New analogues of 13-hydroxyocatdecadienoic acid and 12-hydroxyeicosatetraenoic acid block human blood platelet aggregation and cyclooxygenase-1 activity
- Taghreed Hirz†1,
- Ali Khalaf†2, 3,
- Nehme El-Hachem1,
- May F Mrad1,
- Hassan Abdallah2,
- Christophe Créminon4,
- René Grée3,
- Raghida Abou Merhi5,
- Aïda Habib1Email author,
- Ali Hachem2Email author and
- Eva Hamade5
© Hirz et al.; licensee Chemistry Central Ltd. 2012
Received: 24 September 2012
Accepted: 3 December 2012
Published: 10 December 2012
Thromboxane A2 is derived from arachidonic acid through the action of cyclooxygenases and thromboxane synthase. It is mainly formed in blood platelets upon activation and plays an important role in aggregation. Aspirin is effective in reducing the incidence of complications following acute coronary syndrome and stroke. The anti-thrombotic effect of aspirin is obtained through the irreversible inhibition of cyclooxygenases. Analogues of 12-hydroxyeicosatetraenoic acid and 13-hydroxyocatdecadienoic acid were shown previously to modulate platelet activation and to block thromboxane receptors.
Results and discussion
We synthesized 10 compounds based on the structures of analogues of 12-hydroxyeicosatetraenoic acid and 13-hydroxyocatdecadienoic acid and evaluated their effect on platelet aggregation triggered by arachidonic acid. The structure activity relationship was evaluated. Five compounds showed a significant inhibition of platelet aggregation and highlighted the importance of the lipidic hydrophobic hydrocarbon chain and the phenol group. Their IC50 ranged from 7.5 ± 0.8 to 14.2 ± 5.7 μM (Mean ± S.E.M.). All five compounds decreased platelet aggregation and thromboxane synthesis in response to collagen whereas no modification of platelet aggregation in response to thromboxane receptor agonist, U46619, was observed. Using COS-7 cells overexpressing human cyclooxygenase-1, we showed that these compounds are specific inhibitors of cyclooxygenase-1 with IC50 ranging from 1.3 to 12 μM. Docking observation of human recombinant cyclooxygenase-1 supported a role of the phenol group in the fitting of cyclooxygenase-1, most likely related to hydrogen bonding with the Tyr 355 of cyclooxygenase-1.
In conclusion, the compounds we synthesized at first based on the structures of analogues of 12 lipoxygenase metabolites showed a role of the phenol group in the anti-platelet and anti-cyclooxygenase-1 activities. These compounds mediate their effects via blockade of cyclooxygenase-1.
KeywordsCyclooxygenase-1 Anti-thrombotic Inhibitors Polyunsaturated fatty acid Thrombosis
Results and discussion
Effect on platelet aggregation
Biological activity of compounds of type I and type II
Plt aggregation IC50 ± S.E.M (μM)b
C = NOH
7.5 ± 0.8
14.2 ± 5.7
97.6 ± 83.5
41.2 ± 0.7
8.5 ± 1.2
13.4 ± 0.5
11.3 ± 4.8
We next tested compounds of type II (Table 1). Compounds 7 and 8 exhibit significant inhibitory activities (Figure 2C and 2D, respectively) with compound 8 showing a more linear dose–response inhibition of the platelet aggregation (Figure 2D). Introduction of a methyl group on the carbinol centre (R3 group) did not change the inhibitory effect since compound 9 exhibited also the same range of inhibition with a similar linear dose–response inhibition (Figure 2E). On the contrary, replacing the hydroxyl group by a methoxy group in compound 10, completely abolished the inhibitory activity (Figure 3B). These results support a role of the OH group in position R1. The IC50 of type II compounds were also compared with ibuprofen (Table 1). We conclude from these results that for type I molecules the hydroxyl group at R1 position is critical for the inhibitory effect and that the length of the R3 hydrocarbon chain is appropriate between 4 and 5 carbons, as described for compounds 3 and 4, respectively. Type II compounds 7 and 9 are the most appropriate inhibitory compounds of this group with a hydroxyl group at R1 and a methoxy group at R2 position.
Effect on human COX-1 overexpressed in COS-7 cells
Effect on cyclooxygenase-1 activity
IC50 ± S.E.M (μM)a
2.2 ± 0.7
12.1 ± 2.3
1.3 ± 0.5
6.5 ± 0.2
8.1 ± 1.7
Effect on collagen- and thromboxane receptor - dependent platelet aggregation
We first designed series of new aromatic compounds based on the structure of analogues of polyunsaturated fatty acid metabolites, 12-HETE and 13-HODE, we reported earlier . These new compounds have important structural differences with the initial molecules analogues A and B. Five compounds were shown to have inhibitory effects on arachidonic acid- and collagen- induced platelet aggregation. These compounds mediate these effects via blockade of COX and not as antagonist of the TX receptor. Our results support a role of the phenol group in the inhibitory effect of these compounds, shown also by the docking observation of the molecules in the human recombinant COX-1. The addition of OH at R1 conferred additional properties and fitting in COX-1. There is a direct interaction of the compounds with COX-1 as revealed by the structure-activity relationship data, which showed an important role for the OH in position R1, most likely related to hydrogen bonding with the Tyr 355 of COX-1. In conclusion, the compounds we synthesized at first based on the structures of analogues of 12-HETE and 13-HODE show a role of the phenol group in the anti-platelet and anit-COX-1 activities. These molecules, although structurally different from the initial analogues of 12- HETE and 13-HODE compounds, have anti-platelet effect and anti- COX activities and will help to design more potent analogues. Studies are undertaken in our group and will be reported in due course.
COS-7 cells were obtained from the American Type Culture Collection (ATCC, Massas, VA). Ibuprofen was from EMD-Calbiochem (San Diego, CA). Arachidonic acid, monoclonal antibody anti-bactin and BSA were from Sigma-Aldrich (St Louis, MO). Collagen was from Chrono-Log Corp (Havertown, PA). PGE2 and TXB2 reagents were from Cayman Chemicals (Ann Arbor, MI). WST-1 viability assay and FuGENE® 6 transfection reagent were from Roche Applied Science (Indianapolis, IN.). All drugs were dissolved in DMSO and final concentration did not exceed 0.2%. Vehicle used was 0.2% DMSO and was added in controls or arachidonic treated platelets and COS-7 cells. All other chemicals and electrophoresis reagents were of high pure grade and were obtained from Amresco (Solon, OH) and BioRad (Hercules, CA).
General procedure for preparation of compounds 3, 4, 5, 6, 7, 8, 9, and 10
In a glass vial equipped with a magnetic stirring bar and flashed with nitrogen gas aldehyde/ketone (1 equiv.) was dissolved in dry THF. The mixture was cooled at −15°C before the drop wise addition of the Organomagnesium reagent (1.3 equiv.). The reaction mixture was stirred under nitrogen gas for 1 hour, while the temperature rising slowly to room temperature. The mixture was treated with a saturated NH4Cl solution, extracted by ethyl acetate, dried over MgSO4, and concentrated in Vacuo. The adducts were obtained after silica gel column chromatography in (45 - 70% yield) with high purity.
Preparation of compound 1
In a glass vial equipped with a magnetic stirring bar, hydroxy-hexyl-benzaldehyde (0.39 g, 1.86 mmol), NH2OH, HCl (0.2 g, 1.5 equiv.) and pyridine (1.2 ml) were dissolved in ethanol (8 ml). The mixture was refluxed overnight. After cooling to room temperature, the mixture was acidified by diluted HCl solution, extracted by ethyl acetate, dried over MgSO4, and concentrated in Vacuo. After silica gel column chromatography, oxime 6 was obtained as white crystals (0.31 g, 75% yield); melting point: 80°C. 1H NMR (CDCl3, 300 MHz) δ, ppm: 0.78 (t, 3H, J = 6.6Hz); 1.12-1.35 (m, 6H); 1.54-1.70 (m, 2H); 2.82 (bs, 1H); 4.57 (dd, 1H, J = 6.2, 7.0 Hz, 7.17-7.34 (m, 3H); 7.44 (s, 1H), 8.01 (s, 1H); 9.15 (bs, 1H). 13C NMR (CDCl3, 75 MHz) δ, ppm: 13.96; 22.50; 25.37; 31.66; 38.85; 74.42; 124.55; 126.22; 127.66; 128.80; 132.12; 145.45; 150.36.
Preparation of compound 2
AgNO3 (200 mg) was dissolved in water before the addition of NaOH and formation of precipitates. Few drops of NH3 were added and the precipitates were totally dissolved. This mixture was added to aldehyde 3 in DMSO (3 ml) and stirred for 30 minutes. The mixture was acidified by diluted HCl solution and filtrated. The filtrate was extracted by ethyl acetate, dried over MgSO4, and concentrated in Vacuo. After silica gel column flash chromatography acid 10 was obtained as white crystals (70 mg, 44% yield); melting point: 110°C. 1H NMR (acetone, 300 MHz) δ, ppm: 0.73 (t, 3H, J = 7.05 Hz);1.10-1.39 (m, 6H); 1.49-1.67 (m, 2H); 4.61 (dd, 1H, J = 5.7, 7.2 Hz); 7.29-7.34 (m, 1H); 7.474-7.51 (m, 1H); 7.77 (td, 1H, J = 1.5, 7.7Hz); 7.93 (t, 1H, J = 1.7 Hz). 13C NMR (acetone, 75 MHz) δ, ppm: 14.29; 23.28; 26.13; 32.51; 40.48; 73.76; 127.96; 128.86; 129.03; 131.28; 131.34; 147.93; 167.83.
Spectral and characterization data of compounds 3, 5, 6, 7, 8, 9 and 10
Compound 3:1H NMR (CDCl3, 300 MHz) δ, ppm: 0.79 (t, 3H, J = 6.1 Hz); 1.20-1.36 (m, 6H); 1.53-1.62 (m, 1H); 1.64-1.72 (m, 1H); 3.80 (s, 3H); 4.49 (t, 1H, J = 6.7 Hz); 5.65 (s, 1H); 6.67-6.81 (m, 3H). 13C NMR (d6 acetone, 75 MHz) δ, ppm: 14.0; 22.6; 25.6; 31.7; 39.0; 55.9; 74.7; 108.4; 114.1; 119.0; 137.0; 145.0;146.6. MS: Found m/z: 224.
Compound 5 : 1H NMR (CDCl3, 300 MHz) δ, ppm: 0.77 (t, 3H, J = 6.85Hz); 1.09-1.21 (m, 14H); 1.37-1.46 (m, 1H); 1.61-1.74 (m, 1H); 3.80 (s, 1H); 3.86 (dd, J = 5.70, 7.66 Hz); 5.68 (s, 1H); 6.58 (dd, 1H, J 1.8 Hz, 8.0 Hz); 6.76 (d, 1H, J = 1.8 Hz); 6.78 (d, 1H, J = 8.0Hz). 13C NMR (CDCl3, 75 MHz) δ, ppm: 14.1; 22.7; 26.2; 29.3; 29.5; 29.6;31.9; 38.9; 55.8; 78.2; 109.0; 113.9; 120.3; 135.2; 144.9; 146.7.
Compound 6: 1H NMR (CDCl3, 300 MHz) δ, ppm: 1.45-2.01 (m, 9H); 3.83 (s, 3H); 4.25 (d, 1H, J = 11.38Hz), 5.51 (s, 1H); 6.73 (dd, 1H, J = 1.78, 8.06 Hz); 6.80 (d, 1H, J = 8.06 Hz); 6.83 (d, 1H, J = 1.78 Hz). 13C NMR (CDCl3, 75 MHz) δ, ppm: 25.4; 25.6; 29.5; 29.8; 30.9; 47.6; 55.9; 79.2; 108.8; 114.0; 119.5; 136.6; 145.0; 146.6. MS: Found m/z: 222.
Compound 7: 1H NMR (CDCl3, 300 MHz) δ, ppm: 2.45 (s, 1H); 3.70 (s, 3H); 5.65 (s, 1H), 6.74-6.85 (m, 3H); 7.17-7.33 (m, 5H). 13C NMR (CDCl3, 75 MHz) δ, ppm: 55.9; 76.0; 109.3; 114.2; 119.7; 126.4; 127.4; 128.4; 136.1; 144.0; 145.1; 146.7. MS: Found m/z: 230.
Compound 8: 1H NMR (CDCl3, 300 MHz) δ, ppm: 3.80 (s, 3H); 4.87 (d, 1H, J = 3.93 Hz); 5.77 (d, 1H, J = 3.78 Hz); 6.79 (d, 1H, J = 8.10 Hz); 6.84 (d, 1H, J = 8.10 Hz); 7.04-7.10 (m, 3H); 7.43-7.48 (m, 2H). 13C NMR (CDCl3, 75 MHz) δ, ppm: 56.2; 75.38; 110.9; 115.4 (d, 2C, JC-F = 21.3 Hz); 115.5; 120.2; 129.1 (d, 2C; JC-F = 8.0 Hz); 137.8; 142.8 (d, 1C, JC-F =3.0 Hz); 146.6; 148.2; 162.6 (d, 1C, JC-F = 242.6 Hz). MS: Found m/z: 248.
Compound 9 : 1H NMR (d6 acetone, 300 MHz) δ, ppm: 1.92 (s, 3H); 3.19 (s, 1H); 3.77 (s, 3H); 4.60 (s, 1H); 6.78 (d, 1H, J = 8.25 Hz); 6.91 (dd, 1H, J = 2.07, 8.25 Hz); 7.13 (d, 1H, J = 2.07 Hz); 7.15-7.21 (m, 1H); 7.26-7.31 (m, 2H); 7.48-7.52 (m, 2H). 13C NMR (d6 acetone, 75 MHz) δ, ppm: 31.5; 56.3; 75.9; 110.9; 115.0; 119.5; 126.7; 127.0; 128.6; 141.9; 146.0; 147.8; 150.7. MS: Found: m/z =244.
Compound 10 : 1H NMR (CDCl3, 300 MHz) δ, ppm: 3.76 (s, 3H); 3.78 (s, 3H); 5.71 (s, 1H); 6.74 (d, 1H, J = 8.19 Hz); 6.79-6.85 (m, 2H); 7.18-7.29 (m, 5H). 13C NMR (CDCl3, 75 MHz) δ, ppm: 55.8; 55.9; 75.9; 109.8; 110.9; 119.0; 126.4; 127.5; 128.5; 136.6; 143.9; 148.4; 149.0. MS: Found m/z: 244.
Blood collection, platelet preparation and analysis
Venous blood was obtained from healthy volunteers who had not ingested any drugs for the last 14 days and after informed consent in accordance with the Institutional Review Board (IRB) of the American University of Beirut (Approval # BioCh.AH.03). Washed platelets were prepared as described previously [21, 22]. Briefly, 20 ml of peripheral blood was withdrawn on ACD-C (1 volume for 9 volumes of blood) and centrifuged at 120 g for 15 min at room temperature (RT) to obtain platelet-rich plasma. Platelet-rich plasma was further centrifuged at 1,200 g for 15 min at RT, and the platelet pellet obtained was washed by Tyrode buffer solution containing 0.1 μM of PGE1 and further centrifuged at 1,200 g for 15 min at RT. The pellet was resuspended in Hanks buffer, pH 7.4 containing 1 mg/ml of bovine serum albumin. Aggregation of washed-platelets was determined using light transmittance aggregometry (Chrono-Log Corp., Havertown, PA). 400 μl of platelets (0.4 x 109plt/ml) were preincubated for 1 min at 370C in the absence or presence of inhibitors prior to the addition of 25 μM of arachidonic acid, which was controlled as optimal in our conditions. In some experiments, platelets were triggered with 1 μM U46619, a TX receptor agonist, or 0.5 μg/ml collagen, a concentration we checked in our conditions to induce COX-dependent platelet aggregation. TXB2 was measured in the supernatant by enzyme immunoassay .
Overexpression of human recombinant COX-1 in COS-7 cells and COX-1 activity
COS-7 cells were grown using DMEM-medium containing 10% fetal bovine serum (FBS). Cells were transfected in suspension with pcDNA3-COX-1 plasmid using FuGENE® 6 transfection reagent at a ratio of 3:1 (FuGENE: DNA, v/w) and then cultured in DMEM media + 10% FBS in 12-well plates. For COX-1 activity, cells were incubated 48 hours post transfection in the absence or presence of different concentrations of compounds 3, 4, 7, 8 and 9 or ibuprofen in Hanks buffer, pH 7.4, containing 1 mg/ml BSA for 30 minutes prior to the addition of 25 μM arachidonic acid for 30 minutes. PGE2 was measured in the supernatants by enzyme immunoassay . Cells were washed twice with PBS and lysed in lysis buffer. COX-1 protein was determined by western blot as previously described . Briefly, SDS polyacrylamide electrophoresis was performed using 8% gels followed by protein transfer using a semi-dry transfer machine. Immunoblot analysis was performed using selective monoclonal antibody anti COX-1 (COX-111) (1/1000)  and a monoclonal antibody anti-β-actin (1/2000). The effect of 25 and 50 μM of compounds 3, 4, 7, 8 and 9 on cell viability of COS-7 cells overexpressing COX-1 was evaluated using WST-1 assay and showed an absence of toxicity of these compounds.
Aggregation data were expressed after defining the slope for each aggregation curve, which better reflects the rate of the platelet reaction, using the Born’s method . Curve fitting and calculation of the IC50 values were done using Grafit 7 software (Erithacus software, Staines, UK). Results of TXB2 and PGE2 measurement were expressed as the mean ± S.E.M. for at least 3 different experiments and statistical analysis was performed using Sigma Plot (Systat Software Inc., San Jose, CA). Autoradiograms obtained from western blot analyses were scanned using Epson 1680 pro scanner and densitometric analysis was performed using Scion Image (Scion Corporation, MD).
Target selection and preparation
The 3D structure of the ovine COX-1 complexed with ibuprofen (PDBID: 1EQG) was selected for docking simulations . Water and other heteratoms were removed from the structure. Chain A was retained including ibuprofen and heme group. Hydrogen atoms were added, atom typing and partial charges were assigned using AMBER forcefield . The coordinates of the binding site were extracted using the co-crystallized ligand, Ibuprofen. Docking and scoring: low energy conformations of the chemical compounds were generated using Catalyst (Accelrys, Inc.). The (R)- enantiomers of compounds 3, 7, and 10 were selected, docking simulations were carried out using Autodock 4.2 , each docking simulation was achieved with 10 docking runs with 150 individuals using the Lamarckian genetic algorithm implemented in Autodock and 250000 energy evaluations. The binding energies were estimated from a new free-energy scoring function based on the AMBER forcefield, an updated charge-based desolvation term and improved models of the unbound state. The best poses were analyzed and visualized with Disovery Studio visualizer (Accelrys, Inc.).
Non-steroidal anti-inflammatory drugs
Fetal bovine serum
This work was supported by grants from the Lebanese National Council for Scientific Research (Grant 03-04-09), the Lebanese University (Ecole doctorale des Sciences et Technologies) and the American University of Beirut (Aida H). R. G. and A.K. thank CNRS and University of Rennes 1 for financial support. We are grateful to Dr. Ayad Jaffa (American University of Beirut) and Dr. Bianca Rocca (Pharmacology department, Universita Cattolica del Sacro Cuore, Roma, Italy) for their comments and critical reading of the manuscript.
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