D. Hesk

Evernimicin Binds Exclusively to the 50S Ribosomal Subunit and Inhibits Translation in Cell-Free Systems Derived from both Gram-Positive and Gram-Negative Bacteria

ABSTRACT 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 inStaphylococcus aureus. Using a susceptibleEscherichia coli strain, we demonstrated that evernimicin also inhibited protein synthesis in E. coli. In cell-free translation assays with extracts from either E. coli orS. 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. [14C]evernimicin specifically bound to the 50S subunit from E. coli. Nonlinear regression analysis of binding data generated with 70S ribosomes from E. coli andS. aureus and 50S subunits from E. colireturned 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 [14C]evernimicin to ribosomes.

Estimation of the Extent of Oral Absorption in Animals from Oral and Intravenous Pharmacokinetic Data in Drug Discovery

Oral administration is the most desirable route of drug delivery for systemically active drugs. Oral drugs must possess a certain level of oral bioavailability, which is a product of oral absorption and first-pass effect. Low oral bioavailability may be attributed to poor absorption and/or high first-pass hepatic elimination. In the lead optimization stage of drug discovery, if the relative contribution of oral absorption and metabolism could be discerned for poorly bioavailable compounds, a path forward for remedy would be possible. This report describes an approach utilizing oral/intravenous pharmacokinetic data to estimate oral absorption. The fraction of dose absorbed is calculated as the ratio of the actual bioavailable fraction to the maximum bioavailable fraction estimated from systemic clearance. An arbitrary classification was devised where low absorption encompasses compounds whose extent of absorption is <or=20%, moderate is for those between 21% and 69%, and high is for those that show >or=70% absorption. There was approximately 78% concordance in rats, 65% in monkeys and almost complete concordance in dogs. This approach correctly identified the cause for low oral bioavailability for 11 out of 13 compounds evaluated, and therefore it could be used prospectively with nonradiolabeled compounds during the lead optimization process.

Synthesis of 3H and 14C labelled SCH 48461

3 H-Sch 47949, racemic 3 H-Sch 48461, was prepared at a specific activity of 40 mCi/mmole by Pt catalysed exchange with tritiated water. 3 H-Sch 48461 was prepared at a specific activity of 64.6 Ci/mmole by a Pd/C catalysed reduction of an olefinic intermediate. 14 C-Sch 48461 was prepared in 8 steps from 14 C-potassium cyanide with an overall radiochemical yield of 18.5%.

Synthesis of 3H and 14C‐SCH 27899 by fermentation and evaluation of in vivo label stability

3H and 14 C-Sch 27899 have been prepared by fermentation using the Micromonospora carbonacea organism. In the case of 3 H-Sch 27899, the label was incorporated by a single addition of 100 mCi of 70Ci/mmole L-[3H-methyl]-methionine to two flasks. For 14 C-Sch 27899, 48 mCi of 55 mCi/mmole L-[14C-methyl]-methionine was added in five aliquots to five flasks over a five day period. Both batches were isolated by solvent extraction, oxidized and purified by column chromatography and hplc. An overall incorporation of 7.8% was found from L-[3H-methyl]-methionine and 18.7% from L-[14C-methyl]-methionine. The in vivo stability of label of 3 H and 14 C-Sch 27899 was determined, with 14 C-Sch 27899 found to be a better choice for use in in vivo metabolism studies.

Isolation of circulating metabolites in drug discovery using high-performance liquid chromatography, and their identification by liquid chromatography coupled with tandem mass spectrometry and nuclear magnetic resonance spectroscopy

One of the major components of modern drug discovery is the structural determination and the assessment of biological activity of plasma metabolites. LC-MS/MS has played a prominent role in the identification of metabolites; however, fragmentation patterns alone may not be sufficient for identification. Consequently, it may be necessary to isolate the metabolites for NMR or LC-NMR analysis. This report describes the isolation and identification of the major plasma metabolites of two lead compounds (SCH X and SCH Y). The major metabolite of SCH X in monkey plasma constituted 65% of total compound-derived materials. incubation of rat liver microsomes with SCH X gave the mono-hydroxylated metabolite found in monkey plasma; however, the yield was low. Incubations with microsomes from rats pre-treated with various cytochrome P450 inducers showed that the highest yield was obtained from pregnenolone 16a-carbonitrile (PCN)-induced animals. For SCH Y, two metabolites were found in bile and plasma of both rats and monkeys. Various in vitro systems did not produce amounts sufficient for isolation. Therefore, the metabolites of SCH X and SCH Y were isolated from PCN-induced rat liver microsomal incubation and rat bile, respectively. The chemical structures of the metabolites were unambiguously determined using LC-NMR analyses.

Synthesis of 3H-SCH 51048 and 14C-SCH 56592

3H-SCH 51048 and 14C-SCH 56592 have been synthesized. 3H-SCH 51048 was prepared in two steps by acid catalyzed tritium exchange while 14C-SCH 56592 was prepared in three steps from 14C-formamidine acetate in an overall 21% radiochemical yield.

Synthesis of 3H, 13C2, 2H4 14C-SCH 430765 and 35S-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 of3H,2H4, and14C-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.