9,11-epoxy-9-homoprosta-5-enoic acid analogues as thromboxane A2 receptor antagonists.

A novel bicyclic prostaglandin analogue, (1S)-[1 alpha, 2 alpha(Z),3 alpha(1E,3S*,4R*),4 alpha]-7-[3-(3-hydroxy-4-phenyl-1-pentenyl)-7- oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (4), was found to be a potent and selective thromboxane A2 (TxA2) receptor antagonist. Alcohol 4 was the only member in a series of allylic alcohols which did not display direct contractile activity in the rat stomach strip model. Alcohol 4 was effective in the inhibition of (a) arachidonic acid induced platelet aggregation of human platelet-rich plasma (I50 = 0.65 +/- 0.1 microM); (b) 11,9-epoxymethano-PGH2 induced contraction of guinea pig trachea (pA2 = 8.0 +/- 0.2) or rat aorta (pA2 = 8.1 +/- 0.2); and (c) arachidonic acid induced bronchoconstriction in the anesthetized guinea pig (1 mg/kg iv). A radioiodinated analogue of 4 bound in a specific and saturable manner to human platelet membranes with a Kd = 2.3 +/- 0.9 nM. Modification of the alpha-chain, in an attempt to minimize in vivo metabolism, resulted in TxA2 receptor antagonists of reduced in vitro potency.

Photoaffinity labelling of the human platelet thromboxane A2/prostaglandin H2 receptor.

The synthesis, binding and photoincorporation of a thromboxane A2/prostaglandin H2 (TXA2/PGH2) analog (9,11-dimethylmethano-11,12-methano-16-(3-[125I]iodo-4-azidophenyl )-13,14- dihydro-13-aza-15 alpha beta-omega-tetranor-TXA2) [( 125I]PTA-Azido) to washed human platelets was characterized. Kinetic analysis of the binding of [125I]PTA-Azido at 30 degrees C yielded a k1 of 1.83.10(7) M-1.min-1 and k -1 of 0.195 min-1, Kd = k -1/k1 = 11 nM. Incubation of washed human platelets with [125I]PTA-Azido followed by photolysis resulted in the radiolabelling of a number of platelet proteins as assessed by SDS-PAGE autoradiography. The radiolabelling of three of these protein bands could be either uniformly blocked or reduced with a series of structurally dissimilar TXA2/PGH2 receptor antagonists or agonists and corresponded to proteins with a molecular mass of 43, 39 and 27 kDa. In addition, the incorporation of [125I]PTA-Azido into the three proteins was stereoselectively blocked by a pair of optically active stereoisomers that are TXA2/PGH2 receptor antagonists. Two-dimensional gel electrophoresis indicated that the 43 kDa protein possessed a pI value of 5.6 and that the 27 kDa protein exists in at least three isoforms with pI values of 4.9, 5.1 and 5.3. The labelling pattern was not altered by a mixture of proteinase inhibitors. The data suggest that one or more of these specifically radiolabelled proteins may represent the human platelet TXA2/PGH2 receptor.

Synthesis and structure of the platelet aggregation factor thromboxane A2.

In 1975, Hamberg et al. reported evidence for the existence of an unstable platelet-aggregating factor which they named thromboxane A2 (TXA2) and for which they proposed a novel bicyclic oxetane structure (1, below) based on the short half-life of the factor (t1/2 (37 degrees C) = 32 s at pH 7.4) and the isolation of degradation products related to thromboxane (TXB2) (2, below). As natural TXA2 has not yet been isolated and characterized as a pure compound, we have synthesized the proposed structure (1) from TXB2 and compared its biological properties with those of authentic, biologically generated material. Here we present evidence that synthetic material having structure (1) is indistinguishable from platelet-derived TXA2 in various biological assays and that the proposed structure (1) for TXA2 is correct.

Synthesis and biological properties of pinane-thromboxane A2, a selective inhibitor of coronary artery constriction, platelet aggregation, and thromboxane formation.

Pinane-thromboxane A2 (PTA2, [1alpha,2 beta(Z),-3 alpha (1E,3R*),5 alpha]-7-(3-(3-hydroxy-1-octenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-yl)-5-heptenoic acid) has been synthesized and tested for biological activity in systems responsive to thromboxane A2, stable prostaglandin endoperoxide (PGH2) analogs, and prostatacyclin (PGI2). At low concentrations, PTA2 inhibited cat coronary artery constriction induced by stable prostaglandin endoperoxide analogs, and it stabilized liver lysosomes. At slightly higher concentrations, it inhibited platelet aggregation. At still higher concentrations, PTA2 inhibited thromboxane synthetase, but it had no effect on prostacyclin synthetase. The analog also had no effect on the inhibition of platelet aggregation by PGI2 or prostaglandin D2. It is suggested that PTA2 has a suitable biochemical profile for use as an antithrombotic agent.