Novel hepatotrophic prodrugs of the antiviral nucleoside 9-(2-phosphonylmethoxyethyl)adenine with improved pharmacokinetics and antiviral activity.

The device of new hepatotrophic prodrugs of the antiviral nucleoside 9-(2-phosphonylmethoxyethyl)adenine (PMEA) with specificity for the asialoglycoprotein receptor on parenchymal liver cells is described. PMEA was conjugated to bi- and trivalent cluster glycosides (K(GN)(2) and K(2)(GN)(3), respectively) with nanomolar affinity for the asialoglycoprotein receptor. The liver uptake of the PMEA prodrugs was more than 10-fold higher than that of the parent drug (52+/-6% and 62+/-3% vs. 4.8+/-0.7% of the injected dose for PMEA) and could be attributed for 90% to parenchymal cells. Accumulation of the PMEA prodrugs in extrahepatic tissue (e.g., kidney, skin) was substantially reduced. The ratio of parenchymal liver cell-to-kidney uptake-a measure of the prodrugs therapeutic window-was increased from 0.058 +/- 0.01 for PMEA to 1.86 +/- 0.57 for K(GN)(2)-PMEA and even 2.69 +/- 0.24 for K(2)(GN)(3)-PMEA. Apparently both glycosides have a similar capacity to redirect (antiviral) drugs to the liver. After cellular uptake, both PMEA prodrugs were converted into the parent drug, PMEA, during acidification of the lysosomal milieu (t(1/2) approximately 100 min), and the released PMEA was rapidly translocated into the cytosol. The antiviral activity of the prodrugs in vitro was dramatically enhanced as compared to the parent drug (5- and 52-fold for K(GN)(2)-PMEA and K(2)(GN)(3)-PMEA, respectively). Given the 15-fold enhanced liver uptake of the prodrugs, we anticipate that the potency in vivo will be similarly increased. We conclude that PMEA prodrugs have been developed with greatly improved pharmacokinetics and therapeutic activity against viral infections that implicate the liver parenchyma (e.g., HBV). In addition, the significance of the above prodrug concept also extends to drugs that intervene in other liver disorders such as cholestasis and dyslipidemia.     

Drug targeting: anti-HSV-1 activity of mannosylated polymer-bound 9-(2-phosphonylmethoxyethyl)adenine

The antiviral drug, 9-(2-phosphonylmethoxyethyl) adenine (PMEA) was linked to a synthetic and neutral polymer bearing mannosyl residues to allow its internalization by macrophages via membrane lectins. PMEA bound to the mannosylated polymer was more efficient in vitro than free PMEA in preventing lysis of human macrophages by herpes virus.     

5-Phosphoribosyl 1-pyrophosphate synthetase converts the acyclic nucleoside phosphonates 9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine and 9-(2-phosphonylmethoxyethyl)adenine directly to their antivirally active diphosphate derivatives.

The acyclic nucleoside phosphonates 9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA) and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) are potent inhibitors of DNA viruses and retroviruses, respectively. Unlike nucleoside triphosphates, the metabolically active (diphosphorylated) forms of HPMPA and PMEA (designated HPMPApp and PMEApp) are synthesized in a reversible reaction in which the pyrophosphate group of 5-phosphoribosyl 1-pyrophosphate (PRPP) is directly transferred to HPMPA and PMEA by purified PRPP synthetase. In this respect, PRPP synthetase does not act stereospecifically in that it recognizes both the S-enantiomer and the R-enantiomer of HPMPA as substrate. PRPP synthetase also recognizes other acyclic adenine and 2,6-diaminopurine riboside phosphonates as a substrate. It is now imperative to evaluate the potential role of PRPP synthetase, as activating enzyme, in the antiviral action of this type of molecules in intact cells.     

Ultra sensitive method for the determination of 9-(2-phosphonylmethoxyethyl)adenine in human serum by liquid chromatography-tandem mass spectrometry

An ultra sensitive method for the direct measurement of 9-(2-phosphonylmethoxyethyl)adenine (PMEA), an antiviral agent for hepatitis B, in human serum using high performance liquid chromatography/tandem mass spectrometry (LC-MS/MS) has been developed. This method involves the addition of [13C]PMEA (contains 5 13C) as internal standard, the purification and enrichment by a MCX solid phase extraction (SPE) cartridge, and quantitative analysis using LC-MS/MS. The MS/MS is selected to monitor the m/z 272 --> 134 and m/z 277 --> m/z 139 transitions for PMEA and [13C]PMEA, respectively, using negative electrospray ionization. The MS/MS response is linear over a concentration of 0.1-10 ng/ml with a lower limit of quantitation (LLOQ) of 0.1 ng/ml. The mean inter-assay accuracy (%Bias) for quality control (QC) at 0.1, 0.25, 1.0, and 10 ng/ml are 10, 1.6, -0.8, and 0.0%, respectively. The mean inter-assay precision (%CV) for the corresponding QCs is 3.9, 3.8, 5.3, and 3.4%, respectively. The method has been used to determine PMEA concentration in human serum following a single oral administration of a PMEA pro-drug at dose of 10 and 30 mg.     

Interaction of herpes simplex virus-induced DNA polymerase with 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate

The triphosphate of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) competitively inhibits incorporation of dGTP into DNA catalyzed by DNA polymerases specified by both type 1 and type 2 herpes simplex virus. K1 values were estimated to be 33 nM for type 1 and 46 nM for type 2-specified DNA polymerase. DHPG acted as an alternate substrate to dGTP for the virus-specified DNA polymerase. Incorporation of DHPG into DNA resulted in the slowing down of the rate of DNA synthesis. The position of DHPG incorporation was analyzed, and it was found to enter both internal and terminal linkages. DNA which contained DHPG at termini was found to competitively inhibit utilization of activated DNA as primer. DNA polymerase alpha and DNA polymerases from several phosphonoformic acid-resistant herpes simplex virus type 1 strains were examined for sensitivity to 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate. A lack of correlation between the in vivo sensitivities of the virus mutants and the K1 values of the DNA polymerases was noted.     

X-ray study of an antiviral agent (S)-9-(2,3-dihydroxypropyl)adenine

The molecular and crystal structures of the antiviral compound, (S)-9-(2,3-dihydroxypropyl)adenine, was established. The space group is P21, unit-cell parameters a 5,546(1), b 8,381(1), c 10,119(1), beta 91,979(9) degrees, Z2. The structure was solved by the direct method and refined by a full-matrix least-squares procedure to R 4,2%. All non-hydrogen atoms of this compound are concentrated in two planes. The first one involves the atoms of the purine moiety and N(6) and C(1'), while the second one accommodates C(2'), C(3'), O(2') and O(3'). The angle between these planes is 54,3 degrees. The conformation of the compound in crystal was compared with that deduced from theoretical analysis.     

Inhibition of herpes simplex virus-induced DNA polymerase activity and viral DNA replication by 9-(2-hydroxyethoxymethyl)guanine and its triphosphate.

The effect of the nucleoside analog 9-(2-hydroxyethoxymethyl)guanine (acycloguanosine) on herpes simplex virus type 1 DNA synthesis was examined. Acycloguanosine inhibited herpesvirus DNA synthesis in virus-infected cells. The synthesis of host cell DNA was only partially inhibited in actively growing cells at acycloguanosine concentrations several hundred-fold greater than the 50% effective dose for herpes simplex virus type 1. Studies using partially purified enzymes revealed that the triphosphate of this compound inhibited the virus-induced DNA polymerases (DNA nucleotidyltransferases) to a greater degree than the DNA polymerase of the host cell, that the inhibition was dependent upon the base composition of the template, and that the triphosphate was a better substrate for the virus-induced polymerases than for the alpha cellular DNA polymerases.     

Inhibition of cellular DNA polymerase alpha and human cytomegalovirus-induced DNA polymerase by the triphosphates of 9-(2-hydroxyethoxymethyl)guanine and 9-(1,3-dihydroxy-2-propoxymethyl)guanine.

The triphosphates of 9-(2-hydroxyethoxymethyl)guanine and 9-(1,3-dihydroxy-2-propoxymethyl)guanine were examined for their inhibitory effect on highly purified cellular DNA polymerase alpha and human cytomegalovirus (Towne strain)-induced DNA polymerase. These two nucleoside triphosphates competitively inhibited the incorporation of dGMP into DNA catalyzed by the DNA polymerases. The virus-induced DNA polymerase had greater binding affinity for the triphosphate of 9-(2-hydroxyethoxymethyl)guanine (Ki, 8 nM) than for the triphosphate of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (Ki, 22 nM), although the nucleoside of the latter compound was strikingly more effective against human cytomegalovirus replication in cell cultures than the nucleoside of the former. The Ki values of these two nucleoside triphosphates for alpha polymerase were 96 and 146 nM, respectively, and were 7- to 12-fold higher than those for the virus-induced enzyme. These data indicated that virus-induced DNA polymerase was more sensitive to inhibition by these two nucleoside triphosphates than was the cellular alpha enzyme.     

Synthesis of 9-[1-(substituted)-2-(phosphonomethoxy)ethyl]adenine derivatives as possible antiviral agents

Various C-1'-substituted acyclic N9 adenine nucleosides were prepared from 9-[(1-hydroxymethyl)(3-monomethoxytrityloxy)propyl]-N6-monomethoxytrityladenine. The hydroxymethyl was modified to the phosphonomethoxy derivative, and the 3-monomethoxytrityloxy was converted to hydroxyl, methoxy, azido, and amino. Other substituents, such as ethyl and ea-hydroxyethyl were also prepared. The resulting phosphonomethoxy derivatives were converted to prodrugs.     

Synthesis and in vitro antiviral activity evaluation of 9-(2-azido-2,3-dideoxy-beta-D-threo-pentofuranosyl)adenine derivatives

9-(2-Azido-2,3-dideoxy-beta-D-threo-pentofuranosyl)adenine derivatives (1a-e) containing a lipophilic function at the N-6 position in the purine ring were prepared and evaluated for their antiviral activity. The compounds 1a-e turned out to be inactive as antiviral agents.     

Herpes simplex virus DNA polymerase as the site of phosphonoacetate sensitivity: temperature-sensitive mutants.

Temperature-sensitive (ts) mutants in a number of complementation groups of herpes simplex virus type 1 (HSV-1) are deficient in DNA polymerase induction at the restrictive temperature. Twenty-two mutants in 15 complementation groups were tested for sensitivity to phosphonoacetate (PAA), a compound that inhibits HSV replication in vivo and the DNA polymerase in vitro. One mutant, tsD9, was resistant to PAA (Pr), whereas all others were sensitive. Revertants of tsD9 to the ts+ phenotype simultaneously lost PAA resistance. Additional Pr mutants were isolated from ts mutants belonging to several complementation groups of HSV-1. Double mutants (ts Pr phenotype) were used in three-factor recombination analyses to locate the PAA locus on the genetic map at a position indistinguishable from the ts lesion in tsD9. In all cases, resistance or sensitivity to PAA in vivo was correlated with resistance or sensitivity of DNA polymerase in vitro. These data are compatible with the temperature-sensitive lesion of tsD9 and the determinant of PAA sensitivity both residing in the structural gene for DNA polymerase.     

Intramolecular stacking interactions in ternary copper(II) complexes formed by a heteroaromatic amine and 9-[2-(2-phosphonoethoxy)ethyl]adenine, a relative of the antiviral nucleotide analogue 9-[2-(phosphonomethoxy)ethyl]adenine

The stability constants of the mixed-ligand complexes formed between Cu(Arm)2+, where Arm=2,2'-bipyridine (Bpy) or 1,10-phenanthroline (Phen), and the dianions of 9-[2-(2-phosphonoethoxy)ethyl]adenine (PEEA2-) and (2-phosphonoethoxy)ethane (PEE2-), also known as [2-(2-ethoxy)ethyl]phosphonate, were determined by potentiometric pH titrations in aqueous solution (25 degrees C; I=0.1 M, NaNO3). The ternary Cu(Arm)(PEEA) complexes are considerably more stable than the corresponding Cu(Arm)(R-PO3) species, where R-PO3(2-) represents a phosph(on)ate ligand with a group R that is unable to participate in any kind of interaction within the complexes. The increased stability is attributed to intramolecular stack formation in the Cu(Arm)(PEEA) complexes and also, to a smaller extent, to the formation of 6-membered chelates involving the ether oxygen atom present in the -CH2-O-CH2-CH2-PO3(2-) residue of PEEA2-. This latter interaction is separately quantified by studying the ternary Cu(Arm)(PEE) complexes which can form the 6-membered chelates but where no intramolecular ligand-ligand stacking is possible. Application of these results allows a quantitative analysis of the intramolecular equilibria involving three structurally different Cu(Arm)(PEEA) species; e.g., of the Cu(Bpy)(PEEA) system about 11% exist with the metal ion solely coordinated to the phosphonate group, 4% as a 6-membered chelate involving the ether oxygen atom of the -CH2-O-CH2CH2-PO3(2-) residue, and 85% with an intramolecular stack between the adenine moiety of PEEA2- and the aromatic rings of Bpy. In addition, the Cu(Arm)(PEEA) complexes may be protonated, leading to Cu(Arm)(H;PEEA)+ species for which it is concluded that the proton is located at the phosphonate group and that the complexes are mainly formed (50 and 70%) by a stacking adduct between Cu(Arm)2+ and the adenine residue of H(PEEA)-. Finally, the stacking properties of adenosine 5'-monophosphate (AMP2-), of the dianion of 9-[2-(phophonomethoxy)ethyl]adenine (PMEA2-) and of several of its analogues (=PA2-) are compared in their ternary Cu(Arm)(AMP) and Cu(Arm)(PA) systems. Conclusions regarding the antiviral properties of several acyclic nucleoside phosphonates are shortly discussed.     

Herpes simplex virus type 1 DNA polymerase. Mechanism of inhibition by acyclovir triphosphate

Acyclovir triphosphate (ACVTP) was a substrate for herpes simplex virus type 1 (HSV-1) DNA polymerase and was rapidly incorporated into a synthetic template-primer designed to accept either dGTP or ACVTP followed by dCTP. HSV-1 DNA polymerase was not inactivated by ACVTP, nor was the template-primer with a 3'-terminal acyclovir monophosphate moiety a potent inhibitor. Potent inhibition of HSV-1 DNA polymerase was observed upon binding of the next deoxynucleoside 5'-triphosphate coded by the template subsequent to the incorporation of acyclovir monophosphate into the 3'-end of the primer. The Ki for the dissociation of dCTP (the "next nucleotide") from this dead-end complex was 76 nM. In contrast, the Km for dCTP as a substrate for incorporation into a template-primer containing dGMP in place of acyclovir monophosphate at the 3'-primer terminus was 2.6 microM. The structural requirements for effective binding of the next nucleotide revealed that the order of potency of inhibition of a series of analogs was: dCTP much greater than arabinosyl-CTP greater than 2'-3'-dideoxy-CTP much greater than CTP, dCMP, dCMP + PPi. In the presence of the next required deoxynucleotide (dCTP), high concentrations of dGTP compete with ACVTP for binding and thus retard the formation of the dead-end complex. This results in a first-order loss of enzyme activity indistinguishable from that expected for a mechanism-based inactivator. The reversibility of the dead-end complex was demonstrated by steady-state kinetic analysis, analytical gel filtration, and by rapid gel filtration through Sephadex G-25. Studies indicated that potent, reversible inhibition by ACVTP and the next required deoxynucleoside 5'-triphosphate also occurred when poly(dC)-oligo(dG) or activated calf thymus DNA were used as the template-primer.     

Synthesis of 9-[1-(substituted)-3-(phosphonomethoxy)propyl]adenine derivatives as possible antiviral agents

Acyclic N9 adenine nucleosides substituted at C-1' position were prepared by the Mitsunobu reaction of 1-tert-butyldimethylsilyl-4-pivaloylbutan-1,2,4-triol (5) with adenine. Pivaloyl hydroxyl was modified to the phosphonomethoxy derivatives, and the tert-butyldimethylsilyl hydroxyl was converted to methoxy, azido, amino, fluoro, and c-hydroxyethyl and was eliminated to give vinyl. The resulting phosphonic acids were converted to prodrugs also.     

Inhibition of African swine fever virus DNA synthesis by (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine

The acyclic nucleotide analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA] is a potent and selective inhibitor of African swine fever virus (ASFV) replication. Using the DNA-DNA hybridization technique with plasmid pRPEL-2 as probe, we have shown that (S)-HPMPA exerts a specific, dose-dependent, inhibitory effect on viral DNA synthesis. Also, (S)-HPMPA inhibits the production of late viral proteins, especially IP-73, in ASFV-infected MS and Vero cells. When evaluated under the same experimental conditions, phosphonoacetic acid (PAA) also caused an inhibition of viral DNA and late viral protein synthesis but only so at a concentration which was 10- to 20-fold higher than that required for (S)-HPMPA.     

Fluorinated methylenecyclopropane analogues of nucleosides. Synthesis and antiviral activity of (Z)- and (E)-9-{[(2-fluoromethyl-2-hydroxymethyl)-cyclopropylidene]methyl}adenine and -guanine

Synthesis and antiviral activity of the title fluoromethylenecyclopropane analogues 15a, 15b, 16a, and 16b is described. Methylenecyclopropane carboxylate was first transformed to 2,2-bis-hydroxymethylmethylenecyclopropane. Selective monoacetylation followed by introduction of fluorine gave 2-acetoxymethyl-2-fluoromethylmethylenecyclopropane as the key intermediate. The synthesis of analogues 15a, 15b, 16a, and 16b then followed alkylation-elimination procedure as described previously for other methylenecyclopropane analogues [corrected] Compounds 15a, 15b, 16a and 16b were not active against Epstein-Barr virus (EBV) [corrected] Analogue 15a inhibited hepatitis C virus by virtue of its cytotoxicity and it moderately inhibited replication of the Towne strain of human cytomegalovirus (HCMV). The E-isomer 16a was a substrate for adenosine deaminase, whereas the Z-isomer 15a was not deaminated.     

In vitro Dimroth rearrangement of 1-(2-carboxyethyl) adenine to N6-(2-carboxyethyl)adenine in single-stranded calf thymus DNA.

The new adduct N6-(2-carboxyethyl)adenine (N6-CEA) was prepared from 1-(2-carboxyethyl)adenine (1-CEA) by base catalyzed (Dimroth) rearrangement of 1-CEA. The structure of N6-CEA was assigned on the basis of UV spectra and electron impact and isobutane chemical ionization mass spectra. When the carcinogen beta-propiolactone was reacted in vitro with calf thymus DNA, 1-CEA but not N6-CEA was detected on paper chromatograms following acid hydrolysis of the DNA. When BPL-reacted single-stranded DNA was incubated at pH 11.7 (37 degrees C, 18 h) prior to acid hydrolysis, it was found that 1-CEA was completely converted to N6-CEA in DNA by Dimroth rearrangement, whereas no conversion occurred at pH 7.5. The extent of Dimroth rearrangement at various pHs and temperatures was determined for 1-CEA, 1-methyladenine (1-MeA), 1-(2-carboxyethyl)-deoxyadenosine-5'-monophosphoric acid (1-CEdAdo5'P) and the phosphodiester 5'-O-(2-carboxyethyl)phosphono-1-(2-carboxyethyl)deoxyadenosine (1-CE-Ado-5'-P-CE).     

Stereospecific syntheses of 9-(S)-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA)

9-(S)-(3-Hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA) was prepared from 9-(S)-(2,3-dihydroxypropyl)adenine (DHPA) via its 3-O-chloromethanephosphonate. The latter compound is obtained by treatment of DHPA with chloromethanephosphonyl dichloride and the 3'-isomer separated from its 2'-congener by ion-exchange chromatography. The 3'-isomer is prepared selectively by the same method starting from 2',6-dibenzoyl derivative of DHPA. The 3'-ester is transformed to HPMPA by treatment with aqueous alkali. Alternatively, 9-(S)-(2-hydroxy-3-triphenylmethoxypropyl)-N6-benzoyladenine can be converted to HPMPA by reaction with dialkyl p-tolylsulfonyloxymethane-phosphonates in the presence of NaH followed by successive acid and alkaline treatment.