Application of phosphoramidate ProTide technology significantly improves antiviral potency of carbocyclic adenosine derivatives.

We report the application of phosphoramidate pronucleotide (ProTide) technology to the antiviral agent carbocyclic L-d4A (L-Cd4A). The phenyl methyl alaninyl parent ProTide of L-Cd4A was prepared by Grignard-mediated phosphorochloridate reaction and resulted in a compound with significantly improved anti-HIV (2600-fold) and HBV activity. We describe modifications of the aryl, ester, and amino acid regions of the ProTide and how these changes affect antiviral activity and metabolic stability.

Application of phosphoramidate pronucleotide technology to abacavir leads to a significant enhancement of antiviral potency.

We report the first application of pronucleotide (ProTide) technology to the antiviral agent abacavir (Ziagen), used for the treatment of HIV infection. The phenylmethoxyalaninyl phosphoramidate of abacavir was prepared in good yield in one step. Also prepared was the corresponding phosphoramidate of the guanine nucleoside analogue "carbovir".

Phosphoramidate protides of carbocyclic 2',3'-dideoxy-2',3'-didehydro-7-deazaadenosine with potent activity against HIV and HBV.

Synthesis of phosphoramidate protides of carbocyclic D- and L-2',3'-dideoxy-2',3'-didehydro-7-deazaadenosine by treatment of the nucleoside with phosphorochloridates in the presence of pyridine and t-BuMgCl is described. Several of these protides showed significantly improved antiviral potency over the parent nucleosides against both HIV and HBV.

Membrane permeation characteristics of abacavir in human erythrocytes and human T-lymphoblastoid CD4+ CEM cells: comparison with (-)-carbovir.

Abacavir, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol, is a novel purine carbocyclic nucleoside analogue that has been approved by the FDA for the treatment of HIV (as Ziagen trade mark [abacavir sulfate]). Chemically, abacavir and (-)-carbovir (CBV) differ only at the 6-position of the purine ring; abacavir contains a cyclopropylamino moiety in place of the 6-lactam functionality of CBV. Intracellularly both are ultimately metabolized to CBV triphosphate.

Improved antiviral activity of the aryloxymethoxyalaninyl phosphoramidate (APA) prodrug of abacavir (ABC) is due to the formation of markedly increased carbovir 5'-triphosphate metabolite levels.

The anti-human immunodeficiency virus (HIV) activity of abacavir (ABC; 1-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol) could be markedly enhanced by administering the aryloxymethoxyalaninyl phosphoramidate prodrug derivative of ABC (pro-ABC-MP) to virus-infected cell cultures. Metabolic studies with radiolabeled ABC and pro-ABC-MP in human T-lymphocyte and primary macrophage cell cultures revealed a significantly increased delivery of the activated (phosphorylated) metabolite of ABC (ABC-MP) by pro-ABC-MP, and the concomittant appearance of markedly higher intracellular levels of carbovir 5'-triphosphate (CBV-TP), which represents the eventual antivirally active metabolite of ABC. The intracellular amounts of ABC-MP and appearance of CBV-TP closely correlated with the extracellular pro-ABC-MP concentrations that were administered to the cell cultures within a concentration range between 0.

Phosphoramidate protides of 2',3'-dideoxy-3'-fluoroadenosine and related nucleosides with potent activity against HIV and HBV.

Syntheses of phosphoramidate protides of several 2',3'-dideoxy-3'-fluoroadenosine derivatives by treatment of the nucleoside with phosphorochloridates in the presence of pyridine and t-BuMgCl is described. Several of these protides showed significantly improved antiviral potency over the parent nucleoside against HIV and HBV. Especially marked was the improvement in potency of phosphoramidate protides of 2',3'-dideoxy-3'-fluoroadenosine against both HIV and HBV.

Synthesis of novel 8-substituted carbocyclic analogs of 2',3'-dideoxyadenosine with activity against hepatitis B virus.

Synthesis and antiviral activity of several new 8-substituted carbocyclic analogs of D-2',3'-dideoxyadenosine are described. The new 8-substituted analogs were synthesized via lithiation of carbocyclic 2',3'-dideoxyadenosine followed by quenching with electrophiles. This methodology allows for a divergent synthesis of a variety of 8-substituted analogs from carbocyclic 2',3'-dideoxyadenosine in high yields.

Synthesis of carbocyclic analogs of 2',3'-dideoxysangivamycin, 2',3'-dideoxytoyocamycin, and 2',3'-dideoxytriciribine.

Syntheses and antiviral activity of new carbocyclic analogs of 2', 3'-dideoxysangivamycin, 2',3'-dideoxytoyocamycin and 2',3'-dideoxytriciribine is described. The key intermediate, carbocyclic 4-chloro-5-iodopyrrolopyrimidine. was synthesized in good yield via a novel iodination method using I2 and CF3COOAg.

An efficient, scalable synthesis of the HIV reverse transcriptase inhibitor Ziagen (1592U89).

Ziagen, (1S,cis)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]- 2-cyclopentene-1-methanol, was synthesized from (1S,4R)-azabicyclo[2.2.1]hept-5-en-3-one by efficient processes which bypass problematic steps in earlier routes.

Studies designed to increase the stability and antiviral activity (HCMV) of the active benzimidazole nucleoside, TCRB.

The potent activity of 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) against Human Cytomegalovirus with the concomitant low cellular toxicity at concentrations that inhibit viral growth prompted considerable interest in this research area. This interest was moderated by the pharmacokinetic studies of TCRB in rats and monkeys that revealed the instability of TCRB in vivo. These studies suggested that the instability was due to a cleavage of the glycosidic bond in vivo which released the heterocycle (2,5,6-trichlorobenzimidazole) into the bloodstream.

Unique intracellular activation of the potent anti-human immunodeficiency virus agent 1592U89.

The anabolism of 1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, a selective inhibitor of human immunodeficiency virus (HIV), was characterized in human T-lymphoblastoid CD4+ CEM cells. 1592U89 was ultimately anabolized to the triphosphate (TP) of the guanine analog (-)-carbovir (CBV), a potent inhibitor of HIV reverse transcriptase. However, less than 2% of intracellular 1592U89 was converted to CBV, an amount insufficient to account for the CBV-TP levels observed.

1592U89, a novel carbocyclic nucleoside analog with potent, selective anti-human immunodeficiency virus activity.

1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, is a carbocyclic nucleoside with a unique biological profile giving potent, selective anti-human immunodeficiency virus (HIV) activity. 1592U89 was selected after evaluation of a wide variety of analogs containing a cyclopentene substitution for the 2'-deoxyriboside of natural deoxynucleosides, optimizing in vitro anti-HIV potency, oral bioavailability, and central nervous system (CNS) penetration. 1592U89 was equivalent in potency to 3'-azido-3'-deoxythymidine (AZT) in human peripheral blood lymphocyte (PBL) cultures against clinical isolates of HIV type 1 (HIV-1) from antiretroviral drug-naive patients (average 50% inhibitory concentration [IC50], 0.

DNA polymerase activity of hepatitis B virus particles: differential inhibition by L-enantiomers of nucleotide analogs.

DNA polymerase activity was assayed in hepatitis B virus (HBV) and core particles isolated from chronic producer lines. The particle-associated DNA polymerase activity, which was found to be limited to incorporation of only a few nucleotides, was inhibited by the 5'-triphosphates of nucleoside analogs. The 1-beta-L (1S,4R) and 1-beta-D (1R,4S) enantiomers of antiviral nucleoside analogs were compared for the ability to inhibit incorporation of natural nucleoside triphosphates into the viral DNA.

5-Chloro-2',3'-dideoxy-3'-fluorouridine (935U83), a selective anti-human immunodeficiency virus agent with an improved metabolic and toxicological profile.

5-Chloro-2',3'-dideoxy-3'-fluorouridine (935U83) is a selective anti-human immunodeficiency virus (HIV) agent. When tested in phytohemagglutinin-stimulated normal human peripheral blood lymphocytes against fresh clinical isolates of HIV type 1 (HIV-1) obtained from patients naive to AZT (3'-azido-3'-deoxythymidine [zidovudine]), 935U83 inhibited virus growth with an average 50% inhibitory concentration (IC50) of 1.8 microM; corresponding IC50s were 0.

Evaluation of the potent anti-hepatitis B virus agent (-) cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine in a novel in vivo model.

A murine model was developed to investigate the in vivo activity of anti-hepatitis B virus (HBV) agents. Mice with subcutaneous tumors of HBV-producing 2.2.

Varicella-zoster virus thymidine kinase. Characterization and substrate specificity.

The varicella-zoster virus (VZV) thymidine kinase (TK) EC 2.7.2.

Baquiloprim, a new antifolate antibacterial: in vitro activity and pharmacokinetic properties in cattle.

During examination of the half-lives in cattle of a series of 5-substituted diaminobenzyl-pyrimidines, it was found that replacement of the phenyl ring of trimethoprim (TMP) by bicyclic structures, particularly a quinolyl group, led to increases in half-life. The presence of a dimethylamino group on the quinolyl ring of the compound baquiloprim (BQP) conferred a half-life of about 10 hours. In contrast to TMP (half-life about one hour), BQP was well absorbed from the gastrointestinal tract in all ages of cattle, plasma concentrations reaching a plateau on the day after dosing followed by a slow decline.

Phosphorylation of carbovir enantiomers by cellular enzymes determines the stereoselectivity of antiviral activity.

Two enantiomers of carbovir, a carbocyclic analog of 2',3'-dideoxyguanosine, were compared with respect to their phosphorylation and the phosphorylation of their nucleotides by mammalian enzymes. 5'-Nucleotidase catalyzed the phosphorylation of (-)-carbovir, which is active against HIV (human immunodeficiency virus), but did not phosphorylate (+)-carbovir. (-)-Carbovir monophosphate was 7,000 times more efficient as a substrate for GMP kinase than was (+)-carbovir monophosphate.

Enantiomeric selectivity of carbovir transport.

Carbovir (9-[4 alpha-(hydroxymethyl)cyclopent-2-ene-1 alpha-yl]guanine) (CBV) is a carbocyclic analogue of 2',3'-dideoxyguanosine that exhibits potent and selective in vitro activity against human immunodeficiency virus. Antiviral activity is associated with only the (-)-enantiomer. The transport characteristics of both (-)-CBV and (+)-CBV were investigated in human erythrocytes at 37 degrees C using a papaverine-stop assay.

Potent and selective activity of a new carbocyclic nucleoside analog (carbovir: NSC 614846) against human immunodeficiency virus in vitro.

Carbocyclic 2',3'-didehydro-2',3'-dideoxyguanosine (Carbovir: NSC 614846), a novel nucleoside analog, emerged as a potent and selective anti-HIV agent from a large screening program conducted by the National Cancer Institute and its contractors. Its hydrolytic stability and its ability to inhibit the infectivity and replication of HIV in T-cells at concentrations of approximately 200- to 400-fold below toxic concentrations make carbovir a top-priority candidate for development as a potential antiretroviral agent in the treatment of AIDS patients.

125I-BW-A844U, an antagonist radioligand with high affinity and selectivity for adenosine A1 receptors, and 125I-azido-BW-A844U, a photoaffinity label.

3-(4-Amino)phenethyl-1-propyl-8-cyclopentylxanthine (BW-A844U) has been synthesized and shown to bind with high affinity to adenosine A1 receptors of bovine brain membranes (KD = 0.23 nM). This compound is highly selective for A1 receptors; the KI for binding to A2 receptors of human platelet membranes is 2.

Photoaffinity labeling adenosine A1 receptors with an antagonist 125I-labeled aryl azide derivative of 8-phenylxanthine.

We have derivatized a series of 125I-labeled 8-phenylxanthines with photoactive aryl azide groups on the 1- or 3-position of the xanthine ring. A 3-azidophenethyl derivative was found to be optimal for use as an antagonist photoaffinity label for adenosine A1 receptors. Following photoactivation, radioactivity was covalently and specifically incorporated into a 34,000-dalton and, to a lesser extent, into a 24,000-dalton polypeptide of rat brain membranes.

125I-labeled 8-phenylxanthine derivatives: antagonist radioligands for adenosine A1 receptors.

A series of 8-phenylxanthine derivatives has been synthesized with oxyacetic acid on the para phenyl position to increase aqueous solubility and minimize nonspecific binding and iodinatable groups on the 1- or 3-position of the xanthine ring. The structure-activity relationship for binding of these compounds to A1 adenosine receptors of bovine and rat brain and A2 receptors of human platelets was examined. The addition of arylamine or photosensitive aryl azide groups to the 3-position of xanthine had little effect on A1 binding affinity with or without iodination, whereas substitutions at the 1-position caused greatly reduced A1 binding affinity.

Carbocyclic puromycin: synthesis and inhibition of protein biosynthesis.

The carbocyclic analogue of puromycin was prepared by the coupling of N-(benzyloxycarbonyl)-p-methoxy-L-phenylalanine to the racemic aminonucleoside (+/-)-9-[3 beta-amino-2 beta-hydroxy-4 alpha-(hydroxymethyl)cyclopent-1 alpha-yl]-6-(dimethylamino)purine, followed by separation of the diastereomers and subsequent removal of the Cbz blocking group. Kinetic studies indicate that carbocyclic puromycin is an excellent substrate for the peptidyltransferase reaction with both prokaryotic and eukaryotic ribosomes. A comparison of carbocyclic puromycin with previously synthesized analogues indicate that the furanosyl ring oxygen and the hydroxymethyl group of puromycin do not contribute to ribosomal binding, but both moieties contribute to the rate of product formation from the enzyme-substrate complex.

Carbocyclic analogues of xylofuranosylpurine nucleosides. Synthesis and antitumor activity.

(+/-)-4 alpha-Amino-2 alpha,3 beta-dihydroxy-1 alpha-cyclopentanemethanol (6), the carbocyclic analogue of xylofuranosylamine, was synthesized from the previously reported 4 alpha-acetamido-2 alpha,3 alpha-epoxycyclopentane-1 alpha-methanol. Amine 6 was converted to (+/-)-4 alpha-[(5-amino-6-chloro-4-pyrimidinyl)amino]-2 alpha,3 beta-dihydroxy-1 alpha-cyclopentanemethanol (7) by condensation with 5-amino-4,6-dichloropyrimidine. From 7, the carbocyclic analogues of xylofuranosyladenine and xylofuranosyl-8-azaadenine were prepared.