Difluorothromboxane A 2 and stereoisomers : Stable derivatives of thromboxane A 2 with differential effects on platelets and blood vessels ( receptors / contraction / aggregation / prostaglandins / 10 , 10-difluorothromboxane A 2 and analogues )

The present study reports on the selective effects on human platelets and canine saphenous veins of four stable difluorinated analogues and thromboxane A2 (TXA2), in which the characteristic 2,6-dioxa[3.1. 1]bicycloheptane structure of TXA2 has been retained. The four compounds differ in their stereochemistry of the 5,6 double bond and/or the 15-hydroxyl group. Only 10,10-difluoro-TXA2 (compound I) with the natural stereochemistry of TXA2 was an agonist in both platelets and canine saphenous veins (EC50 = 36 ± 3.6nM and 3.7 ± 0.8 nM, respectively). (15R)-10,10-Difluoro-TXA2 (compound II), (5E)-10,10-difluoro-TXA2 (compound IM), and (5E,15R)-10,10-difluoro-TXA2 (compound IV) were antagonists of platelet aggregation stimulated by compound I (Kd = 98 ± 46 nM, 140 ± 42 nM, and 1450 ± 350 nM, respectively). However, compounds 11,1, and IV stimulated contraction of canine saphenous veins (EC50 = 36 ± 4.4 nM, 31 ± 6.8 nM, and 321 ± 50 nM, respectively). All four compounds could displace the TXA2/prostaglandin H2 antagonist 9,11-dimethylmethano-11,12-methano-16-(3-'251-4-hydroxyphenyl)-13,14-dihydro-13-aza-15a13-w-tetranor-TXA2 from its platelet receptor (Kd values = 100 ± 30 nM, compound I; 280 ± 60 nM, compound II; 230 ± 70 nM, compound HI; and 1410 ± 1020 nM, compound IV). These results support the existence of two subtypes of TXA2/prostaglandin H2 receptors and emphasize the importance of the stereochemical requirements of these TXA2 analogues for interaction with these receptors. These stable fluorinated TXA2 analogues should prove useful tools for the further characterization of these and other TXA2/prostaglandin H2 receptors. Thromboxane A2 (TXA2), and prostaglandin H2 (PGH2) cause aggregation of platelets and constriction of vascular smooth muscle (1-4) by means of cell-surface receptors. To study the specific events accompanying the interaction of these autacoids with their receptors, it is necessary to use stable analogues because both TXA2 and PGH2 are unstable, their half-lives under physiological conditions being 30 sec and 5 min, respectively. For these reasons many compounds have been synthesized that are related in structure to both PGH2 and TXA2 and that act as either agonists or antagonists for TXA2/PGH2 receptors on platelets and vascular smooth muscle (5-12). Studies of these analogues have provided evidence that the TXA2/PGH2 receptors on vascular smooth muscle differ from those of platelets. For example, a carbocyclic analogue ofTXA2 [2,3(Z),3a-(lE,3R)-3-(3-hydroxy(1-octenyl)-bicyclo[3.1. 1]hept-2-yl-5-heptenoic acid; CTA2] has been shown to constrict cat coronary arteries, whereas in human platelets this analogue is an antagonist (13). Other studies, also using different species, have shown differences in receptors in the two cell types (14, 15). Because different species were used as the source of platelets and blood vessels, these differences may be species-selective rather than represent actual differences in the receptors. Recent studies, however, have provided stronger evidence for different TXA2/PGH2 receptors in platelets and in blood vessels (12, 16, 17). The platelet and vascular receptors also differ in their steric requirements for the orientation of the 15-hydroxyl group (17). In saphenous veins orientation of the 15-hydroxyl group influenced the potency of the compounds as antagonists in the vessels, whereas in platelets the orientation had no influence. The platelet receptor has been designated the (TXA2/PGH2)a receptor, a for aggregation, and the vascular receptor (TXA2/PGH2), r for tone (12, 16, 17). Most compounds used in these studies possessed the [2.2. 1]bicycloheptane skeleton of PGH2; less attention had been paid to analogues of TXA2, which possess the [3.1. 1]bicycloheptane system. In both structural types increased stability was imparted to the molecules by substitution of one or both of the oxygen atoms by carbon, sulfur, or nitrogen. However, these systems still represent significant structural changes compared with TXA2 or PGH2. Indeed, some of these analogues do not mimic the actions ofTXA2 in platelets (13) and blood vessels. The current studies report on the selective effects on human platelets and canine saphenous veins of a difluoro analogue of TXA2 and three of its stereoisomers in which the 2,6-dioxabicyclo structure of TXA2 has been retained except for the substitution of two hydrogen atoms by fluorine (18). MATERIALS AND METHODS The syntheses of the four difluorinated TXA2 derivatives, compounds I-IV (Fig. 1), used in this study are described elsewhere (18). 9,11-Dimethy-lmethano-11,12-methano16-(3-iodo-4-hydroxyphenyl)-13 ,14-dihydro-13-aza-15a,Bw-tetranor-TXA2 (I-PTA-OH) and [1251]PTA-OH were synthesized as described (11). U-46619 was a gift from Upjohn. Platelet Aggregation. Blood was drawn via venipuncture from normal human volunteers, who had not taken any Abbreviations: PGH2, prostaglandin H2; TXA2, thromboxane A2; CTA2, 2,B(Z),3a-(lE,3R)-3-(3-hydroxy(1-octenyl)-bicyclo[3.1. 1Ihept-2-yl-5-heptenoic acid; I-PTA-OH, 9,11-dimethylmethano11,12-methano-16-(3-iodo-4-hydroxyphenyl)-13,14-dihydro-13-aza15a13-co-tetranor-TXA2; U-46619, (15S)-hydroxy-lla,9a-(epoxymethano)prosta-(5Z,13E)-dienoic acid; SQ 29548, -{(lS)-[la,2.8(5Z),3p,4a]}-7-[3-({2-[(phenylamino)carbonyl]hydrazino}methyl)7-oxabicyclo[2.2. 1]hept-2-yl]-5-heptenoic acid; STA2, 9,11-epithio11,12-methano-TXA25600 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 86 (1989) 5601