Activation of peroxisome proliferator-activated receptor isoforms and inhibition of prostaglandin H(2) synthases by ibuprofen, naproxen, and indomethacin.

A series of nonsteroidal anti-inflammatory drugs (NSAIDs) [S(+)-naproxen, ibuprofen isomers, and indomethacin] were evaluated for their activation of peroxisome proliferator-activated receptor (PPAR) alpha and gamma isoforms in CV-1 cells co-transfected with rat PPAR alpha and gamma, and peroxisome proliferator response element (PPRE)-luciferase reporter gene plasmids, for stimulation of peroxisomal fatty acyl CoA beta-oxidase activity in H4IIEC3 cells, and for comparative inhibition of ovine prostaglandin endoperoxide H synthase (PGHS)-1 and PGHS-2 and arachidonic acid-induced human platelet activation. Each drug produced a concentration-dependent activation of the PPAR isoforms and fatty acid beta-oxidase activity, inhibition of human arachidonic acid-induced platelet aggregation and serotonin secretion, and inhibition of PGHS-1 and PGHS-2 activities. For PPARalpha activation in CV-1 and H4IIEC3 cells, and the stimulation of fatty acyl oxidase activity in H4IIEC3 cells, the rank order of stereoselectivity was S(+)- ibuprofen > R(-)-ibuprofen; S(+)-ibuprofen was more potent than indomethacin and naproxen on these parameters. On PPARgamma, the rank order was S(+)-naproxen > indomethacin > S(+)-ibuprofen > R(-)-ibuprofen. Each drug inhibited PGHS-1 activity and platelet aggregation with the same rank order of indomethacin > S(+)-ibuprofen > S(+)-naproxen > R(-)-ibuprofen. Notably, the S(+)-isomer of ibuprofen was 32-, 41-, and 96-fold more potent than the R(-)-isomer for the inhibition of PGHS-1 activity, human platelet aggregation, and serotonin secretion, respectively. On PGHS-2, the ibuprofen isomers showed no selectivity, and indomethacin, S(+)-ibuprofen, and S(+)-naproxen were 6-, 27-, and 5-fold more potent as inhibitors of PGHS-1 than PGHS-2 activity. These results demonstrate that the mechanisms of action of NSAIDs on these cell systems are different, and we propose that the pharmacological effects of NSAIDs may be related to both their profile of inhibition of PGHS enzymes and the activation of PPARalpha and/or PPARgamma isoforms.

Beta-adrenergic receptor and platelet inhibitory activities of a new series of trimetoquinol and related benzazepine analogs.

1. A series of dimethoxy and methylenedioxy analogs of trimetoquinol (TMQ) and structurally related 7-membered ring benzazepines (BA) were evaluated for their pharmacological effects in beta-adrenergic (atria, trachea) and thromboxane A2/prostaglandin H2 receptor systems (aorta, platelets). 2. Results show that both the 6,7-dihydroxy (catechol) moiety of trimetoquinol and an intact tetrahydroisoquinoline nucleus are essential for maintaining potent beta-stimulating and antithromboxane A2 activities. 3. By contrast, ring enlargement, as in the BA analogs, or masking of the catechol with dimethoxy or methylenedioxy functional groups enhanced the potency of inhibitors on thromboxane A2-independent activation of human platelets induced by bacterial phospholipase C (PLC). 4. The selective blockade of this pathway by these compounds suggests that they may represent a new and novel class of antiplatelet drugs.

Synthesis and evaluation of trimetoquinol derivatives: novel thromboxane A2/prostaglandin H2 antagonists with diminished beta-adrenergic agonist activity.

Trimetoquinol (TMQ, 1) is a unique catecholamine with a strong stereodependence for agonism at beta-adrenergic (S > > R) and antagonism at thromboxane A2/prostaglandin H2 (TP; R > > S) receptors. Our laboratory has reported the effects of N-alkylation and modification of the trisubstituted benzyl group in these receptor systems. For iodinated derivative 5, maintaining potency in TP receptor systems (112%) was coupled with maintaining limited potency in beta-adrenergic receptor systems (34% for beta 1 and 47% for beta 2). In this study, several diverse TMQ derivatives were prepared to probe for binding interactions specific to a particular receptor system. Planar amidine 2, which was designed to explore the importance of TMQ's chiral center, showed a dramatic loss of potency (< 1%) in each receptor system. Likewise, the homologation of a previously described N-benzyl derivative (3) to the N-phenylethyl derivative 4 also showed reduced potency (< 3%) in both receptor systems. However, modification of the trimethoxybenzyl group of TMQ to a 4-hydroxy-3-nitrobenzyl group (7) provided a unique lead for TMQ derivatives with significant potency in TP receptor systems (91%) and reduced potency in beta-adrenergic receptor systems (4% for beta 1 and 19% for beta 2).

Differential eudismic ratios in the antagonism of human platelet function by phenoxy- and thiophenoxyacetic acids.

The antilipidemic drug clofibric acid (CPIB) exhibits antiplatelet effects. In order to examine the role of enantioselectivity and hydrophobicity, the mono(desmethyl) enantiomers of 2-(4-chlorophenoxy)propanoic acid (CPPA), related butanoate homologs, 2-(4-chlorophenoxy)butanoic acid (CPBA), thioether relatives, 2-(4-chlorothiophenoxy)propanoic acid (CTPA) and corresponding butanoate homologs, 2-(4-chlorothiophenoxy)butanoic acid (CTBA) were studied. All compounds inhibit prostaglandin-dependent human platelet aggregation and serotonin secretion induced by ADP. For the phenoxy acid series, (+)-(R)-CPPA is 5-fold more potent than either (-)-(S)-CPPA or CPIB giving a rank order potency of (+)-(R)-CPPA > (+)-(R)-CPBA > (-)-(S)-CPPA > (-)-(S)-CPBA. With the exception of (-)-(S)-CTPA, all thiophenoxy acid enantiomers are less potent than their respective phenoxy acid isomers [(+)-(R)-CTPA > (-)-(S)-CTPA > (+)-(R)-CTBA > (-)-(S)-CTBA]. The same rank order of potencies are observed against responses induced by arachidonic acid (AA) with the exception of CPIB which is inactive. However, inhibition of thrombin-induced [3H]-AA release by phenoxyacetic acids is not stereoselective but correlates with hydrophobicity.

A structure-activity relationship study of benzylic modifications of 4-[1-(1-naphthyl)ethyl]-1H-imidazoles on alpha 1- and alpha 2-adrenergic receptors.

The naphthalene analog of medetomidine (1), 4-[1-(1-naphthyl)ethyl]-1H- imidazole (2), is a highly potent, selective alpha 2-adrenoceptor agonist. We have initiated a structure-activity relationship study of the replacement of the methyl group on the carbon bridge between the naphthalene and imidazole rings of 2 with a hydrogen, hydroxy, methoxy, carbonyl, or trifluoromethyl group and compared their biological activities with medetomidine 1 and the optical isomers of 2. Analogs of 2 were antagonists of alpha 2A-adrenoceptor-mediated human platelet aggregation and agonists on alpha 1- and alpha 2-adrenoceptors in guinea pig ileum. The rank order and potencies of these analogs on platelets (alpha 2A-subtype) and guinea pig ileum (alpha 1-subtype) were nearly the same, whereas racemic and S-(+)-2, desmethyl, and hydroxy analogs were potent agonists on alpha 2-adrenoceptors in guinea pig ileum. With the exception of the desmethyl analog 5, none of the other analogs were as potent as the parent drug 2 on alpha 2A- (human platelets), alpha 1- (guinea pig ileum), or alpha 2- (guinea pig ileum) adrenergic receptor systems. As with analog 2, the desmethyl- and methoxy-substituted analogs retained a greater alpha 2/alpha 1-selectivity in both functional (agonist activity) and biochemical (receptor displacement) studies. Receptor binding studies indicate that S-(+)-2 possessed greater affinity than the R-(-)-isomer on both alpha 1- and alpha 2-adrenoceptors in rat brain. In addition, R-(-)-2 did not show agonist activity in alpha 2-adrenoceptors of guinea pig ileum and was 10-fold more potent than S-(+)-2 as an antagonist of alpha 2A-adrenoceptors in human platelets. Thus, the nature of the substituent and the chirality at the carbon bridge between the naphthalene and imidazole rings play an important role in maintaining potent alpha 2-adrenoceptor activity and high alpha 2/alpha 1-selectivity within the 4-substituted imidazole class.

Interactions of nonprostanoid trimetoquinol analogs with thromboxane A2/prostaglandin H2 receptors in human platelets, rat vascular endothelial cells and rat vascular smooth muscle cells.

Trimetoquinol (TMQ), a nonprostanoid compound, inhibits thromboxane A2 agonist-induced responses in platelets and vascular smooth muscle. Sixteen TMQ analogs were used to examine the stereochemical requirements of the interaction with thromboxane A2/prostaglandin H2 (TP) receptor sites in human platelets (HP), and cultured rat vascular endothelial (RVEC) and smooth muscle (RVSMC) cells. [3H]SQ 29548 was used as the ligand for TP receptors. The receptor binding affinities of these TMQ analogs for TP receptors in HP, RVEC and RVSMC were highly correlated with each other, and to their reported inhibitory potency values against U46619-induced HP aggregation and serotonin secretion, and contraction of rat aorta. TP receptor binding affinities of TMQ and 8-fluoro TMQ isomers were highly stereoselective (R-isomer > S-isomer), and only the 8-fluoro TMQ isomers gave qualitatively different functional responses in rat aorta. The affinity of TMQ for TP receptors was increased by addition of iodine and fluorine atoms at the 5- and 8-positions of the catechol ring or by replacement of the methoxy groups with iodine atoms on the 1-benzyl ring system. The results indicate that: 1) the stereochemical requirements of TMQ analogs for interaction with TP receptors in these cell systems are the same; 2) although TMQ analogs act as TP receptor antagonists, differences in functional responses by 8-fluoro TMQ isomers in platelets and aorta are not explained by their relative binding affinities to TP receptors; and 3) asymmetric halogenated TMQ analogs should be useful as affinity probes for further characterization of TP receptors.

Halogen-substituted trimetoquinol analogs as thromboxane A2 receptor antagonists in platelets and aorta.

Trimetoquinol (TMQ) is a non-prostanoid compound that blocks prostaglandin H2/thromboxane A2 (TXA2) receptor-mediated responses initiated by a prostaglandin (PG) H2 analog, U46619, in human platelets and rat aorta. Ring fluorine-substituted TMQ analogs selectively antagonized PG-dependent human platelet activation induced by U46619, arachidonic acid, collagen, ADP or epinephrine; and were about 300-fold less potent as inhibitors of PG-independent responses mediated by thrombin or bacterial phospholipase C. For each inducer of the PG-dependent pathway, the rank order of inhibitory potency was identical (TMQ > 8-fluoro-TMQ > 5-fluoro- TMQ). Iodine substitution yielded a similar rank order of antagonism against U46619-induced platelet activation (TMQ > 8-iodo-TMQ > 5-iodo-TMQ), and all TMQ analogs inhibited platelet aggregation in whole blood as well as in platelet-rich plasma. Inhibition of specific [3H]SQ 29,548 binding by TMQ analogs was highly correlated with inhibition of functional responses to U46619. Radioligand binding experiments using TMQ analogs with rat platelets showed no interspecies difference in comparison with human platelets. The rank order of inhibitory potencies for the fluorinated (but not iodinated) TMQ analogs changed in rat thoracic aorta with 8-fluoro-TMQ > TMQ > or = 5-fluoro-TMQ as antagonists of U46619-induced vascular contraction. These findings demonstrate that the primary mechanism of antiplatelet action of TMQ analogs is related to a blockade of TXA2 receptor sites, and ring-halogenated TMQ analogs distinguish between TXA2-mediated functional responses in vascular smooth muscle and platelets.

Synthesis of halogenated trimetoquinol derivatives and evaluation of their beta-agonist and thromboxane A2 (TXA2) antagonist activities.

The 5,8-difluoro (4), 5-iodo (5), 8-iodo (6), and 5-trifluoromethyl (7) derivatives of trimetoquinol (TMQ, 1) have been synthesized and evaluated for their ability to stimulate beta 1 (guinea pig atria) and beta 2 (guinea pig trachea) adrenoceptors as well as for their inhibitory activity against U46619 [a thromboxane A2 (TXA2) mimetic]-mediated contraction of rat thoracic aorta and human platelet aggregation. Both 5 and 6 were considerably less active than TMQ on both beta-adrenergic systems and gave a rank order of stimulatory potency of 1 much greater than 6 greater than or equal to 5. Similarly, iodine substitution at either position also caused a reduction in TXA2 antagonist activity with a rank order potency of 1 greater than 6 much greater than 5. Compared to 1, however, 5-iodo-TMQ (5) showed a marked selectivity for blockade of U46619 responses in rat aorta over human platelets. On beta-systems, 4 had reduced potency compared to TMQ and was similarly nonselective. Introduction of a trifluoromethyl group at the 5-position of TMQ completely abolished both beta 1- and beta 2-adrenergic agonist activities while imparting weak antagonist activity on beta 1 receptors. On TXA2 systems, both 4 and 7 possessed significantly decreased inhibitory activity compared to TMQ. The synthetic approaches to the synthesis of 8-(trifluoromethyl)-TMQ (8) are also described. The enantiomers of the 8-fluoro derivative (3) of TMQ were separated on a preparative Chiralcel OD column and evaluated on beta-adrenergic systems and TXA2 systems. On beta-adrenergic systems, (S)-(+)-8-fluoro-TMQ was at least 10-fold more potent than (R)-(-)-8-fluoro-TMQ. Conversely, (R)-(-)-8-fluoro-TMQ was approximately 14-fold more potent as an antagonist of TXA2-mediated aggregation in human platelets than (S)-(+)-8-fluoro-TMQ. In contrast to platelets, (S)-(+)-8-fluoro-TMQ was an agonist in rat aorta whereas (R)-(-)-8-fluoro-TMQ was an antagonist.

Synthesis and alpha-adrenergic activities of 2- and 4-substituted imidazoline and imidazole analogues.

Seven analogues of medetomidine and naphazoline were synthesized and evaluated for their alpha 1 (aorta) and alpha 2 (platelet) activities. The analogues were composed of 2- and 4-substituted imidazoles and imidazolines attached through a methylene bridge to either the 1- or 2-naphthalene ring system. In general the 1-naphthalene analogues were the most potent inhibitors of epinephrine-induced platelet aggregation. Of considerable interest was the fact that the 1-naphthalene analogues (2, 5-7) were partial agonists while the 2-naphthalene analogues (3, 8, 9) were antagonists in an alpha 1-adrenergic system (aorta). Thus, appropriately substituted naphthalene analogues of medetomidine and naphthazoline provide a spectrum of alpha 1-agonist, alpha 1-antagonist, and alpha 2-antagonist activity.

Resolution and adrenergic activities of the optical isomers of 4-[1-(1-naphthyl)ethyl]-1H-imidazole.

Recently we synthesized a naphthalene analog of medetomidine, 4-[1-(1-naphthyl)ethyl]-1H-imidazole hydrochloride (1), and found it to be highly potent in adrenergic systems. The separation of optical isomers of this naphthalene analog was achieved by using the isomers of tartaric acid. The optical purities of the isomers were determined by HPLC using a chiral column. Using X-ray analysis the (+)-isomer was determined to have the S absolute configuration. It has been reported that the (+)-isomer of medetomidine (2) is the most potent enantiomer on alpha 2-adrenergic receptors. There were both qualitative and quantitative differences in biological activities of the optical isomers of 1 in alpha 1- and alpha 2-adrenergic receptor systems of guinea pig ileum and human platelets. (+)-(S)-1, but not (-)-(R)-1 was a selective agonist of alpha 2-mediated responses in ileum whereas (-)-(R)-1 was more potent than (+)-(S)-1 as an inhibitor of alpha 2-mediated platelet aggregation.

Structure-activity studies of new imidazolines on adrenoceptors of rat aorta and human platelets.

Potencies of new aromatic substituted fluoro or iodo analogues of catecholimidazolines on functional responses in rat aorta (alpha 1) and platelets (alpha 2) were quantified. (1) When compared either on the basis of EC50 or the dissociation constant (KA), 5-fluorocatecholimidazoline was as potent as the reference alpha 1-adrenoceptor agonist, phenylephrine in the vascular tissue. The maximum contraction of aorta produced by the fluoro analogue was, however, 17% higher than that of phenylephrine. The time required for 1/2 relaxation of the tissue after 5-fluoro hydroxy imidazoline was at least twice as long as that of the phenylephrine. The catechol moiety as well as fluorine substitution at the critical 5-position of the aromatic ring is essential for higher alpha 1 adrenoceptor-mediated potency. (2) As compared to the fluoro analogues, the adrenoceptor-mediated potencies of iodo-analogues were relatively weak on vascular tissue. Naphazoline and its analogues were partial agonists on vascular tissue with dissociation constants which ranged from 110 to 2600 nmol/l. (3) Imidazole analogues were generally less potent agonist than the imidazolines by one order of magnitude. (4) The vacular effects of all agonists were competitively blocked by prazosin with KB values which ranged from 0.04 to 0.48 nmol/l. Since the variation in KB values were within normal limits, the action of new imidazolines on rat aorta appears to be mediated mainly by the activation of the alpha 1-adrenoceptor. Prazosin 10 nmol/l abolished the vascular response of some partial agonists. This indicates a slightly different mode of interaction of agonists with the transduction process. (5) Carbon 4-substituted imidazolines produced little or no alpha 1 adrenoceptor-mediated intrinsic activity, but competitive receptor blocking potency was comparable to that of phentolamine. (6) Medetomidine was a partial agonist on the rat aorta with a KA of 260 nmol/l. When investigated as a blocker, the KB of medetomidine against phenylephrine was approximately 5600 nmol/l. The variation in the latter value was high. (7) In acetylsalicylic acid-treated human platelets, the alpha 2-adrenoceptor-mediated aggregatory effect of all fluoro analogues was weak. Iodo or naphazoline analogues did not initiate platelet aggregation but blocked the aggregation induced by epinephrine. The affinity of naphazoline for the alpha 2-adrenoceptor was 1100 nmol/l. The IC50 of medetomidine for platelet anti-aggregatory effect was 3300 nmol/l, which compares favorably with other imidazoline type of blockers of platelet aggregation. (8) Sympathomimetic vasoconstrictor actions and platelet aggregation effects of these compounds can be dissociated.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacologic antagonism of thromboxane A2 receptors by trimetoquinol analogs in vitro and in vivo.

Although (-)-(S)-trimetoquinol [1-(3,4,5-trimethoxy-benzyl)- 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; TMQ] is recognized as a potent bronchodilator, (+)-(R)-TMQ is a selective antagonist of human platelet aggregation and serotonin secretion induced by thromboxane A2 (TXA2) agonists. To confirm the pharmacological actions of TMQ analogs, the interaction of the drugs with TXA2 receptors was examined in human platelets and in a mouse sudden death model. The inhibitory potencies of TMQ analogs (pIC50 values) for displacement of [3H]SQ 29,548 binding to platelets showed excellent correlation with the respective pIC50 (-log IC50) values for U46619-induced aggregation (r = 0.99, P less than 0.01) and serotonin secretion (r = 0.99, P less than 0.01) in human platelet-rich plasma and for whole blood aggregation (r = 0.99, P less than 0.01). In each system, the rank order of inhibitory potencies was rac-iodoTMQ greater than or equal to (+)-(R)-TMQ greater than rac-TMQ much greater than (-)-(S)-TMQ. Antithrombotic effects of TMQ analogs were evaluated in a mouse sudden death model. In vivo antithrombotic potencies of these compounds were consistent with the in vitro potencies as TXA2 receptor antagonists in platelet systems. Administration of rac-iodoTMQ, (+)-(R)-TMQ and rac-TMQ 15 min before the injection of U46619 (800 micrograms/kg, iv) protected mice against U46619-induced sudden death. On the other hand, (-)-(S)-TMQ did not protect animals against death. Protection of U46619-induced cardiopulmonary thrombosis by TMQ analogs was seen at doses of 3-100 mg/kg.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological properties of novel bicyclic isoquinoline analogs in isolated guinea pig atria, trachea and in human platelets: relationship to trimetoquinol.

1. Antiplatelet and beta-adrenoceptor activities of a set of secondary and tertiary N-methyl substituted amine analogs of trimetoquinol (TMQ, I and II, respectively) and 5,8-ethano-l-(p-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquin oline (bicyclic isoquinoline compounds III and IV, respectively) were examined. 2. Compounds III and IV induced relaxations of guinea pig trachea which were blocked by propranolol whereas neither compound acted as an agonist nor antagonist of beta-adrenoceptors (chronotropy) in guinea pig atria. TMQ analogs (I and II) were agonists in both beta-adrenoceptor systems. 3. When tested in human platelets, compounds III and IV, like the TMQ analogs, blocked several inducers of the prostaglandin-dependent and -independent pathways, and the alpha 2-adrenoceptor-mediated pathway of platelet activation. 4. The bicyclic isoquinoline analogs (III and IV) possessed more selective beta 2-adrenoceptor stimulatory activity and equal or greater inhibitory activity against inducers of the prostaglandin-independent pathways of platelet function than the corresponding TMQ analogs (I and II). 5. These chemically novel lipophilic bicyclic compounds provide a new lead to the development of agents useful for the treatment of asthma and thrombotic disorders.

Pharmacological evaluation of the beta-adrenoceptor agonist and thromboxane receptor blocking properties of 1-benzyl substituted trimetoquinol analogues.

The beta 1- and beta 2-adrenoceptor agonist and thromboxane A2 (TXA2) antagonist properties of trimetoquinol (TMQ, I) and 1-benzyl substituted TMQ analogues [3'-iodo-4',5'-dimethoxy TMQ, II; 3',5'-diiodo-4'-dimethoxy TMQ, III; 3',4'-dimethoxy-5'-nitro TMQ, IV; 3',4'-dimethoxy-5'-amino TMQ; V; and 3',4'-dimethoxy TMQ, VI] were studied in guinea pig atria (beta 1) and trachea (beta 2), and in rat thoracic aorta and human platelets, respectively. The rank order of agonist activities in beta 1- and beta 2-adrenoceptor tissues was IV greater than or equal to I greater than II greater than V greater than III greater than VI and I greater than II = IV = V greater than VI greater than III, respectively. An increase of beta 2/beta 1-selectivity (2- to 3-fold) was observed for analogues V and VI as compared to TMQ. The rank order of inhibitory potency against U46619-induced contraction of rat aorta and human platelet aggregation and secretion was the same (I = II = III greater than IV greater than V greater than VI). The results show that varying the substituents at the 3'- and 5'-positions of the trimethoxybenzyl group of TMQ produces compounds which give different profiles of biological activity for beta-adrenoceptor agonism versus TXA2 antagonism. Certain TMQ analogues, notably analogue V, showed a greater selectivity as beta 2-receptor agonists and TXA2 antagonists in vascular smooth muscle than the parent drug (TMQ), and the iodinated analogues (II and III) have promise as potential radioligands or photoaffinity probes for thromboxane A2 receptors.

Synthesis and alpha 2-adrenoceptor effects of substituted catecholimidazoline and catecholimidazole analogues in human platelets.

It is known that the steric requirements for the interactions of catecholamines and catecholimidazolines with alpha 1- and alpha 2-adrenoceptors are different. New analogues of desoxycatecholimidazoline (1), desoxycatecholimidazole (3), benzylic hydroxyl substituted imidazole (4), and the aromatic fluorine substitution analogues of 1 at the 2 (5), 5 (6), and 6 (7) positions, and a set of asymmetric 4-substituted catecholimidazolines, S-8 and R-8, were prepared and tested for interaction with alpha 2-adrenoceptors in human platelets. With the exception of 3, all compounds were selective for alpha-adrenoceptor-mediated responses in human platelets. Introduction of a double bond in imidazoline 1 to give an imidazole 3 or the introduction of a benzylic hydroxyl group to 3, as in 4, reduced the inhibition of platelet aggregation with a rank order potency of 1 greater than 3 greater than 4. Fluorine atom substitution at the 2-, 5-, or 6-positions only slightly modified the inhibitory activity of 1. Each analogue (1, 3-7) produced alpha 2-mediated inhibition of platelet adenylate cyclase and can be classified as a partial agonist. The inhibition potency of S-8 and R-8 against epinephrine-induced aggregatory responses were greatly different, and only R-8 and 4 were alpha 2-agonists on human platelet function. Our studies provide further evidence for the differential interaction of catecholamines and catecholimidazolines in alpha 1- and alpha 2-adrenoceptor systems.

Potent antiplatelet effects of cyclic clofibric acid (CPIB) analogs on human platelets.

CPIB possesses antiplatelet as well as hypolipidemic activities. Two cyclic CPIB analogs, 6-phenylchroman-2-carboxylic acid (PCCA) and 6-cyclohexylchroman-2-carboxylic acid (CHCCA) were also found to be antagonists of the prostaglandin (PG)-dependent pathway of human platelet activation. PCCA and CHCCA were inhibitors of aggregation (AGG) and secretion (SEC) induced by ADP or epinephrine (E) [second waves only] and arachidonic acid (AA) with IC50 values ranging from 2.3-8.7 microM for PCCA and 3.7-12.1 microM for CHCCA. Neither compound antagonized the proaggregatory effects of the thromboxane A2 (TXA2) receptor agonist, U46619. CPIB blocked ADP and E-induced AGG and SEC (IC50's greater than 1200 microM) but not AA- or U46619-induced responses. Only CPIB blocked thrombin-induced AA release. Data showed that PCCA and CHCCA inhibited AA-induced malondialdehyde formation (IC50's = 9.3 and 11.3 microM, respectively) and thrombin-induced production of prostaglandin E2, prostaglandin F2 alpha, and TXB2 with IC50's ranging from 2.9 to 13.4 microM. PCCA and CHCCA were at least 200- to 500-fold more potent than CPIB as inhibitors of the PG-dependent pathway of human platelet activation. We conclude that PCCA and CHCCA act by inhibiting platelet cyclooxygenase activity whereas CPIB blocks the activity of phospholipase A2. Hypolipidemic PCCA and CHCCA represent a potent class of cyclooxygenase inhibitors which may be more useful than CPIB for treatment of atherosclerotic and thrombotic disorders.

Inhibition of human platelet aggregation and secretion by ant venom and a compound isolated from venom.

Venom from the tropical ant, Pseudomyrmex triplarinus, has activity against rheumatoid arthritis. Since platelets are involved in inflammatory responses, they were employed to study the effects of venom on prostaglandin-dependent human platelet aggregation and secretion. The assay is very sensitive and uses microliter volumes, which makes it useful as a screen during isolation and characterization of venom components. Whole venom inhibited arachidonic acid- and U46619-induced platelet aggregation with IC50s of 45 and 39 micrograms/ml, respectively. This suggested that venom prevented the action of prostaglandins. Pure venom was fractionated by gel filtration and at least three materials with antiplatelet activity were detected. The smallest component (factor F) was most active and was purified by additional molecular filtration and characterized by UV absorbance, thin-layer chromatography, nuclear magnetic resonance spectra, and activity to platelets. Factor F was identified as adenosine, which is known to stimulate platelet adenylate cyclase and has not been previously reported to be a component of insect venom.

Characterization of the inhibition of U46619-mediated human platelet activation by the trimetoquinol isomers. Evidence for endoperoxide/thromboxane A2 receptor blockade.

Sites of inhibition for the trimetoquinol (TMQ) isomers on 15S-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619)-, 12-O-tetradecanoylphorbol 13-acetate (TPA)- and A23187-induced human platelet activation were investigated. Experiments using washed human platelets were designed to characterize relationships among functional (aggregation, secretion) and biochemical (protein phosphorylation, metabolism of inositol phospholipids and radioligand displacement analysis) processes of platelet activation by U46619 and the specificity of inhibition by the TMQ isomers. Thromboxane A2 receptor stimulation by U46619 in human platelets resulted in a time- and concentration-dependent breakdown of inositol phospholipids [phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol (PI)], phosphatidic acid (PA) accumulation, phosphorylation of 20 and 45 kD proteins, aggregation and serotonin secretion. The TMQ isomers stereoselectively inhibited all U46619-mediated platelet activation processes. R(+)-TMQ was 40- and 22-fold more potent than S(-)-TMQ as an inhibitor of U46619-induced platelet aggregation and serotonin secretion respectively. In addition, R(+)-TMQ blocked U46619-induced 20 kD protein phosphorylation, 45 kD protein phosphorylation, PIP2, PIP and PI breakdown, and PA accumulation with a potency which was 8-, 13-, 45-, 37-, 33- and 33-fold greater than the S(-)-isomer respectively. In contrast to S(-)-TMQ, R(+)-TMQ produced a concentration-dependent inhibition of specific [3H]U46619 binding to endoperoxide/thromboxane A2 receptor sites in washed platelets. In other experiments, S(-)-TMQ was more potent than R(+)-TMQ as an inhibitor of TPA- and A23187-induced platelet aggregation and serotonin secretion, and of TPA-induced phosphorylation of 45 and 20 kD proteins. The inhibitory potencies of S(-)-TMQ against TPA- or A23187-induced responses were similar to those needed for antagonism of U46619-mediated platelet activation. In contrast, much higher concentrations of R(+)-TMQ were required for blockade of TPA or A23187 versus U46619-mediated responses in human platelets. Taken collectively, the data show that the TMQ isomers interfered with the endoperoxide/thromboxane A2 receptor-mediated phospholipase C-signal cascade of inositol phospholipid hydrolysis, calcium mobilization, and protein phosphorylation leading to platelet aggregation and secretion. R(+)-TMQ acted as a pharmacologically selective and highly stereospecific [R(+)-TMQ much greater than S(-)-TMQ] antagonist of endoperoxide/thromboxane A2 receptor sites in platelets.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis and thromboxane A2 antagonist activity of N-benzyltrimetoquinol analogues.

It is currently believed that the platelet thromboxane A2 (TXA2/PGH2) receptor is different from the vascular TXA2/PGH2 receptor. While the majority of TXA2 receptor antagonists are structurally related to the prostaglandins, trimetoquinol (TMQ) represents a unique nonprostanoid antagonist. TMQ also possesses beta-adrenergic activity; however, an N-benzyl substituent on TMQ has been shown to impart some selectivity for platelet antiaggregatory activity versus beta-adrenergic activity. In this study, we examined the synthesis and TXA2 antagonist activity of a series of substituted N-benzyl analogues of TMQ. While these analogues showed an apparent direct correlation between platelet antiaggregatory activity and electron-donating ability of the N-benzyl substituents, no such correlation could be demonstrated for the inhibition of contractile responses. Thus, nonprostanoid TXA2 antagonists can be used to demonstrate differences between platelet and vascular TXA2/PGH2 responses.

Synthetic aci-reductones: 3,4-dihydroxy-2H-1-benzopyran-2-ones and their cis- and trans-4a,5,6,7,8,8a-hexahydro diastereomers. Antiaggregatory, antilipidemic, and redox properties compared to those of the 4-substituted 2-hydroxytetronic acids.

Synthetic procedures for the elaboration of aci-reductones belonging to the 6- or 7-mono- or bis-substituted-3,4-dihydroxy-2H-1-benzopyran-2-ones (6-10) and their cis- and trans-4a,5,6,7,8,8a-hexahydro diastereomers (11, 12) are described. hexahydrobenzopyranone aci-reductones were conveniently prepared by using Meldrum's synthon (2,2-dimethyl-1,3-dioxane-4,6-dione, 49). Certain of these substances were evaluated for antilipidemic activity in the cholesterol-fed rat model, and all analogues were studied for their ability to inhibit aggregation of human platelets. Results are compared to aci-reductones belonging to the 4-aryl- and 4-spiroalkyl-2-hydroxytetronic acid systems (4,5a,b). Redox potentials for all aci-reductones were determined with cyclic voltammetry. It would appear that the 4-aryl-2-hydroxytetronic acids represent leads for further study as antiatherosclerotic drugs owing to their favorable antilipidemic and antiaggregatory properties whereas the benzopyranones are of most interest as probes for platelet antiaggregatory mechanism studies.