Antitumor potential of acyclic nucleoside phosphonates.

Acyclic nucleoside phosphonates such as HPMPC (cidofovir) and PMEA (adefovir) have been identified as broad-spectrum antiviral agents that are effective against herpes-, retro- and hepadnavirus infections (PMEA) and herpes-, pox-, adeno-, polyoma-, and papillomavirus infections (HPMPC). Here we show that HPMPC and PMEA also offer great potential as antitumor agents, through the induction of tumor cell differentiation (PMEA), inhibition of angiogenesis (HPMPC) and induction of apoptosis (HPMPC). In vivo tumor regressions have been noted for choriocarcinoma (PMEA) in rats, hemangioma (HPMPC) in rats and papillomatous lesions (HPMPC) in humans. Acyclic nucleoside phosphonates can be considered as a new dimension to the discipline of chemotherapy. They have a unique mode of action that is targeted at (viral or tumoral) DNA synthesis. They exhibit a pronounced and prolonged anti-viral and/or tumoral activity that can persist for days or weeks after a single administration. Most importantly, they have a uniquely broad spectrum of indications for clinical use, encompassing both DNA- and retrovirus infections, as well as various forms of cancer of both viral and non-viral origin.     

Antitumor Potential of Acyclic Nucleoside Phosphonates

Abstract

Acyclic nucleoside phosphonates such as HPMPC (cidofovir) and PMEA (adefovir) have been identified as broad-spectrum antiviral agents that are effective against herpes-, retro- and hepadnavirus infections (PMEA) and herpes-, pox-, adeno-, polyoma-, and papillomavirus infections (HPMPC). Here we show that HPMPC and PMEA also offer great potential as antitumor agents, through the induction of tumor cell differentiation (PMEA), inhibition of angiogenesis (HPMPC) and induction of apoptosis (HPMPC). In vivo tumor regressions have been noted for choriocarcinoma (PMEA) in rats, hemangioma (HPMPC) in rats and papillomatous lesions (HPMPC) in humans. Acyclic nucleoside phosphonates can be considered as a new dimension to the discipline of chemotherapy. They have a unique mode of action that is targeted at (viral or tumoral) DNA synthesis. They exhibit a pronounced and prolonged anti-viral and/or tumoral activity that can persist for days or weeks after a single administration. Most importantly, they have a uniquely broad spectrum of indications for clinical use, encompassing both DNA- and retrovirus infections, as well as various forms of cancer of both viral and non-viral origin.     

Evaluation of Novel Acyclic Nucleoside Phosphonates against Human and Animal Gammaherpesviruses Revealed an Altered Metabolism of Cyclic Prodrugs upon Epstein-Barr Virus Reactivation in P3HR-1 Cells

Acyclic nucleoside phosphonates (ANPs), such as (S)-1-[(3-hydroxy-2-phosphonomethoxy)propyl)]cytosine (HPMPC), are an important group of broad-spectrum antiviral agents with activity against DNA viruses. In this report, we present the in vitro potencies of novel ANPs against gammaherpesviruses, including Kaposi''s sarcoma-associated herpesvirus, Epstein-Barr virus (EBV), and three animal gammaherpesviruses. 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine (HPMP-5-azaC), (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-3-deazaadenine (3-deaza-HPMPA), and their cyclic derivatives have emerged as highly potent antigammaherpesvirus agents. Interestingly, cyclic prodrugs of ANPs exhibited reduced activities against EBV strain P3HR-1, but not against EBV strain Akata. Cell culture metabolism studies with HPMPC and cyclic HPMPC revealed that these differences were attributable to an altered drug metabolism in P3HR-1 cells after EBV reactivation and, more specifically, to a reduced hydrolysis of cyclic HPMPC by cyclic CMP phosphodiesterase. We did not correlate this effect with phosphodiesterase downregulation, or to functional mutations. Instead, altered cyclic AMP levels in P3HR-1 cells indicated a competitive inhibition of the phosphodiesterase by this cyclic nucleotide. Finally, both HPMPC and HPMP-5-azaC emerged as highly effective inhibitors in vivo through significant inhibition of murine gammaherpesvirus replication and dissemination. With the current need for potent antigammaherpesvirus agents, our findings underline the requirement of appropriate surrogate viruses for antiviral susceptibility testing and highlight HPMP-5-azaC as a promising compound for future clinical development.     

Therapeutic Potential of HPMPC (Cidofovir), PMEA (Adefovir) and Related Acyclic Nucleoside Phosphonate Analogues as Broad-Spectrum Anttviral Agents

Abstract

This article reviews the antiviral features of the acyclic nucleoside phosphonate (ANP) analogues, with a special focus on the most recent findings concerning the biochemistry and clinical efficacy of HPMPC [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine; cidofovir; Vistide®] and PMEA [9-(2-phosphonyl-methoxyethyl)adenine; adefovir].     

Pharmacokinetics of salicylate ester prodrugs of cyclic HPMPC in dogs

A series of aryl ester prodrugs of cyclic HPMPC have been synthesized and their physicochemical properties, pharmacokinetics and metabolism have been evaluated. Chemical stability was dependent on the orientation of the exo-cyclic ligand; the equatorial isomers were 5.4 to 9.4 fold more reactive than the axial isomers. The oral bioavailability of cyclic HPMPC from the aryl ester prodrugs ranged from 11.2% for o-pentylphenyl cyclic HPMPC to 46.3% for butylsalicylyl cyclic HPMPC. Cyclic HPMPC was the major metabolite observed for all the salicylyl ester prodrugs. Cidofovir accounted for 2 to 12% of the total plasma AUC for butyl-, cyclohexyl- and phenethyl-salicylyl esters of cyclic HPMPC. Intact prodrug or the corresponding monosalicylyl esters of cidofovir each accounted for less than 10% of the total AUC for salicylyl ester prodrugs.     

PHARMACOKINETICS OF SALICYLATE ESTER PRODRUGS OF CYCLIC HPMPC IN DOGS

A series of aryl ester prodrugs of cyclic HPMPC have been synthesized and their physicochemical properties, pharmacokinetics and metabolism have been evaluated. Chemical stability was dependent on the orientation of the exo-cyclic ligand; the equatorial isomers were 5.4 to 9.4 fold more reactive than the axial isomers. The oral bioavailability of cyclic HPMPC from the aryl ester prodrugs ranged from 11.2% for o-pentylphenyl cyclic HPMPC to 46.3% for butylsalicylyl cyclic HPMPC. Cyclic HPMPC was the major metabolite observed for all the salicylyl ester prodrugs. Cidofovir accounted for 2 to 12% of the total plasma AUC for butyl-, cyclohexyl- and phenethyl-salicylyl esters of cyclic HPMPC. Intact prodrug or the corresponding monosalicylyl esters of cidofovir each accounted for less than 10% of the total AUC for salicylyl ester prodrugs.     

Cidofovir resistance in vaccinia virus is linked to diminished virulence in mice

Cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC)] is recognized as a promising drug for the treatment of poxvirus infections, but drug resistance can arise by a mechanism that is poorly understood. We show here that in vitro selection for high levels of resistance to HPMPC produces viruses encoding two substitution mutations in the virus DNA polymerase (E9L) gene. These mutations are located within the regions of the gene encoding the 3'-5' exonuclease (A314T) and polymerase (A684V) catalytic domains. These mutant viruses exhibited cross-resistance to other nucleoside phosphonate drugs, while they remained sensitive to other unrelated DNA polymerase inhibitors. Marker rescue experiments were used to transfer A314T and/or A684V alleles into a vaccinia virus Western Reserve strain. Either mutation alone could confer a drug resistance phenotype, although the degree of resistance was significantly lower than when virus encoded both mutations. The A684V substitution, but not the A314T change, also conferred a spontaneous mutator phenotype. All of the HPMPC-resistant recombinant viruses exhibited reduced virulence in mice, demonstrating that these E9L mutations are inextricably linked to reduced fitness in vivo. HPMPC, at a dose of 50 mg/kg of body weight/day for 5 days, still protected mice against intranasal challenge with the drug-resistant virus with A314T and A684V mutations. Our studies show that proposed drug therapies offer a reasonable likelihood of controlling orthopoxvirus infections, even if the viruses encode drug resistance markers.     

Synthesis and Antiherpesvirus Activity of (S)-1-((3-Hydroxy-2-Phosphon-Ylmethoxy)Propyi.)Cytosine (HPMPC) and Related Nucleotide Analogues

Abstract

Of a series of phosphonate nucleotide analogues, HPMPC (2) showed the greatest in vivo therapeutic index against herpes simplex viruses.     

An efficient process for the synthesis of cyclic HPMPC

1-[((S)-2-Hydroxy-2-oxo-1,4,2-dioxaphosphorinan-5-yl)methyl] cytosine (cyclic HPMPC) was readily synthesized in gram to multi-kilogram quantities by treating a DMF suspension of HPMPC with four molar equivalents of ethyl chloroformate. This dehydrative intramolecular cyclization process typically afforded cHPMPC in 94% isolated yield and high purity. Benign by-products and solvents were easily removed.     

AN EFFICIENT PROCESS FOR THE SYNTHESIS OF CYCLIC HPMPC

1-[((S)-2-Hydroxy-2-oxo-1,4,2-dioxaphosphorinan-5-yl)methyl] cytosine (cyc-lic HPMPC) was readily synthesized in gram to multi-kilogram quantities by treating a DMF suspension of HPMPC with four molar equivalents of ethyl chloroformate. This dehydrative intramolecular cyclization process typically afforded cHPMPC in 94% isolated yield and high purity. Benign by-products and solvents were easily removed.     

Stable expression of a recombinant sodium-dependent, pyrimidine-selective nucleoside transporter (CNT1) in a transport-deficient mouse leukemia cell line.

Previous studies of nucleoside transport in mammalian cells have identified two types of activities: the equilibrative nucleoside transporters and concentrative, Na+-nucleoside cotransporters. Characterization of the concentrative nucleoside transporters has been hampered by the presence in most cells and tissues of multiple transporters with overlapping permeant specificities. With the recent cloning of cDNAs encoding rat and human members of the concentrative nucleoside transporter (CNT) family, it is now possible to study the concentrative transporters in isolation by use of functional expression systems. We report here the isolation of a nucleoside transport-deficient subline of L1210 mouse leukemia (L1210/DNC3) that is a suitable recipient for stable expression of cloned nucleoside transporter cDNAs. We have used L1210/DNC3 as the recipient in gene transfer studies to develop a stable cell line (L1210/DU5) that produces the recombinant concentrative nucleoside transporter with selectivity for pyrimidine nucleosides (CNT1) that was initially identified in rat intestine (Q.Q. Huang, S.Y. Yao, M.W. Ritzel, A.R.P. Paterson, C.E. Cass, and J.D. Young. 1994. J. Biol. Chem. 269: 17,757-17,760). L1210/DU5 was used to examine the permeant selectivity of recombinant rat CNT1 by comparing a series of nucleoside analogs with respect to (i) inhibition of inward fluxes of [3H]thymidine, (ii) initial rates of transport of 3H-analog, and (iii) cytotoxicity to L1210/DU5 versus the parental transport-deficient cell line. By all three criteria, recombinant CNT1 transported 5-fluoro-2'-deoxyuridine and 5-fluorouridine well and cytosine arabinoside poorly. Although some purine nucleosides (2'-deoxyadenosinedeoxyadeno-2'-deoxyadenosine, 7-deazaadenosine) were potent inhibitors of CNT1, they were poor permeants when uptake was measured directly by analysis of isotopic fluxes or indirectly by comparison of cytotoxicity ratios. We conclude that comparison of analog cytotoxicity to L1210/DU5 versus L1210/DNC3 is a reliable indirect predictor of transportability, suggesting that cytotoxicity assays with a panel of such cell lines, each with a different recombinant nucleoside transporter, would be a valuable tool in the development of antiviral and antitumor nucleoside analogs.     

Point mutations in the DNA polymerase gene of human cytomegalovirus that result in resistance to antiviral agents.

Three independently isolated mutants of human cytomegalovirus strain AD169 were found to be resistant to ganciclovir at a 50% effective dose of 200 microM. Phosphorylation of ganciclovir was reduced 10-fold in mutant-infected cells compared with AD169-infected cells. All three mutants were also determined to be resistant to the nucleotide analogs (S)-1-[(3-hydroxy-2- phosphonylmethoxy)propyl]adenine (HPMPA) and (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine (HPMPC) and hypersensitive to thymine-1-D-arabinofuranoside (AraT). Single base changes resulting in amino acid substitutions were demonstrated in the nucleotide sequence of the DNA polymerase gene of each mutant. The polymerase mutation contained in one of the mutants was transferred to the wild-type AD169 background. Ganciclovir phosphorylation in cells infected with the recombinant virus produced by this transfer was found to be equivalent to that of AD169-infected cells. The ganciclovir resistance of the recombinant was reduced fourfold compared with that of the parental mutant; however, the recombinant remained resistant to HPMPA and HPMPC and hypersensitive to AraT. The ganciclovir resistance of the mutants therefore appears to result from mutations in two genes: (i) a kinase which phosphorylates ganciclovir and (ii) the viral DNA polymerase.     

Computerized Interpretation of the Electron Ionization Mass Spectra of Nucleoside TMS Derivatives

Abstract

A computer program has been developed for the automated interpretation of mass spectra of TMS derivatives of nucleosides found in human urine. The m/z values in the unknown spectrum are compared to m/z values of 3 different ion series commonly observed in the mass spectra of nucleoside TMS derivatives.¹ If a correlation exists, the unknown spectra are marked with color according to the scheme: 1) blue—molecular ion series, 2) red—base ion series and 3) yellow—sugar ion series. The program suggests a structural assignment for each of the marked ions and calculates a series related ion current. The calculated ion current is used to assign the of sugar contained in the unknown nucleoside.     

Synthesis, conformation analysis, and antiviral activity of benzimidazole ribofuranosides

To study the structure-biological effect correlation in the series of nucleoside analogues containing deazapurines, a number of 2-R-benzimidazole 1-beta-D-ribofuranosides (R = H, CF3, SCF3, CH2SCF3, CH2Ph, CH2CN) have been prepared by the modified silyl method. On the basis of CD and PMR data it was shown that the compounds exist in solution mainly as syn-conformers. Calculation of the furanose ring pseudorotation parameters in terms of N-S model indicates the predominance of S-population. In contrast to acyclonucleosides, the ribofuranosides obtained are nonactive against entheroviruses and more cytotoxic.     

Synthesis of a series of NAD<Superscript>+</Superscript> analogues,potential inhibitors of PARP 1, using ADP conjugates functionalized at the terminal phosphate group

A convenient approach has been proposed to the synthesis of nicotinamide adenine dinucleotide (NAD⁺) mimetics, which comprise morpholino analogues of nucleosides. The approach is based on the use of ADP conjugates containing an amino group, which is tethered to the terminal phosphate through the aliphatic linker by the phosphodiester bond. We have synthesized four series of the NAD⁺ mimetics, which differ in the type of the modified nucleoside (2-aminomethylmorpholine (Mor) or 2-aminomethyl-4-carboxymethylmorpholine (MorGly) derivatives), in the linker length, and in the manner of the nucleoside attachment to the ADP derivative. We have studied the efficiency of NAD⁺ mimetics in the inhibition of the auto-poly(ADP-ribosyl)ation by the PARP 1 enzyme. The linker length, the mode of the attachment of the morpholino nucleoside analogue, and the nature of the heterocyclic base of the modified nucleoside were shown to inf luence the inhibition efficiency.     

Synthesis and structural analysis of oxadiazole carboxamide deoxyribonucleoside analogs

Two novel C-linked oxadiazole carboxamide nucleosides 5-(2'-deoxy-3',5'-beta-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-5-carboxamide (1) and 5-(2'-deoxy-3',5'-beta-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-3-carboxamide (2) were successfully synthesized and characterized by X-ray crystallography. The crystallographic analysis shows that both unnatural nucleoside analogs 1 and 2 adapt the C2'-endo ("south") conformation. The orientation of the oxadiazole carboxamide nucleobase moiety was determined as anti (conformer A) and high anti (conformer B) in the case of the nucleoside analog 1 whereas the syn conformation is adapted by the unnatural nucleoside 2. Furthermore, nucleoside analogs 1 and 2 were converted with high efficiency to corresponding nucleoside triphosphates through the combination chemo-enzymatic approach. Oxadiazole carboxamide deoxyribonucleoside analogs represent valuable tools to study DNA polymerase recognition, fidelity of nucleotide incorporation, and extension.     

Synthesis of azole nucleoside 5'-monophosphate mimics (P1Ms) and their inhibitory properties of IMP dehydrogenases

IMPDH inhibitors have potential antimicrobial, anticancer and immunomodulatory effects. Nucleoside inhibitors of IMPDH exert their inhibitory effects via nucleoside 5'-MPs. Conversion of nucleoside analogs to NMPs by cellular nucleoside kinases is not assured, and usually is inefficient. In order to bypass cellular phosphorylation, a series of azole nucleoside 5'-MP mimics (P1Ms) based on ribavirin, EICAR and bredinin were synthesized and screened against human and C. albicans IMP dehydrogenises. P1Ms 8, 16, 25, 28 and 29 demonstrated substantial IMPDH inhibition with Ki values in low micromolar range.     

Synthesis and biological activities of new carbaacyclonucleosides and 1'-oxaacyclonucleosides related to clitocine

We describe the synthesis of two series of acyclonucleosides:carbaacyclonucleosides and 1'-oxaacyclonucleosides which possess the same aglycone as clitocine 3 which is a natural nucleoside exhibiting interesting biological properties. These compounds have been obtained by condensation of 4-aminobutanol or 3-silyloxypropoxyamine with 4,6-dichloro-5-nitropyrimidine. Structural modifications have been made on the heterocyclic base and the side chain to enhance their potential activity. All these compounds have been tested against different viruses: HIV-1, HSV-1, HSV-2, CMV, VZV, EBV. The carbaacyclonucleoside 10 was associated with an anti-EBV activity (EC50 = 0.86 microg/mL).