ABSTRACT
Mechanism-based inhibitors of human cytomegalovirus (HCMV) protease have been designed based on the pyrrolidine-5,5-trans-lactam ring system. New routes to the beta-methyl-, desmethyl-, and alpha-methyl-pyrrolidine-5,5-trans-lactam templates have been developed from 2,4-diaminobutyric acid. ESI/MS studies have shown that these inhibitors can bind covalently and reversibly to the viral enzyme in a time-dependent manner by a mechanism which is consistent with acylation of HCMV deltaAla protease at the active site nucleophile Ser 132. SAR in this series of pyrrolidine-5, 5-trans-lactams has defined the relative stereochemisty of the methyl substituent adjacent to the lactam carbonyl, the functionality on the lactam nitrogen, and the mechanism of action of this novel series of serine protease inhibitors against the HCMV deltaAla protease. Activity decreases on moving from the alpha-methyl to the desmethyl to the beta-methyl series. This selectivity is the opposite of that observed for these templates against the elastase and thrombin enzymes. The activity against HCMV deltaAla protease is the greatest with inhibitors based on the Cbz-protected alpha-methyl-5,5-trans-lactam template which have low micromolar activity against the viral enzyme.
Subject(s)
Cytomegalovirus/chemistry , Lactams/chemical synthesis , Pyrroles/chemical synthesis , Serine Endopeptidases/chemistry , Serine Proteinase Inhibitors/chemical synthesis , Drug Design , Humans , Lactams/chemistry , Pyrroles/chemistry , Serine Proteinase Inhibitors/chemistry , Stereoisomerism , Structure-Activity RelationshipABSTRACT
An investigation into the interaction between human cytomegalovirus (HCMV) protease and several beta-lactams, with characterization of the resulting acylenzymes using mass spectrometry, is reported. The time dependence of the inhibitors is highlighted by making comparisons of values obtained for inhibition and acylation. Analysis of inactivated HCMV protease revealed a beta-lactam: protease stoichiometry of 1. Subsequent enzymatic digestion with trypsin, peptide mapping using liquid chromatography coupled with electrospray ionization mass spectrometry and sequencing by nanoelectrospray tandem mass spectrometry (NanoES-MS/MS) allowed the identification of the site of covalent modification and confirmed Ser 132 as the active site hydroxyl nucleophile. Further, treatment of the protease with a peptide chloromethylketone and sequence analysis using NanoES-MS/MS of the alkylated enzyme confirmed His 63 as the active site imidazole nucleophile.
Subject(s)
Cytomegalovirus/enzymology , Serine Endopeptidases/chemistry , Amino Acid Chloromethyl Ketones/chemistry , Amino Acid Chloromethyl Ketones/pharmacology , Amino Acid Sequence , Base Sequence , Catalytic Domain/genetics , Chromatography, Liquid , Cloning, Molecular , Cytomegalovirus/genetics , DNA, Viral/genetics , Humans , Mass Spectrometry/methods , Molecular Sequence Data , Mutagenesis, Site-Directed , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Saccharomyces cerevisiae/genetics , Serine Endopeptidases/genetics , Trypsin , beta-Lactams/chemistry , beta-Lactams/pharmacologyABSTRACT
Mechanism based inhibitors of HCMV protease have been designed based on the monocyclic beta-lactam nucleus, which have been shown to acylate the viral enzyme in a time dependent manner. SAR in a series of monocyclic beta-lactam N-ureas, has defined the size and relative stereochemistry of the C-3 substituent producing a low micromolar inhibitor 17b with good aqueous stability and selectivity over the mammalian serine proteases.
Subject(s)
Protease Inhibitors/chemistry , Serine Endopeptidases/drug effects , beta-Lactams/chemistry , Drug Stability , Humans , Isomerism , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Structure-Activity Relationship , Substrate Specificity , beta-Lactams/chemical synthesis , beta-Lactams/pharmacologyABSTRACT
A novel series of tetrahydrobenzodioxinopyrroles has been identified as potent and selective alpha 2-adrenoceptor antagonists. Convergent syntheses have been developed that allowed the preparation of analogues and their enantiomers. A compound of particular interest is the 5-fluoro substituted analogue (fluparoxan).
Subject(s)
Adrenergic alpha-2 Receptor Antagonists , Adrenergic alpha-Antagonists/chemical synthesis , Adrenergic alpha-Antagonists/pharmacology , Pyrroles/chemical synthesis , Pyrroles/pharmacology , Adrenergic alpha-Antagonists/chemistry , Animals , In Vitro Techniques , Male , Mice , Molecular Structure , Piperoxan/analogs & derivatives , Piperoxan/chemical synthesis , Piperoxan/chemistry , Piperoxan/pharmacology , Pyrroles/chemistry , Rats , StereoisomerismABSTRACT
A series of four isomeric 2'- and 6'-fluorocarbocyclic guanosine analogues have been prepared and evaluated as potential anti-herpes agents. The racemic 2' beta-fluoro isomer 2-amino-1,9-dihydro- 9-[(1 alpha, 2 alpha, 3 beta, 4 alpha)-2-fluoro-3-hydroxy-4- (hydroxymethyl)cyclopentyl]-6H-purin-6-one (11a, C-AFG) and its 2' alpha-fluoro epimer 11b plus the chiral 6' beta-fluoro isomer 2-amino-1,9-dihydro-9-[[1S-(1 alpha, 2 alpha, 3 alpha, 4 beta)]- 2-fluoro-4-hydroxy-3-(hydroxymethyl)cyclopentyl]-6H-purine-6-one (11c) and its 6' alpha-fluoro epimer 11d were prepared from their respective fluoro amino diol hydrochlorides (6a,d). For comparison, the furanosyl compound 9-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)guanine (17, AFG) was prepared by coupling 2-amino-6-chloropurine with 2-deoxy-2-fluoro-3,5-di-O-benzoyl-alpha- D-arabinofuranosyl bromide followed by base hydrolysis. The 6' alpha-fluoro derivative 11d exhibited comparable activity to that of acyclovir (ACV) against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in vitro but was greater than 30-fold more active than ACV against HSV-1 and HSV-2 in vivo in the mouse systemic model. The 2' beta-fluoro derivative (11a, C-AFG) was extremely potent in vitro against HSV-1 and HSV-2 (ID50 0.006 and 0.05 micrograms/mL) and in vivo it was greater than 2 orders of magnitude more potent than ACV against HSV-1 and 70-fold more potent against HSV-2. The 2' alpha-fluoro 11b and 6' beta-fluoro 11c isomers were much less active.
Subject(s)
Antiviral Agents/chemical synthesis , Deoxyguanosine/analogs & derivatives , Herpes Simplex/drug therapy , Simplexvirus/physiology , Antiviral Agents/chemistry , Antiviral Agents/therapeutic use , Chemical Phenomena , Chemistry , Deoxyguanosine/chemical synthesis , Deoxyguanosine/chemistry , Deoxyguanosine/pharmacology , Deoxyguanosine/therapeutic use , Magnetic Resonance Spectroscopy , Molecular Structure , Structure-Activity Relationship , Virus Replication/drug effectsABSTRACT
The racemic carbocyclic 2'-fluoroarabinosyl pyrimidine nucleosides 8, 9 (C-FIAU), 12, and 13 (C-FMAU) and the 2'-fluororibosyl pyrimidine nucleosides 17, 20, and 21 were prepared from their respective protected 2'-fluoro amino diols 5 and 14. The carbocyclic 2'-2'-difluorothymidine analogue 27 was obtained from the protected difluoro amino diol 24 which was prepared from the ketone 23 and (diethylamino)sulfur trifluoride (DAST). The chiral carbocyclic 2'-deoxy-6'-fluorouridines 33, 34, 38, and 39 were synthesized from the protected 6'-fluoro amino diols 30 and 36, which were prepared by reduction of the azides 28 and 35. C-FMAU (13) and C-FIAU (9) were active in vitro against HSV-1 with ID50 values of 4.4 and 11 micrograms/mL, respectively, but they were inactive against HSV-2. The cytidine analogues 12 and 20 displayed modest activity in vitro against HSV-1 and HSV-2 but were inactive against human influenza A virus.
