Synthesis of 3 H, 13 C2 , 2 H414 C-SCH 430765 and 35 S-SCH 500946, potent and selective inhibitors of the NPY5 receptor.

SCH 430765 and SCH 500496 are potent and selective antagonists of the NPY5 receptor. NPY5 receptor antagonists have the potential for the treatment of obesity. [35 S]SCH 500946 was prepared for a competition binding assay which led to the identification of SCH 430765. Three distinct isotopically labelled forms of SCH 430765 were synthesized. [3 H]SCH 430765 was prepared for a preliminary absorption, distribution, metabolism and excretion data evaluation of the compound and [14 C]SCH 430765 for more definitive absorption, distribution, metabolism and excretion data work. In addition, [13 C2 ,2 H4 ]SCH 430765 was prepared as an internal standard for a LC-MS bioanalytical method. The paper discusses the synthesis of 3 isotopically labelled forms of SCH 430765 and [35 S]SCH 500946.

Synthesis of 3 H, 2 H4 , and 14 C-MK 3814 (preladenant).

MK 3814 is a potent and selective antagonist of the A2a receptor. A2a receptor antagonists have the potential for the treatment of Parkinson disease. Three distinct isotopically labelled forms of MK 3814 were synthesized. [3 H]MK 3814 was prepared for a preliminary absorption, distribution, metabolism, and excretion data (ADME) evaluation of the compound and [14 C]MK 3814 for more definitive ADME work, including an absorption, metabolism, and excretion study in man. In addition, [2 H4 ]MK 3814 was prepared as an internal standard for a liquid chromatography mass spectrometry bioanalytical method. This paper discusses the synthesis of 3 isotopically labelled forms of MK 3814.

Synthesis of (3) H, (2) H4 and (14) C-SCH 417690 (Vicriviroc).

Vicriviroc or SCH 417690 is a potent and selective antagonist of the CCR5 receptor. CCR5 receptor antagonists have the potential for the treatment of HIV infections. Four distinct isotopically labelled forms of SCH 417690 were synthesized. Low specific activity [(3) H]SCH 417690 was prepared for a preliminary absorption, distribution, metabolism and excretion evaluation of the compound and [(14) C]SCH 417690 for more definitive absorption, distribution, metabolism and excretion work, including an absorption, metabolism and excretion study in man. In addition, high specific activity [(3) H]SCH 417690 was prepared for CCR5 receptor binding work and [(2) H4 ]SCH 417690 was prepared as an internal standard for a liquid chromatography-mass spectrometry bioanalytical method. The paper discusses the synthesis of four isotopically labelled forms of SCH 417690.

Synthesis of 3H, 13C,2H3,15N and 14C-labelled SCH 466036, a histamine 3 receptor antagonist.

The synthesis of [(3)H]SCH 466036, [Me-(3)H3]SCH 466036, [(13)C,(2)H3,(15)N]SCH 466036 and [(14)C]SCH 466036 is described. [(3)H]SCH 466036 was prepared in two steps via Raney Ni-catalysed exchange with tritiated water. [Me-(3)H3]SCH 466036 was prepared in a single step from [(3)H]methyl iodide in 45% yield. [(13)C,(2)H3,(15)N]SCH 466036 was prepared in two steps from [(15)N]hydroxylamine and [(13)C,(2)H3]methyl iodide with an overall yield of 16%. [(14)C]SCH 466036 was prepared in seven steps from [(14)C]potassium cyanide in an overall yield of 13%.

SCH 503034, a mechanism-based inhibitor of hepatitis C virus NS3 protease, suppresses polyprotein maturation and enhances the antiviral activity of alpha interferon in replicon cells.

Cleavage of the hepatitis C virus (HCV) polyprotein by the viral NS3 protease releases functional viral proteins essential for viral replication. Recent studies by Foy and coworkers strongly suggest that NS3-mediated cleavage of host factors may abrogate cellular response to alpha interferon (IFN-alpha) (E. Foy, K. Li, R. Sumpter, Jr., Y.-M. Loo, C. L. Johnson, C. Wang, P. M. Fish, M. Yoneyama, T. Fujita, S. M. Lemon, and M. Gale, Jr., Proc. Natl. Acad. Sci. USA 102:2986-2991, 2005, and E. Foy, K. Li, C. Wang, R. Sumpter, Jr., M. Ikeda, S. M. Lemon, and M. Gale, Jr., Science 300:1145-1148, 2003). Blockage of NS3 protease activity therefore is expected to inhibit HCV replication by both direct suppression of viral protein production as well as by restoring host responsiveness to IFN. Using structure-assisted design, a ketoamide inhibitor, SCH 503034, was generated which demonstrated potent (overall inhibition constant, 14 nM) time-dependent inhibition of the NS3 protease in cell-free enzyme assays as well as robust in vitro activity in the HCV replicon system, as monitored by immunofluorescence and real-time PCR analysis. Continuous exposure of replicon-bearing cell lines to six times the 90% effective concentration of SCH 503034 for 15 days resulted in a greater than 4-log reduction in replicon RNA. The combination of SCH 503034 with IFN was more effective in suppressing replicon synthesis than either compound alone, supporting the suggestion of Foy and coworkers that combinations of IFN with protease inhibitors would lead to enhanced therapeutic efficacy.

Evernimicin binds exclusively to the 50S ribosomal subunit and inhibits translation in cell-free systems derived from both gram-positive and gram-negative bacteria.

Evernimicin (SCH 27899) is a new antibiotic with activity against a wide spectrum of gram-positive bacteria and activity against some gram-negative bacteria. Previous metabolic labeling studies indicated that evernimicin specifically inhibited protein synthesis in Staphylococcus aureus. Using a susceptible Escherichia coli strain, we demonstrated that evernimicin also inhibited protein synthesis in E. coli. In cell-free translation assays with extracts from either E. coli or S. aureus, evernimicin had a 50% inhibitory concentration of approximately 125 nM. In contrast, cell-free systems derived from wheat germ and rabbit reticulocytes were inhibited only by very high levels of evernimicin. Evernimicin did not promote transcript misreading. [(14)C]evernimicin specifically bound to the 50S subunit from E. coli. Nonlinear regression analysis of binding data generated with 70S ribosomes from E. coli and S. aureus and 50S subunits from E. coli returned dissociation constants of 84, 86, and 160 nM, respectively. In binding experiments, performed in the presence of excess quantities of a selection of antibiotics known to bind to the 50S subunit, only the structurally similar drug avilamycin blocked binding of [(14)C]evernimicin to ribosomes.

Binding of a thrombin receptor tethered ligand analogue to human platelet thrombin receptor.

A thrombin receptor-radioligand binding assay was developed using [3H]A(pF-F)R(ChA)(hR)Y-NH2 ([3H]haTRAP), a high affinity thrombin receptor-activating peptide (TRAP), and human platelet membranes. Scatchard analysis of saturation binding data indicated that [3H]haTRAP bound to platelet membranes with a Kd of 15 nM and a Bmax of 5.2 pmol/mg of protein. The binding was reduced by GPPNHP, a nonmetabolizable GTP analogue. Various TRAPs and a TRAP antagonist, but not other receptor agonists, displaced [3H]haTRAP from the binding sites. SFLLRN-NH2, a thrombin receptor-tethered ligand analogue, and [3H]haTRAP exhibited competitive binding for the same binding sites. The relative affinity of these peptides for the binding site paralleled their EC50 or IC50 values for platelet aggregation. These data indicate that [3H]haTRAP binds specifically and saturably to the functioning G protein-linked thrombin (tethered ligand) receptor in human platelet membranes.

A rapid method for isolating glandular trichomes.

A physical method is described for the rapid isolation of plant trichomes, with emphasis on stalked glandular types. The technique involved breaking frozen trichomes with powdered dry ice and collection of glandular heads by sieving from larger tissue fragments. This method was applied to several plants that bear similar stalked trichomes: geranium (Pelargonium), potato (Solanum tuberosum), tomato (Lycopersicon esculentum), squash (Cucurbita pepo), and velvetleaf (Abutilon theophrasti). The tissue preparation was of sufficient quality without further purification for biochemical and molecular studies. The preparation maintained the biochemical integrity of the trichomes for active enzymes and usable nucleic acids. A large quantity of tissue can be harvested; for example, 351 milligrams dry weight of glandular trichomes were harvested from geranium pedicels in 12 hours. The utility of the technique was demonstrated by examining the fatty acid composition of tall glandular trichomes of geraniums, Pelargonium xhortorum L.H. Bailey. These purified cells contained high concentrations of unusual omega5-unsaturated fatty acids, proportionally 23.4% of total fatty acids in the trichomes. When the trichomes were removed, the supporting tissue contained no omega5-fatty acids, thereby unequivocally localizing omega5-fatty acids to the trichomes. Because omega5-fatty acids are unique precursors for the biosynthesis of omega5-anacardic acids, we conclude that anacardic acid synthesis must occur in the glandular trichomes.

Comparison of anacardic acid biosynthetic capability between insect-resistant and-susceptible geraniums.

The garden geranium (Pelargonium xhortorum) has been shown to secrete anacardic acids in the form of a viscous sticky exudate from tall glandular trichomes, and this exudate provides a sticky trap defense against small pest species. The anacardic acids from genetically related pest-resistant and -susceptible plants have been characterized, and resistance has been shown to depend upon the presence ofω5 unsaturated anacardic acids. In this study, the biosynthesis of these anacardic acids was comparatively investigated by incubating [(14)C]methyl palmìtate, margarate, stearate, oleate and linoleate on floral buds of resistant and susceptible plants. In addition, the incorporation of [(14)C]valine, -isoleucine, and -leucine into anacardic acids was also studied. Nineteen anacardic acids were quantitated utilizing an improved HPLC technique. Fatty acids and, to a much lesser extent, amino acids were incorporated into anacardic acids. There are at least two pathways of biosynthesis operating: direct elongation, and ß-oxidation with reincorporation of the [(14)C]acetate, the latter being more prevalent in the resistant plant. The amino acids were processed into branched chain anacardic acids, isoleucine being the precursor of the anteiso compounds, and valine the iso branched ones. The major difference between resistant and susceptible plants was the ability of resistant plants, but not the susceptible plants, to synthesizeω5 unsaturated anacardic acids. Both types of plants were capable of directly incorporating(14)C-labeled fatty acid methy esters into anacardic acids regardless of the plant's normal anacardic acid composition, thus bypassing the plant's tightly controlled regulation of the chemical structures of anacardic acids. No evidence was found forω5 desaturation of saturated anacardic acids. A revised biosynthesis scheme is presented.

Regiospecific deuteriation and tritiation of various drugs using a homogeneous rhodium trichloride catalyst.

The deuteriation and tritiation of a number of drugs containing carboxyl, amide, aralkylamine, and anilide functional groups have been investigated using homogeneous rhodium trichloride as a catalyst. Good incorporation of deuterium was observed and the regiospecificity for ortho exchange was very high for most of the drugs studied. Similarly, with tritium, good incorporation (specific activities) and regiospecificities were achieved in many cases. Satisfactory results were also obtained from the small number of heterocyclic-containing drugs that were included in the present study.

Inhibition of lipoxygenase and prostaglandin endoperoxide synthase by anacardic acids.

C22:1 omega 5-anacardic acid was found to be a good inhibitor of both potato lipoxygenase and ovine prostaglandin endoperoxide synthase with approximate IC50's of 6 and 27 microM, respectively. Very similar inhibition was seen with the crude exudate, rich in omega 5-anacardic acids, from glandular trichomes of an arthropod-resistant strain of geranium, Pelargonium xhortorum. The saturated anacardic acid (C22:0 sat), abundant in the trichome exudate of susceptible strains, was nearly as inhibitory toward both prostaglandin endoperoxide synthase and lipoxygenase as the omega 5-unsaturated compound. However, the dimethyl derivative of C22:1 omega 5-anacardic acid was a poor inhibitor of prostaglandin endoperoxide synthase and caused only moderate (32%) inhibition of lipoxygenase even at 135 microM. The possible role of prostaglandin endoperoxide synthase and lipoxygenase inhibition in the enhanced pest resistance of geraniums which produce the omega 5-AnAs is discussed.

The fate of 4-cyanoacetanilide in rats and mice; mechanism of formation of a novel electrophilic metabolite.

1. The metabolic fate of 4-cyanoacetanilide (CAA), labelled with 14C and 13C in the N-acetyl group, was studied in rats (oral dose, 22.5 mg/kg) and mice (oral dose 21.7 mg/kg). 2. The metabolic profile in the urine of rats was compared with that obtained previously with 4-cyano-N,N-dimethylaniline (CDA) and confirms the intermediacy of CAA in the metabolism of CDA. 3. The precursor of a major metabolite of CDA and CAA (the mercapturic acid N-acetyl-S-[2-keto-2-(4-cyanoanilino)ethyl]cysteine, metabolite C) was identified in the urine of CAA-dosed rats as the O-sulphate conjugate of N-(4-cyanophenyl)glycolamide. 4. Pretreatment of rats with the sulphotransferase inhibitor pentachlorophenol reduced the yield of the mercapturic acid metabolite C, further indicating the intermediacy of a sulphate conjugate. 5. Metabolite C was not formed from CAA by mice; thus, this species difference, also observed with CDA, occurs at the level side-chain (acetyl) hydroxylation as well as at N-acetylation of 4-cyanoaniline as previously proposed. 6. The significance of this pathway as a bioactivation reaction of CDA, CAA and other acetanilides is discussed.

The fate of 4-cyano-N,N-dimethylaniline in mice: the occurrence of a novel metabolite during N-demethylation of an aromatic amine.

4-Cyano-N,N-dimethylaniline (CDA), when administered as a single oral dose to mice (18.5 mg/kg), was rapidly absorbed and eliminated. The major route of elimination was the urine (78% dose in 24h). The residues in the tissues 48 h after dosing, as microgram equiv. of CDA/g, were: liver, 0.19; kidney, 0.10; testes, 0.01; fat, 0.10; blood, 0.02. The major metabolite was 2-amino-5-cyanophenyl sulphate, with the N-methyl analogue as a minor metabolite. A novel metabolite, N-acetyl-S-(4-cyanoanilinomethyl)cysteine, was also a significant urinary metabolite, indicating that an electrophilic intermediate is generated during the N-demethylation of CDA. The implications are that N-demethylation may have important toxicological consequences.