Mass spectrometric analysis of platelet-activating factor after isolation by solid-phase extraction and direct derivatization with pentafluorobenzoic anhydride.

Platelet-activating factor is the term used to denote a class of extremely potent lipid mediators that consist predominantly of 1-O-alkyl- and 1-O-acyl-2-acetyl-sn-glycero-3-phosphocholines. A method has been devised for rapid isolation of these acetylated phospholipids by solid-phase extraction prior to direct derivatization with pentafluorobenzoic anhydride and analysis by gas chromatography (GC)/electron-capture mass spectrometry. Recovery through the entire method (lipid isolation, derivatization, and purification) typically ranged from 70% to 85%. Using the direct derivatization procedure described here, the practical limit of detection for each of the standard alkyl- and acyl-platelet-activating factor homologs was 1 fmol injected into the GC. Results from the application of the method to the analysis of alkyl and acyl homologs of platelet-activating factor isolated from stimulated human umbilical vein endothelial cells are presented, exhibiting excellent accuracy and precision for a wide range of tissue levels of this class of potent autacoids.

Molecular heterogeneity of PAF in normal human mixed saliva: quantitative mass spectral analysis after direct derivatization of PAF with pentafluorobenzoic anhydride.

Platelet-activating factor (PAF), a family of phospholipid autacoids with potent pro-inflammatory activities, is present in saliva. The current study has quantitated various species of PAF isolated from normal human mixed saliva. Choline-containing, sn-2 acetylated phospholipids with sn-1 ether- or ester-linked fatty alcohol/acid moieties (alkyl-PAF or acyl-PAF, respectively) were evaluated after direct derivatization with pentafluorobenzoic (PFB) anhydride. Individual species of PFB-derivatized PAF were separated by gas chromatography prior to mass spectral analysis; quantitative estimates of six different species of PAF in saliva were made by comparison to corresponding authentic, synthetic PAF standards. In each saliva sample, all six species of PAF were readily detected by this facile procedure. The predominant PAF was 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine or 16:0-alkyl-PAF (0.75 +/- 0.09 pmol/ml saliva; mean +/- S.E.; n = 5) which represented only 30.4 +/- 1.5% of the total PAF. Substantial amounts of 18:1- and 18:0-alkyl-PAF and 16:0-acyl-PAF were also identified (0.52 +/- 0.07, 0.35 +/- 0.06, and 0.35 +/- 0.02 pmol/ml saliva, respectively). In summary, mass spectrometric analysis of PAF after direct derivatization with PFB anhydride has revealed that at least six different species of PAF are present in normal human mixed saliva. This structural diversity may represent an important aspect of homeostasis in the healthy oral cavity.

Cholesterol immobilization via ether-linked sepharose gels.

Cholesterol has been immobilized on Sepharose-6B via oxyether linkages to the 3- or 25-position. The 3- or 25-hydroxysterol methanesulfonates were coupled with epoxy-Sepharose-6B at 80 degrees C for 24 h. Approximately 2% of the ligand was incorporated into the gel. These types of affinity columns may be useful in purifying proteins that specifically bind or metabolize cholesterol.

An approach to trapping gamma-glutamyl radical intermediates proposed for vitamin K dependent carboxylase: alpha,beta-methyleneglutamic acid.

The vitamin K dependent carboxylase activates the glutamyl gamma-CH of substrate peptides for carboxylation by producing a gamma-glutamyl free radical, a gamma-glutamyl carbanion, or through a concerted carboxylation. We propose to intercept the putative gamma-glutamyl free radical by the intramolecular rearrangement of a substrate containing the alpha,beta-cyclopropane analogue of glutamic acid. The rearrangement of cyclopropylcarbinyl radicals into 2-butenyl radicals is rapid, exothermic, and considered diagnostic of free-radical formation. 1-Amino-2-(carboxymethyl)cyclopropane-1-carboxylate, the beta-cyclopropane analogue of glutamic acid, was synthesized starting from diethyl alpha-ketoglutarate. The alpha-keto ester was first treated with benzonitrile in sulfuric acid, to yield diethyl alpha,alpha-dibenzamidoglutarate. The alpha,alpha-dibenzamido acid was cleaved to produce the alpha,beta-dehydroamino acid and benzamide on treatment with p-toluenesulfonic acid in hot benzene. Diazomethane addition to the dehydroamino acid resulted in cycloaddition of diazomethane and production of the pyrazoline, which upon irradiation lost N2 to give the protected cyclopropane-containing amino acid analogue. Acidic hydrolysis of the N-benzoyl-alpha,beta-methyleneglutamate diethyl ester resulted in the production of the unprotected amino acid, alpha,beta-methyleneglutamic acid, in high yield. A single dehydroamino acid and a single methyleneglutamic acid isomer were produced in this synthesis; both are identified as the Z isomer, the former by NMR using the nuclear Overhauser effect and the latter through X-ray crystallographic analysis of N-benzoyl-alpha,beta-methyleneglutamate diethyl ester. Saponification of a N-protected methyleneglutamic acid dialkyl ester using limiting alkali was shown to selectively yield the alpha-alkyl ester gamma-acid. The reaction was used to produce alpha,beta-cyclopropane-containing analogues of the carboxylase substrates N-t-Boc-L-glutamic acid alpha-benzyl ester and N-benzoyl-L-glutamic acid alpha-ethyl ester. The cyclpropane-containing analogues were tested and found to be neither substrates for nor inhibitors of the rat liver microsomal vitamin K dependent carboxylase. The inability of the enzyme to recognize these substrate analogues is attributed to the alpha-alkyl substitution, which apparently abolishes substrate binding.

A novel method for the analysis of platelet-activating factor: direct derivatization of glycerophospholipids.

A novel, facile, and sensitive method for the quantitative and complete structure-proof analysis of platelet-activating factor (PAF) and other glycerophospholipids is described. 1-O-Alkyl/acyl-2-acyl-3-glycerophospholipids were treated with heptafluorobutyric anhydride in a one-step reaction to yield 1-O-alkyl/acyl-2-acyl-3-heptafluorobutyroyl-sn-glycerols as gas-liquid chromatography (GLC)-compatible derivatives. Furthermore, the components of the polar head group were also analyzed from the aqueous extract of the same reaction mixture as t-butyldimethylsilyl derivatives. Thus, this new method eliminates the need for phospholipase C treatment and subsequent purification procedures. Moreover, the direct derivatization of PAF homologs and analogs with hepatofluorobutyric anhydride does not result in positional isomerization of the product, providing increased specificity for gas-liquid chromatography-mass spectrometric (MS) analysis. It has also been shown that the heptafluorobutyroyl (HFB) derivative can easily be converted to the respective t-butyldimethylsilyl analog in a one-step reaction using t-butyldimethylsilyl chloride/imidazole reagent. Analogous to the formation of heptafluorobutyroyl derivatives, PAF also was reacted with pentafluorobenzoyl chloride to generate the pentafluorobenzoyl derivative. Therefore, this method has wide applicability for the formation of GLC-compatible derivatives of various glycerophospholipids. Our successful HFB derivatization and GLC-MS detection of subnanogram quantities of PAF indicate that this analytical procedure will greatly facilitate complete and quantitative identification of each of the molecular species of biologically derived PAF.

Synthesis and CNS activities of some 2-alkyl-3(2-substituted indol-3-yl)-methyl, 6-substituted-quinazolin-4(3H)-ones.

Nineteen title compounds (1-3, Fig. 1) have been synthesised by Mannich reaction of the corresponding 2,6-disubstituted-quinazolones, 2-substituted-indole and formaldehyde. Some of them have been found to be CNS stimulants while some others were CNS depressants. Compounds 2c and 2d have been found to possess good anticonvulsant activity in MES protection test at the dose level of 100 mg/kg.

Newer phenothiazines as anticonvulsants and monoamine oxidase inhibitors.

Eight new compounds have been synthesised in the series of 10-(alpha-aminoacylhydrazinoacetyl)phenothiazines. Their structures have been confirmed by elemental analysis and IR spectroscopic studies. The compounds have been found to possess the protection of mice against chemoshock- and electroshock-induced seizures and the monoamineoxidase (MAO) inhibitory activity. A good correlation between these two activities and the structure of the compounds has been found.

2-(2'-Hydroxyphenyl)-4-aryl-1,5-benzodiazepines as CNS active agents.

1-Aryl-3(2'-hydroxyphenyl)-propane-1,3-diones (III) have been prepared by the Baker-Venkataraman transformation of the corresponding 2-substituted benzoyl-oxyacetophenones (II). The aforesaid propane-1,3-diones (III) were condensed with o-phenylene diamine to give eight benzodiazepines (IV) which have been found to be very good MAO inhibitors (in vitro), and anticonvulsant and hypnotic (in vivo) agents. Some chemical leads on pharmacological activities have been observed.

Some new 1-(2'-methyl-6'-methoxy-quinoline-4'-yl amino)-2-methyl-4-arylidene-imidazolin-5-ones as anticonvulsant and analgesic agents.

Seventeen new 1-(2'-methyl-6'-methoxyquinoline-4'-yl-amino)-2-methyl-4-arylideneimidazol-5-ones have been synthesized by the condensation between the respective azlactones (prepared by the well known Erlenmeyer azlactone synthesis) and 2-methyl-4 hydrazino-6-methoxy-quinolines. The compounds have been found to possess anticonvulsant activity against maximal electroshock seizures and chemoshock induced seizures and the analgesic activity. Some qualitative structure activity relationship has been established.

Monoamine oxidase inhibitory and CNS activities of some quinazolinones.

16 new compounds have been synthesised in the series of 3-aryl-2-(1'-aryl-piperazin-4'-yl-carboxamidomethyl)-mercapto-quinazolinones. The compounds showed interesting results in anticonvulsant, hypnotic and monoamine oxidase (MAO) inhibitory screenings and are non-toxic. SAR were evaluated.

Some new 5-substituted benzylidene-1-phenyl-3-(2'-pyridinobutan-4'-yl) thiobarbituric acids as psychopharmacological agents.

Ten substituted thiobarbiturates have been prepared in order to evaluate their anticonvulsant activity in vivo and MAO inhibitory activity in vitro. All the compounds have been found good MAO and convulsion inhibitors. 5-(4-N,N-dimethylaminobenzylidene)-1-phenyl-3-(2'-pyridinobutan-4'-yl)thiobarbi turic acid has been found to be best in both these activities.

Search for CNS active agents: synthesis of N,N'-bis-[potassium-alkyl/aryl carboxylate]-5-arylidene barbituric acids.

5-Arylidene barbituric acids (1--5, Fig. 1) were obtained by the Perkin's condensation between the corresponding aryl aldehyde and active methylene group of barbituric acid. By the condensation of these 5-arylidene barbituric acids with different alkyl/aryl-chloroacids, fifteen new N,N'bis[potassium-alkyl/aryl carboxylate]-5-arylidene barbituric acids (6--8 Fig. 1) were prepared. Eight of the fifteen final compounds were screened for their pharmacological actions on CNS. They have shown irregular behavioral changes on the CNS; some of them were the CNS stimulants, whereas others were CNS depressants at 1/5 of ALD50 dose level (316--1000 g/kg).

New indolic anti-tremor agents.

Twenty eight compounds have been synthesized in the series 2-substituted-3-[(N-alkanol-N-imidazolin-2'-ylamino)methyl]indoles. Some of them have been shown to protect mice from tremorine-induced tremors. The compounds are central nervous system depressants and relatively non-toxic.

New anti-mycobacterial hydantoins.

Ten 1-(4-substituted-thiazol-2-yl)hydantoins have been synthesized. Some of them have shown antitubercular activity against Mycobacterium smegmatis.

Search for non-steroidal anti-inflammatory agents. Part 1: Synthesis of 2-alkyl/aryl-3-benzimidazolylalkylquinazolon-4(3H)-ones.

Sixteen title compounds have been synthesized and some of them have shown interesting results in antiinflammatory screening against carrageenin-induced inflammation. In addition, the compounds were found to be central nervous system depressants and relatively non-toxic.