Optimization of potency and pharmacokinetics of tricyclic indole derived inhibitors of HCV NS5B polymerase. Identification of ester prodrugs with improved oral pharmacokinetics.

HCV infections are the leading causes for hepatocellular carcinoma and liver transplantation in the United States. Recent advances in drug discovery have identified direct acting antivirals which have significantly improved cure rates in patients. Current efforts are directed towards identification of novel direct acting antiviral targeting different mechanism of actions which could become part of all oral therapies. We recently disclosed the identification of a novel tricyclic indole derived inhibitors of HCV NS5B polymerase that bound to the enzyme close to the active site. In this manuscript we describe further optimization of potency and pharmacokinetics (PK) of these inhibitors to identify compounds in low nM potency against gt-1b. These analogs also demonstrate excellent PK in rats and monkeys when administered as a dimethyl ethyl amino ester prodrug.

Tricyclic thienopyridine-pyrimidones/thienopyrimidine-pyrimidones as orally efficacious mGluR1 antagonists for neuropathic pain.

Introduction of small unsaturated alkylamino groups at the 4-position of the A-ring of the tricyclic framework (triazafluorenone) afforded extremely potent and selective mGluR1 antagonists with desirable properties. Compounds 11q and 11s are active in the SNL pain model with ED(50)s 3.3 and 6.4 mg/kg respectively. Metabolic outcome of propargyl amino moiety was studied.

Isolation, structural determination, synthesis and quantitative determination of impurities in Intron-A, leached from a silicone tubing.

Investigation of unexpected levels of impurities in Intron product has revealed the presence of low levels of impurities leached from the silicone tubing (Rehau RAU-SIK) on the Bosch filling line. In order to investigate the effect of these compounds (1a, 1b and 2) on humans, they were isolated identified and synthesized. They were extracted from the tubing by stirring in Intron placebo at room temperature for 72 h and were enriched on a reverse phase CHP-20P column, eluting with gradient aqueous ACN and were separated by HPLC. Structural elucidation of 1a, 1b and 2 by MS and NMR studies demonstrated them to be halogenated biphenyl carboxylic acids. The structures were confirmed by independent synthesis. Levels of extractable impurities in first filled vials of actual production are estimated to be in the range of 0.01-0.55 microg/vial for each leached impurity. Potential toxicity of these extractables does not represent a risk for patients under the conditions of clinical use.

Posaconazole (Noxafil, SCH 56592), a new azole antifungal drug, was a discovery based on the isolation and mass spectral characterization of a circulating metabolite of an earlier lead (SCH 51048).

Posaconazole (SCH 56592) is a novel triazole antifungal drug that is marketed in Europe and the United States under the trade name 'Noxafil' for prophylaxis against invasive fungal infections. SCH 56592 was discovered as a possible active metabolite of SCH 51048, an earlier lead. Initial studies have shown that serum concentrations determined by a microbiological assay were higher than those determined by HPLC from animals dosed with SCH 51048. Subsequently, several animals species were dosed with (3)H-SCH 51048 and the serum was analyzed for total radioactivity, SCH 51048 concentration and antifungal activity. The antifungal activity was higher than that expected based on SCH 51048 serum concentrations, confirming the presence of active metabolite(s). Metabolite profiling of serum samples at selected time intervals pinpointed the peak that was suspected to be the active metabolite. Consequently, (3)H-SCH 51048 was administered to a large group of mice, the serum was harvested and the metabolite was isolated by extraction and semipreparative HPLC. LC-MS/MS analysis suggested that the active metabolite is a secondary alcohol with the hydroxyl group in the aliphatic side chain of SCH 51048. All corresponding monohydroxylated diastereomeric mixtures were synthesized and characterized. The HPLC retention time and LC-MS/MS spectra of the diastereomeric secondary alcohols of SCH 51048 were similar to those of the isolated active metabolite. Finally, all corresponding individual monohydroxylated diasteriomers were synthesized and evaluated for in vitro and in vivo antifungal potencies, as well as pharmacokinetics. SCH 56592 emerged as the candidate with the best overall profile.

Ultra-performance liquid chromatography/tandem mass spectrometric determination of diastereomers of SCH 503034 in monkey plasma.

This paper describes the development and qualification of a fast, sensitive and specific ultra-performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method for the determination of diastereomers of SCH 503034 in monkey plasma. The analytical method involves direct protein precipitation with a mixture of methanol/acetonitrile (10/90) containing an internal standard, followed by separation of the stereoisomers on an Acquity UPLC C(18) column and detected by selected reaction monitoring (SRM) in positive ionization mode using atmospheric pressure chemical ionization (APCI). The effects of ion-pairing agents on separation and ionization efficiency were investigated. The two diastereomers were well separated (R=1.3) with a runtime of 5 min under an isocratic condition. The method was qualified. The linear concentration range was 1-2500 ng/ml for the both stereoisomers. Inter-assay mean bias and relative standard deviation (R.S.D.) were in the range of -1.2% to 3.6% and 2.8-10%, respectively. Intra-assay mean bias and R.S.D. were in the range of -1.3% to 5.5% and 2.3-7.8%, respectively. Recoveries of the stereoisomers at concentration levels of 2.5, 50 and 1000 ng/ml were 87.2-90.0%, 89.1-90.4% and 92.3-94.3%, respectively. The LLOQ for this assay was 1 ng/ml. No matrix interferences were observed in six different sources of blank monkey plasma.

Posaconazole is a potent inhibitor of sterol 14alpha-demethylation in yeasts and molds.

Posaconazole (POS; SCH 56592) is a novel triazole that is active against a wide variety of fungi, including fluconazole-resistant Candida albicans isolates and fungi that are inherently less susceptible to approved azoles, such as Candida glabrata. In this study, we compared the effects of POS, itraconazole (ITZ), fluconazole (FLZ), and voriconazole (VOR) on sterol biosynthesis in strains of C. albicans (both azole-sensitive and azole-resistant strains), C. glabrata, Aspergillus fumigatus, and Aspergillus flavus. Following exposure to azoles, nonsaponifiable sterols were extracted and resolved by liquid chromatography and sterol identity was confirmed by mass spectroscopy. Ergosterol was the major sterol in all but one of the strains; C. glabrata strain C110 synthesized an unusual sterol in place of ergosterol. Exposure to POS led to a decrease in the total sterol content of all the strains tested. The decrease was accompanied by the accumulation of 14alpha-methylated sterols, supporting the contention that POS inhibits the cytochrome P450 14alpha-demethylase enzyme. The degree of sterol inhibition was dependent on both dose and the susceptibility of the strain tested. POS retained activity against C. albicans isolates with mutated forms of the 14alpha-demethylase that rendered these strains resistant to FLZ, ITZ, and VOR. In addition, POS was a more potent inhibitor of sterol synthesis in A. fumigatus and A. flavus than either ITZ or VOR.

Microwave enhanced Akabori reaction for peptide analysis.

The Akabori reaction, devised in 1952 for the identification of C-terminus amino acids, involves the heating of a linear peptide in the presence of anhydrous hydrazine in a sealed tube for several hours. We report here a modified Akabori reaction that rapidly identifies the C-terminus amino acid in a polypeptide including its amino acid sequence information at both the C-terminus and the N-terminus. This modified methodology demonstrates the fundamentals of microwave chemistry applied to bioanalytical problems. In this modified process, hydrazinolysis has been accelerated by the application of microwave irradiation. In our reaction, the linear peptide and hydrazine solution, contained in a loosely covered conical flask, was exposed to a few minutes of irradiation using an unmodified domestic microwave oven. While the classical Akabori reaction required several hours, the microwave assisted reaction takes just minutes. If dimethyl sulfoxide is added to dilute the reaction mixture, the process is retarded enough to allow aliquots of the reaction mixture to be drawn every few minutes over a period of about an hour in order to study the progress of hydrazinolysis. Reaction products were monitored by mass spectrometry-primarily FAB-MS. In addition to providing sequence information, the microwave enhanced Akabori reaction quickly detects the presence of arginine (Arg) by converting each Arg to ornithine (Orn). Furthermore, certain amino acids, containing beta-SH, CO2H, and CONH2 groups in their side chain, are susceptible to modification by hydrazine, thereby, providing rapid confirmation of the presence of these amino acid residues. In these preliminary studies, the following oligopeptides were analyzed to demonstrate the effectiveness of our approach; the dipeptide (Trp-Phe), the tripeptide (Tyr-Gly-Gly), the tetrapeptide (Pro-Phe-Gly-Lys), the heptapeptide (Ala-Pro-Arg-Leu-Arg-Phe-Tyr), and a N-terminal blocked tripeptide (N-acetyl-Met-Leu-Phe).

Mechanism of azole antifungal activity as determined by liquid chromatographic/mass spectrometric monitoring of ergosterol biosynthesis.

A liquid chromatography/mass spectrometry (LC/MS) method for separation and characterization of ergosterol biosynthetic precursors was developed to study the effect of Posaconazole on sterol biosynthesis in fungi. Ergosterol biosynthetic precursors were characterized from their electron ionization mass spectra acquired by a normal-phase chromatography, particle beam LC/MS method. Fragment ions resulting from cleavage across the D-ring and an abundant M - 15 fragment ion were diagnostic for methyl substitution at C-4 and C-14. Comparison of the sterol profile in control and treated Candida albicans incubations showed depletion of ergosterol and accumulation of C-4 and C-14 methyl-substituted sterols following treatment with Posaconazole. These C-4 and C-14 methyl sterols are known to be incapable of sustaining cell growth. The results demonstrate that Posaconazole exerts its antifungal activity by inhibition of ergosterol biosynthesis. Furthermore, Posaconazole appears to disrupt ergosterol biosynthesis by inhibition of lanosterol 14alpha-demethylase.