Novel HCV NS5B polymerase inhibitors derived from 4-(1',1'-dioxo-1',4'-dihydro-1'lambda(6)-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones. Part 5: Exploration of pyridazinones containing 6-amino-substituents

The synthesis of 4-(1',1'-dioxo-1',4'-dihydro-1'lambda(6)-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones bearing 6-amino substituents as potent inhibitors of the HCV RNA-dependent RNA polymerase (NS5B) is described. Several of these agents also display potent antiviral activity in cell culture experiments (EC(50)<0.10 microM). In vitro DMPK data (microsome t(1/2), Caco-2 P(app)) for many of the compounds are also disclosed, and a crystal structure of a representative inhibitor complexed with the NS5B protein is discussed.     

Novel HCV NS5B polymerase inhibitors derived from 4-(1',1'-dioxo-1',4'-dihydro-1'lambda6-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones: Part 4. Optimization of DMPK properties

5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as potent inhibitors of genotype 1 HCV NS5B polymerase focusing on the optimization of their drug metabolism and pharmacokinetics (DMPK) profiles. This investigation led to the discovery of potent inhibitors with improved DMPK properties.     

Novel HCV NS5B polymerase inhibitors derived from 4-(1',1'-dioxo-1',4'-dihydro-1'lambda(6)-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones. Part 2: Variation of the 2- and 6-pyridazinone substituents

5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as inhibitors of genotype 1 HCV NS5B polymerase. The structure-activity relationship (SAR) associated with variation of the pyridazinone 2- and 6-substituents is discussed. The synthesis and metabolic stability of this new class of compounds are also described.     

Novel HCV NS5B polymerase inhibitors derived from 4-(1',1'-dioxo-1',4'-dihydro-1'lambda6-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones. Part 1: exploration of 7'-substitution of benzothiadiazine

5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as inhibitors of genotype 1 HCV NS5B polymerase. The synthesis, structure-activity relationships (SAR), metabolic stability, and structure-based design approach for this new class of compounds are discussed.     

4-(1,1-Dioxo-1,4-dihydro-1lambda6-benzo[1,4]thiazin-3-yl)-5-hydroxy-2H-pyridazin-3-ones as potent inhibitors of HCV NS5B polymerase

4-(1,1-Dioxo-1,4-dihydro-1lambda(6)-benzo[1,4]thiazin-3-yl)-5-hydroxy-2H-pyridazin-3-one analogs were discovered as a novel class of inhibitors of HCV NS5B polymerase. Structure-based design led to the identification of compound 3a that displayed potent inhibitory activities in biochemical and replicon assays (1b IC(50)<10 nM; 1b EC(50)=1.1 nM) as well as good stability toward human liver microsomes (HLM t(1/2)>60 min).     

Synthesis of novel 2-phenylsulfonylhydrazono-3-(2',3',4',6'-tetra-O-acetyl-beta-d-glucopyranosyl)thiazolidine-4-ones from thiosemicarbazide precursors

To develop novel biologically active organic compounds possessing a sugar moiety, a series of 2-phenylsulfonylhydrazono-3-(2',3',4',6'-tetra-O-acetyl-beta-d-glucopyranosyl)thiazolidine-4-one were synthesized via reaction of the thiosemicarbazide with ethyl bromoacetate. Their chemical structures were characterized by (1)H and (13)C NMR spectroscopy, elemental analysis and MS. The bioassay results indicated that some of these compound exhibit moderate fungicidal and herbicidal activities. Furthermore, the effect of various solvents at reflux temperature on the reactions of ethyl bromoacetate with the related thiosemicarbazides was investigated.     

Synthesis of 5'-C-methyl-1',3'-dioxolan-4'-yl nucleosides

Novel racemic 5'-C-methyl-1',3'-dioxolan-4'-yl nucleosides were synthesized from the key intermediate, 2-benzoyloxymethyl-4-oxo-5-C-methyl-1,3-dioxolane, which was prepared from racemic lactic acid.     

Stereoselective reactions of (20R)-3,20-dihydroxy-(3',4'-dihydro-2'H-pyranyl)-5-pregnene derivatives form 27-nor-3,20,23,26-tetrahydroxy-cholesten-22-ones and related bromo ketones

The previously reported analog of pregnenolone having a 3,4-dihydro-2H-pyran attached via a Cz.sbnd;C bond to the C-20 position (1), stereoselectively reacts with m-chloroperoxybenzoic acid in methanol at -5 degrees C. Acid-catalyzed hydrolysis of the isolated intermediates gives good yields of mostly a new 27-norcholesterol analog: (20R,23R)-3,20,23,26-tetrahydroxy-27-norcholest-5-en-22-one-3-acetate (2a, and a smaller amount of its 23S enantiomer 2b). Three different conditions of epoxidation and methanolysis followed by acid-catalyzed hydrolysis typically produce approximately 2:1 ratios of the 23R:23S diastereoisomers with a C-23 hydroxy group at the new asymmetric center. Bromine also reacts stereoselectively with (20R)-3,20-dihydroxy-(3',4'-dihydro-2'H-pyranyl)-5-pregnene (4) giving mostly (20R,23R)-23-bromo-3,20,26-trihydroxy-27-norcholest-5-en-22-one (7a). Thus both major steroidal products 2a and 7a have the same C-23R configuration. Assignment of molecular structures and the absolute configurations to 1 and 2a were based on elemental analysis, mass spectra, nuclear magnetic resonance, FTIR infrared spectroscopic analysis and X-ray crystallography. Mechanisms are discussed for stereochemical selectivity during epoxidation and bromination of the 3,4-dihydro-2H-pyranyl ring in 1 and 4.     

Inhibitors of HCV NS5B polymerase. Part 1: Evaluation of the southern region of (2Z)-2-(benzoylamino)-3-(5-phenyl-2-furyl)acrylic acid

A novel series of nonnucleoside HCV NS5B polymerase inhibitors were prepared from (2Z)-2-(benzoylamino)-3-(5-phenyl-2-furyl)acrylic acid, a high throughput screening lead. SAR studies combined with structure based drug design focusing on the southern heterobiaryl region of the template led to the synthesis of several potent and orally bioavailable lead compounds. X-ray crystallography studies were also performed to understand the interaction of these inhibitors with HCV NS5B polymerase.     

2-(3-Thienyl)-5,6-dihydroxypyrimidine-4-carboxylic acids as inhibitors of HCV NS5B RdRp

A series of 2-(3-thienyl)-5,6-dihydroxypyrimidine-4-carboxylic acid inhibitors of the hepatitis C virus (HCV) NS5B polymerase enzyme are reported. Sulfonyl urea substituted analogs in this series proved to be the most potent active site non-nucleoside inhibitors of NS5B reported to date. These compounds had low nanomolar enzyme inhibition across HCV genotypes 1–3 and showed single digit micromolar inhibition in the HCV replicon assay. This improved cell-based activity allowed the binding mode of these compounds to be probed by selection of resistant mutations against compound 21. The results generated are in broad agreement with the previously proposed binding model for this compound class.     

Microbial hydroxylation of (+/-)- and (-)-(2Z,4E)-5-(1',2'-epoxy-2',6',6'-trimethylcyclohexyl)-3-methyl-2,4-pentadienoic acid into (+/-)- and (-)-xanthoxin acid by Cunninghamella echinulata

Microbial hydroxylation of (+/-)-(2Z,4E)-5-(1',2'-epoxy-2',6',6'-trimethylcyclohexyl)-3-methyl-2,4-pentadienoic acid (3a) with Cercospora cruenta, a fungus producing (+)-abscisic acid, gave a four-stereoisomeric mixture consisting of (+)- and (-)-xanthoxin acid (4a), and (+)- and (-)-epi-xanthoxin acid (5a) by an HPLC analysis with a chiral column. Screening of the microorganisms capable of oxidizing (+/-)-3a showed that Cunninghamella echinulata stereoselectively oxidized (+/-)-3a to xanthoxin acid (4a) with the some degree of enantioselectivity as (-)-3a to (-)-4a.     

Synthesis of 3-[4'-(p-chlorophenyl)-thiazol-2'-yl]-2-[(substituted azetidinone/thiazolidinone)-aminomethyl]-6-bromoquinazolin-4-ones as anti-inflammatory agent

N-Chloroacetyl-5-bromoanthranilic acid (1), 3-[4'-(p-chlorophenyl)-thiazol-2'-yl]-2-chloromethyl-6-bromoquinazolin-4-one (2), 3-[4'-(p-chlorophenyl)-thiazol-2'-yl]-2-hydrazinomethyl-6-bromoquinazolin-4-one (3), 3-[4'-(p-chlorophenyl)-thiazol-2'-yl]-2-substitutedbenzylidene aminomethyl-6-bromoquinazolin-4-ones (4-11), 2-[(4'-oxo-3'-chloro-2'-phenylazetidin-1'-yl)aminomethyl]-3-[4'-(p-chlorophenyl)thiazol-2'-yl]-6-bromoquinazolin-4-ones (12-19) and 2-(4'-oxo-2'-phenyl-thiazolidin-3'-yl-aminomethyl)- 3-[4'-(p-chlorophenyl)-thiazol-2'-yl]-6-bromoquinazolin-4-ones (20-27) have been synthesized. All the compounds have been screened for their anti-inflammatory and analgesic activities at the dose of 50mg/kg po. Compound 21 showed maximum anti-inflammatory (38.35%) and analgesic (37.36%) activities. Compound 21 was also tested for ulcerogenic activity and the UD(50) value was found to be 195.6mg/kg po. The structure of all compounds has been evaluated by elemental analysis (C, H, N) and spectral analysis (IR, (1)H NMR and mass spectrometry).     

Synthesis and antiviral activity assay of novel (E)-3',5'-diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine

(E)-3',5'-diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. In contrast with BVDU, compound 5 did not show activity against herpes simplex virus or varicella-zoster virus.     

Synthesis and antimicrobial activity of 4-hydroxy-4-(pyridyl)alk-3-en-2-ones

4-Hydroxy-4-(pyridyl)alk-3-en-2-ones were prepared by base-mediated condensation of ketones with pyridinecarboxylates. Several derivatives show weak antimicrobial activity against Gram-positive and Gram-negative bacteria.     

2'-Deoxy-3'-O-(4-benzoylbenzoyl)- and 3'(2')-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate, fluorescent photoaffinity analogues of adenosine 5'-diphosphate. Synthesis, characterization, and interaction with myosin subfragment 1

Two new fluorescent nucleotide photoaffinity labels, 3'(2')-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate (Bz2 epsilon ADP) and 2'-deoxy-3'-O-(4-benzoylbenzoyl)-1,N6-ethenoadenosine 5'-diphosphate [3'(Bz2)2'd epsilon ADP], have been synthesized and used as probes of the ATP binding site of myosin subfragment 1 (SF1). These analogues are stably trapped by the bifunctional thiol cross-linker N,N'-p-phenylenedimaleimide (pPDM) at the active site in a manner similar to that of ATP [Wells, J.A., & Yount, R.G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966-4970], and nonspecific photolabeling can be minimized by removing free probe by gel filtration prior to irradiation. Both probes covalently photoincorporate with high efficiency (40-50%) into the central 50-kDa heavy chain tryptic peptide, as found previously for the nonfluorescent parent compound 3'(2')-O-(4-benzoylbenzoyl)adenosine diphosphate [Mahmood, R., & Yount, R.G. (1984) J. Biol. Chem. 259, 12956-12959]. The solution conformations of Bz2 epsilon ADP and 3'(Bz2)-2'd epsilon ADP were analyzed by steady-state and time-resolved fluorescence spectroscopy. These data indicated that the benzoylbenzoyl rings in both analogues were stacked over the epsilon-adenine ring. The degree of stacking was greater with the 2' isomer than with the 3' isomer. Fluorescence quantum yields and lifetimes were measured for Bz2 epsilon ADP and 3'(Bz2)2'd epsilon ADP reversibly bound, stably trapped, and covalently photoincorporated at the active site of SF1. These values were compared with those for 3'(2')-O-[[(phenylhydroxymethyl)phenyl]carbonyl]-1,N6-ethenoadenos ine diphosphate (CBH epsilon ADP) and 2'-deoxy-3'-O-[[(phenylhydroxymethyl)phenyl]carbonyl]-1,N6- ethenoadenosine diphosphate [3'(CBH)2'd epsilon ADP]. These derivatives were synthesized as fluorescent analogues of the expected product of the photochemical reactions of Bz2 epsilon ADP and 3'(Bz2)2'd epsilon ADP, respectively, with the active site of SF1. The fluorescence properties of the carboxybenzhydrol derivatives trapped at the active site by pPDM were compared with those of the Bz2 nucleotide-SF1 complexes. These properties were consistent with a photoincorporation mechanism in which the carbonyl of benzophenone was converted to a tertiary alcohol attached covalently to the protein. The specific, highly efficient photoincorporation of Bz2 epsilon ADP at the active site will allow it to be used as a donor in distance measurements by fluorescence resonance energy transfer to acceptor sites on actin.     

Synthesis and stability studies of 2',3',5'-tri-O-acetyl-2-amino(-N6-cyclopentyl)-1-deazaadenosines

In this article, we report on the synthesis of 2',3',5'-tri-O-acetyl-2-amino-1-deazaadenosine and of 2',3',5'-tri-O-acetyl-2-amino-N(6)-cyclopentyl-1-deazaadenosine, which are very versatile intermediates for the preparation of 2-substituted 1-deazaadenosine derivatives. The two synthesized compounds showed to be quite unstable, with the N(6)-substituted derivatives being less stable than the N(6)-unsubstituted counterpart, according to the calculated HOMO-LUMO energy gap. Stability studies were performed through HPLC-MS analysis.     

Synthesis of 5-(4-hydroxy-3-methylphenyl) -5- (substituted phenyl)-4, 5-dihydro-1H-1-pyrazolyl-4-pyridylmethanone derivatives with anti-viral activity

A series of N1-nicotinoyl-3- (4-hydroxy-3-methyl phenyl)-5-(substituted phenyl)-2-pyrazolines were synthesized by the reaction between isoniazid (INH) and chalcones and were tested for their in vitro anti-viral activity. Among the compounds, the electron withdrawing group substituted analogues 5-(4-chlorophenyl)-3-(4-hydroxy-3-methylphenyl)-4, 5-dihydro-1H-1- pyrazolyl-4-pyridylmethanone (4b), 5-(2-chlorophenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro-1H-1-pyrazolyl-4-pyri- dylmethanone (4i), 5-(4-fluorophenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro- 1H-1-pyrazolyl-4-pyridylmethanone (4h) and 5-(2,6-dichlorophenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro- 1H-1-pyrazolyl-4-pyridyl methanone (4j) were the most promising and the halogeno function appeared to be essential for antiviral activity.