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1.
Bioorg Med Chem Lett ; 22(18): 5924-9, 2012 Sep 15.
Article in English | MEDLINE | ID: mdl-22892115

ABSTRACT

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.


Subject(s)
Antiviral Agents/pharmacology , Hepacivirus/drug effects , Nucleotides, Cyclic/pharmacology , Prodrugs/pharmacology , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Dose-Response Relationship, Drug , Drug Stability , Humans , Liver/drug effects , Microbial Sensitivity Tests , Models, Molecular , Molecular Structure , Nucleotides, Cyclic/chemical synthesis , Nucleotides, Cyclic/chemistry , Prodrugs/chemical synthesis , Prodrugs/chemistry , Rats , Structure-Activity Relationship , Virus Replication/drug effects
2.
Antimicrob Agents Chemother ; 56(7): 3767-75, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22526308

ABSTRACT

PSI-352938 is a novel cyclic phosphate prodrug of ß-D-2'-deoxy-2'-α-fluoro-2'-ß-C-methylguanosine-5'-monophosphate with potent anti-HCV activity. In order to inhibit the NS5B RNA-dependent RNA polymerase, PSI-352938 must be metabolized to the active triphosphate form, PSI-352666. During in vitro incubations with PSI-352938, significantly larger amounts of PSI-352666 were formed in primary hepatocytes than in clone A hepatitis C virus (HCV) replicon cells. Metabolism and biochemical assays were performed to define the molecular mechanism of PSI-352938 activation. The first step, removal of the isopropyl group on the 3',5'-cyclic phosphate moiety, was found to be cytochrome P450 (CYP) 3A4 dependent, with other CYP isoforms unable to catalyze the reaction. The second step, opening of the cyclic phosphate ring, was catalyzed by phosphodiesterases (PDEs) 2A1, 5A, 9A, and 11A4, all known to be expressed in the liver. The role of these enzymes in the activation of PSI-352938 was confirmed in primary human hepatocytes, where prodrug activation was reduced by inhibitors of CYP3A4 and PDEs. The third step, removal of the O(6)-ethyl group on the nucleobase, was shown to be catalyzed by adenosine deaminase-like protein 1. The resulting monophosphate was consecutively phosphorylated to the diphosphate and to the triphosphate PSI-352666 by guanylate kinase 1 and nucleoside diphosphate kinase, respectively. In addition, formation of nucleoside metabolites was observed in primary hepatocytes, and ecto-5'-nucleotidase was able to dephosphorylate the monophosphate metabolites. Since CYP3A4 is highly expressed in the liver, the CYP3A4-dependent metabolism of PSI-352938 makes it an effective liver-targeted prodrug, in part accounting for the potent antiviral activity observed clinically.


Subject(s)
Antiviral Agents/metabolism , Cyclic P-Oxides/metabolism , Hepacivirus/drug effects , Nucleosides/metabolism , Cells, Cultured , Cytochrome P-450 CYP3A/metabolism , Guanylate Kinases/metabolism , Hepatocytes/metabolism , Humans , Nucleoside-Diphosphate Kinase/metabolism , Phosphoric Diester Hydrolases/metabolism
3.
Nucleosides Nucleotides Nucleic Acids ; 30(11): 886-96, 2011 Nov.
Article in English | MEDLINE | ID: mdl-22060553

ABSTRACT

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.


Subject(s)
Antiviral Agents/chemistry , Cyclic P-Oxides/chemistry , Hepacivirus/drug effects , Nucleosides/chemistry , Prodrugs/chemistry , Uridine Monophosphate/analogs & derivatives , Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Cyclic P-Oxides/chemical synthesis , Cyclic P-Oxides/pharmacology , Halogenation , Hepatitis C/drug therapy , Humans , Isotope Labeling/methods , Nucleosides/chemical synthesis , Nucleosides/pharmacology , Prodrugs/chemical synthesis , Prodrugs/pharmacology , Sofosbuvir , Uridine Monophosphate/chemical synthesis , Uridine Monophosphate/chemistry , Uridine Monophosphate/pharmacology
4.
J Org Chem ; 76(20): 8311-9, 2011 Oct 21.
Article in English | MEDLINE | ID: mdl-21916475

ABSTRACT

Prodrugs of therapeutic nucleoside monophosphates masked as phosphoramidate derivatives have become an increasingly important class of antiviral drugs in pharmaceutical research for delivering nucleotides in vitro and in vivo. Conventionally, phosphoramidate derivatives are prepared as a mixture of two diastereomers. We report a class of stable phosphoramidating reagents containing an amino acid ester and two phenolic groups, one unsubstituted and the other with electron-withdrawing substituents. The reagents can be isolated as single diastereomers and reacted with the 5'-hydroxyl group of nucleosides through selective nucleophilic displacement of the substituted phenol to prepare single diastereomer phosphoramidate products. This method has been used to prepare the HCV clinical candidate PSI-7977 in high yield and high diastereomeric purity.


Subject(s)
Amides/chemistry , Antiviral Agents/chemical synthesis , Chemistry, Pharmaceutical/methods , DNA-Directed RNA Polymerases/antagonists & inhibitors , Hepacivirus/drug effects , Hepatitis C/drug therapy , Phosphoric Acids/chemistry , Uridine Monophosphate/analogs & derivatives , Viral Proteins/antagonists & inhibitors , Amino Acids/chemistry , Antiviral Agents/pharmacology , Chromatography, High Pressure Liquid , DNA-Directed RNA Polymerases/metabolism , Electrons , Esters/chemistry , Hepacivirus/enzymology , Hepatitis C/virology , Humans , Magnetic Resonance Spectroscopy , Nucleosides/chemistry , Nucleotides/chemistry , Phenols/chemistry , Prodrugs/chemical synthesis , Prodrugs/pharmacology , Sofosbuvir , Stereoisomerism , Structure-Activity Relationship , Uridine Monophosphate/chemical synthesis , Uridine Monophosphate/pharmacology , Viral Proteins/metabolism
5.
J Org Chem ; 76(10): 3782-90, 2011 May 20.
Article in English | MEDLINE | ID: mdl-21469736

ABSTRACT

PSI-352938 is a novel 2'-deoxy-2'-α-fluoro-2'-ß-C-methyl 3',5'-cyclic phosphate nucleotide prodrug currently under investigation for the treatment of hepatitis C virus (HCV) infection. PSI-352938 demonstrated superior characteristics in vitro that include broad genotype coverage, superior resistance profile, and high levels of active triphosphate in vivo in the liver compared to our first and second generation nucleoside inhibitors of this class. Consequently, PSI-352938 was selected for further development and an efficient and scalable synthesis was sought to support clinical development. We report an improved, diastereoselective synthesis of a key 1'-ß-nucleoside intermediate 13 via S(N)2 displacement of 1-α-bromo ribofuranose sugar 16 with the potassium salt of 6-chloro-2-amino purine and an efficient method to prepare cis-Rp cyclic phosphate (PSI-352938) in a highly stereoselective manner without any chromatographic purification. The 1-α-bromo sugar 16 was stereospecifically prepared from the corresponding 1-ß-lactol in high yield under mild bromination conditions using CBr(4)/PPh(3) (Appel reaction). The desired cis-Rp 3',5'-cyclic phosphate construction was accomplished using isopropyl phosphorodichloridate readily obtained from POCl(3) and isopropyl alcohol. The base combination of Et(3)N/NMI was identified as a key factor for producing PSI-352938 as the major (>95%) diastereomer (cis-Rp) in high yield after the final cyclization step. The current route described in this article was successfully used to produce PSI-352938 on multikilogram scale.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/chemical synthesis , Cyclic P-Oxides/chemistry , Cyclic P-Oxides/chemical synthesis , Hepacivirus/drug effects , Nucleosides/chemistry , Nucleosides/chemical synthesis , Prodrugs/chemistry , Prodrugs/chemical synthesis , Antiviral Agents/pharmacology , Cyclic P-Oxides/pharmacology , Cyclization , Nucleosides/pharmacology , Prodrugs/pharmacology , Stereoisomerism , Substrate Specificity
6.
ACS Med Chem Lett ; 2(2): 130-5, 2011 Feb 10.
Article in English | MEDLINE | ID: mdl-24900291

ABSTRACT

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.

7.
Bioorg Med Chem Lett ; 20(24): 7376-80, 2010 Dec 15.
Article in English | MEDLINE | ID: mdl-21050754

ABSTRACT

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).


Subject(s)
Antiviral Agents/chemistry , Cyclic P-Oxides/chemistry , Enzyme Inhibitors/chemistry , Hepacivirus/enzymology , Nucleosides/chemistry , Nucleotides, Cyclic/chemistry , Prodrugs/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Administration, Oral , Animals , Antiviral Agents/pharmacokinetics , Antiviral Agents/toxicity , Cell Line, Tumor , Crystallography, X-Ray , Cyclic P-Oxides/pharmacokinetics , Cyclic P-Oxides/toxicity , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/toxicity , Humans , Molecular Conformation , Nucleosides/pharmacokinetics , Nucleosides/toxicity , Nucleotides, Cyclic/chemical synthesis , Nucleotides, Cyclic/toxicity , Prodrugs/pharmacokinetics , Prodrugs/pharmacology , Rats , Structure-Activity Relationship , Viral Nonstructural Proteins/metabolism
8.
J Med Chem ; 53(19): 7202-18, 2010 Oct 14.
Article in English | MEDLINE | ID: mdl-20845908

ABSTRACT

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.


Subject(s)
Antiviral Agents/chemical synthesis , Hepacivirus/drug effects , Prodrugs/chemical synthesis , Uridine Monophosphate/analogs & derivatives , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Antiviral Agents/pharmacokinetics , Antiviral Agents/pharmacology , Cell Line , Crystallography, X-Ray , Dogs , Drug Resistance, Viral , Esters , Hepacivirus/genetics , Hepatocytes/metabolism , Humans , In Vitro Techniques , Liver/metabolism , Macaca fascicularis , Mutation , Prodrugs/pharmacokinetics , Prodrugs/pharmacology , Rats , Replicon , Sofosbuvir , Stereoisomerism , Structure-Activity Relationship , Uridine Monophosphate/chemical synthesis , Uridine Monophosphate/pharmacokinetics , Uridine Monophosphate/pharmacology , Viral Nonstructural Proteins/genetics
9.
Chem Commun (Camb) ; (8): 844-6, 2006 Feb 28.
Article in English | MEDLINE | ID: mdl-16479286

ABSTRACT

The regioselective and enantiospecific rhodium-catalyzed allylic amination of secondary allylic carbonates with N3-benzoyl thymine in conjunction with a stereoselective free radical cyclization provides a convenient method for the construction of a new conformationally rigid nucleoside.


Subject(s)
Nucleosides/chemical synthesis , Rhodium/chemistry , Thymine/chemistry , Amination , Catalysis , Nucleic Acid Conformation , Stereoisomerism
11.
J Am Chem Soc ; 125(48): 14702-3, 2003 Dec 03.
Article in English | MEDLINE | ID: mdl-14640634

ABSTRACT

The enantioselective total synthesis of the annonaceous acetogenin (-)-mucocin (1) was accomplished using a triply convergent 12-step sequence (longest linear sequence) in 13.6% overall yield. This represents the first application of the temporary silicon-tethered (TST) ring-closing metathesis (RCM) cross-coupling reaction and the enantioselective alkyne/aldehyde addition to the synthesis of a complex annonaceous acetogenin. Moreover, all three fragments required for the coupling reactions are conveniently prepared in 5-6 steps from two readily available enantiomerically enriched epoxides. Finally, this synthesis stimulated the development of a new approach for the construction of 3-hydroxy-2,6-disubstituted tetrahydropyrans, using the bismuth tribromide-mediated reductive etherification reaction, which represents a motif that is prevalent in a wide range of pharmacologically significant natural products.


Subject(s)
Antineoplastic Agents, Phytogenic/chemical synthesis , Lactones/chemical synthesis , Pyrans/chemical synthesis , Cyclization , Rollinia/chemistry , Silicon/chemistry , Stereoisomerism
12.
J Org Chem ; 64(5): 1650-1656, 1999 Mar 05.
Article in English | MEDLINE | ID: mdl-11674232

ABSTRACT

Thermolysis of the benzoenyne-allene 20 in 1,4-cyclohexadiene (1,4-CHD) at 75 degrees C produced the cycloaromatized adduct 23 in 22% yield. The reaction presumably proceeds through a cascade sequence involving an initial Myers cyclization reaction to form the biradical 21. The subsequent trapping of the aryl radical center in 21 with the tetraarylallenic moiety intramolecularly affords 22, having two stabilized triarylmethyl radical centers. Hydrogen-atom abstraction from 1,4-CHD by 22, manifesting its radical character, then produces 23. Similarly, thermolysis of benzoenyne-allenes 25 in 1,4-CHD furnished fluoranthenes 27 in essentially quantitative yields. The presence of the fused five-membered ring in 20 and 25 is necessary to direct the initial biradical-forming step toward the Myers cyclization reaction. Without the five-membered ring as in the cases of 31, the C2-C6 cyclization reaction became the preferred pathway, leading to benzofluorenes 34.

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