Approach to dual-acting platelet activating factor (PAF) receptor antagonist/thromboxane synthase inhibitor (TxSI) based on the link of PAF antagonists and TxSIs

A series of compounds (22-36) which possess dual-acting PAF antagonist/TxSI have been generated by the approach of linking the known PAF antagonists and TxSIs, such as Ridogrel (1).     

Novel agents combining platelet activating factor (PAF) receptor antagonist with thromboxane synthase inhibitor (TxSI)

Compounds 1 or 2 which possess dual-acting PAF antagonist/TxSI in a previous paper were modified and evaluated for the dual-acting activity. It was found that several compounds were potent dual-acting PAF antagonist/TxSI in and ex vivo. 6-(2-Chlorophenyl)-3-[4-[(E/Z)-6-ethoxycarbonyl-1-(3-pyridyl)-1-hexenyl]phenylmethyl]-8,11-dimethyl-2,3,4,5-tetrahydro-8H-pyrido[4',3': 4,5]thieno[3,2-f]triazolo[4,3-a]diazepine (12) is excellent orally dual-acting PAF antagonist/TxSI.     

The platelet-activating factor receptor antagonist L-659,989 inhibits phospholipase D activity.

The platelet-activating factor (PAF) receptor antagonist L-659,989 [(+/-)-trans-2-(3-methoxy-5-methylsulfonyl-4-propoxyphenyl)-5-(3,4, 5-trimethoxyphenyl)tetrahydrofuran)] has been reported to be a specific inhibitor of the PAF receptor and as such, it is widely used for assessment of PAF receptor mediated biological effects. We report here that L-659,989 may not be as specific as previously reported because it is also a potent inhibitor of phospholipase D activity. At concentrations of 30 micrograms/ml, L-659,989 inhibited basal and agonist-stimulated phospholipase D activity by about 55% and 70-100% respectively, through a mechanism that may involve the generation of intracellular ceramides. Another PAF receptor antagonist, WEB-2086, did not affect phospholipase D activity at concentrations up to 50 micrograms/ml. Either of these inhibitors when present at 20 micrograms/ml are reported to fully block the effects of PAF. Furthermore, L-659,989 directly inhibited the activity of bacterial PLD in vitro. These results indicate that caution is required in the interpretation of results derived from the use of L-659,989.     

The Effect of Lyso-PAF on Ciliary Activity of Human Paranasal Sinus Mucosa in vitro

The effect of lyso-PAF on ciliated cells was investigated in vitro. Normal mucosa was surgically obtained from human paranasal sinuses and incubated in the form of tissue culture. Ciliated epithelium was magnified under an inverted microscope, and ciliary movement was photo-electrically measured. Ciliary activity was significantly inhibited by 10(-8) M lyso-PAF and could be restored. The effect of lyso-PAF was completely blocked by CV-6209, a specific PAF antagonist. The PAF concentration in the incubation medium of lyso-PAF was determined by radioimmunoassay, because PAF is a well known inhibitor of ciliary activity. PAF gradually increased and after 20 min reached its maximal level. These findings indicated the existence of an enzyme in the paranasal sinus mucosa, by which lyso-PAF is converted to PAF, and that lyso-PAF can inhibit ciliary activity by means of PAF.     

The platelet-activating factor receptor antagonist L-659,989 inhibits phospholipase D activity

The platelet-activating factor (PAF) receptor antagonist L-659,989 [(±)-trans-2-(3-methoxy-5-methylsulfonyl-4-propoxyphenyl)-5-(3,4,5-trimethoxyphenyl)tetrahydrofuran)] has been reported to be a specific inhibitor of the PAF receptor and as such, it is widely used for assessment of PAF receptor mediated biological effects. We report here that L-659,989 may not be as specific as previously reported because it is also a potent inhibitor of phospholipase D activity. At concentrations of 30 μg/ml, L-659,989 inhibited basal and agonist-stimulated phospholipase D activity by about 55% and 70–100% respectively, through a mechanism that may involve the generation of intracellular ceramides. Another PAF receptor antagonist, WEB-2086, did not affect phospholipase D activity at concentrations up to 50 μg/ml. Either of these inhibitors when present at 20 μg/ml are reported to fully block the effects of PAF. Furthermore, L-659,989 directly inhibited the activity of bacterial PLD in vitro. These results indicate that caution is required in the interpretation of results derived from the use of L-659,989.     

Conformational considerations in the design of dual antagonists of platelet-activating factor (PAF) and histamine.

Following the discovery of the first dual antagonist of platelet-activating factor (PAF) and histamine, 1-acetyl-4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin- 11-ylidene)piperidine, Sch 37370, 1, a related series of structures, exemplified by (+/-)-1-acetyl-4-(8-chloro-5,6-dihydro-11H-benzo[5,6]-cyclohepta[1,2-b] pyridin-11-yl)piperazine, Sch 40338, 2, were prepared. Interestingly, the compounds exhibited a parallel structure antiallergy activity relationship, suggesting that the two series may adopt a common conformation at the PAF receptor. Conformational analysis led to a proposal for this bioactive conformation accessible to both series. The synthesis of novel conformationally constrained analogues that might mimic the proposed bioactive conformation of these compounds, and the evaluation of their in vitro antiallergy activity form the subject matter of this report.     

Effect of Platelet-activating Factor on in vitro and in vivo Interleukin-6 Production

The aim of the present study was to investigate the possible effect of platelet-activating factor (PAF), by comparison with interleukin-1beta and polyriboinositic/polyribocytidylic (poly I-C) acid, on IL-6 production by L 929 mouse fibroblasts. At concentrations above 1 muM PAF, the production of IL-6 by mouse fibroblasts was enhanced in a dose dependent fashion. At 5 muM PAF, the peak increase (60.1 +/- 19.4 U/ml) was similar to that induced by 50 mug/ml poly I-C (60.0 +/- 35.0 U/ml) and higher than the one evoked by 100 U/ml IL-1beta (3.8 +/- 1.8 U/ml). The increase of 11-6 activity induced by 5 muM PAF was maximal after a 22 h incubation period with L 929 cells. Lyso-PAF (5 muM) also increased IL-6 activity from fibroblasts to a similar extent compared with 5 muM PAF. In addition, the IL-6 activity induced by 5 muM PAF was still observed when the specific PAF antagonist, BN 52021 (10 muM), was added to the incubation medium of L 929 cells. The result suggests that the production of IL-6 by L 929 cells evoked by PAF in vitro is not receptor mediated. The in vivo effect of PAF on IL-6 production was also investigated in the rat. Two hours after intravenous injection of PAF (2 to 4 mug/kg), a dramatic increase of IL-6 activity in rat serum was observed, this effect being dose dependent. The increase of IL-6 induced by 3 mug/kg PAF was not observed when the animals were treated with the PAF antagonist, BN 52021 (1 to 60 mg/kg0. These results demonstrate that PAF modulates IL-6 production and that the in vivo effect is receptor mediated.     

The effect of platelet-activating factor and its antagonist--BN 52021--on immune reactions in vivo in mice and in vitro

The effect produced by the injection of platelet activation factor (PAF) and its antagonist BN 52021 on the intensity of humoral immune response in (CBA x C57BL)F1 mice was studied. PAF was found to stimulate the formation of antibodies to sheep red blood cells. In addition PAF stimulated the phagocytic activity of mouse peritoneal macrophages. The stimulation of immune response under the action of PAF may be attributed to an increase in the phagocytic activity of macrophages. The stimulating effect of PAF on immune response in vivo was abolished by the injection of BN 52021, the antagonist of PAF. At the same time the dose-dependent decrease of immune response was observed after the injection of BN 52021. Indomethacin, an inhibitor of prostaglandin synthesis, when administered to mice treated with BN 52021, abolished the BN 52021-induced suppression of humoral immune response. Mouse peritoneal macrophages, treated in vitro with BN 52021, were found to produce significantly more prostaglandin E than control macrophages. Thus, BN 52021 induced the suppression of humoral immune response in vivo; this suppression was probably due to the action of prostaglandin E2, a messenger of the second order. Besides, the PAF antagonist BN 52021 significantly decreased leukotriene B4 production by macrophages in vitro. BN 52021 may be supposed to switch over the synthesis and/or secretion of arachidonic acid from the lipoxygenase pathway to the cycloxygenase one.     

Syntheses and bioactivities of novel carbamates combining platelet activating factor (PAF) receptor antagonist with thromboxane synthase inhibitor (TxSI)

Synthesis of carbamates 3b which possess dual-acting PAF antagonist/TxSI using unstable esters 1, diazepines 2, K2CO3 and 18-crown-6 is described.     

Effects of platelet activating factor on the chemotaxis of normodense eosinophils from normal subjects

Highly purified eosinophils were obtained from normal subjects, and their chemotactic responses to platelet activating factor (PAF) were evaluated. PAF induced both eosinophil chemotactic and chemokinetic responses, and was 100 fold more potent eosinophil chemotactic factor as compared to eosinophil chemotactic factor of anaphylaxis. On the other hand, leukotriene B4 did not show any eosinophil chemotactic activity. A selective PAF antagonist, BN52021, inhibited eosinophil chemotaxis in a dose dependent manner. Preincubation of eosinophils with PAF induced the deactivation of eosinophils for further chemotactic responses to PAF. These findings suggest that PAF is a potent chemotactic factor for normal eosinophils.     

Platelet activating factor (PAF) involvement in endotoxin-induced hypotension in rats. Studies with PAF-receptor antagonist kadsurenone

Evidence from three types of experiments indicates that platelet activating factor (PAF)1 is an important mediator of endotoxin-induced hypotension in rats. a) Endotoxin infusion stimulates the time-dependent appearance of PAF in the blood. b) PAF infusion results immediately (less than 30 sec) in hypotension while endotoxin-induced hypotension takes 3-5 min to occur, allowing time for PAF production. c) Infusion of the specific PAF-receptor antagonist kadsurenone (2.2 mumole/kg bolus, 0.9 mumoles/min/kg continuous infusion), which inhibits PAF-induced hypotension by 67%, causes a 67% reversal of endotoxin-elicited hypotension. An additional finding of this study is that rats respond hypotensively to each of a series of low-dose PAF infusions but only to the first low-dose endotoxin infusion. These endotoxin-refractory rats do respond to subsequent PAF infusions.     

Dual antagonists of platelet activating factor and histamine 3. synthesis, biological activity and conformational implications of substituted N-acyl-bis-arylcycloheptapiperazines

A series of N-acyl-4-(5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)piperazines is described that are dual antagonists of PAF and histamine. The structural requirements for activity in this series parallel those of their previously reported piperidinylidene counterparts. Whereas their global minimum energy conformations are different for both series of compounds, computer assisted molecular modeling suggests that a common bioactive conformation is possible.     

Synthesis of bicyclic thiazolidine PAF antagonists

PAF antagonist 1 is susceptible to thiazolidine ring fragmentation in vitro and in vivo. The search for a more stable compound prompted the synthesis of a series of bicyclic analogs. Three classes of bicyclic thiazolidines (2: X = 0, CH₂, NCH₃) were prepared using a common synthetic pathway which generated all the possible diastereomers. The most potent PAF antagonists were the oxygen-substituted analogs which possessed receptor binding affinities largely dependent on stereochemistry.     

Antagonism of PAF-induced death in mice

The ability of three platelet activating factor (PAF) antagonists, BN52021, L652, 731 and 48740RP, and the leukotriene antagonist FPL55712 to block iv PAF-induced death was tested in mice. PAF-induced sudden death was been previously characterized as a model of systemic anaphylaxis and circulatory shock related its hypotensive actions. Of the drugs, BN52021 and L652, 731 provided dose-dependent protection against PAF toxicity, whereas the others had no effect. 48740RP was, however active against PAF-induced rabbit platelet aggregation. BN52021 was inactive in three other mouse sudden death models in which arachidonic acid, U46619 or collagen combined with epinephrine is injected iv to provoke a thrombotic/ischemic sudden death. In contrast, the TXA₂ antagonist SQ29548 inhibited the acute toxicity of two of these latter challenges (arachidonic acid and thromboxane agonist U46619), but was inactive against PAF lethality.These results suggest that PAF toxicity in mice is a specific model for PAF agonism, and is not mediated by TXA₂ or peptido-leukotrienes. Further, PAF-induced mortality should be a simple and useful technique for testing potential PAF antagonists for activity by various routes of administration.     

Mechanism and regulation of neutrophil priming by platelet-activating factor

Although a weak direct stimulus of superoxide anion (O₂^?) production, platelet-activating factor (PAF) markedly enhances responses to chemotactic peptides (such as n-formyl-met-leu-phe, FMLP) and phorbol esters (such as phorbol myristate acetate, PMA) in human neutrophils. The mechanism of priming was explored first through inhibition of steps in the signal transduction pathway at and following PAF receptor occupation. Priming was not altered by pertussis toxin or intracellular calcium chelation, but the PAF receptor antagonist WEB 2086 and the protein kinase C (PKC) inhibitors sphinganine and staurosporine significantly inhibited the primed response. In order to study the regulation of PAF priming, the effect of PAF alone was desensitized by exposure to escalating doses of PAF prior to exposure to the secondary stimuli. The priming effect of PAF was not desensitized under these conditions. The role of PKC in desensitization was also studied. Prior exposure to PAF also desensitized the increase in membrane PKC activity evoked by a single concentration of PAF. However, when the PAF response was desensitized, PKC priming of the response to FMLP or PMA still occurred, suggesting that PKC activity may play a role in the maintenance of the primed state despite PAF desensitization. These data suggest that: (1) PAF priming is receptor- and PKC-mediated but is independent of pertussis toxin-inhibitable G-proteins or intracellular calcium, (2) during migration in vivo, neutrophils may be desensitized to the direct effects of PAF but maintain the capacity for enhanced responses to other stimuli, (3) desensitization of PAF-induced particulate PKC activity also occurs, but PAF primes PKC activity despite PAF desensitization, and (4) distinct mechanisms govern the direct and priming effects of PAF on oxidative metabolism. © 1993 Wiley-Liss, Inc.     

Platelet-activating factor (PAF) activates casein kinase 2 in the protozoan parasite Herpetomonas muscarum muscarum

Herpetomonas muscarum muscarum is a flagellate parasite of the family Trypanosomatidae, whose cell differentiation can be triggered by the lipid mediator, PAF. In this study we demonstrate for the first time that PAF effect relies on the activation of casein kinase 2 (CK2). The classical antagonist of PAF receptor, WEB 2086, abrogated PAF-enhanced CK2 activity. CK2 activation by PAF was also inhibited when parasite extracts were assayed in the presence of modulators of PKC, MAPK, and both Ser/Thr and Tyr phosphatases. Finally, a cell permeable inhibitor of CK2 (DRB) suppressed PAF-induced cell differentiation in a dose-dependent manner.     

Platelet activating factor activity in the phospholipids of bovine spermatozoa

Platelet activating factor (PAF) has been detected in sperm from several mammalian species and can affect sperm motility and fertilization. Because bovine sperm contain a high percentage of ether-linked phospholipid precursors required for PAF synthesis, a study was undertaken to determine the PAF activity of bovine sperm phospholipids. Total lipids of washed, ejaculated bull sperm were extracted, and phospholipids were fractionated by thin-layer chromatography. Individual phospholipid fractions were assayed for PAF activity on the basis of [3H]serotonin release from equine platelets. PAF activity was detected in the PAF fraction (1.84 pmol/mumol total phospholipid) and in serine/inositol (PS/PI), choline (CP), and ethanolamine phosphoglyceride (EP) and cardiolipin (CA) fractions. Activity was highest in the CP fraction (8.05 pmol/mumol total phospholipid). Incomplete resolution of PAF and neutral lipids may have contributed to the activity in the PS/PI and CA fractions, respectively. Phospholipids from nonsperm sources did not stimulate serotonin release. Platelet activation by purified PAF and by sperm phospholipid fractions was inhibited by the receptor antagonist SRI 63-675. These results indicate that bovine sperm contain PAF and that other sperm phospholipids, especially CP and EP, which are high in glycerylether components, are capable of receptor-mediated platelet activation.     

The effect of PAF in the cochlea of guinea pigs

The influence of 10(-10) and 10(-9) M PAF/animal given into the jugular vein over 30 sec on inner ear potentials, i.e. endolymphatic potential (EP), summating potential (SP) and cochlear microphonics (CM) was investigated. The EP showed the most pronounced changes. When infusing a specific PAF receptor antagonist, ginkgolide B, or the TXA2 receptor antagonist, sulotraban, before the the infusion of PAF, the changes in cochlear potentials could be completely prevented. A second TXA2 receptor antagonist, daltroban, did not effectively prevent PAF actions. It is hypothesized that these PAF effects are due to an interference with ion transport in the non-sensory structures of the inner ear.     

Cooperativity between platelet-activating factor and collagen in aggregation of bovine platelets III

In cooperative aggregation of bovine platelets induced by simultaneous addition of PAF and collagen at subthreshold concentrations the following observations were made. (i) Formation of phosphatidic acid and arachidonic acid metabolites, which characterize PAF and collagen alone-induced aggregation, respectively, was observed very obviously. (ii) A thromboxane antagonist did not completely block the cooperative aggregation. The extent of residual aggregation activity was dependent on concentration of collagen used in the simultaneous administration with PAF. These results suggest that both signal transduction pathways activated by PAF and collagen alone at high concentrations are attained by simultaneous addition of both agonists at subthreshold concentrations through unknown mechanisms.     

PAF对肺内小动脉平滑肌细胞TXA_2，PGI_2乃Ca~（2＋）－ATPase的影响

本工作采用分离培养家兔肺内小动脉平滑肌细胞（ＰＡＳＭＣｓ），观察了外源性血小板活化因子（ｐｌａｔｅｌｅｔａｃｔｉｖａｔｉｎｇｆａｃｔｏｒ，ＰＡＦ）、ＢＮ５２０２１（ＰＡＦ受体拮抗剂）、吲哚美辛、维拉帕米对ＰＡＳＭＣｓ产生血栓素Ａ_２（ＴｘＡ_２）、前列环素（ＰＧＩ_２）及对细胞膜Ｃａ~（２＋）－ＡＴＰａｓｅ活力的影响。结果表明：（１）基础状态下ＰＡＳＭＣｓ存在花生四烯酸（ＡＡ）代谢。（２）外源性ＰＡＦ通过受体后途径激活环加氧酶促进ＡＡ代谢致ＴＸＡ_２及ＰＧＩ－２增加，ＴＸＡ_２／ＰＧＩ_２比值无明显变化。（３）外源性ＰＡＦ能直接抑制Ｃａ~（２＋）－ＡＴＰａｓｅ活力。（４）维拉帕米可逆转ＰＡＦ抑制ＰＡＳＭＣｓ膜Ｃａ~（２＋）－ＡＴＰａｓｅ活力的效应。