Synthesis of a highly active new anti-HIV agent 2',3'-didehydro-3'-deoxy-4'-ethynylthymidine.

Compounds having methyl, vinyl, and ethynyl groups at the 4'-position of stavudine (d4T: 2',3'-didehydro-3'-deoxythymidine) were synthesized. The compounds were assayed for their ability to inhibit the replication of HIV in cell culture. The 4'-ethynyl analogue (15) was found to be more potent and less toxic than the parent compound stavudine.

Synthesis and biological evaluation of L- and D-configurations of 2',3'-dideoxy-4'-C-methyl-3'-oxacytidine analogues.

Novel L- and D-configuration 2',3'-dideoxy-4'-C-methyl-3'-oxacytidine and their 5-fluoro analogues have been synthesized from 1-benzyloxy-2-propanone and L-ascorbic acid in eight steps and evaluated for biological activity.

Synthesis of halogen-substituted 3-deazaadenosine and 3-deazaguanosine analogues as potential antitumor/antiviral agents.

Various 2-halogen-substituted analogues (38, 39, 43 and 44), 3-halogen-substituted analogues (51 and 52), and 2',3'-dihalogen-substituted analogues (57-60) of 3-deazaadenosine and 3-halogen-substituted analogues (61 and 62) of 3-deazaguanosine have been synthesized as potential anticancer and/or antiviral agents. Among these compounds, 3-deaza-3-bromoguanosine (62) showed significant cytotoxicity against L1210, P388, CCRF-CEM and B16F10 cell lines in vitro, producing IC50 values of 3, 7, 9 and 7 microM, respectively. Several 3-deazaadenosine analogues (38, 51, 57 and 59) showed moderate to weak activity against hepatitis B virus.

Anti-Epstein-Barr virus (EBV) activity of beta-L-5-iododioxolane uracil is dependent on EBV thymidine kinase.

beta-L-5-Iododioxolane uracil was shown to have potent anti-Epstein-Barr virus (EBV) activity (50% effective concentration = 0.03 microM) with low cytotoxicity (50% cytotoxic concentration = 1,000 microM). It exerts its antiviral activity by suppressing replicative EBV DNA and viral protein synthesis. This compound is phosphorylated in cells where the EBV is replicating but not in cells where the EBV is latent. EBV-specific thymidine kinase could phosphorylate beta-L-5-iododioxolane uracil to the monophosphate metabolite. The K(m) of beta-L-5-iododioxolane uracil with EBV thymidine kinase was estimated to be 5.5 microM, which is similar to that obtained with thymidine but about fivefold higher than that obtained with 2' fluoro-5-methyl-beta-L-arabinofuranosyl uracil, the first L-nucleoside analogue discovered to have anti-EBV activity. The relative V(max) is seven times higher than that of thymidine. The anti-EBV activity of beta-L-5-iododioxolane uracil and its intracellular phosphorylation could be inhibited by 5'-ethynylthymidine, a potent EBV thymidine kinase inhibitor. The present study suggests that beta-L-5-iododioxolane uracil exerts its action after phosphorylation; therefore, EBV thymidine kinase is critical for the antiviral action of this drug.

Metabolism and mode of inhibition of varicella-zoster virus by L-beta-5-bromovinyl-(2-hydroxymethyl)-(1,3-dioxolanyl)uracil is dependent on viral thymidine kinase.

A nonnaturally occurring L-configuration nucleoside analog, L-beta-5-bromovinyl-(2-hydroxymethyl)-1,3-(dioxolanyl)uracil (L-BVOddU) selectively inhibited varicella-zoster virus growth in human embryonic lung (HEL) 299 cell culture with an EC(50) of 0.055 microM, whereas no inhibition of CEM and HEL 299 cell growth or mitochondrial DNA synthesis was observed at concentrations up to 200 microM. L-BVOddU was phosphorylated by viral thymidine kinase but not by human cytosolic thymidine kinase, and the antiviral activity of this compound is dependent on the viral thymidine kinase. Unlike other D-configuration bromovinyl deoxyuridine analogs, such as E-5-(2-bromovinyl)-2'-deoxyuridine and 1-beta-arabinofuranosyl-E-5-(2-bromovinyl)uracil, this compound was metabolized only to its monophosphate metabolite. The di- or triphosphate metabolites were not detected. This suggested that the inhibitory mechanism may be unique and different from other anti-herpesvirus nucleoside analogs.

Synthesis and biological evaluation of L- and D-configuration 1,3-dioxolane 5-azacytosine and 6-azathymine nucleosides.

Novel L- and D-configuration dioxolane 5-azacytosine and 6-azathymine nucleosides have been synthesized and evaluated for biological activity. (-)-(2S,4S)-1-[2-(Hydroxymethyl)-1,3-dioxolan-4-yl]-5-azacytosine (6) showed significant activity against HBV, whereas the D-configuration analogue (14) has been found to exhibit potent anti-HIV activity.

Synthesis and biological evaluation of 1,3-oxathiolane 5-azapyrimidine, 6-azapyrimidine, and fluorosubstituted 3-deazapyrimidine nucleosides.

(2R,5S)-5-Amino-2-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]- 1,2,4-triazine-3(2H)-one (8) and (2R,5R)-5-amino-2-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2,4-tr iazine-3(2H)-one (9) have been synthesized via a multi-step procedure from 6-azauridine. (2R,5S)-4-Amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,3, 5-triazine-2(1H)-one (11) and (2R,5R)-4-amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]- 1,3,5-triazine-2(1H)-one (12), and the fluorosubstituted 3-deazanucleosides (19-24) have been synthesized by the transglycosylation of (2R,5S)-1-[2-[[(tert-butyldiphenylsilyl) oxy]methyl]-1,3-oxathiolan-5-yl] cytosine (2) with silylated 5-azacytosine and the corresponding silylated fluorosubstituted 3-deazacytosines, respectively, in the presence of trimethylsilyl trifluoromethanesulfonate as the catalyst in anhydrous dichloroethane, followed by deprotection of the blocking groups. These compounds were tested in vitro for cytotoxicity against L1210, B16F10, and CCRF-CEM tumor cell lines and for antiviral activity against HIV-1 and HBV.

Synthesis of 2'-methylene-substituted 5-azapyrimidine, 6-azapyrimidine, and 3-deazaguanine nucleoside analogues as potential antitumor/antiviral agents.

2'-Deoxy-2'-methylene-6-azauridine (5) and 2'-deoxy-2'-methylene-6-azacytidine (8) have been synthesized via a multi-step procedure from 6-azauridine. 2'-Deoxy-2'-methylene-5-azacytidine (14a) and 2'-deoxy-2'-methylene-3-deazaguanosine (19a) and their corresponding alpha-anomers (14b and 19b) have been synthesized by the transglycosylation of 3',5'-O-(1,1,3,3- tetraisopropyldisiloxane-1,3-diyl)-2'-deoxy-2'-methyleneu ridine (12) with silylated 5-azacytosine and silylated N2-palmitoyl-3-deazaguanine, respectively, in the presence of trimethylsilyl trifluoromethanesulfonate as the catalyst in anhydrous dichloroethane, followed by separation of the isomers and deprotection of the blocking groups. These compounds were tested for cytotoxicity against B16F10, L1210, and CCRF-CEM tumor cell lines and for antiviral activity against HIV-1, HSV-1, and HSV-2.

Metabolism of 2',3'-dideoxy-2',3'-didehydro-beta-L(-)-5-fluorocytidine and its activity in combination with clinically approved anti-human immunodeficiency virus beta-D(+) nucleoside analogs in vitro.

2',3'-Dideoxy-2',3'-didehydro-beta-L(-)-5-fluorocytidine [L(-)Fd4C] has been reported to be a potent inhibitor of the human immunodeficiency virus (HIV) in cell culture. In the present study the antiviral activity of this compound in two-drug combinations and its intracellular metabolism are addressed. The two-drug combination of L(-)Fd4C plus 2',3'-didehydro-2'-3'-dideoxythymidine (D4T, or stavudine) or 3'-azido-3'-deoxythymidine (AZT, or zidovudine) synergistically inhibited replication of HIV in vitro. Additive antiviral activity was observed with L(-)Fd4C in combination with 2',3'-dideoxycytidine (ddC, or zalcitabine) or 2',3'-dideoxyinosine (ddI, or didanosine). This beta-L(-) nucleoside analog has no activity against mitochondrial DNA synthesis at concentrations up to 10 microM. As we previously reported for other beta-L(-) nucleoside analogs, L(-)Fd4C could protect against mitochondrial toxicity associated with D4T, ddC, and ddI. Metabolism studies showed that this drug is converted intracellularly to its mono-, di-, and triphosphate metabolites. The enzyme responsible for monophosphate formation was identified as cytoplasmic deoxycytidine kinase, and the K(m) is 100 microM. L(-)Fd4C was not recognized in vitro by human mitochondrial deoxypyrimidine nucleoside kinase. Also, L(-)Fd4C was not a substrate for deoxycytidine deaminase. L(-)Fd4C 5'-triphosphate served as an alternative substrate to dCTP for incorporation into DNA by HIV reverse transcriptase. The favorable anti-HIV activity and protection from mitochondrial toxicity by L(-)Fd4C in two-drug combinations favors the further development of L(-)Fd4C as an anti-HIV agent.

Telomerase from human leukemia cells: properties and its interaction with deoxynucleoside analogues.

Telomerase is a unique reverse transcriptase involved in the maintenance of genomic integrity. In an attempt to understand the properties of this enzyme and to study the effect of deoxynucleoside analogues, we have isolated and partially purified telomerase from the blast cells of a patient with acute myelogenous leukemia. During the course of purification of telomerase, three characteristic forms of this enzyme activity were separated. Two processive forms and one less processive form were noted. All forms of the enzyme activities could be abolished by RNase A and proteinase K treatments, implying that they are ribonucleoproteins. The major form of telomerase was characterized with respect to divalent ion requirements, effect of salt and nonionic detergents. The Km of deoxynucleoside triphosphates was determined with a modified telomerase repeat array protocol assay. Studies with deoxynucleoside analogues indicated that 3'-azido-3'deoxythymidine triphosphate is much more inhibitory than 2',3'-dideoxy 2',3'didehydrothymidine triphosphate, and the cytidine analogue ddCTP was not inhibitory. ddGTP was the most potent inhibitor among all dideoxynucleosides studied.

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.

Favorable interaction of beta-L(-) nucleoside analogues with clinically approved anti-HIV nucleoside analogues for the treatment of human immunodeficiency virus.

The combination of L(-)-2',3'-dideoxy-3'-thiacytidine (L(-)SddC, 3TC), L(-)-2',3'-dideoxy-5-fluorocytidine (L(-)FddC), or L(-)-2',3'-dideoxy-5-fluoro-3'-thiacytidine (L(-)(FTC) with 3'-azido-3'-deoxythymidine (AZT) synergistically inhibited replication of human immunodeficiency virus (HIV) in vitro. Similar synergistic activity was also obtained when these compounds were used in combination with 2',3'-didehyro-2',3'-dideoxythymidine (D4T). In terms of 2',3'- dideoxyinosime (ddI) and 2',3'-dideoxycytidine (ddC), only additive anti-HIV activity was observed. None of the beta-L(-) nucleoside analogues had additive toxicity in cell culture, and they could protect against the delayed mitochondrial toxicity associated with AZT, D4T, ddC, and ddI in drug-treated cells. Thus, combinations of beta-L(-) nucleoside analogues with any of the approved anti-HIV drugs could have a potentially beneficial outcome.

Action of uracil analogs on human immunodeficiency virus type 1 and its reverse transcriptase.

Three structural analogs of 5-ethyl-1-benzyloxymethyl-6-(phenylthio)uracil (E-BPU) inhibited human immunodeficiency virus type 1 (HIV-1) replication without cytotoxicity in vitro and were more potent than azidothymidine and were as potent as E-BPU. The target of these compounds is HIV-1 reverse transcriptase. Reverse transcriptases resistant to nevirapine (tyrosine at position 181 to cysteine) and TIBO R82150 (leucine at position 100 to isoleucine) are cross resistant to E-BPU analogs. Nevirapine- or TIBO R82150-resistant HIV-1 were cross resistant to E-BPU analogs but were inhibited at concentrations 11- to 135-fold lower than the cytotoxic doses.

Characterization of a hydroxyurea-resistant human KB cell line with supersensitivity to 6-thioguanine.

Hydroxyurea (HU) is currently used in the clinic for the treatment of chronic myelogenous leukemia, head and neck carcinoma, and sarcoma. One of its drawbacks, however, is the development of HU resistance. To study this problem, we developed a HU-resistant human KB cell line which exhibits a 15-fold resistance to HU. The characterization of this HU-resistant phenotype revealed a gene amplification of the M2 subunit of ribonucleotide reductase (RR), increased levels of M2 mRNA and protein, and a 3-fold increase of RR activity. This HU-resistant cell line also expressed a "collateral sensitivity" to 6-thioguanine (6-TG), with a 10-fold decrease in the dose inhibiting cell growth by 50% as compared to the KB parental line. The mechanism responsible for this supersensitivity to 6-TG is believed to be related to an increasingly efficient conversion of 6-TG to its triphosphate form, which is subsequently incorporated into DNA. After passage of the resistant cells in the absence of HU, the cell line reverts. The revertant cells lose their resistance to HU and concomitantly their sensitivity to 6-TG. This phenomenon is due to the return of RR to levels comparable to that of the KB parental cell line. These observations and their relevance to cancer chemotherapy will be discussed in this paper. Our results suggest that a clinical protocol could be designed which would allow for a lower dose of 6-TG to be used by taking advantage of the increased RR activity in HU-refractory cancer patients. Two drugs which display collateral sensitivity are known as a "Ying-Yang" pair. Alternate treatment with two different Ying-Yang pairs is the rationale for the "Ying-Yang Ping-Pong" theory in cancer treatment. This rationale allows for effective cancer chemotherapy with reduced toxicity.

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.

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.

Syntheses and biological evaluations of 3'-deoxy-3'-C-branched-chain-substituted nucleosides.

Various 3'-deoxy-3'-C-(hydroxymethyl)-, 3'-deoxy-3'-C-(fluoromethyl)-, 3'-deoxy-3'-C-(azidomethyl)-, and 3'-deoxy-3'-C-(aminomethyl)-substituted nucleosides (total 12 compounds) have been synthesized and evaluated against L1210, P388, S-180, and CCRF-CEM cells and HSV-1, HSV-2, and HIV-1 in culture. Only 3'-deoxy-3'-C-(hydroxymethyl)thymidine (36) was found to show significant anticancer activity against L1210, P388, S-180, and CCRF-CEM cells with ED50 values of 50, 5, 10, and 1 microM, respectively. None of these compounds demonstrated significant antiviral activity against HSV-1, HSV-2, or HIV-1. These compounds were also evaluated against thymidine kinases derived from HSV-I (strain KOS), HSV-2 (strain 333), and mammalian (K562) cells. The thymidine kinase (HSV-1 strain KOS) was inhibited significantly by both 3'-deoxy-3'-C-(hydroxymethyl)- and 3'-deoxy-3'-C-(fluoromethyl)thymidine.

A single conservative amino acid substitution in the reverse transcriptase of human immunodeficiency virus-1 confers resistance to (+)-(5S)-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5, 1- jk][1,4]benzodiazepin-2(1H)-thione (TIBO R82150).

Tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione (TIBO) derivatives (e.g., R82150) are potent, human immunodeficiency virus-1 (HIV-1)-specific, inhibitors of reverse transcriptase (RT) that are undergoing initial evaluation in clinical trials. Because HIV-1 has become resistant to other RT inhibitors, we investigated the potential for viral resistance to TIBO R82150 by serial in vitro passage of HIV-1IIIB in the presence of drug. R82150-resistant variants (> 100-fold increase in IC50) dominated the replicating virus population after only three or four passages. R82150-resistant virus was partially cross-resistant to other HIV-1-specific RT inhibitors, including nevirapine (approximately 10-fold increase in IC50) and 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (approximately 3.5-fold increase) but remained susceptible to 2',3'-dideoxynucleosides and phosphonoformate. DNA sequencing of cloned resistant RT, combined with site-specific mutational analyses and construction of mutant recombinant proviruses, demonstrated that a single, conservative amino acid substitution (Leu100 to Ile) in HIV-1 RT is responsible for high level R82150 resistance and partial nevirapine resistance. These studies indicate that a subtle mutation in HIV-1 RT can dramatically affect viral susceptibility to an HIV-1-specific RT inhibitor. The clinical efficacy of TIBO derivatives and other HIV-1-specific RT inhibitors may be limited by the emergence of drug-resistant viral strains.