2-Hydroxy-5-(3,5,7-trihydroxy-4-oxo-4 H -chromen-2-yl)phenyl ( E )-3-(4-hydroxy-3-methoxyphenyl)acrylate: Synthesis, In Silico Analysis and In Vitro Pharmacological Evaluation

: Quercetin and ferulic acid are two phytochemicals extensively represented in the plant kingdom and daily consumed in considerable amounts through diets. Due to a common phenolic structure, these two molecules share several pharmacological properties, e.g., antioxidant and free radical scavenging, anti-cancer, anti-inﬂammatory, anti-arrhythmic, and vasorelaxant. The aim of the present work was the combination of the two molecules in a single chemical entity, conceivably endowed with more efﬁcacious vasorelaxant activity. Preliminary in silico studies herein described suggested that the new hybrid compound bound spontaneously and with high afﬁnity on the K Ca 1.1 channel. Thus, the synthesis of the 3 (cid:48) -ferulic ester derivative of quercetin was achieved and its structure conﬁrmed by 1 H- and 13 C-NMR spectra, HSQC and HMBC experiments, mass spectrometry, and elementary analysis. The effect of the new hybrid compound on vascular K Ca 1.1 and Ca V 1.2 channels revealed a partial loss of the stimulatory activity that characterizes the parent compound quercetin. Therefore, further studies are necessary to identify a better strategy to improve the vascular properties of this ﬂavonoid


Introduction
Plant-derived secondary metabolites are non-nutrient compounds that aroused research interest due to their heterogeneous chemical structures complementary to a myriad of pharmacological activities. In this scenario, quercetin, one of the most studied and wellknown phytochemicals, is the flavonol that exhibits many interesting healthy properties, particularly on the cardiovascular system [1]. A growing body of evidence indicates that these beneficial effects are driven by a direct modulation of ion channel function and/or channel protein expression, trafficking, and signaling [2][3][4][5]. In particular, the modulatory effects of quercetin on Ca V 1.2 and K Ca 1.1 channels, playing a fundamental role in the regulation of vascular smooth muscle tone, have been comprehensively described [6,7]. In this context, drugs endowed with vascular bifunctional activity (namely, stimulation of K Ca 1.1 and inhibition of Ca V 1.2 channels) are of potential interest for the treatment of systemic hypertension. In fact, high blood pressure triggers an arterial "ion channel remodeling", a phenomenon involving the upregulation of Ca V 1.2 channels [8] coupled to the down-regulation of voltage-dependent K + (K V ) channels. Consequently, a compensatory overexpression of K Ca 1.1 channels occurs, which helps avert local vasospasm [9]. Therefore, 2 of 12 designing vasodilator drugs, which target and counterbalance disease-specific changes of ion channel expression, would seem a logical approach for developing antihypertensive agents. Noticeably, a close relationship between chemical modifications and activity within quercetin family is reported and exploited both to generate functional diversity and to overcome stability and bioavailability limits [10]. In the search for novel flavonoid derivatives [11][12][13][14], the acylation approach came out as an effective tool for obtaining hybrid compounds selectively decorated with a natural acid residue in the polyhydroxylated 2-phenyl-4H-chromen-4-one core. Accordingly, ferulic acid, a cinnamic-derived plant metabolite abundant in foods, is considered a protective agent with a very versatile and safe pharmacological profile [15]. Its endothelial protective effects [16] prompted us to hypothesize that combination of this molecule with quercetin in a single chemical entity might give rise to a potent vasoactive compound with better physico-chemical properties. Indeed, the synthesis of 3 -ferulic ester derivative of quercetin, i.e., 2-hydroxy-5-(3,5,7trihydroxy-4-oxo-4H-chromen-2-yl)phenyl (E)-3-(4-hydroxy-3-methoxyphenyl)acrylate 1, is here reported together with the assessment of its vascular ion channel modulatory activity both in silico and in vitro.
The effects of compound 1 on Ba 2+ current through Ca V 1.2 channels (I Ba1.2 ) and on K + current through K Ca 1.1 channels (I KCa1.1 ), which play a fundamental role in the regulation of vascular smooth muscle tone, were investigated in rat tail artery myocytes using the whole-cell patch-clamp method.

In Silico Analysis
An in silico docking approach was followed to evaluate the potential activity of both mono-ferulic ester derivatives of quercetin, compound 1, i.e., 3 -O-feruloyl derivative, and compound 2, i.e., the 4 -O-feruloyl derivative, on K Ca 1.1 channels. A rational docking simulation was carried out within the K Ca 1.1 channel binding site proposed by Carullo and colleagues [12]. The docking result showed that both compounds bound spontaneously to the target, exhibiting a not significantly different binding free energy (−8.7 kcal/mol for compound 1 and −9.1 kcal/mol for compound 2) and a docking pose very similar within the binding pocket ( Figure 1B). The interaction network analysis was evaluated by the protein-ligand interaction profile (PLIP) tool. Compound 1 was able to bind in a sensing region of the K Ca 1.1 channel, triggering several interactions with the target key residues [12,17] (Figure 1C and Table S1). Analogously, compound 2 triggered different interactions inside the target binding site ( Figure 1D), forming an interaction network with residues identified as crucial for K Ca 1.1 channel activity [12,17] (Table S1). The high binding affinity and the wide interaction network of compounds 1 and 2 in a sensing region of the K Ca 1.1 channel located in the S6/RCK linker [18], would support the hypothesis of a potential modulating activity of our derivatives against K Ca 1.1 channel. The binding residues are colored in cyan sticks and balls. Hydrogen bonds, hydrophobic interactions, π-stacking and π-cationic interactions are represented as blue, grey, green and orange dotted lines, respectively.

Chemistry
Regioselective synthesis of mono-O-substituted quercetin analogues is a challenging task as the results reported in the literature demonstrate [19][20][21]. In fact, quercetin hydroxyls are not equivalent in terms of chemical reactivity and slight modifications in reaction conditions, such as temperature, timing, order of the reagent addition, and nature of the acylating/alkylating agent, can lead to different mono-O-derivatives or complex reaction mixtures. Therefore, the enzymatic approach is often preferred to obtain selective acylation of the phenolic compound [22][23][24] and, specifically, Kumar and collaborators reported the regioselective synthesis of quercetin ferulate using Rhizopus oryzae lipase [25]. Nevertheless, in a previous work [12] a simple and mild one-step procedure to obtain the 4′-regioselective acylation of quercetin was used, by reacting controlled amounts of the appropriate acids and the natural flavonol in the presence of a water-soluble carbodiimide as carboxyl activating agent. The direct acylation procedure of quercetin under strictly controlled conditions was also successfully reported by Veverka and collaborators [21] as a time-saving alternative. Thus, by modifying our previous procedure to partially overcome the solubility issues of the starting flavonol, the 3′-ferulic ester derivative of quercetin was selectively obtained in acceptable yield (28.0%). In fact, compound 1 was obtained by reacting quercetin (Que) dihydrate with ferulic acid (stoichiometric ratio of 1:1) in the presence of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDCI) and 1-hydroxybenzotriazole (HOBt) in dry dichloromethane/N,N-dimethylformamide (DCM/DMF, procedure A) or acetonitrile (procedure B, Scheme 1) at room temperature. Although the addition of DMF to DCM resulted in a clear yellow solution, acetonitrile alone is an excellent alternative when the reagents are poorly soluble in dichloromethane

Chemistry
Regioselective synthesis of mono-O-substituted quercetin analogues is a challenging task as the results reported in the literature demonstrate [19][20][21]. In fact, quercetin hydroxyls are not equivalent in terms of chemical reactivity and slight modifications in reaction conditions, such as temperature, timing, order of the reagent addition, and nature of the acylating/alkylating agent, can lead to different mono-O-derivatives or complex reaction mixtures. Therefore, the enzymatic approach is often preferred to obtain selective acylation of the phenolic compound [22][23][24] and, specifically, Kumar and collaborators reported the regioselective synthesis of quercetin ferulate using Rhizopus oryzae lipase [25]. Nevertheless, in a previous work [12] a simple and mild one-step procedure to obtain the 4 -regioselective acylation of quercetin was used, by reacting controlled amounts of the appropriate acids and the natural flavonol in the presence of a water-soluble carbodiimide as carboxyl activating agent. The direct acylation procedure of quercetin under strictly controlled conditions was also successfully reported by Veverka and collaborators [21] as a time-saving alternative. Thus, by modifying our previous procedure to partially overcome the solubility issues of the starting flavonol, the 3 -ferulic ester derivative of quercetin was selectively obtained in acceptable yield (28.0%). In fact, compound 1 was obtained by reacting quercetin (Que) dihydrate with ferulic acid (stoichiometric ratio of 1:1) in the presence of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDCI) and 1-hydroxybenzotriazole (HOBt) in dry dichloromethane/N,N-dimethylformamide (DCM/DMF, procedure A) or acetonitrile (procedure B, Scheme 1) at room temperature. Although the addition of DMF to DCM resulted in a clear yellow solution, acetonitrile alone is an excellent alternative when the reagents are poorly soluble in dichloromethane and allowed to eliminate part of the un-reacted quercetin from the reaction mixture by simple filtration of the partially concentrated suspension. The purification of the raw reaction material represented the limiting step to obtaining the desired product in high yield. However, the C-3 ferulic ester of quercetin was the only coupling product isolated as demonstrated by NMR experiments ( 1 H, 13  Concerning the correctness of the proposed structure of compound 1, the five quercetin hydroxyls are known to have a different reactivity and steric hindrance. Specifically, 3-OH and 5-OH hydroxyl groups are the least reactive being involved in an intramolecular hydrogen bond with the oxygen of the carbonyl in position 4 [26]; among the other three hydroxyl groups, the reactivity order strongly depends on the reaction conditions used, even if the results reported in the literature are non-homogeneous and often inconsistent [21,23,27,28]. The mild reaction conditions (a water-soluble carbodiimide as coupling agent, a molar equivalent of the acid, high dilution, and room temperature) promoted the acylation in position 3 . The whole assignment of the NMR signals was confirmed by HSQC and HMBC analyses while the site of acylation was confirmed by comparing upfield and/or downfield shifts in NMR spectra between ester derivative 1 and quercetin signals (see Table 1). and allowed to eliminate part of the unreacted quercetin from the reaction mixture by simple filtration of the partially concentrated suspension. The purification of the raw reaction material represented the limiting step to obtaining the desired product in high yield. However, the C-3′ ferulic ester of quercetin was the only coupling product isolated as demonstrated by NMR experiments ( 1 H, 13 [26]; among the other three hydroxyl groups, the reactivity order strongly depends on the reaction conditions used, even if the results reported in the literature are non-homogeneous and often inconsistent [21,23,27,28]. The mild reaction conditions (a water-soluble carbodiimide as coupling agent, a molar equivalent of the acid, high dilution, and room temperature) promoted the acylation in position 3′. The whole assignment of the NMR signals was confirmed by HSQC and HMBC analyses while the site of acylation was confirmed by comparing upfield and/or downfield shifts in NMR spectra between ester derivative 1 and quercetin signals (see Table 1).
Specifically, in the 2-phenyl-4H-chromen-4-one system the chemical shift values of carbons 3, 5, and 7 as well as those of the protons in 6 and 8 are unchanged (Table 1), excluding the hydroxyls in 7, 5, and 3 as acylation sites. Acylation at 3′ position was supported by the significant upfield shift of the 3′ carbon value (138.8 ppm of compd. 1 vs. 145.5 ppm of Que) associated with the significant downfield shift of the 4′ carbon value (151.6 ppm of compd. 1 vs. 148.2 ppm of Que, Table 1). Moreover, the presence of the ferulic portion moved the carbon and proton chemical shifts of all neighboring protons/carbons (2′, 5′ and 6′) to lower field and among these both the 2′-and 6′-CH values were the most affected (Table 1). According to data reported in the literature [21,29], the assignment of signals is consistent with the proposed structure. The mass spectrum of compound 1 also provided the expected [M − H] − peak (100% intensity) for the assigned structure, also confirmed by the elementary analysis.  Specifically, in the 2-phenyl-4H-chromen-4-one system the chemical shift values of carbons 3, 5, and 7 as well as those of the protons in 6 and 8 are unchanged (Table 1), excluding the hydroxyls in 7, 5, and 3 as acylation sites. Acylation at 3 position was supported by the significant upfield shift of the 3 carbon value (138.8 ppm of compd. 1 vs. 145.5 ppm of Que) associated with the significant downfield shift of the 4 carbon value (151.6 ppm of compd. 1 vs. 148.2 ppm of Que, Table 1). Moreover, the presence of the ferulic portion moved the carbon and proton chemical shifts of all neighboring protons/carbons (2 , 5 and 6 ) to lower field and among these both the 2 -and 6 -CH values were the most affected (Table 1). According to data reported in the literature [21,29], the assignment of signals is consistent with the proposed structure. The mass spectrum of compound 1 also provided the expected [M − H] − peak (100% intensity) for the assigned structure, also confirmed by the elementary analysis. The derivative stimulated current amplitude in a concentration-dependent manner and in particular, at 70 mV, 100 µM compound 1 increased I KCa1.1 by 115%, less than that observed with quercetin (210%) under similar experimental conditions [7]. Figure 2B shows the time course of current amplitude stimulation induced by compound 1 that, at the maximal concentration assessed, did not significantly modify the time course of activation of the current evoked by the depolarizing pulses to 70 mV ( Figure 2B inset). Under control conditions, in fact, τ of activation was 45.3 ± 5.8 ms (n = 5) and a similar value was recorded in the presence of 100 µM compound 1 (38.5 ± 6.9). Similar results were observed with quercetin [7]. Taken together, these data demonstrated that the 3 -ferulic acid ester derivative halves the stimulatory efficacy of the flavonoid towards I KCa1.1 .  Figure 2A shows the current-voltage relationships of IKCa1.1 elicited with clamp pulses in the range −20 mV and 70 mV from a Vh of −40 mV, under control conditions and after the cumulative addition of 30-100 µ M compound 1. The derivative stimulated current amplitude in a concentration-dependent manner and in particular, at 70 mV, 100 µ M compound 1 increased IKCa1.1 by 115%, less than that observed with quercetin (210%) under similar experimental conditions [7]. Figure 2B shows the time course of current amplitude stimulation induced by compound 1 that, at the maximal concentration assessed, did not significantly modify the time course of activation of the current evoked by the depolarizing pulses to 70 mV ( Figure 2B inset). Under control conditions, in fact, τ of activation was 45.3 ± 5.8 ms (n = 5) and a similar value was recorded in the presence of 100 µ M compound 1 (38.5 ± 6.9). Similar results were observed with quercetin [7]. Taken together, these data demonstrated that the 3′-ferulic acid ester derivative halves the stimulatory efficacy of the flavonoid towards IKCa1.1.  Figure 3A shows recordings of the inward current elicited with a clamp pulse to 0 mV from a Vh of −50 mV under control conditions and after the addition of cumulative concentrations of the hybrid derivative. Compound 1 showed a biphasic  Figure 3A 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 the hybrid derivative. Compound 1 showed a biphasic behavior, first stimulating (maximal effect of 141% at 30 µM concentration) and then bringing current amplitude back to control values ( Figure 3B). Under similar experimental conditions quercetin stimulated I Ba1.2 with an EC 50 value of 8.1 µM and a maximal effect of 227% at 50 µM concentration [6]. These findings demonstrate that quercetin derivatization with a ferulic acid moiety attached to the 3 position, as occurred in compound 1, gave rise to a novel agent endowed of a weaker Ca V 1.2 channel stimulating activity as compared to the parent flavonoid. I Ba1.2 evoked at 0 mV from a V h of −50 mV activated and then declined with a time course that could be fitted by a monoexponential function. Compound 1 significantly slowed the τ of activation, showing a bell-shaped pattern ( Figure 3C,D). This behavior almost overlapped that observed on current amplitude. The marked delay in channel activation caused by compound 1 affected τ of inactivation measurement, at least in the concentration range 3-30 µM. Under similar experimental conditions quercetin also slowed down significantly the τ of activation without affecting that of inactivation [6]. Taken together, these findings demonstrate that the derivatization here pursued did not modify the capability of the parent compound to slow down the transition of Ca V 1.2 channels from the closed to the open state or, in some other way, to modify the gating mechanism.
Molbank 2021, 2021, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/molbank behavior, first stimulating (maximal effect of 141% at 30 µ M concentration) and then bringing current amplitude back to control values ( Figure 3B). Under similar experimental conditions quercetin stimulated IBa1.2 with an EC50 value of 8.1 µ M and a maximal effect of 227% at 50 µ M concentration [6]. These findings demonstrate that quercetin derivatization with a ferulic acid moiety attached to the 3′ position, as occurred in compound 1, gave rise to a novel agent endowed of a weaker CaV1.2 channel stimulating activity as compared to the parent flavonoid. IBa1.2 evoked at 0 mV from a Vh of −50 mV activated and then declined with a time course that could be fitted by a monoexponential function. Compound 1 significantly slowed the τ of activation, showing a bell-shaped pattern ( Figure 3C,D). This behavior almost overlapped that observed on current amplitude. The marked delay in channel activation caused by compound 1 affected τ of inactivation measurement, at least in the concentration range 3-30 µ M. Under similar experimental conditions quercetin also slowed down significantly the τ of activation without affecting that of inactivation [6]. Taken together, these findings demonstrate that the derivatization here pursued did not modify the capability of the parent compound to slow down the transition of CaV1.2 channels from the closed to the open state or, in some other way, to modify the gating mechanism.

In Silico Methods
The homology 3D model of the Rattus norvegicus K Ca 1.1 channel was obtained as reported in a previous work [5]. The 3D structure of compound 1 was drawn using Chemdraw software and then optimized using the Dock Prep [30]. To examine the potential binding pose of the compound on the target, a flexible docking simulation was performed with AutoDock/VinaXB [31]. The pdbqt file of the ligand was obtained by Open Babel tool v. 2.4.1 [32], while pdbqt format of the receptor was generated by using the Molecular Graphics Laboratory software (MGLTools) [33]. Multiple ligand-protein interaction maps were generated using the Protein-Ligand Interaction Profiler (PLIP) [34]. PyMOL v. 2.2 was used as molecular graphic system (The PyMOL Molecular Graphics System, Version 2.2, Schrödinger, LLC, New York, NY, USA).

General
Reagents and solvent were purchased from Merck (Milan, Italy) and used without further purification. Anhydrous conditions were obtained by a positive pressure flow of dry nitrogen into oven-dried glassware and dried solvents were bought (dry DMF) or freshly prepared (dry DCM) using the proper drying agents. Organic solutions were dried over anhydrous sodium sulfate and concentrated using a rotary evaporator (Büchi R-110, Büchi, Milan, Italy) equipped with a KNF N 820 FT 18 vacuum pump. The final compound was purified by column chromatography (Merck 60 silica gel, 230-400 mesh, Milan, Italy) and checked for purity by thin layer chromatography (TLC plates, Merck 60 F254 aluminum silica plates, Milan, Italy). Melting points were measured with a Kofler hot stage apparatus (K) and are uncorrected. Its identity was unambiguously verified by nuclear magnetic resonance and mass spectroscopy. 1 H-NMR and 13 C-NMR spectra as well as 2D-NMR experiments (HSQC and HMBC) were recorded on a Bruker Advance (Milan, Italy) operating at 600 MHz in the indicated solvent at 25 • C, and chemical shifts were expressed as δ (ppm) and the coupling constants (J) in Hz. Mass spectra were acquired in positive mode (mass range m/z of 150-1500) using a chromatography-mass spectrometry (LC-MS) apparatus (Agilent 1100, supplied with a binary high-pressure gradient pump (flow rate of 0.4 mL/min, solvent system of 95/5 methanol/water), a solvent degassing unit, and a UV detection (254 nm). Nitrogen (purity 99.995%) was used as nebulizer and drying gas. Elemental analyses were performed on a Perkin-Elmer PE 2004 elemental system (Perkin Elmer, Milan, Italy) and the data for C, H, and N are within 0.4% of the theoretical values.
Procedure B. In a round-bottomed flask equipped with a calcium chloride drying tube, to a stirred suspension of quercetin dihydrate (100.0 mg, 0.32 mmol) in dry acetonitrile (40 mL) were added in the order ferulic acid (62.0 mg, 0.32 mmol) and HOBt (52.0 mg, 0.38 mmol, 1.2 equiv.); then, a solution of EDCI (82.4 mg, 0.43 mmol, 1.5 equiv.) in dry acetonitrile (5 mL) was added dropwise and the reaction was left to stand overnight. Afterwards, the reaction mixture was partially concentrated, filtered and then, concentrated to dryness. Purification of the raw material by silica gel column chromatography, using a gradient elution (dichloromethane/methanol 50/4, 50/5, 50/6), furnished compound 1 as a yellow solid. Yield: 28.0%.

Cell Isolation Procedure
The tail main artery was dissected free of its connective tissue and smooth muscle cells were freshly isolated under the following conditions. A 5-mm long piece of artery was incubated at 37 • C for 40-45 min in 2 mL of 0.1 mM Ca 2+ external solution (in mM: 130 NaCl, 5.6 KCl, 10 HEPES, 20 glucose, 1.2 MgCl 2 , and 5 Na-pyruvate; pH 7.4) containing 20 mM taurine, which replaced an equimolar amount of NaCl, 1.35 mg/mL collagenase (type XI), 1 mg/mL soybean trypsin inhibitor, and 1 mg/mL bovine serum albumin. This solution was gently bubbled with a 95% O 2 -5% CO 2 gas mixture to stir the enzyme solution and cells isolated as previously described [35]. Cells stored in 0.05 mM Ca 2+ 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 [36].

Whole-Cell Patch Clamp Recordings
The conventional whole-cell patch-clamp method was employed to voltage clamp smooth muscle cells. Recording electrodes were pulled from borosilicate glass capillaries (WPI, Berlin, Germany) and fire-polished to obtain a pipette resistance of 2-5 MΩ when filled with internal solution. An Axopatch 200B patch-clamp amplifier (Molecular Devices Corporation, 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. Cells were continuously superfused using a peristaltic pump (LKB 2132, Bromma, Sweden) at a flow rate of 400 µL/min. 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 (20-22 • C).
3.3.4. I KCa1.1 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 h of −40 mV. This V h limited the contribution of K V channels to the overall whole-cell current. Data were collected once the current amplitude had been stabilized (usually 8-10 min after the whole-cell configuration had been obtained). I KCa1.1 current did not run down during the following 20-30 min under the present experimental conditions [7]. The external solution for I KCa1 Cells were continuously superfused with external solution containing 0.1 mM Ca 2+ and 30 mM tetraethylammonium (TEA). The internal solution (pCa 8.4) consisted of (in mM): 100 CsCl, 10 HEPES, 11 EGTA, 2 MgCl 2 , 1 CaCl 2 , 5 Na-pyruvate, 5 succinic acid, 5 oxaloacetic acid, 3 Na 2 ATP, and 5 phosphocreatine; the pH was adjusted to 7.4 with CsOH. I Ba1.2 , recorded in an external solution containing 30 mM TEA and 5 mM Ba 2+ , was elicited with 250 ms clamp pulses (0.067 Hz) to 0 mV from a V h of −50 mV. Data were collected once the current amplitude had been stabilized (usually 7-10 min after the whole-cell configuration had been obtained). Under these conditions, the current did not run down during the following 40 min [37]. 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 I Ba1.2 . The osmolarity of the 30 mM TEA-and 5 mM Ba 2+ -containing external solution and that of the internal solution were 320 mOsmol and 290 mOsmol, respectively.

Drugs and Chemicals
The chemicals used included: collagenase (type XI), trypsin inhibitor, bovine serum albumin, TEA chloride, taurine, nicardipine, and nifedipine (Merck, Milan, Italy). All other substances were of analytical grade and used without further purification. Nifedipine and nicardipine, dissolved directly in ethanol, and compound 1, dissolved directly in DMSO, were diluted at least 1000 times prior to use. Control experiments confirmed that no response was induced in vascular preparations when DMSO or ethanol, at the final concentration used in the above dilutions (0.1%, v/v), was added alone (data not shown). Final drug concentrations are stated in the text.

Statistical Analysis
Analysis of data was accomplished by using pClamp 9.2.1.8 software (Molecular Devices Corporation, San Jose, CA, USA) and GraphPad Prism version 5.04 (GraphPad Software Inc., San Diego, CA, USA). Data are reported as mean ± s.e.m.; n is the number of cells analyzed (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 paired samples (two tailed) were obtained using GraphPad Prism version 5.04 (GraphPad Software Inc., San Diego, CA, USA). In all comparisons, p < 0.05 was considered significant.

Conclusions
In this short note, the design and synthesis of the 3 -ferulic ester derivative of quercetin were reported along with the assessment of its effects on vascular K Ca 1.1 and Ca V 1.2 channels. A simple and time-saving synthetic protocol, characterized by the direct acylation of quercetin under mild and strongly controlled reaction conditions followed by careful chromatographic purification with gradient elution, was developed. In silico analysis pointed out the ability of the new hybrid compound to bind with high affinity on the K Ca 1.1 channel, justifying a possible vascular ion channel modulatory activity. Pharmacological findings suggest that the introduction of a 3 -ferulic ester moiety in quercetin structure caused a partial reduction of its stimulatory activity on both K Ca 1.1 and Ca V 1.2 channels.
It is conceivable, therefore, that its vasorelaxant activity is not improved, though only functional experiments performed on isolated vessels may clarify this point. In the light of the results presented here, it cannot be ruled out that reduction of both activities may be due either to the presence of the ferulic acid or to the loss of the quercetin 3 -OH group, or both. It is well known, in fact, that the number and/or position of OH groups in the flavonoid scaffold are crucial determinants of their pharmacological activity. The design strategy aimed to improve quercetin beneficial vascular activity, therefore, this needs to be re-designed either positioning ferulic acid in a different position or replacing it with different substituents, as recently shown for the same flavonoid [4] or its analogue morin [5].  Figures S9-S10: HSQC spectra of compound 1, Figures S11-S12: HMBC spectra of compound 1, Figure S13: Table S1 reporting K Ca 1.1 channel-compound interaction network.