1-(Arylpiperazinylamidoalkyl)-pyrimidones: orally active inhibitors of lipoprotein-associated phospholipase A(2).

The lipophilic 1-substituent in a series of 1-((amidolinked)-alkyl)-pyrimidones, inhibitors of recombinant lipoprotein-associated phospholipase A(2), has been modified to give inhibitors of high potency in human plasma and enhanced physicochemical properties. Phenylpiperazineacetamide derivative 23 shows very promising oral activity.

The identification of a potent, water soluble inhibitor of lipoprotein-associated phospholipase A2.

Modification of the pyrimidone 5-substituent in a series of 1-((amidolinked)-alkyl)-pyrimidones, lipophilic inhibitors of lipoprotein-associated phospholipase A2, has given inhibitors of nanomolar potency and improved physicochemical properties. Compound 23 was identified as a potent, highly water soluble. CNS penetrant inhibitor suitable for intravenous administration.

N-1 substituted pyrimidin-4-ones: novel, orally active inhibitors of lipoprotein-associated phospholipase A2.

From two related series of 2-(alkylthio)-pyrimidones, a novel series of 1-((amidolinked)-alkyl)-pyrimidones has been designed as nanomolar inhibitors of human lipoprotein-associated phospholipase A2. These compounds show greatly enhanced activity in isolated plasma. Selected derivatives such as compounds 51 and 52 are orally active with a good duration of action.

2-(Alkylthio)pyrimidin-4-ones as novel, reversible inhibitors of lipoprotein-associated phospholipase A2.

Starting from two weakly active hits from high throughput screening, a novel series of 2-(alkylthio)-pyrimidin-4-ones with high potency and selectivity for lipoprotein-associated phospholipase A2 has been designed. In contrast to previously known inhibitors, these have been shown to act by a non-covalent and substrate competitive mechanism.

Lipoprotein-associated phospholipase A(2), platelet-activating factor acetylhydrolase, is expressed by macrophages in human and rabbit atherosclerotic lesions.

We studied the expression of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), an enzyme capable of hydrolyzing platelet-activating factor (PAF), PAF-like phospholipids, and polar-modified phosphatidylcholines, in human and rabbit atherosclerotic lesions. Oxidative modification of low-density lipoprotein, which plays an important role in atherogenesis, generates biologically active PAF-like modified phospholipid derivatives with polar fatty acid chains. PAF is known to have a potent proinflammatory activity and is inactivated by its hydrolysis. On the other hand, lysophosphatidylcholine and oxidized fatty acids released from oxidized low-density lipoprotein as a result of Lp-PLA(2) activity are thought to be involved in the progression of atherosclerosis. Using combined in situ hybridization and immunocytochemistry, we detected Lp-PLA(2) mRNA and protein in macrophages in both human and rabbit atherosclerotic lesions. Reverse transcriptase-polymerase chain reaction analysis indicated an increased expression of Lp-PLA(2) mRNA in human atherosclerotic lesions. In addition, approximately 6-fold higher Lp-PLA(2) activity was detected in atherosclerotic aortas of Watanabe heritable hyperlipidemic rabbits compared with normal aortas from control rabbits. It is concluded that (1) macrophages in both human and rabbit atherosclerotic lesions express Lp-PLA(2), which could cleave any oxidatively modified phosphatidylcholine present in the lesion area, and (2) modulation of Lp-PLA(2) activity could lead to antiatherogenic effects in the vessel wall.

Lipoprotein-associated phospholipase A2, platelet-activating factor acetylhydrolase, generates two bioactive products during the oxidation of low-density lipoprotein: use of a novel inhibitor.

A novel and potent azetidinone inhibitor of the lipoprotein-associated phospholipase A2 (Lp-PLA2), i.e. platelet-activating factor acetylhydrolase, is described for the first time. This inhibitor, SB-222657 (Ki=40+/-3 nM, kobs/[I]=6. 6x10(5) M-1.s-1), is inactive against paraoxonase, is a poor inhibitor of lecithin:cholesterol acyltransferase and has been used to investigate the role of Lp-PLA2 in the oxidative modification of lipoproteins. Although pretreatment with SB-222657 did not affect the kinetics of low-density lipoprotein (LDL) oxidation by Cu2+ or an azo free-radical generator as determined by assay of lipid hydroperoxides (LOOHs), conjugated dienes and thiobarbituric acid-reacting substances, in both cases it inhibited the elevation in lysophosphatidylcholine content. Moreover, the significantly increased monocyte chemoattractant activity found in a non-esterified fatty acid fraction from LDL oxidized by Cu2+ was also prevented by pretreatment with SB-222657, with an IC50 value of 5.0+/-0.4 nM. The less potent diastereoisomer of SB-222657, SB-223777 (Ki=6.3+/-0.5 microM, kobs/[I]=1.6x10(4) M-1.s-1), was found to be significantly less active in both assays. Thus, in addition to generating lysophosphatidylcholine, a known biologically active lipid, these results demonstrate that Lp-PLA2 is capable of generating oxidized non-esterified fatty acid moieties that are also bioactive. These findings are consistent with our proposal that Lp-PLA2 has a predominantly pro-inflammatory role in atherogenesis. Finally, similar studies have demonstrated that a different situation exists during the oxidation of high-density lipoprotein, with enzyme(s) other than Lp-PLA2 apparently being responsible for generating lysophosphatidylcholine.

Purine derivatives as competitive inhibitors of human erythrocyte membrane phosphatidylinositol 4-kinase.

The possibility of deriving a potent, cell-penetrating inhibitor of human erythrocyte PI 4-kinase, competitive with respect to ATP, has been investigated in a series of purine derivatives and analogues. The purine nucleus is not essential for binding to the ATP site but offers the advantage of synthetic accessibility to its derivatives. The optimum substitution pattern in purine was found to be an electron-releasing substituent in the 6-position (e.g. amino, as in adenine, 1) and a compact, lipophilic group in either the 8-position or, preferably, the 9-position, suggesting the importance of the N-1 lone pair and hydrophobic contributions of the 8- and 9-substituents to binding. The most potent inhibitor synthesized was 9-cyclohexyladenine (54), which has an apparent Ki value of 3.7 microM.

Studies on the mechanism of action of omeprazole.

The effects of omeprazole on preparations of pig gastric (H+ + K+)-ATPase have been studied. Omeprazole was found to inhibit the (H+ + K+)-ATPase activity in a time-dependent manner. Inhibition was more pronounced at pH 6.1 compared with pH 7.4 and decreased as the concentration of (H+ + K+)-ATPase preparation increased. The potency of omeprazole was therefore highly dependent upon the conditions used. When pre- incubated with (H+ + K+)-ATPase preparation (30 micrograms protein/ml) for 30 min at 37 degrees and pH 6.1, omeprazole inhibited the (H+ + K+)-ATPase activity with an IC50 of 3.9 microM. This inhibition was shown to be irreversible in nature. Whilst omeprazole itself was not very potent as an inhibitor of the (H+ + K+)-ATPase activity at pH 7.4 (IC50 = 36 microM), transient acidification of omeprazole resulted in the formation of a compound(s) which produced marked inhibition at this pH (IC50 = 5.2 microM). The effects of omeprazole in the absence of acidification may have resulted from the rate-limiting formation of this compound. Radiolabelled omeprazole was shown to incorporate into the (H+ + K+)-ATPase preparation in a time-dependent and pH-dependent manner. Omeprazole, radiolabelled in three separate positions (the sulphur atom and the two adjacent carbon atoms), incorporated with equivalent time courses suggesting that the incorporation did not involve a fragmentation of the omeprazole molecule. Under conditions shown to produce a 50% inhibition of (H+ + K+)-ATPase activity, [14C] omeprazole had incorporated to a level of 4-5 nmoles/mg protein. Incorporation continued beyond the point required to produce 100% inhibition of (H+ + K+)-ATPase activity and reached 30 nmoles/mg protein after 5 hr. Prior acidification of the omeprazole resulted in a more rapid initial rate of incorporation although the final level of incorporation was lower than for omeprazole. Omeprazole was also shown to interact with the (Na+ + K+)-ATPase from dog kidney. Omeprazole inhibited the (Na+ + K+)-ATPase activity (IC50 = 186 microM). Acid-degraded omeprazole inhibited the (Na+ + K+)-ATPase activity with greater potency (IC50 = 19 microM) and was also shown to incorporate into this enzyme preparation.

The effects of histamine H2-receptor antagonists on PHA induced lymphocyte proliferation.

Histamine H2-receptor antagonists must be used with caution to define the pharmacology of histamine effects on lymphocyte mitogenesis induced by PHA, because they can enhance and/or suppress in their own right, because these effects are similar to those of histamine itself, because mitogenic doses of PHA can release significant amounts of histamine from supposedly pure mononuclear cell preparations.

Histamine-induced inhibition of lymphocyte proliferation and lysosomal enzyme release from polymorphs may not be mediated via H1- or H2-receptors.

Histamine, selective histamine H1- and H2-receptor agonists, and chemical analogues of these compounds lacking activity at histamine receptors, were tested as inhibitors of phytohaemagglutinin-induced human lymphocyte proliferation and zymosan-induced release of lysosomal enzymes from human polymorphs. No correlation was found between their inhibitory potency in these systems and their relative activity at histamine H1- or H2-receptors.