)-1H-indol-1-yl)acetamide (IIb)
Compound IIb was synthesized from N-(2-(1H-indol-3-yl)ethyl)-4-methylbenzenesulfonamide (8c) and
2-chloro-N-(4-chlorophenyl)acetamide (10b) with the procedure described for compound IIa (white solid,
36.57%). Mp = 192-193 °C.
IR(KBr): 552, 736, 1086, 1093, 1156, 1387, 1534, 1597, 1697, 2296, 3259 cm-1. 1H-NMR (300 MHz, DMSO)
δ 10.44 (1H, s), 7.69-7.65 (3H, m), 7.62-7.59 (2H, m), 7.41-7.34 (6H, m), 7.15 (1H, s), 7.10 (1H, t, J = 8.10 Hz),
7.00 (1H, t, J = 7.20 Hz), 4.97 (2H, s), 3.02-2.95 (2H, m), 2.82-2.72 (2H, m), 2.36 (3H, s). MS (ESI) m/z 481 [M+].
Anal. (C25H24ClN3O3S) C, H, N. Calcd for: 62.30, 5.02, 8.72; Found: 62.13, 4.94, 8.38.
4.1.44. N-(4-methoxyphenyl)-2-(3-(2-(4-methoxyphenylsulfonamido)ethyl)-1H-indol-1-yl)acetamide (IIc)
Compound IIc was synthesized from N-(2-(1H-indol-3-yl)ethyl)-4-methoxybenzenesulfonamide (8b) and
2-chloro-N-(4-methoxyphenyl)acetamide (10a) with the procedure described for compound IIa (white solid,

28.91%). Mp = 155-156 °C.
IR(KBr): 562, 736, 832, 1153, 1248, 1326, 1514, 1534, 1676, 3287 cm-1. 1H-NMR (300 MHz, DMSO) δ 10.16
(1H, s), 7.72 (2H, d, J = 9.00 Hz), 7.59 (1H, t, J = 5.70 Hz), 7.50-7.47 (2H, m), 7.41-7.34 (2H, m), 7.15-7.06 (4H,
m), 7.02-6.98 (1H, m), 6.90-6.86 (2H, m), 4.92 (2H, s), 3.81 (3H, s), 3.71 (3H, s), 3.02-2.94 (2H, m), 2.82-2.73
(2H, m). MS (ESI) m/z 493 [M+]. Anal. (C26H27ClN3O5S) C, H, N. Calcd for: 63.27, 5.51, 8.51; Found: 63.02, 5.55,
8.44.
4.1.45. N-(4-chlorophenyl)-2-(3-(2-(4-methoxyphenylsulfonamido)ethyl)-1H-indol-1-yl)acetamide (IId)
Compound IId was synthesized from N-(2-(1H-indol-3-yl)ethyl)-4-methoxybenzenesulfonamide (8b) and
2-chloro-N-(4-chlorophenyl)acetamide (10b) with the procedure described for compound IIa (white solid,
88.90%). Mp = 166-167 °C.
1
IR(KBr): 738, 832, 1153, 1328, 1493, 1537, 1599, 1688, 2922, 3268 cm-1. H-NMR (300 MHz, DMSO) δ
10.45 (1H, s), 7.72 (2H, d, J = 9.00 Hz), 7.63-7.57 (3H, m), 7.42-7.34 (4H, m), 7.16-7.05 (4H, m), 7.03-6.98 (1H,
m), 4.97 (2H, s), 3.81 (3H, s), 3.01-2.94 (2H, m), 2.82-2.73 (2H, m). MS (ESI) m/z 497 [M+]. HR-MS ([M + H] +)
Calcd for C25H24N3O5S, 498.1249; Found: 498.1260. HPLC purity: 99.64%; retention time: 3.615 min (mobile
phase: methanol/water 98:2, v/v)
4.1.46. N-(4-methoxyphenyl)-2-(3-(2-(4-butoxyphenylsulfonamido)ethyl)-1H-indol-1-yl) acetamide (IIe)
Compound IIe was synthesized from N-(2-(1H-indol-3-yl)ethyl)-4-butoxybenzenesulfonamide (8a) and
2-chloro-N-(4-methoxyphenyl)acetamide (10a) with the procedure described for compound IIa (white solid,
74.25%). Mp = 160-161 °C.
IR(KBr): 584, 739, 829, 1154, 1319, 1524, 1600, 1673, 2954, 3306, 3414 cm-1. 1H-NMR (300 MHz, DMSO) δ
10.16 (1H, s), 7.70 (2H, d, J = 8.70 Hz), 7.58 (1H, t, J = 5.70 Hz), 7.49 (2H, d, J = 9.00 Hz), 7.41-7.34 (2H, m),
7.15-7.05 (4H, m), 7.02-6.97 (1H, m), 6.879 (2H, d, J = 9.00 Hz), 4.92 (2H, s), 4.02 (2H, t, J = 6.60 Hz), 3.71 (3H,
s), 3.01-2.94 (2H, m), 2.81-2.76 (2H, m), 1.75-1.65 (2H, m), 1.46-1.38 (2H, m), 0.92 (3H, t, J = 7.50 Hz). MS (ESI)
m/z 535 [M+]. Anal. (C29H33N3O5S) C, H, N. Calcd for: 65.03, 6.21, 7.84; Found: 64.98, 6.39, 7.73.
4.1.47. N-(4-chlorophenyl)-2-(3-(2-(4-butoxyphenylsulfonamido)ethyl)-1H-indol-1-yl) acetamide (IIf)
Compound IIf was synthesized from N-(2-(1H-indol-3-yl)ethyl)-4-butoxybenzenesulfonamide (8a) and
2-chloro-N-(4-chlorophenyl)acetamide (10b) with the procedure described for compound IIa (white solid,
17.30%). Mp = 151-153 °C.
IR(KBr): 588, 743, 830, 1153, 1326, 1526, 1596, 1683, 2956, 3272, 3311 cm-1. 1H-NMR (300 MHz, DMSO) δ
10.45 (1H, s), 7.71 (2H, d, J = 8.70 Hz), 7.63-7.56 (3H, m), 7.42-7.34 (4H, m), 7.15-6.97 (5H, m), 4.98 (2H, s),

4.02 (2H, t, J = 6.30 Hz), 3.01-2.95 (2H, m), 2.79 (2H, t, J = 6.90 Hz), 1.75-1.65 (2H, m), 1.46-1.38 (2H, m), 0.92
(3H, t, J = 7.50 Hz). MS (ESI) m/z 539 [M+]. Anal. (C28H30ClN3O4S) C, H, N. Calcd for: 62.27, 5.60, 7.78; Found:
62.16, 5.46, 7.45.
4.1.48. N-phenyl-2-(3-((2-tosylhydrazono)methyl)-1H-indol-1-yl)acetamide (IIIa)
Compound IIIa was synthesized from N'-((1H-indol-3-yl)methylene)-4-methylbenzenesulfonohydrazide (12a)
and 2-chloro-N-phenylacetamide (10c) with the procedure described for compound IIa (yellow solid, 23.59%).
Mp = 150 °C (dec).
1
IR(KBr): 550, 691, 742, 1162, 1303, 1444, 1539, 1600, 1671, 3035, 3192, 3328, 3441 cm-1. H-NMR (300
MHz, DMSO) δ 10.89 (1H, s), 10.40 (1H, s), 8.08 (1H, s), 7.99 (1H, d, J = 7.20 Hz), 7.80 (2H, d, J = 8.10 Hz),
7.74 (1H, s), 7.58-7.55 (2H, m), 7.45-7.28 (5H, m), 7.19-7.16 (2H, m), 7.09-7.00 (1H, m), 5.06 (2H, s), 2.33 (3H,
s). 13C-NMR (75MHz, CDCl3) δ 165.7, 144.4, 143.1, 138.6, 137.5, 136.3, 134.3, 129.4, 128.8, 127.3, 124.5, 123.5,
122.8, 121.7, 120.8, 199.1, 110.7, 110.1, 49.1, 20.9. MS (ESI) m/z 445 ([M - H]+), 447 ([M + H]+). Anal.
(C24H22N4O3S) C, H, N. Calcd for: 64.56, 4.97, 12.55; Found: 64.23, 5.05, 12.36.
4.1.49. N-(2,4-dimethylphenyl)-2-(3-((2-tosylhydrazono)methyl)-1H-indol-1-yl)acetamide (IIIb)
Compound IIIb was synthesized from N'-((1H-indol-3-yl)methylene)-4-methylbenzenesulfonohydrazide (12a)
and 2-chloro-N-(2,4-dimethylphenyl)acetamide (10d) with the procedure described for compound IIa (white solid,
21.10%). Mp = 213-214 °C (dec).
1
IR(KBr): 553, 682, 742, 814, 1164, 1311, 1467, 1529, 1616, 1669, 3169, 3304 cm-1. H-NMR (300 MHz,
DMSO) δ 10.81 (1H, s), 9.62 (1H, s), 8.08 (1H, s), 7.99 (1H, d, J = 7.80 Hz), 7.81-7.75 (3H, m), 7.47 (1H, d, J =
7.50 Hz), 7.38 (2H, d, J = 8.10 Hz), 7.26-7.14 (3H, m), 7.01 (1H, s), 6.95-6.92 (1H, m), 5.07 (2H, s), 2.33 (3H, s),
2.22 (3H, s), 2.13 (3H, s). MS (ESI) m/z 475 ([M + H]+). Anal. (C26H26N4O3S) C, H, N. Calcd for: 65.80, 5.52,
11.87; Found: 66.17, 5.80, 11.85.
4.1.50. N-(2,5-dimethylphenyl)-2-(3-((2-tosylhydrazono)methyl)-1H-indol-1-yl)acetamide (IIIc)
Compound IIIc was synthesized from N'-((1H-indol-3-yl)methylene)-4-methylbenzenesulfonohydrazide (12a)
and 2-chloro-N-(2,5-dimethylphenyl)acetamide (10e) with the procedure described for compound IIa (white solid,
15.85%). Mp = 219-220 °C (dec).
1
IR(KBr): 583, 743, 813, 948, 1164, 1310, 1467, 1534, 1670, 3182, 3303, 3470 cm-1. H-NMR (300 MHz,
DMSO) δ 10.81 (1H, s), 9.63 (1H, s), 8.09 (1H, s), 7.99 (1H, d, J = 7.50 Hz), 7.82-7.76 (3H, m), 7.47 (1H, d, J =
8.10 Hz), 7.38 (2H, d, J = 8.10 Hz), 7.25-7.17 (3H, m), 7.09-7.07 (1H, m), 6.90-6.88 (1H, m), 5.09 (2H, s), 2.33
(3H, s), 2.21 (3H, s), 2.14 (3H, s). MS (ESI) m/z 473 ([M - H]+), 475 ([M + H]+). Anal. (C26H26N4O3S) C, H, N.

Calcd for: 65.80, 5.52, 11.87; Found: 65.64, 5.68, 11.82.
4.1.51. N-(3,4-dimethylphenyl)-2-(3-((2-tosylhydrazono)methyl)-1H-indol-1-yl)acetamide (IIId)
Compound IIId was synthesized from N'-((1H-indol-3-yl)methylene)-4-methylbenzenesulfonohydrazide (12a)
and 2-chloro-N-(3,4-dimethylphenyl)acetamide (10f) with the procedure described for compound IIa (white solid,
25.30%). Mp = 220-221 °C (dec).
1
IR(KBr): 583, 742, 814, 949, 1092, 1164, 1528, 1669, 2832, 3168, 3305, 3472 cm-1. H-NMR (300 MHz,
DMSO) δ 10.88 (1H, s), 10.23 (1H, s), 8.08 (1H, s), 7.99 (1H, d, J = 7.50 Hz), 7.82-7.79 (2H, m), 7.74 (1H, s),
7.16-7.40 (7H, m), 7.06-7.03 (1H, m), 5.02 (2H, s), 2.34 (3H, s), 2.15 (6H, s). MS (ESI) m/z 473 ([M - H]+), 497
([M + Na]+). Anal. (C26H26N4O3S) C, H, N. Calcd for: 65.80, 5.52, 11.87; Found: 66.00, 5.86, 11.90.
4.1.52. N-(4-methoxyphenyl)-2-(3-((2-tosylhydrazono)methyl)-1H-indol-1-yl)acetamide (IIIe)
Compound IIIe was synthesized from N'-((1H-indol-3-yl)methylene)-4-methylbenzenesulfonohydrazide (12a)
and 2-chloro-N-(4-methoxyphenyl)acetamide (10a) with the procedure described for compound IIa (white solid,
18.92%). Mp = 217-219 °C (dec).
1
IR(KBr): 593, 714, 832, 1034, 1303, 1512, 1555, 1666, 2355, 2960, 3195, 3685, 3849 cm-1. H-NMR (300
MHz, DMSO) δ 10.89 (1H, s), 10.27 (1H, s), 8.08 (1H, s), 7.99 (1H, d, J = 7.50 Hz), 7.82-7.79 (3H, m), 7.49-7.37
(5H, m), 7.22-7.16 (2H, m), 6.89-6.86 (2H, m), 5.03 (2H, s), 3.07 (3H, s), 2.34 (3H, s). MS (ESI) m/z 475 ([M -
H]+), 477 ([M + H]+). Anal. (C25H24N4O4S) C, H, N. Calcd for: 63.01, 5.08, 11.76; Found: 62.85, 5.10, 12.10.
4.1.53. N-(4-(trifluoromethoxy)-2-(3-((2-tosylhydrazono)methyl)-1H-indol-1-yl) phenyl)acetamide (IIIf)
Compound IIIf was synthesized from N'-((1H-indol-3-yl)methylene)-4-methylbenzenesulfonohydrazide (12a)
and 2-chloro-N-(4-(trifluoromethoxy)phenyl)acetamide (10g) with the procedure described for compound IIa
(white solid, 21.77%). Mp = 220-222 °C (dec).
IR(KBr): 550, 665, 743, 844, 1397, 1470, 1510, 1694, 2228, 2932, 3068, 3198, 3366 cm-1. 1H-NMR (300 MHz,
DMSO) δ 10.89 (1H, s), 10.60 (1H, s), 8.08 (1H, s), 7.99 (1H, d, J = 7.50 Hz), 7.81 (2H, d, J = 8.10 Hz), 7.75 (1H,
s), 7.68 (2H, d, J = 9.00 Hz), 7.45-7.31 (5H, m), 7.24-7.14 (2H, m), 5.08 (2H, s), 2.34 (3H, s). MS (ESI) m/z 531
([M + H]+). Anal. (C25H21F3N4O4S) C, H, N. Calcd for: 56.60, 3.90, 10.56; Found: 56.31, 3.99, 10.43.
4.1.54. N-(4-chlorophenyl)-2-(3-((2-tosylhydrazono)methyl)-1H-indol-1-yl)acetamide (IIIg)
Compound IIIg was synthesized from N'-((1H-indol-3-yl)methylene)-4-methylbenzenesulfonohydrazide (12a)
and 2-chloro-N-(4-chlorophenyl)acetamide (10b) with the procedure described for compound IIa (white solid,
41.74%). Mp = 229-230 °C (dec).
1
IR(KBr): 550, 742, 953, 1091, 1396, 1491, 1540, 1600, 1617, 1740, 3053, 3180, 3358 cm-1. H-NMR (300

MHz, DMSO) δ 10.89 (1H, s), 10.53 (1H, s), 8.08 (1H, s), 7.99 (1H, d, J = 7.20 Hz), 7.82-7.74 (3H, m), 7.60 (2H,
d, J = 8.70 Hz), 7.45-7.35 (5H, m), 7.22-7.17 (2H, m), 5.07 (2H, s), 2.34 (3H, s). MS (ESI) m/z 481 ([M + H]+).
Anal. (C24H21ClN4O3S) C, H, N. Calcd for: 59.93, 4.40, 11.65; Found: 59.80, 4.38, 11.77.
4.1.55. N-(2-chlorophenyl)-2-(3-((2-tosylhydrazono)methyl)-1H-indol-1-yl)acetamide (IIIh)
Compound IIIh was synthesized from N'-((1H-indol-3-yl)methylene)-4-methylbenzenesulfonohydrazide (12a)
and 2-chloro-N-(2-chlorophenyl)acetamide (10h) with the procedure described for compound IIa (white solid,
22.89%). Mp = 216-217 °C (dec).
1
IR(KBr): 749, 951, 1159, 1303, 1468, 1537, 1681, 2356, 2925, 3175, 3308, 3849 cm-1. H-NMR (300 MHz,
DMSO) δ 10.89 (1H, s), 9.91 (1H, s), 8.09 (1H, s), 7.99 (1H, d, J = 7.80 Hz), 7.82-7.69 (4H, m), 7.52-7.45 (2H, m),
7.40-7.37 (2H, m), 7.33-7.15 (4H, m), 5.17 (2H, s), 2.33 (3H, s). MS (ESI) m/z 479 ([M - H]+), 481 ([M + H]+).
Anal. (C24H21ClN4O3S) C, H, N. Calcd for: 59.93, 4.40, 11.65; Found: 59.81, 4.44, 11.68.
4.1.56. N-(2,4-difluorophenyl)-2-(3-((2-tosylhydrazono)methyl)-1H-indol-1-yl)acetamide (IIIi)
Compound IIIi was synthesized from N'-((1H-indol-3-yl)methylene)-4-methylbenzenesulfonohydrazide (12a)
and 2-chloro-N-(2,4-difluorophenyl)acetamide (10i) with the procedure described for compound IIa (white solid,
14.53%). Mp = 201-202 °C (dec).
IR(KBr): 590, 753, 970, 1181, 1385, 1555, 1617, 1673, 3284, 3409, 3473 cm-1. 1H-NMR (300 MHz, DMSO) δ
10.89 (1H, s), 10.20 (1H, s), 8.08 (1H, s), 7.99 (1H, d, J = 7.80 Hz), 7.85-7.74 (3H, m), 7.45-7.31 (4H, m),
7.25-7.14 (2H, m), 7.07-7.02 (2H, m), 5.13 (2H, s), 2.33 (3H, s). MS (ESI) m/z 481 ([M - H]+). Anal.
(C24H20F2N4O3S) C, H, N. Calcd for: 59.74, 4.18, 11.61; Found: 59.59, 4.15, 11.63.
4.1.57. 4-(2-(3-((2-tosylhydrazono)methyl)-1H-indol-1-yl)acetamido)phenyl propionate (IIIj)
Compound IIIj was synthesized from N'-((1H-indol-3-yl)methylene)-4-methylbenzenesulfonohydrazide (12a)
and 4-(2-chloroacetamido)phenyl propionate (10j) with the procedure described for compound IIa (white solid,
27.25%). Mp = 209-210 °C (dec).
1
IR(KBr): 550, 737, 859, 1284, 1549, 1620, 1675, 1714, 2939, 3060, 3125, 3189, 3458 cm-1. H-NMR (300
MHz, DMSO) δ 10.89 (1H, s), 10.74 (1H, s), 8.09 (1H, s), 7.99 (1H, d, J = 6.90 Hz), 7.94-7.91 (2H, m), 7.82-7.69
(5H, m), 7.47-7.38 (3H, m), 7.24-7.15 (2H, m), 5.25 (2H, s), 4.24 (2H, q, J = 6.90 Hz), 2.33 (3H, s), 1.30 (3H, t, J
= 7.20 Hz). MS (ESI) m/z 519 ([M - H]+), 517 ([M + H]+). Anal. (C27H26N4O5S) C, H, N. Calcd for: 62.53, 5.05,
10.80; Found: 62.23, 4.68, 10.63.
4.1.58. N-phenyl-2-(5-methoxy-3-((2-tosylhydrazono)methyl)-1H-indol-1-yl)acetamide (IIIk)
Compound
IIIk
was
synthesized
from
N'-((5-methoxy-1H-indol-3-yl)methylene)-4-

methylbenzenesulfonohydrazide (12b) and 2-chloro-N-phenylacetamide (10c) with the procedure described for
compound IIa (white solid, 30.47%). Mp = 207-208 °C (dec).
1
IR(KBr): 549, 690, 1054, 1163, 1258, 1309, 1536, 1667, 2826, 2932, 3295, 3306, 3457 cm-1. H-NMR (300
MHz, DMSO) δ 10.87 (1H, s), 10.36 (1H, s), 8.08 (1H, s), 7.80 (2H, d, J = 8.10 Hz), 7.70 (1H, s), 7.56 (2H, d, J =
7.80 Hz), 7.48 (1H, d, J = 2.40 Hz), 7.41-7.27 (5H, m), 7.08-7.03 (1H, m), 6.83 (1H, d, J = 2.40 Hz), 5.02 (2H, s),
3.76 (3H, s), 2.39 (3H, s). MS (ESI) m/z 475 ([M - H]+), 477 ([M + H]+). HR-MS ([M + H]+) Calcd for
C25H24N4O4S, 477.1591; Found: 477.1596. HPLC purity: 99.74%, retention time: 2.682min (mobile phase:
methanol/water 98:2, v/v).
4.1.59. N-(4-chlorophenyl)-2-(5-methoxy-3-((2-tosylhydrazono)methyl)-1H-indol-1-yl)acetamide (IIIl)
Compound
IIIl
was
synthesized
from
N'-((5-methoxy-1H-indol-3-yl)methylene)-4-
methylbenzenesulfonohydrazide (12b) and 2-chloro-N-(4-chlorophenyl)acetamide (10b) with the procedure
described for compound IIa (white solid, 26.49%). Mp = 226-228 °C (dec).
1
IR(KBr): 548, 670, 812, 1092, 1163, 1229, 1304, 1490, 1533, 1673, 2927, 3199, 3310 cm-1. H-NMR (300
MHz, DMSO) δ 10.87 (1H, s), 10.49 (1H, s), 8.08 (1H, s), 7.82-7.79 (2H, m), 7.69 (1H, s), 7.61-7.58 (2H, m),
7.48-7.47 (1H, m), 7.41-7.32 (5H, m), 6.86-6.82 (1H, m), 5.02 (2H, s), 3.76 (3H, s), 2.35 (3H, s). MS (ESI) m/z
511 ([M]+). Anal. (C25H23ClN4O4S) C, H, N. Calcd for: 58.76, 4.54, 10.96; Found: 58.54, 4.50, 10.87.
4.1.60. N-(2,4-difluorophenyl)-2-(5-methoxy-3-((2-tosylhydrazono)methyl)-1H-indol-1-yl)acetamide (IIIm)
Compound
IIIm
was
synthesized
from
N'-((5-methoxy-1H-indol-3-yl)methylene)-4-
methylbenzenesulfonohydrazide (12b) and 2-chloro-N-(2,4-difluorophenyl)acetamide (10i) with the procedure
described for compound IIa (white solid, 14.62%). Mp = 221-222 °C (dec).
1
IR(KBr): 548, 676, 844, 949, 1163, 1314, 1485, 1543, 1616, 1702, 2809, 2971, 3231, 3380 cm-1. H-NMR
(300 MHz, DMSO) δ 10.89 (1H, s), 10.18 (1H, s), 8.07 (1H, s), 7.85-7.77 (3H, m), 7.70 (1H, s), 7.48 (1H, s),
7.47-7.31 (4H, m), 7.08-7.02 (1H, m), 6.87-6.84 (1H, m), 5.09 (2H, s), 3.77 (3H, s), 2.34 (3H, s). MS (ESI) m/z
513 ([M + H]+). Anal. (C25H22F2N4O4S) C, H, N. Calcd for: 58.59, 4.33, 10.93; Found: 58.64, 4.05, 11.15.
4.2. Biological evaluation
4.2.1. Whole-cell patch lamp technique
The HEK 293 cell line that stably expressed hKv1.5 potassium channel and the CHO cell line that stably
expressed hERG (IKr) potassium channel were kindly gifts 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
37
Kong, SAR, China). Transfected HEK 293 cells and transfected CHO cells (stably expressed hERG potassium

channel) 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.
38
Isolated cells from ventricular muscle were prepared as described previously.
Briefly, male guinea pigs
(weight about 350 g) were sacrificed by cervical dislocation. The hearts were dissected and perfused retrograde via
the aorta at 37 °C with nominally Ca2+-free Tyrode solution (in mM: 117 NaCl, 5.4 KCl, 0.5 MgCl2, 1.2 NaH2PO4,
10 glucose, 20 taurine, pH adjusted to 7.4 with NaOH), then with Tyrode solution containing 50 μmol/L CaCl2, 0.3
mg/mL collagenase (type II; Worthington, Lakewood, NJ), 1 mg/mL bovine serum albumin (Sunshine, Nanjing,
China). After 10-15 min of treatment, the digested left ventricular free wall was placed in Kraftbruhe (KB) solution
(in mM: L-glutamic acid 50, KCl 40, KOH 70, taurine 20, KH2PO4 20, MgCl2 3, glucose 10, EGTA 0.5, HEPES
10, pH adjusted to 7.4 with KOH) and pipetted gently to produce a cell suspension. The solution was agitated with
95% O2 + 5% CO2. The entire enzyme isolation procedure was performed at 37 °C. Isolated myocytes were kept at
room temperature (25 °C) in medium for 1 h. A small aliquot (2 mL) of the solution containing the isolated cells
was placed in an open perfusion chamber and mounted on the stage of an inverted microscope. Only quiescent and
rod-shaped cells showing clear striations were selected for experiments. 39
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: 500-ms depolarizing pulse steps to +60 mV from a
holding potential of -80 mV at 0.1 Hz; 39 IKr: the holding potential was -70 mV and a family of voltage steps from
-60 to +50 mV for 2 sec., in 10-mV increments to evoke step currents, followed by a re-polarisation step to -40
40
mV for 2 sec., to induce tail currents (stimulation frequency of 0.1 Hz);
INa: 30-ms depolarizing pulse to
41
potentials ranging from -80 mV to +40 mV in 10-mV increments at 0.5 Hz, the holding potential was -90 mV.
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. The data are presented as the mean and
standard deviation (x ± SD), n = 3. All experiments were performed at 25 °C.

4.2.2. The effect of compounds IIIi and IIIl on ERP in isolated rat hearts
SD rats (male, 230 ± 30 g) were anesthetized with 10% chloral hydrate (intraperitoneal injection). Hearts
were rapidly removed through a midline sternal incision, and left atrium and right ventricular were isolated
immediately and put into Krebs-Henseleit solution bath (composition in mmol/L: NaCl 118, KCl 4.7, CaCl2 2.5,
MgSO4 1.2, KH2PO4 1.2, NaHCO3 24, Glucose 10 and the pH was adjusted to 7.4) gassed with 95% O2-5% CO2.
Compounds IIIi and IIIl were added to the solution bath in serial concentrations of 0.1, 1.0, 10 and 50 μmol/L,
allowing 10 minutes for equilibration. Recording the ERP (BL-420 system of Tai Meng, Cheng Du) of left atrium
and right ventricular before and after compounds added.
Data were analyzed using Student’s t test and are presented as the mean (x ± SEM), and n = 3.
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 State Key Laboratory of Natural Medicines and Jiang Su
Key Laboratory of Drug Design and Optimization, 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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