Anti-inflammatory constituents of the red alga Gracilaria verrucosa and their synthetic analogues.

A chemical study on the anti-inflammatory components of the red alga Gracilaria verrucosa led to the isolation of new 11-deoxyprostaglandins ( 1- 4), a ceramide ( 5), and a C 16 keto fatty acid ( 6), along with known oxygenated fatty acids ( 7- 14). Their structures were elucidated on the basis of NMR and MS data. The absolute configurations of compounds 1- 5 were determined by Mosher's method. The anti-inflammatory activity of the isolated compounds ( 1- 14) was evaluated by determining their inhibitory effects on the production of pro-inflammatory mediators (NO, IL-6, and TNF-alpha) in lipopolysaccharide (LPS)-activated RAW 264.7 murine macrophage cells. Compounds 9 and 10 exhibited the most potent activity. In the evaluation of these two compounds and derivatized analogues ( 15- 40), the anti-inflammatory activity was enhanced in some synthetic analogues. These enone fatty acids were investigated as potential anti-inflammatory leads for the first time.

Total synthesis of 17-isoLevuglandin E4 and the structure of C22-PGF4alpha.

The isolevuglandin 17-isoLGE4 (10-acetyl-11-formyl-14-hydroxynonadeca-4(Z),7(Z),12(E),16(Z)-tetr aenoic acid) is a levulinaldehyde derivative that is expected to be generated during the free radical-induced oxidation of docosahexaenoic acid. A total synthesis was executed to facilitate detection and identification of 17-isoLGE4 in biological samples. Conjugate addition of a higher order vinyl cyanocuprate to a gamma-alkoxy enone achieved the final carbon-carbon bond formation to complete a convergent elaboration of the 17-isoLGE4 carbon skeleton. Attempted construction of the requisite vinyl nucleophile synthon using hydrostannylation of an alkyne was foiled by tri-n-butylstannyl radical-promoted isomerization of a cis to a trans double bond. Derivatization of 17-isoLGE4 with methoxylamine under anhydrous or wet conditions generated bismethoximes of 17-isoLGE4 or the isomerized delta11-17-isoLGE4 respectively. Analysis of the mass spectrum of a bismethoxime-pentafluorobenzyl ester-trimethylsilyl ether derivative of 17-isoLGE4 provided presumptive evidence that an incorrect structure was proposed earlier for C22-PGF4alpha, the only F4-isoprostane which is produced enzymatically. We conclude that the 22-carbon analogue of PGF2alpha, produced from docosahexaenoic acid by a cyclooxygenase from trout gill, does not have the same side chains as 17-isoLGE4. Furthermore, we now propose that mass spectral data reported for "C22-PGF4alpha" support a 14-PGF4alpha structure rather than the 17-PGF4alpha structure suggested previously.

Characterization of the lysyl adducts formed from prostaglandin H2 via the levuglandin pathway.

Prostaglandin H(2) has been demonstrated to rearrange to gamma-ketoaldehyde prostanoids termed levuglandins E(2) and D(2). As gamma-dicarbonyl molecules, the levuglandins react readily with amines. We sought to characterize the adducts formed by synthetic levuglandin E(2) and prostaglandin H(2)-derived levuglandins with lysine. Using liquid chromatography/electrospray mass spectrometry, we found that the reaction predominantly produces lysyl-levuglandin Schiff base adducts that readily dehydrate to form lysyl-anhydrolevuglandin Schiff base adducts. These adducts were characterized by examination of their mass spectra, by analysis of the products of their reaction with sodium cyanide, sodium borohydride, and methoxylamine and by the mass spectra derived from collision-induced dissociation in tandem mass spectrometry. The Schiff base adducts also are formed on peptide-bound lysyl residues. In addition, synthetic levuglandin E(2) and prostaglandin H(2)-derived levuglandins produced pyrrole-derived lactam and hydroxylactam adducts upon reaction with lysine as determined by tandem mass spectrometry. A marked time dependence in the formation of these adducts was observed: Schiff base adducts formed very rapidly and robustly, whereas the lactam and hydroxylactam adducts formed more slowly but accumulated throughout the time of the experiment. These findings provide a basis for investigating protein modification induced by oxygenation of arachidonic acid by the cyclooxygenases.

Oxidation of low-density lipoproteins produces levuglandin-protein adducts.

Free-radical oxidation of human low-density lipoprotein (LDL) produces levuglandin (LG)-protein adducts that were detected with an enzyme-linked immunosorbent assay using LGE2-KLH antibodies which recognize LGE2-derived pyrroles. The level of immunoreactivity increases with time of oxidation and reaches a maximum by 8 h. The yield of pyrrole varies nonlinearly with the level of LG adduction to LDL. At low LG:LDL ratios, such as those detected in oxidized LDL, the reaction of primary amino groups with LGE2 produces mostly non-pyrrole adducts that are not immunoreactive. Concomitant phospholipolysis must occur if the generation of immunoreactive epitopes in LDL involves oxidation of arachidonyl phospholipids. Thus, since a protein adduct prepared from synthetic LGE2-2-lysophosphatidylcholine ester showed, at most, only 0.5% cross-reactivity with the LGE2-KLH antibodies, the epitopes detected in oxidized LDL are almost certainly not protein adducts of LG-phospholipid esters. As expected, hydrolysis of the carboxylic ester in the protein adduct of LGE2-2-lysophosphatidylcholine ester by treatment with phospholipase A2 produced a fully immunoreactive LGE2-protein adduct.

Biosynthesis of prostaglandins from 17(18)epoxy-eicosatetraenoic acid, a cytochrome P-450 metabolite of eicosapentaenoic acid.

Eicosapentaenoic acid (20:5(n - 3)) is oxygenated to 17S(18R)epoxyeicosatetraenoic acid (EpETE) by microsomes of monkey seminal vesicles, which also are rich in prostaglandin (PG) H synthase. The metabolism of racemic [14C]17(18)EpETE by PGH synthase of sheep vesicular glands was investigated in the present report. The two main metabolites were identified by GC-MS as 17(18)epoxyprostagland E2 (17(18)EpPGE2) and 17(18)EpPGF2 alpha. The structures were confirmed by chemical synthesis of these prostaglandins from PGE3. 17(18)EpPGE1 was synthesized from 17,18-dehydro-PGE1 by the same method. Alkali treatment of 17(18)EpPGE2 yielded 17(18)EpPGB2, which could be resolved by RP-HPLC into the 17R(18S) and 17S(18R) stereoisomers. The 17S(18R) stereoisomer was identified by co-chromatography with [14C]17S(18R)EpPGB2, which was formed by PGH synthase from biosynthetic [14C]17S(18R)EpETE. The 17(18)epoxyprostaglandins were found to be relatively unstable during acidic extractive isolation. 17(18)EpPGE1 and 17(18)EpPGE2 could not be detected in seminal vesicles of the cynomolgus monkey in significant amounts relative to 19-hydroxy-PGE1. Nevertheless, biosynthesis of 17(18)epoxyprostaglandins should be considered when the biological effects of 17S(18R)EpETE are investigated.

[Total synthesis and properties of prostaglandins. XXXI. Synthesis of pyranylated and glycosidated omega-hydroxyalkyl ethers of 11- deoxyprostaglandin E1]. / Total'nyi sintez i svoistva prostaglandinov. XXXI. Sintez piranilirvannykh i glikozidirovannykh omega-gidroksialkilovykh éfirov 11-dezoksiprostaglandina E1.

Four methods of the synthesis of model glycosides with 11-deoxyprostaglandin E1 and a connecting polymethylene chain as the aglycone are compared. Interaction of potassium salt of prostaglandin PG with omega-iodoalkylglycosides is the most promising approach.

Prostaglandin endoperoxides 21. Covalent binding of levuglandin E2 with proteins.

Levuglandin E2 (LGE2), a gamma-ketoaldehyde produced by rearrangement of the prostaglandin endoperoxide PGH2 under the aqueous conditions of its biosynthesis, binds covalently with ram seminal vesicle microsomes. Totally synthetic 5,6-ditritio-LGE2 was prepared and used to determine that rapid covalent binding of LGE2 (initially 800 microM) occurs with 6.4 microM bovine serum albumin (greater than 10 equiv within 1 min) which approaches saturation (approximately 16 equiv) after 40 min at 37 degrees C.

Hormone-responsive cells derived from human dental papilla: characterization in vitro and in vivo in diffusion chambers.

Cells of the dental papilla are capable of odontoblastic, fibroblastic, and endothelial differentiation and formation of dentin and the dental pulp. In the present study dental papilla cells, obtained from human tooth buds (HDP cells), were cultured in vitro through 3 to 7 passages. After exposure to prostaglandin E2 there was a marked decrease in intracellular cyclic AMP (cAMP) levels as compared to hormone-free controls. Parathyroid hormone and calcitonin had stimulatory effects with 1 and 2 log increases in cAMP, respectively. The HDP cells showed moderate activity of alkaline phosphatase, 1 log higher than that of hamster kidney fibroblasts (BHK 13) and 1 log lower than that of osteoblastic osteosarcoma cells (ROS 17/2). When cultured for 4 or 8 wk in diffusion chambers (DC) implanted in athymic mice, many of the HDP cells underwent odontoblastic morphodifferentiation with very long, single processes extending into the matrix. This matrix contained banded and unbanded collagen fibers. Neither light nor electron microscopy of the DC content revealed mineral deposits. These results suggest that HDP cells have an intrinsic potential for partial odontoblastic differentiation; inductive signals like those originating from odontogenic epithelium are probably essential for the completion of hard tissue formation.

Synthesis of macrolide prostaglandin analogs.

Prostaglandin analogs of the E- and F2 alpha-functional type, which are constrained to conformations in which the side-chains are close in space and specifically aligned in the terminal portions by covalent bonding, have been synthesized. These analogs are 1, (omega-1)-macrolides. The syntheses proceeded from aldehyde intermediate I via the Emmon's condensation with dimethyl n-(dimethyl-t-butylsilyloxy)2-oxoalkylphosphonate anions (II a or b). The macrolide closures were performed using 2, 2'-dipyridyl disulfide. For the synthesis of 9-ketoprostaglandin macrolides, a free 9-hydroxy is available for oxidation after macrolide closure, so long as the 9-position is protected as the acetate rather than benzoate. Chiroptical data revealed that the conformations of the macrolide prostaglandins are unchanged (relative to the natural unconstrained prostaglandins) in the vicinity of the five-membered ring and the allyl alcohol unit by the formation of the macrolide linkage.

Synthesis and chiroptical characterization of prostacyclin diastereomers.

In the mercuri- and halo-cyclizations of PGF2 alpha methyl ester and its 11,15-bis(alpha- ethoxyethyl)-ether (or other protected forms) the exo-PGI1 derivative predominates independent of reagent and degree of protection of the PGF2 alpha sample used. Diastereomerically pure samples of exo- and endo-PGI1 and prostacycline (PGI2) were prepared. PGI0 epimers were prepared: catalytic hydrogenation of PGI2 Me ester provides exclusively the endo isomer. PGI2 methyl ester was found to be stable to extensive chromatography on silica, and to storage for at least a year in anhydrous ethanol at -20 degrees C. At pH 7.4 in 2:1 H2O:EtOH, the ester has a half-life in excess of 5 hr at 25 degrees C. A reproducible small scale (0.4-3 mg) synthesis of prostacyclin uses a modification of Whittaker's iodocyclization followed by DBN treatment. This procedure, developed with 15-3H-PGF2 alpha, proved widely applicable to PGF2 alpha analogs and diastereomers. The following prostacyclins (in the Me ester and Na salt forms) bearing the 5-en-6-yl ether unit were prepared in this way: ent-PGI2, rac-PGI2, 15-epi-PGI2, ent-15-epi-PGI2, 11-epi-PGI2, 8,9,12-epi-PGI2, E-PGI2, 13,14-dihydro-PGI2, and 13,14-dihydro-15-epi-PGI2. NMR comparisons for the methyl esters reveal that of the resonances (H-5,9,UU, 15) that appear at delta 4.0 +/- 0.6 ppm, the most deshielded is H-9 so long as the 5.6-olefin is Z. The 8R,9S-6,9-oxido-Z-5,6-ene unit is most readily characterized by its strong positive dichroic absorption at 210-230 nm. CD spectroscopy not only serves to confirm the presence of this unit in analogs, but also can be used for quantitative analysis of PGI2 solutions and for monitoring the rate of hydrolytic cleavage of these enol ethers.

Hydrolysis of an orally active platelet inhibitory prostanoid amide in the plasma of several species.

The prostanoid 3-oxa-4,5,6-trinor-3,7-inter-m-phenylene-PGE1-amide (OI-PGE1-amide) has a prolonged duration of oral platelet aggregation inhibitory activity when compared to the parent free acid (OI-PGE1) in the rat. When incubated in rat plasma at 1 microgram/ml for 30 seconds prior to addition of ADP, OI-PGE1-amide inhibits in vitro rat platelet aggregation approximately 50%. OI-PGE1 inhibits at 1 ng/ml. Inhibition of platelet aggregation by plasma incubated with OI-PGE1-amide (1 microgram/ml) increases with time and the rate of this increase differs with species. Incubation of OI-PGE1 in plasma does not result in an increase of platelet inhibitory activity with time. The increase of platelet inhibitory activity was assumed to indicate hydrolysis of OI-PGE1-amide to the more active OI-PGE1. A compound, different from OI-PGE1-amide, was isolated by an ion exchange/silica gel separation sequence from an incubation of OI-PGE1-amide in rat plasma. It had potent platelet aggregation inhibitory activity. This material was shown to be OI-PGE1 by thin-layer chromatography, gas chromatography and mass spectral analysis. Studies with [3H]-OI-PGE1-amide confirmed the formation of OI-PGE1 in plasma incubations. Amide hydrolytic activity was significantly different between species, the rank order being: rat greater than guine pig greater than monkey = human greater than dog. This relationship corresponded with that determined by measuring the increase in platelet inhibitory activity with time in plasma incubations of OI-PGE1-amide reported above. Present data indicate that (a) OI-PGE1-amide is hydrolyzed to the parent acid by plasma enzymes of several species and (b) hydrolytic activity of plasma varies widely between species.

Synthesis of the sultam analog of 11-deoxy PGE2.

As an extension of our work on the series of N-alkylmethanesulfonamidoheptanoic acids, we have prepared the cyclic-sulfonamide (sultam) analog of 11-deoxy PGE2. Although numerous publications have described the introduction of heteroatoms into the 5-membered ring of the prostaglandins, the cyclic-sulfonamides have remained a heretofore unexplored class. The synthetic scheme for the preparation of this unique PG analog is presented in this paper.

Prostaglandins and congeners. 14. Synthesis and bronchodilator activity of dl-16,16-trimethyleneprostaglandins.

The interesting bronchodilator activity of novel dl-16,16-trimethyleneprostaglandin congeners and their preparation via the conjugate addition of the appropriate vinyl lithiocuprate reagent to several cyclopentenones are described. Also discussed is the preparation of the key intermediate vinyl iodine 7.

Synthesis of diastereomeric bis-unsaturated prostaglandins.

With the report given herein all diastereomers of PGF2, PGE2, and PGD2 which bear the naturally recognized 15-S hydroxylated center, whether in the natural or entprostanoic acid skeleton, have been prepared by a route involving initial introduction of the carboxyl (alpha) chain (1). A major advantage of the initial alpha-ylation route is the facile reduction of the 13, 14-en-15-one system with methanolic NaBH4 which proceeds without competing 1,4-reduction. The products are thus free of 13,14-dihydro-PG2 contaminants (2). The initial pharmacological evaluation of these diastereomers will be submitted for publication in this journal (3).

Prostaglandins and congeners XIII (1). The synthesis of dl-erythro-16-methoxyprostaglandins.

dl-Erythro-16-methoxy-PGE2, PGA2, PGF2alpha, 11-deoxy PGE1, and 11-deoxy PGF1alpha have been prepared via the cuprate conjugate addition procedure. These congeners are less potent than the parent prostaglandins as stimulators of isolated gerbil colon contractions and as bronchodilators in the guinea pig Konzett assay.