Synthesis and in-vitro activity of 4'-modified analogues of ddA as potent anti-HIV agents.

This paper reports the synthesis of novel 4'-hydrophobic pocket deoxythreosyl C-nucleosides. The key threose-like intermediates 9 and 14 were constructed from acyclic ketone derivatives, respectively. The antiviral activities of the synthesized compounds against the HIV-1, HSV-1, HSV-2, and HCMV viruses were evaluated. The 9-deaza-adenine derivatives 10 and 20 showed good anti-HIV activity without exhibiting significant cytotoxicity.

Synthesis of 9-(2,3-dideoxy-2,3-difluoro-beta-D-arabinofuranosyl)adenine.

Convergent synthesis of 9-(2,3-dideoxy-2,3-difluoro-beta-D-arabinofuranosyl)adenine is described starting from methyl 5-O-benzyl-2-deoxy-2-fluoro-alpha-D-arabinofuranoside.

Azido-iodo-phenyl-analogs of 2',5'-dideoxy-adenosine as photoaffinity ligands for adenylyl cyclase.

Azidoiodophenyl-analogs of 2',5'-dideoxyadenosine were synthesized and tested as potential 'P'-site selective affinity probes for adenylyl cyclases. The 3'-substituted analogs included: 1: 3'-[(4-nitrophenyl)-acetyl]-2',5'-dideoxy-adenosine 2: 3'-[(4-nitrophenyl)-butyryl]-2',5'-dideoxyadenosine 3: 3'-[(4-azido-3-iodophenyl)-acetyl]-2',5'-dideoxyadenosine and 4: 3'-[(4-azido-3-iodophenyl)-butyryl]-2',5'-dideoxyadenosine. The azidoiodo-phenyl-analogs inactivated adenylyl cyclase irreversibly and in a light-dependent manner. This was observed with detergent-dispersed enzyme from rat brain, purified native enzyme from bovine brain, and recombinant Type I bovine adenylyl cyclase expressed in membranes from fall army worm ovarian (Sf9) cells. Inactivation of the recombinant enzyme was inversely dependent on ATP concentration and was not completely prevented by 2',5'-dideoxyadenosine. Inhibition kinetics with the recombinant enzyme in the absence of light suggested two sites of inhibition, whereas with the native Type I enzyme inhibition kinetics exhibited a straightforward noncompetitive mechanism. Occupation of either or both sites by ligand protected the enzyme against denaturation by UV-irradiation per se. The data are consistent with inactivation of the recombinant enzyme occurring both through the 'P'-site and the catalytic active site, but suggest that this is a characteristic of the recombinant enzyme and is not dependent on the probes per se. The data suggest the potential for independent interactions of such ligands with different sites on a given enzyme and also with other enzymes containing adenosine or adenine nucleotide binding domains.

[Synthesis of a novel L-nucleoside, beta-L-D4A and its inhibition on the replication of hepatitis B virus in vitro].

AIM: Nucleoside analogues have become the most promising candidates of anti-HBV drugs. In this study, beta-L-D4A was synthesized and explored its inhibitiory action against hepatitis B virus (HBV) in 2. 2. 15 cells derived from HepG2 cells transfected with HBV genome. METHODS: beta-L-D4A was stereo-controlled synthesized from D-glutamic acid, and the structure was identified by IR, 1H NMR and MS. 2. 2. 15 Cells were placed at a density of 5 x 10(4) per well in 12-well tissue culture plates, and treated with various concentrations of beta-L-D4A for 6 days. At the end, medium was processed to obtain virions by a polyethlene glycol precipitation method. At the same time, intracellular DNA was also extracted and digested with Hind III. Both of the above DNA were subjected to Southern blot, hybridized with a 32P-labeled HBV probe and autoradiographed. The intensity of the autoradiographic bands was quantitated by densitometric scans of computer and EC50 was calculated. 2. 2. 15 cells were also seeded in 24-well tissue culture plates, and cytotoxicity with different concentrations was examined by MTT method. IC50 was calculated. RESULTS: The synthesized compound structure conformed with beta-L-D4A; Autoradiographic bands showed similar for supernatant and intracellular HBV DNA. Episomal HBV DNA was inhibited in a dose-dependent manner. EC50 0.2 micromol x L(-1). The experiment of cytotoxicity gained IC50 200 micromol x L(-10. CONCLUSION: beta-L-D4A has been synthesized successfully. beta-L-D4A possessed potent inhibitory effect on replication of HBV in vitro with low cytotoxicity, TI value was 1 000. It is expected to be developed clinically into a new anti-HBV drug.

Synthesis of 2',5'-dideoxy-2-fluoroadenosine and 2',5'-dideoxy-2,5'-difluoroadenosine: potent P-site inhibitors of adenylyl cyclase.

Glycosylation of 2-fluoroadenine with the appropriate protected thioglycoside derivatives, followed by deprotection and anomer separation, produced the alpha- and beta-anomers of 2',5'-dideoxy-2-fluoroadenosine (1), 2',5'-dideoxy-2,5'-difluoroadenosine (2), and 2'-deoxy-2-fluoroadenosine (3). These were examined as P-site inhibitors of adenylyl cyclase. The presence of fluorine on the purine ring increased potency of inhibition, and the most potent compound, beta-2',5'-dideoxy-2-fluoroadenosine (1b), was 3 times more potent than beta-2',5'-dideoxyadenosine.

Discovery of type II (covalent) inactivation of S-adenosyl-L-homocysteine hydrolase involving its "hydrolytic activity": synthesis and evaluation of dihalohomovinyl nucleoside analogues derived from adenosine.

Treatment of the 5'-carboxaldehyde derived by Moffatt oxidation of 6-N-benzoyl-2',3'-O-isopropylideneadenosine (1) with the "(bromofluoromethylene)triphenylphosphorane" reagent and deprotection gave 9-(6-bromo-5, 6-dideoxy-6-fluoro-beta-d-ribo-hex-5-enofuranosyl)adenine (4). Parallel treatment with a "dibromomethylene Wittig reagent" and deprotection gave 9-(6,6-dibromo-5, 6-dideoxy-beta-d-ribo-hex-5-enofuranosyl)adenine (7), which also was prepared by successive bromination and dehydrobromination of the 6'-bromohomovinyl nucleoside 8. Bromination-dehydrobromination of the 5'-bromohomovinyl analogue 11 and deprotection gave (E)-9-(5, 6-dibromo-5,6-dideoxy-beta-d-ribo-hex-5-enofuranosyl)adenine (15). Compounds 4, 7, and 15 were designed as putative substrates of the "hydrolytic activity" of S-adenosyl-l-homocysteine (AdoHcy) hydrolase. Enzyme-mediated addition of water across the 5,6-double bond could generate electrophilic acyl halide or alpha-halo ketone species that could undergo nucleophilic attack by proximal groups on the enzyme. Such type II (covalent) mechanism-based inactivation is supported by protein labeling with 8-[3H]-4 and concomitant release of bromide and fluoride ions. Incubation of AdoHcy hydrolase with 7 or 15 resulted in irreversible inactivation and release of bromide ion. In contrast with type I mechanism-based inactivation, reduction of enzyme-bound NAD+ to NADH was not observed. Compounds 4, 7, and 15 were not inhibitory to a variety of viruses in cell culture, and weak cytotoxicity was observed only for CEM cells.

Doubly homologated dihalovinyl and acetylene analogues of adenosine: synthesis, interaction with S-adenosyl-L-homocysteine hydrolase, and antiviral and cytostatic effects.

Treatment of the 6-aldehyde derived by Moffatt oxidation of 3-O-benzoyl-1,2-O-isopropylidene-alpha-D-ribo-hexofuranose (2c) with the dibromo- or bromofluoromethylene Wittig reagents generated in situ with tetrabromomethane or tribromofluoromethane, triphenylphosphine, and zinc gave the dihalomethyleneheptofuranose analogues 3b and 3d, respectively. Acetolysis, coupling with adenine, and deprotection gave 9-(7,7-dibromo-5,6, 7-trideoxy-beta-D-ribo-hept-6-enofuranosyl)adenine (5a) or its bromofluoro analogue 5b. Treatment of 5a with excess butyllithium provided the acetylenic derivative 9-(5,6, 7-trideoxy-beta-D-ribo-hept-6-ynofuranosyl)adenine (6). The doubly homologated vinyl halides 5a and 5b and acetylenic 6 adenine nucleosides were designed as putative substrates of the "hydrolytic activity" of S-adenosyl-L-homocysteine (AdoHcy) hydrolase. Incubation of AdoHcy hydrolase with 5a, 5b, and 6 resulted in time- and concentration-dependent inactivation of the enzyme (K(i): 8.5 +/- 0.5, 17 +/- 2, and 8.6 +/- 0.5 microM, respectively), as well as partial reduction of enzyme-bound NAD(+) to E-NADH. However, no products of the "hydrolytic activity" were observed indicating these compounds are type I mechanism-based inhibitors. The compounds displayed minimal antiviral and cytostatic activity, except for 6, against vaccinia virus and vesicular stomatitis virus (IC(50): 15 and 7 microM, respectively). These viruses typically fall within the activity spectrum of AdoHcy hydrolase inhibitors.

Enzymatic properties of the unnatural beta-L-enantiomers of 2',3'-dideoxyadenosine and 2',3'-didehydro-2',3'-dideoxyadenosine.

The beta-L-enantiomers of 2',3'-dideoxyadenosine and 2',3'-didehydro-2',3'-dideoxyadenosine have been stereospecifically synthesized. In an attempt to explain the previously reported antiviral activities of these compounds, their enzymatic properties were studied with respect to adenosine kinase, deoxycytidine kinase, adenosine deaminase, and purine nucleoside phosphorylase. Adenosine deaminase was strictly enantioselective and favored beta-D-ddA and beta-D-d4A, whereas adenosine kinase and purine nucleoside phosphorylase had no apparent substrate properties for the D- or L-enantiomers of beta-ddA or beta-d4A. Human deoxycytidine kinase showed a remarkable inversion of the expected enantioselectivity, with beta-L-ddA and beta-L-d4A having better substrate efficiencies than their corresponding beta-D-enantiomers. Our results demonstrate the potential of beta-L-adenosine analogues as antiviral agents and suggest that deoxycytidine kinase has a strategic importance in their cellular activation.

An industrial process for synthesizing Lodenosine (FddA).

Two industrial synthetic approaches to Lodenosine (1, FddA, 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl) adenine) via a purine riboside or a purine 3'-deoxyriboside are described. Several novel applications of deoxygenation and fluorination methods are compared considering reaction yields, economy, safety and environmental concerns.

A facile synthetic method for 3'-alpha-fluoro-2',3'-dideoxyadenosine.

A facile method for the synthesis of 3'-alpha-fluoro-2',3'-dideoxyadenosine (5) has been developed using a novel rearrangement of 3'-beta-bromine to the 2'-beta position during 3'-alpha fluorination.

The "beta-fluorine effect" in the non-metal hydride radical deoxygenation of fluorine-containing nucleoside xanthates.

An alternative method to conduct a Barton-McCombie deoxygenation in nucleosides is described. The utility of the procedure is limited to structures with an electronegative substituent, particularly fluorine, in the beta-position relative to the radical center. The process is radical in nature and triggered by peroxides. The abstraction of hydrogen from the solvent is favorably influenced by the presence of a beta-fluorine.

Cyclopropyl and related analogs of the anti-HIV compound, isodideoxyadenosine.

Novel 3'-substituted isonucleoside analogs were designed on the basis of the similarities of their electrostatic potential with the active anti-HIV compound, (S,S)-isodideoxy-adenosine. The key synthetic step involved coupling between the dideoxygenated sugar derivatives, 10 and 14, and adenine under Mitsunobu conditions. Anti-HIV data are mentioned.

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. 8-Methyl and 8-halogenated carbocyclic 2',3'-dideoxyadenosine analogs showed 6-25 fold more activity against hepatitis B virus than the unsubstituted carbocyclic D-2',3'-dideoxyadenosine.

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.

Synthesis and antiviral evaluation of 3'-C-trifluoromethyl nucleoside derivatives bearing adenine as the base.

3'-deoxy-3'-C-trifluoromethyl- (3), 2',3'-dideoxy-3'-C-trifluoromethyl- (5) and 2',3'-dideoxy-2',3'-didehydro-3'-C-trifluoromethyladenosine (6) derivatives have been synthesized and their antiviral properties examined. All these derivatives were stereospecifically prepared by glycosylation of adenine with a trifluoromethyl sugar precursor (1), followed by appropriate chemical modifications. The prepared compounds were tested for their activity against HIV, but they did not show an antiviral effect.

Synthesis and biological evaluation of endocyclic 2',3'-didehydro-2',3'-dideoxymethanocarba adenosine.

This is the first report describing the synthesis and conformation of methanocarba nucleosides incorporating an endo (beta-face) cyclopropyl at the 2',3' position of 2',3'-didehydro-2',3'-dideoxy carbocyclic nucleosides. These nucleoside isosteres have been shown to exist in a unique extreme eastern conformation. This prediction was confirmed by x-ray crystallography and high resolution NMR spectroscopy. As expected, the methanocarba adenosine compound was neither a substrate nor an inhibitor of adenosine deaminase. However, some of the compounds synthesized demonstrated moderate antiviral activity against HSV-1. The methanocarba adenosine and its triphosphate form were evaluated as inhibitors of HIV-1 reverse transcriptase.

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.

[Inhibition of HIV reproduction in cell culture by 5'-phosphonates of 3'-azido-2',3'-dideoxynucleosides]. / Ingibirovanie reproduktsii virusa immunodefitsita cheloveka v kul'ture kletok 5'-fosfonatami 3'-azido-2',3'-dideoksinukleozidov.

The antiviral activity of 3'-azido-2',3'-dideoxynucleoside 5'-phosphate analogues: 5'-phosphonomethylene-3'-azido-2',3'-dideoxythymidine, 5'-methylphosphonate and 5'-phosphite of 3'-azido-2',3'-dideoxythymidine, 5'-phosphites of 3'-azido-2',3'-dideoxyadenosine and guanosine was investigated in HIV-infected cell cultures (human lymphoblastoid cells). The effectivity of inhibition of HIV-reproduction in cells by these substances was close or even higher than that for the corresponding 3'-azido-2',3'-dideoxynucleosides, whereas their toxicity was lower than that of nucleosides. These substances are supposed to be transported into the cells and to be transformed into the corresponding 5'-triphosphate analogues under the action of cell kinases. It is possible that such agents are terminator substrates of virus reverse transcriptases and thus inhibit the biosynthesis of DNA chains.

Synthesis of 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)adenine (FddA) via a purine 3'-deoxynucleoside.

A synthesis of 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)adenine (1, FddA) via a 6-chloro-9-(3-deoxy-beta-D-erythro-pentofuranosyl)-9H-purine (9), which was readily obtained from inosine (5), is described. Fluorination at the C2'-beta position of the purine 3'-deoxynucleoside with diethylaminosulfur trifluoride was improved by the introduction of a 6-chloro group and proceeded in moderate yield. Purine 3'-deoxynucleoside derivatives were also subjected to nucleophilic reactions with triethylamine trihydrofluoride and gave the desired fluorinated nucleoside in good yield. The safety and yield of the fluorination process were greatly improved by the use of triethylamine trihydrofluoride. The influence of the sugar ring conformation and 6-chloro group on the rate of the nucleophilic reaction against elimination are also discussed.