Synthesis and Biological Evaluation of Resveratrol Methoxy Derivatives

Resveratrol, a naturally occurring stilbene, exhibits numerous beneficial health effects. Various studies have demonstrated its diverse biological actions, including anti-oxidant, anti-inflammatory, and anti-platelet properties, thereby supporting its potential for cardio protection, neuroprotection, and anti-cancer activity. However, a significant limitation of resveratrol is its weak bioavailability. To overcome this challenge, multiple research groups have investigated the synthesis of new resveratrol derivatives to enhance bioavailability and pharmacological activities. Nevertheless, there are limited data on the effects of resveratrol derivatives on platelet function. Therefore, the objective of this study was to synthesize resveratrol methoxy derivatives and evaluate their anti-platelet and anti-proliferative activity. Platelet-rich plasma (PRP) obtained from healthy volunteers was utilized to assess the derivatives’ ability to inhibit platelet aggregation induced by platelet activating factor (PAF), adenosine diphosphate (ADP), and thrombin receptor activating peptide (TRAP). Additionally, the derivatives’ anti-tumor activity was evaluated against the proliferation of PC-3 and HCT116 cells. The results revealed that some methoxy derivatives of resveratrol exhibited comparable or even superior anti-platelet activity compared to the original compound. The most potent derivative was the 4′-methoxy derivative, which demonstrated approximately 2.5 orders of magnitude higher anti-platelet activity against TRAP-induced platelet aggregation, indicating its potential as an anti-platelet agent. Concerning in silico studies, the 4′-methyl group of 4′-methoxy derivative is oriented similarly to the fluorophenyl–pyridyl group of Vorapaxar, buried in a hydrophobic cavity. In terms of their anti-tumor activity, 3-MRESV exhibited the highest potency in PC-3 cells, while 3,4′-DMRESV and TMRESV showed the greatest efficacy in HCT116 cells. In conclusion, methoxy derivatives of resveratrol possess similar or improved anti-platelet and anti-cancer effects, thereby holding potential as bioactive compounds in various pathological conditions.


Introduction
Resveratrol is a naturally occurring stilbene with several beneficial health effects [1,2]. It is most commonly in grapes and wine but also in other plants such as polygonum cuspidatum, peanuts, and red fruits [2]. It exists in two diastereoisomeric forms found in nature: transform (E-1,2-diphenylethylene) and cis-form (Z-1,2-triphenylethylene), with the former being the bioactive arrangement. One hydroxyl group is present in one ring, in position 4 , and two hydroxyl groups are present in the other ring, in positions 3 and 5, respectively.

Biological Assays
Anti-platelet activity of methoxy resveratrol derivatives.
The anti-platelet activity of methoxy derivatives of resveratrol (Scheme 1) was evaluated by testing their ability to inhibit platelet aggregation in PRP from healthy volunteers.

Biological Assays
Anti-platelet activity of methoxy resveratrol derivatives.
The anti-platelet activity of methoxy derivatives of resveratrol (Scheme 1) was evaluated by testing their ability to inhibit platelet aggregation in PRP from healthy volunteers.
Three agonists of platelet aggregation were used, namely, PAF, ADP, and TRAP, and several concentrations of resveratrol and its methoxy derivatives were tested. The results are presented as IC 50 values calculated either from the amplitude or from the AUC of the agonist and shown in Table 1.
Resveratrol was found to inhibit the aggregation of all agonists but not to the same extent (p < 0.000). It was observed that resveratrol inhibited platelet aggregation induced by PAF (p < 0.000) and TRAP (p = 0.001) more effectively than that induced by ADP. No significant difference was detected between its ability to inhibit PAF-and TRAP-induced platelet aggregation. Similar results were obtained when the IC 50 values were calculated based on the AUC of aggregation instead of the amplitude, but lower IC 50 values were revealed in all cases (p < 0.02). Methylation of resveratrol did not improve its ability to inhibit ADP-induced platelet aggregation. When methylation occurred in the 3-hydroxyl group (p < 0.000) or both in the 3and 5-hydroxyl groups (p = 0.003) a significant decrease in inhibitory activity was observed compared to the initial compound. When methylation occurred in the 4 -hydroxyl group, i.e., the 4 derivative (p = 0.13), the 3,4 derivative (p = 1.0), and the 3,4 ,5 derivative (p = 1.0), the IC 50 values were higher but not significantly different from the initial compound. When the IC 50 values were calculated based on the AUC of aggregation instead of amplitude, only methylation occurring in the 3 -hydroxyl group (p < 0.000) revealed a significant increase in IC 50 value compared to resveratrol.
Regarding PAF as the agonist, methylation also worsened the ability of resveratrol to inhibit platelet aggregation (p < 0.000). Specifically, methylation in both aromatic rings (3-and 4 -hydroxyl groups) resulted in significantly higher IC 50 values compared to the initial compound, as did trimethylation (3,4 -DMRESV p < 0.000, TMRESV p = 0.01). When methylation occurred only in one ring, no significant change was observed in IC 50 values, although a slight increase was observed (3-MRESV p = 0.5, 4 -MRESV p = 0.4, 3,5-DMResv p = 0.3). Similar results were obtained when the IC 50 values were calculated based on the AUC of aggregation instead of the amplitude (3,4-DMRESV p = 0.002, TMRESV p = 0.06, all the others p > 0.05).
In contrast, when TRAP was used as the agonist, methylation of resveratrol improved its ability to inhibit platelet aggregation. The monomethylated derivatives exhibited the best inhibitory ability, with almost one magnitude lower IC 50 values, namely 3-MRESV (p < 0.000) and two 4 -MRESV (p < 0.000), respectively, compared to resveratrol. Dimethylation occurring in different rings resulted in a lower IC 50 value (3,4 -DMRESV, p = 0.01), whereas when it occurred in the same ring, a similar IC 50 was observed to the initial compound's anti-platelet activity (3,5-DMRESV, p = 0.1). Figure 1 shows a characteristic inhibition of TRAP-induced platelet aggregation by 4 -MRESV. The trimethylation of resveratrol dramatically diminished its inhibitory activity, and the IC 50 value could not be calculated. When the IC 50 values were calculated based on AUC of aggregation instead of amplitude, similar results were obtained. resveratrol dramatically diminished its inhibitory activity, and the IC50 value could not be calculated. When the IC50 values were calculated based on AUC of aggregation instead of amplitude, similar results were obtained. Anti-cancer effect of methoxy resveratrol derivatives The anti-proliferative activity of the new compounds was evaluated in vitro against the PC-3 human prostate cancer cell line and the HCT116 colon cancer cell line. The results of the MTT dye reduction assay, expressed as 50% inhibitory concentrations (IC50) in µM, are depicted in Table 2. Each experiment was performed in triplicate and mean values ± SD are reported. Among the tested compounds, 3-MRESV is found to be more potent than resveratrol in androgen-independent prostate cancer cells (PC-3). In addition, 3,5-DMRESV and 3,4′-DMRESV exhibited significant anti-cancer properties to a lesser extent. Furthermore, 3,4′-DMRESV and TMRESV were found to be more potent than resveratrol in colon cancer cells (HCT116).  Anti-cancer effect of methoxy resveratrol derivatives The anti-proliferative activity of the new compounds was evaluated in vitro against the PC-3 human prostate cancer cell line and the HCT116 colon cancer cell line. The results of the MTT dye reduction assay, expressed as 50% inhibitory concentrations (IC 50 ) in µM, are depicted in Table 2. Each experiment was performed in triplicate and mean values ± SD are reported. Among the tested compounds, 3-MRESV is found to be more potent than resveratrol in androgen-independent prostate cancer cells (PC-3). In addition, 3,5-DMRESV and 3,4 -DMRESV exhibited significant anti-cancer properties to a lesser extent. Furthermore, 3,4 -DMRESV and TMRESV were found to be more potent than resveratrol in colon cancer cells (HCT116).

Molecular Calculations
To better understand the selectivity of the different methylated analogues of resveratrol, we run molecular docking simulations utilizing the induced-fit algorithm as implemented on Schrodinger Suite 2019. We selected the crystallographic structure of human protease-activated receptor 1 (PAR1) bound with the antagonist vorapaxar (PDB: 3VW7) as the starting structure. Vorapaxar, unlike ligands for other GPCRs, does not penetrate deeply into the transmembrane core. It is composed of two hydrophobic cores connected with a double bond, showing moderate similarity to resveratrol scaffold.
All synthesized analogues were docked inside the binding cavity with average binding affinity (9-11 Kcal/mol) (Figure 2A,B). The 4 -methyl group of 4 -MRESV is oriented similarly to the fluorophenyl-pyridyl group of Vorapaxar, buried in a hydrophobic cavity ( Figure 2C). The 2 hydroxyl groups are oriented next to the extracellular part of PAR1, forming hydrogen bonds with H255 and S344 ( Figure 2C). 3-MRESV is oriented with the A phenyl group in the hydrophobic pocket. The 5 OH group forms hydrogen bonds with Y187 and the backbone of G233, while the 4 OH group forms hydrogen bond with H336 ( Figure 2D). Moreover, 3,4 DMRESV is oriented deeper to the binding cavity because of the second O-Me group, which is placed next to Y353 and A349, while the phenolic OH group forms hydrogen bonds with H336 and Y337 ( Figure 2E). 3,5-DMRESV and TMRESV are placed inside the binding cavity with the 3,5 dimethyl groups facing the extracellular aqueous environment, decreasing the overall binding affinity. In the case of 3,5-DMRESV, the OH group is placed inside the hydrophobic cavity, and although it is forming hydrogen bonds with Y187 and the backbone of G233, it is unfavorable to pass through such a hydrophobic channel and finally reach those residues. Resveratrol shows the lowest binding energy and has the 4 OH group placed inside the hydrophobic cavity which is unfavorable as was explained earlier.

Discussion
Resveratrol is a natural molecule that has attracted much scientific interest in recent decades [2]. Several biological activities have been attributed to resveratrol, including anti-oxidant, anti-inflammatory, and anti-cancer effects. However, its low availability is a primary concern about its in vivo activity. In this study, methoxy derivatives of resveratrol were synthesized to increase its hydrophobicity and were found to have similar, worse, or better anti-platelet activity than the initial molecule depending on the position of the substitution. The 4′ methoxy and 3 methoxy derivatives efficiently inhibit TRAP-induced platelet aggregation, especially in two orders and one order of magnitude, respectively, more potently than the initial compound.

Discussion
Resveratrol is a natural molecule that has attracted much scientific interest in recent decades [2]. Several biological activities have been attributed to resveratrol, including anti-oxidant, anti-inflammatory, and anti-cancer effects. However, its low availability is a primary concern about its in vivo activity. In this study, methoxy derivatives of resveratrol were synthesized to increase its hydrophobicity and were found to have similar, worse, or better anti-platelet activity than the initial molecule depending on the position of the substitution. The 4 methoxy and 3 methoxy derivatives efficiently inhibit TRAP-induced platelet aggregation, especially in two orders and one order of magnitude, respectively, more potently than the initial compound.
Platelets are key cells in many physio-pathological mechanisms [14]. Several antagonists, such as thrombin, collagen, ADP, PAF, serotonin, epinephrine, and thromboxane A2 (TXA2), induce platelet activation and aggregation. Many reports indicate that resveratrol inhibits thrombin, collagen, PAF or ADP-induced platelet adhesion or aggregation [18,26]. In addition, resveratrol inhibited thromboxane B2-induced platelet aggregation by inhibiting protein kinase C activity [18]. The inhibitory capacity of resveratrol ranges between tens and hundreds of µM depending on the use of PRP or washed platelet preparations, the agonist used, and the platelets' origin (human or rabbit). It should be noted that a lower concentration is needed when washed platelets are used to determine the anti-platelet effect. In the present study, human PRP was used instead of washed platelets because we believe that its matrix is more representative of the cells' in vivo environment. The IC 50 value of resveratrol against PAF and TRAP was similar, whereas against ADP, it was significantly higher, indicating a greater capacity of resveratrol to interfere with PAF and TRAP agonists instead of ADP.
Methylation has been shown to significantly affect platelet aggregation, depending on the number of methoxy groups and the specific methylated ring or position. Furthermore, the influence of methylation on platelet aggregation varies depending on the agonist used to induce it. ADP was the first low-molecular-weight compound identified as a platelet agonist [31] and stimulates primary platelet aggregation. Released by the storage granules, ADP is responsible for the secondary wave of aggregation and it is also involved in platelet stimulation by other agonists [32]. ADP-induced platelet aggregation is mediated by the activation of platelet P2Y1 and P2Y12 receptors [33]. However, resveratrol's methylation did not improve the ability to inhibit ADP-induced platelet aggregation. In fact, when the substitution was carried out in the A ring, either in one or both hydroxyl groups, the anti-platelet activity of the initial compound worsened. In addition, the methylation in both the 3-and 5-hydroxy groups, in ring A has a lower impact on the second wave of platelet aggregation, as the IC 50 value calculated based on AUC, instead of amplitude, was not significantly different from that of the initial compound. On the other hand, isorhapontigenin (3,4 ,5-trihydroxy-3 -methoxystilbene), another derivative of resveratrol with an extra methylated hydroxyl group, has been reported to inhibit ADP-induced platelet aggregation more efficiently, which was also supported by molecular interactions of isorhapontigenin and resveratrol with P2Y12 receptor as predicted by in silico studies [34].
PAF, also known as acetyl-glyceryl-ether-phosphorylcholine, is an ether phospholipid that functions as a potent inducer of inflammation and platelet aggregation [12]. PAF exerts its effects through its specific receptor, PAFR, which is a G-protein coupled receptor with seven transmembrane domains. In our previous report, we demonstrated that acetylated derivatives inhibit PAF-induced platelet aggregation [26]. Specifically, mono-and triacetylation of resveratrol showed similar anti-platelet activity to resveratrol itself, while di-acetylation resulted in improved anti-platelet activity, indicating that the presence of two acetyl groups, but not three, is necessary for enhancing inhibitory activity. In the present study, methylation of resveratrol did not enhance its anti-platelet activity. Methylation of one ring (either A or B) did not show significant differences compared to the original compound. However, when methylation was performed on both rings (3,4 -DMRESV and TMRESV), resveratrol's ability to inhibit PAF-induced aggregation was reduced. These findings suggest that the specific group used for substitution (acetoxy or methoxy) in resveratrol is more crucial than the increase in lipophilicity achieved by both substitutions.
Thrombin, a serine protease, is another enzyme that induces platelet aggregation and triggers the release of active substances, including ADP. This process requires the presence of calcium ions. Thrombin elicits signaling, partly through G-protein-linked protease-activated receptors (PARs), with human platelets expressing PAR1 and PAR4 [35]. PAR1 is considered to be the high-affinity thrombin receptor responsible for the initial platelet response, while PAR4 requires higher thrombin concentrations and participates in the late-phase response [36]. Consequently, PAR1 has attracted scientific interest as a novel anti-platelet target, but only one PAR1 antagonist, vorapaxar, is currently approved for clinical use [37]. In the present study, instead of thrombin, TRAP was used as agonist, a synthetic peptide that corresponds to the new N-terminal amino acid sequence of the "tethered ligand" generated after thrombin hydrolysis of the PAR1. The results revealed that, in some cases, methylation of resveratrol resulted in favorable alterations of its antiplatelet action. Specifically, dimethylation in the A ring (3,5-DMRESV, TMRESV) revealed compounds with similar or no anti-platelet activity against TRAP compared to resveratrol. In contrast, methyl substitution in the 3-or 4'-hydroxyl groups, alone or in combination, resulted in compounds with significantly enhanced anti-platelet activity. The dimethyl derivative (3,4 -DMRESV), the monomethyl derivative in the A ring (3-MRESV), and the monomethylated derivative in the B ring (4 -MRESV) exhibited IC 50 values almost half, 3.5 times, and 25 times lower than resveratrol, respectively. To our knowledge, no similar data exists to compare our results. However, a recent study reported that the 3,5,2 ,4tetramethoxystilbene selectively inhibits PAR4-mediated human platelet aggregation, ATP secretion, integrin αIIbβ3 activation, and signaling pathways [27].
The activation of platelets by all tested agonists involves the activation of phopholipase C, which hydrolyzes the membrane-associated phosphatidylinositol 4,5 bisphosphate and produces the second messenger inositol 1,4,5-irisphosphate (IP3), contributing to Ca 2+ mobilization from internal stores [35]. Additionally, the production of membrane-associated diacylglycerol activates protein kinase C, which participates in the phosphorylation of specific substrates [35]. While the anti-platelet mechanisms of methoxy derivatives cannot be identified based on the design of this study, their distinct actions against different agonists suggest interference in specific pathways related to each agonist. In silico studies were conducted to further investigate the binding of resveratrol methoxy derivatives with PAR1, supporting the biological experiments and revealing that 4 -MRESV exhibits greater binding affinity toward the PAR1 receptor than resveratrol.
Among the new compounds, 3MRESV appears to be worth investigating in prostate cancer cells, along with 3,5-DMRESV and 3,4 -DMRESV, which have IC 50 [40]. Furthermore, the para-and ortho-resveratrol derivatives appeared as the most active RSV derivatives in inhibiting cell viability in a panel of pancreatic cancer cell lines with distinct genetic profiles compared to resveratrol [41]. Finally, the resveratrol analogues 4,4 -(ethane-1,2-diyl) bis(2-methoxyphenol) and 4-(3hydroxy-4-methoxyphenethyl)-2-methoxyphenol had very similar structures but exerted different cytotoxicity in non-small lung cancer cells. The IC50 values were 108.6 ± 10.82 and more than 200 µM in the A549 cell line and 103.5 ± 6.08 and more than 200 µM in H23 cells, respectively, while resveratrol itself exhibited more than 200 µM in both cell lines [42].
Finally, aspirin (acetylsalicylic acid) has emerged as a promising agent for cancer prevention. However, there are suggestions that anti-platelet treatment may promote the development of solid tumors. Given the complex and bidirectional interaction between tumor cells and platelets in the blood and tumor microenvironment, further investigation is needed to understand the potential of anti-platelet agents in both cancer prevention and treatment [28,29].

3,5-Dimethoxybenzyl alcohol (2)
To a suspension of LiAlH 4 (1.52 g, 40 mmol) in dry THF (50 mL), at 0 • C, under argon, was added dropwise a solution of acid 1 (1.82 g, 10 mmol) in dry THF (10 mL) and the resulting solution was stirred at rt for 2 h. After completion of the reaction, the mixture was cooled at 0 • C and carefully triturated with 10% aqueous NaOH solution and the resulting precipitate was filtrated through a Celite pad and washed with a small amount of THF. The filtrate was diluted with water, extracted with EtOAc (3 × 100 mL), dried over anhydrous Na 2 SO 4 , and concentrated to dryness, to afford the title compound 2 (1.2 g, 72%) which was used for the next step without any further purification [30]. 1

3,5-Dimethoxybenzyl bromide (3)
To a solution of alcohol 2 (0.84 g, 5 mmol) in dry CH 2 Cl 2 (100 mL), at 0 • C, under argon, was added PPh 3 (5.24 g, 20 mmol, 1.2 eq.), and CBr 4 (4.98 g, 15 mmol) and the resulting solution was stirred at rt for 2 h. After completion of the reaction, the volatiles were vacuum evaporated and the crude product was purified by column chromatography (silica gel), using a mixture of cyclohexane-EtOAc 5-1 as the eluent, to afford 728 mg (63%) of the title compound 3 [30]. 1 16 g, 5 mmol, 3) and Bu 4 NI (184 mg, 0.5 mmol) in PO(OEt) 3 (10 mL) was stirred at 120 • C for 12 h, under argon. After completion of the reaction, the volatiles were vacuum evaporated to afford the title compound 4, which was used for the next step without any further purification [30]. 1  To a solution of diethyl 3,5-dimethoxybenzylphosphonate (1.15 g, 4 mmol, 4) and p-anisaldehyde (558 mg, 4.1 mmol) in dry THF (10 mL), at 0 • C, under argon, was added portion-wise NaH (180 mg, 4.5 mmol, 60% in mineral oil) and the resulting mixture was stirred at rt for 16 h. After completion of the reaction, the mixture was cooled at 0 • C, carefully triturated with methanol, and evaporated to dryness. The residue was dissolved in CH 2 Cl 2 , washed with water, brine, dried over anhydrous Na 2 SO 4 , and evaporated to dryness [30]. 1  (E)-5-(4-Hydroxystyryl)benzene-1,3-diol or resveratrol (6) To a solution of 5 (2.16 g, 8 mmol) in dry CH 2 Cl 2 (45 mL) at −78 • C, under argon, was added dropwise a solution of BBr 3 (25 mL, 25 mmol, 1 M solution in CH 2 Cl 2 ) and the resulting mixture was stirred at rt for 4 h. After completion of the reaction, the mixture was cooled at 0 • C and carefully poured into ice. The organic phase was separated, dried over Na 2 SO 4 , and concentrated under reduced pressure. The aqueous phase was filtered, the filtrate was washed with water and air-dried. The crude product (from aqueous and organic phase) was purified by column chromatography (silica gel) using a mixture of cyclohexane-EtOAc 1-1 as the eluent, to obtain pure compound 6 (1.44 g, 79%). 1

Synthesis of acetylated resveratrol compounds 7a-e
A suspension of resveratrol (950 mg, 4.17 mmol, 6), acetic anhydride (553 µL, 5.84 mmol) and triethylamine (1.8 mL, 11.68 mmol) in anhydrous CH 2 Cl 2 (100 mL), was stirred at room temperature for 12 h. The reaction mixture was then vacuum evaporated, extracted with CH 2 Cl 2 and water, the organic layer was dried over Na 2 SO 4 and evaporated to dryness. The residue was purified by column chromatography (silica gel) using a mixture of CH 2 Cl 2 -EtOAc 100-4 as the eluent, to afford the title compounds 7a-e [43].

Blood Sample Collection
The study was conducted at the Metabolic Unit of the Department of Nutrition and Dietetics, Harokopio University, according to the guidelines laid down in the Declaration of Helsinki. Volunteers gave informed consent and the University Ethics Committee approved the experimental protocol (number: 57/15 September 2017). Informed consent was obtained from all subjects involved in the study. Healthy volunteers who had not taken aspirin or non-steroidal anti-inflammatory drugs for at least 2 weeks were used. Twelve hour fasting venous blood was collected from the brachial vein of the volunteers using citrate acid as anticoagulant. Blood was centrifuged at 170× g for 15 min in order to collect the supernatant platelet-rich plasma (PRP). Platelet-poor plasma (PPP) was obtained by recentrifugation of the pellet at 1500× g for 10 min.

Platelet Aggregation in Plasma-Rich Platelets (PRP)
The effect of phenolic compounds on platelet aggregation against PAF, ADP, and TRAP (Thrombin analogue) was determined on plasma-rich platelets (PRP) from healthy volunteers. The molecules were dissolved in DMSO (1.6% maximum DMSO final concentration). Platelet count of PRP was adjusted to 300.000/mL with PPP. Samples were incubated at 37 • C, with a stirring rate of 1.000 rpm. A Chronolog Aggregometer (Model 440VS) was used for determining aggregation responses based on the light transmittance method against various concentrations of PAF, ADP, and thrombin receptor-activating peptide (TRAP), a PAR-1 (Protease-Activated Receptor) selective activating peptide. Optical aggregation results were expressed as a percentage of aggregation at a given time interval from agonist addition; aggregation was defined as the difference between the 0% (PRP) baseline and the 100% (PPP) baseline. The aggregation was induced by final concentra-tion of PAF ranging between 1-4 µM (dissolved in bovine serum albumin 2.5 mg/mL), of ADP ranging between 50-100 µM (dissolved in saline) and of TRAP ranging between 4-7 µM (dissolved in saline) with respect to each volunteer. In addition, 0% inhibition was considered as platelet aggregation without the addition of the examined compound. The plot of percentage inhibition (ranging from 20% to 80%) vs. different concentrations of the examined compound is linear and it was used to calculate the concentration of the sample that induced 50% inhibition against each agonist. This value was defined as the IC 50 , namely, inhibitory concentration producing 50% inhibition.

MTT Assay
To test the inhibitory activities of compounds using a cell-based protocol, MTT assay was performed for cell viability. HCT116 cells were plated at a density of 1500 per well, while PC-3 cells were plated at a density of 750 per well in a 96-well plate. After 24 h, cells were treated with the compounds in a dose-dependent manner for 72 h and 96 h. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma M-5655) was added at a final concentration of 0.5 mg/mL directly to each well for 4 h at 37 • C. The medium was aspirated and the blue MTT formazan precipitate was dissolved in dimethyl sulfoxide (DMSO). Absorbance was determined in a Powerwave microplate spectrophotometer (Biotek Instruments, Inc., Winooski, VT, USA) at 540 nm. Viable cell numbers were determined by tetrazolium conversion to its formazan dye. Each experiment was performed in triplicate [50].

Protein Preparation
The protein (PDB entry 3VW7) was prepared for the docking calculations using the Protein Preparation Workflow (Schrödinger Suite 2021 Protein Preparation Wizard) implemented in Schrödinger suite and accessible from within the Maestro program (Maestro, version 12.8, Schrödinger, LLC, New York, NY, USA, 2021). Briefly, the hydrogen atoms were added and the orientation of the hydroxyl groups, Asn, Gln, and the protonation state of His were optimized to maximize hydrogen bonding. Finally, the ligand-protein complex was refined with a restrained minimization performed by Impref utility, which is based on the Impact molecular mechanics engine (Impact version 9.1, Schrödinger, LLC, New York, NY, USA, 2021) and the OPLS_2005 force field, setting a max rmsd of 0.30. Ligand preparation for docking was performed with the LigPrep (Schrödinger, LLC, New York, NY, USA, 2021) application which consists of a series of steps that perform conversions, apply corrections to the structure, generate ionization states and tautomers, and optimize the geometries.

Ligand Preparation
All ligands were designed using Maestro software (Maestro, version 12.8, Schrödinger, LLC, New York, NY, USA, 2021). Furthermore, LigPrep (Schrödinger, LLC, New York, NY, USA, 2021) was used to generate tautomeric, stereochemical, and ionization variations for all ligands. Finally, partial charges were predicted from the force field OPLS2005.

Docking Simulations
The induced-fit docking algorithm was utilized for molecular docking as implemented on Schrödinger Suite 2021. For calculating the grid box size, the center of the grid box was taken to be the center of the ligand in the crystal structure, and the length of the grid box for the receptor was twice the distance from the ligand center to its farthest ligand atom plus 10 Å in each dimension. The scoring calculations were performed using standard precision (SP).

Statistical Analysis
Statistical analysis was completed by using SPSS 18 (SPSS Inc., Chicago, Il, USA) software. Kolmogorov-Smirnov criterion was used for testing normality. All variables were normally distributed and presented as mean values ± standard deviation. Significance level for p-values is 5%.

Conclusions
In conclusion, the findings of this study support the notion that certain methoxy derivatives of resveratrol may have comparable or even better anti-platelet effects compared to the original compound. The most potent derivative observed was the 4 -methoxy derivative, which exhibited approximately 2.5 orders of magnitude greater anti-platelet activity against TRAP-induced platelet aggregation, suggesting its potential as an anti-platelet agent. Using in silico docking simulations, we have discovered that all compounds exhibit a binding mode comparable to vorapaxar. Furthermore, our investigation has allowed us to elucidate the correlation between binding modes and the potency of the 4 -methoxy derivative. It is also noteworthy that methoxy derivatives with similar activity to resveratrol could be potential anti-platelet agents as they may undergo in vivo bio-transformation, leading to improved bioavailability compared to resveratrol. These encouraging preliminary findings will provide valuable insights for our ongoing rational drug design campaign targeting human protease-activated receptor 1 (PAR1).
Author Contributions: E.F. was responsible for the conceptualization, methodology and supervision of anti-platelet properties of compounds as well as data curation and writing of the manuscript. I.K.K. was responsible for the methodology and supervision of synthesis of the methoxy derivatives of resveratrol as well as reviewing and editing the manuscript. R.T. was responsible for the methodology and supervision of the anti-cancer properties of compounds as well as reviewing and editing the manuscript. G.L. was responsible for the methodology and docking calculations. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.