Pyrazolopyrimidinones, a novel class of copper-dependent bactericidal antibiotics against multi-drug resistant S. aureus.

The treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections poses a therapeutic challenge as even last resort drugs become increasingly ineffective. As the demand for antibiotics with novel modes of action is growing, new approaches are needed to probe a greater spectrum of antimicrobial activities for their potential efficacy against drug-resistant pathogens. The use of copper (Cu) by the innate immune system to mount an antimicrobial response against bacterial invaders has created an opportunity to explore a role for Cu in antimicrobial therapy. Here we describe pyrazolopyrimidinones (PZP) as novel copper-dependent inhibitors (CDI) of S. aureus. 5-Benzyl-3-(4-chlorophenyl)-2-methyl-4H,7H-pyrazolo[1,5-a]pyrimidin-7-one (PZP-915) showed potent bactericidal properties at sub-micromolar concentrations and activity against clinical MRSA isolates and biofilms cultures. This cupricidal activity is founded on the molecule's ability to coordinate Cu and induce accumulation of Cu ions inside S. aureus cells. We demonstrate that exposure to 915 + Cu led to an almost instantaneous collapse of the membrane potential which was accompanied by a complete depletion of cellular ATP, loss of cell-associated K+, a substantial gain of cell associated Na+, and an inability to control the influx of protons in slightly acidic medium, while the integrity of the cell membrane remained intact. These findings highlight PZP-915 as a novel membrane-directed metalloantibiotic against S. aureus that is likely to target a multiplicity of membrane associated protein functions rather than imposing physical damage to the membrane structure.

Design and Synthesis of Nonpeptide Inhibitors of Hepatocyte Growth Factor Activation.

In this letter we report first nonpeptide inhibitors of hepatocyte growth factor (HGF) activation. These compounds inhibit the three proteases (matriptase, hepsin, and HGF activator) required for HGF maturation. We show that 6, 8a, 8b, and 8d block activation of fibroblast-derived pro-HGF, thus preventing fibroblast-induced scattering of DU145 prostate cancer cells. Compound 6 (SRI 31215) is very soluble (91 µM) and has excellent microsome stability (human t 1/2 = 162 min; mouse t 1/2 = 296 min). In mouse 6 has an in vivo t 1/2 = 5.8 h following IV administration. The high solubility of 6 and IV t 1/2 make this compound a suitable prototype "triplex inhibitor" for the study of the inhibition of HGF activation in vivo.

Discovery of a novel class of potent HCV NS4B inhibitors: SAR studies on piperazinone derivatives.

HTS screening identified compound 2a (piperazinone derivative) as a low micromolar HCV genotype 1 (GT-1) inhibitor. Resistance mapping studies suggested that this piperazinone chemotype targets the HCV nonstructural protein NS4B. Extensive SAR studies were performed around 2a and the amide function and the C-3/C-6 cis stereochemistry of the piperazinone core were essential for HCV activity. A 10-fold increase in GT-1 potency was observed when the chiral phenylcyclopropyl amide side chain of 2a was replaced with p-fluorophenylisoxazole-carbonyl moiety (67). Replacing the C-6 nonpolar hydrophobic moiety of 67 with a phenyl moiety (95) did not diminish the GT-1 potency. A heterocyclic thiophene moiety (103) and an isoxazole moiety (108) were incorporated as isosteric replacements for the C-6 phenyl moiety (95), resulting in significant improvement in GT-1b and 1a potency. However, the piperazonone class of compounds lacks GT-2 activity and, consequently, were not pursued further into development.

ß-D-2'-α-F-2'-ß-C-Methyl-6-O-substituted 3',5'-cyclic phosphate nucleotide prodrugs as inhibitors of hepatitis C virus replication: a structure-activity relationship study.

The 3',5'-cyclic phosphate prodrug 9-[ß-d-2'-deoxy-2'-α-fluoro-2'-ß-C-methylribofuranosyl]-2-amino-6-ethoxypurine, PSI-352938 1, has demonstrated promising anti-HCV efficacy in vitro and in human clinical trials. A structure-activity relationship study of the nucleoside 3',5'-cyclic phosphate series of ß-d-2'-deoxy-2'-α-fluoro-2'-ß-C-methylribofuranosyl nucleoside prodrugs was undertaken and the anti-HCV activity and in vitro safety profile were assessed. Cycloalkyl 3',5'-cyclic phosphate prodrugs were shown to be significantly more potent as inhibitors of HCV replication than branched and straight chain alkyl 3',5'-cyclic phosphate prodrugs. No cytotoxicity and mitochondrial toxicity for prodrugs 12, 13 and 19 were observed at concentrations up to 100 µm in vitro. Cycloalkyl esters of 3',5'-cyclic phosphate nucleotide prodrugs demonstrated the ability to produce high levels of active triphosphate in clone-A cells and primary human hepatocytes. Compounds 12, 13 and 19 also demonstrated the ability to effectively deliver in vivo high levels of active nucleoside phosphates to rat liver.

Rapid differentiation of nucleotide phosphoramidate diastereomers by electrospray ionization tandem mass spectrometry.

RATIONALE: Nucleotide phosphoramidates are prodrugs which effectively deliver the active nucleotide to target tissues. It was shown that the individual phosphoramidate diastereomers have different antiviral activity, although the active nucleotide is the same. Therefore, a fast and simple analytical method is needed to characterize the individual diastereomeric phosphoramidate prodrugs. METHODS: Stock solutions of diastereomeric nucleotide phosphoramidate prodrugs, i.e., 5'-phosphate derivatives of the ß-D-2'-deoxy-2'-α-fluoro-2'-ß-C-methyluridine nucleotide, were made in 25% acetonitrile to achieve a final concentration of 10 µg/mL. The samples were studied using high-performance liquid chromatography (HPLC) coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS). RESULTS: The MS/MS spectra of diastereomeric pairs showed substantial differences in the relative abundances of a characteristic ion in negative mode, which is proposed to be a cyclic phosphoramidate ion. Results were confirmed by the MS/MS spectrum of an analog without the NH proton and deuterium exchange experiment. Furthermore, the diastereomer-specific fragmentation behavior in negative ESI-MS was used to characterize a series of nucleotide phosphoramidates with different amino acid and aromatic substituents. CONCLUSIONS: An HPLC/MS/MS method was developed for the differentiation of the diastereomers of phosphoramidate prodrugs. In negative mode MS/MS spectra, the cyclic phosphoramidate ions yielded unambiguous distinction. This method presented a rapid and simple way for the characterization of nucleotide phosphoramidates.

Phosphoramidate prodrugs of (-)-ß-D-(2R,4R)-dioxolane-thymine (DOT) as potent anti-HIV agents.

BACKGROUND: Nucleoside reverse transcriptase inhibitors (NRTIs) are an effective class of agents that has played a vital role in the treatment of HIV infections. (-)-ß-D-(2R,4R)-dioxolane-thymine (DOT) is a thymidine analogue that is active against wild-type and NRTI-resistant HIV-1 mutants. It has been shown that the anti-HIV activity of DOT is limited due to poor monophosphorylation. METHODS: To further enhance the anti-HIV activity of DOT, an extensive structure-activity relationship analysis of phosphoramidate prodrugs of DOT monophosphate was undertaken. These prodrugs were evaluated for anti-HIV activity using Hela CD4 ß-gal reporter cells (P4-CCR5 luc cells). RESULTS: Among the synthesized prodrugs, the 4-bromophenyl benzyloxy l-alanyl phosphate derivative of DOT was the most potent, with a 50% effective concentration of 0.089 µM corresponding to a 75-fold increase in activity relative to the parent nucleoside DOT with no increased cytotoxicity. The metabolic stability of a selected number of potent DOT phosphoramidates was also evaluated in simulated gastric fluid, simulated intestinal fluid, human plasma and liver S9 fractions. CONCLUSIONS: A series of new phosphoramidate prodrugs of DOT were prepared and evaluated as inhibitors of HIV replication in vitro. Metabolic stability studies indicated that these DOT phosphoramidate derivatives have the potential to show acceptable stability in the gastrointestinal tract, but they metabolize rapidly in the liver.

A liquid chromatography-tandem mass spectrometry method for the quantitative determination of diastereomers of a phosphoramidate nucleotide prodrug (PSI-7851) in human plasma.

A rapid and stereospecific method using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for the separation and determination of PSI-7851 diastereomers in human K2EDTA plasma has been developed. The analytical method involves direct protein precipitation with acetonitrile, followed by separation of the diastereomers on a Luna C18 column, positive mode electrospray ionization and selected reaction monitoring mode mass spectrometry detection. The mobile phase composition and pH were investigated for the resolution of the two diastereomers of PSI-7851. The optimized method showed good resolution (R(s) = 4.8) within short analysis time (approximately 8 min). The assay range was 5-2500 ng/mL for both diastereomers using a 1/x² weighted linear regression analysis for standard curve fitting. Replicate sample analysis indicated that intra- and inter-day accuracy and precision were within ±15.0%. The recovery of diastereomers from human plasma was greater than 85% and no significant matrix effect was observed. The method was demonstrated to be sensitive, selective and robust, and was successfully used to support clinical studies.

Synthesis of stable isotope labeled analogs of the anti-hepatitis C virus nucleotide prodrugs PSI-7977 and PSI-352938.

In order to support bioanalytical LC/MS method development and plasma sample analysis in preclinical and clinical studies of the anti-hepatitis C-virus nucleotides, PSI-7977 and PSI-352938, the corresponding stable isotope labeled forms were prepared. These labeled compounds were prepared by addition reaction of the freshly prepared Grignard reagent (13)CD(3)MgI to the corresponding 2 '-ketone nucleosides followed by fluorination of the resulting carbinol with DAST. As expected, these 2 '-C-(trideuterated-(13)C-methyl) nucleotide prodrugs showed similar anti-HCV activity to that of the corresponding unlabeled ones.

Discovery of PSI-353661, a Novel Purine Nucleotide Prodrug for the Treatment of HCV Infection.

Hepatitis C virus afflicts approximately 180 million people worldwide, and the development of direct acting antivirals may offer substantial benefit compared to the current standard of care. Accordingly, prodrugs of 2'-deoxy-2'-fluoro-2'-C-methylguanosine monophosphate analogues were prepared and evaluated for their anti-HCV efficacy and tolerability. These prodrugs demonstrated >1000 fold greater potency than the parent nucleoside in a cell-based replicon assay as a result of higher intracellular triphosphate levels. Further optimization led to the discovery of the clinical candidate PSI-353661, which has demonstrated strong in vitro inhibition against HCV without cytotoxicity and equipotent activity against both the wild type and the known S282T nucleoside/tide resistant replicon. PSI-353661 is currently in preclinical development for the treatment of HCV.

2'-deoxy-2'-α-fluoro-2'-ß-C-methyl 3',5'-cyclic phosphate nucleotide prodrug analogs as inhibitors of HCV NS5B polymerase: discovery of PSI-352938.

A series of novel 2'-deoxy-2'-α-fluoro-2'-ß-C-methyl 3',5'-cyclic phosphate nucleotide prodrug analogs were synthesized and evaluated for their in vitro anti-HCV activity and safety. These prodrugs demonstrated a 10-100-fold greater potency than the parent nucleoside in a cell-based replicon assay due to higher cellular triphosphate levels. Our structure-activity relationship (SAR) studies provided compounds that gave high levels of active triphosphate in rat liver when administered orally to rats. These studies ultimately led to the selection of the clinical development candidate 24a (PSI-352938).

Discovery of a ß-d-2'-deoxy-2'-α-fluoro-2'-ß-C-methyluridine nucleotide prodrug (PSI-7977) for the treatment of hepatitis C virus.

Hepatitis C virus (HCV) is a global health problem requiring novel approaches for effective treatment of this disease. The HCV NS5B polymerase has been demonstrated to be a viable target for the development of HCV therapies. ß-d-2'-Deoxy-2'-α-fluoro-2'-ß-C-methyl nucleosides are selective inhibitors of the HCV NS5B polymerase and have demonstrated potent activity in the clinic. Phosphoramidate prodrugs of the 5'-phosphate derivative of the ß-d-2'-deoxy-2'-α-fluoro-2'-ß-C-methyluridine nucleoside were prepared and showed significant potency in the HCV subgenomic replicon assay (<1 µM) and produced high levels of triphosphate 6 in primary hepatocytes and in the livers of rats, dogs, and monkeys when administered in vivo. The single diastereomer 51 of diastereomeric mixture 14 was crystallized, and an X-ray structure was determined establishing the phosphoramidate stereochemistry as Sp, thus correlating for the first time the stereochemistry of a phosphoramidate prodrug with biological activity. 51 (PSI-7977) was selected as a clinical development candidate.

Mechanism of activation of PSI-7851 and its diastereoisomer PSI-7977.

A phosphoramidate prodrug of 2'-deoxy-2'-α-fluoro-ß-C-methyluridine-5'-monophosphate, PSI-7851, demonstrates potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. PSI-7851 is a mixture of two diastereoisomers, PSI-7976 and PSI-7977, with PSI-7977 being the more active inhibitor of HCV RNA replication in the HCV replicon assay. To inhibit the HCV NS5B RNA-dependent RNA polymerase, PSI-7851 must be metabolized to the active triphosphate form. The first step, hydrolysis of the carboxyl ester by human cathepsin A (CatA) and/or carboxylesterase 1 (CES1), is a stereospecific reaction. Western blot analysis showed that CatA and CES1 are both expressed in primary human hepatocytes. However, expression of CES1 is undetectable in clone A replicon cells. Studies with inhibitors of CatA and/or CES1 indicated that CatA is primarily responsible for hydrolysis of the carboxyl ester in clone A cells, although in primary human hepatocytes, both CatA and CES1 contribute to the hydrolysis. Hydrolysis of the ester is followed by a putative nucleophilic attack on the phosphorus by the carboxyl group resulting in the spontaneous elimination of phenol and the production of an alaninyl phosphate metabolite, PSI-352707, which is common to both isomers. The removal of the amino acid moiety of PSI-352707 is catalyzed by histidine triad nucleotide-binding protein 1 (Hint1) to give the 5'-monophosphate form, PSI-7411. siRNA-mediated Hint1 knockdown studies further indicate that Hint1 is, at least in part, responsible for converting PSI-352707 to PSI-7411. PSI-7411 is then consecutively phosphorylated to the diphosphate, PSI-7410, and to the active triphosphate metabolite, PSI-7409, by UMP-CMP kinase and nucleoside diphosphate kinase, respectively.

Simultaneous determination of rifampicin and levofloxacin concentrations in catheter segments from a mouse model of a device-related infection by liquid chromatography/electrospray ionization tandem mass spectrometry.

The aim of this study was to develop a specific and sensitive liquid chromatography mass spectrometry (LC/MS) method for the determination of rifampicin and levofloxacin concentrations from infected tissues within teflon catheter segments which were subcutaneously implanted in mice. A solid-phase extraction procedure was used to extract analytes from tissue homogenates of the catheter segments and reverse-phase HPLC combined with positive electrospray ionization mass spectrometry was used for analyte separation and quantification. The assay was found to be linear over the concentration range of 0.02-2 microg/g for rifampicin and levofloxacin in tissues and provided good validation data for accuracy and precision. The intra-day accuracy as determined by the relative error was -1.3% for levofloxacin and 6.1% for rifampicin, and precision was evaluated by R.S.D.s with a maximum of 5.1% for levofloxacin and 8.1% for rifampicin. The inter-day accuracy was -3.3% for levofloxacin and -4.6% for rifampicin, and precision was 8.6% for levofloxacin and 7.1% for rifampicin. The assay uses less tissue than previously described methods and can be applied to determine the penetration of rifampicin and the fluoroquinolone in catheter segments from a mouse model of a device-related infection. Finally, the HPLC-MS assay should be applicable to studies of rifamycin+quinolone combination therapies in other animal models of bacterial infection.

Determination of tacrine metabolites in microsomal incubate by high performance liquid chromatography-nuclear magnetic resonance/mass spectrometry with a column trapping system.

A column trapping system has been incorporated into high performance liquid chromatography-nuclear magnetic resonance-mass spectrometry (HPLC-NMR-MS) to reduce data acquisition time of NMR experiments. The system uses a trapping column to capture analytes after the HPLC column and back flush trapped analyte to the flow cell of the NMR probe for detection. A dilution solvent is mixed with eluent from HPLC column to reduce the influence of the organic content in the mobile phase before column trapping. The trapping column is also coupled with a mass spectrometer (MS) to get complementary MS data on the same peak. Studies on 1-hydroxylated 9-amino-1,2,3,4-tetrahydro-acridine (1-OH tacrine), indomethacin and testosterone with the column trapping system showed good recovery of analytes and over 3-fold mean increase in UV-VIS signal intensity. The time saving on NMR experiments with the column trapping system was demonstrated by the analysis of dog microsomal incubate with tacrine.