3'-Carbon-substituted pyrimidine nucleosides having a 2',3'-dideoxy and 2',3'-didehydro-2',3'-dideoxy structure: synthesis and antiviral evaluation.

The bis(tributylstannyl) derivative of 2',3'-didehydro-2',3'-dideoxyuridine (d4U) underwent an anionic 5'-O-->3'-C stannyl migration to yield the 3'-tributylstannyl-d4U. This compound, with its vinylstannane structure, allowed ready access to the preparation of 3'-carbon-substituted analogues through the Stille reaction. A conventional transformation of the uracil moiety of these d4U analogues led to the corresponding 2',3'-didehydro-2',3'-dideoxycytidine (d4C) counterparts. Some 2',3'-dideoxycytidine (ddC) analogues were also synthesized. Antiviral evaluation revealed that none of these analogues showed activity against HIV, hepatitis B virus, herpes simplex virus-1 (HSV-1) and HSV-2.

Synthesis of phosphorothioamidites derived from 3'-thio-3'-deoxythymidine and 3'-thio-2',3'-dideoxycytidine and the automated synthesis of oligodeoxynucleotides containing a 3'-S-phosphorothiolate linkage.

The synthesis of N4-benzoyl-5'-O-dimethoxytrityl-2',3'-dideoxy-3'-thiocytidine and its phosphorothioamidite is described for the first time, together with a shortened procedure for the preparation of 5'-O-dimethoxytrityl-3'-deoxy-3'-thiothymidine and its corresponding phosphorothioamidite. The first fully automated coupling procedure for the incorporation of a phosphorothioamidite into a synthetic oligodeoxynucleotide has been developed, which conveniently uses routine activators and reagents. Coupling yields using this protocol were in the range of 85-90% and good yields of singularly modified oligonucleotides were obtained. Coupling yields were also equally good when performed on either a 0.2 or 1 micro mol reaction column, thus facilitating large scale syntheses required for mechanistic studies.

Inhibition of human immunodeficiency virus type 1 replication by phosphonoformate- and phosphonoacetate-2',3'-dideoxy-3'-thiacytidine conjugates.

The synthesis of potential "combined prodrugs" where phosphonoformic acid (PFA) or phosphonoacetic acid (PAA) was attached to the 5'-O or N4 position of 2',3'-dideoxy-3'-thiacytidine (BCH-189) is described. The anti-HIV-1 activity of 11 analogues which included carboxylic ester or phosphoric ester linkages of PFA or PAA to BCH-189 was determined in MT-4 cells. Of these compounds, the IC50 of analogues 3, 4, 6, and 7 ranged from 0.2 to 100 microM, while IC50 for BCH-189 in this system was 0.1 microM. In vitro hydrolysis of the various esters or amides in human plasma indicated that these agents were relatively stable in the presence of plasma esterases with t1/2 values of up to 120 min. Moreover, lipophilicity of these compounds (partition coefficient) was determined in order to establish correlation between lipophilicity and diffusion of BCH-189 analogues into the cells. The active compounds may exert their effects by extracellular or intracellular hydrolysis to the corresponding antiviral agent BCH-189, but intrinsic anti-HIV-1 activity of some of PAA and PFA adducts, themselves, may also be involved.

Highly water-soluble lipophilic prodrugs of the anti-HIV nucleoside analogue 2',3'-dideoxycytidine and its 3'-fluoro derivative.

The synthesis of a novel series of lipophilic prodrug derivatives of the anti-HIV drugs 2',3'-dideoxycytidine (ddC, 1) and 3'-fluoro-ddC (2), involving N4-substitution with (N,N-dialkylamino)methylene side chains, is described. The increase in the partition coefficients for the prodrug series, compared to those of the parent drugs 1 and 2, ranged from 1.5- to 122-fold and from 1.6- to 175-fold, respectively. At pH 7.4, 37 degrees C, the hydrolytic t1/2 values ranged from 2 to 52 h, the diisopropyl derivatives (3d and 4d) being most stable in the series. 3d and 4d were greater than or equal to 4-fold and 1.7-fold more soluble in water than 1 and 2, respectively. The in vitro antiretroviral activities of 3d, 4a, and 4d were evaluated; the results indicate efficient prodrug-to-drug conversion under the assay conditions. The results of this investigation demonstrate that it is indeed feasible to chemically modify certain nucleoside analogues with inferior solubility properties to simultaneously achieve significantly enhanced lipid and water solubility.

Synthesis and biological evaluation of 2',3'-didehydro-2',3'- dideoxy-5-fluorocytidine (D4FC) analogues: discovery of carbocyclic nucleoside triphosphates with potent inhibitory activity against HIV-1 reverse transcriptase.

The discovery of a novel cytosine nucleoside, beta-D-2', 3'-didehydro-2',3'-dideoxy-5-fluorocytidine (D-D4FC), as a potent antihuman immunodeficiency virus (HIV) agent led us to synthesize a series of analogues and derivatives of beta-D-D4FC that could be more selective and also possess increased glycosidic bond stability. The synthesized D-D4FC analogues were evaluated for anti-HIV-1 activity, anticancer activity, and cytotoxicity in various cells. The biological data demonstrated that the 5-substitution of beta-D-D4FC with bromine (6c) and iodine (6d) resulted in the loss of antiviral activity, and the alpha-D anomer (7a) of D-D4FC was also devoid of activity. The 5-fluorouracil analogues (6b and 7b) of D-D4FC were less potent and more cytotoxic than the parent compound, whereas the beta-L-D4FU (11) showed both potent anti-HIV-1 activity and cytotoxicity. N4- and 5'-O-acyl derivatives (17, 15a-c) of beta-D-D4FC exhibited comparable antiviral activity to beta-D-D4FC. In contrast, the N4-isopropyl derivative (20) of beta-D-D4FC was not active against HIV-1, even at 100 microM. The carbocyclic analogues (26a,b) of D4FC demonstrated weak activity against HIV-1 and no toxicity in various cells. The triphosphates (27a,b) of the carbocyclic nucleosides demonstrated potent inhibitory activity against recombinant HIV-1 reverse transcriptase at submicromolar concentrations. Of the compounds tested as potential anticancer agents, beta-D-, alpha-D-, and beta-L-D4FU (6b, 7b, 11) showed inhibitory activity against rat glioma and modest activity against human lung carcinoma, lymphoblastoid, and skin melanoma cells.

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.

Synthesis and anti-HIV-1 activity of 2'-"up"-fluoro analogues of active anti-AIDS nucleosides 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (DDC).

1-(3-Azido-2,3-dideoxy-2-fluoro-beta-D-arabinofuranosyl)thymine (6, F-AZT) and 1-(2,3-dideoxy-2-fluoro-beta-D-threopentofuranosyl)cytosine (12, F-DDC) were synthesized from the potent antiherpes virus nucleosides 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)thymine (1, FMAU) and 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC) in the hope that introduction of a 2-"up"-fluoro substituent might potentiate the anti-HIV activity of AZT and DDC. FMAU (1) was converted in three steps into 2,3'-anhydro-1-(2-fluoro-2-deoxy-5-O-trityl-beta-D-lyxofuranosyl)thymine (4), which when treated with NaN3 followed by detritylation afforded 6. F-DDC was prepared by two methods. Tritylation of FIAC followed by treatment of the product with thiocarbonyldimidazole afforded the 5'-O-trityl-3'-O-(imidazolyl)thiocarbonyl nucleoside 9. Upon radical reduction of 9 with Bu3SnH and AIBN, 5'-O-trityl-DDC 10 was obtained. Compound 10 was detritylated to give 12, which (when obtained by this procedure) resisted crystallization, but the diacetate 12' was obtained in crystalline form. Alternatively, FAC (14) was converted into N4,O5'-dibenzoyl derivative 15, which was treated with thiocarbonyldiimidazole. Reduction of 16 with Bu3SnH/AIBN followed by debenzoylation afforded 12, which was obtained in crystalline form. F-AZT did not exhibit any significant activity against the human immunodeficiency virus (HIV) in vitro. F-DDC, however, showed activity against HIV-1, but the therapeutic index is much inferior to that of AZT.

Synthesis and antiviral activity of monofluoro and difluoro analogues of pyrimidine deoxyribonucleosides against human immunodeficiency virus (HIV-1).

A range of 2'-fluoro and 2',3'-difluoro analogues of pyrimidine deoxyribonucleosides have been synthesized and evaluated against human immunodeficiency virus (HIV-1) in a human lymphoblastoid cell line. Among these compounds, 1-(2,3-dideoxy-2-fluoro-beta-D-threopentofuranosyl)cytosine (12), 2',3'-didehydro-2',3'-dideoxy-2'-fluorocytidine (35), 1-(2,3-dideoxy-2,3-difluoro-beta-D-arabinofuranosyl)cytosine (41), and 3'-deoxy-2',3'-didehydro-2'-fluorothymidine (45) were found to have significant antiviral activity, with IC50 values of 0.65, 10, 10, and 100 microM, respectively. The structure-activity relationships are discussed.

Synthesis and anti-HIV activity of several 2'-fluoro-containing pyrimidine nucleosides.

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.

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 biological evaluation of 2',3'-dideoxy-L-pyrimidine nucleosides as potential antiviral agents against human immunodeficiency virus (HIV) and hepatitis B virus (HBV).

Various 2',3'-dideoxy-L-cytidine,2',3'-dideoxy-L-uridine, and 3'-deoxy-L-thymidine analogues have been synthesized and evaluated in vitro as potential anti-HIV and anti-HBV agents. Coupling of 1-O-acetyl-5-O-(tert-butyldimethylsilyl)-2,3-dideoxy-L-ribofuranose (1) with silylated derivatives of 5-fluorocytosine, cytosine, 5-fluorouracil, uracil, and thymine in the presence of ethylaluminum dichloride gave the corresponding nucleosides 2, 3, 4, 5, 10, 11, 12, 16, 17, and 18 as a mixture of alpha- and beta-anomers, which were then deblocked to yield the corresponding 2',3'-dideoxy-L-5-fluorocytidine derivatives, 6 and 7, 2',3'-dideoxy-L-cytidine derivatives, 8 and 9, 2',3'-dideoxy-beta-L-fluorouridine (13), 2',3'-dideoxy-beta-L-uridine (14), and 3'-deoxy-L-thymidine derivatives, 15 and 19. Among these 2',3'-dideoxy-L-nucleoside analogues, 2',3'-dideoxy-beta-L-5-fluorocytidine (6, beta-L-FddC) was found to be the most active against HIV-1, which is approximately 3 and 4 times more active against HIV-1 in vitro than 2',3'-dideoxy-beta-D-cytidine (ddC) and 2',3'-dideoxy-beta-D-5-fluorocytidine (beta-D-FddC) with ED50 values of 0.5, 1.5, and 2 microM, respectively. The dose-limiting toxicity of ddC is severe neuropathy which may be caused by the inhibition of the synthesis of mitochondrial DNA. ddC has an IC50 value of 0.022 microM against host mitochondrial DNA synthesis. Conversely, the IC50 values for beta-L-FddC and beta-L-ddC are > 100 microM; therefore, neuropathy may not present itself to be a problem with beta-L-FddC and beta-L-ddC as chemotherapeutic agents. In addition, beta-L-FddC and 2',3'-dideoxy-beta-L-cytidine (8, beta-L-ddC) demonstrated equally potent activity against HBV in vitro by having the same ED50 value of 0.01 microM. Both beta-L-FddC and beta-L-ddC, which have an "unnatural" L-configuration in the sugar moiety, are approximately 1000 and 280 times more potent, respectively, against HBV than the D-configuration beta-D-FddC and ddC which have an ED50 values of 10 and 2.8 microM. In view of the potent antiviral activity of beta-L-FddC against both HIV-1 and HBV and potent antiviral activity of beta-L-ddC against HBV in vitro, their low cytotoxicity, and especially the negligible inhibitory effect on host mitochondrial DNA synthesis, beta-L-FddC and beta-L-ddC merit further development as potential anti-HIV and anti-HBV agents.

A 4'-C-ethynyl-2',3'-dideoxynucleoside analogue highlights the role of the 3'-OH in anti-HIV active 4'-C-ethynyl-2'-deoxy nucleosides.

4'-C-ethynyl-2'-deoxynucleosides belong to a novel class of nucleoside analogues endowed with potent activity against a wide spectrum of HIV viruses, including a variety of resistant clones. Although favorable selectivity indices were reported for several of these analogues, some concern still exists regarding the 3'-OH group and its role in cellular toxicity. To address this problem, we removed the 3'-OH group from 4'-C-ethynyl-2'-deoxycytidine (1a). This compound was chosen because of its combined high potency and low selectivity index. The removal of the 3'-OH was not straightforward; it required a different synthetic approach from the one used to synthesize the parent compound. Starting with glycidyl-4-methoxyphenyl ether, the target 4'-C-ethynyl-2',3'-dideoxycytidine analogue (rac-1h) was obtained after 13 steps. In a cellular assay, rac-1h was completely inactive (0.001-10 microM) against HIV(LAI), demonstrating the critical importance of the 3'-OH for antiviral activity. To determine whether the role of the 3'-OH was essential for the phosphorylation of the compound by cellular kinases or for inhibition of DNA polymerization, we synthesized and tested the 5'-triphosphate (rac-1h-TP) for its ability to inhibit HIV reverse transcriptase (RT). rac-1h-TP was slightly more potent than AZT-5'-triphosphate against wild-type HIV RT, suggesting that the role of the 3'-OH is crucial only for the activation of the drug by cellular kinases. The lipase-catalyzed resolution of rac-1h into ent-1h (beta-D-dideoxyribo) and ent-14 (beta-L-dideoxyribo) and the synthesis of the corresponding 5'-triphosphates established the stereochemical assignment based on HIV RT's preference for the beta-D-enantiomer, which was confirmed by assaying against the M184V variant, an RT mutant with a marked preference for incorporating nucleosides in the D-configuration.

Antiviral activity of 2',3'-dideoxy-beta-L-5-fluorocytidine (beta-L-FddC) and 2',3'-dideoxy-beta-L-cytidine (beta-L-ddC) against hepatitis B virus and human immunodeficiency virus type 1 in vitro.

2',3'-Dideoxy-beta-L-5-fluorocytidine (beta-L-FddC) and 2',3'-dideoxy-beta-L-cytidine (beta-L-ddC), two nucleosides with "unnatural L-configuration," have been synthesized and found to have potent antiviral activity against hepatitis B virus (HBV) and human immunodeficiency virus type 1 (HIV-1) in vitro with very little toxicity. At 1 microM, both beta-L-ddC and beta-L-FddC inhibited the growth of HBV by more than 90%, while at the same concentration the D-configuration counterparts, 2',3'-dideoxy-beta-D-cytidine (ddC) and 2',3'-dideoxy-beta-D-5-fluorocytidine (beta-D-FddC), did not show antiviral activity against HBV. The order of anti-HIV-1 activity was beta-L-FddC > ddC; beta-D-FddC > beta-L-ddC. The dose-limiting toxicity of ddC is neuropathy which is believed to be caused by the inhibition of the synthesis of mitochondrial DNA. ddC severely inhibited the mitochondrial DNA synthesis of CEM cells yielding an IC50 value of 0.022 microM. Conversely, both beta-L-FddC and beta-L-ddC did not demonstrate any inhibition against mitochondrial DNA synthesis up to 100 microM concentration.

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.

Antiviral activity of phosphatidyl-dideoxycytidine in hepatitis B-infected cells and enhanced hepatic uptake in mice.

Dideoxycytidine (ddC) inhibits the replication of hepatitis B virus (HBV) but its clinical use is limited by peripheral neuropathy. We synthesized dioleoylphosphatidyl-ddC (DOP-ddC), a phospholipid prodrug of ddC which forms lipid bilayers and is readily incorporated into liposomes. The 90% effective dose (ED90) of DOP-ddC was 18 microM vs. 7 microM for ddC. However, in HBV-infected human hepatoma cells (2.2.15 cells), DOP-ddC was less toxic in vitro. When liposomal DOP-[5,6-3H]ddC was administered intraperitoneally to mice, drug levels in liver were 40 times greater than [5,6-3H]ddC when expressed as area under curve. Liposomal DOP-ddC also provided higher levels of drug in lymph nodes and spleen, important accessory sites of HBV replication. Plasma levels of drug remained above the ED90 six times longer with DOP-ddC than with ddC. DOP-ddC levels in sciatic nerve, the major site of toxicity, were not significantly different from levels observed with free ddC. The phospholipid prodrug approach is a general one which may readily be applied to other antiviral nucleosides for HBV.

Synthesis and biological evaluation of dinucleoside methylphosphonates of 3'-azido-3'-deoxythymidine and 2', 3'-dideoxycytidine.

The 5'----5' dinucleoside methylphosphonates of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (DDC) were prepared and evaluated for their inhibitory properties against different viruses, including human immunodeficiency virus (HIV). The synthesis of the compounds was achieved by reaction of AZT or N4-(4-monomethoxytrityl)-2',3'-dideoxycytidine with in situ prepared methylphosphonic bis (triazolide), followed in the latter case by an acidic treatment. The two title compounds showed in vitro anti-HIV activity, that was 200- to 450-fold less pronounced that that shown by the corresponding monomeric nucleosides AZT and DDC. The decreased antiviral activity may be ascribed to nuclease resistance of the methylphosphonate linkage.

N4-acyl-modified D-2',3'-dideoxy-5-fluorocytidine nucleoside analogues with improved antiviral activity.

A series of 2',3'-dideoxy (D2) and 2',3'-didehydro-2',3'-dideoxy (D4) 5-fluorocytosine nucleosides modified with substituted benzoyl, heteroaromatic carbonyl, cycloalkylcarbonyl and alkanoyl at the N4-position were synthesized and evaluated for anti-human immunodeficiency virus type 1 (HIV-1) and anti-hepatitis B virus (HBV) activity in vitro. For most D2-nucleosides, N4-substitutions improved the anti-HIV-1 activity markedly without increasing the cytotoxicity. In the D4-nucleosides series, some of the substituents at the N4-position enhanced the anti-HIV-1 activity with a modest increase in the cytotoxicity. The most potent and selective N4-modified nucleoside for the D2-series was N4-p-iodobenzoyl-D2FC, which had a 46-fold increase in anti-HIV-1 potency in MT-2 cells compared to the parent nucleoside D-D2FC. In the D4-series, N4-p-bromobenzoyl-D4FC was 12-fold more potent in MT-2 cells compared to the parent nucleoside D-D4FC. All eight N4-p-halobenzoyl-substituted D2- and D4-nucleosides evaluated against HBV in HepAD38 cells demonstrated equal or greater potency than the two parental compounds, D-D2FC and D-D4FC. The N4-modification especially in the D2-nucleoside series containing the N4-nicotinoyl, o-nitrobenzoyl and n-butyryl showed a significant reduction in mitochondrial toxicity relative to the parent nucleoside analogue. Although the 5'-triphosphate of the parent compound (D-D4FC-TP) was formed from the N4-acyl-D4FC analogues in different cells, the levels of the 5'-triphosphate nucleotide did not correlate with the cell-derived 90% effective antiviral concentrations (EC90), suggesting that a direct interaction of the triphosphates of these N4-acyl nucleosides was involved in the antiviral activity.

Synthesis and evaluation of novel prodrugs of foscarnet and dideoxycytidine with a universal carrier compound comprising a chemiluminescent and a photochromic conjugate.

To facilitate intracellular delivery of hydrophilic drugs, a general lipophilic carrier molecule was designed and synthesized. The carrier comprised a chemiluminescent-photochromic conjugate that potentiates diffusion across cell membranes to enhance intracellular uptake of the drug. The designed mechanism involves activation of the chemiluminescent moiety by intracellular oxygen free radicals and intermolecular energy transfer of the excited state energy to the photochromic moiety to result in release of the drug to allow the desired pharmacological effect to occur. Prodrugs of foscarnet and dideoxycytidine with several carriers caused suppression of a human immunodeficiency virus infection in human cultured macrophages that was up to five times more effective than the drug alone. Successful in vivo efficacy testing of prodrug has been accomplished by demonstrating the suppression of a retroviral infection of Friend leukemia virus in mice. Acute toxicity studies of the carrier indicated that it was nontoxic.

Synthesis of 2',3'-dideoxy-2',3'-alpha-methanocytidine.

A six-stage synthesis of 2',3'-dideoxy-2',3'-alpha-methanocytidine (3) from (5S)-5-benzoyloxymethyl-(5H)-furan-2-one (5) is described. The key step involved the stereoselective formation of (1R,4S,5S)-4-benzoyloxymethyl-3-oxabicyclo[3.1.0]hexan-2-one (7) via 1,3-dipolar cycloaddition of diazomethane to 5 followed by photoinduced elimination of nitrogen. Reduction of 7 to the corresponding lactol followed by acetylation yielded primarily 1-O-acetyl-5-O-benzoyl-2,3-dideoxy-beta-D-ribofuranose (8). Reaction of 8 with 2,4-bis(trimethylsilyl)cytosine and EtAlCl2 followed by deprotection and chromatography gave 3, which exhibited only weak activity against the human immunodeficiency virus (HIV).

[Synthesis and anti-retroviral activity of various new 2',3'-dideoxynucleoside analogs]. / Synthese en antiretrovirale activiteit van enige nieuwe 2', 3'-dideoxynucleoside analogen.

About 40 new 2',3'-dideoxynucleoside analogues with different basic structures have been synthetized with the aim of finding more potent and selective inhibitors of HIV and the associated pathogenicity. Some new synthesis techniques were developed and existing ones were used for the preparation of these analogues. Among the 3'-fluorinated 2',3'-dideoxynucleosides several potent inhibitors of HIV were found and especially 3'-fluoro-2',3'-dideoxy-5-chloro-uridine (81) is endowed with a high selectivity index, comparable to the selectivity displayed by 3'-azido-3'-deoxythymidine (AZT). Because of its substantially reduced toxicity profile compared to AZT, this compound deserves further evaluation as a possible alternative for the treatment of AIDS. Likewise, the 5-chlorinated cytidine analogues 102 and 103 should be further examined because of their lack of toxicity in vitro.