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A series of 5'-derivatives and modified pyrimidine analogues of 2',3'-didehydro-3'-deoxythymidine (d4T, stavudine, 1) were synthesized to determine their potential as oral prodrugs of d4T. Utilizing a screen developed for the rapid evaluation of a variety of prodrugs in mice, it was determined that 5'-acetate 2 provided comparable plasma levels of d4T after oral administration of the prodrug to that when d4T was administered alone. The relative oral bioavailability of methoxy acetate 3 and cyclohexyl carbonate 5 was 79 and 41%, respectively. Dihydropyridine ester 6 did not provide detectable levels of d4T up to 1 h after oral administration of 6. Thiopyrimidines 8 and 9, as well as aminopyrimidine 10 also failed to provide measurable levels of d4T after oral administration. 5'-Derivatives 3, 5, and 6 showed similar activity to that of d4T against HIV and MuLV, as did 5'-benzoyl-4-thio derivative 8. However, the corresponding 4-thio 5'-alcohol 9 was inactive.

Novel phosphonate isosteres of acyclovir (ACV) and ganciclovir (DHPG) monophosphates were found to be potent and selective antiherpesvirus agents. In the series of phosphonate analogues of ACV monophosphate, only the guanine analogue 20 exhibited activity against herpesviruses, similar to the structure-activity relationship observed for base modification of ACV analogues. The phosphonate isostere of ACV monophosphate (20) was more effective than ACV in the HSV-1 infected mouse model. The 3'-carba analogues of 9-[3-hydroxy-2-(phosphonomethoxy)propyl]purines/pyrimidines (adenine, HPMPA; guanine, HPMPG; cytosine, HPMPC) are devoid of antiherpesvirus activity. This result confirms that the beta-oxygen atom of the phosphonomethyl ether functionality in HPMP-purines/pyrimidines plays a critical role for activity against herpesviruses.

Various 3'-deoxy pyrimidine nucleoside analogues have been synthesized for evaluation as potential anticancer and antiviral agents. Among these compounds, 1-(3-deoxy-beta-D-threo-pentofuranosyl)cytosine (10, 3'-deoxy-ara-C) and 3'-deoxycytidine (22) had significant anticancer activity against CCRF-CEM, L1210, P388, and S-180 cancer cell lines in vitro, producing ED50 values of 2, 10, 5, and 34 microM, respectively, for 3'-deoxy-ara-C (10); and 25, 5, 2.5, and 15 microM, respectively, for 3'-deoxycytidine (22). Thus, 3'-deoxy-ara-C (10) was 12.5 times more active against CCRF-CEM cells than 3'-deoxycytidine (22). The 2'-O-acetyl, 5'-O-acetyl, and 2',5'-di-O-acetyl derivatives of 3'-deoxy-ara-C (10), compounds 34, 31, and 30, demonstrated anticancer activity in the same range as 3'-deoxy-ara-C (10) against CCRF-CEM, L1210, P388, and S-180 cells. The 5'-O-acetyl derivative (31) had significantly greater activity against CCRF-CEM with an ED50 value of 0.4, but this compound also showed similar activity, as did 3'-deoxy-ara-C, against L1210, P388, and S-180 with ED50 values of 3, 3, and 13 microM, respectively. 3'-Deoxy-ara-C was also evaluated in vitro against HSV-2, HCMV, and GPCMV viruses and was found to be not very active with respective IC50 values of 110, 220, and 1000 microM. The single-crystal structure of 3'-deoxy-ara-C (10) was determined by X-ray crystallography. There are two molecules of the nucleoside and one molecule of water in the asymmetric unit. The sugar moieties of the two nucleoside molecules adopt different conformations. In molecule A, the ring pucker is C3'-endo with P = 18.7 degrees and tau m = 37.3 degrees, while the CH2OH side chain is gauche+. In molecule B, the ring pucker is C2'-endo with P = 156.8 degrees and tau m = 37.8 degrees and the side chain is trans.

Several 2'-fluoroarabino-2',3'-dideoxy- and 2'-fluoro-2',3'-unsaturated 2',3'-dideoxy pyrimidine nucleoside analogues are reported. The saturated analogues 1-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)thymine (2'-threo-FddT, 33), 1-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)uracil (2'-threo-FddU, 22) were readily prepared from the corresponding 2'-deoxy-2'-fluoroarabinosyl nucleoside analogue by radical deoxygenation of the 3'-OH. The unsaturated compounds 1-(2,3-dideoxy-2-fluoro-beta-D-glycero-pent-2-enofuranosyl)thymine (2'-Fd4T, 40) and 1-[5-O-(mono-methoxytrityl)-2-fluoro-2,3-dideoxy-beta-D-glycero-pen t-2- enofuranosyl]uracil (39) were synthesized by an elimination reaction of the O-2,3'-anhydro-2'-fluoro-lyxo derivatives under basic conditions. The cytidine analogues 28 and 41 were prepared by amination of the corresponding uridine derivatives; compounds 28 and 41 were deprotected to give 1-(2,3-dideoxy-2-fluoro-beta-D-arabinofuranosyl)cytidine (2'-threo-FddC, 29) and 1-(2,3-dideoxy-2-fluoro-beta-D-glycero-pent-2- enofuranosyl)cytosine (2'-Fd4C, 42), respectively. All of these novel compounds were evaluated in vitro against human immunodeficiency virus (HIV) (LAV isolate). 2'-threo-FddC (29) was the most active of the newly synthesized substances against HIV with an ID50 of 0.8 microgram/mL; ddC had an ID50 of 0.007 micrograms/mL. Because of its potency in the initial tests, 29 was further evaluated in both T cells and macrophage/monocyte cell lines, with several different isolates of HIV. Although 2'-threo-FddC (29) exhibited good antiviral activity in these systems it was less active than AZT in these assays. At 1 microM the inhibition of CFU-GM by 29 was found to be 35-40%; this is slightly higher than seen with AZT.

A series of 9-(phosphonoalkyl)purines, which are analogues of 9-[2-(phosphonomethoxy)ethyl]purines (guanine, PMEG, 1; adenine, PMEA, 2), were synthesized. The analogues were tested for activity against herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), human cytomegalovirus (HCMV), Rauscher murine leukemia virus (R-MuLV), and human immunodeficiency virus type 1 (HIV-1). With variations in the length of the alkyl chain, the optimal activity was achieved with two carbons between the purine base and the phosphonomethoxy functionality. Despite the structural similarity and the close pKa2 value of 8 to that of PMEG, no phosphorylation of 8 was observed by the bovine brain guanylate kinase. Since all isosteric analogues of PMEG (7-9) were not inhibitory against HSV-1 and HSV-2, the presence of the 3'-oxygen atom in the PME purines proved critical for anti-HSV activity. Introduction of the 1'-methyl group on the PMEG side chain significantly reduced its anti-HSV activity. Analogue 11, which is a mimic of the phosphate by incorporation of the alpha,alpha-difluoro carbon, was ineffective against HSV-1 and HSV-2. These results suggest that the structural requirements of PME purines for anti-HSV activity appear to be very strict.

Our studies have shown that the acyclic nucleotide analogues PMEA and HPMPC are able to penetrate into cells and are then activated to mono- and diphosphate derivatives. The latter correspond to triphosphate analogues and presumably serve an important role in the biological activity exerted by these antiviral agents. In support of this idea, the inhibitory effect of PMEApp on HIV reverse transcriptase has been demonstrated with both RNA and DNA template-primer systems. Further studies will be undertaken to determine the effect of HPMPCpp on viral DNA polymerases. Whereas the metabolism of PMEA in CEM cells gives rise to only PMEAp and PMEApp, additional metabolites were obtained in MRC-5 cells; the identity of these metabolites remains to be determined. In the case of HPMPC, a third metabolite was obtained in addition to HPMPCp and HPMPCpp, which has been tentatively assigned as a phosphate-choline adduct by analogy with activation of cytosine-based nucleoside derivatives. The metabolism of HPMPC was unchanged between uninfected and infected cells, indicating that viral enzymes are not necessary for the activation of HPMPC. The long intracellular half-lives of the HPMPC metabolites may have implications for the antiviral efficacy of this compound. The persistence of activated metabolites suggests that infrequent dosing may be possible due to a prolonged antiviral effect. Our results on the effectiveness of infrequent dosing schedules with HPMPC in the treatment of HSV 2 infections in mice support this hypothesis. It is also possible that HPMPCp-choline may serve as a reservoir for HPMPC and therefore for the presumed active metabolite HPMPCpp.

The acyclic nucleotide analogue (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl] cytosine (2, HPMPC) was prepared on a multigram scale in 18% overall yield starting from (R)-2,3-O-isopropylideneglycerol. The key step in the nine-step synthetic route is coupling of cytosine with the side-chain derivative 8 which bears a protected phosphonylmethyl ether group. In vitro data showed that HPMPC has good activity against herpes simplex virus types 1 and 2, although it was 10-fold less potent than acyclovir [AVC, 9-[(2-hydroxyethoxy)methyl]guanine]. By comparison, HPMPC exhibited greater activity than ACV against a thymidine kinase deficient strain of HSV 1 and was more potent than ganciclovir [DHPG, 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine] against human cytomegalovirus. In vivo, HPMPC showed exceptional potency against HSV 1 systemic infection in mice, having an ED50 of 0.1 mg/kg per day (ip) compared with 50 mg/kg per day for ACV. HPMPC was also more efficacious than ACV in the topical treatment of HSV 1 induced cutaneous lesions in guinea pigs.

Various 3'-azido, 3'-amino, 2',3'-unsaturated, 2',3'-dideoxy, and 5-substituted analogues of pyrimidine deoxyribonucleosides have been prepared and tested against Moloney-murine leukemia virus (M-MULV), a mammalian T-lymphotropic retrovirus in vitro. Among these compounds, the 3'-azido analogues of thymidine, 2'-deoxy-5-bromouridine, and 2'-deoxy-5-iodouridine, the 2',3'-unsaturated analogue of thymidine and and 2'-deoxycytidine, and 2',3'-dideoxycytidine were found to be most active, with ED50 values of 0.02, 1.5, 3.0, 2.5, 3.7, and 4.0 microM, respectively. These active compounds were nontoxic to the host SC-1 cells up to 100 microM concentration. The 3'-azido analogues of thymidine and 2'-deoxy-5-bromouridine were also tested in vitro against HTLV-III/LAV/AAV ("AIDS" virus) and found to be significantly active, with ED50 values of 0.23 and 2.3 microM, respectively. The structure-activity relationships are discussed.

The nucleoside analogue 1-(2,3-dideoxy-beta-D-glycero-pent-2-enofuranosyl)thymine (d4T, 1) was prepared by ring opening of the 3',5'-anhydro compound 5. This method has been refined such that it can be used to prepare d4T on a large scale. The triphosphate of d4T was also synthesized from 1 in order to examine the mode of action. The in vitro inhibitory activity of d4T was found to be comparable to that of AZT in HIV-infected CEM cells. The triphosphate of d4T (8) and that of AZT inhibited the HIV reverse transcriptase with poly(rA):oligo(dT) as the template:primer with Ki values of 0.032 and 0.007 microM, respectively. The in vitro toxicity of d4T against normal human hematopoietic progenitor cells (CFU-GM) was measured in comparison to AZT. While d4T reduces colony-forming units by 50% at a concentration of 100 microM, it takes only 1 microM AZT to have a similar toxic effect. With erythrocyte burst forming units (BFU-E) the in vitro toxicities for d4T and AZT have comparable ID50 values of 10 and 6.7 microM, respectively.

Syngeneic tumor cell lines free of endogenous type C virus or viral antigen antigen expression were derived from spontaneously occurring tumors of the BALB/cCr mouse. Two cell lines free of endogenous type C virus were examined and found to be highly tumorigenic in tumor growth kinetic studies. In vitro inoculation of these cell lines with Rauscher-murine leukemia virus (R-MuLV) resulted in their chronic infection in which 95 to 100% of the cells were scored as virus positive. These infected lines showed a highly significant increase in their immunogenicity as compared to their uninfected controls. Animals in which these virus-positive tumors regressed were then shown to be highly resistant to challenge with the uninfected tumor cell lines as well as to live R-MuLV. This observed resistance to uninfected tumor cell lines could not be induced by immunization of the mouse with uninfected tumor cells and R-MuLV simultaneously at the same injection site, nor could it be induced with lethally irradiated virus-infected tumor cells, subtumorigenic doses of uninfected cells, or inactivated R-MuLV or Gross leukemia virus (G-MuLV). Cell-mediated cytotoxicity studies revealed that spleen cells obtained from animals whose virus-infected tumors regressed were cytotoxic to homologous infected and uninfected tumor cells as well as to other uninfected tumor cell lines syngeneic to the BALB/c mouse. Correlation of in vitro cytotoxicity with in vivo immunity was provided by the Winn assay, by inoculation into susceptible mice of immune and nonimmune spleen cells premixed with uninfected tumor cells. The immune cells were highly effective in preventing this tumor cell transplantation. It was concluded that type-C virus infection of these syngeneic tumor cells resulted in their acquiring strong transplantation antigens that were in part due to the virion, but were at least in part due to alterations of antigens or haptens that are present in a less immunogenic form on the uninfected tumor cell.

The protected nucleoside 1-(2-deoxy-2-fluoro-3,5-di-O-benzoyl-beta-D-arabinofuranosyl)-5-ethylura cil (10) was prepared by condensation of 3,5-dibenzoyl-2-deoxy-2-fluoro-alpha-D-arabinofuranosyl bromide (9) with 2,4-bis-O-(trimethylsilyl)-5-ethyluracil (8). The ratio in this coupling reaction has been raised to 17:1 in favor of the desired beta-anomer. Deprotection by aminolysis gave 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-ethyluracil (FEAU, 1) in 67% isolated yield from the bromo sugar 9. In vitro data show that FEAU has activity against herpes simplex virus types 1 and 2 comparable to that of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAU, 2), 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU, 3), and acyclovir (ACV, 12). The cellular toxicity of FEAU was found to be much lower than that of the other nucleoside analogues. Biochemical experiments indicate that FEAU has similar affinity toward thymidine kinases encoded by HSV 1 and 2 and a much lower affinity for cellular thymidine kinase than thymidine. The in vivo antiviral effects of FEAU, FMAU, FIAU, and ACV were evaluated against herpes infection in a systemic mouse encephalitis model and a cutaneous guinea pig model. While FEAU showed activity comparable to that of ACV in the systemic infection model, it was superior in the cutaneous herpes infection model.

A review of our current progress in C-type virus vaccine research is presented. This includes the findings of C-type virus or its antigen expressions in every naturally occurring tumor of two strains of "low-incidence" laboratory mice, the BALB/cCr mouse and the NIH Swiss mouse. Vaccine preparation methods are described including the inactivation of C-type virus infectivity with optimal maintenance of the antigen titers of at least two of the polypeptides of the C-type virus, gp69/71 and p30. The cell-mediated immune response of the mouse to C-type virus vaccines, as measured by a footpad assay for delayed-type hypersensitivity and an in vitro lymphocyte transformation assay, is described. Studies with two murine C-type viruses (Rauscher leukemia and Gross leukemia) a simian C-type virus, and an avian C-type virus (avian myeloblastosis virus) showed that the cell-mediated immune response of the animal includes type-specific, group-specific, and interspecies-specific reactivity. The mouse gave a cell-mediated immune response to at least one of the polypeptides of the C-type virus, the gp69/71, whether this polypeptide was presented to the immune system of the mouse as whole virus, Tween-ether-treated virus, or a purified polypeptide. One measure of the effectiveness of the C-type virus vaccines was provided by immunization of the mouse with Rauscher leukemia virus preparation that induced resistance to challenge with both live Rauscher leukemia virus and a naturally occurring BALB/c leukemia virus. Evidence is presented that the C-type virus can act as an effective transplantation antigen in syngeneic tumor cell lines resulting in the immunogenicity and loss of tumorigenicity of these cell lines. An approach to the viral immunoprevention of spontaneously occurring tumors is discussed.

Several 2,5'-anhydro analogues of 3'-azido-3'-deoxythymidine (AZT), 3'-azido-2'3'-dideoxyuridine (AZU), 3'-azido-2'3'-dideoxy-5-bromouridine, 3'-azido-2',3'-dideoxy-5-iodouridine, and 3'-deoxythymidine and the 3'-azido derivative of 5-methyl-2'-deoxyisocytidine have been synthesized for evaluation as potential anti-HIV (human immunodeficiency virus) agents. These 2,5'-anhydro derivatives, compounds 13-17, demonstrated significant anti-HIV-1 activity with IC50 values of 0.56, 4.95, 26.5, 27.1, and 48 microM, respectively. Compared to that of the parent compounds AZT and AZU, the respective 2,5'-anhydro analogues, compounds 13 and 14, were somewhat less active. Whereas AZT was cytotoxic with a TCID50 of 29 microM, the toxicity of the 2,5'-anhydro derivative of AZT, compound 13, was reduced considerably to a TCID50 value of greater than 100 microM. The 2,5'-anhydro analogue of 5-methyl-2'-deoxyisocytidine also demonstrated anti-HIV-1 activity with an IC50 value of 12 microM. These compounds were also evaluated against Rauscher-Murine leukemia virus (R-MuLV) in cell culture. Among them, AZT, 3'-azido-2',3'-dideoxy-5-iodouridine, 3'-azido-2',3'-dideoxy-5-bromouridine, and 2,5'-anhydro-3'-azido-3'-deoxythymidine (13) were found to be most active, with IC50 values of 0.023, 0.21, 0.23, and 0.27 microM, respectively.

Syntheses of 5-(2-haloethyl)-2'-deoxyuridines, 5-(3-chloropropyl)-2'-deoxyuridines, and 5-(2-chloroethyl)-2'-deoxycytidine are described. The antiviral activities of these compounds were determined in cell culture against herpes simplex virus types 1 and 2. All compounds were shown to possess significant and selective antiviral activity. The most potent derivative, 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), inhibited HSV-1 at concentrations below 0.1 microgram/mL. It exerted measurable inhibitory effects on cell proliferation only at concentrations higher than 100 micrograms/mL. In vivo CEDU reduced the mortality rate of HSV-1-infected mice at concentrations lower than 5 mg/kg per day when given intraperitoneally and orally. Thus, it proved to be more effective in this in vivo model than the reference compounds (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 9-[(2-hydroxyethoxy)methyl]guanine (ACV).