Phosphorylation of ganciclovir phosphonate by cellular GMP kinase determines the stereoselectivity of anti-human cytomegalovirus activity.

A racemic mixture of ganciclovir phosphonate was resolved by stereoselective phosphorylation using GMP kinase. The R-enantiomer of ganciclovir phosphonate was active against human cytomegalovirus but the S-enantiomer was less active. We show that enantiomeric selectivity of antiviral for ganciclovir phosphonate was conferred by stereoselective phosphorylations by mammalian enzymes, not by stereoselective inhibition of DNA polymerase from human cytomegalovirus.

Guanine, pyrazolo[3,4-d]pyrimidine, and triazolo[4,5-d]pyrimidine (8-azaguanine) phosphonate acyclic derivatives as inhibitors of purine nucleoside phosphorylase.

Phosphonate acyclic derivates of guanines, pyrazolo[3,4-d]pyrimidines, and triazolo[4,5-d]-pyrimidines (8-azaguanines) are inhibitors of the enzyme purine nucleoside phosphorylase (PNPase) with Ki' values ranging from 0.05 to 1.6 microM. These compounds are enzymatically stable congeners of the potent PNPase inhibitor acyclovir diphosphate (53).

Metabolism and pharmacokinetics of a double prodrug of ganciclovir in the rat and monkey.

Ganciclovir (GCV), which is used in the treatment of human cytomegalovirus infections, is poorly absorbed orally. A double prodrug of GCV, the dipivalate ester of 6-deoxy-GCV (6-dGCV) (called 6-dGCV-DPiv), was given orally to rats (25 mg/kg) and resulted in a nearly 7-fold enhancement of GCV bioavailability compared with administration of GCV alone and a 2-fold increase compared with administration of 6-dGCV. The prodrug was rapidly hydrolyzed and extensively oxidized by first-pass metabolism in such a way that only GCV, 6-dGCV, and a small amount of the monopivalate ester of 6-dGCV were observed in rat plasma. In cynomolgus monkey was given the prodrug orally (22.5 mg/kg), two additional metabolites were observed--the 8-hydroxy analogs of GCV and dGCV. The double prodrug approach demonstrated the potential for enhanced oral delivery of GCV in humans.

An enantiospecific synthesis of the human cytomegalovirus antiviral agent [(R)-3-((2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy)-4- hydroxybutyl]phosphonic acid.

The racemic isosteric phosphonate of ganciclovir monophosphate (BW2482U89, SR3745, [3-((2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methoxy)-4- hydroxybutyl]phosphonic acid, 1) has potent and selective in vitro activity against human cytomegalovirus. An enantiospecific synthesis of the R-enantiomer of compound 1 starting from L-arabinose was developed. The synthesis involved (1) the preparation of a chiral acyclic moiety, (2) the coupling of the chiral acyclic moiety to diacetylguanine, (3) the introduction of phosphorus, and (4) the final deprotection. The R-enantiomer, which has stereochemistry analogous to the natural compound GMP, was tested against human cytomegalovirus and had an IC50 of 1.7 microM, which was approximately 2-fold more active than the racemic material. Both racemic and chiral compounds were less toxic than ganciclovir to bone marrow progenitor cells in an in vitro assay.

Review of research leading to new anti-herpesvirus agents in clinical development: valaciclovir hydrochloride (256U, the L-valyl ester of acyclovir) and 882C, a specific agent for varicella zoster virus.

Research leading to the new anti-herpesvirus compounds discussed here has come from three approaches. The first approach was directed towards improving the bioavailability of acyclovir by examining the potential of a variety of prodrugs, leading to the new compound valaciclovir hydrochloride. The second approach was to examine a large number of 5-substituted pyrimidines for activity against those viruses which were not as potently inhibited by acyclovir as are herpes simplex viruses, i.e., varicella zoster virus (VZV) and human cytomegalovirus (HCMV). This research led to the new chemical entity 882C for VZV. A third approach has been to examine drug combinations with acyclovir. This research led to the compound 348U, an inhibitor of herpes simplex virus ribonucleotide reductase which acts synergistically in combination with acyclovir. This manuscript will focus on the first two approaches leading to new compounds valaciclovir hydrochloride and 882C since Dr. Safrin details such background for 348U/acyclovir. Attempts to improve the bioavailability of acyclovir began a decade ago. Early prodrugs were compounds with alterations in the 6-substituent of the purine ring of acyclovir. The 6-amino congener required the cellular enzyme adenosine deaminase for conversion to acyclovir and the 6-deoxycongener was dependent on cellular xanthine oxidase for conversion. Neither of these prodrugs had a chronic toxicity profile in laboratory animals as good as acyclovir. Efforts were directed towards simpler esters and 18 amino acid esters were made. The pharmacokinetic profile of each prodrug was determined in rats by measuring the recovery of acyclovir in urine after oral dosing.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleotide analogue inhibitors of purine nucleoside phosphorylase.

The diphosphate of the antiherpetic agent acyclovir [9-[(2-hydroxyethoxy)methyl]guanine] has been shown to inhibit purine nucleoside phosphorylase with unique potency (Tuttle, J. V., and Krenitsky, T. A. (1984) J. Biol. Chem. 259, 4065-4069). A major factor contributing to the superior inhibition by this diphosphate over the corresponding mono- and triphosphates is revealed here. Homologues of acyclovir mono- and diphosphate that extend the ethoxy moiety by one to four methylene groups were synthesized. These homologues were evaluated for their ability to inhibit human purine nucleoside phosphorylase. Within the diphosphate series, the Ki values increased progressively with increasing chain length. With the monophosphates, the Ki values reached a minimum with the homologue containing a pentoxy moiety. A plot of chain length versus Ki values for both mono- and diphosphates showed that both series had similar optimal distances between the aminal carbon and the terminal oxygen anion. Monophosphates with optimal positioning were somewhat less potent than diphosphates with similar positioning. Nevertheless, it was clear that a major factor in determining potency of inhibition was the distance of the terminal phosphate from the guanine moiety.

Effect of acyclic pyrimidines related to 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine on herpesviruses.

A series of pyrimidines related to the potent antiherpetic agent 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (1, BW B759U), all containing the same acyclic chain, have been synthesized. Some of the compounds were derivatives of the naturally occurring bases, cytosine, uracil, and thymine; others included compounds in which the 5-position of the cytosine and uracil moieties were substituted by bromo, iodo, fluoro, methyl, and amino groups. Other variations of the cytosine derivatives were the 5-aza, 2-mercapto, 4-methylamino, 4-dimethylamino, and isocytosine congeners. A 4-aminopyrimidine adduct was also made. Antiviral testing showed that 1-[(1,3-dihydroxy-2-propoxy)methyl]cytosine (18, BW A1117U) was equivalent to the guanine analogue in potency against human cytomegalovirus and Epstein Barr virus. Other compounds in the series were largely inactive in antiviral screening against the herpesviruses.

Modifications on the heterocyclic base of acyclovir: syntheses and antiviral properties.

A group of compounds was prepared in which variations of the ring portion of the acyclovir (ACV) structure were made. These modifications included monocyclic (isocytosine, triazole, imidazole), bicyclic (8-azapurine, pyrrolo[2,3-d]pyrimidine, pyrazolo[3,4-d]pyrimidine) and tricyclic (linear benzoguanine) congeners. The derivatives were evaluated against herpes simplex virus type 1 (HSV-1) by the plaque-inhibition and plaque-reduction methods with only the 8-azapurine analogue 28 showing some activity. In a test measuring the ability of these compounds to inhibit the HSV-1 thymidine kinase, 28 and the tricyclic derivative 38 exhibited competition with ACV for binding to the enzyme. The inability of the group to exert significant antiherpetic action is attributed to their lack of phosphorylation to the requisite triphosphate stage.

6-Deoxyacyclovir: a xanthine oxidase-activated prodrug of acyclovir.

Acyclovir [9-[(2-hydroxyethoxy)methyl]guanine] is an acyclic guanine nucleoside analogue that is widely used clinically as an antiherpetic agent. Its limited absorption in humans after oral administration prompted the search for prodrugs. A congener, referred to as 6- deoxyacyclovir [2-amino-9-[(2-hydroxyethoxy)methyl]-9H-purine], was synthesized and found to be 18 times more water soluble than was acyclovir. Surprisingly, this congener was readily oxidized to acyclovir by xanthine oxidase (EC 1.2.3.2). It was also oxidized by aldehyde oxidase (EC 1.2.3.1) largely to 8-hydroxy-6- deoxyacyclovir [2-amino-8-hydroxy-9-[(2-hydroxyethoxy)methyl]-9H-purine] and then to 8- hydroxyacyclovir [2-amino-6,8-dihydroxy-9[(2-hydroxyethoxy)methyl]-9H-purine]. 6- Deoxyacyclovir and the major products of its oxidation by aldehyde oxidase lacked appreciable activity against herpes simplex type I in vitro. On the basis of these results, it was apparent that the success of 6- deoxyacyclovir as a prodrug in vivo would depend upon how well its desired activation by xanthine oxidase competed with the nonactivating oxidations by aldehyde oxidase. In rats dosed orally with 6- deoxyacyclovir , absorption was extensive and the major urinary metabolite was acyclovir. In two human volunteers, urinary excretions of acyclovir were 5-6 times greater than those typically observed after administration of equivalent doses of acyclovir itself. The areas under the plasma concentration-time curves for acyclovir were also 5-6 times greater. Plasma levels of acyclovir peaked soon after ingestion of the prodrug, indicating rapid absorption and metabolic conversion. These results suggested that 6- deoxyacyclovir might have clinical usefulness as a prodrug of acyclovir suitable for oral administration.

A bicycle ergometer for high altitude investigations.

The basic Monark bicycle ergometer was machined into twelve pieces which allowed it to be transported in backpacks to high altitudes. The machining, reassembly and calibration of the adapted ergometer are discussed. To determine whether the functional characteristics of the bicycle ergometer had been affected by the modifications, 18 subjects randomly pedalled a standard bicycle ergometer or the modified model at workloads of 300, 600 and 900 kpm at 50 rpm for 6 minutes at each level, while their heart rates and oxygen uptake values were collected. Subsequent analysis revealed no significant differences between bicycles for either heart rate or oxygen uptake. This adapted ergometer proved to be very functional for carrying out high altitude investigations in which a controlled work load is required.

9-(2-hydroxyethoxymethyl) guanine activity against viruses of the herpes group.

Of a series of nucleoside analogues synthesised, 9-(2-hydroxyethoxymethyl) guanine was found to have marked antiviral activity in animal models of herpes virus infections, associated with very low toxicity.

Selectivity of action of an antiherpetic agent, 9-(2-hydroxyethoxymethyl) guanine.

A guanine derivative with an acyclic side chain, 2-hydroxyethoxymethyl, at position 9 has potent antiviral activity [dose for 50% inhibition (ED(50)) = 0.1 muM] against herpes simplex virus type 1. This acyclic nucleoside analog, termed acycloguanosine, is converted to a monophosphate by a virus-specified pyrimidine deoxynucleoside (thymidine) kinase and is subsequently converted to acycloguanosine di- and triphosphates. In the uninfected host cell (Vero) these phosphorylations of acycloguanosine occur to a very limited extent. Acycloguanosine triphosphate inhibits herpes simplex virus DNA polymerase (DNA nucleotidyltransferase) 10-30 times more effectively than cellular (HeLa S3) DNA polymerase. These factors contribute to the drug's selectivity; inhibition of growth of the host cell requires a 3000-fold greater concentration of drug than does the inhibition of viral multiplication. There is, moreover, the strong possibility of chain termination of the viral DNA by incorporation of acycloguanosine. The identity of the kinase that phosphorylates acycloguanosine was determined after separation of the cellular and virus-specified thymidine kinase activities by affinity chromatography, by reversal studies with thymidine, and by the lack of monophosphate formation in a temperature-sensitive, thymidine kinase-deficient mutant of the KOS strain of herpes simplex virus type 1 (tsA1).