Chemistry and anti-HIV properties of 2'-fluoro-2',3'-dideoxyarabinofuranosylpyrimidines.

The synthesis, chemistry, biochemistry, and anti-HIV activity of a series of 1-(2,3-dideoxy-2-fluoro-beta-D-threopentofuranosyl)pyrimidines have been studied in an attempt to find useful anti-AIDS drugs. Synthesis is carried out via a 2,3-dideoxyribose intermediate which facilitates the preparation of analogues by removing the sugar 3'-hydroxyl group prior to, rather than after, condensation with a uracil or cytosine aglycon. The 2'-F-dd-uridine analogues 7a-d (with H, F, Cl, and CH3 substitution in the 5-position) as well as the 4-deoxy compound (12b) are nonprotective to ATH8 or CEM cells infected with HIV-1. In the corresponding cytidine series, the 5-chloro analogue (11) is inactive. However, 2'-fluoro-2',3'-dideoxyarabinosylcytosine, 10a, and its 5-fluoro analogue, 10b, are both active. While neither compounds is a potent as ddC or 5-F-ddC (2b), 10b gives complete protection against the cytopathic effects of HIV in both host cell lines. 2'-Fluoro substitution confers increased chemical and enzymatic stability on dideoxynucleosides. Even though dideoxy pyrimidine nucleosides are inherently more stable than the corresponding purine analogues toward acid-catalyzed cleavage of the glycosidic bond, 2'-fluoro substitution (10a) still increases stabilization relative to ddC (2b). No detectable deamination by partially purified cytidine deaminase is observed with the 2'-fluoro compounds 10a, 10b, or 11 under conditions which rapidly deaminate cytidine. A small amount of 2'-F-dd-ara-U (7a) is formed from 10a in monkey plasma after greater than 24 h of exposure. The octanol-water partition coefficients for the dideoxynucleosides in this study indicate their hydrophilic character, with log P values varying from -0.28 to -1.18.     

5-Halogeno-3'-fluoro-2',3'-dideoxyuridines as inhibitors of human immunodeficiency virus (HIV): potent and selective anti-HIV activity of 3'-fluoro-2',3'-dideoxy-5-chlorouridine

The novel 5-chloro-, 5-bromo-, and 5-iodo-derivatives of 3'-fluoro-2',3'-dideoxyuridine (FddUrd), designated FddCIUrd, FddBrUrd, and FddIUrd, respectively, have been synthesized and evaluated for their antiretrovirus activity against human immunodeficiency virus (HIV) and murine Moloney sarcoma virus. All three 5-halogeno-FddUrd analogues inhibited HIV-1 replication in MT4 cells with an effective dose (ED50) of about 0.2-0.4 microM. However, FddCIUrd was markedly more selective in its anti-HIV-1 activity than FddBrUrd or FddIUrd. The selectivity index of FddCIUrd was similar to that of 3'-azido-2',3'-dideoxythymidine (AZT) when evaluated in parallel (1408 and 1603, respectively). The FddUrd derivatives also had a marked inhibitory effect on HIV-2 replication in MT4 cells and HIV-1 induced antigen expression in HUT-78 cells. However, neither FddUrd nor its 5-halogeno derivatives were inhibitory to Moloney sarcoma virus-induced transformation of murine C3H cells. The anti-HIV-1 activity of FddUrd, FddCIUrd, FddBrUrd, and FddIUrd was reversed by the addition of thymidine and 2'-deoxycytidine. The 5-halogeno-FddUrd analogues had a markedly higher affinity for MT4 thymidine kinase than FddUrd (Ki/Km, 4.0-4.7, as compared with 302 for FddUrd).     

Disposition in animals of a new anti-HIV agent: 2',3'-didehydro-3'-deoxythymidine

The pharmacokinetics and CNS penetration of the anti-human immunodeficiency virus agent 2',3'-didehydro-3'-deoxythymidine have been examined in CD-1 mice. The drug was rapidly cleared from plasma with a terminal half-life of 17 min after an iv bolus dose at 25 mg/kg. Oral absorption of 2',3'-didehydro-3'-deoxythymidine was rapid and complete (98% bioavailable) with plasma levels approximately the same as those measured after iv administration. Estimates of the total body clearance and apparent volume of distribution were 43 ml/hr and 19 ml, respectively. In the mouse, entry into the central nervous system was rapid but the concentrations were somewhat low. However, drug concentrations which were reported to be effective in inhibiting replication of the virus in cell culture, greater than 0.01 microM, could be measured in the brain after a single oral dose at 25 mg/kg. A study to examine the urinary excretion of the drug in CD rats, beagle dogs and cynomolgus monkeys showed that 2',3'-didehydro-3'-deoxythymidine was primarily renally excreted unchanged.     

Single dose and multiple dose pharmacokinetics of 2'-fluoro-2',3'-dideoxyadenosine and 2'-fluoro-2',3'-dideoxyinosine, anti-HIV agents, in rats.

A single and multiple dose pharmacokinetic (PK) study was conducted in rats following oral administration of 2'-fluoro-2',3'-dideoxyadenosine (FddA) and 2'-fluoro-2',3'-dideoxyinosine (FddI) at three dose levels. Six rats/gender were assigned to one of the three FddA or FddI dose levels: 40, 250, and 1000 mg/kg/day. Three rats/gender were assigned to the PK study on day 1, while the remaining 3 rats/gender were assigned to the PK study on day 14. The rats received the appropriate doses of either FddA or FddI orally by gavage once a day for 14 days. Serial blood samples up to 24 h and cumulative urine samples (0-24 h) were collected on both days 1 and 14. Plasma and urine samples were analyzed for the concentrations of intact FddA and/or FddI using a validated assay. The data were subjected to non-compartmental PK analyses. Over the dose range of 40-1000 mg/kg. both FddA and FddI exhibited dose dependent pharmacokinetics in rats. Following FddA administration, there was a rapid and extensive in vivo conversion of FddA to FddI; FddI was the major circulating moiety as reflected by Cmax and AUC values (generally 2-3-fold greater than those of FddA at each dose level) as well as the amount excreted (%UR) in the urine. In contrast, following FddI administration, Cmax, AUC, and %UR values were 2-5-fold lower as compared to the FddI generated from FddA administration at each dose level, which also suggested that FddI was not absorbed as extensively as FddA. Based on the findings of this study, FddA is an excellent prodrug of FddI.     

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.     

5'-O-phosphonomethyl-2',3'-dideoxynucleosides: synthesis and anti-HIV activity.

5'-O-Phosphonomethylation of different pyrimidine 2',3'-dideoxynucleosides was accomplished by reaction of the latter with diethyl [(p-toylsulfonyl)oxy]methanephosphonate (1) in the presence of sodium hydride. The base-phosphonomethylated (15-19) and sugar-phosphonomethylated (8-12) derivatives could be readily distinguished by 1H and 13C NMR and MS analysis. Protection of the uracil or thymine residue with a N3-benzoyl group failed to prevent base modification. However, O4-methyl-protected 2',3'-dideoxyuridine readily afforded the 5'-O-phosphonomethylated derivative 12, which was converted to both the 2',3'-didoxyuridine analogue 27 and the 2',3'-dideoxycytidine counterpart 29. The 5'-O-phosphonomethyl derivatives of 3'-deoxythymidine (23), 2',3'-dideoxyuridine, (27), 2',3'-dideoxycytidine (29), 3'-O-methylthymidine (26), and 3'-amino-3'-deoxythymidine (28) did not show an appreciable anti-HIV activity in MT-4 cells. In contrast, the 5'-O-phosphonomethyl derivatives of 3'-deoxy-3'-fluorothymidine (24) and 3'-azido-3'-deoxythymidine (25) inhibited HIV-1 cytopathogenicity by 50% at a concentration of approximately 1 microM.     

Synthesis, structure-activity relationships, and drug resistance of beta-d-3'-fluoro-2',3'-unsaturated nucleosides as anti-HIV Agents

Our recent studies demonstrated that d- and l-2'-fluoro-2',3'-unsaturated nucleosides (d- and l-2'-F-d4Ns) display moderate to potent antiviral activities against HIV-1 and HBV. As an extension of these findings, beta-d-3'-fluoro-2',3'-unsaturated nucleosides were synthesized as potential antiviral agents. The key intermediate (2S)-5-(1,3-dioxolan)-1-benzoyloxy-3,3-difluoropentan-2-ol 6 was prepared from 2,3-O-isopropylidene-d-glyceraldehyde 1, which was converted to 5-O-benzoxy-d-2-deoxy-3,3-difluoropentofuranosyl acetate 7 by the ring-closure reaction under acidic conditions. The acetate 7 was condensed with silylated purine and pyrimidine bases, which produced the alpha and beta isomers. The 3',3'-difluoro nucleosides were then treated with t-BuOK to give the desired 3'-fluoro-unsaturated nucleosides. We studied the structure-activity relationships of d-3'-fluoro-2',3'-unsaturated nucleosides against HIV-1 in human peripheral blood mononuclear cells, from which we found that the cytosine derivative 26 was the most potent among the synthesized compounds. To understand the mode of action and drug resistance profile, with particular regard to the role of fluorine, we performed the molecular modeling studies of the cytidine analogue d-3'F-d4C and found a good correlation between calculated relative binding energies and activity/resistance data. Our model also shows interactions of the 3'-fluorine and the 2',3' double bond, which can be correlated to the observed biological data. Differences between fluorine substitution at the 3' and 2' positions may account for the higher cross-resistance with lamivudine observed in the 2'-fluorinated series.     

Potent and selective activity of 3'-azido-2,6-diaminopurine-2',3'-dideoxyriboside, 3'-fluoro-2,6-diaminopurine-2',3'-dideoxyriboside, and 3'-fluoro-2',3'-dideoxyguanosine against human immunodeficiency virus

Several sugar-modified 2,6-diaminopurine and guanine 2',3'-dideoxyribosides were synthesized and evaluated in vitro for their ability to inhibit the cytopathic effect and replication of human immunodeficiency virus (HIV), the causative agent of acquired immunodeficiency syndrome (AIDS). 3'-Azido-2,6-diaminopurine-2',3'-dideoxyriboside (AzddDAPR), 3'-fluoro-2,6-diaminopurine-2',3'-dideoxyriboside (FddDAPR), and 3'-fluoro-2',3'-dideoxyguanosine emerged as potent and selective anti-HIV agents in MT4 cells (50% effective antiviral dose: 0.3-4.5 microM). Their selectivity indexes, based on the ratio of the 50% cytotoxic dose to the 50% antiviral effective dose, were 157, 80, and 96, respectively, as compared to 106 for 2,6-diaminopurine-2',3'-dideoxyriboside (ddDAPR) and 132 for 2',3'-dideoxyadenosine (ddAdo), two other potent anti-HIV agents. The 9-beta-D-arabinoside and 9-beta-D-2'-deoxyxyloside derivatives of 2,6-diaminopurine were devoid of any antiretrovirus activity. Both AzddDAPR and FddDAPR, like the parent compounds ddDAPR and ddAdo, proved susceptible to deamination by beef intestine adenosine deaminase (Km, 11, 148, 29, and 73 microM, respectively). 2'-Deoxycoformycin, a potent inhibitor of adenosine deaminase, decreased the antiretrovirus and cytostatic activity of ddDAPR and FddDAPR to a greater extent than that of AzddDAPR. This suggests that ddDAPR and FddDAPR are primarily active as their guanine analogues, whereas AzddDAPR may be potentially active as a 2,6-diaminopurine derivative as well.     

The HIV replication inhibitor 3'-fluoro-3'-deoxythymidine blocks sialylation of N-linked oligosaccharides.

The ability of 3'-fluoro-3'-deoxythymidine (FLT) to interfere with glycosylation was investigated in an experimental system, where the effects on the herpes simplex virus type 1-specified glycoprotein gC were determined. By adding FLT to HSV-infected cells after the peak of DNA synthesis, it was possible to segregate possible effects on nucleic acid metabolism from the effects on glycosylation of gC. It was found that FLT treatment of HSV-infected cells at concentrations of 20-500 micrograms/ml resulted in a significant increase in the electrophoretic mobility of gC, indicating a reduction of the amount of carbohydrates incorporated into gC. Lectin-binding assays demonstrated that the FLT treatment blocked addition of sialic acid to complex type N-linked glycans. The effects on glycosylation were observed in cells infected with an HSV mutant, deficient in thymidine kinase (TK), but not in cells infected with wild type virus. The cells infected with the wild type virus contained five times more total FLT metabolites than the cells infected with the TK-deficient mutant, whereas the latter cell type contained significantly higher amounts of unmetabolized FLT. This result indicates that FLT itself, and not a metabolite, was responsible for the effects on glycosylation.     

Anti-retrovirus activity of 3'-fluoro- and 3'-azido-substituted pyrimidine 2',3'-dideoxynucleoside analogues

The 3'-fluoro-and 3'-azido-substituted derivatives of 2',3'-dideoxythymidine (ddThd), 2',3'-dideoxyuridine (ddUrd), 2',3'-dideoxy-5-ethyluridine (ddEtUrd) and 2',3'-dideoxycytidine (ddCyd) have been synthesized and evaluated for their anti-retrovirus activity [against human immunodeficiency virus (HIV) and murine Moloney sarcoma virus (MSV)]. Based on their 50% effective doses the most potent inhibitors of HIV replication in human MT4 lymphocytes were: FddThd (0.001 microM), AzddThd (0.004 microM), FddUrd (0.04 microM) and AzddUrd (0.36 microM). Their selectivity indexes were 197, 5000, 500 and 677, respectively. In contrast, none of the 3'-substituted ddEtUrd derivatives had a marked antiviral effect. The 2',3'-dideoxynucleoside analogues showed poor, if any, substrate affinity for (bacterial) dThd phosphorylase. AzddThd and FddThd inhibited human dThd kinase to a much greater extent (Ki/Km: 0.66 and 3.4, respectively) than did AzddUrd or FddUrd (Ki/Km: 71 and 171, respectively). The Ki/Km values of FddCyd and AzddCyd for human dCyd kinase were about 60. Although phosphorylation is a prerequisite for the anti-retrovirus activity of the 2',3'-dideoxynucleoside derivatives, there is no close correlation between the anti-retrovirus potency of the 3'-fluoro- and 3'-azido-substituted ddUrd, ddThd, ddEtUrd and ddCyd derivatives and their affinity for dThd kinase or dCyd kinase.     

The anti-HTLV-III (anti-HIV) and cytotoxic activity of 2',3'-didehydro-2',3'-dideoxyribonucleosides: a comparison with their parental 2',3'-dideoxyribonucleosides

A series of 2',3'-didehydro-2',3'-dideoxyribonucleosides (ddeNs) [i.e., 2',3'-dideoxythymidinene (ddeThd), 2',3'-dideoxyuridinene (ddeUrd), 2',3'-dideoxycytidinene (ddeCyd), and 2',3'-dideoxyadenosinene (ddeAdo)] has been synthesized and the individual members compared in terms of their in vitro antiviral, antimetabolic, and cytostatic properties to their 2',3'-saturated counterparts (ddNs) (i.e., ddThd, ddUrd, ddCyd and ddAdo). All ddeNs except ddeUrd are potent and/or selective inhibitors of human immunodeficiency virus (HIV) in vitro, ddeCyd being the most potent (MIC50, 0.30 microM). The inhibitory effect of ddeCyd on ATH8 cell proliferation and HIV-induced cytopathogenicity is comparable to that of ddCyd. ddeThd is a more potent anti-HIV agent than ddThd (MIC50, 3.4 microM and 84 microM, respectively), but also more cytostatic (ID50, 172 microM and greater than 2000 microM, respectively). However, its in vitro chemotherapeutic index is higher than that of 3'-azido-2',3'-dideoxythymidine, a drug which has recently proven effective in the treatment of acquired immunodeficiency syndrome. ddeAdo has a weaker anti-HIV and a stronger cytostatic effect than ddAdo. Neither ddeUrd nor ddUrd shows significant anti-retroviral activity at 500 microM. In contrast to their anti-retroviral activity, both ddNs and ddeNs lack any appreciable inhibitory activity against a series of nononcogenic RNA and DNA viruses, pointing to their selectivity as anti-retroviral agents. All ddeNs show a progressive loss of anti-retroviral effect upon prolonged incubation with virus-infected cells. This phenomenon is most likely due to the chemical instability of these compounds, and not to a preferential enzymatic phosphorolytic cleavage of the ddeNs. Evidence is presented that ddeCyd and ddCyd, and ddeThd and ddThd are phosphorylated by cellular dCyd kinase and dThd kinase, respectively. However, the Ki values as alternate substrate inhibitors for their respective kinases are high (greater than 500 microM), indicating poor substrate activity and, thus, poor anabolism in ATH8 cells.     

Synthesis and anti-HIV activity of 4'-thio-2',3'-dideoxynucleosides.

A series of 2',3'-dideoxy-4'-thionucleoside analogues of purines and pyrimidines, including 4'-thioddI (17), 4'-thioddC (27), and 4'-thioAZT (34), were synthesized and evaluated for their inhibitory activity against human immunodeficiency virus (HIV). A stereospecific synthesis of the 2,3-dideoxy-4-thioribofuranosyl carbohydrate precursor 11 starting with L-glutamic acid is described. 2',3'-Dideoxy-4'-thiocytidine (27) displayed significant, but modest activity in vitro against human immunodeficiency virus.     

Stereoselective synthesis and antiviral activity of D-2',3'-didehydro-2',3'-dideoxy-2'-fluoro-4'-thionucleosides

As 2',3'-didehydro-2',3'-dideoxy-2'-fluoronucleosides have exhibited interesting antiviral effects against HIV-1 as well as HBV, it is of interest to synthesize the isosterically substituted 4'-thionucleosides in which 4'-oxygen is replaced by a sulfur atom. To study structure-activity relationships, various pyrimidine and purine nucleosides were synthesized from the key intermediate (2R,4S)-1-O-acetyl-5-O-(tert-butyldiphenylsilyl)-2,3-dideoxy-2-fluoro-2-phenylselenyl-4-thio-beta-D-ribofuranoside 8, which was prepared from the 2,3-O-isopropylidene-D-glyceraldehyde 1 in 13 steps. The antiviral activity of the synthesized compounds were evaluated against HIV-1 in human peripheral blood mononuclear (PBM) cells, among which cytidine 17, 5-fluorocytidine 18, adenosine 24, and 2-fluoroadenosine 32 showed moderate to potent anti-HIV activities (EC(50) 1.3, 11.6, 8.1, and 1.2 microM, respectively). It is noteworthy that 2-fluoroadenosine analogue 32 showed antiviral potency as well as high cytotoxicity (IC(50) 1.5, 1.1, and 7.6 microM for PBM, CEM, and Vero, respectively) whereas no other compound showed cytotoxicity up to 100 microM. The cytidine 17 and 5-fluorocytidine 18 analogues showed significantly decreased antiviral activity against the clinically important lamivudine-resistant variants (HIV-1(M184V)), whereas the corresponding D-2'-Fd4 nucleosides showed limited cross-resistance. Molecular modeling studies demonstrated that the larger van der Waals radius as well as the close proximity to Met184 of the 4'-sulfur atom of D-2'-F-4'-Sd4C (17) may be the reasons for the decreased antiviral potency of synthesized 4'-thio nucleosides against the lamivudine-resistant variants (HIV-1(M184V)).     

Synthesis and antiviral activity evaluation of 3'-fluoro-3'-deoxyribonucleosides: broad-spectrum antiviral activity of 3'-fluoro-3'-deoxyadenosine

Five 3'-fluorinated ribonucleosides were prepared and evaluated for their inhibitory properties against different viruses. The synthesis of these compounds was achieved by treatment of 2',5'-di-O-tritylated nucleoside analogues possessing a xylo-configuration with diethylaminosulfur trifluoride, followed by deprotection. 3'-Fluoro-3'-deoxyadenosine was active against a broad range of viruses, encompassing both DNA viruses [pox (vaccinia)], single-stranded (+) RNA viruses [picorna (polio, Coxsackie B), toga (sindbis, Semliki Forest)] and double-stranded RNA viruses (reo). In its antiviral activity spectrum 3'-fluoro-3'-deoxyadenosine clearly differed from those adenosine analogues that are known as inhibitors of S-adenosylhomocysteine hydrolase. 3'-Fluoro-3'-deoxyadenosine also proved effective in vivo, in inhibiting tail lesion formation in mice inoculated intravenously with vaccinia virus.     

Synthesis and anti-HIV activity of carbocyclic 2',3'-didehydro-2',3'-dideoxy 2,6-disubstituted purine nucleosides

(+-)-cis-[4-[(2,5-Diamino-6-chloropyrimidinyl)amino]-2- cyclopentenyl]carbinol (5a) was synthesized from 2-amino-4,6-dichloropyrimidine and cis-4-(hydroxymethyl)cyclopentenylamine (2a) by subsequent preparation of the 5-[(4-chlorophenyl)azo] derivative of the resulting pyrimidine (3a) and reduction of the azo moiety with zinc and acetic acid. The carbocyclic analogue of 2',3'-didehydro-2',3'-dideoxy 2-amino-6-chloropurine (6a) and the corresponding 8-azapurine (9a) were prepared from 5a. The carbocyclic 2',3'-didehydro-2',3'-dideoxy analogues of guanine (7a) and 2,6-diaminopurine (8a), and 8-azaguanine (10a) and 8-aza-2,6-diaminopurine (11a) were prepared from 6a and 9a, respectively. The corresponding 2',3'-saturated series of 2-amino-6-substituted-purine carbocyclic nucleosides was prepared following the same scheme starting with cis-4-(hydroxymethyl)cyclopentylamine (2b). Carbocyclic 2',3'-didehydro-2',3'-dideoxyguanosine (carbovir, 7a) emerged as a potent and selective anti-HIV agent. Its hydrolytic stability and its ability to inhibit the infectivity and replication of HIV in T-cells at concentrations of approximately 200-400-fold below toxic concentrations make carbovir an excellent candidate for development as a potential antiretroviral agent.     

Synthesis and anti-HIV activity of some novel arylphosphate and H-phosphonate derivatives of 3'-azido-2',3'-dideoxythymidine and 2',3'-didehydro-2',3'-dideoxythymidine.

Phosphate and H-phosphonate derivatives of anti-HIV nucleoside analogues (AZT and d4T) were prepared as potential prodrugs of the bio-active free nucleotide and they were evaluated for their inhibitory effects on the replication of HIV-1 in several cell culture systems. One compound exhibited an important anti-HIV-1 activity and proved to be significantly more efficient than the parent nucleoside.