A novel synthesis and biological activity of several 5-halo-5'-amino analogues of deoxyribopyrimidine nucleosides

A novel synthetic procedure has been developed for the large-scale synthesis of 5-chloro-, 5-bromo-, and 5-iodo-5'-amino-2',5'-dideoxyuridine (4c-e) as well as of two new analogues, 5-iodo-5'-amino-2',5'-dideoxycytidine and 5-fluoro-5'-amino-2',5'-dideoxyuridine (4a and 4b), in good yield. The starting materials, 5-halo-2'-deoxyuridine and 5-halo-2'-deoxycytidine, are readily available and the method is straightforward. This report describes the synthesis and the biologial activities of these compounds.     

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).     

Synthesis and biological activities of 5-trifluoromethyl-5'-azido-2',5'-dideoxyuridine and 5-trifluoromethyl-5'-amino-2',5'-dideoxyuridine.

5-Trifluoromethyl-2'-deoxyuridine (1) was tosylated with p-toluenesulfonyl chloride in dry pyridine at 3 degrees to give 5-trifluoromethyl-5'-O-(p-tolylsulfonyl)-2'-deoxyuridine (2), which was converted to 5-trifluoromethyl-5'-azido-2',5'-dideoxyuridine (3) by reacting with lithium azide in N,N-dimethylformamide at 85-90 degrees for 2 h. Compound 3 was then hydrogenated in ethanol-water (1:1, v/v) at room temperature and 35 psi of hydrogen pressure, using 10% palladium on charcoal as cstalyst, to yield 5-trifluoromethyl-5'-amino-2',5'-dideoxyuridine (4). Compound 4 is about fourfold less potent than compound 1 as an antiviral agent but is about 40-fold less toxic to the host Vero cells. Thus the therapeutic index of compound 1 has been improved by a factor of 10 by replacement of the 5'-hydroxyl with an amino group. Compound 1, however, is more than 100-fold more inhibitory to Sarcoma 180 cells in culture relative to compound 4. Compound 3 is markedly less potent than compound 1 or 4 as either an antiviral or an antineoplastic compound.     

Inhibition of poly(ADP-ribose)polymerase activity by nucleoside analogs of thymidine.

The poly ADP-ribosylation of proteins catalyzed by poly(ADP-ribose)polymerase (PARP) is involved in a number of important cellular metabolic activities. We evaluated various analogs of deoxythymidine and deoxyuridine as inhibitors of PARP. Most of these compounds have antiviral and/or anticancer activities. The structural requirements for these nucleoside analogs to be inhibitors of PARP were determined. The compounds evaluated had various substitutions on the 2-, 4- and/or 5-position of the pyrimidine ring, as well as on the 2'-, 3'- and/or 5'-position of the pentose moiety. Inhibition of PARP was strongly dependent on the size of the alkyl or halogen substituent on the 5-position of the pyrimidine ring. Whereas the 5-position of the pyrimidine ring could be varied, alteration of the 2- or 4-position drastically decreased the inhibition of PARP. Kinetic analysis was performed with concentrations of 1-10 microM NAD+. The Ki values for many compounds were five to seven times lower than the Ki for 3-aminobenzamide, a previously described potent inhibitor of PARP. Compounds with combined substituents at both the 5-position of the pyrimidine ring and the 3'- or 5'-position of deoxyribose generally were potent inhibitors of PARP, as for example 3'-amino-2', 3'-dideoxy-(E)-5-(2-bromovinyl)uridine (Ki = 0.7 microM), or 5'-azido-2',5'-dideoxy-5-ethyluridine (Ki = 0.8 microM). The 5-halogenated analogs had Ki values of 18, 35, 110 and greater than 1000 microM for 5-iodo-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, 5-chloro-2'-deoxyuridine, and 5-fluoro-2'-deoxyuridine, respectively, and the 5-alkyl analogs had Ki values of 45, 2.2, 7, 16 and 180 microM for 5-methyl-2'-deoxyuridine, 5-ethyl-2'-deoxyuridine, 5-propyl-2'-deoxyuridine, 5-butyl-2'-deoxyuridine and 5-pentyl-2'-deoxyuridine, respectively. Two other compounds with substituents in the 5-position of the pyrimidine moiety also had potent activities: (E)-5-(2-bromovinyl)-2'-deoxyuridine (Ki = 6 microM) and 5-trifluoromethyl-2'-deoxyuridine (Ki = 1.6 microM). Compounds substituted in the 2'-, 3'- and/or 5'-position of the deoxyribose moiety were investigated and 5'-azido-5'-deoxythymidine, 5'-amino-5'-deoxythymidine, 3'-azido-3'-deoxythymidine and 3'-deoxythymidine (d2T) and Ki values of 12, 16, 18 and 30 microM, respectively.     

Inhibition of Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium avium by novel dideoxy nucleosides

The prevalence of tuberculosis (TB) and mutidrug-resistant tuberculosis (MDR-TB) has been increasing, leading to serious infections, high mortality, and a global health threat. Here, we report the identification of a novel class of dideoxy nucleosides as potent and selective inhibitors of Mycobacterium bovis, Mycobacterium tuberculosis, and drug-resistant Mycobacterium tuberculosis. A series of 5-acetylenic derivatives of 2',3'-dideoxyuridine (3-8) and 3'-fluoro-2',3'-dideoxyuridine (22-27) were synthesized and tested for their antimycobacterial activity against M. bovis, M. tuberculosis, and M. avium. 2',3'-Dideoxyuridine possessing 5-decynyl, 5-dodecynyl, 5-tridecynyl, and 5-tetradecynyl substituents (4-7) exhibited the highest antimycobacterial activity against all three mycobacteria. In contrast, in the 3'-fluoro-2',3'-dideoxyuridine series, a 5-tetradecynyl analogue (26) displayed the most potent activity against these mycobacteria. Among other derivatives, 5-bromo-2',3'-dideoxycytidine (11), 5-methyl-2',3'-dideoxycytidine (12), and 5-chloro-4-thio-2',3'-dideoxyuridine (19) exhibited modest inhibition of M. bovis and M. tuberculosis. In the series of dideoxy derivatives of adenosine, guanosine, and purines, 2-amino-6-mercaptoethyl-9-(2,3-dideoxy-beta-d-glyceropentofuranosyl)purine (32) and 2-amino-4-fluoro-7-(2,3-dideoxy-beta-d-glyceropentofuranosyl)pyrrolo[2,3-d]pyrimidine (35) were the most efficacious against M. bovis and M. tuberculosis, and M. avium, respectively.     

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).     

Production of herpes simplex virus type 1 thymidine kinase in the presence of thymidine analogues

Treatment of herpes simplex virus type 1 (HSV-1) infected Vero, BHK, BHKtk- and LMtk- cells with 5-iodo-5'-amino-2',5'-dideoxyuridine (AIdUrd) caused increased synthesis of ICP36 and an increase in HSV-1 thymidine kinase (tk) activity at late times of infection. The overproduced ICP36 was identified as the HSV-1 encoded tk protein by immunoprecipitation. Whereas the thymidine analogue 5'-amino-5'-deoxythymidine (AdThd) caused an increase in HSV-1 tk synthesis and activity in wild type Vero and BHK cells, 5-iodo-2'-deoxyuridine (IdUrd) caused a similar increase only in tk- cells (LMtk-, BHKtk-). In vivo and in vitro stabilization studies using a [35S]methionine pulse-chase experiment or heat inactivation studies with purified HSV-1 tk revealed that stabilization of tk by the analogues could not account for the extent of the observed increase. Since overproduction of tk is observed only at late times of infection, it is suggested that the presence of these thymidine analogues in either the viral DNA or the cellular nucleotide pools is responsible for the observed differential effects.     

Structure-activity analysis of antagonism of the feedback inhibition of thymidine kinase

The effects of a variety of 5-, 5'-, and 3'-substituted deoxyuridine derivatives on the cytoplasmic thymidine kinase (EC 2.7.1.21) purified from a human colon carcinoma cell line, HCT 116, were determined. Of particular interest was elucidation of the structural features important for antagonism of the feedback inhibition of thymidine kinase exerted by thymidine triphosphate. Substitutions at the 5-position altered the potency of the 5'-modified compounds. The replacement of the 5-hydrogen with a methyl group or an iodine greatly increased the affinity of compounds for the thymidine kinase. This was evident for enzyme substrates with 5'-hydroxyl groups [2'-deoxyuridine (dUrd), 2'-deoxythymidine (dThd) and 5-iodo-2'-deoxyuridine (IdUrd)], feedback inhibitors with 5'-triphosphate substitutions (dUTP, dTTP and IdUTP), and for 5'-amino derivatives [5'-amino-2',5'-dideoxyuridine (5'-AdUrd), 5'-amino-2'-5'-dideoxythymidine (5'-AdThd) and 5-iodo-5'-amino-2',5'-dideoxyuridine (5'-AIdUrd)]. Qualitatively, however, the 5-substitutions did not affect the nature of the interactions with dThd kinase. For example, in the presence of dTTP, 5'-AdUrd stimulated dThd kinase activity as much as 5'-AdThd, but approximately a 100-fold greater concentration of 5'-AdUrd was required. Similar results were obtained using intact cells in which substitutions at the 5-position affected the potency, but not the efficacy, of the 5'-amino derivatives to stimulate dThd phosphorylation. In contrast, substitutions at the 5'-position did alter the nature of the interaction with dThd kinase. Thus, the 5'-hydroxyl compounds, dUrd, dThd and IdUrd, did not reverse the enzyme inhibition produced by dTTP nor did they stimulate dThd uptake in intact cells. 5'-Deoxy-5'-(ethylthio)thymidine, 5'-deoxy-5'-[(2-hydroxyethyl)thio]thymidine, and dTMP, but not dTDP, also antagonized the inhibition of dThd kinase produced by dTTP. In comparison to 5'-AdThd, the 3'-amino derivatives, 3'-AdThd and 3'-5'-diAdThd, were much less potent, but still efficacious, antagonists of feedback inhibition. These results indicate that a wide range of dUrd derivatives can disrupt the regulation of dThd kinase and provide leads for the development of new nucleotide analogues.     

Synthesis and biological activity of various 3'-azido and 3'-amino analogues of 5-substituted pyrimidine deoxyribonucleosides

Various new 5-substituted 3'-azido- and 3'-amino derivatives of 2'-deoxyuridine and 2'-deoxycytidine have been synthesized and biologically evaluated. Among these compounds, 3'-amino-2',3'-dideoxy-5-fluorouridine (3), 3'-amino-2',3'-dideoxycytidine (7a), and 3'-amino-2',3'-dideoxy-5-fluorocytidine (7c) were found to be the most active against murine L1210 and sarcoma 180 neoplastic cells in vitro, with an ED50 of 15 and 1 microM, 0.7 and 4 microM, and 10 and 1 microM, respectively. The 3'-azido derivatives, 2 and 6c, were less active in comparison with their 3'-amino counterparts. In addition, the 5-fluoro-3'-amino nucleosides, 3 and 7c, were tested against L1210 leukemia bearing CDF1 mice. Our preliminary findings indicate that compound 7c (6 X 200 mg/kg) was as active as the positive control, 5-fluorouracil (6 X 20 mg/kg), yielding a T/C X 100 of 146 and 129, respectively. However, 3 was found to be inactive in this experiment.     

3'-Amino-2',3'-dideoxyribonucleosides of some pyrimidines: synthesis and biological activities

3'-Amino-2',3'-dideoxyribonucleosides of thymine, uracil, and 5-iodouracil (1-3) were synthesized from the corresponding 2'-deoxyribonucleosides via the threo-3'-chloro and the erythro-3'-azido derivatives. Corresponding aminonucleosides of 5-bromouracil, 5-chlorouracil, and 5-fluorouracil (4-6) were synthesized enzymatically with 3'-amino-2',3'-dideoxythymidine as the aminopentosyl donor and thymidine phosphorylase (EC 2.4.2.4) as the catalyst. 3'-Amino-2',3'-dideoxycytidine (7) was synthesized by amination of the 3'-azido precursor of 3'-amino-2',3'-dideoxyuridine. The biological activity of 3'-amino-2',3'-dideoxy-5-fluorouridine (6) was notable among this group of aminonucleosides. It had an ED50 of 10 microM against adenovirus and was not appreciably cytotoxic to mammalian cells in culture. It also had activity against some Gram-positive bacteria but not against a variety of Gram-negative bacteria. The other aminonucleosides (1-5 and 7) lacked or exhibited weak antiviral and antibacterial activities. The only compounds in this group that were appreciably toxic to mammalian cells in culture were the thymidine and deoxycytidine analogues (1 and 7).     

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.     

Effects of nucleoside analogues on the expression of herpes simplex type 1-induced proteins

Exposure of herpes simplex virus type 1 (HSV-1)-infected Vero cells to the nucleoside analogues 5-iodo-5'-amino-2',5'-dideoxyuridine (AIdUrd), 5-iodo-2'-deoxyuridine (IdUrd) or 5'-amino-2',5'-dideoxythymidine (5'-AdThd) resulted in altered expression of HSV-1-induced proteins. Infected cell proteins (ICPs) synthesized in the presence of the nucleoside analogues were compared by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis to ICPs from non-drug-treated cells and it was found that there was no effect on HSV-1-induced alpha proteins but beta and gamma proteins were reduced as much as 60%. There were three exceptions: ICP 35 (Mr = 46,000) and ICP 39 (Mr = 36,000) were not reduced and ICP 36 (Mr = 42,000) was increased during drug treatment. Progeny virions were isolated from drug-treated infected Vero cells and were compared to progeny isolated from control cells with respect to their polypeptide make-up and for their ability to induce HSV-1 proteins in non-drug-treated Vero cells. The progeny virus from drug-treated cells exhibited altered protein patterns on SDS-polyacrylamide gels with respect to control HSV-1. The progeny virions from AIdUrd- or IdUrd- but not from 5'-AdThd-treated cells were defective in their abilities to induce proteins upon subsequent infection of non-drug-treated Vero cells. Two unusual phosphoproteins were detected; one with an apparent molecular weight of 30,000 was induced by progeny virus from AIdUrd-treated cells and another at approximately 69,000 was induced by progeny virus from 5'-AdThd-treated cells.     

3'-Fluoro-2',3'-dideoxy-5-chlorouridine: most selective anti-HIV-1 agent among a series of new 2'- and 3'-fluorinated 2',3'-dideoxynucleoside analogues

A series of 2'- and 3'-fluorinated 2',3'-dideoxynucleosides and 3'-azido-2',3'-dideoxynucleosides were synthesized and evaluated for their inhibitory activity against human immunodeficiency virus-1 (HIV-1) replication in MT-4 cells. Neither conversion of 3'-fluoro- or 3'-azido-2',3'-dideoxyadenosine to the corresponding inosine derivatives nor 8-bromination of 2',3'-dideoxyadenosine resulted in increased anti-HIV-1 activity. Nor did introduction of a 2'-fluorine in the erythro or threo configuration lead to improved anti-HIV-1 activity of the parent 2',3'-dideoxynucleosides. 1-(2-Fluoro-2,3-dideoxy-beta-D-threo-pentofuranosyl)cytosine and 1-(2-fluoro-2,3-dideoxy-beta-D-erythropentofuranosyl)thymine were only marginally active. However, 3'-fluoro-2',3'-dideoxyuridine (FddUrd) proved to be potent and a relatively nontoxic inhibitor of HIV-1. 5-Halogenated derivatives of FddUrd were prepared in attempts to further increase its anti-HIV potency and selectivity. Of these 5-halogenated derivatives, 3'-fluoro-2',3'-dideoxy-5-chlorouridine emerged as the most selective inhibitor of HIV-1 replication. Its selectivity index was comparable to that of azidothymidine when evaluated under the same conditions.     

Efficiency and selectivity of (E)-5-(2-bromovinyl)-2'-deoxyuridine and some other 5-substituted 2'-deoxypyrimidine nucleosides as anti-herpes agents

A number of novel 5-substituted 2'deoxypyrimidine nucleosides exhibited antiviral activity against herpes simplex virus type 1 strain V3 (HSV-1-V3) when assayed under one-step conditions in primary human lung fibroblast j(PHLF) cell cultures, and compared with the reference compounds cytosine arabinoside (ara-C), 5-iodo-2'-deoxyuridine (IUdR), and 5-iodo-5'amino-2',5'-dideoxyuridine (AIU). The most effective of these were (in order of decreasing activity): (E)-5-(2-bromovinyl)-UdR (BrVUdR) greater than ara-C greater than IUdR greater than 5-azidomethyl-UdR (AMeUdR) greater than 5-formyl-UdR (fUdR) greater than 5-hydroxymethyl-UdR (HMeUdR) greater than AIU greater than 5-mercaptomethyl-UdR (MMeUdR) = 5-hydroxymethyl-2'-deoxy-cytidine (HMeCdR) greater than 5-benzyloxymethyl-UdR (BOMeUdR). In a multistep virus replication experiment (plaque reduction assay on Vero cells) the order of decreasing activity was as follows: BrVUdR = ara-C greater than HMeUdR greater than fUdR IUdR greater than HMeCdR greater than BOMeUdR greater than AMeUdR greater than AIU greater than MMeUdR. BrVUdR effected a 50% reduction in plaque formation of different strains of HSV-1 at a concentration of 0.06-0.22 microM, of pseudorabies virus (PRV) at 0.02-0.23 microM, and of herpes simplex virus type 2 (HSV-2) at 8 microM, whereas the ID50 values for adenovirus type 2 and type 5 were 100 and 50-100 microM, respectively. The growth of synchronied baby hamster kidney cells in suspension cultures was inhibited by 50% at concentrations of 100, 70, 20, 4, 8, and 0.2 microM for BrVUdR, HMeCdR, IUdR, fUdR, BOMeUdR, and HMeUdR, respectively.     

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.     

Structure-activity relationships of pyrimidine nucleosides as antiviral agents for human immunodeficiency virus type 1 in peripheral blood mononuclear cells

The structure-activity relationships of several pyrimidine nucleosides related to 3'-azido-3'-deoxythymidine (AZT) were determined in human immunodeficiency virus type 1 (HIV-1) infected human peripheral blood mononuclear cells. These studies indicated that nucleosides with a 3'-azido group on the sugar ring exhibited the most potent antiviral activity. Substitution at C-5 with H, CH3, and C2H5 produced derivatives with the highest potency, whereas alkyl functions greater than C2, including bromovinyl substitution reduced the antiviral potency significantly. Changing the 3'-azido function to an amino or iodo group reduced the antiviral activity. Replacement of the uracil ring by cytosine or 5-methylcytosine produced analogues with high potency and low toxicity. Modification of the 5'-hydroxy group markedly reduced the antiviral activity. Similarly, various C-nucleoside analogues related to AZT and 2',3'-dideoxycytidine were inactive and nontoxic. From these systematic studies 3'-azido-2',3'-dideoxyuridine (5a), 3'-azido-5-ethyl-2',3'-dideoxyuridine (5c), and 3'-azido-2',3'-dideoxycytidine (7a) and its 5-methyl analogue (7b) were identified as potent and selective anti-HIV-1 agent in primary human lymphocytes.     

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.     

Synthesis of 5-[1-hydroxy(or methoxy)-2-bromo(or chloro)ethyl]-2'-deoxyuridines and related halohydrin analogues with antiviral and cytotoxic activity

A series of new 5-(1-hydroxy-2-haloethyl)-2'-deoxyuridines (3, 6, 8) were synthesized in 60-70% yields by addition of HOX (X = Br, Cl, I) to the vinyl substituent of the respective 5-vinyl-2'-deoxyuridines (2, 5, 7). Treatment of 3a,b with methanolic sulfuric acid afforded the corresponding 5-(1-methoxy-2-haloethyl)-2'-(deoxyuridines (4a,b). The 5-(1-hydroxy-2-chloroethyl) (3b), 5-(1-methoxy-2-bromoethyl) (4a), 5-(1-hydroxy-2-bromo-2-(ethoxycarbonyl)ethyl) (6a), and 5-(1-hydroxy-2-iodo-2-(ethoxycarbonyl)ethyl) (6b) derivatives exhibited in vitro antiviral activity (ID50 = 0.1-1 microgram/mL range) against herpes simplex virus type 1 (HSV-1). 5-(1-Hydroxy-2-bromo-2-(ethoxycarbonyl)-ethyl)-2'-deoxyuridine (6a) was the most active cytotoxic agent in the in vitro L1210 screen exhibiting an ED50 of 11 micrograms/mL relative to melphalan (ED50 = 0.15 micrograms/mL).     

Acquisition of human concentrative nucleoside transporter 2 (hcnt2) activity by gene transfer confers sensitivity to fluoropyrimidine nucleosides in drug-resistant leukemia cells

CEM-ARAC leukemia cells with resistance to cytarabine were shown to lack equilibrative transporter (hENT1) expression and activity. Stable transfer of hCNT2 cDNA into CEM-ARAC enabled Na(+)-dependent transport of purine and pyrimidine nucleoside analogs and provided a unique in vitro model for studying hCNT2. Analysis of [(3)H]uridine inhibitory activity by test substances in hCNT2 transfectant ARAC/D2 revealed structural requirements for interaction with hCNT2: 1) ribosyl and 2'-deoxyribosyl nucleosides were better inhibitors than 3'-deoxyribosyl, 2',3'-dideoxyribosyl or arabinosyl nucleosides; 2) uridine analogs with halogens at position 5 were better inhibitors than 5-methyluridine or thymidine; 3) 2-chloroadenosine was a better inhibitor than 2-chloro-2'-deoxyadenosine (cladribine); and 4) cytosine-containing nucleosides, 7-deazaadenosine and nucleobases were not inhibitors. Quantification of inhibitory capacity yielded K(i) values of 34-50 microM (5-halogenated uridine analogs, 2'-deoxyuridine), 82 microM (5-fluoro-2'-deoxyuridine), 197-246 microM (5-methyluridine < 5-bromo-2'-deoxyuridine < 5-iodo-2'-deoxyuridine), and 411 microM (5-fluoro-5'-deoxyuridine, capecitabine metabolite). Comparisons of hCNT2-mediated transport rates indicated halogenated uridine analogs were transported more rapidly than halogenated adenosine analogs, even though hCNT2 exhibited preference for physiologic purine nucleosides over uridine. Kinetics of hCNT2-mediated transport of 5-fluorouridine and uridine were similar (K(m) values, 43-46 microM). The impact of hCNT2-mediated transport on chemosensitivity was assessed by comparing antiproliferative activity of nucleoside analogs against hCNT2-containing cells with transport-defective, drug-resistant cells. Chemosensitivity was restored partially for cladribine, completely for 5-fluorouridine and 5-fluoro-2'-deoxyuridine, whereas there was little effect on chemosensitivity for fludarabine, 7-deazaadenosine, or cytarabine. These studies, which demonstrated hCNT2 uptake of halogenated uridine analogs, suggested that hCNT2 is an important determinant of cytotoxicity of this class of compounds in vivo.     

2'-azido-2',3'-dideoxypyrimidine nucleosides. Synthesis and antiviral activity against human immunodeficiency virus

A series of four 2'-azido-2',3'-dideoxypyrimidine nucleosides were synthesized and their activity against human immunodeficiency virus was explored. 2,2'-Anhydro-5-O-benzoyluridine (6a) was prepared from 5-O-benzoyluridine (5a) and converted into 3'-deoxy analogue 8a by imidazolylthiocarbonylation followed by Bu3SnH reduction. Treatment of 8a with LiN3 in DMF followed by saponification afforded 2'-azido-2',3'-dideoxyuridine (1a). The 5'-O-benzoylated nucleoside 9a was further converted into the 5-bromo and 5-iodo analogues (1b and 1c) by halogenation and debenzoylation. 2',3'-O-Isopropylideneuridine (3) was converted in two steps into the thymine nucleoside, which was benzoylated and de-O-isopropylidenated to afford 5'-O-benzoyl-5-methyluridine (5d). 2'-Azido-2',3'-dideoxy-5-methyluridine (1d) was synthesized from 5d in a similar manner as that used for the synthesis of 1a from 5a. These new nucleosides, closely related to AZT, however, did not exhibit any significant anti-HIV activity in tissue culture using H9 cells.