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).     

Synthesis and anti-HIV activity of different sugar-modified pyrimidine and purine nucleosides

A series of base-modified pyrimidine 3'-azido-2',3'-dideoxynucleosides and 3'-substituted purine and pyrimidine 2',3'-dideoxynucleosides have been synthesized and evaluated for their inhibitory activity against human immunodeficiency virus (HIV) replication in MT-4 cells. The following pyrimidine derivatives emerged as the most potent and/or selective inhibitors of HIV-induced cytopathogenicity (in order of decreasing selectivity: 3'-azido-3'-deoxythymidine (AZT), 3'-azido-2',3'-dideoxyuridine (AzddUrd), 3'-azido-2',3'-dideoxy-5-methylcytidine (AzddMeCyd), 3'-fluoro-ddUrd (FddUrd), 3'-fluoro-ddThd (FddThd), the N4-hydroxylated derivative of AzddMeCyd and the N4-methylated derivative of AzddMeCyd. Among the purine 2',3'-dideoxynucleosides, 3'-azido-2',3'-dideoxyguanosine (AzddGuo), 3'-fluoro-ddGuo (FddGuo), and 3'-fluoro-2,6-diaminopurine 2',3'-dideoxynucleoside (FddDAPR) were the most selective inhibitors of HIV replication.     

Synthesis and anti-HIV evaluation of 2',3'-dideoxyribo-5-chloropyrimidine analogues: reduced toxicity of 5-chlorinated 2',3'-dideoxynucleosides

In view of the selective anti-HIV activity of 2',3'-dideoxy-3'-fluoro-5-chlorouridine (11), a series of eight 2',3'-dideoxy-5-chloropyrimidines were synthesized and evaluated for their inhibitory activity against human immunodeficiency virus type 1 (HIV-1) replication in MT-4 cells. A marked improvement in selectivity was noted for the 5-chlorouracil derivatives of 2,3-dideoxyribofuranose, 3-azido-2,3-dideoxyribofuranose, and 3-fluoro-2,3-dideoxyribofuranose, mainly due to decreased toxicity of the compounds for the host cells. While chlorination of 2',3'-dideoxycytidine removed the anti-HIV activity, introduction of a chlorine at the C-5 position of 3'-fluoro-, 3'-azido- or 2',3'-didehydro-2',3'-dideoxycytidine led to reduced cytotoxicity with only slightly reduced anti-HIV activity. X-ray analysis showed compound 11 to have two molecules in the asymmetric unit with chi = -168.8 (3) degrees and -131.3 (3) degrees and P = 179 (1) degree and 163 (1) degree, respectively; thus revealing no close resemblance to 3'-azido-3'-deoxythymidine (AZT).     

Inhibition of hepatitis B virus production by modified 2',3'-dideoxy-thymidine and 2',3'-dideoxy-5-methylcytidine derivatives. In vitro and in vivo studies.

The effect of analogues of both 2',3'-dideoxy-3'-fluorothymidine (FddThd) [2',3'-dideoxy-3'-fluorouridine (FddUrd), 2',3'-dideoxy-3'-fluoro-5-chlorouridine (FddClUrd), 2',3'-dideoxy-3'- fluoro-5-bromouridine (FddBrUrd) and 2',3'-dideoxy-3'-fluoro-5-bromovinyluridine (FddBVUrd)] and 2',3'-dideoxy-3'-fluorocytidine (FddCyt) [2',3'-dideoxy-3'-fluoro-5-fluorocytidine (FddFCyt), 2',3'-dideoxy-3'-fluoro-5-chlorocytidine (FddClCyt), 2',3'-dideoxy-3'-fluoro-5-methylcytidine (FddMeCyt), 2',3'-dideoxy-3'-fluoro-5-ethylcytidine (FddEtCyt), 2',3'-dideoxy-3'-chloro-5-methylcytidine (ClddMeCyt), 2',3'-dideoxy-3'-amino-5-methylcytidine (AmddMeCyt), 2',3'-dideoxy-3'-azido-5- methylcytidine (AzddMeCyt) and arabinosyl-5-methylcytosine (AraMeCyt)] were tested for their potential antiviral activity in vitro using the human hepatoblastoma cell line, Hep G2 2.2.15, which was transfected with a vector containing hepatitis B virus (HBV). It was found that FddThd, FddMeCyt, FddEtCyt, ClddMeCyt, AmddMeCyt and AraMeCyt display cytostatic activity at concentrations (CD50 values) between 0.54 (FddMeCyt) and 3.93 microM (FddEtCyt), while FddUrd, FddClUrd, FddBrUrd, FddBVUrd, FddCyt, FddFCyt, FddClCyt and AzddMeCyt do not affect cell growth at concentrations of up to 25 microM. Among the thymidine analogues tested, FddThd is the most effective antiviral agent: at a concentration of 0.03 microM a more than 90% reduction of HBV DNA synthesis was measured. On the other hand, the antiviral indexes displayed by FddClUrd, FddBrUrd and FddBVUrd are higher than tht of FddThd; FddUrd was completely inactive. The most powerful antiviral agents in the group of cytidine analogues tested in vitro were FddMeCyt (more than 90% reduction of HBVDNA synthesis at 0.10 microM) and ClddMeCyt (0.10 microM); FddClCyt, FddEtCyt, AmddMeCyt and AraMeCyt were of intermediate activity. None of the negligible antiviral activity was determined for FddUrd, FddCyt, FddFCyt and AzddMeCyt. FddThd and FddMeCyt displayed in vivo an antiviral effect in the duck/duck HBV (DHBV) animal system. Administration of 10 or 20 mg/kg (total daily dose) of FddThd and 5 or 10 mg/kg of FddMeCyt (i.m. daily) to ducks infected with DHBV for 12 days blocked virus production. Termination of treatment with FddThd of infected animals led to reappearance of the virus in the serum though at lower levels. The in vitro and the in vivo data suggest that FddThd and FddMeCyt might be promising antiviral agents for the treatment of infection caused by HBV in humans.     

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.     

TSAO analogues. Stereospecific synthesis and anti-HIV-1 activity of 1-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro -5'- (4'-amino-1',2'-oxathiole 2',2'-dioxide) pyrimidine and pyrimidine-modified nucleosides.

Several analogues of a new lead for anti-HIV-1 agents [1-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-thymine] -3'-spiro-5'-(4'-amino-1',2'-oxathiole 2',2'-dioxide) (TSAO) modified at positions N-3, O-4 and C-5 of the thymine moiety, have been prepared and evaluated as inhibitors of HIV-1 replication. A new stereoselective synthetic procedure is described. Reaction of 1,2-di-O-acetyl-5-O-benzoyl-3-C-cyano-3-O-mesyl-D-ribofuranose with pyrimidine bases, followed by treatment with Cs2CO3 afforded stereoselectively, beta-D-ribofuranosyl-3'-spiro nucleosides. 2',5'-O-Deacylation and subsequent treatment with tert-butyldimethylsilyl chloride gave the TSAO derivatives. Only those analogues having a tBDMSi group at both the C-5' and C-2' positions of the ribose moiety showed potent anti-HIV-1 activity. The activity ranged from 0.060 microM to 1.0 microM. Introduction of an alkyl or alkenyl function at N-3 of the thymine ring markedly decreased cytotoxicity without affecting the antiviral activity. While markedly active against HIV-1, the TSAO derivatives had no activity against HIV-2 or SIV. They represent the first example of nucleoside analogues with an intact ribose moiety that discriminate between HIV-1 and other retroviruses.     

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.     

Synthesis and antiretrovirus properties of 5'-isocyano-5'-deoxythymidine, 5'-isocyano-2',5'-dideoxyuridine, 3'-azido-5'-isocyano-3',5'-dideoxythymidine, and 3'-azido-5'-isocyano-2',3',5'-trideoxyuridine

The 5'-azidonucleosides 3 and 4 were obtained by treating thymidine and 2'-deoxyuridine with TPP/DEAD/HN3. The 3'-O-silylated 5'-azido-5'-deoxythymidine 5 and the corresponding 2'-deoxyuridine derivative 6 were transformed to the formamides (7 and 8, respectively) and dehydrated to the protected 5'-isocyano derivatives 9 and 10; deblocking gave 5'-isocyano-5'-deoxythymidine (11) and 5'-isocyano-2',5'-dideoxyuridine (12). 2,3'-Anhydro-5'-formamido derivatives of thymidine and 2'-deoxyuridine (19 and 20, respectively) were prepared by three different ways. In the most direct synthesis 3 and 4 were transformed to the 2,3'-anhydro-5'- azidonucleosides 17 and 18 by using TPP/DEAD; following the reaction with TPP/HCO2COCH3 gave 19 and 20. Nucleophilic opening reaction with LiN3 yielded the 3'-azido-5'-formylamino derivatives 21 and 22. Dehydration to 3'-azido-5'-isocyano-3',5'-dideoxythymidine (23) and 3'-azido-5'-isocyano-2',3',5'-trideoxyuridine (24) was achieved with tosyl chloride/pyridine. In contrast with 3'-azido-3'-deoxythymidine, compounds 11, 12, 23, and 24 were devoid of any marked inhibitory effect against DNA and RNA viruses including human immunodeficiency virus type I (HIV).     

Side-chain derivatives of biologically active nucleosides. 1. Side-chain analogs of 3'-azido-3'-deoxythymidine (AZT).

Starting from 3-O-mesyl-1,2-O-isopropylidene-alpha-D-allofuranose (9) the anomeric mixtures of the requisite carbohydrates 1,2-di-O-acetyl-6-O-benzoyl-5-deoxy-3-O-mesyl-D-allofuranoses++ + 17A alpha/beta, 1,2-di-O-acetyl-5,6-di-O-benzoyl-3-O-mesyl-D-allofuranoses 17B alpha/beta, and 1,2-di-O-acetyl-5,6-di-O-benzoyl-3-O-mesyl-L-talofuranoses 17C alpha/beta were synthesized. 1,2-Di-O-acetyl-5-O-benzoyl-6-deoxy-3-O-mesyl-D-allofuranoses++ + 17D alpha/beta and the corresponding L-talofuranoses 17E alpha/beta were obtained from 6-deoxy-3,5-di-O-benzoyl-1,2-O-isopropylidene-alpha-D- allofuranose (12) and the corresponding beta-L-talofuranose 13. Coupling of these sugar derivatives with thymine gave the beta-nucleoside derivatives 18A-E. Treatment of compounds 18A-E with DBU produced the corresponding 2,3'-anhydro nucleosides 19A-E with a free 2'-OH group. After deoxygenation of 2'-O-[[(4-methylphenyl)oxy]thiocarbonyl] compounds 20A-E with tributyltin hydride the 2,3'-anhydro bridge of the 2'-deoxynucleosides 21A-E was opened with LiN3 to produce the protected 3'-azido-2,3'-dideoxynucleoside derivatives 22A-G. Saponification with NaOCH3 gave 1-(3'-azido-2',3',5'-trideoxy-beta-D-allofuranosyl)thymine (2; homo-AZT), the 5'-C-(hydroxymethyl) derivatives of AZT 1-(3'-azido-2',3'- dideoxy-beta-D-allofuranosyl)thymine (3) and 1-(3'-azido-2',3'-dideoxy-alpha-L-talofuranosyl)thymine (4), and the 5'-C-methyl derivatives of AZT 1-(3'-azido-2',3',6'-trideoxy-beta-D-allofuranosyl)thymine (5) and 1-(3'-azido-2',3',6'-trideoxy-alpha-L-talofuranosyl)thymine (6). Compounds 2-6 were evaluated for their inhibitory effect on human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) replication in MT-4 cells and found inactive at subtoxic concentrations. Compounds 2-4 and 6 are not effective against herpes simplex virus type 1 (HSV-1) and type 2 (HIV-2), vaccinia virus (VV), and vesicular stomatitis virus (VSV) at 400 micrograms/mL. 5 is slightly active against HSV-1, HSV-2 and VV at 150, 300, and 300 micrograms/mL, respectively.     

Synthesis and antiviral activity of several 2,5'-anhydro analogues of 3'-azido-3'-deoxythymidine, 3'-azido-2',3'-dideoxyuridine, 3'-azido-2',3'-dideoxy-5-halouridines, and 3'-deoxythymidine against human immunodeficiency virus and Rauscher-murine leukemia virus

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.     

Novel [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]- 3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2" -dioxide) derivatives with anti-HIV-1 and anti-human-cytomegalovirus activity

New [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-d-ribofuranosyl]-3'-spiro-5' '-(4'-amino-1',2'-oxathiole-2',2'-dioxide) (TSAO) derivatives substituted at the 4' '-amino group of the spiro moiety with different carbonyl functionalities have been designed and synthesized. Various synthetic procedures, on the scarcely studied reactivity of the 3'-spiroaminooxathioledioxide moiety, have been explored. The compounds were evaluated for their inhibitory effect on both wild-type and TSAO-resistant HIV-1 strains, in cell culture. The presence of a methyl ester (10) or amide groups (12) at the 4'-position conferred the highest anti-HIV-1 activity, while the free oxalyl acid derivative (11) was 10- to 20-fold less active against the virus. In contrast, the presence at this position of (un)substituted ureido or acyl groups markedly diminished or annihilated the anti-HIV-1 activity. Surprisingly, some of the target compounds also showed inhibition of human cytomegalovirus (HCMV) replication at subtoxic concentrations. This has never been observed previously for TSAO derivatives. In particular, compound 26 represents the first TSAO derivative with dual anti-HIV-1 and -HCMV activity.     

Catabolic disposition of 3'-azido-2',3'-dideoxyuridine in hepatocytes with evidence of azido reduction being a general catabolic pathway of 3'-azido-2',3'-dideoxynucleosides.

3'-Azido-2',3'-dideoxyuridine (AzddU, CS-87) is a potent inhibitor of human immunodeficiency virus replication in vitro with low bone marrow toxicity. Although AzddU is currently being evaluated in clinical trials, its catabolic disposition is unknown. Pharmacokinetic studies in rhesus monkeys have demonstrated that a 5'-O-glucuronide is excreted in urine. The present study examined the catabolic disposition of AzddU is isolated rat hepatocytes, a model for the study at the cellular level of biosynthetic, catabolic and transport phenomena in the liver. Following exposure of cells to 10 microM [3H]AzddU, low intracellular levels of two catabolites, identified as 3'-azido-2',3'-dideoxy-5'-beta-D-glucopyranosyluridine (GAzddU) and 3'-amino-2',3'-dideoxyuridine (AMddU), were detected. Studies using rat microsomes demonstrated that GAzddU formation was only detected in the presence of uridine 5'-diphosphoglucuronic acid, and that the rate of AMddU formation increased significantly in the presence of NADPH. Under similar conditions, reduction of the 3'-azido function was also demonstrated herein with 3'-azido-2',3'-dideoxycytidine (AzddC), 3'-azido-2',3'-dideoxy-5-methylcytidine (AzddMeC) and 3'-azido-2',3'-dideoxyguanine (AzddG), suggesting that enzymatic reduction to a 3'-amino derivative is a general catabolic pathway of 3'-azido-2',3'-dideoxynucleosides at the hepatic site.     

3'-substituted 2',3'-dideoxynucleoside analogues as potential anti-HIV (HTLV-III/LAV) agents

A series of 2',3'-unsaturated and 3'-substituted 2',3'-dideoxynucleoside analogues of purines and pyrimidines have been synthesized and evaluated for their inhibitory activity against human immunodeficiency virus (HIV). The 2',3'-unsaturated analogues of 2',3'-dideoxycytidine (ddeCyd) and 2',3'-dideoxythymidine (ddeThd), 3'-azido-2',3'-dideoxythymidine (AzddThd), 3'-fluoro-2',3'-dideoxythymidine, 2',3'-dideoxycytidine (ddCyd), and 2',3'-dideoxyadenosine (ddAdo) emerged as the most potent inhibitors of HIV-induced cytopathogenicity in the human T lymphocyte cell lines ATH8 and MT4. In ATH8 cells ddCyd, ddeCyd, and ddAdo had the highest therapeutic index whereas in MT4 cells AzddThd, ddThd, ddCyd, and ddAdo were the most selective. Derivatives from ddThd in which the substituent group was linked to the 3'-carbon atom via a thio, sulfonyl, or oxygen bridge were far less inhibitory to HIV than was AzddThd.     

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.     

Synthesis and antiviral and cytostatic properties of 3'-deoxy-3'-fluoro- and 2'-azido-3'-fluoro-2',3'-dideoxy-D-ribofuranosides of natural heterocyclic bases

A series of 3'-deoxy-3'-fluoro- and 2'-azido-2',3'-dideoxy-3'-fluoro-D-ribofuranosides of natural heterocyclic bases have been synthesized with the use of universal carbohydrate precursors, viz., 1-O-acetyl-2,5-di-O-benzoyl-3-deoxy-3-fluoro-D-ribofuranose and methyl 2-azido-5-O-benzoyl-2,3-dideoxy-3-fluoro-beta-D-ribofuranoside, respectively. The cytostatic and antiviral activity of the compounds was evaluated against a variety of tumor cell lines and DNA/RNA viruses, respectively. As the most active compound, from both a cytostatic and antiviral activity viewpoint, emerged 3'-deoxy-3'-fluoroadenosine. It inhibited the proliferation of some tumor cell lines (i.e. murine leukemia L1210 and human T-lymphocyte MT-4) at a concentration of 0.2-2 micrograms/mL, and proved inhibitory to the replication of positive-stranded RNA viruses (i.e. polio, Coxsackie, Sindbis, Semliki forest), double-stranded RNA viruses (i.e. reo), and some DNA viruses (i.e. vaccinia) at a concentration of 1-4 micrograms/mL, which is well below the cytotoxicity threshold (40 micrograms/mL).     

3'-Spiro nucleosides, a new class of specific human immunodeficiency virus type 1 inhibitors: synthesis and antiviral activity of [2'-5'-bis-O-(tert-butyldimethylsilyl)-beta-D-xylo- and -ribofuranose]-3'-spiro-5"-[4"-amino-1",2"-oxathiole 2",2"-dioxide] (TSAO) pyrimidine nucleosides.

A series of 3'-spiro nucleosides have been synthesized and evaluated as anti-HIV-1 agents. Reaction of O-mesylcyanohydrins of furanos-3'-ulosyl nucleosides with base afforded [1-[2',5'-bis-O- (tert-butyldimethylsilyl)-beta-D-xylo- and -ribofuranosyl]]-3'-spiro-5"- [4"-amino-1",2"-oxathiole 2",2"-dioxide] derivatives of thymine, uracil and 4-N-acetylcytosine 11 and 12. Desilylation of 11 and 12 gave the full deprotected 3'-spiro xylo- and ribofuranosyl nucleosides 13 and 14 or the partially 5'-O-deprotected-3'-spiro beta-D-xylo- and -ribo-nucleosides 15 and 16, or 2'-O-deprotected-3'-spiro beta-D-ribo-nucleoside 17. 2'-Deoxygenation of 17 afforded 2'-deoxy-3'-spiro beta-D-erythro-pentofuranosyl derivative 18. These 3'-spiro derivatives were evaluated for their anti-HIV-1 activity. All 3'-spiro nucleosides having a xylo configuration did not show any anti-HIV-1 activity. 3'-Spiro ribo-nucleosides with none or only one silyl group at C-2' or C-5' or the 2'-deoxy derivative were also inactive at subtoxic concentrations. However, 3'-spiro ribo-nucleosides having two silyl groups at C-2' and C-5' were potent and selective inhibitors of HIV-1. None of the nucleoside analogues that showed anti-HIV-1 activity proved inhibitory to the replication of HIV-2 or SIV.     

Potential prodrug derivatives of 2',3'-didehydro-2',3'-dideoxynucleosides. Preparations and antiviral activities.

The preparations and antiviral activities of a series (4-17) of potential prodrug forms of the antivirals 2',3'-didehydro-2',3'-dideoxyadenosine (D4A) and 2',3'-didehydro-2',3'-dideoxycytosine (D4C) are reported. The 5'-phenyl- and 5'-methylphosphonates (4, 6, 8, and 10) and their phosphonothionate congeners (5, 7, 9, and 11), with the exception of 10, were inactive in vitro against HIV-1 and HIV-2. However, the 5'-phenyl, 5'-methyl, and 5'-(3'-thymidyl) phosphate diesters (12-17) demonstrated inhibition of the cytopathic effect of HIV-1 and HIV-2 (EC50 approximately 1-60 microM) and cytotoxicities (CC50 approximately 35-200 microM) at concentration levels comparable to those of their parent compounds, D4A and D4C. This strongly suggests that the diesters are hydrolyzed to the nucleosides D4A and D4C and/or their 5'-monophosphates. The facile hydrolysis of 12 and 13 to these products was demonstrated in a medium containing 10% fetal calf serum. The molecules can serve as ready prodrug sources of the free nucleosides and their 5'-monophosphates. Evidently, the phosphonates and phosphonothionates are not similarly cleaved, nor are they phosphorylated to form antivirally active or cytotoxic products. The importance of intracellular formation of these products in the activation of 12-17 is less clear. Potential prodrugs 4-17 are all stable in aqueous solution for hours with the exception of 14. Conjugates 4-17 showed no activity against a series of DNA and RNA viruses.