Aspirin Hybrid Molecules with Improved Antiplatelet Properties Designed As New Drug Candidates to Prevent Atherothrombosis,
Authors: Santos Jean Leandro , de Oliveira Ednir Vizioli , Pires Maria Elisa Lopes , de Melo Thais Regina Ferreira , Costa Fernando Ferreira , Chin Chung Man 
Published: October 2019

Abstract
Abstract 3364

Introduction.
Platelets are involved in atherothrombosis as a consequence of their inappropriate and sustained activation, being thus considered an important target for pharmacological interventions. Aspirin (ASA) is the current drug standard of care of most patients; however the therapy is limited and presents several inconveniences such as lack of effectiveness and bleeding. The use of hybridization strategy allows obtaining compounds with pharmacokinetic and pharmacodynamic properties improved. In this work, we describe new compounds with significant antiplatelet activity induced by ADP. These compounds 2-(2-((4-methyl-1,2,5-oxadiazole-2-oxide)methylene)hydrazinecarbonyl) phenyl acetate (Lapdesf 1) and 2-(2-((4-phenyl-1,2,5-oxadiazole-2-oxide)methylene)hydrazinecarbonyl) phenyl acetate (Lapdesf 2) were structurally planned by modification of acetyl salicylic acid (ASA) based on the use of acylhydrazone subunit, previously reported to contribute to inhibit platelet aggregation. Furthermore, 1,2,5-oxadiazole N-oxide subunit known by its NO-donor properties were included in the hybrid structure in order to contribute for the antiplatelet activity. Methods. 1. Detection of nitrite. A solution of the appropriate compound (20 μL) in DMSO was added to 2 mL of a mixture of 50 mM phosphate buffer (pH 7.4) and methanol (1:1, v:v), containing 5 mM of L-cysteine. The final concentration of the compound was 10–4M. After 1 h at 37 °C, 1 mL of the reaction mixture was treated with 250 μL of Griess reagent. After 10 min at room temperature, the absorbance was measured at 540 nm using a spectrophotometer. Standard sodium nitrite solutions (10–80 nmol/mL) were used to construct the calibration curve. The yields of nitrite are expressed as % NO2– (mol/mol). 2. Antiplatelet activity. Blood was withdrawn from mice central artery and mixed with 3.8% trisodium citrate (9:1 v/v). Platelet-rich plasma (PRP) was prepared by centrifugation at 375 × g for 10 min at room temperature. The platelet-poor plasma (PPP) was prepared by centrifugation of the pellet at 1800 × g for 10 min at room temperature. Platelet aggregation was monitored by the turbidimetric method of Born and Cross using aggregometer. PRP (300 μL) was incubated at 37 °C for 1 min with compounds before addition of the aggregating agent. Platelet aggregation was induced by ADP (10 μM). Compounds and ASA used as control were evaluated at 150 μM. 3. Tail vein bleeding time model. Mice were anesthetized (ketamine 75mg/kg; medetomidine 1 mg/kg IP). A transverse incision is made with a scalpel over a lateral tail vein at a position where the diameter of the tail is 2.25–2.5 mm. The tail was immersed in normal saline (37°C) in a hand-held test tube. This last one is rotated to prevent the shed blood from obscuring the incision site. The time from the incision to the cessation of bleeding is recorded. The results are expressed as mean time + SEM of 6 animals per group. The data were analyzed statistically with Student's t test at a significance level of P < 0.05. Results. 1. Detection of nitrite: Isosorbide dinitrate used as the control, induced 10.9 + 0.5 % of nitrite (mol/mol) formation while compound Lapdesf 1 and Lapdesf 2 induced 1.3 + 0.1 and 4.7 + 0.2, respectively. 2.Antiplatelet activity. The compounds inhibited platelet aggregation induced by ADP. The aggregation percentage in presence of Lapdesf 1 and Lapdesf 2 were 3.1 + 0,2 and 32 + 0,4 respectively. The aggregation percentage of control (ADP) and ASA were 40.3 + 0,3 and 57.5 + 0,4 respectively. 3. Tail vein bleeding time model. The control used showed an average of 52 + 15 seconds to stop bleeding while ASA demonstrated an average of 73 + 20 seconds. Lapdesf 1 and Lapdesf 2 showed an average of 113 + 25 seconds and 192 + 31 seconds. Conclusions. Results demonstrate that molecular hybridization approach used allows us to obtain new ASA derivatives with property of inhibited platelet aggregation induced by ADP. Lapdesf 1 and 2 demonstrated different NO-donor properties related to methyl and phenyl substitutions. Furthermore, both compounds prolonged tail vein bleeding time using mice model. These compounds could be new drug candidates, alternative to ASA, useful to prevent atherothrombosis events.

Disclosures:
No relevant conflicts of interest to declare.