Synthesis of Imine Congeners of Resveratrol and Evaluation of Their AntiPlatelet Activity

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a cardioprotective phytochemical occurring in many plant products. In this study, a new series of imine congeners of resveratrol has been synthesized in which the imine moiety replaced the double bond in the structure of resveratrol. In addition, the in vitro antiplatelet activity of these resveratrol derivatives has been evaluated against adenosine diphosphate (ADP), arachidonic acid (AA), and collagen as platelet aggregation inducers. In general, the synthesized compounds were active as antiplatelet agents, and, therefore, the imine functional group may be considered as an effective replacement for a double bond in resveratrol for developing new and promising antiplatelet drugs.


Introduction
Cardiovascular diseases (CVDs) are recognized as the first global cause of death.It has been reported by the World Health Organization (WHO) that 17.7 million people died from CVDs in 2015 [1,2].Platelet aggregation plays an essential role in the process of blood clotting and CVDs.However, many antiplatelet drugs such as aspirin and clopidogrel, which are available in clinics, are associated with some side effects such as bleeding and drug resistance that limit their usage [3,4].Therefore, the search for new antiplatelet agents with fewer side effects and higher efficacy is among the priorities of medicinal chemists.
Therefore, the present research was aimed at the synthesis of a new series of imine congeners of resveratrol in which the imine moiety replaced the double bond in the structure of resveratrol (Figure 1) [28], and the evaluation of their in vitro antiplatelet activity against adenosine diphosphate (ADP), arachidonic acid (AA), and collagen as platelet aggregation inducers.

Chemistry
The designed compounds were synthesized by the reaction of different aniline derivatives with appropriate aldehydes in water as a green solvent without any catalyst (Figure 2).The synthesized derivatives (3a-3r) were obtained with high yields (>89%).Structure of the synthesized compounds was characterized by LC-MS 1 H-NMR and 13 C-NMR.The 1 H-NMR spectra of the synthesized compounds exhibited a singlet peak for the CH=N proton between 8.51 and 8.96 ppm.A literature review revealed that C=N moiety is present in many structures with antiplatelet activity.
Therefore, the present research was aimed at the synthesis of a new series of imine congeners of resveratrol in which the imine moiety replaced the double bond in the structure of resveratrol (Figure 1) [28], and the evaluation of their in vitro antiplatelet activity against adenosine diphosphate (ADP), arachidonic acid (AA), and collagen as platelet aggregation inducers.

Chemistry
The designed compounds were synthesized by the reaction of different aniline derivatives with appropriate aldehydes in water as a green solvent without any catalyst (Figure 2).The synthesized derivatives (3a-3r) were obtained with high yields (>89%).Structure of the synthesized compounds was characterized by LC-MS 1 H-NMR and 13 C-NMR.The 1 H-NMR spectra of the synthesized compounds exhibited a singlet peak for the CH=N proton between 8.51 and 8.96 ppm.

Chemistry
The designed compounds were synthesized by the reaction of different aniline derivatives with appropriate aldehydes in water as a green solvent without any catalyst (Figure 2).The synthesized derivatives (3a-3r) were obtained with high yields (>89%).Structure of the synthesized compounds was characterized by LC-MS 1 H-NMR and 13 C-NMR.The 1 H-NMR spectra of the synthesized compounds exhibited a singlet peak for the CH=N proton between 8.51 and 8.96 ppm.

Anti-Platelet Activity
The anti-platelet activity of the synthesized derivatives against ADP, AA, and collagen as platelet aggregation inducers were evaluated, according to the Born method [29,30].The obtained data are presented in Table 1.
Table 1.Anti-platelet activity of the synthesized derivatives.Adenosine diphosphate (ADP), arachidonic acid (AA), and collagen were used as a platelet aggregation inducer at a final concentration of 5 µM, 1.35 µM, and 2.5 µg•mL −1 , respectively.The results are expressed as the mean ± standard error of mean (SEM) from three independent experiments.The anti-platelet activity of the synthesized derivatives against ADP, AA, and collagen as platelet aggregation inducers were evaluated, according to the Born method [29,30].The obtained data are presented in Table 1.

Structure Activity Relationship
The data reported in Table 1 show that all the compounds (3a-3r) at the concentration of 1 mM inhibited platelet aggregation induced by ADP, AA, and collagen.The inhibition range for ADP and collagen were 30%-84.6%and 15.9%-93.4%,respectively.When AA was used as a platelet aggregation inducer, the inhibition was increased and ranged from 74.7% to 100%.
Compound 3m with three-methoxy group on ring A inhibited platelet aggregation induced by all the three platelet inducers above 85%.
Table 1.Anti-platelet activity of the synthesized derivatives.Adenosine diphosphate (ADP), arachidonic acid (AA), and collagen were used as a platelet aggregation inducer at a final concentration of 5 µM, 1.35 µM, and 2.5 µg•mL −1 , respectively.The results are expressed as the mean ± standard error of mean (SEM) from three independent experiments.Since all compounds at concentration of 1 mM were able to completely inhibit platelet aggregation induced by AA, the IC50 values for these compounds were calculated (Table 1).
As shown in

Structure Activity Relationship
The data reported in Table 1 show that all the compounds (3a-3r) at the concentration of 1 mM inhibited platelet aggregation induced by ADP, AA, and collagen.The inhibition range for ADP and collagen were 30%-84.6%and 15.9%-93.4%,respectively.When AA was used as a platelet aggregation inducer, the inhibition was increased and ranged from 74.7% to 100%.
Compound 3m with three-methoxy group on ring A inhibited platelet aggregation induced by all the three platelet inducers above 85%.
Since all compounds at concentration of 1 mM were able to completely inhibit platelet aggregation induced by AA, the IC 50 values for these compounds were calculated (Table 1).
As shown in Table 1, all the compounds with hydroxyl substituent on the B ring show high activity (IC 50 < 69.1 µM) except 3o.The results demonstrated that all the compounds with Schiff base and phenolic hydroxyl groups at the ortho position of ring A or B show IC 50 values ranging between 19.8 µM and 30.7 µM except for 3c and 3p.Compounds 3i and 3q exhibited satisfactory activity with IC 50 values of 29.9 µM and 30.7 µM, respectively.Compound 3r with IC 50 value of 19.8 µM was the most active compound.

General Procedure for the Preparation of 3a-3r
The mixture of aromatic amine (1 mmol) and aldehyde (1 mmol) in water was stirred at room temperature.After completion of the reaction indicated by TLC (thin-layer chromatography), the obtained precipitate was filtered off and washed with water.The obtained precipitate was recrystallized from the appropriate solvent.

Anti-Platelet Assay
The anti-platelet aggregation activity of the synthesized compounds was evaluated on an APACT 4004 aggregometer (LABiTec, Ahrensburg, Germany), according to the method described before [29,40,41].Compounds (3a-3r) were added to platelet-rich plasma (PRP) and were incubated for 5 min at 37 • C. Adenosine diphosphate (ADP), arachidonic acid, and collagen were added separately as platelet aggregation inducers at a final concentration of 5 µM, 1.35 µM, and 2.5 µg•mL −1 , respectively.The aggregation procedure was monitored for 5 min.Compounds were screened at a concentration of 1 mM in DMSO.The IC 50 values against AA were determined for the synthesized compounds.Each experiment was carried out in triplicate and the results are shown as a mean ± standard error of mean (SEM).

Table 1
, all the compounds with hydroxyl substituent on the B ring show high activity (IC50 < 69.1 µM) except 3o.The results demonstrated that all the compounds with Schiff base a Inhibition of platelet aggregation was assessed at 1 mM concentration.