Synthesis and biological evaluation of L- and D-configurations of 2',3'-dideoxy-4'-C-methyl-3'-oxacytidine analogues.

Novel L- and D-configuration 2',3'-dideoxy-4'-C-methyl-3'-oxacytidine and their 5-fluoro analogues have been synthesized from 1-benzyloxy-2-propanone and L-ascorbic acid in eight steps and evaluated for biological activity.

Synthesis of halogen-substituted 3-deazaadenosine and 3-deazaguanosine analogues as potential antitumor/antiviral agents.

Various 2-halogen-substituted analogues (38, 39, 43 and 44), 3-halogen-substituted analogues (51 and 52), and 2',3'-dihalogen-substituted analogues (57-60) of 3-deazaadenosine and 3-halogen-substituted analogues (61 and 62) of 3-deazaguanosine have been synthesized as potential anticancer and/or antiviral agents. Among these compounds, 3-deaza-3-bromoguanosine (62) showed significant cytotoxicity against L1210, P388, CCRF-CEM and B16F10 cell lines in vitro, producing IC50 values of 3, 7, 9 and 7 microM, respectively. Several 3-deazaadenosine analogues (38, 51, 57 and 59) showed moderate to weak activity against hepatitis B virus.

Synthesis and antiviral activity of oxaselenolane nucleosides.

As dioxolane and oxathiolane nucleosides have exhibited promising antiviral and anticancer activities, it was of interest to synthesize isoelectronically substituted oxaselenolane nucleosides, in which the 3'-CH(2) is replaced by a selenium atom. To study structure-activity relationships, various pyrimidine and purine oxaselenolane nucleosides were synthesized from the key intermediate, (+/-)-2-benzoyloxymethyl-1,2-oxaselenolane 5-acetate (6). Among the synthesized racemic nucleosides, cytosine and 5-fluorocytosine analogues exhibited potent anti-HIV and anti-HBV activities. It was of interest to obtain the enantiomerically pure isomers to determine if they have differential antiviral activities. However, due to the difficult and time-consuming nature of enantiomeric synthesis, a chiral HPLC separation was performed to obtain optical isomers from the corresponding racemic mixtures. Each pair of enantiomers of Se-ddC and Se-FddC was separated by an amylose chiral column using a mobile phase of 100% 2-propanol. The results indicate that most of the anti-HIV activity of both cytosine and fluorocytosine nucleosides resides with the (-)-isomers.

Metabolism and mode of inhibition of varicella-zoster virus by L-beta-5-bromovinyl-(2-hydroxymethyl)-(1,3-dioxolanyl)uracil is dependent on viral thymidine kinase.

A nonnaturally occurring L-configuration nucleoside analog, L-beta-5-bromovinyl-(2-hydroxymethyl)-1,3-(dioxolanyl)uracil (L-BVOddU) selectively inhibited varicella-zoster virus growth in human embryonic lung (HEL) 299 cell culture with an EC(50) of 0.055 microM, whereas no inhibition of CEM and HEL 299 cell growth or mitochondrial DNA synthesis was observed at concentrations up to 200 microM. L-BVOddU was phosphorylated by viral thymidine kinase but not by human cytosolic thymidine kinase, and the antiviral activity of this compound is dependent on the viral thymidine kinase. Unlike other D-configuration bromovinyl deoxyuridine analogs, such as E-5-(2-bromovinyl)-2'-deoxyuridine and 1-beta-arabinofuranosyl-E-5-(2-bromovinyl)uracil, this compound was metabolized only to its monophosphate metabolite. The di- or triphosphate metabolites were not detected. This suggested that the inhibitory mechanism may be unique and different from other anti-herpesvirus nucleoside analogs.

Synthesis and biological evaluation of L- and D-configuration 1,3-dioxolane 5-azacytosine and 6-azathymine nucleosides.

Novel L- and D-configuration dioxolane 5-azacytosine and 6-azathymine nucleosides have been synthesized and evaluated for biological activity. (-)-(2S,4S)-1-[2-(Hydroxymethyl)-1,3-dioxolan-4-yl]-5-azacytosine (6) showed significant activity against HBV, whereas the D-configuration analogue (14) has been found to exhibit potent anti-HIV activity.

Synthesis and enantioselectivity of the antiviral effects of (R,Z)-,(S,Z)-methylenecyclopropane analogues of purine nucleosides and phosphoralaninate prodrugs: influence of heterocyclic base, type of virus and host cells.

A series of R and S enantiomers of 2-aminopurine methylenecyclopropane analogues of nucleosides was synthesized. Two diastereoisomeric lipophilic phosphate prodrugs derived from R and S enantiomers of 2,6-diaminopurine analogue were also prepared. Enantioselectivity (diastereoselectivity in case of prodrugs) of in vitro antiviral effects was investigated with human and murine cytomegalovirus (HCMV and MCMV, respectively), herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively), human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), Epstein-Barr virus (EBV) and varicella zoster virus (VZV). Strong differences in enantioselectivity were found between the R and S enantiomers of adenine analogue and enantiomeric 2-aminopurine analogues. Thus, the enantiomers of adenine analogue were equipotent against HCMV but not MCMV, where the S enantiomer is strongly preferred. The same S preference was found throughout the 2-aminopurine series for both HCMV and MCMV. In contrast, R-synadenol in HIV-1 assays was the best agent, whereas the S enantiomers of moderately effective 2-amino-6-cyclopropylamino and 2-amino-6-methoxypurine analogues were preferred. Little enantiomeric preference was found for R and S enantiomers of synadenol and the corresponding enantiomers of 2,6-diaminopurine analogue against HBV. A mixed pattern of enantioselectivity was observed for EBV depending on the type of host cells and assay. Against VZV, the R and S enantiomers of adenine analogue were equipotent or almost equipotent, but throughout the series of 2-aminopurine analogues a distinct preference for the S enantiomers was found. The stereoselectivity pattern of both diastereoisomeric prodrugs mostly followed enantioselectivity of the parent analogues. The varying enantioselectivities in the series of purine methylenecyclopropane analogues are probably a consequence of differences in the mechanisms of action in different virus/host cell systems.

Structure-activity relationships of (E)-5-(2-bromovinyl)uracil and related pyrimidine nucleosides as antiviral agents for herpes viruses.

A series of (E)-5-(2-bromovinyl)uracil analogues and related nucleosides was synthesized, and their antiviral activities were evaluated. (E)-5-(2-Bromovinyl)-2'-deoxy-L-uridine (L-BVDU, 2), 1-(beta-L-arabinofuranosyl)-(E)-5-(2-bromovinyl)uracil (L-BVAU, 4), (E)-5-(2-bromovinyl)-1-(2-deoxy-2-fluoro-beta-L-ribofuranosyl)uracil (L-FBVRU, 8) and (E)-5-(2-bromovinyl)-1-(2-deoxy-2-fluoro-beta-L-arabinofuranosyl)urac il (L-FBVAU, 10) were synthesized via appropriate 5-iodouracil analogues from L-arabinose. D- and L-Oxathiolane and -dioxolane derivatives 13, 16, 20, 21, and 29-34 were prepared by glycosylation reaction of the oxathiolane and dioxolane intermediates with silylated uracil analogues using TMSI as the coupling agent. The synthesized compounds were evaluated in cell cultures infected with the following viruses: varicella zoster virus (VZV), Epstein Barr virus (EBV), and herpes simplex virus types 1 and 2 (HSV-1 and HSV-2). Among the tested compounds, beta-L-CV-OddU (29), beta-L-BV-OddU (31), and beta-L-IV-OddU (33) exhibited potent in vitro antiviral activity against VZV with EC(50) values of 0.15, 0. 07, and 0.035 microM, respectively, and against EBV with EC(50) values of 0.49, 0.59, and 3.91 microM, respectively.

Synthesis and biological evaluation of 1,3-oxathiolane 5-azapyrimidine, 6-azapyrimidine, and fluorosubstituted 3-deazapyrimidine nucleosides.

(2R,5S)-5-Amino-2-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]- 1,2,4-triazine-3(2H)-one (8) and (2R,5R)-5-amino-2-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2,4-tr iazine-3(2H)-one (9) have been synthesized via a multi-step procedure from 6-azauridine. (2R,5S)-4-Amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,3, 5-triazine-2(1H)-one (11) and (2R,5R)-4-amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]- 1,3,5-triazine-2(1H)-one (12), and the fluorosubstituted 3-deazanucleosides (19-24) have been synthesized by the transglycosylation of (2R,5S)-1-[2-[[(tert-butyldiphenylsilyl) oxy]methyl]-1,3-oxathiolan-5-yl] cytosine (2) with silylated 5-azacytosine and the corresponding silylated fluorosubstituted 3-deazacytosines, respectively, in the presence of trimethylsilyl trifluoromethanesulfonate as the catalyst in anhydrous dichloroethane, followed by deprotection of the blocking groups. These compounds were tested in vitro for cytotoxicity against L1210, B16F10, and CCRF-CEM tumor cell lines and for antiviral activity against HIV-1 and HBV.

Methylenecyclopropane analogues of nucleosides: synthesis, absolute configuration, and enantioselectivity of antiviral effect of (R)-(-)- and (S)-(+)-synadenol.

Synthesis, absolute configuration and antiviral activity of enantiomeric antiviral agents (R)-(-)- and (S)-(+)-synadenol (2 and 3a) are described.

Structure-activity relationships of L-dioxolane uracil nucleosides as anti-Epstein Barr virus agents.

A series of 1,3-dioxolanyluracil analogues was prepared from the dioxolane intermediates 2, and their anti-Epstein Barr virus (anti-EBV) activities were determined. The potency of L-dioxolane uracil nucleosides against EBV replication is dependent on the substituents at the 5-position in the following decreasing order: I > Br > Cl > CH3 > CF3 > F. The most active and selective analogue was the iodo derivative (L-I-OddU) with an EC50 value of 0.03 microM and an EC90 value of 0.16 microM. There was no cytotoxicity or depletion of mitochondrial DNA in cells after exposure to L-I-OddU at 50 microM. The action against EBV replication in H1 cells is time-dependent, and EBV DNA in cells treated with L-I-OddU could rebound to pretreatment levels once the drug was removed. In view of the potent antiviral activity plus favorable toxicity profiles, L-I-OddU may be potentially useful for the treatment of EBV-related infectious diseases as well as for delaying the onset or decreasing the incidence of EBV-associated cancers.

Synthesis and anti-HIV and anti-HBV activities of 2'-fluoro-2', 3'-unsaturated L-nucleosides.

The synthesis of L-nucleoside analogues containing 2'-vinylic fluoride was accomplished by direct condensation method, and their anti-HIV and anti-HBV activities were evaluated in vitro. The key intermediate 8, the sugar moiety of our target compounds, was prepared from 1,2-O-isopropylidene-L-glyceraldehyde via (R)-2-fluorobutenolide intermediate 5 in five steps. Coupling of the acetate 8 with the appropriate heterocycles (silylated uracil, thymine, N4-benzoylcytosine, N4-benzoyl-5-fluorocytosine, 6-chloropurine, and 6-chloro-2-fluoropurine) in the presence of Lewis acid afforded a series of 2'-fluorinated L-nucleoside analogues (15-18, 23-26, 36-45). The newly synthesized compounds were evaluated for their antiviral activities against HIV-1 in human peripheral blood mononuclear (PBM) cells and HBV in 2.2.15 cells. Cytosine 23, 5-fluorocytosine 25, and adenine 36 derivatives exhibited moderate to potent anti-HIV (EC50 0.51, 0.17, and 1.5 microM, respectively) and anti-HBV (EC50 0.18, 0.225, and 1.7 microM, respectively) activities without significant cytotoxicity up to 100 microM in human PBM, Vero, CEM, and HepG2 cells.

(R)-(-)- and (S)-(+)-Synadenol: synthesis, absolute configuration, and enantioselectivity of antiviral effect.

Synthesis of (R)-(-)- and (S)-(+)-synadenol (1a and 2a, 95-96% ee) is described. Racemic synadenol (1a + 2a) was deaminated with adenosine deaminase to give (R)-(-)-synadenol (1a) and (S)-(+)-hypoxanthine derivative 5. Acetylation of the latter compound gave acetate 6. Reaction with N, N-dimethylchloromethyleneammonium chloride led to 6-chloropurine derivative 7. Ammonolysis furnished (S)-(+)-synadenol (2a). Absolute configuration of 1a was established by two methods: (i) synthesis from (R)-methylenecyclopropanecarboxylic acid (8) and (ii) X-ray diffraction of a single crystal of (-)-synadenol hydrochloride. Racemic methylenecyclopropanecarboxylic acid (10) was resolved by a modification of the described procedure. The R-enantiomer 8 was converted to ethyl ester 13 which was brominated to give vicinal dibromides 14. Reduction with diisobutylaluminum hydride then furnished alcohol 15 which was acetylated to the corresponding acetate 16. Alkylation-elimination procedure of adenine with 16 yielded acetates 17 and 18. Deprotection with ammonia afforded a mixture of Z- and E-isomers 1a and 19 of the R-configuration. Comparison with products 1a and 2a by chiral HPLC established the R-configuration of (-)-synadenol (1a). These results were confirmed by X-ray diffraction of a single crystal of (-)-synadenol hydrochloride. The latter forms a pseudosymmetric dimer with adenine-adenine base pairing in the lattice with the nucleobase in an anti-like conformation. Enantiomers 1a and 2a exhibit varied enantioselectivity toward different viruses. Both enantiomers are equipotent against human cytomegalovirus (HCMV) and varicella zoster virus (VZV). The S-enantiomer 2a is somewhat more effective than R-enantiomer 1a in herpes simplex virus 1 and 2 (HSV-1 and HSV-2) assays. By contrast, enantioselectivity of antiviral effect is reversed in Epstein-Barr virus (EBV) and human immunodeficiency virus type 1 (HIV-1) assays where the R-enantiomer 1a is preferred. In these assays, the S-enantiomer 2a is less effective (EBV) or devoid of activity (HIV-1).

Metabolism of 2',3'-dideoxy-2',3'-didehydro-beta-L(-)-5-fluorocytidine and its activity in combination with clinically approved anti-human immunodeficiency virus beta-D(+) nucleoside analogs in vitro.

2',3'-Dideoxy-2',3'-didehydro-beta-L(-)-5-fluorocytidine [L(-)Fd4C] has been reported to be a potent inhibitor of the human immunodeficiency virus (HIV) in cell culture. In the present study the antiviral activity of this compound in two-drug combinations and its intracellular metabolism are addressed. The two-drug combination of L(-)Fd4C plus 2',3'-didehydro-2'-3'-dideoxythymidine (D4T, or stavudine) or 3'-azido-3'-deoxythymidine (AZT, or zidovudine) synergistically inhibited replication of HIV in vitro. Additive antiviral activity was observed with L(-)Fd4C in combination with 2',3'-dideoxycytidine (ddC, or zalcitabine) or 2',3'-dideoxyinosine (ddI, or didanosine). This beta-L(-) nucleoside analog has no activity against mitochondrial DNA synthesis at concentrations up to 10 microM. As we previously reported for other beta-L(-) nucleoside analogs, L(-)Fd4C could protect against mitochondrial toxicity associated with D4T, ddC, and ddI. Metabolism studies showed that this drug is converted intracellularly to its mono-, di-, and triphosphate metabolites. The enzyme responsible for monophosphate formation was identified as cytoplasmic deoxycytidine kinase, and the K(m) is 100 microM. L(-)Fd4C was not recognized in vitro by human mitochondrial deoxypyrimidine nucleoside kinase. Also, L(-)Fd4C was not a substrate for deoxycytidine deaminase. L(-)Fd4C 5'-triphosphate served as an alternative substrate to dCTP for incorporation into DNA by HIV reverse transcriptase. The favorable anti-HIV activity and protection from mitochondrial toxicity by L(-)Fd4C in two-drug combinations favors the further development of L(-)Fd4C as an anti-HIV agent.

Synthesis and comparative evaluation of two antiviral agents: beta-L-Fd4C and beta-D-Fd4C.

The synthesis of beta-D-Fd4C was achieved in a stereoselective fashion from D-xylose. The antiviral activity and cytotoxicity of beta-D-Fd4C was compared with that of beta-L-Fd4C and 3TC (Lamivudine). Of the three agents compared, beta-L-Fd4C was found to be the most potent antiviral agent.

Favorable interaction of beta-L(-) nucleoside analogues with clinically approved anti-HIV nucleoside analogues for the treatment of human immunodeficiency virus.

The combination of L(-)-2',3'-dideoxy-3'-thiacytidine (L(-)SddC, 3TC), L(-)-2',3'-dideoxy-5-fluorocytidine (L(-)FddC), or L(-)-2',3'-dideoxy-5-fluoro-3'-thiacytidine (L(-)(FTC) with 3'-azido-3'-deoxythymidine (AZT) synergistically inhibited replication of human immunodeficiency virus (HIV) in vitro. Similar synergistic activity was also obtained when these compounds were used in combination with 2',3'-didehyro-2',3'-dideoxythymidine (D4T). In terms of 2',3'- dideoxyinosime (ddI) and 2',3'-dideoxycytidine (ddC), only additive anti-HIV activity was observed. None of the beta-L(-) nucleoside analogues had additive toxicity in cell culture, and they could protect against the delayed mitochondrial toxicity associated with AZT, D4T, ddC, and ddI in drug-treated cells. Thus, combinations of beta-L(-) nucleoside analogues with any of the approved anti-HIV drugs could have a potentially beneficial outcome.