Cytosine deoxyribonucleoside anti-HIV analogues: a small chemical substitution allows relevant activities.

The search for new nucleoside analogue compounds targeting the virally encoded reverse transcriptase was developed by modifying the nucleoside structure to create inhibitor compounds. In this review, the structure-activity relationship of antiviral compounds synthesised from the naturally existing cytosine deoxyribonucleoside (dC) was evaluated. The line of research starting from dC led to the synthesis of 2',3'-dideoxycytidine (ddC; zalcitabine), 2',3'-dideoxy-3'-thiacytidine (3TC; lamivudine) and 2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC; emtricitabine) and looks very interesting because each product comes from a single small change in the chemical structure of the former compound, resulting in a progressive improvement in terms of activity, pharmacokinetics, tolerability and emergence of resistance.

The triphosphate of beta-D-4'-C-ethynyl-2',3'-dideoxycytidine is the preferred enantiomer substrate for HIV reverse transcriptase.

The enantioselective synthesis of the beta-d (1) enantiomer of 4'-C-ethynyl-2',3'-dideoxycytidine confirms an earlier stereochemical assignment that was strictly based on the ability of HIV reverse transcriptase and its M184V mutant to discriminate between the d- and l-configuration of nucleoside 5'-triphosphates.

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.

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.

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 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.

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.

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)).

DPC 817: a cytidine nucleoside analog with activity against zidovudine- and lamivudine-resistant viral variants.

Highly active antiretroviral therapy (HAART) is the standard treatment for infection with the human immunodeficiency virus (HIV). HAART regimens consist of protease inhibitors or nonnucleoside reverse transcriptase inhibitors combined with two or more nucleoside reverse transcriptase inhibitors (NRTIs). DPC 817, 2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine (PSI 5582 D-D4FC) is a potent inhibitor of HIV type 1 replication in vitro. Importantly, DPC 817 retains activity against isolates harboring mutations in the reverse transcriptase gene that confer resistance to lamivudine (3TC) and zidovudine (AZT), which are frequent components of initial HAART regimens. DPC 817 combines this favorable resistance profile with rapid uptake and conversion to the active metabolite DPC 817-triphosphate, which has an intracellular half-life of 13 to 17 h. Pharmacokinetics in the rhesus monkey suggest low clearance of parent DPC 817 and a plasma half-life longer than that of either AZT or 3TC. Together, these properties suggest that DPC 817 may be useful as a component of HAART regimens in individuals with resistance to older NRTI agents.

Synthesis and antiviral evaluation of C-4-hydrazide derivatives of 2',3'-dideoxycytidine.

Syntheses of three hitherto unknown derivatives of 2',3'-dideoxycytidine, namely C-4-(salicylic hydrazide)-ddC, C-4-(N-butyloxycarbonyl-isoleucine hydrazide)-ddC and its N-unprotected chlorhydrate salt have been carried out. These compounds do not induce inhibition of HIV-1 replication in cell culture experiments. Nevertheless, the modifications on the base moiety increased in all cases the lipophilicity of the parent molecule with an acceptable water solubility compared to ddC.

Synthesis, in vitro anti-HIV and anti-hepatitis B activities and pharmacokinetic properties of amphiphilic heterodinucleoside phosphates containing ddC and AZT.

Amphiphilic heterodinucleoside phosphates containing AZT and ddC as antiviral monomer were synthesized according to the hydrogenphosphonate method and evaluated in vitro against HIV. dT-N4-pamddC was the most active (IC50 = 40 microM, EC50 = 80 nM) and least toxic (TI = 524) dimer and it exhibited also strong antiviral effects against eight AZT-resistant HIV strains. The ddC-containing heterodimers additionally inhibited HBV replication by 50-80% at 50 microM in Hep G2 2.2.15 cells.

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.

Efficient synthesis of N-4 alkyl derivatives of 2',3'-dideoxycytidine (ddC).

The C-4 triisopropylphenylsulfonyl (TPS) group of the 2,3-dideoxyuridine derivative 2 is readily displaced in situ by nitrogen nucleophiles forming N-4 substituted ddC in acceptable yields.

Interaction of beta-L-2',3'-dideoxy-2',3'-didehydro-5-fluoro-CTP with human immunodeficiency virus-1 reverse transcriptase and human DNA polymerases: implications for human immunodeficiency virus drug design.

The work reported in this article has evaluated the relative molecular activity of the 5'-triphosphate of a novel beta-L-nucleoside with an unsaturated ribose residue, beta-L-2', 3'-dideoxy-2',3'-didehydro-5-fluorocytidine (beta-L-Fd4CTP), with that of beta-L-2',3'-dideoxy-5-fluorocytidine (beta-L-FddCTP) and 2', 3'-dideoxycytidine (ddCTP), on DNA strand elongation by human immunodeficiency virus-1 reverse transcriptase (HIV RT) and human DNA polymerases alpha (pol alpha), beta (pol beta), gamma (pol gamma), and epsilon (pol epsilon). The concentrations of beta-L-Fd4CTP that inhibited the yield of products by 50% were 0.20 micro M, 1.8 micro M, and 4.0 micro M for HIV RT, pol gamma, and pol beta, respectively. The beta-L-Fd4CTP at a concentration as high as 40 micro M had no inhibitory effect on pol epsilon, but could inhibit pol alpha by 10-20% at 20 micro M. The Km and relative Vmax values of beta-L-Fd4CTP, beta-L-FddCTP, and ddCTP for incorporation into the standing start point of 5'-[32P]-oligonucleotide primer annealed with M13mp19 phage DNA by HIV RT and human DNA polymerases were evaluated. The efficiency of incorporation (Vmax/Km) of beta-L-Fd4CTP by HIV RT was about 4-fold and 12-fold higher than that of ddCTP and beta-L-FddCTP, respectively. In contrast, the Vmax/Km ratio of beta-L-Fd4CTP for pol gamma was 7-fold lower than that of ddCTP, but 4-fold higher than that of beta-L-FddCTP. Pol alpha could use beta-L-Fd4CTP as a substrate, but only at a high concentration (>20 micro M). Incorporation of beta-L-Fd4CTP by pol epsilon could not be detected. A hypothesis about the preferable recognition of the 2',3'-dideoxy-2',3'-didehydro- structure of beta-L-Fd4CTP to that of the 2',3'-dideoxy-structure of beta-L-FddCTP by HIV RT is discussed.

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

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

[Which is the strategy? The latest in the use of antiretrovirals]. / Cual es la estrategia? Lo mas reciente en el uso de antiretrovirales.

AIDS: The four main antiretroviral drugs, or nucleoside analogs, used in AIDS treatment are AZT, ddI, ddC and d4T. AZT is generally prescribed first, adding combinations of ddC of ddI if resistance or intolerance appear. Researchers are determining, in vitro, the effects of combining three of these drugs to significantly reduce the reproduction of HIV. The combination of AZT and 3TC in studies has produced a potent antiretroviral combination, showing prolonged suppression of HIV production, and an increase in T4 cells. A new group of drugs, protease inhibitors, shows promise because protease is needed for HIV to reproduce in infected cells. In preliminary studies, these drugs reduce virus levels in blood and may be more effective and less toxic than current drugs. Saquinavir, L-524, and ABT538 are in advanced stages of development. Studies which measure their impact on T4 cells, viral reproduction, or AIDS progress are not available. Some problems include rapid development of viral resistance, limited availability, and difficulty in production of these medications.^ieng

Synthesis and antileukemic activity of chymotrypsin-activated derivatives of 3'-amino-2',3'-dideoxycytidine. (Synthetic nucleosides and nucleotides. XXXIII.

3'-Amino-2',3'-dideoxycytidine (8) was directly synthesized from 2'-deoxycytidine. 2',3'-Dideoxy-3'-(N-acyl-L-phenylalanylamino)cytidines (acyl = butoxycarbonyl (9a), acetyl (9b), benzoyl (9c), and n-hexanoyl (9d)) were synthesized as chymotrypsin-activated prodrugs of 8. This N-protection was required for activation by chymotrypsin to 8. In vitro, compound 8 showed high cytotoxic activity against P388 cells, but the prodrugs 9a-d were ineffective. In vivo, however, these prodrugs showed much higher activity than 8 in mice bearing P388 cells.