Design, synthesis and pharmacological evaluation of ester-based quercetin derivatives as selective vascular KCa1.1 channel stimulators

Article abstract of DOI:10.1016/j.bioorg.2020.104404

Quercetin represents one of the most studied dietary flavonoids; it exerts a panel of pharmacological activities particularly on the cardiovascular system. Stimulation of vascular K_Ca1.1 channels contributes to its vasorelaxant activity, which

Full text of DOI:10.1016/j.bioorg.2020.104404

Bioorganic Chemistry 105 (2020) 104404
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and pharmacological evaluation of ester-based quercetin
derivatives as selective vascular K 1.1 channel stimulators
Ca
a
,
b
c
b
b
b
Gabriele Carullo , Amer Ahmed , Alfonso Trezza , Ottavia Spiga , Antonella Brizzi ,
c
,
*
b
,
*
a
Simona Saponara , Fabio Fusi , Francesca Aiello
a
Department of Pharmacy, Health and Nutritional Sciences, DoE 2018-2022, University of Calabria, Edificio Polifunzionale, 87036 Rende (CS), Italy
Department of Biotechnology, Chemistry and Pharmacy, DoE 2018-2022, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy
Department of Life Sciences, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
Ca 1.2 channel
Ca1.1 channel
Quercetin represents one of the most studied dietary flavonoids; it exerts a panel of pharmacological activities
particularly on the cardiovascular system. Stimulation of vascular KCa1.1 channels contributes to its vasorelaxant
V
K
V
activity, which is, however, counteracted in part by its concomitant stimulation of Ca 1.2 channels. Therefore,
Molecular docking
Quercetin
several quercetin hybrid derivatives were designed and synthesized to produce a more selective KCa1.1 channel
stimulator, then assessed both in silico and in vitro. All the derivatives interacted with the KCa1.1 channel with
similar binding energy values. Among the selected derivatives, 1E was a weak vasodilator, though displaying an
Vasoactivity
Lipoic acid
V
interesting Ca 1.2 channel blocking activity. The lipoyl derivatives 1F and 3F, though showing pharmacological
Hypertension
Ester-based derivatives
and electrophysiological features similar to those of quercetin, seemed to be more effective as KCa1.1 channel
stimulators as compared to the parent compound. The strategy pursued demonstrated how different chemical
V
substituents on the quercetin core can change/invert its effect on Ca 1.2 channels or enhance its KCa1.1 channel
stimulatory activity, thus opening new avenues for the synthesis of efficacious vasorelaxant quercetin hybrids.
1
. Introduction
Quercetin is a natural-occurring flavonoid that has captured the in-
Moreover, Corvitin stabilizes the necrotic zone and reduces the mass of
the necrotized myocardium [9]. Administration of quercetin (either
before the onset of ischemia or during whole reperfusion) improves the
recovery of cardiac function after global ischemia and reperfusion [10].
Quercetin supplementation for 14 days protects rats from isoproterenol-
induced acute myocardial injury: cardioprotective effects include a
significant attenuation of oxidative stress and inflammation, preserva-
tion of heart architecture, along with downregulation of calpain
expression. Moreover, rats treated with quercetin do not show any sign
of progression of experimental autoimmune myocarditis to dilated car-
diomyopathy [11]. This was ascribed to the decrease of myocardial
levels of endoplasmic reticulum stress and fibrosis markers via modu-
lation of endothelin-1/MAPK signalling cascade.
terest of scientific community, as it possesses several beneficial prop-
erties. Furthermore, it is easily isolated from a variety of food sources,
such as cherries, apple, red wine, capers, and red onion [1]. Quercetin as
well as many other flavonoids modulate several physiological functions,
exerting anti-inflammatory [2], anti-infectious, anticancer/chemo-
preventive [3], neuroprotective, wound regenerating [4,5], blood
glucose lowering [6,7] and anti-hypertensive/vasorelaxant activities
[
8]. Focusing on cardiovascular diseases, clinical studies demonstrate
that Corvitin® (a water-soluble form of quercetin for i.v. use) has
beneficial effects on clinical forms of ischemic heart disease, including
myocardial infarction. The antiarrhythmic action of Corvitin may be the
result of its membrane-stabilizing action, as well as of the improvement
of intracardiac hemodynamics due to the reduction of myocardial stress.
Various mechanisms underpin the cardiovascular properties of
quercetin. In particular, quercetin is capable, along with many other
flavonoids, to stimulate KCa1.1 channels [12], activated by membrane
Abbreviations: CHCl
3
, chloroform; CMC, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide; DCM, dichloromethane; DMF, N,N-dimethylformamide; DMSO,
, inositol trisphosphate;
dimethyl sulphoxide; HOBt, N-hydroxybenzotriazole; HPLC, high-pressure liquid chromatography; IKCa1.1, KCa1.1 channel currents; IP
3
MAPK, mitogen activated protein kinase; MeOH, methanol; PKA, protein kinase A; PKG, protein kinase G; TEA, tetraethylammonium; TLC, thin-layer
chromatography.
*
Corresponding authors.
E-mail addresses: simona.saponara@unisi.it (S. Saponara), fabio.fusi@unisi.it (F. Fusi).
https://doi.org/10.1016/j.bioorg.2020.104404
Received 3 September 2020; Received in revised form 8 October 2020; Accepted 19 October 2020
Available online 22 October 2020
0
045-2068/© 2020 Elsevier Inc. All rights reserved.

G. Carullo et al.
Bioorganic Chemistry 105 (2020) 104404
depolarization and/or changes in the sub-plasmalemmal Ca2 concen-
tration, and controlled by numerous intracellular chemical ligands and
kinases.
+
compared to that measured with quercetin alone) indeed prevails over
quercetin-induced myorelaxation, thus causing smooth muscle
contraction [24]. Finally, low µM concentrations of the flavonoid are
Noticeably a reduced KCa1.1 channel function and/or expression,
leading to vasoconstriction, has been associated to a number of patho-
logical conditions including hypertension, type 2 diabetes mellitus,
ischemia/reperfusion or brain injury, haemorrhagic shock and athero-
sclerosis [13]. Therefore, KCa1.1 channels represent important targets
for therapeutic intervention [14].
capable to counteract Ca
V
1.2 channel stimulation operated by Bay K
1.2 channels can anyhow affect its
8644 [25]. As this effect on Ca
V
vasorelaxant activity, the aim of the present work was to design quer-
cetin derivatives as more selective modulators of KCa1.1 channels,
V
somehow devoid of the stimulatory activity on or even blocking Ca 1.2
channels, in order to produce novel selective vasorelaxing agents. New
hybrid compounds, ester-derivatives were thus designed choosing acyl
donors of natural source, because of their known vasodilating properties
[26] and structural similarities with other KCa1.1 channel ligands such
Quercetin causes coronary vasodilation due, at least in part, to a
2 2
H O -mediated increase of the iberiotoxin-sensitive KCa1.1 channel
currents (IKCa1.1) [15]. This effect has been ascribed to the pro-oxidant
activity of the flavonoid that manifests under certain experimental
conditions. Furthermore, quercetin increases the frequency of sponta-
4
as MTx, SKA 31, and DIBAC (3) (Fig. 1) [27,28]. Synthesized com-
pounds were assessed for their vascular and ion channel modulating
activity in intact tissue and isolated myocytes, while analyzing in depth
their interaction with the channel protein in silico. Findings indicate
future strategies to improve quercetin selectivity towards KCa1.1 chan-
neous transient IKCa1.1, which are triggered by Ca2 sparks, and hyper-
polarizes the cell membrane, thus potentiating a fundamental feedback
mechanism in vascular function that contrasts membrane depolarization
and vasoconstriction. It is noteworthy that this effect on IKCa1.1 is already
significant at low concentrations, similar to those observed in human
plasma after the ingestion of quercetin-rich foods [16]. Quercetin
stimulates vascular IKCa1.1 with a mechanism that seems to be tissue-
specific. In fact, in the rat tail main artery this effect partly occurs via
a protein kinase G (PKG)-mediated, catalase-independent mechanism
+
V
nels as compared to Ca 1.2 channels.
2. Results and discussion
2.1. In silico screening
[
17]. Additionally, in this tissue the flavonoid decreases both the fre-
Semi-synthetic hybrids were designed, by using different acyl donors
(Fig. 1), derived from natural sources, with reported vasoactive prop-
erties, as just demonstrated [26,29].
quency and the amplitude of spontaneous transient IKCa1.1 by reducing
2
+
the amount of Ca releasable from the sarcoplasmic reticulum [18].
2
+
Among Ca
+
channels, Ca
V
channels constitute the predominant
Twenty-four quercetin derivatives (see Table 1 Supporting infor-
mation) were designed according to the key chemically reactive centers
Ca2 influx route in vascular smooth muscle cells and support several
functions in the cardiovascular system: action potential generation and
pacemaking activity, cardiac inotropism and atrial excitability, arterial
myogenic tone and vascular resistance. Surprisingly, though behaving as
′
of the molecule, namely C-4 , C-3 and C-7 [30,31]. To select the best
leads among the series an in silico docking approach was followed.
Firstly, a blind docking was performed for each compound to identify a
potential binding region on the KCa1.1 channel. All the molecules
localized, with high frequency, in a region proximal to the S6 segment
(Fig. 2A). A rational docking simulation was then carried out to obtain
the most accurate binding mode for each ligand. Interestingly, all de-
rivatives located in the same binding pocket, assuming a comparable
binding pose (Fig. 2 B-C). Their binding affinity for the target was high,
V
a vasodilator in vitro as well as in vivo, quercetin stimulates Ca 1.2
current in clonal rat pituitary GH4C1 cells, via cAMP-induced activation
of protein kinase A (PKA) [19], in guinea-pig gut [20] and in rat vascular
smooth muscle cells, where stimulation is rather specific, since quercetin
does not affect the co-expressed Ca
V
3.1 channels [21–23]. However,
Ca 1.2 channel stimulation by quercetin causes an influx of extracellular
V
2
+
ꢀ 1
ꢀ 1
Ca that is not sufficient to overcome its myorelaxing activity. This
hypothesis is corroborated by the observation that maximal activation of
ranging from ꢀ 10.2 kcal mol (1E) to ꢀ 7.6 kcal mol (3F) (see Table 1
Supporting information). A careful analysis of the ΔG values suggested
that, in general, the C-3 substitution gave rise to molecules with lower
1.2 channels by Bay K 8644 (i.e. larger influx of extracellular Ca2 as
+
Ca
V
Fig. 1. Design of new quercetin derivatives as selective KCa1.1 channel stimulators starting from quercetin and already described KCa1.1channel stimulators (MTx,
SKA31, and DIBAC (3)).
4
2

G. Carullo et al.
Bioorganic Chemistry 105 (2020) 104404
Table 1
The interaction network analysis indicated that the three ligands
formed different bonds within the binding pocket (Fig. 3 A-C), only a few
interactions being conserved. In particular, all compounds shared three
hydrophobic interactions with Tyr-402, Lys-458, and Phe-461, three
K
Ca1.1 channel-compound interaction network summary. The consensus
binding residues are highlighted in bold.
Compound
Hydrophobic
interaction
Hydrogen
bond
π
-Stacking
π
-Cationic
ΔG
(kcal
hydrogen bonds with Lys-300, Tyr-398, and Tyr-402, and a -stacking
π
ꢀ
1
mol
)
interaction with Tyr-398. The region identified as the potential binding
pocket of quercetin derivatives plays a key role in KCa1.1 channel acti-
vation [32].
1
1
E
F
Arg-395, Tyr-
Lys-300,
Tyr-398,
Tyr-402,
Glu-454,
Phe-466,
Tyr-467
Lys-300,
Asn-394,
Tyr-398,
Tyr-402,
Lys-458,
Val-464,
Phe-466
Lys-300,
Ser-383,
Tyr-398,
Tyr-402,
Glu-454
Tyr-398
Lys-458
ꢀ 10.2
4
02, Lys-458,
Phe-461, Glu-
65
In fact, Gessner et al. [28] showed through in vitro mutagenesis that
residues located in this region, particularly Tyr-402, are crucial both for
the activation of the channel and for the modulator binding. Therefore,
the hydrogen bond formed by all derivatives with Tyr-402 may trigger a
conformational change of S6/RCK linker, inducing KCa1.1 channel
stimulation. Noticeably, the three hydrophobic interactions with Tyr-
4
Arg-395, Tyr-
02, Lys-458,
Tyr-398,
Lys-458
ꢀ 9.3
4
Phe-461
Phe-461
4
02, Lys-458, and Phe-461, the other two hydrogen bonds with Lys-
300 and Tyr-398, and the -stacking interaction with Tyr-398, might
π
represent novel consensus binding sites for KCa1.1 channel modulators.
In silico results showed that the three derivatives were able to bind
with high affinity inside the KCa1.1 channel binding pocket located in
the S6/RCK linker. Furthermore, the KCa1.1 channel-compound inter-
action network suggested that the potential mechanism of action of the
ligands might be based on the hydrogen bond formed with Tyr-402.
3
F
Lys-397, Tyr-
Tyr-398,
ꢀ 7.6
4
02, Lys-458,
Phe-461, Glu-
65
Phe-461
4
′
ΔG values as compared to the C-7 and C-4 substituted ones. Therefore,
the two compounds with the highest and lowest ΔG values, respectively,
were selected as the most representative of the series. Among the in-
2
.2. Chemistry
′
termediate ΔG values, a C-4 substituted derivative (1F) was preferred to
attain also SAR indications (Table 1).
According to these computational data, three derivatives named 1E,
1
F, and 3F were synthesized by simple chemical procedures (Scheme 1).
Fig. 2. KCa1.1 channel-compound docked poses overview. (A) The KCa1.1 channel protein is represented as a transparent cyan surface cartoon while the binding
pocket is shown in cyan surface. Compounds 1E, 1F, and 3F are depicted as gold, purple, and magenta sticks and balls. The membrane bilayer is delimited by two
black dotted lines (B,C) Enlarged and twisted view of docked poses of compounds into the KCa1.1 channel binding pocket. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
3

G. Carullo et al.
Bioorganic Chemistry 105 (2020) 104404
Fig. 3. KCa1.1 channel binding residues-ligand interaction network. (A) 1E, (B) 1F, and (C) 3F are displayed in gold, purple, and magenta balls and sticks,
whereas the KCa1.1 channel binding residues are in cyan. Hydrophobic interactions, hydrogen bonds, -stacking, and -cationic interactions are depicted as grey,
π
π
blue, green, and orange dotted lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Scheme 1. Synthesis of the selected quercetin derivatives as KCa1.1 channel stimulators. Reagents and conditions: a) 3,4-(methylenedioxy)cinnamic acid (for
◦
◦
1
E),
α
-lipoic acid (for 1F), HOBt, CMC, DCM, DMF, 0 C, 15 min, then rt, 24 h; b) acetic anhydride, pyridine, reflux, 5 h; c) imidazole, DCM, ꢀ 15 C, then rt, 2 h; d)
◦
α
-lipoic acid, HOBt, CMC, DCM, DMF, 0 C, 15 min, then rt, 24 h; e) 6 N aqueous HCl, CH
3
CN.
4

G. Carullo et al.
Bioorganic Chemistry 105 (2020) 104404
1
E and 1F were synthesized through a Steglich reaction performed
1.2 channels and the ensuing Ca2 influx from the extracellular
+
of Ca
V
by using 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC), and
N-hydroxybenzotriazole (HOBt): 1E and 1F were thus obtained in
acceptable yield (32% and 46%, respectively). The NMR analysis,
space. In particular, the two KCl concentrations employed here (namely
25 mM and 60 mM) represented standardized experimental settings to
+
2+
detect K channel openers and Ca antagonists, respectively [33]. In
fact, the former should be more efficacious on 25 mM KCl-induced
contraction whereas the latter on that induced by 60 mM KCl. As no
significant differences were observed between the two experimental
settings, it can be hypothesized that the vasorelaxant activity of quer-
′
identified only C-4 esters. Anything else, if present, was considered as a
side product.
A different approach was followed for the synthesis of 3F, because
the OH in C-7 position is the third in order of reactivity of quercetin core
[
7]. Firstly, quercetin was converted to its penta-acetylated derivative
V
cetin and its derivatives cannot be ascribed only to a Ca 1.2 channel
Que1, which was selectively deacetylated in C-7 position with imid-
azole. The product Que2 was then subjected to Steglich conditions to
obtain the ester Que3, which was converted to the final compound 3F in
acidic conditions. 1E, 1F, and 3F were then assessed for their vascular
activity.
blockade or to a KCa1.1 channel stimulation. Of note two findings: the
weak myorelaxant activity displayed by 1E and the higher efficacy of 3F
as compared to quercetin in rings depolarized with 60 mM KCl.
The effects of 1E, 1F, and 3F were further investigated on rings pre-
contracted with the
α
-adrenergic receptor agonist phenylephrine.
Fig. 4C shows that 3F and quercetin relaxed aorta ring preparations in a
concentration-dependent manner, with a similar pattern of potency and
efficacy. 1F was less active, whereas 1E spasmolytic activity accounted
for a mere 10% relaxation at the maximal concentration assessed.
2
.3. Pharmacological evaluation
In a first series of experiments, 1E, 1F, and 3F were tested on rings
Phenylephrine-induced contraction is the result of Ca2 release from
the sarcoplasmic reticulum triggered by inositol trisphosphate (IP ) and
influx from the extracellular space through receptor-, store-
operated, and Ca 1.2 channels [34]. Therefore, quercetin and 3F, 1F
+
pre-contracted with high KCl, which depolarizes the membrane, thus
causing Ca 1.2 channels to open. Quercetin was also assessed as positive
V
3
2
+
control. In rings depolarized with 25 mM KCl, 1F, 3F, and quercetin
caused a concentration dependent relaxation of equal potencies and
efficacy (Fig. 4A). However, 1E was only partially effective, showing a
relaxant effect of 27.5 ± 6.3% (n = 4). In preparations depolarized with
Ca
V
to a lesser extent, but not 1E, likely affect one or more of these pathways
leading to vasorelaxation.
6
0 mM KCl, 1F and quercetin showed a vasorelaxant activity superim-
Taken together, aorta ring findings suggest that the introduction of a
posable to that recorded on 25 mM KCl-stimulated rings (Fig. 4B). The
spasmolytic activity of 3F was significantly greater than that of the
parent compound quercetin. Furthermore, 1E efficacy was further
reduced. High KCl-induced contraction is essentially due to the opening
lipoyl moiety in C-7 position leaves unaltered or even improves quer-
′
cetin vasorelaxant activity, whereas that in C-4 position does not
change or slightly reduces it. The latter observation is further supported
by the marked reduction of vasorelaxant activity characterizing 1E that
Fig. 4. Effects of quercetin and its derivatives on rat aorta rings. Endothelium-deprived rings were pre-contracted with (A) 25 mM KCl (K25), (B) 60 mM KCl (K60),
or (C) 0.3 µM phenylephrine (phe). On the plateau of the contraction, cumulative concentrations of quercetin, 1E, 1F or 3F were added. On the ordinate axis muscle
tension is reported as percentage of the initial tone evoked by either KCl or phenylephrine. Data points represent mean ± s.e.m. (n = 4–6). *P < 0.05 vs. quercetin,
Student’s t test for unpaired samples.
5

G. Carullo et al.
Bioorganic Chemistry 105 (2020) 104404
′
bears a 3,4-methyelenedioxycynnamoyl moiety in C-4 position.
These results prompted us to investigate the effects of the most and
observed with quercetin under similar experimental conditions (i.e.
78.6%) [21].
the less active vasorelaxants, i.e. 3F and 1E, respectively, on Ba2 cur-
+
Fig. 7 (right column) shows the time course of current amplitude
stimulation induced by the two derivatives. No significant differences
were observed in the kinetics of stimulation. Furthermore, 1F and 3F, at
the maximal concentration assessed, did not modify the kinetics of
channel activation measured at the depolarizing pulses to 70 mV
V
rent through Ca 1.2 channels (IBa1.2), which play a fundamental role in
the regulation of vascular smooth muscle tone.
Fig. 5 shows recordings of the inward current elicited with a clamp
pulse to 0 mV from a V
h
of ꢀ 50 mV under control conditions and after
the addition of cumulative concentrations of 1E (panel A) and 3F (panel
B). Surprisingly, 1E inhibited peak IBa1.2 in a concentration-dependent
manner, inhibition being around 40% at the maximal concentration
assessed (100 µM). In contrast, 3F showed a biphasic behaviour, first
stimulating with a pEC50 value of 5.58 ± 0.07 M (n = 5) and then
inhibiting IBa1.2. Under similar experimental conditions quercetin stim-
ulated IBa1.2 with a pEC50 value of 5.09 M [21]. These findings
demonstrate that quercetin derivatization with a 3,4-methylendioxycyn-
namoyl moiety as occurred in 1E gave rise to a novel agent not only
(Fig. 7). Under control conditions,
τ of activation were 44.69 ± 5.23 ms
and 40.95 ± 4.64 ms (n = 5). Similar values were recorded in the
presence of 30 µM 1F (50.41 ± 8.19 ms) or 10 µM 3F (37.12 ± 4.72 ms;
P > 0.05).
3. Conclusions
The present findings suggest that the introduction of a lipoyl moiety
in quercetin structure only marginally affected its IKCa1.1 stimulatory
activity, particularly when present in C-7 position. Furthermore, the OH
V
deprived of Ca 1.2 channel stimulating activity, but also possessing a
2
+
′
moderate but significant Ca antagonist effect. On the contrary, 3F,
bearing a lipoyl moiety in C-7 position, did not affect the IBa1.2 stimu-
latory activity of the parent flavonoid.
group in C-7 and C-4 positions does not seem crucial to this activity. The
design strategy pursued along with the in silico analysis gave rise to
quercetin derivatives either endowed of Ca2 antagonist features
(compound 1E) or displaying KCa1.1 channel stimulatory activity and
vasorelaxant effects comparable to that of the parent compound (com-
pounds 1F and 3F). These findings open new avenues for the derivati-
zation of quercetin to improve its beneficial vascular activity.
+
I
Ba1.2 evoked at 0 mV from a V
declined with a time course that could be fitted by a monoexponential
function. The two derivatives slowed significantly the of activation,
though with different concentration-dependent patterns, bell-shaped for
F, exponential-like for 1E (Fig. 6). These behaviours paralleled those
observed on current amplitude. 3F, at 10 µM concentration, slowed
significantly also the of inactivation.
Under similar experimental conditions also quercetin slowed down
significantly the of activation without affecting that of inactivation
21]. Taken together, these findings demonstrate that the two different
derivatizations did not modify the capability of the parent compound to
slow down the transition of Ca 1.2 channels from the closed to the open
h
of ꢀ 50 mV activated and then
τ
3
4. Experimental section
τ
4.1. Docking studies
τ
[
The homology 3D model of the Rattus Norvegicus KCa1.1 channel was
used as previously described [12]. The chemical structures of the
designed compounds (Table 1) were drawn using Chemdraw software,
and optimized prior to the docking procedure by using the Dock Prep. To
study the binding mode of compounds to the channel, a docking simu-
lation was performed carrying out a flexible side chain protocol with
AutoDock/VinaXB [35]. The pdbqt files of ligands were obtained by
Open Babel tool v. 2.4.1 [36], while pdbqt format of the receptor was
created by AutoDock Vina v. 1.1.2 tools [37]. The Protein–Ligand
Interaction Profiler (PLIP) generated multiple ligand–protein interaction
maps [38]. PyMOL v. 2.2 was used as molecular graphics system (The
PyMOL Molecular Graphics System, Version 2.2 Schr o¨ dinger, LLC).
V
state or, in some other way, to modify the gating mechanism. Further-
more, the presence of a lipoyl moiety in C-7 (3F) gave rise to a derivative
that slowed down Ca
pulse.
V
1.2 channel inactivation during the depolarizing
Given the weak vasorelaxant activity of 1E, the present work focused
on 3F and its analogue 1F, which showed myorelaxant effects compa-
rable to those of quercetin. The final aim was to verify whether the lipoyl
moiety and its position might affect quercetin stimulation of KCa1.1
channels. Fig. 7 (left column) shows the current-voltage relationships of
I
KCa1.1 elicited with clamp pulses in the range ꢀ 20 mV and 70 mV from a
V
h
of ꢀ 40 mV, under control conditions and after the cumulative
4
.2. General chemical informations
addition of 10–30 µM 1F (panel A), or 3–10 µM 3F (panel B). The two
derivatives stimulated current amplitude in a concentration-dependent
manner with a similar efficacy.
Commercially available reagents purchased from Merck (Milan,
Italy) were used without further purification, and all solvents were of
HPLC quality. Reactions under nitrogen atmosphere were performed in
oven- or flame-dried glassware and anhydrous solvents, and then
In fact at 70 mV, 10 µM 1F or 3F increased IKCa1.1 amplitude by
3.4% and 68.3%, respectively. These results are in line with that
5
Fig. 5. Effects of 1E and 3F on IBa1.2 recorded in rat tail artery myocytes. Concentration-dependent effect of (A) 1E and (B) 3F. On the ordinate scale IBa1.2 amplitude
is reported as percentage of that recorded just before drug addition. Data points are means ± s.e.m. (n = 4–5). Insets: current traces (averaged from 5 cells) recorded
with a clamp pulse to 0 mV from a V
h
of ꢀ 50 mV in the absence (ctrl) or presence of various concentrations (µM) of quercetin derivatives.
6

G. Carullo et al.
Bioorganic Chemistry 105 (2020) 104404
Fig. 6. Effects of 1E and 3F on IBa1.2 kinetics recorded in rat tail artery myocytes. Concentration-dependent effect of (A) 1E and (B) 3F on time constant of current
activation ( act; open columns) or inactivation (
inact; closed columns). Columns are means ± s.e.m. (n = 5) *P < 0.05, repeated measures ANOVA and Dunnett’s post-
test. Right: current traces (averaged from 5 cells) sized so that the peak amplitude of the traces in presence of the drug matched that of the control.
τ
τ
monitored by thin-layer chromatography (TLC) Merck 60 F254 silica
plates. Analytical TLC was conducted on Merck aluminum sheets
covered with silica (C60). The plates were either visualized under UV-
light (254 nm) or stained by dipping in a developing agent followed
4.2.1.1. 2-hydroxy-4-(3,5,7-trihydroxy-4-oxo-4H-chromen-2-yl)phenyl
1
(E)-3-(benzo[d][1,3]dioxol-5-yl)acrylate 1E. Yellow resin, 32% yield. H
NMR (DMSO‑d
): δ 8.10–7.90 (m, 3H), 7.72 (d, 1H, J = 4.6 Hz),
7.66–7.59 (m, 1H), 7.52–7.41 (m, 2H), 7.35 (d, 1H, J = 4.6 Hz), 6.90
6
1
3
by heating with KMnO
4
[3 g in water (300 mL) along with K
2
CO
3
(20 g)
(dd, 2H, J = 3.0, 15.0 Hz), 6.10 (s, 2H). C NMR (DMSO‑d
6
): δ 176.2,
and 5% aqueous NaOH (5 mL)]. Flash column chromatography was
166.5, 164.0, 162.0, 159.0, 152.2, 148.0 (x3C), 139.9, 136.4, 127.0,
performed using silica gel 60 (0.040–0.063 mm). The synthesized
126.1, 123.0, 122.4, 121.6, 116.1 (x2C), 115.3, 108.0, 105.9, 104.0,
1
13
compounds were characterized by H NMR, C NMR (Bruker Advance
16
100.2, 98.3, 94.1. Anal. Calcd for C25H O10: C, 63.03; H, 3.39. Found:
operating at 300 MHz) and melting point (m.p.) when applicable. Unless
C, 62.89; H, 3.27.
◦
otherwise stated, all NMR spectra were recorded at 25 C. The chemical
shifts (δ) are reported in parts per million (ppm) and the coupling con-
4.2.1.2. 2-hydroxy-4-(3,5,7-trihydroxy-4-oxo-4H-chromen-2-yl)phenyl 5-
1
stants (J) in Hz. For spectra recorded in CDCl
measured relative to the signal for CHCl
(7.26 ppm for 1H NMR and
7.16 ppm for 13C NMR). For spectra recorded in DMSO‑d
, signal po-
sitions were measured relative to the signal for DMSO (δ 2.50 ppm for H
NMR and 39.52 ppm for 13C NMR). For spectra recorded in acetone‑d
3
, signal positions were
(1,2-dithiolan-3-yl)pentanoate 1F. Yellow resin, 46% yield. H NMR
3
(DMSO‑d ): δ 7.59 (s, 1H), 7.50–7.40 (m, 1H), 6.80–6.70 (m, 1H), 6.34
6
7
6
(s, 1H), 6.10 (s, 1H), 3.60–3.50 (m, 2H), 3.20–3.01 (m, 2H), 2.50–2.30
1
13
(m, 1H), 2.10–1.80 (m, 1H) 1.70–1.40 (m, 7H). C NMR (DMSO‑d ): δ
6
6
,
176.1, 172.0, 166.4, 161.8, 158.8, 150.8, 146.9, 139.2, 136.5, 127.0,
124.0, 120.8, 114.3, 104.5, 98.3, 94.0, 61.4, 45.2, 43.3, 39.6, 38.2, 29.6,
24.6. Anal. Calcd for C23H22O S : C, 56.32; H, 4.52; S, 13.07. Found: C,
signal positions were measured relative to the signal for acetone (δ 2.60
ppm for 1H NMR and 30.0 and 206 ppm for 13C NMR). Anhydrous
8
2
quercetin (99%, HPLC), CMC, HOBt,
α
-lipoic acid, and acetic anhydride
55.62; H, 5.01; S, 12.08.
were purchased from Merck (Milan, Italy).
4
.2.2. Procedure b): Synthesis of 2-(3,4-diacetoxyphenyl)-4-oxo-4H-
4
.2.1. Procedure a): Synthesis of compounds 1E and 1F
chromene-3,5,7-triyl triacetate Que1
A solution of carboxylic acid (1 equiv.), quercetin (1 equiv.), and
Quercetin (1 equiv.), acetic anhydride (20 equiv.), and pyridine (15
mL) were stirred at reflux for 5 h. Then, a mixture of ice-water (50 g) was
added. The resulting precipitate was filtered and washed with cold
EtOAc to obtain Que1. White solid, 79% yield. Spectroscopic data are in
agreement with those previously reported [40].
HOBt (1.2 equiv.) in anydrous DCM/DMF (65:35) (5 mL) was cooled at
◦
0
C and stirred under nitrogen atmosphere for 15 min as previously
described [39]. Thereafter, a solution of CMC (1.5 equiv.) in dry DCM
3.5 mL) was added dropwise. The mixture was stirred at rt for 24 h,
washed twice with a 5% aqueous NaHCO solution and finally once with
brine. The collected organic layers, dried on anhydrous Na SO , were
(
3
2
4
4.2.3. Procedure c): Synthesis of 4-(3,5-diacetoxy-7-hydroxy-4-oxo-4H-
chromen-2-yl)-1,2-phenylene diacetate Que2
concentrated and the pure compound obtained after flash-column
chromatography (eluent DCM/MeOH 15/1).
A solution of imidazole (2 equiv.) in DCM (5 mL) was added
7

G. Carullo et al.
Bioorganic Chemistry 105 (2020) 104404
Fig. 7. Effects of 1F and 3F on IKCa1.1 recorded in rat tail artery myocytes. Left: concentration-dependent effect of (A) 1F and (B) 3F. On the ordinate scale IKCa1.1
amplitude is reported in pA/pF. Data points are means ± s.e.m. (n = 5). *P < 0.05, repeated measures ANOVA and Dunnett’s post-test. Right: time-course of current
stimulation caused by (A) 1F and (B) 3F. On the ordinate scale IKCa1.1 is reported as percentage of that recorded just before the addition of the first concentration of
the derivative. The effect of 1 mM TEA, which blocked IKCa1.1, is also shown. Insets: current traces (averaged from 5 cells) recorded in the absence (control) or
presence of (A) 30 µM 1F, or (B) 10 µM 3F. IKCa1.1 was elicited with a clamp pulse to 70 mV from a V of ꢀ 40 mV, delivered every 10 s.
h
◦
dropwise to a solution of Que1 (1 equiv.) in DCM (10 mL) at –15 C in an
4.2.5. Procedure e): Synthesis of 2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-
ice/acetone bath. The mixture was allowed to warm to rt and stirred for
4-oxo-4H-chromen-7-yl 5-(1,2-dithiolan-3-yl)pentanoate 3F
2
3
h. The reaction mixture was diluted in DCM (50 mL) and washed with
3
Que3 (1 equiv.) was added to CH CN (20 mL) and 6 N aqueous HCl
M aqueous HCl (3 × 50 mL). The organic layer was then dried over
(10 mL). The resulting solution was stirred at reflux for 1.5 h. Then
EtOAc (100 mL) and water (100 mL) were added. The organic layer was
washed with 3 N aqueous HCl (3 × 100 mL), dried over anhydrous
2 4
anhydrous Na SO and filtered. The solvent was evaporated under
reduced pressure. The resulting residue was purified through flash-
column chromatography (eluent: CHCl /MeOH, 97/3). White solid,
7% yield. Spectroscopic data are in agreement with those previously
reported [40].
3
2 4
Na SO and filtered. The solvent was evaporated under reduced pres-
8
sure. 3F was obtained after precipitation of the residue with DCM.
Yellow resin, 80% yield. 1H NMR (Acetone‑d
): δ 12.30 (bs, OH–(C5),
6
′
′
1
H), 9.80 (bs, OH–(C4 ), 1H), 8.70 (bs, OH–(C3 ), 1H), 8.40 (bs, OH–
4
.2.4. Procedure d): Synthesis of 7-((5-(1,2-dithiolan-3-yl) pentanoyl)
(C3), 1H), 7.83 (s, 1H), 7.70–7.50 (m, 1H), 7.01–6.90 (m, 1H), 6.51 (s,
1H), 6.28 (s, 1H), 3.60–3.50 (m, 2H), 3.20–3.01 (m, 2H), 2.50–2.30 (m,
oxy)-2-(3,4-diacetoxyphenyl)-4-oxo-4H-chromene-3,5-diyl diacetate Que3
1
3
This product was obtained with the same procedure used for com-
1H), 2.10–1.80 (m, 1H) 1.70–1.40 (m, 7H). C NMR (Acetone‑d ): δ
6
pounds 1E and 1F. Yellow resin, 78% yield. 1H NMR (CDCl
): δ
.80–7.60 (m, 1H), 7.40–7.32 (m, 2H), 6.92–6.86 (m, 2H), 3.30–3.10
180.8, 169.2, 166.5, 161.9, 152.6, 151.2, 150.0, 146.9, 141.0, 136.0,
128.0, 125.7, 120.4, 120.0, 108.4, 103.4, 98.7, 61.4, 45.2, 43.3, 39.6,
3
7
1
3
(
m, 1H), 2.80–2.30 (m, 15H), 2.01–1.40 (m, 9H). C NMR (CDCl
3
): δ
38.2, 29.6. Anal. Calcd for C23
Found: C, 56.20; H, 4.32.
22 8 2
H O S : C, 56.32; H, 4.52; S, 13.07.
1
1
3
78.6, 169.3, 167.9 (x 3C), 167.8, 154.2, 150.3, 144.3, 126.4, 125.0,
24.0, 123.8, 122.3, 115.5, 114.1, 107.9, 56.2, 40.2, 38.5, 34.5, 33.7,
3.5, 28.6, 24.5, 24.3, 21.1, 21.0, 20.7, 20.6, 20.5.
8

G. Carullo et al.
Bioorganic Chemistry 105 (2020) 104404
mM TEA- and 5 mM Ba2 -containing external solution (320 mOsmol)
and that of the internal solution (290 mOsmol) were measured with an
osmometer (Osmostat OM 6020, Menarini Diagnostics, Florence, Italy).
+
4
4
.3. Pharmacological analysis
.3.1. Animals
All experimental protocols were approved by the Animal Care and
Ethics Committee of the University of Siena and Italian Department of
Health (7DF-19.N.TBT) and carried out in accordance to the European
Union Guidelines for the Care and the Use of Laboratory Animals (Eu-
ropean Union Directive 2010/63/EU). Male Wistar rats, weighing
4.3.5. IKCa1.1 current measurement
I
KCa1.1 (registration period 500 ms) was measured over a range of test
potentials from ꢀ 20 to 70 mV from a V of ꢀ 40 mV. This V limited the
contribution of K channels to the overall whole-cell current. Data were
h
h
V
3
25–450 g were purchased from Charles River Italia (Calco, Italy) and
collected once the current amplitude had been stabilized (usually 8–10
min after the whole-cell configuration had been obtained) [46]. IKCa1.1
current did not run down during the following 20–30 min under the
present experimental conditions. The external solution for IKCa1.1 re-
cordings contained (in mM): 145 NaCl, 6 KCl, 10 glucose, 10 HEPES, 5
◦
maintained in an animal house facility at 25 ± 1 C and 12:12 h dark-
light cycle with free access to standard chow diet and water. Animals
2
were anaesthetized with an isoflurane (4%) and O gas mixture by using
Fluovac (Harvard Apparatus, Holliston, Massachusetts, USA), decapi-
tated and exsanguinated. The thoracic aorta and tail (cleaned of skin)
were immediately removed and placed in physiological solution
Na-pyruvate, 1.2 MgCl
ternal solution contained (in mM): 90 KCl, 10 NaCl, 10 HEPES, 10 EGTA,
1 MgCl , 6.41 CaCl (pCa 7.0); pH 7.4. The osmolarity of the external
2 2
, 0.1 CaCl , 0.003 nicardipine (pH 7.4). The in-
[
namely modified Krebs-Henseleit solution (KHS)] or external solution
2
2
(
see below for compostion), respectively.
and internal solutions were 310 mosmol and 265 mosmol, respectively.
The current-voltage relationships were calculated on the basis of the
values recorded during the last 200 ms of each test pulse (leakage cor-
rected). IKCa1.1 was isolated from other currents as well as corrected for
leakage using 1 mM TEA, a specific blocker of KCa1.1 channels [16].
4
.3.2. Cell isolation procedure
The tail main artery was dissected free of its connective tissue and
smooth muscle cells were freshly isolated under the following condi-
◦
tions. A 5-mm long piece of artery was incubated at 37 C for 40–45 min
in 2 mL of 0.1 mM Ca2 external solution (in mM: 130 NaCl, 5.6 KCl, 10
+
4.4. Functional experiments
HEPES, 20 glucose, 1.2 MgCl
2
, and 5 Na-pyruvate; pH 7.4) containing
2
0 mM taurine, which replaced an equimolar amount of NaCl, 1.35 mg
4.4.1. Preparation of rat aorta rings
ꢀ 1
ꢀ 1
mL collagenase (type XI), 1 mg mL soybean trypsin inhibitor, and 1
The thoracic aorta was immediately removed, gently cleaned of
adherent tissues, and cut into 3.0-mm wide rings. The endothelium was
removed by gently rubbing the lumen of the ring with forceps tip. Rings
were mounted in organ baths between two parallel L-shaped stainless
steel hooks, one fixed in place and the other connected to an isometric
transducer. Rings were allowed to equilibrate for 60 min in a modified
KHS (composition in mM: 118 NaCl; 4.75 KCl; 1.19 KH PO ; 1.19
ꢀ
1
mg mL bovine serum albumin. This solution was gently bubbled with
a 95% O -5% CO gas mixture to stir the enzyme solution and cells
isolated as previously described [41]. Cells stored in 0.05 mM Ca
2
2
2
+
ꢀ 1
external solution containing 20 mM taurine and 0.5 mg mL bovine
◦
serum albumin at 4 C under air were used for experiments within two
days after isolation [42].
2
4
4 3 2 2
MgSO ; 25 NaHCO ; 11.5 glucose; 2.5 CaCl ; gassed with a 95% O -5%
4
.3.3. Whole-cell patch clamp recordings
CO2 gas mixture to create a pH of 7.4) under a passive tension of 1 g
[47]. Isometric tension was recorded using a digital PowerLab data
acquisition system (PowerLab 8/30; ADInstruments) and analyzed by
using LabChart 7.3.7 Pro (PowerLab; ADInstruments). Rings viability
was assessed by recording the response to 0.3 µM phenylephrine. The
lack of response (relaxation ≤ 10%) to the addition of 10 µM acetyl-
choline at the plateau of phenylephrine-induced contraction denoted the
absence of a functional endothelium [48].
An Axopatch 200B patch-clamp amplifier (Molecular Devices Cor-
poration, Sunnyvale, CA, USA) was used to generate and apply voltage
pulses to the clamped cells and record the corresponding membrane
currents. At the beginning of each experiment, the junction potential
between the pipette and bath solution was electronically adjusted to
zero. Current signals, after compensation for whole-cell capacitance and
series resistance (between 70% and 75%), were low-pass filtered at 1
kHz and digitized at 3 kHz prior to being stored on the computer hard
+
disk. Electrophysiological responses were tested at room temperature
4.4.2. Effect of quercetin and its derivatives on phenylephrine- and high K -
◦
(
20–22 C).
induced contraction
Aorta rings were stimulated pharmacomechanically with 0.3 µM
phenylephrine, or electromechanically with 25 mM or 60 mM KCl [49].
Once vessel tone reached a stable plateau, the drug was added cumu-
latively to the organ bath to assess its spasmolytic activity by con-
structing a concentration-response curve. At the end of these protocols,
100 µM sodium nitroprusside alone (phenylephrine-induced contrac-
tion) or 1 µM nifedipine followed by sodium nitroprusside (KCl-induced
contraction) were added to prove the functional integrity of smooth
muscle. The spasmolytic activity of each drug was calculated as a per-
centage of the maximum contraction induced by phenylephrine or KCl
(taken as 100%).
4
.3.4. IBa1.2 recording
Cells were continuously superfused with external solution containing
+
.1 mM Ca2 and 30 mM tetraethylammonium (TEA) using a peristaltic
0
ꢀ
1
pump (LKB 2132, Bromma, Sweden) at a flow rate of 400 µl min . The
conventional whole-cell patch-clamp method was employed to voltage
clamp smooth muscle cells. Recording electrodes were pulled from bo-
rosilicate glass capillaries (WPI, Berlin, Germany) and fire-polished to
obtain a pipette resistance of 2–5 MΩ when filled with internal solution.
The internal solution (pCa 8.4) consisted of (in mM): 100 CsCl, 10
HEPES, 11 EGTA, 2 MgCl
2 2
, 1 CaCl , 5 Na-pyruvate, 5 succinic acid, 5
oxaloacetic acid, 3 Na ATP, and 5 phosphocreatine; the pH was adjusted
2
to 7.4 with CsOH. Ba2 current through Ca
ded in an external solution containing 30 mM TEA and 5 mM Ba , was
elicited with 250 ms clamp pulses (0.067 Hz) to 0 mV from a V
of ꢀ 50
mV. Data were collected once the current amplitude had been stabilized
+
1.2 channels (IBa1.2) recor-
V
4.5. Drugs and chemicals
2
+
h
The chemicals used included: collagenase (type XI), trypsin inhibitor,
bovine serum albumin, TEA chloride, HEPES, taurine, phenylephrine,
acetylcholine, quercetin, and nifedipine (Merck, Milan, Italy); sodium
nitroprusside (Riedel-De Ha ¨e n AG). All other substances were of
analytical grade and used without further purification. Phenylephrine
was solubilized in 0.1 M HCl. Nifedipine and nicardipine, dissolved
directly in ethanol, and quercetin and its derivatives, dissolved directly
in DMSO, were diluted at least 1000 times prior to use. Control
(
usually 7–10 min after the whole-cell configuration had been obtained)
43]. Under these conditions, the current did not run down during the
[
+
following 40 min [44,45] K currents were blocked with 30 mM TEA in
+
the external solution and Cs in the internal solution. Current values
were corrected for leakage and residual outward currents using 10 µM
nifedipine, which completely blocked IBa1.2. The osmolarity of the 30
9

G. Carullo et al.
Bioorganic Chemistry 105 (2020) 104404
experiments confirmed that no response was induced in vascular prep-
arations when DMSO or ethanol, at the final concentration used in the
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.6. Statistical analysis
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number of cells or rings analysed (indicated in parentheses), isolated
from at least three animals. Statistical analysis and significance, as
measured by repeated measures ANOVA (followed by Dunnett’s post
hoc test), or Student’s t test for unpaired samples (two tailed) were
obtained using GraphPad Prism version 5.04 (GraphPad Software Inc.).
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Declaration of Competing Interest
K
Ca1.1 channels, Acta Physiol. 208 (2013) 329–339, https://doi.org/10.1111/
apha.12083.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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Acknowledgment
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K. T o¨ rnquist, H. Vuorela, Effects of simple aromatic compounds and flavonoids on
We wish to thank Dr. Carmelo Spinella for the assistance in some
preliminary experiments. Authors from the Department of Pharmacy,
Health and Nutritional Sciences – University of Calabria, and Depart-
ment of Biotechnology, Chemistry and Pharmacy – University of Siena
acknowledge Ministero dell’Istruzione, dell’Universit a` e della Ricerca
for grant Dipartimento di Eccellenza 2018-2022, L. 232/2016.
2
+
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https://doi.org/10.1016/S0014-2999(01)00774-9.
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Appendix A. Supplementary material
v
5
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2020.104404.
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