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Canine hemangiosarcoma (HSA) has an extremely poor prognosis, making it necessary to develop new systemic treatment methods. MicroRNA-214 (miR-214) is one of many microRNAs (miRNA) that can induce apoptosis in HSA cell lines. Synthetic miR-214 (miR-214/5AE), which showed higher cytotoxicity and greater nuclease resistance than mature miR-214, has been developed for clinical application. In this study, we evaluated the effects of miR-214/5AE on stage 2 HSA in a mouse model. Mice intraperitoneally administered with miR-214/5AE (5AE group) had significantly fewer intraperitoneal dissemination tumor foci (median number: 72.5 vs. 237.5; p < 0.05) and a lower median foci weight (0.26 g vs. 0.61 g; p < 0.05). Mice in the 5AE group had increased expression of p53 and cleaved caspase-3, and a significantly lower proportion of Ki-67-positive cells, than those in the non-specific miR group. Notably, no significant side effects were observed. These results indicate that intraperitoneal administration of miR-214/5AE exhibits antitumor effects in an intraperitoneal dissemination mouse model of HSA by inducing apoptosis and suppressing cell proliferation. These results provide a basis for future studies on the antitumor effect of miR-214/5AE for HSA., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Chronic hepatitis B virus (HBV) infection is a major public health problem that affects millions of people worldwide. Nucleoside analogue reverse transcriptase (RT) inhibitors, such as entecavir (ETV) and lamivudine (3TC), serve as crucial anti-HBV drugs. However, structural studies of HBV RT have been hampered due to its unexpectedly poor solubility. Here, we show that human immunodeficiency virus type-1 (HIV-1) with HBV-associated amino acid substitutions Y115F/F116Y/Q151M in its RT (HIV Y115F/F116Y/Q151M ) is highly susceptible to ETV and 3TC. Additionally, we experimentally simulated previously reported ETV/3TC resistance for HBV using HIV Y115F/F116Y/Q151M with F160M/M184V (L180M/M204V in HBV RT) substituted. We determined crystal structures for HIV-1 RT Y115F/F116Y/Q151M :DNA complexed with 3TC-triphosphate (3TC-TP)/ETV-triphosphate (ETV-TP)/dCTP/dGTP. These structures revealed an atypically tight binding conformation of 3TC-TP, where the Met184 side-chain is pushed away by the oxathiolane of 3TC-TP and exocyclic methylene of ETV-TP. Structural analysis of RT Y115F/F116Y/Q151M/F160M/M184V :DNA:3TC-TP also demonstrated that the loosely bound 3TC-TP is misaligned at the active site to prevent a steric clash with the side chain γ-methyl of Val184. These findings shed light on the common structural mechanism of HBV and HIV-1 resistance to 3TC and ETV and should aid in the design of new agents to overcome drug resistance to 3TC and ETV.

Hepatitis B virus (HBV) infection is a major worldwide health problem that requires the development of improved antiviral therapies. Here, a series of 4'-Azido-thymidine/4'-Azido-2'-deoxy-5-methylcytidine derivatives ( 6 , 10-15 ) were synthesized, and their anti-HBV activities evaluated. Compounds 10-15 were synthesized via an S N Ar reaction of 18 , in which the 4-position of the thymine moiety was activated as the 2,4,6-triisopropylbenzenesulfonate. Compounds 11-15 showed no antiviral activity. However, 4'-Azido thymidine ( 6 ) and 4'-Azido-2'-deoxy-5-methylcytidine ( 10 ) displayed significant anti-HBV activity (EC 50 = 0.63 and 5.99  µ M, respectively) with no detectable cytotoxicity against MT-2 cells up to 100  µ M.

We designed, synthesized, and characterized a novel nucleoside analog, (1 S ,3 S ,5 S )-3-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-5-hydroxy-1-(hydroxymethyl)-2-methylene-cyclopentanecarbonitrile, or 4'-cyano-methylenecarbocyclic-2'-deoxyguanosine (CMCdG), and evaluated its anti-hepatitis B virus (anti-HBV) activity, safety, and related features. CMCdG's in vitro activity was determined using quantitative PCR and Southern blotting assays, and its cytotoxicity was determined with a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, while its in vivo activity and safety were determined in human liver-chimeric mice infected with wild-type HBV genotype Ce (HBV WT Ce ) and an entecavir (ETV)-resistant HBV variant containing the amino acid substitutions L180M, S202G, and M204V (HBV ETV-R L180M/S202G/M204V ). CMCdG potently inhibited HBV production in HepG2.2.15 cells (50% inhibitory concentration [IC 50 ], ∼30 nM) and HBV WT Ce plasmid-transfected Huh7 cells (IC 50 , 206 nM) and efficiently suppressed ETV-resistant HBV ETV-R L180M/S202G/M204V (IC 50 , 2,657 nM), while it showed no or little cytotoxicity (50% cytotoxic concentration, >500 μM in most hepatocytic cells examined). Two-week peroral administration of CMCdG (1 mg/kg of body weight/day once a day [q.d.]) to HBV WT Ce -infected human liver-chimeric mice reduced the level of viremia by ∼2 logs. CMCdG also reduced the level of HBV ETV-R L180M/S202G/M204V viremia by ∼1 log in HBV ETV-R L180M/S202G/M204V -infected human liver-chimeric mice, while ETV (1 mg/kg/day q.d.) completely failed to reduce the viremia. None of the CMCdG-treated mice had significant drug-related changes in body weights or serum human albumin levels. Structural analyses using homology modeling, semiempirical quantum methods, and molecular dynamics revealed that although ETV triphosphate (TP) forms good van der Waals contacts with L180 and M204 of HBV WT Ce reverse transcriptase (RT), its contacts with the M180 substitution are totally lost in the HBV ETV-R L180M/S202G/M204V RT complex. However, CMCdG-TP retains good contacts with both the HBV WT Ce RT and HBV ETV-R L180M/S202G/M204V RT complexes. The present data warrant further studies toward the development of CMCdG as a potential therapeutic for patients infected with drug-resistant HBV and shed light on the further development of more potent and safer anti-HBV agents., (Copyright © 2019 American Society for Microbiology.)

Nucleoside analogue reverse transcriptase (RT) inhibitors (NRTIs) are major antiviral agents against hepatitis B virus (HBV) and human immunodeficiency virus type-1 (HIV-1). However, the notorious insoluble property of HBV RT has prevented atomic-resolution structural studies and rational anti-HBV drug design. Here, we created HIV-1 RT mutants containing HBV-mimicking sextuple or septuple amino acid substitutions at the nucleoside-binding site (N-site) and verified that these mutants retained the RT activity. The most active RT mutant, HIV-1 RT 7MC , carrying Q151M/G112S/D113A/Y115F/F116Y/F160L/I159L was successfully crystallized, and its three-dimensional structure was determined in complex with DNA:dGTP/entecavir-triphosphate (ETV-TP), a potent anti-HBV guanosine analogue RT inhibitor, at a resolution of 2.43 Å and 2.60 Å, respectively. The structures reveal significant positional rearrangements of the amino acid side-chains at the N-site, elucidating the mechanism underlying the differential susceptibility of HIV-1 and HBV against recently reported 4'-modified NRTIs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA/MK-8591), a nucleoside reverse transcriptase inhibitor (NRTI) under clinical trials, is a potent and promising long-acting anti-HIV type 1 (HIV-1) agent. EFdA and its derivatives possess a modified 4'-moiety and potently inhibit the replication of a wide spectrum of HIV-1 strains resistant to existing NRTIs. Here, we report that EFdA and NRTIs with a 4'-ethynyl- or 4'-cyano-moiety exerted activity against HIV-1 with an M184V mutation and multiple NRTI-resistant HIV-1s, whereas NRTIs with other moieties (e.g., 4'-methyl) did not show this activity. Structural analysis indicated that EFdA and 4'-ethynyl-NRTIs (but not other 4'-modified NRTIs), formed strong van der Waals interactions with critical amino acid residues of reverse transcriptase. Such interactions were maintained even in the presence of a broad resistance-endowing M184V substitution, thus potently inhibiting drug-resistant HIV-1 strains. These findings also explain the mechanism for the potency of EFdA and provide insights for further design of anti-HIV-1 therapeutics., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

4'-cyano-2'-deoxyinosine (SK14-061a), a novel nucleoside analog based on inosine, has antiviral activity against the human immunodeficiency virus type 1 that has the ability to acquire resistance against many types of reverse transcriptase inhibitors based on nucleosides. The aim of this study was to investigate the pharmacokinetics studies after its oral administration to rats. For this purpose, we first developed and validated an analytical method for quantitatively determining SK14-061a levels in biological samples by a UPLC system interfaced with a TOF-MS system. A rapid, simple and selective method for the quantification of SK14-061a in biological samples was established using liquid chromatography mass spectrometry (LC-MS) with solid phase extraction. The pharmacokinetic properties of SK14-061a in rats after oral administration were then evaluated using this LC-MS method. SK14-061a was found to be relatively highly bioavailable, is rapidly absorbed from the intestinal tract, and is then mainly distributed to the liver and then ultimately excreted via the urine in an unchanged form. Furthermore, the simultaneous administration of SK14-061a with the nucleoside analog, entecavir, led to a significant alteration in the pharmacokinetics of SK14-061a. These results suggest that the SK14-061a has favorable pharmacokinetic properties with a high bioavailability with the potential for use in oral pharmaceutical formulations, but drug-drug interactions should also be considered., Competing Interests: The authors have declared that no competing interests exist.

Objectives: 4'-cyano-2'-deoxyguanosine (CdG), a novel nucleoside analogue, has a high degree of antiviral activity against the chronic hepatitis B virus (HBV). The objective of this study was to develop an analytical method for quantitatively determining CdG levels in biological samples by liquid chromatography-mass spectrometry (LC/MS) and to investigate the pharmacokinetic properties of CdG in rats after intravenous and oral administration., Methods: An analytical method using a UPLC system interfaced with a TOF-MS system was developed and validated. The pharmacokinetic properties after the intravenous and oral administration of CdG to rats were evaluated. In vivo pharmacokinetic interactions between CdG and entecavir were also investigated., Key Findings: A rapid, simple and selective method for the quantification of CdG in biological samples was established using LC/MS with solid-phase extraction. In vivo pharmacokinetic studies of CdG in rats demonstrated that CdG is highly bioavailable, is rapidly absorbed from the intestinal tract, is then distributed to the liver rather than kidney and is ultimately excreted via the urine in an unchanged form. The co-administration of CdG and entecavir led to pharmacokinetic interactions with each other., Conclusions: The data generated in this study provide support for the clinical development of CdG for use in the treatment of HBV., (© 2018 Royal Pharmaceutical Society.)

Hepatitis B virus (HBV) reverse transcriptase (RT) is essential for viral replication and is an important drug target. Nonetheless, the notorious insolubility of HBV RT has hindered experimental structural studies and structure-based drug design. Here, we demonstrate that a Q151M substitution alone at the nucleotide-binding site (N-site) of human immunodeficiency virus type-1 (HIV-1) RT renders HIV-1 highly sensitive to entecavir (ETV), a potent nucleoside analogue RT inhibitor (NRTI) against HBV. The results suggest that Met151 forms a transient hydrophobic interaction with the cyclopentyl methylene of ETV, a characteristic hydrophobic moiety of ETV. We thus solved the crystal structures of HIV-1 RT Q151M :DNA complex with bound dGTP or ETV-triphosphate (ETV-TP). The structures revealed that ETV-TP is accommodated at the N-site slightly apart from the ribose ring of the 3'-end nucleotide, compared to the position of bound dGTP and previously reported NRTI/dNTP. In addition, the protruding methylene group of bound ETV-TP directly pushes the side-chain of Met184 backward. Met184 is a key residue that confers ETV resistance upon substitution with smaller Ile/Val. These results provide novel insights into NRTI binding to the N-site and further provide important clues for the development of novel anti-HBV/HIV-1 RT inhibitors to overcome critical drug resistance.

Synthesis of a novel 2'-deoxy-guanine carbocyclic nucleoside 4 constructed with spiro[2.4]heptane core structure in the aglycon moiety was carried out. Radical-mediated 5-exo-dig mode cyclization and following cyclopropanation proceeded efficiently to furnish the spiro alcohol 10. Subsequent Mitsunobu-type glycosylation between 13 and 14, deoxygenation of the 2'-hydroxyl group of 16 and deprotection of 17 gave the title compound 4. Compound 4 demonstrated moderate anti-HBV activity (EC 50 value of 0.12 ± 0.02 µM) and no cytotoxicity against HepG2 cells was observed up to 100 µM.

A method for the diastereoselective synthesis of 6″-(Z)- and 6″-(E)-fluorinated analogues of the anti-HBV agent entecavir has been developed. Construction of the methylenecyclopentane skeleton of the target molecules has been accomplished by radical-mediated 5-exo-dig cyclization of the selenides 6 and 15 having the phenylsulfanylethynyl structure as a radical accepting moiety. In the radical reaction of the TBS-protected precursor 6, (Z)-anti-12 was formed as a major product. On the other hand, TIPS-protected 15 gave (E)-anti-12. The sulfur-extrusive stannylation of anti-12 furnished a mixture of geometric isomers of the respective vinylstannane, whereas benzoyl-protected 17 underwent the stannylation in the manner of retention of configuration. Following XeF2-mediated fluorination, introduction of the purine base and deoxygenation of the resulting carbocyclic guanosine gave the target (E)- and (Z)-3 after deprotection. Evaluation of the anti-HBV activity of 3 revealed that fluorine-substitution at the 6″-position of entecavir gave rise to a reduction in the cytotoxicity in HepG2 cells with retention of the antiviral activity.

Unlabelled: Certain nucleoside/nucleotide reverse transcriptase (RT) inhibitors (NRTIs) are effective against human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV). However, both viruses often acquire NRTI resistance, making it crucial to develop more-potent agents that offer profound viral suppression. Here, we report that 4'-C-cyano-2-amino-2'-deoxyadenosine (CAdA) is a novel, highly potent inhibitor of both HBV (half maximal inhibitory concentration [IC50 ] = 0.4 nM) and HIV-1 (IC50  = 0.4 nM). In contrast, the approved anti-HBV NRTI, entecavir (ETV), potently inhibits HBV (IC50  = 0.7 nM), but is much less active against HIV-1 (IC50  = 1,000 nM). Similarly, the highly potent HIV-1 inhibitor, 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA; IC50  = 0.3 nM) is less active against HBV (IC50  = 160 nM). Southern analysis using Huh-7 cells transfected with HBV-containing plasmids demonstrated that CAdA was potent against both wild-type (IC50  = 7.2 nM) and ETV-resistant HBV (IC50  = 69.6 nM for HBVETV-RL180M/S202G/M204V), whereas ETV failed to reduce HBVETV-RL180M/S202G/M204V DNA even at 1 μM. Once-daily peroral administration of CAdA reduced HBVETV-RL180M/S202G/M204V viremia (P = 0.0005) in human-liver-chimeric/ HBVETV-RL180M/S202G/M204V-infected mice, whereas ETV completely failed to reduce HBVETV-RL180M/S202G/M204V viremia. None of the mice had significant drug-related body-weight or serum human-albumin concentration changes. Molecular modeling suggests that a shallower HBV-RT hydrophobic pocket at the polymerase active site can better accommodate the slightly shorter 4'-cyano of CAdA-triphosphate (TP), but not the longer 4'-ethynyl of EFdA-TP. In contrast, the deeper HIV-1-RT pocket can efficiently accommodate the 4'-substitutions of both NRTIs. The ETV-TP's cyclopentyl ring can bind more efficiently at the shallow HBV-RT binding pocket., Conclusion: These data provide insights on the structural and functional associations of HBV- and HIV-1-RTs and show that CAdA may offer new therapeutic options for HBV patients., (© 2015 by the American Association for the Study of Liver Diseases.)

Exomethylene acycloguanine nucleosides 4, 6 and its monophosphate derivatives 5, 7, and 8 have been synthesized. Mitsunobu-type coupling of 2-N-acetyl-6-O-diphenylcarbamoylguanine (11) with primary alcohols proceeded regioselectively to furnish the desired N(9)-substituted products in moderate yield. Evaluation of 4-8 for anti-HBV activity in HepG2 cells revealed that the phosphonate derivative 8 was found to exhibit moderated activity (EC50 value of 0.29 μM), but cytotoxicity (CC50 value of 39 μM) against the host cells was also observed.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), a recently discovered nucleoside reverse transcriptase inhibitor, exhibits activity against a wide spectrum of wild-type and multidrug-resistant clinical human immunodeficiency virus type 1 (HIV-1) isolates (50% effective concentration, 0.0001 to 0.001 microM). In the present study, we used human peripheral blood mononuclear cell-transplanted, HIV-1-infected NOD/SCID/Janus kinase 3 knockout mice for in vivo evaluation of the anti-HIV activity of EFdA. Administration of EFdA decreased the replication and cytopathic effects of HIV-1 without identifiable adverse effects. In phosphate-buffered saline (PBS)-treated mice, the CD4+/CD8+ cell ratio in the spleen was low (median, 0.04; range, 0.02 to 0.49), while that in mice receiving EFdA was increased (median, 0.65; range, 0.57 to 1.43). EFdA treatment significantly suppressed the amount of HIV-1 RNA (median of 9.0 x 10(2) copies/ml [range, 8.1 x 10(2) to 1.1 x 10(3) copies/ml] versus median of 9.9 x 10(4) copies/ml [range, 8.1 x 10(2) to 1.1 x 10(3) copies/ml]; P < 0.001), the p24 level in plasma (2.5 x 10(3) pg/ml [range, 8.2 x 10(2) to 5.6 x 10(3) pg/ml] versus 2.8 x 10(2) pg/ml [range, 8.2 x 10(1) to 6.3 x 10(2) pg/ml]; P < 0.001), and the percentage of p24-expressing cells in the spleen (median of 1.90% [range, 0.33% to 3.68%] versus median of 0.11% [range, 0.00% to 1.00%]; P = 0.003) in comparison with PBS-treated mice. These data suggest that EFdA is a promising candidate for a new age of HIV-1 chemotherapy and should be developed further as a potential therapy for individuals with multidrug-resistant HIV-1 variants.

One of the formidable challenges in therapy of infections by human immunodeficiency virus (HIV) is the emergence of drug-resistant variants that attenuate the efficacy of highly active antiretroviral therapy (HAART). We have recently introduced 4'-ethynyl-nucleoside analogs as nucleoside reverse transcriptase inhibitors (NRTIs) that could be developed as therapeutics for treatment of HIV infections. In this study, we present 2'-deoxy-4'-C-ethynyl-2-fluoroadenosine (EFdA), a second generation 4'-ethynyl inhibitor that exerted highly potent activity against wild-type HIV-1 (EC50 approximately 0.07 nM). EFdA retains potency toward many HIV-1 resistant strains, including the multi-drug resistant clone HIV-1A62V/V75I/F77L/F116Y/Q151M. The selectivity index of EFdA (cytotoxicity/inhibitory activity) is more favorable than all approved NRTIs used in HIV therapy. Furthermore, EFdA efficiently inhibited clinical isolates from patients heavily treated with multiple anti-HIV-1 drugs. EFdA appears to be primarily phosphorylated by the cellular 2'-deoxycytidine kinase (dCK) because: (a) the antiviral activity of EFdA was reduced by the addition of dC, which competes nucleosides phosphorylated by the dCK pathway, (b) the antiviral activity of EFdA was significantly reduced in dCK-deficient HT-1080/Ara-Cr cells, but restored after dCK transduction. Further, unlike other dA analogs, EFdA is completely resistant to degradation by adenosine deaminase. Moderate decrease in susceptibility to EFdA is conferred by a combination of three RT mutations (I142V, T165R, and M184V) that result in a significant decrease of viral fitness. Molecular modeling analysis suggests that the M184V/I substitutions may reduce anti-HIV activity of EFdA through steric hindrance between its 4'-ethynyl moiety and the V/I184 beta-branched side chains. The present data suggest that EFdA, is a promising candidate for developing as a therapeutic agent for the treatment of individuals harboring multi-drug resistant HIV variants.

We examined the intracytoplasmic anabolism and kinetics of antiviral activity against human immunodeficiency virus type 1 (HIV-1) of a nucleoside reverse transcriptase inhibitor, 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), which has potent activity against wild-type and multidrug-resistant HIV-1 strains. When CEM cells were exposed to 0.1 microM [(3)H]EFdA or [(3)H]3'-azido-2',3'-dideoxythymidine (AZT) for 6 h, the intracellular EFdA-triphosphate (TP) level was 91.6 pmol/10(9) cells, while that of AZT was 396.5 pmol/10(9) cells. When CEM cells were exposed to 10 microM [(3)H]EFdA, the amount of EFdA-TP increased by 22-fold (2,090 pmol/10(9) cells), while the amount of [(3)H]AZT-TP increased only moderately by 2.4-fold (970 pmol/10(9) cells). The intracellular half-life values of EFdA-TP and AZT-TP were approximately 17 and approximately 3 h, respectively. When MT-4 cells were cultured with 0.01 microM EFdA for 24 h, thoroughly washed to remove EFdA, further cultured without EFdA for various periods of time, exposed to HIV-1(NL4-3), and cultured for an additional 5 days, the protection values were 75 and 47%, respectively, after 24 and 48 h with no drug incubation, while those with 1 microM AZT were 55 and 9.2%, respectively. The 50% inhibitory concentration values of EFdA-TP against human polymerases alpha, beta, and gamma were >100 microM, >100 microM, and 10 microM, respectively, while those of ddA-TP were >100 microM, 0.2 microM, and 0.2 microM, respectively. These data warrant further development of EFdA as a potential therapeutic agent for those patients who harbor wild-type HIV-1 and/or multidrug-resistant variants.

Working hypotheses to solve the critical problems of the existing highly active anti-retroviral therapy were proposed. The study based on the hypotheses proved the validity of the hypotheses and resulted in the development of 2'-deoxy-4'-C-ethynyl-2-fluoroadenosine, a nucleoside reverse transcriptase inhibitor, with highly potent activity against all HIV-1, very favorable toxic profiles, and stability in plasma. The nucleoside will prevent or delay the emergence of drug-resistant HIV-1 variants and be an ideal therapeutic agent for both HIV-1 and HBV infections.

A working hypothesis to solve the critical problems of existing HAART was proposed. The study based on the hypothesis proved the validity of the hypothesis and resulted in the development of 2'-deoxy-4'-C-ethynyl-2-fluoro-adenosine (4'Ed2FA), a nucleoside reverse transcriptase inhibitor (NRTI) with highly potent activity against all HIV-1 strains, very favourable toxic profiles, and stability in plasma.

Extensive efforts have been made to identify nucleoside reverse transcriptase inhibitors (NRTIs). Eight NRTIs have now been approved for clinical use; however, variants of HIV-1 resistant to these antiviral agents have emerged in patients even when they are treated with combinations [highly active antiretroviral therapy (HAART)]. Thus, the development of novel compounds that are active against drug-resistant HIV-1 variants and that prevent or delay the emergence of resistant HIV-1 variants is urgently needed. Previously, 4'-C-substituted nucleosides (4'-SNs) were designed as new types of NRTIs. They were synthesized and examined as potential therapeutic agents against HIV infection. Among them, several 4'-substituted-2'-deoxynucleosides (4'-SdNs), especially those that bear an ethynyl group, were shown to be active against various laboratory and clinical HIV-1 strains including known drug-resistant variants. These results were recently reported by our collaborators. In this review, we summarize the design, synthesis and demonstrations of the anti-HIV activity of 4'-SNs, and then consider 4'-SNs as potential therapeutic agents for HIV-1.

We investigated the potential of 4'-C-substituted nucleosides for the treatment of HIV-1 and HBV. Of the nucleosides we prepared, several 4'-C-ethynyl-2'-deoxypurine nucleosides showed the most potent anti-HIV activity. However, two candidates, 4'-C-ethynyl-2'-deoxyguanosine and 9-(2-deoxy-4-C-ethynyl-beta-D-ribo-pentofuranosyl)-2,6-diaminopurine, were very toxic during in vivo study. On the other hand, lamivudine (3TC) is known to show remarkable activity against HIV and HBV with lower cytotoxicity. Therefore, we attempted to synthesize the L-enantiomer of 4'-C-ethynyl-2'-deoxypurine nucleosides in 20-21 steps. These methods consisted of preparing 4-C-ethynyl-L-sugar, starting from D-arabinose and then condensing the L-sugar derivative with 2,6-diaminopurine. 4'-C-Ethynyl-2'-deoxyguanosine was also prepared by enzymatic deamination from the 2,6-diaminopurine derivative. The compounds' antiviral activity against HIV and HBV was then evaluated. Unfortunately, they demonstrated no activity and no cytotoxicity.

Some 4'-C-ethynyl-2'-deoxy purine nucleosides showed the most potent anti-HIV activity among the series of 4'-C-substituted 2'-deoxynucleosides whose 4'-C-substituents were methyl, ethyl, ethynyl and so on. Our hypothesis is that the smaller the substituent at the C-4' position they have, the more acceptable biological activity they show. Thus, 4'-C-cyano-2'-deoxy purine nucleosides, whose substituent is smaller than the ethynyl group, will have more potent antiviral activity. To prove our hypothesis, we planned to develop an efficient synthesis of 4'-C-cyano-2'-deoxy purine nucleosides (4'-CNdNs) and 4'-C-ethynyl-2'-deoxy purine nucleosides (4'-EdNs). Consequently, we succeeded in developing an efficient synthesis of six 2'-deoxy purine nucleosides bearing either a cyano or an ethynyl group at the C-4' position of the sugar moiety from 2'-deoxyadenosine and 2,6-diaminopurine 2'-deoxyriboside. Unfortunately, 4'-C-cyano derivatives showed lower activity against HIV-1, and two 4'-C-ethynyl derivatives suggested high toxicity in vivo.

Purine 2'-deoxynucleosides bearing an ethynyl or a cyano group at C-4' of the sugar moiety were synthesized from the corresponding 2'-deoxynucleosides. These compounds exhibited very potent anti-HIV activity, and remained active against drug resistant HIV strains.

The L-enantiomer of 4'-C-ethynyl-2'-deoxycytidine (2) was synthesized, but did not show any activity against HIV-1 up to 100 microM.

A series of 4'-ethynyl (4'-E) nucleoside analogs were designed, synthesized, and identified as being active against a wide spectrum of human immunodeficiency viruses (HIV), including a variety of laboratory strains of HIV-1, HIV-2, and primary clinical HIV-1 isolates. Among such analogs examined, 4'-E-2'-deoxycytidine (4'-E-dC), 4'-E-2'-deoxyadenosine (4'-E-dA), 4'-E-2'-deoxyribofuranosyl-2,6-diaminopurine, and 4'-E-2'-deoxyguanosine were the most potent and blocked HIV-1 replication with 50% effective concentrations ranging from 0.0003 to 0.01 microM in vitro with favorable cellular toxicity profiles (selectivity indices ranging 458 to 2,600). These 4'-E analogs also suppressed replication of various drug-resistant HIV-1 clones, including HIV-1(M41L/T215Y), HIV-1(K65R), HIV-1(L74V), HIV-1(M41L/T69S-S-G/T215Y), and HIV-1(A62V/V75I/F77L/F116Y/Q151M). Moreover, these analogs inhibited the replication of multidrug-resistant clinical HIV-1 strains carrying a variety of drug resistance-related amino acid substitutions isolated from HIV-1-infected individuals for whom 10 or 11 different anti-HIV-1 agents had failed. The 4'-E analogs also blocked the replication of a non-nucleoside reverse transcriptase inhibitor-resistant clone, HIV-1(Y181C), and showed an HIV-1 inhibition profile similar to that of zidovudine in time-of-drug-addition assays. The antiviral activity of 4'-E-thymidine and 4'-E-dC was blocked by the addition of thymidine and 2'-deoxycytidine, respectively, while that of 4'-E-dA was not affected by 2'-deoxyadenosine, similar to the antiviral activity reversion feature of 2',3'-dideoxynucleosides, strongly suggesting that 4'-E analogs belong to the family of nucleoside reverse transcriptase inhibitors. Further development of 4'-E analogs as potential therapeutics for infection with multidrug-resistant HIV-1 is warranted.

4'-C-Ethynyl-beta-D-arabino- and 4'-C-ethynyl-2'-deoxy-beta-D-ribo-pentofuranosylpyrimidine and -purine nucleosides were synthesized and evaluated for their in vitro anti-HIV activity. The key intermediate, 4-C-ethynyl- or 4-C-triethylsilylethynyl-D-ribo-pentofuranose, was prepared from D-glucose and glycosidated with various pyrimidine or purine bases. The arabinopyrimidine derivatives were prepared from the corresponding ribo derivatives via O(2),2'-anhydro nucleosides. The 2'-deoxy-ribo derivatives were synthesized by radical reduction of 2'-bromo or 2'- phenoxythiocarbonyloxy nucleosides. Among these 4'-C-ethynyl nucleosides, seven analogues proved to be potent against HIV-1 in vitro with EC(50) values ranging from 0.0003 to 0. 03 microM. These compounds also exerted activity against clinical and multi-dideoxy-nucleoside-resistant HIV-1 strains with comparable EC(50) values. Three such 4'-C-ethynyl-2'-deoxypurine analogues including 4'-C-ethynyl-2'-deoxyadenosine and 4'-C-ethynyl-2, 6-diamino-2'-deoxypurine were less cytotoxic [selectivity indices (SIs): 975-2733] than three 4'-C-ethynyl-2'-deoxycytidine analogues (SIs: 63-363). 4'-C-Ethynyl-5-fluoro-2'-deoxycytidine was least toxic (SI: >3333) and potent against all HIV strains tested.

4'-C-Ethynyl-beta-D-arabino-pentofuranosyl thymine (14) and cytosine (16), and 4'-C-ethynyl-2'-deoxy-beta-D-ribo-pentofuranosyl thymine (25) and cytosine (27) were synthesized by properly protected 4'-C-hydroxy-methyl-3,5-di-O-benzyl-alpha-D-ribo-pentofuranose (1) from D-glucose. Among them, 2'-deoxy derivatives 25 and 27 exhibited antiviral activity, while cytidine derivatives 16 and 27 inhibited the growth of neoplastic cells.

The 4'-C-ethynyl-β-D-ribo-pentofuranosylpyrimidines were prepared from D-glucose through properly protected 4'-C-formyl-D-ribo-ribofuranose as the key intermediate, and preliminary biological tests against some viruses and tumor cells showed that the compounds were not active.

The effects of four kinds of 2'-deoxy-L-nucleoside 5'-triphosphates and L-ATP, which are enantiomers of natural D-dNTPs and D-ATP, on deoxycytidine kinase (dCK) partially purified from mouse leukemic P388 cells were investigated. Only L-dCTP did not act as a phosphate donor while other L-dNTPs and L-ATP showed 15-30% of the activity of the corresponding D-dNTP or D-ATP. L-dCTP inhibited dCK non-competitively with 2'-deoxycytidine (D-dCyd) and competitively with phosphate donor D-ATP. These inhibitory effects of L-dCTP on dCK were similar to the results of earlier studies using D-dCTP. Thus, L-dCTP was shown to be capable of serving as a feedback inhibitor for dCK instead of D-dCTP. Mouse dCK was also able to phosphorylate L-dCyd, as demonstrated in the case of human dCK. The present results suggest that the chirality of not only dCyd as the substrate but also nucleotides as the substrate or effector is not strictly discriminated by dCK.

2'-Deoxyuridine derivatives bearing an activated ester at C-5 position were synthesized and was examined their use for the preparation of modified oligodeoxyribonucleotides (ODNs) by a post-modification method. The ODNs containing cyanomethyl ester at C-5 position of the deoxyuridine residue reacted easily with a primary amine of several functional molecules under the mild condition to give the corresponding modified ODNs.