Rottlerin: Structure Modifications and KCNQ1/KCNE1 Ion Channel Activity

Article abstract of DOI:10.1002/cmdc.202000083

The slow delayed rectifier potassium current (I_Ks) is formed by the KCNQ1 (K_v7.1) channel, an ion channel of four α-subunits that modulates KCNE1 β-subunits. I_Ks is central to the repolarization of the cardiac action potential. Loss of function mutation reducing ventricular cardiac I_Ks cause the long-QT syndrome (LQTS), a disorder that predisposes patients to arrhythmia and sudden death. Current therapy for LQTS is inadequate. Rottlerin, a natural product of the kamala tree, activates I_Ks and has the potential to provide a new strategy for rational drug therapy. In this study, we show that simple modifications such as penta-acetylation or penta-methylation of rottlerin blunts activation activity. Total synthesis was used to prepare side-chain-modified derivatives that slowed down KCNQ1/KCNE1 channel deactivation to different degrees. A binding hypothesis of rottlerin is provided that opens the way to improved I_Ks activators as novel therapeutics for the treatment of LQTS.

Full text of DOI:10.1002/cmdc.202000083

Accepted Article
Title: Rottlerin: Structure Modifications and KCNQ1/KCNE1 Ion
Channel Activity
Authors: Marco Lübke, Julian A. Schreiber, Thang Le Quoc, Florian
Körber, Jasmin Müller, Sivatharushan Sivanathan, Veronika
Matschke, Janina Schubert, Nathalie Strutz-Seebohm,
Guiscard Seebohm, and Jürgen Scherkenbeck
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To be cited as: ChemMedChem 10.1002/cmdc.202000083
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1/2020

ChemMedChem
10.1002/cmdc.202000083
FULL PAPER
Rottlerin: Structure Modifications and KCNQ1/KCNE1
Ion Channel Activity
a*
b*
c*
a
a
Marco Lübke , Dr. Julian A. Schreiber , Dr. Thang Le Quoc , Dr. Florian Körber , Dr. Jasmin Müller ,
a
d
b
Dr. Sivatharushan Sivanathan , Dr. Veronika Matschke , Janina Schubert , Prof. Nathalie Strutz-
Seebohm , Prof. Guiscard Seebohm , Prof. Jürgen Scherkenbeck
b
b*
a*
a
University Wuppertal, Faculty of Mathematics and Natural Sciences, 42119 Wuppertal, Germany
University Hospital Münster, Institut für Genetik von Herzerkrankungen, Albert-Schweitzer-Campus 1, 48149 Münster, Germany
Hue University, Department of Chemistry, 34 Le Loi St., Hue City, Vietnam
b
c
d
Ruhr University Bochum, Medical Faculty, Institute of Anatomy, Department of Cytology, 44801 Bochum, Germany
*
these authors contributed equally to the study.
Abstract: The slow delayed rectifier potassium current (IKs) is
formed by the KCNQ1 (K 7.1) channel, an ion channel bearing
Inhibitory activities have been reported for several kinases such
as p38-regulated/activated protein kinase (PRAK), mitogen-
activated protein kinase-activated protein kinase 2 (MAP-KAPK-
v
four α-subunits and modulating KCNE1 β-subunits. IKs is central
in the repolarization of the cardiac action potential. Loss of
function mutation reducing ventricular cardiac IKs cause the
long-QT syndrome (LQTS), a disorder that predisposes patients
to arrhythmia and sudden death. Current therapy for LQTS is
inadequate. Rottlerin, a natural product of the kamala tree,
activates IKs and has the potential to provide a new strategy for
rational drug therapy. In this study, we show that simple
modifications such as penta-acetylation or penta-methylation of
rottlerin blunts activation activity. A total synthetic approach was
used for the preparation of side chain modified derivatives, that
slowed down KCNQ1/KCNE1 channel deactivation to different
degrees. A binding hypothesis of rottlerin is provided, which
opens the path for improved IKs activators as novel therapeutics
for the treatment of LQTS.
2
+
2), protein kinase B (PKB, Akt), Ca /calmodulin-dependent
protein kinase (CaMK), and protein kinase C-delta (PKC-δ).[ 8,9] In
2005 Zakharov and coworkers first described an agonistic activity
on a BK potassium channel (SLO-1).[ Recently, we identified
rottlerin as a potent KCNQ1 and KCNQ4 activator.[ There is a
10]
11]
V
well-recognized need for improved therapies in K 7 based
channelopathies.[ A drug that activates the respective KCNQ1
12]
+
K channel, might be useful for treatment of inherited forms of
potentially lethal LQTS, high blood pressure, diabetes and
hearing impairment.[12,13] Currently, there is no selective, highly
potent published KCNQ1/KCNE1 activator known, which is
available for the clinical use of LQTS. In this paper, we describe
the first rottlerin derivatives, obtained by derivatization of the
natural product and total synthesis. KCNQ1/KCNE1 activities as
well as first structure activity relationships are studied and
discussed.
Introduction
The kamala tree, Mallotus philippensis (Lam.) Muell. Arg. var. Results and Discussion
philippensis (Euphorbiaceae) is distributed widely in Southeast
Asia. The granular hairs on the surface of the fruits of M.
philippensis are covered with a reddish powder, which is used
since ancient times in traditional Indian medicine and as a natural
red dye.[1,2] Numerous chemical constituents have been isolated
from kamala powder, the majority of which belongs to the family
of polyphenols.[3] A diverse range of biological activities has been
reported for kamala powder and extracts thereof, including
Syntheses of rottlerin derivatives
Though, the structure of rottlerin is known for decades, the first
two total syntheses were published only recently. Both syntheses
rely on a convergent, two building-block approach, consisting of a
chromene and a phloroglucinol unit which are coupled in the final
steps via a methylene bridge. In the Wang[14] synthesis three
different protecting groups and two different starting materials are
used, which renders this route less straightforward compared to
the Kumar[15] route. Since rottlerin is known to be unstable under
intermediate acidic and basic conditions, the choice of the
protective group strategy and the method for the introduction of
the methylene bridge between the chromene and phloroglucinol
building blocks are essential for success.[16,17]
[4-
anthelmintic, antibacterial, antiplasmodial, and cytotoxic effects.
7
]
Among various dimeric phloroglucinol compounds, isolated
from M. philippensis, only the major constituent rottlerin (1), also
known as mallotoxin, has been studied more intensively for its
biological potential (Figure 1).
After manifold efforts to develop a more efficient rottlerin
synthesis, we finally embarked with minor modifications on the
Kumar synthesis for the preparation of rottlerin derivatives 5b-d
(
Scheme 1). Due to our experience, only the MOM-group was
compatible with all steps of the synthesis, even if the yields (5a-
d) of the deprotection reaction were not optimal (Table 1). The
acidic conditions (3M HCl) led to dimerization and decomposition
of the chromene units, especially in case of the electron-rich
methoxy substituent (5d). With more diluted HCl the deprotection
Figure 1. Structures of rottlerin (1) and rottlerone (2).
1
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ChemMedChem
10.1002/cmdc.202000083
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became inacceptable slow, again with concomitant formation of
side products. Remarkably, even the structurally related MEM-
protective group turned out to be unsuitable. With TFA or HCl
all formaldehyde based standard procedures for coupling
symmetric polyphenol building blocks under basic or acidic
conditions failed for the unsymmetrical rottlerin.[23,24] We generally
observed either decomposition or the preferred formation of
symmetrical products, regardless of whether the methylol group
was pre-formed on the chromene or phloroglucinol moiety or a
mixture of building blocks was used. Trials to introduce a
chloromethyl or bromomethyl group into methylether protected
acetyl phloroglucinol and subsequent palladium catalysed cross-
coupling reactions to introduce the chromene unit were
unsuccessful, too. Already the formation of halogenomethyl
derivatives failed due to an unexpected replacement of the acetyl
group by a second chloromethyl or bromomethyl group. Also, the
use of MOMCl as a one-carbon source under acidic conditions
was not successful.[25] Only Eschenmoser’s salt, used by Kumar,
gave satisfactory yields in the coupling reaction of the
phloroglucinol and the chromene moieties.[23,26] Noticeably, the
dimethylaminomethyl group has to be introduced into the
chromene unit. The corresponding phloroglucinol derivative is
accessible only in low yields and does not couple to the chromene
unit.
(
6M) either no reaction or decomposition of the chromene unit
was observed. Cleavage with BBr led to the unexpected
3
formation of rottlerone (2, Figure 1) by intermediate formation of
an oxonium species, which attacks a second chromene molecule.
Both, methyl ether or silyl ether protective groups provided
problems with selective cleavage and/or stability in different
stages of the synthesis. Anyway, the construction of chromene
building blocks 5a-f and phloroglucinol 7 (Scheme 1) is standard
chemistry in polyphenol and flavonoid synthesis.[18-22]
Finally, we were able to synthesize rottlerin in multi-milligram
amounts and the first derivatives (8b-d) with modified side chain
arenes. However, rottlerin can be obtained more straightforward
in gram-quantities by extraction from natural kamala powder,
following a literature procedure.[27] Reaction of the natural product
with dimethylsulfate yielded pentamethyl rottlerin (9) in 73% yield.
Reaction with acetic anhydride gave pentaacetyl rottlerin (10) in
92% yield, respectively (Figure 2).
Figure 2. Rottlerin derivatives by modification of the natural product.
Compounds 8b-d increase steady-state activation of
KCNQ1/KCNE1 channels.
Scheme 1: Total synthesis of rottlerin (1) and derivatives 8b-f.
Compounds 8b-d were tested at heteromeric KCNQ1/KCNE1
channels, expressed in Xenopus laevis oocytes. Channel
sensitivity to tested compounds was determined using the two-
electrode voltage clamp (TEVC) technique. KCNQ1/KCNE1
channels were activated by sequentially applied pulse protocols.
Compounds 8b-d were tested at concentrations of 0.1 - 30 µM
(see materials and methods). Only concentrations of more than
10 µM 8b-d could significantly increase activating currents (Table
2). Therefore, only 10 µM and 30 µM of compounds 8b-d were
compared with the activity of rottlerin (1) to study the influence of
newly incorporated substituents (Figure 3A-3D). All three
derivatives and rottlerin are nearly equally effective (Figure 3E-3I).
Only at lower test pulses of -20 mV and 0 mV, 8b and 8c
increased activation current significantly more than rottlerin
Table 1: yields (%) for chromene
derivatives and final coupling steps.
4
5
6
8*
1*
a
b
c
d
e
73
89
62
58
31
-
32
27
17
36
-
79
98
83
53
23
25
17
19
f
*
low yields due to losses during
chromatographic purification
(
Figure 3F-3G). Interestingly, only compound 8c shows a
Though, many polyphenolic natural products with a methylene
bridge are known and have been synthesized, the introduction of
significant dose-dependent rise of activation current in the range
of –40 mV to +20 mV for the tested concentration levels
suggesting a right-shift of the dose response curve (Figure 3E-3I).
an appropriate one-carbon linker has developed into
a
considerable challenge for the synthesis of rottlerin. In our hands,
2
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ChemMedChem
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Table 2. normalized currents of KCNQ1/KCNE1 expressing oocytes in absence and presence of compounds 8b-d or rottlerin (1).
Conc.
µM]
ctr
Comp.
(n)
Voltage
[
-40 mV
-20 mV
0 mV
20 mV
40 mV
- (127)
0.00 ± 0.00; -
0.03 ± 0.00; -
0.03 ± 0.01; ns
0.04 ± 0.01; ns
0.05 ± 0.01; ns
0.03 ± 0.01; ns
0.03 ± 0.01; ns
0.03 ± 0.01; ns
0.04 ± 0.01; ns
0.03 ± 0.00; ns
0.03 ± 0.01; ns
0.03 ± 0.02; ns
0.03 ± 0.01; ns
0.03 ± 0.01; ns
0.06 ± 0.01; *
0.10 ± 0.03; ***
0.07 ± 0.01; ***
0.10 ± 0.02; ***
0.11 ± 0.01; ***
0.15 ± 0.02; ***
0.16 ± 0.03; ***
0.11 ± 0.02; ***
0.16 ± 0.01; -
0.50 ± 0.01; -
0.56 ± 0.07; ns
0.54 ± 0.07; ns
0.65 ± 0.04; ns
0.53 ± 0.07; ns
0.49 ± 0.05; ns
0.55 ± 0.06; ns
0.58 ± 0.07; ns
0.59 ± 0.06; ns
0.55 ± 0.04; ns
0.49 ± 0.10; ns
0.55 ± 0.04; ns
0.56 ± 0.07; ns
0.73 ± 0.08; **
0.90 ± 0.06; ***
0.67 ± 0.05; ns
0.89 ± 0.08; ***
0.82 ± 0.04; ***
0.89 ± 0.07; ***
0.96 ± 0.05; ***
0.88 ± 0.08; ***
1.00 ± 0.02; -
1.02 ± 0.10; ns
1.00 ± 0.10; ns
1.17 ± 0.05; ns
0.98 ± 0.10; ns
1.04 ± 0.05; ns
1.06 ± 0.07; ns
1.05 ± 0.09; ns
1.16 ± 0.12; ns
1.04 ± 0.07; ns
0.92 ± 0.13; ns
1.06 ± 0.06; ns
1.08 ± 0.11; ns
1.28 ± 0.11; **
1.41 ± 0.07; ***
1.16 ± 0.08; ns
1.48 ± 0.09; ***
1.35 ± 0.05; ***
1.37 ± 0.08; ***
1.51 ± 0.05; ***
1.40 ± 0.11; ***
8b (5)
0.00 ± 0.00; ns
0.00 ± 0.00; ns
0.00 ± 0.00; ns
0.00 ± 0.00; ns
0.00 ± 0.00; ns
0.00 ± 0.00; ns
0.00 ± 0.00; ns
0.00 ± 0.00; ns
0.00 ± 0.00; ns
-0.01 ± 0.01; ns
-0.01 ± 0.00; ns
0.00 ± 0.00; ns
0.00 ± 0.00; ns
0.01 ± 0.00; ns
0.00 ± 0.00; ns
0.00 ± 0.01; ns
0.01 ± 0.00; ***
0.02 ± 0.01; ***
0.02 ± 0.01; ***
0.01 ± 0.00; ns
0.20 ± 0.04; ns
0.20 ± 0.04; ns
0.24 ± 0.02; ns
0.19 ± 0.04; ns
0.15 ± 0.04; ns
0.18 ± 0.04; ns
0.22 ± 0.04; ns
0.19 ± 0.02; ns
0.19 ± 0.02; ns
0.18 ± 0.05; ns
0.18 ± 0.02; ns
0.18 ± 0.04; ns
0.26 ± 0.00; *
0
0
1
3
.1
8c (5)
8
d (4)
b (5)
8
.3
.0
.0
8c (5)
8
d (3)
b (5)
8
8c (5)
8
d (5)
b (5)
8
8c (5)
8
1
8
8
8
1
8
8
8
d (5)
(5)
b (5)
c (5)
d (4)
(18)
0.44 ± 0.04; ***
0.29 ± 0.03; ***
0.41 ± 0.05; ***
0.39 ± 0.03; ***
0.46 ± 0.05; ***
0.50 ± 0.04; ***
0.42 ± 0.05; ***
1
0.0
0.0
b (5)
c (5)
d (4)
3
Values are given as mean of 3 – 127 independent oocytes ± SEM. Number of independent oocytes (n) is given for each compound and
concentration. Significance of mean differences compared to absence of compound (ctr) was determined by one-way ANOVA and post
hoc mean comparison Tukey test (p > 0.05 ns; p < 0.05 *; p < 0.01 **; p < 0.001 ***).
Figure 3. A-D. Voltage dependent activation of KCNQ1/KCNE1 channels in absence (control, ctr) and presence of rottlerin (1) and analogs 8b-d. Compounds were
applied at 10 µM and 30 µM and the effect on steady state activated current amplitude was assessed at different voltages. Current amplitudes were normalized to
the mean of amplitudes at +40 mV in absence of test compounds (control) E-I. Mean of normalized current amplitudes ± SEM of different voltage steps in absence
(ctr) and presence of rottlerin (1) and 8b-d. Number of independent experimental data points are given in Table 2. Significance of mean differences was determined
by one-way ANOVA and posthoc mean comparison Tukey Test (ns p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001).
3
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Compounds 8b-d impact deactivation of KCNQ1/KCNE1
channels.
KCNQ1 channels coexpressed with the KCNE1 subunit
deactivate slowly in a voltage dependent manner and deactivation
was reported to be slowed by native rottlerin.[11,28] Therefore, we
tested how rottlerin analogs 8b-d impacted deactivation of
KCNQ1/KCNE1 channels. Time dependent decay, indicative of
channel deactivation was analyzed by single-exponential fitting in
absence and presence of compounds 8b-d and compared to
deactivation in presence of rottlerin (1) (see materials and
methods). At concentrations of 10 µM, the ratio of deactivation
constants of analogs 8b-d shows no significant differences
compared to 1, indicating similar efficiency at KCNQ1/KCNE1
channels (Figure 4). However, at concentrations of 30 µM time
constant ratio of compound 8c was significantly higher than the
ratio for 30 µM rottlerin (1) indicating an increased slowdown of
channel deactivation.
Figure 5. Normalized current amplitudes of KCNQ1/KCNE1 expressing
oocytes under presence of 30 µM of compounds 9 and 10. Effect of
derivatives on KCNQ1/KCNE1 currents was determined as described
above and are shown as mean ± SEM (n=9-12, unpaired t-test ** p < 0.01,
*
** p < 0.001).
develop a binding-model for rottlerin by in silico binding-site
prediction.[29] These simulations of a single KCNQ1 subunit
suggested, that rottlerin binds to the S4 / S4-S5 region, stabilizing
the channel by hydrogen bond formation to Arg243.
This hypothesis is well supported by the fact that mutation
of Arg243 to alanine made KCNQ1 completely insensitive to
rottlerin.[29] In a seminal paper, MacKinnon provided the structural
basis of human KCNQ1 modulation and gating.[30] In particular,
the exact binding-site of PIP
KCNE3-CaM complex. Essentially, this complex functions as a
PIP ligand-gated ion channel, which on binding of PIP
undergoes large conformational changes and pore-opening.
2
was elucidated in a KCNQ1-
2
2
In our own, foregoing work based on a homology model of
the open tetrameric channel, we suggested that rottlerin binds to
the same or an overlapping binding site around the amino acid
Ile337 as the benzodiazepinone R-L3 (14). A flexible in silico
docking of rottlerin gave the highest docking scores for a crevice,
located between VSD and PD (S5-S6 outer face). This region
overlaps with the R-L3 activator 14 binding-site. Mutation of Ile337
in the center of the R-L3 binding site to valine, rendered the
KCNQ1 channel insensitive to rottlerin.
Figure 4. Relation of KCNQ1/KCNE1 deactivation time constants in
presence of rottlerin (1) and compounds 8b-8d (τcompound) compared to
absence of compounds (τcontrol). Time constants of deactivation were
determined as described in the methods section. Values are given as
mean ± SEM. Significance of mean differences was determined by one-
way ANOVA and posthoc mean comparison Tukey Test (ns p > 0.05; * p
<
0.05; ** p < 0.01; *** p < 0.001).
We first performed an unbiased virtual binding site mapping
Schrödinger modeling package 11.5) on a single subunit of the
To evaluate the influence of pentamethylation and
pentaacetylation, compounds 9 and 10 were also tested at
KCNQ1/KCNE1 expressing oocytes. Interestingly, rottlerin
(
closed channel structure (Xenopus laevis, PDB 5VMS), which has
also been used by DeSilva. The mapping revealed ten potential
binding sites for small molecules, seven of which however were
located at the interfaces between the channel subunit and CaM.
The three remaining binding sites were identified in the S1/S4
domain, between the S4 helix and S4S/S5 linker, including
Arg243, and in the S6/HC region (Figure 6). Remarkably, no
binding-site was identified around Ile337. Similar results were
obtained with the single units of the closed (PDB 6UZZ) and
derivatives
amplitudes suggesting an antagonistic activity instead of agonism
Figure 5). However, these compounds are not suited as LQT-
9 and 10 decreased the normalized current
(
drug candidates since antagonistic activity at KCNQ1/KCNE1
would amplify the pathology of LQT syndrome.
Molecular modeling studies
A cryo-EM structure of a KCNQ1 / calmodulin (CaM) closed pore
v
opened (PDB 6V01) channel structure of hK 7.1.
complex of Xenopus laevis was published by MacKinnon in 2017
28]
(
PDB 5VMS).[ This construct shared 78% sequence homology
with the human KCNQ1 channel and was used by DeSilva to
4
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ChemMedChem
10.1002/cmdc.202000083
FULL PAPER
2
Figure 6. a) Structure of a KCNQ1 subunit and potential binding sites for small molecules. b) open KCNQ1 channel (6V01, PIP bound, human),
c) closed KCNQ1 channel (6UZZ, human).
different tautomers and protonation states between pH 5-9. All
docking experiments were performed with Glide in extra precision
mode.
Our KCNQ1 in silico ligand library consisted of rottlerin (1),
derivatives 8b-d, and known ligands such as the agonistic
benzodiazepinone R-L3 (14), the antagonistic benzodiazepinone
L7 (15), the natural product tanshinone (13), the thiazole ML277
Table 3. Docking scores of ligand library for single KCNQ1 subunit derived
(
16) and others (Figure 7). All ligands were prepared with the
from PDB structure 5VMS.
ligprep routine of the Schrödinger modeling suite, to generate
Compound
Arg243
-6.6
-6.4
-6.2
-6.1
-3.5
-3.2
-2.9
-2.8
-2.3
-1.6
-1.6
-1.4
S1/S4 S6/HA/HB site
rottlerin (1)
-1.8
-2.3
-2.4
-6.2
-2.6
-4.5
-1.9
-3.4
n.f.
-4.5
-4.9
-5.1
-3.4
-2.2
-2.3
-0.7
-1.9
n.f.
thiophene rottlerin (8d)
3
CF Ophenyl rottlerin (8b)
fluorophenyl rottlerin (8c)
BMS204352 (11) [31]
hexachlorophene (12) [32]
pentamethoxy rottlerin (9)
tanshinone (13) [33]
pentaacetoxy rottlerin (10)
benzodiazepinine R-L3 (14) [34]
benzodiazepinone L7 (15) [35]
ML277 (16) [36]
-2.2
-2.1
-3.0
-2.2
-1.5
-2.9
Numbers represent the calculated docking-scores. Docking scores are in
kcal/mol: the difference in binding for two compounds is G = G2 - G1
=
RT * ln (KD2 / KD1); at 25 °C, a difference in binding score of 1.36 kcal/mol
corresponds to a 10-fold difference in K ; more negative values indicate a
D
higher predicted affinity. Docking scores in the range of 4 or below are
Figure 7. Structures of published KCNQ1 ligands
usually not significant. n.f.: not found.
5
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Dockings into the three putative binding sites were performed with
a single KCNQ1 subunit obtained from the PDB structure 5VMS.
The Arg243 site turned out to be specific for rottlerin and
derivatives 8b-d, demonstrated by the significantly higher docking
scores compared to all non-rottlerin compounds. The preferred
poses of rottlerin and derivatives 8b-d form tight clusters, which
underlines the relevance of the Arg243 binding site and thus
confirms the results of DeSilva.[28] The S6/HA/HB site shows only
minor affinity and in particular, the S1/S4 site appears not relevant
for rottlerin binding at all. Cluster formation of rottlerin and
derivatives 8b-d poses in the S1/S4 and the S6/HA/HB site is less
pronounced which again favors the Arg243 site of the KCNQ1
subunit.
The recently published structure of a human KCNQ1-
Figure 8. a) Superposition of thiophene rottlerin 8d and PIP
2
. b)
KCNE3-CaM-PIP
insight into the binding mode of rottlerin as an activator of this
specific potassium channel.[30] Important to note, the PIP
bound
structure 6V01 has an open pore while the KCNQ1 structures
VMS and 6UZZ represent the potassium channels in the closed
2
(PDB 6V01) complex allows a more thorough
Superposition of rottlerin and PIP . c) Orientation of rottlerin (orange) and
2
PIP
PIP
2
2
(green) in the Arg243 site. According to the cryo-EM structure 6V01,
2
contains heptanoic acid in sn1 position and 5E,8E,11E,14Z-
hexadecatetraenoic acid in position sn2.
5
state. Our modeling studies were performed with the complete
tetrameric channel, which is relevant in particular for 6V01, since
the binding pocket of PIP is formed by two neighboring subunits.
2
Rottlerin is fixed in the Arg243 site by hydrogen bonds to Arg192,
Lys196 and a cation- interaction between the styrene phenyl ring
and Lys196. This binding mode offers an obvious explanation for
the low affinities of pentamethoxy rottlerin 9 and the pentaacetoxy
derivative 10. Both rottlerin derivatives lost an important hydrogen
bond and additionally in derivative 10, the electronic situation of
the phenolic oxygens has been altered by acetylation.
Remarkably, derivatives 8b and 8d orient differently in the binding
pocket compared to rottlerin. Since the styrene pocket is too small
to accommodate a substituted benzene ring, the preferred
orientation of both compounds is now an optimal match of the
more lipophilic arene side chains with the unsaturated side chain
Table 4. Docking scores of ligand library for Arg243 and Ile337 sites
performed with PDB structures 6UZZ and 6V01 (complete channels).
Compound
6UZZ
6V01 6UZZ 6V01
Arg243 Arg243 Ile337 Ile337
thiophene rottlerin (8d)
rottlerin (1)
-9.6
-9.0
-9.0
-8.9
-5.6
-5.5
-4.9
-4.6
-4.4
-3.8
-1.7
n.f.
-8.8
-8.2
-8.3
-7.5
-4.5
-4.9
-5.5
-3.6
-4.3
-3.8
-1.8
-3.5
-5.2
-4.5
-5.1
-4.3
-6.7
-5.9
-8.0
-6.3
-6.4
-6.0
-4.8
-3.8
-8.7
-6.3
-9.4
-6.4
-5.8
-4.6
-5.8
-7.0
-6.9
-6.6
n.f.
3
CF Ophenyl rottlerin (8b)
2
of PIP . In this arrangement, the chromene unit serves as a mimic
fluorophenyl rottlerin (8c)
tanshinone (13)
for the inositol-ring with the pyrane oxygen situated close to or in
the pocket of the inositol-4-phosphate group, while the partially
negatively charged trihydroxyphenyl ring imitates the 5-
phosphate residue.
Contrary to the single subunits, the tetrameric KCNQ1
channels from PDB structures 6UZZ and 6V01 form well-defined
binding pockets around Ile337 by contribution of two neighboring
subunits in the open and the closed state. The pocket in the open
hexachlorophene (12)
benzodiazepinone L7 (15)
BMS204352 (11)
ML277 (16)
benzodiazepinone R-L3 (14)
pentamethoxy rottlerin (9)
pentaacetoxy rottlerin (10)
state (PDB 6V01) overlaps with the binding site of the PIP
2
unsaturated chain and has a high affinity for rottlerin and
derivatives 8b-d. It consists of two interconnected crevices, which
accommodate the two aromatic side chains of rottlerin (Figure 9).
In the closed state (PDB 6UZZ), the two sites are smaller and
more separated by Phe335, which protrudes into the channel,
connecting the two sites. This reduces the affinity for rottlerin and
derivatives 8b-d by a factor of 5-10. Instead, the binding of more
compact molecules such as the benzodiazepinones or the natural
product tanshinone (13) is favored. The significance of the open-
channel pocket is also underlined by pentamethoxy and
pentaacetyl rottlerin 9 and 10, both of which are not found as
binders. As we have already demonstrated, mutation of Ile337 to
a sterically more demanding Val changes the shape of the
binding-pocket in a way, that neither the styrene phenyl ring of
rottlerin nor the benzodiazepinone 14 can be accommodated
anymore.
n.f.
In general, the tendencies resemble the docking studies with
VMS. The Arg243 site of both channel structures shows a
5
considerably higher affinity for rottlerin and its phenyl-substituted
derivatives than for other known KCNQ1 ligands. Again,
pentamethoxy-rottlerin 9 and pentaacetoxy rottlerin 10 had either
no or only a modest affinity in silico. Interestingly, rottlerin and
derivatives 8b-d, express a slightly higher affinity to the closed
KCNQ1 potassium channel (Table 4). All poses collected for
rottlerin form a single cluster, which is oriented to the inositol
2
headgroup of PIP . The chromene unit of rottlerin serves as a kind
of pincer, which directs the styrene side chain into a cavity
occupied by the inositol-4-phosphate group (Figure 8). The
electron-rich trihydroxyphenyl side chain of rottlerin is placed into
a cationic binding-pocket in direct neighborhood of the inositol
residue with one phenolic hydroxy group oriented to the inositol
3-hydroxy group. The chromene unit itself imitates the branching
of the two PIP alkyl side chains.
2
6
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.202000083
FULL PAPER
Rottlerin is such an activator of IKs.[11,28] However, further scaffold
optimization is needed to gain higher selectivity, improved kinetic
profile and agonistic activity at KCNQ1/KCNE1 channels.
Synthetic rottlerin and its derivatives are all active as
KCNQ1/KCNE1 channel activators. Interestingly, derivatives 8b-
8d preserve channel activating efficiency, but show kinetic
alterations, suggesting that future chemical modifications may
provide fine-tuned pharmacological effects.
Experimental Section
Isolation of rottlerin (1)
Rottlerin was isolated from Mallotus phillippinensis Kamala powder,
purchased from Kremer Pigmente GmbH, Germany according to a
literature procedure.[27] Kamala powder (15 g) was refluxed in boiling
benzene for 7 h. After filtration of the insoluble material rottlerin was
obtained from the solution by precipitation. Recrystallization yielded
rottlerin (680 mg) of the same purity as a commercial reference sample.
Figure 9. a) open-channel binding-pocket around Ile337 (PDB 6V01) with
bound trifluoromethoxyphenyl rottlerin 8b. Colors code protein chains b)
closed-channel Ile337 binding-pocket (PDB 6UZZ) with bound
benzodiazepinone 14. Colors code protein chains. c) open-channel
structure with residues found important for rottlerin and benzodiazepinone
Syntheses
Abbreviations
Cyh, cyclohexane; DCM, dichloromethane; EDDA, ethylenediamine
diacetate; Et O, diethyl ether; EtOAc, ethyl acetate; MeOH, methanol;
2
MOM, methoxymethyl; sat., saturated; TMS, tetramethylsilane.
14 binding (PDB 6V01). d) closed-channel structure with residues found
essential for rottlerin and benzodiazepinone 14 binding (PDB 6UZZ).
General
1
IR spectra were recorded on a Bruker ALPHA FTIR spectrometer. H- and
13C-NMR spectra were recorded on Bruker Avance III 600 and Bruker
Avance 400 spectrometers operating at 600 and 400 MHz (1H)
Based on these findings it is reasonable to assume, that rottlerin
mimics in a way PIP . Rottlerin binds to the open and / or closed
2
13
respectively and 150 and 100 MHz ( C) respectively. Accurate mass
determinations were achieved with Bruker micrOTOF mass
KCNQ1 potassium channel, which results in channel opening or
keeping the channel open. Rottlerin and even more the
derivatives 8b and 8d show a considerable affinity and distinct
selectivity for the Ile337 site of the open channel. Presumably,
binding to that site exerts a similar effect as the agonistic
benzodiazepinone 14, which stabilizes the open state of the
KCNQ1 channel. Overall, modifications of the phenyl residue of
the styrene side chain appear to be of limited benefit for more
powerful rottlerin derivatives, since the binding-pocket for rottlerin
can accommodate only an unsubstituted benzene ring. Larger
phenyl substituents cause a reorientation of the rottlerin derivative
a
spectrometer. The reactions were monitored by TLC carried out on
Macherey Nagel silica gel plates (60F-254) or Merk silica gel 60 RP-18
F254 plates using UV light and aqueous solution of KMnO , K CO , NaOH
4 2 3
and heat as the visualizing agents. HPLC analysis was conducted on a
PerfectSil Target ODS-3 HD 5 μm 100×4.6 mm column using an Agilent
1100 instrument. Reagents and solvents were purchased from commercial
sources and used without further purification, unless otherwise stated.
DCM was dried with a MB-SPS-800 solvent purification system. MeOH
was redistilled from magnesium turnings. Reactions were stirred
magnetically under an argon atmosphere unless otherwise stated.
in the Arg243 binding site, now mimicking the unsaturated PIP
2
General procedure for preparation of chalcones 4
side chain, which contributes only via weak lipophilic interactions
to the overall binding energy.
To a solution of 1-[7-Hydroxy-5-(methoxymethoxy)-2,2-dimethyl-chromen-
8-yl]ethenone (3) (1 eq.) in ethanol (15 mL) were added potassium
hydroxide (5 eq.) and the aryl aldehyde (1.3 eq.). The reaction mixture was
4
stirred for 3 days at room temperature. At completion, a saturated NH Cl-
solution was added and the mixture war partitioned between water (50 mL)
and ethyl acetate (50 mL). The aqueous layer was extracted with ethyl
acetate (2 × 30 mL). The combined organic extracts were washed with
brine (15 mL), dried over Na SO , and concentrated under reduced
2 4
pressure. The crude products were purified by flash chromatography over
Conclusion
The functional effect, the increment of deactivation time and
activation current for compounds 8b and 8d showed similar
activity at 10 and 30 µM compared to rottlerin (1), while compound
silica gel to provide the desired compound (4).
8
c showed slightly, but not significant reduced activity at 10 µM
(
E)-1-[7-Hydroxy-5-(methoxymethoxy)-2,2-dimethyl-chromen-8-yl]-3-
and slightly increased activity at 30 µM. This might indicate a right-
shifted dose-response curve for 8c and a preference for aromatic
systems with high electron density at this position. However, at
high concentration of 30 µM compound 8c was more potent in
slowing down KCNQ1/KCNE1 channel deactivation.
phenyl-prop-2-en-1-on (4a)
Compound 4a was obtained from 1-[7-hydroxy-5-(methoxymethoxy)-2,2-
dimethyl-chromen-8-yl]ethenone (3) and benzaldehyde. 288 mg, yield:
7
1
3%. H-NMR (400 MHz, CDCl
3
): δ (ppm) = 13.98 (s, 1H), 8.13 (d, J = 15.6
Hz, 1H), 7.80 (d, J = 15.6 Hz, 1H), 7.65-7.62 (m, 2H), 7.47-7.38 (m, 3H),
.65 (d, J = 10.0 Hz, 1H), 6.28 (s, 1H), 5.50 (d, J = 10.0 Hz, 1H), 5.25 (s,
2H), 3.52 (s, 3H), 1.58 (s, 6 H). 13C-NMR (100 MHz, CDCl
): δ (ppm) =
193.0, 166.9, 158.7, 155.9, 142.3, 135.6, 130.1, 129.0, 128.3, 127.5, 124.9,
16.8, 107.0, 103.6, 95.3, 94.3, 78.0, 56.5, 28.0. HPLC-MS (ESI): m/z
6
In the context of therapy of the long QT syndrome, activation of
KCNQ1/KCNE1 channels was discussed as a therapy option.
Due to the lack of drugs on the market that activate the native
KCNQ1/KCNE1 channel, novel compounds would be desired.
3
1
-
-
(%): 365.2 [M -H] (100), 366.2 [M ] (18). HR-MS (ESI): m/z found =
7
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.202000083
FULL PAPER
-
1
3
2
65.1396, calcd for C121
H O
21 5
= 365.1394. IR (cm ): 3068, 2972, 2958,
concentrated under reduced pressure. The crude products were purified
by flash chromatography over silica gel to give the deprotected compounds
907, 2827, 1642, 1582, 1546. Rf: 0.52 (CH/EE 19:1).
5.
(
[
E)-1-[7-Hydroxy-5-(methoxymethoxy)-2,2-dimethyl-chromen-8-yl]-3-
4-(trifluoromethoxy) phenyl]prop-2-en-1-one (4b)
(E)-1-(5,7-Dihydroxy-2,2-dimethyl-chromen-8-yl)-3-phenyl-prop-2-en-
1-one (5a)
Compound 5a was obtained from (E)-1-[7-hydroxy-5-(methoxymethoxy)-
Product 4b was obtained from 1-[7-hydroxy-5-(methoxymethoxy)-2,2-
dimethyl-chromen-8-yl]ethenone (3) and 4-(trifluoromethoxy)-
benzaldehyde. 293 mg, yield: 89%. H-NMR (400 MHz, CDCl ): δ (ppm) =
3.89 (s, 1H), 8.08 (d, J = 15.8 Hz, 1H), 7.75 (d, J = 15.5 Hz, 1H), 7.64 (d,
J = 8.7 Hz, 2H), 7.28 (d, J = 8.1 Hz, 2H), 6,64 (d, J = 9.9 Hz, 1H), 6.28 (s,
H), 5.51 (d, J = 10.0 Hz), 5.25 (s, 2H), 3.52 (s, 3H), 1.57 (s, 6H). 13C-NMR
100 MHz, CDCl ): δ (ppm) = 192.6, 166.9, 158.9, 155.9, 150.2, 140.3,
1
3
2,2-dimethyl-chromen-8-yl]-3-phenyl-prop-2-en-1-one (4a). 78 mg, yield:
1
1
32%. H-NMR (400 MHz, CDCl
3
): δ (ppm) = 14.11 (s, 1H), 8.12 (d, J = 15.6
Hz, 1H), 7.79 (d, J = 15.6 Hz, 1H), 7.65-7.60 (m, 2H), 7.47-7.39 (m, 2H),
6.60 (d, J = 9.5 Hz, 1H), 6.00 (s, 1H), 5.51 (d, J = 9.5 Hz, 1H), 1.58 (s, 6H).
1
(
1
1
13C-NMR (100 MHz, CDCl
135.6, 130.1, 129.0, 128.3, 127.4, 124.9, 116.4, 106.6, 102.4, 96.4, 78.2,
3
3
): δ (ppm) = 193.0, 166.5, 158.0, 156.7, 142.4,
34.2, 129.5, 128.3, 124.9, 121.2, 120.4 (q, J = 258.7 Hz), 116.8, 106.9,
-
-
-
03.6, 95.3, 94.3, 78.1, 56.5, 28.1. HPLC-MS (ESI): m/z (%): 449.2 [M -H]
28.0. HPLC-MS (ESI): m/z (%): 321.2 [M -H] (100), 322.2 [M ] (20). HR-
-
= 321.1132. IR (cm-
(100), 450.2 [M ] (20). HR-MS (ESI): m/z found = 449.1219, calcd for
MS (ESI): m/z found = 321.1130, calcd for C20
): 3233, 2973, 1593, 1546. Rf: 0.27 (CH/EE 8:2).
H O
17 4
-
1
1
C
H F O
23 20 3 6
= 449.1217. IR (cm ): 3115, 3079, 2974, 2958, 2915, 2836,
1637, 1587, 1545. Rf: 0.36 (DCM/CH 1:1).
(
E)-1-(5,7-Dihydroxy-2,2-dimethyl-chromen-8-yl)-3-[4-
(
E)-3-(4-Fluorophenyl)-1-[7-hydroxy-5-(methoxymethoxy)-2,2-
(trifluoromethoxy)phenyl]prop-2-en-1-one (5b)
dimethyl-chromen-8-yl]prop-2-en-1-one (4c)
Product 5b was obtained from (E)-1-[7-hydroxy-5-(methoxymethoxy)-2,2-
Product 4c was obtained from 1-[7-hydroxy-5-(methoxymethoxy)-2,2-
dimethyl-chromen-8-yl]-3-[4-(trifluoromethoxy)phenyl]prop-2-en-1-one
1
dimethyl-chromen-8-yl]ethenone (3) and 4-fluorobenzaldehyde. 194 mg.
(4b). 69 mg, yield: 27%. H-NMR (400 MHz, CDCl
3
): δ (ppm) = 14.02 (s,
1
yield: 62%. H-NMR (400 MHz, CDCl
3
): δ (ppm) = 13.95 (s, 1H), 8.04 (d,
1H), 8.08 (d, J = 15.7 Hz, 1H), 7.74 (d, J = 15.7 Hz), 7.63 (d, J = 8.6 Hz,
2H), 7.28 (d, J = 8.1 Hz, 2H), 6.60 (d, J = 9.9 Hz, 1H), 6.21 (bs, 1H), 6.00
J = 15.7 Hz, 1H), 7.75 (d, J = 15.7 Hz, 1H), 7.59 - 7.63 (m, 2H), 7.10 – 7.16
(
(
1
3
m, 2H), 6.34 (d, J = 10.0, 1H), 6.28 (s, 1H), 5.50 (d, J = 10.0 Hz, 1H), 5.25
s, 2H), 3.52 (s, 3 H), 1.57 (s, 6H). 13C-NMR (100 MHz, CDCl
): δ (ppm) =
92.8, 166.8, 163.4 (d, J = 251.3 Hz), 158.7, 155.8, 141.0, 131.8 (d, J =
.4 Hz), 130.0 (d, J = 8.5 Hz), 127.2, 124.8, 116.8, 116.1 116.1 (d, J = 21.9
(s, 1H), 5.51 (d, J = 9.8 Hz), 1.57 (s, 6H). 13C-NMR (100 MHz, CDCl
3
): δ
3
(ppm) = 192.6, 166.5, 158.3, 156.7, 150.2, 140.4, 134.2, 129.5, 128.3,
124.8, 121.2, 120.4 (q, J = 250.2 Hz), 116.4, 106.6, 102.4, 96.4, 78.3, 28.1.
-
-
HPLC-MS (ESI): m/z (%): 405.2 [M -H] (100), 406.2 [M ] (22). HR-MS
-
1
Hz), 106.9, 103.6, 95.3, 94.3, 78.1, 56.5, 28.0. HPLC-MS (ESI): m/z (%):
16 3 5
(ESI): m/z found = 405.0953, calcd for C21H F O = 405.0955. IR (cm ):
3231, 3129, 2971, 2926, 2854, 1628, 1586, 1505. Rf: 0.24 (CH/EE 6:1).
-
-
3
85.1 [M -H] (100), 386.1 [M ] (19). HR-MS (ESI): m/z found = 407.1264,
-
1
21 1 1 4
calcd for C22H F Na O = 407.1265 IR (cm ): 2974, 2958, 2906, 1634,
1582, 1544, 1507. Rf: 0.24 (CH/EE 19:1).
(E)-1-(5,7-Dihydroxy-2,2-dimethyl-chromen-8-yl)-3-(4-
fluorophenyl)prop-2-en-1-one (5c)
(
(
E)-1-[7-Hydroxy-5-(methoxymethoxy)-2,2-dimethyl-chromen-8-yl]-3-
2-thienyl)prop-2-en-1-one (4d)
Product 5c was obtained from (E)-3-(4-fluorophenyl)-1-[7-hydroxy-5-
(methoxymethoxy)-2,2-dimethyl-chromen-8-yl]prop-2-en-1-one (4c). 14
1
Product 4d was obtained from 1-[7-hydroxy-5-(methoxymethoxy)-2,2-
mg, yield: 17%. H-NMR (400 MHz, CDCl
3
): δ (ppm) = 14.11 (s, 1H), 8.03
dimethyl-chromen-8-yl]ethanon (3) and thiophen-2-carbaldehyde. 148 mg,
(d, J = 15.4, 1H), 7.74 (d, J = 15.4, 1H), 7.75 - 7.72 (m, 2H), 7.15 - 7.10
(m, 2H), 6.60 (d, J = 9.9 Hz, 1H), 6.01 (s, 1H), 5.50 (d, J = 9.9 Hz, 1H),
1
58% yield. H-NMR (400 MHz, CDCl
3
): δ (ppm) = 14.05 (s, 1H), 8.00 (d, J
=
15.3 Hz), 7.94 (d, J = 7.94 Hz), 7.40 (d, J = 4.9 Hz, 1H), 7.32 (d, J = 3.44
1.57 (s, 6H). 13C-NMR (100 MHz, CDCl
3
): δ (ppm) = 192.8, 166.4, 163.8
Hz), 7.10 (dd, J = 5.1 Hz, J = 3.6 Hz, 1H), 6.63 (d, J = 10.4 Hz), 6.26 (s,
H), 5.50 (d, J = 10.4 Hz), 5.24 (s, 2H), 3.51 (s, 3H), 1.61 (s, 6H). 13C-NMR
100 MHz, CDCl ): δ (ppm) = 192.4, 166.9, 158.6, 155.8, 141.4, 135.1,
31.7, 128.3, 128.2, 126.5, 124.5, 116.6, 106.8, 103.6, 95.2, 94.3, 78.1,
(d, J = 251.3 Hz), 158.4, 156.7, 141.1, 131.8 (d, J = 3.6 Hz), 130.0 (d, J =
8.5 Hz), 127.2, 124.7, 116.5, 116.1 (d, J = 21.9 Hz), 106.5, 102.5, 96.4,
78.2, 28.0. HPLC-MS (ESI): m/z (%): 339.2 [M -H] (100), 340.2 [M ] (19).
1
(
1
5
-
-
3
HR-MS (ESI): m/z found = 339.1035, calcd for C20
(cm ): 3524, 3245, 2970, 2925, 1628, 1595, 1506. Rf: 0.17 (CH:EE 8:2).
H
16
F
1
O
4
= 339.1038. IR
-
-
-1
6.5, 27.9. HPLC-MS (ESI): m/z (%): 371.2 [M -H] (100), 372.2 [M ] (17).
= 395.0924.
IR (cm ): 3326, 3032, 2957, 1709, 1585, 1542. Rf: 0.47 (CH/EE 4:1).
1 5 1
HR-MS (ESI): m/z found = 395.0926, calcd for C20H20Na O S
-
1
(E)-1-(5,7-Dihydroxy-2,2-dimethyl-chromen-8-yl)-3-(2-thienyl)prop-2-
en-1-one (5d)
(
(
E)-1-[7-Hydroxy-5-(methoxymethoxy)-2,2-dimethyl-chromen-8-yl]-3-
4-methoxyphenyl)prop-2-en-1-one (4e)
Product 5d was obtained from (E)-1-[7-hydroxy-5-(methoxymethoxy)-2,2-
dimethyl-chromen-8-yl]-3-(2-thienyl)prop-2-en-1-one (4d). 43 mg, yield:
1
Compound 4e was obtained from 1-[7-hydroxy-5-(methoxymethoxy)-2,2-
36%. H-NMR (400 MHz, CDCl
3
): δ (ppm) = 14.19 (s, 1H), 8.01 (d, J = 15.3
dimethyl-chromen-8-yl]ethenone (3) and 4-methoxybenzaldehyde. 11 mg,
Hz, 1H), 7.94 (d, J = 15.4 Hz, 1H), 7.40 (d, J = 5.3 Hz), 7.32 (d, J = 3.5 Hz),
7.10 (dd, J = 5.1 Hz, 3.6 Hz, 1H), 6.60 (d, J = 9.9 Hz, 1H), 6.43 (bs, 1H),
6.00 (s, 1H), 5.51 (d, J = 10.0 Hz, 1H), 1.62 (s, 6H,). 13C-NMR (100 MHz,
1
31% yield. H-NMR (400 MHz, CDCl
3
): δ (ppm) = 14.10 (s, 1H), 8.03 (d, J
=
15.7 Hz, 1H), 7.79 (d, J = 15.7 Hz, 1H), 7.58 (d, J = 8.7 Hz, 2H), 6.96 (d,
J = 8.7 Hz, 2H), 6.64 (d, J = 9.8 Hz, 1H), 6.27 (s, 1H), 5.50 (d, J = 9.9 Hz,
H), 5.24 (s, 2H), 3.88 (s, 3H), 3.51 (s, 3H), 1.58 (s, 6H). 13C-NMR (100
MHz, CDCl ): δ (ppm) = 192.9, 166.9, 161.4, 158.5, 155.8, 142.5, 130.0,
28.4, 125.1, 124.8, 116.8, 114.5, 107.0, 103.6, 95.3, 94.3, 77.9, 56.5,
CDCl
3
): δ (ppm) = 191.2, 166.6, 158.2, 156.6, 141.5, 135.2, 131.2, 128.3,
1
128.2, 126.5, 124.9, 116.4, 106.4, 102.4, 96.4, 78.3, 28.0. HPLC-MS (ESI):
m/z (%): 327.1 [M -H] (100), 328.1 [M ] (18). HR-MS (ESI): m/z found =
326.0695, calcd for C18H O S = 326.0697. IR (cm ): 3244, 3105, 3089,
15 4 1
2964, 2922, 1642, 1593, 1547, 1503. Rf: 0.30 (CH/EE 9:1).
-
-
3
-
1
1
-
-
5
5.4, 28.0. HPLC-MS (ESI): m/z (%): 395.2 [M -H] (100), 396.2 [M ] (26).
-
1
23 6
HR-MS: m/z found = 395.1502, calcd for C23H O = 395.1500. IR (cm ):
3
5
108, 3065, 2992, 2968, 2937, 1683, 1593, 1546, 1508. Rf: 0.38 (CH/EE
:1).
General procedure for reacting Eschenmoser’s salt with chromene
building blocks 5
To a solution of the deprotected chalcone (1 eq.) in chloroform (15 mL)
was added Eschenmoser´s salt (3 eq.). The reaction mixture was stirred
for 3 h. The reaction mixture was then diluted with chloroform and a 1M
HCl solution (10 mL) was added. The aqueous phase was extracted with
ethyl acetate (2 × 25 mL). The combined organic phases were washed
General procedure for the deprotection of the MOM-group
Chalcones 4 were dissolved in methanol (30 mL) and a 3 M HCl solution
(
3 mL) was added. The reaction mixture was refluxed for 2 h. The reaction
was quenched with a saturated NaHCO -solution (15 mL). The reaction
3
mixture was partitioned between water (50 mL) and ethyl acetate (50 mL).
The aqueous layer was extracted with ethyl acetate (2 × 30 mL). The
2 4
with brine (10 mL), dried over Na SO , and concentrated under reduced
pressure. The crude products 6 were used directly without further
combined organic phases were washed with brine, dried over Na
2
SO
4
, and
purification.
8
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.202000083
FULL PAPER
1
03.8, 101.9, 78.2, 32.5, 28.1, 15.8, 7.5. HPLC-MS (ESI): m/z (%): 599.3
-
-
[5,7-Dihydroxy-2,2-dimethyl-8-[(E)-3-phenylprop-2-enoyl]chromen-6-
[M -H] (100), 600.3 [M ] (30). HR-MS (ESI): m/z found = 599.1534, calcd
for C31H F O = 599.134. IR (cm ): 3246, 2961, 2925, 2855, 1630, 1598,
26 3 9
-
1
yl]methyl-dimethyl-ammoniumiodide (6a)
Compound 6a was obtained from (E)-1-(5,7-dihydroxy-2,2-dimethyl-
chromen-8-yl)-3-phenyl-prop-2-en-1-one (5a) and Eschenmoser´s salt. 86
1507. Rf: 0.10 (CH/EE 2:1).
+
+
mg, yield: 79%. HPLC-MS (ESI): m/z (%): 380.2 [M +H-HI] (34), 381.2 [M -
(E)-1-[6-[(3-Acetyl-2,4,6-trihydroxy-5-methyl-phenyl)methyl]-5,7-
dihydroxy-2,2-dimethyl-chromen-8-yl]-3-(4-fluorophenyl)prop-2-en-
1-one (8c)
+
HI] (7), 335.1 [M +H-HI-C
2 8
H N] (100). HR-MS (ESI): m/z found = 380.1856,
calcd for C23H N O
26 1 4
= 380.1856.
Compound 8c was obtained from [8-[(E)-3-(4-fluorophenyl)prop-2-enoyl]-
5,7-dihydroxy-2,2-dimethyl-chromen-6-yl]methyl-dimethylammonium-
iodide (6c) and 1-(2,4,6-trihydroxy-3-methyl-phenyl)ethenone (7). 7 mg,
[
(
5,7-Dihydroxy-2,2-dimethyl-8-[(E)-3-[4-
trifluoromethoxy)phenyl]prop-2-enoyl]chromen-6-yl]methyl-
dimethyl-ammoniumiodide (6b)
yield: 21%. 1H-NMR (400 MHz, CDCl
3
): δ (ppm) = 8.12 (d, J = 15.4 Hz,
compound 6b was obtained from (E)-1-(5,7-dihydroxy-2,2-dimethyl-
chromen-8-yl)-3-[4-(trifluoromethoxy)phenyl]prop-2-en-1-one (5b) and
Eschenmoser´s salt. 64 mg, yield: 98%. HPLC-MS (ESI): m/z (%): 462.2
1H), 7.81 (d, J = 15.5 Hz, 1H), 7.64 - 7.59 (m, 2H), 7.16 – 7.11 (m, 2H),
6.79 (d, J = 9.9 Hz, 1H), 5.50 (d, J = 9.9 Hz, 1H), 3.83 (s, 2H), 2.73 (s, 3H),
2.11 (s, 3H), 1.55 (s, 6H). 13C-NMR (100 MHz, CDCl
3
): δ (ppm) = 204.1,
-
-
-
[M -2H-HI] (100), 463.2[M -H-HI] (23), 464.2 [M -HI] (3). HR-MS (ESI): m/z
192.7, 164.0 (d, J = 251.3 Hz), 162.8, 160.6, 159.6, 158.8, 155.4, 142.0,
131.7 (d, J = 3.2 Hz), 130.2 (d, J = 8.7 Hz), 126.6, 125.0, 117.2, 116.2 (d,
J = 22.1 Hz), 106.5, 106.0, 105.2, 104.2, 103.8, 101.9, 78.2, 32.5, 28.0,
found = 464.1677, calcd for C24 = 464.1679.
25 3 1 5
H F N O
-
-
[
8-[(E)-3-(4-Fluorophenyl)prop-2-enoyl]-5,7-dihydroxy-2,2-dimethyl-
15.8, 7.4. HPLC-MS (ESI): m/z (%): 533.3 [M -H] (100), 534.3 [M ] (30).
HR-MS (ESI): m/z found = 533,1615 calcd for C30 = 533.1617. IR
chromen-6-yl]methyl-dimethyl-ammoniumiodide (6c)
Compound 6c was obtained from (E)-1-(5,7-dihydroxy-2,2-dimethyl-
26 1 8
H F O
-
1
(cm ): 3247, 2954, 2923, 2852, 1599, 1507. 1507. Rf: 0.16 (CH/EE 2:1).
chromen-8-yl)-3-(4-fluorophenyl)prop-2-en-1-one
(5c)
and
Eschenmoser´s salt. 32 mg, yield 83%. HPLC-MS (ESI): m/z (%): 339.2
(E)-1-[6-[(3-Acetyl-2,4,6-trihydroxy-5-methyl-phenyl)methyl]-5,7-
dihydroxy-2,2-dimethyl-chromen-8-yl]-3-(2-thienyl)prop-2-en-1-one
(8d)
-
-
2 8
[M -H-HI-C H N] (100), 340.2 [M -HI] (19). HR-MS (ESI): m/z
16 1 4
found = 339.1035, calcd for C20H F O
= 339.1038.
Compound 8d was obtained from [5,7-dihydroxy-2,2-dimethyl-8-[(E)-3-(2-
thienyl)prop-2-enoyl]chromen-6-yl]methyl-dimethyl-ammoniumiodide (6d)
and 1-(2,4,6-trihydroxy-3-methyl-phenyl)ethenone (7). 13 mg, yield: 35%.
[
5,7-Dihydroxy-2,2-dimethyl-8-[(E)-3-(2-thienyl)prop-2-
enoyl]chromen-6-yl]methyl-dimethyl-ammoniumiodide (6d)
Compound 6d was obtained from (E)-1-(5,7-dihydroxy-2,2-dimethyl-
1H-NMR (400 MHz, CDCl
3
): δ (ppm) = 8.07 (d, J = 15.3 Hz, 1H), 7.98 (d, J
chromen-8-yl)-3-(2-thienyl)prop-2-en-1-one (5d) and Eschenmoser´s salt.
= 15.3 Hz, 1H), 7.43 (d, J = 5.0 Hz, 1H), 7.34 (d, J = 3.4 Hz, 1H), 7.10 (dd,
J = 5.3, 3.4, 1H), 6.67 (d, J = 9.7 Hz, 1H), 5.50 (d, J = 9.7 Hz, 1H), 3.82 (s,
-
3
6 mg, yield: 53%. HPLC-MS (ESI): m/z (%): 384.2 [M -H-HI] (100), 385.2
-
2H), 2.73 (s, 3H), 2.10, (s, 3H), 1.58 (s, 6H). 13C-NMR (100 MHz, CDCl
[M -HI] (26.6). HR-MS (ESI): m/z found
=
386.1420, calcd for
3
):
δ (ppm) = 204.1, 192.0, 162.9, 160.6, 159.6, 158.7, 155.3, 141.4, 136.1,
21 24 1 4 1
C H N O S = 386.1421.
1
32.0, 128.7, 128.4, 125.8, 125.2, 117.1, 106.5, 106.0, 105.1, 104.3, 103.7,
-
General procedure for couplings via Eschenmoser’s salts 6
solution of the salt (1 eq.) and 1-(2,4,6-trihydroxy-3-methyl-
101.9, 78.3, 32.5, 27.9, 15.8, 7.5. HPLC-MS (ESI): m/z (%): 521.2 [M -H]
(100), 522.2 [M ] (30). HR-MS (ESI): m/z found = 521.1275, calcd for
C H O S = 521.1276. IR (cm ): 3235, 2972, 2927, 1736, 1709, 1600,
28 25 8 1
-
A
6
-
1
phenyl)ethenone (1 eq.) in toluene was stirred for 1 h at 110 °C. At
completion, the reaction mixture was concentrated under reduced
pressure. The crude products were purified by flash chromatography over
silica gel to obtain the final compound (1, 8).
1547. Rf: 0.27 (CH/EE 2:1).
Molecular Biology
(
E)-1-[6-[(3-acetyl-2,4,6-trihydroxy-5-methyl-phenyl)methyl]-5,7-
Materials and Methods
dihydroxy-2,2-dimethyl-chromen-8-yl]-3-phenyl-prop-2-en-1-one,
rottlerin (1)
The KCNQ-clones resembles the clone published by NCBI Annotation
Project
Accession
numbers
XM_052604.2
(hKCNQ1)
and
Rottlerin (1) was obtained from 1-(2,4,6-trihydroxy-3-methyl-
phenyl)ethenone (7) and [5,7-dihydroxy-2,2-dimethyl-8-[(E)-3-phenylprop-
NM_001127670.3 (hKCNE1). Molecular biological procedures were those
described previously.[37] In brief, cRNAs were generated by in vitro
transcription with the Ambion T7 mMessage mMachine kit (Life
Technologies. Darmstadt. Germany) from linearized cDNA templates.
2
-enoyl]chromen-6-yl]methyl-dimethyl-ammoniumiodid (6a). 25 mg, yield:
1
29%. H-NMR (400 MHz, CDCl
3
): δ (ppm) = 8.20 (d, 15.8 Hz, 1H), 7.85 (d,
J = 15.8 Hz, 1H), 7.65-7.60 (m, 2H), 7.47-7.39 (m, 3H), 6.68 (d, J = 9.8 Hz,
1
6
1
1
H), 5.50 (d, J = 9,8 Hz), 3.82 (s, 2H), 2.73 (s, 3H), 2.10 (s, 3H), 1.56 (s,
Two-electrode voltage-clamp (TEVC) in Xenopus laevis oocytes
The standard TEVC procedures were similar as previously described.
Defolliculated oocytes were obtained from EcoCyte Bioscience (Dortmund.
Germany). Oocytes were injected with 8 ng KCNQ1-WT and 4 ng KCNE1-
WT and stored for 3-4 days in Bath’s solution containing (mmol L-1): 88
H). 13C-NMR (100 MHz, CDCl
3
): δ (ppm) = 204.1, 192.9, 162.8, 160.6,
59.6, 158.8, 156.6, 155.4, 143.2, 135.5, 130.3, 129.0, 128.4, 126.8, 125.1,
17.2, 106.5, 106.0, 105.3, 104.2, 103.7, 101.9, 78.2, 32.5, 28.0, 15.8, 7.5.
-
-
HPLC-MS (ESI): m/z (%): 515.3 [M -H] (100), 516.3 [M ] (30). HR-MS
-
1
(ESI): m/z found = 515.1713, calcd for C30
H O
28 8
= 515.1711. IR (cm ):
NaCl, 1 KCl, 0.4 CaCl
2 2 4
, 0.33 Ca(NO3) , 0.6 MgSO , 5 TRIS-HCl, 2.4
3234, 2954, 2922, 2851, 1710, 1603, 1555. Rf: 0.24 (CH/EE 2:1).
NaHCO and supplemented with 80 mg L-1 theophylline, 63 mg L-1
3
benzylpenicillin, 40 mg L-1 streptomycin and 100 mg L-1 gentamycin.
Standard TEVC recordings were performed at 22°C with a Turbo Tec
10CX (NPI) amplifier combined with GePulse software for data acquisition.
Macroscopic currents were recorded 3-4 days after injection in recording
(
E)-1-[6-[(3-Acetyl-2,4,6-trihydroxy-5-methyl-phenyl)methyl]-5,7-
dihydroxy-2,2-dimethyl-chromen-8-yl]-3-[4-(trifluoromethoxy)-
phenyl]prop-2-en-1-one (8b)
Compound 8b was obtained from [5,7-dihydroxy-2,2-dimethyl-8-[(E)-3-[4-
2 2
solution ND96 (NaCl 96 mM, KCl 2 mM, CaCl 1.8 mM, MgCl 1 mM,
(
trifluoromethoxy)phenyl]prop-2-enoyl]chromen-6-yl]methyl-dimethyl-
HEPES 5 mM, pH 7.4). Compound solutions were freshly prepared from
10 mM DMSO stock solutions. For maximal compatibility, all recording
solutions including the control solution contained a final DMSO
ammoniumiodid (6b) and 1-(2,4,6-trihydroxy-3-methyl-phenyl)ethenone
(
1
7). 6 mg, 25% yield. H-NMR (400 MHz, CDCl
3
): δ (ppm) = 8.16 (d, J =
1
5.5 Hz, 1H), 7.81 (d, J = 15.7 Hz, 2H), 7.65 (m, 2H), 7.29 (m, 2H), 6.69
d, J = 9.9 Hz, 1H), 5.51 (d, J = 9.9 Hz, 1 H), 3.84 (s, 2H), 2.74 (s, 3H),
concentration of 0.3 %. Pipettes were filled with 3M KCl and had
resistances of 0.5 – 1.5 M. Channel functions were analysed using the
following pulse protocols:
(
2
1
1
.11 (s, 3H), 1.55 (s, 6H). 13C-NMR (100 MHz, CDCl
): δ (ppm) = 204.1,
3
92.5, 162.8, 160.6, 159.6, 159.0, 155.4, 150.4, 141.3, 134.1, 129.7, 127.7,
25.1, 121.2, 120.4 (q, J = 255.0 Hz) 117.2, 106.5, 105.9, 105.3, 104.2,
9
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.202000083
FULL PAPER
KCNQ1/KCNE1 channels deactivate due to hyperpolarization using pulse
protocol tail current. This voltage dependent channel closure can be
analyzed by means of exponential fits. For comparison of deactivation
kinetics in presence of compounds, deactivation current trace was fitted to
following equitation to determine time constant :
−
휏
푡
푌 = 퐴 + 퐴 ∗ 푒
0
1
Deactivation was fitted for current traces with voltage changes from +40
mV to -120 mV for all oocytes in absence (control) and presence of
compounds. After determination of τ for all current traces, compound was
D
normalized to control
:
ꢀꢁ표푚푝표푢푛푑
ꢀꢁ표푛푡푟표푙
Pulse protocol IV was used to assess the voltage dependent activation of
channel currents, while pulse protocol deactivation was used to assess the
voltage dependent deactivation of channel currents. Both pulse protocols
were sequential applied at the same oocyte to record reference currents
Statistics
(
control). After completion, compound solutions were washed in and pulse
Statistical significance of results was evaluated by one-way ANOVA with
posthoc mean comparison Tukey Test or Student’s t test and is indicated
by asterisks (ns p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001).
protocols were applied again under presence of compound solution.
Data analysis
Electrophysiological data were recorded with GePulse and analyzed with
accompanying software Ana (Dr. Michael Pusch. Genova. Italy). Data
analysis was done using OriginPro 2018 (OriginLab Corporation.
Northampton. MA. USA).
Acknowledgements
The authors thank Ms. I. Polanz, Ms. S. Bettinger and Mr. A.
Siebert for measuring the high-resolution MS and NMR spectra.
Steady-state activation
Currents were normalized to the average current of the +40 mV pulses in
absence of compounds (control) at the end of the 9 sec for recordings of
the same experiment (see representative current traces beneath).
Keywords: natural product rottlerin • potassium channel KCNQ1
activator • total synthesis • mode of action
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1
1
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10.1002/cmdc.202000083
FULL PAPER
Entry for the Table of Contents
Rottlerin, a polyphenolic natural product, is isolated from Kamala powder, used for centuries in traditional Indian medicine. Among
other biological effects, rottlerin has been shown to activate KCNQ1 potassium channels. First rottlerin analogues were synthesized
and evaluated electrophysiologically in Xenopus laevis oocytes. A molecular modeling based model was developed to explain the
binding-mode of rottlerin and other KCNQ1 ligands.
1
2
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