Deviated binding of anti-HBV nucleoside analog E-CFCP-TP to the reverse transcriptase active site attenuates the effect of drug-resistant mutations.

While certain human hepatitis B virus-targeting nucleoside analogs (NAs) serve as crucial anti-HBV drugs, HBV yet remains to be a major global health threat. E-CFCP is a 4'-modified and fluoromethylenated NA that exhibits potent antiviral activity against both wild-type and drug-resistant HBVs but less potent against human immunodeficiency virus type-1 (HIV-1). Here, we show that HIV-1 with HBV-associated amino acid substitutions introduced into the RT's dNTP-binding site (N-site) is highly susceptible to E-CFCP. We determined the X-ray structures of HBV-associated HIV-1 RT mutants complexed with DNA:E-CFCP-triphosphate (E-CFCP-TP). The structures revealed that exocyclic fluoromethylene pushes the Met184 sidechain backward, and the resultant enlarged hydrophobic pocket accommodates both the fluoromethylene and 4'-cyano moiety of E-CFCP. Structural comparison with the DNA:dGTP/entecavir-triphosphate complex also indicated that the cyclopentene moiety of the bound E-CFCP-TP is slightly skewed and deviated. This positioning partly corresponds to that of the bound dNTP observed in the HIV-1 RT mutant with drug-resistant mutations F160M/M184V, resulting in the attenuation of the structural effects of F160M/M184V substitutions. These results expand our knowledge of the interactions between NAs and the RT N-site and should help further design antiviral NAs against both HIV-1 and HBV.

Synthesis of novel entecavir analogues having 4'-cyano-6''-fluoromethylenecyclopentene skeletons as an aglycone moiety as highly potent and long-acting anti-hepatitis B virus agent.

Encouraged by our recent findings that 4'-cyano-deoxyguanosine (2), entecavir analogues 4 and 5 are highly potent anti-hepatitis B virus (HBV) agents, we designed and synthesized 6 having a hybridized structure of 4 and 5. The chiral quaternary carbon portion at the 4'-position, which is substituted by cyano- and 5'-hydroxymethyl groups, was stereospecifically constructed by radical-mediated 5-exo-dig mode cyclization of 10. The introduction of the fluorine atom into the 6''-position was achieved by radical-mediated stannylation of sulfide (E)-11 and subsequent electrophilic fluorination of (E)-12. The desired (E)-6 was obtained after the introduction of the guanine base into (E)-18 under Mitsunobu conditions and following global deprotection. The stereoisomer (Z)-6 was also prepared by the same procedure using (Z)-12. Compound (E)-6 showed highly potent anti-HBV activity (EC50 = 1.2 nM) with favorable cytotoxicity (CC50 = 93 µM).

A novel anti-HBV agent, E-CFCP, restores Hepatitis B virus (HBV)-induced senescence-associated cellular marker perturbation in human hepatocytes.

Cellular senescence is a cellular state with a broad spectrum of age-related physiological conditions that can be affected by various infectious diseases and treatments. Therapy of hepatitis B virus (HBV) infection with nucleos(t)ide analogs [NA(s)] is well established and benefits many HBV-infected patients, but requires long-term, perhaps lifelong, medication. In addition to the effects of HBV infection, the effects of NA administration on hepatocellular senescence are still unclear. This study investigated how HBV infection and NA treatment influence cellular senescence in human hepatocytes and humanized-liver chimeric mice chronically infected with live HBV. HBV infection upregulates or downregulates multiple cellular markers including senescence-associated ß-galactosidase (SA-ß-Gal) activity and cell cycle regulatory proteins (e.g., p21CIP1) expression level in hepatocellular nuclei and humanized-mice liver. A novel highly potent anti-HBV NA, E-CFCP, per se did not have significant disturbance on markers evaluated. Besides, E-CFCP treatment restored HBV-infected cells to their physiological phenotypes that are comparable to the HBV-uninfected cells. The results reported here demonstrate that, regardless of the mechanism(s), chronic HBV infection perturbates multiple senescence-associated markers in human hepatocytes and humanized-mice liver, but E-CFCP can restore this phenomenon.

4-Thiofuranoid Glycal: Versatile Glycosyl Donor for the Selective Synthesis of ß-anomer of 4'-thionucleoside and its Biological Activities.

The first highly diastereoselective synthesis of ß-anomers of 4'-thionucleosides has been carried out by means of electrophilic glycosidation utilizing 3,5-O-(di-tertbutylsilylene) (DTBS)-4-thiofuranoid glycal as a glycosyl donor. The resulting glycosides were transformed into ribo-, 2'-deoxy-, and arabinofuranosyl nucleosides through a chemical transformation of the 2'-substituent. The additive Pummerer reaction of the glycal Soxide gave 1,2-di-O-acetyl-3,5-O-DTBS-4-thioribofuranose. The utility of the DTBSprotected 4-thioribofuranose has been demonstrated by the preparation of 4'-thio analogues of pyrimidine- and purine-4'-thioribonucleosides based on the Vorbrüggen glycosidation. Synthesis of 4'-thio-counterpart of C-nucleoside antibiotic tiazofurin has also been carried out. α-Face selective hydroboration of 1-C-aryl- or 1-C-heteroaryl-glycals obtained by cross-coupling of 1-tributylstannylglycal has furnished the respective ß- anomer of 4'-thio-C-ribonucleosides, including 4'-thio analogue of nucleoside antibiotic pseudouridine and 9-deazaadenosine. On the basis of lithiation chemistry, 1-C- and 2-Ccarbon- carbon-substituted 3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3- diyl) (TIPDS)- 4- thiofuranoid glycal were synthesized. These glycals enabled us to prepare 1'-C- and 2'-ß- C-carbon-substituted 2'-deoxy-4'-thionucleosides, including thio-counterpart of antitumor nucleoside antibiotic angustmycin C. Furthermore, 1'-C-methyl-4'-thiothymidine emerged as a potent inhibitor of angiogenesis. In addition, 1'-C-methyl-4'-thiothymidine exhibited more potent inhibitory activity against thymidine kinase-deficient mutant of herpes virus than that of ganciclovir. Among the 4'-substituted 4'-thiothymidines, the 4'- C-cyano- and 4'-C-ethynyl derivatives inhibited replication of HIV variant resistant to 3TC (HIVM184V) as potently as HIV-1IIIB. In terms of the value of selectivity index (SI), 4'-C-cyano-4'-thiothymidine showed a 3-fold selective index (SI) than that of the corresponding thymidine derivative. Furthermore, 4'-C-ethynyl-2'-deoxy-4'-thioguanosine has a 20-fold better value (>18,200) than that of 2'-deoxyguanosine counterpart (933). Furthermore, 4'-azido-4'-thiothymidine emerged as a selective and potent anti-EBV agent. In terms of antineoplastic activity, 4'-azido- and 4'-C-fluoromethyl-2'-deoxy-4'-thiocytidine inhibited proliferation of human B-cell (CCRF-SB) and T-cell leukemia (Molt-4) cell lines, although the parent compound 2'-deoxy-4'-thiocytidine did not exhibit any cytotoxicity up to 100 µM. These facts concerning the biological activities suggested that replacement of the furanose oxygen with a sulfur atom is a promising approach for the development of less toxic antiviral and antineoplastic nucleoside antimetabolites. 4'- Thionucleoside also acts as a monomer for oligonucleotides (ONs) therapeutics, exhibiting superior biological properties. Therefore, this review provides a wide range of potential monomers for antisense ON and siRNA.

Identification of a novel long-acting 4'-modified nucleoside reverse transcriptase inhibitor against HBV.

BACKGROUND & AIMS: While certain nucleos(t)ide reverse transcriptase inhibitors (NRTIs) are efficacious in treating HBV infection, their effects are yet to be optimized and the emergence of NRTI-resistant HBV variants is an issue because of the requirement for lifelong treatment. The development of agents that more profoundly suppress wild-type and drug-resistant HBVs, and that have a long-acting effect, are crucial to improve patient outcomes. METHODS: Herein, we synthesized a novel long-acting 4'-modified NRTI termed E-CFCP. We tested its anti-HBV activity in vitro, before evaluating its anti-HBV activity in HBV-infected human-liver-chimeric mice (PXB-mice). E-CFCP's long-acting features and E-CFCP-triphosphate's interactions with the HBV reverse transcriptase (HBV-RT) were examined. RESULTS: E-CFCP potently blocked HBVWTD1 production (IC50qPCR_cell=1.8 nM) in HepG2.2.15 cells and HBVWTC2 (IC50SB_cell=0.7 nM), entecavir (ETV)-resistant HBVETV-RL180M/S202G/M204V (IC50SB_cell=77.5 nM), and adefovir-resistant HBVADV-RA181T/N236T production (IC50SB_cell=14.1 nM) in Huh7 cells. E-CFCP profoundly inhibited intracellular HBV DNA production to below the detection limit, but ETV and tenofovir alafenamide (TAF) failed to do so. E-CFCP also showed less toxicity than ETV and TAF. E-CFCP better penetrated hepatocytes and was better tri-phosphorylated; E-CFCP-triphosphate persisted intracellularly for longer than ETV-triphosphate. Once-daily peroral E-CFCP administration over 2 weeks (0.02~0.2 mg/kg/day) reduced HBVWTC2-viremia by 2-3 logs in PXB-mice without significant toxicities and the reduction persisted over 1-3 weeks following treatment cessation, suggesting once-weekly dosing capabilities. E-CFCP also reduced HBVETV-RL180M/S202G/M204V-viremia by 2 logs over 2 weeks, while ETV completely failed to reduce HBVETV-RL180M/S202G/M204V-viremia. E-CFCP's 4'-cyano and fluorine interact with both HBVWT-RT and HBVETV-RL180M/S202G-M204 -RT via Van der Waals and polar forces, being important for E-CFCP-triphosphate's interactions and anti-HBV potency. CONCLUSION: E-CFCP represents the first reported potential long-acting NRTI with potent activity against wild-type and treatment-resistant HBV. LAY SUMMARY: Although there are currently effective treatment options for HBV, treatment-resistant variants and the need for lifelong therapy pose a significant challenge. Therefore, the development of new treatment options is crucial to improve outcomes and quality of life. Herein, we report preclinical evidence showing that the anti-HBV agent, E-CFCP, has potent activity against wild-type and treatment-resistant variants. In addition, once-weekly oral dosing may be possible, which is preferrable to the current daily dosing regimens.

An alternative method for the synthesis of 2'-halogeno-1',2'-unsaturated uridine derivatives through syn-elimination of pivalic acid of 2'-halogeno- 2'-deoxy-1'-pivaloyloxyuracil nucleoside: preparation of its 2'-C-branched nucleosides.

An alternative method for the preparation of 2'-bromo- (5b) and 2'-iodo- (5c) 1',2'-unsaturated uracil nucleosides has been developed. The protocol was on the basis of the syn-elimination of pivalic acid from 2'-bromo-(7a,b) and 2'-iodo-(9a,b) 1'-pivaloyloxy-2'-deoxyuridine derivatives, which were derived from the halo-pivaloyloxylation of 3',5'-bis-O-TBDMS-1',2'-unsaturated uridine 1. Compounds 5b and 5c were shown to serve as versatile synthons for the respective 2'-C-branched 1',2'-unsaturated uracil nucleosides, through palladium-catalyzed cross-coupling or halogen-lithium exchange reactions.

Synthesis and evaluation of the anti-hepatitis B virus activity of 4'-Azido-thymidine analogs and 4'-Azido-2'-deoxy-5-methylcytidine analogs: structural insights for the development of a novel anti-HBV agent.

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 SNAr 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 (EC50 = 0.63 and 5.99 µM, respectively) with no detectable cytotoxicity against MT-2 cells up to 100 µM.

Synthesis, Anti-HBV, and Anti-HIV Activities of 3'-Halogenated Bis(hydroxymethyl)-cyclopentenyladenines.

Synthesis of 3'-halogeno analogues (5a-d) of 9-[c-4,t-5-bis(hydroxymethyl)-cyclopent-2-en-r-1-yl]-9H-adenine (BCA, 3) was accomplished by means of dual utilization of the vinyl sulfone functional moieties in both 10 and 16 utilizing a SN2' conjugate-addition reaction and a sulfur-extrusive stannylation, respectively. Evaluation of the antiviral activities of 5a-d revealed that introduction of a halogeno-substituent into the 3'-position of (-)-BCA diminished its anti-HIV-1 activity but increased the inhibitory activity for the reverse transcriptase of HBV in that the 3'-fluorinated BCA 5d exhibited the highest activity without significant cytotoxicity.

A novel entecavir analogue constructing with a spiro[2.4]heptane core structure in the aglycon moiety: Its synthesis and evaluation for anti-hepatitis B virus activity.

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 (EC50 value of 0.12 ± 0.02 µM) and no cytotoxicity against HepG2 cells was observed up to 100 µM.

Pyrolysis of UR-144, a synthetic cannabinoid, augments an affinity to human CB1 receptor and cannabimimetic effects in mice.

Drug abusers most often smoke 'herbal incense' as a cigarette or inhale it using a smoking tool. Smoking may cause pyrolysis of the drug and produce decomposed products of which biological effect has never been investigated. The synthetic cannabinoid UR-144 is known to undergo thermal degradation, giving a ring-opened isomer, so-called UR-144 degradant. The present study demonstrates by using UR-144 as a model drug that the smoke of burned UR-144 contains the UR-144 degradant. The UR-144 degradant showed approximately four fold higher agonist activity to human CB1 receptor and augmented hypothermic and akinetic actions in mice compared to UR-144. These results indicate that smoking behavior may increase psychological actions of the certain synthetic cannabinoids.

Diastereoselective Synthesis of 6″-(Z)- and 6″-(E)-Fluoro Analogues of Anti-hepatitis B Virus Agent Entecavir and Its Evaluation of the Activity and Toxicity Profile of the Diastereomers.

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.

Design, Synthesis, and Evaluation of Anti-HBV Activity of Hybrid Molecules of Entecavir and Adefovir: Exomethylene Acycloguanine Nucleosides and Their Monophosphate Derivatives.

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.

From the chemistry of epoxy-sugar nucleosides to the discovery of anti-HIV agent 4'-ethynylstavudine-Festinavir.

Branched sugar nucleosides have attracted much attention due to their biological activities. We have demonstrated that epoxysugar nucleosides serve as versatile precursor for the stereo-defined synthesis of these nucleoside derivatives on the basis of its ring opening with organoaluminum or organosilicon reagents. In this review article, novel methods for the synthesis of nucleoside analogues branched at the 1' and 4'-position will be described. During this study, we could discover an anti-HIV agent, 4'-ethynylstavudine (Festinavir). Festinavir showed more potent anti-HIV activity than the parent compound stavudine (d4T). Other significant properties of Festinavir are as follows: 1) much less toxic to various cells and also to mitochondorial DNA synthesis than d4T, 2) better substrate for human thymidine kinase than d4T, 3) resistant not only to chemical glycosidic bond cleavage but also to catabolism by thymidine phosphorylase, 4) the activity improves in the presence of a major mutation, K103N, associated with resistance to non-nucleoside reverse transcriptase inhibitors. Detailed profile of the antiviral activities, biology and pharmacology of Festinavir are also described.

Tuning efficiency of the 4-exo-trig cyclization by the electronic effect: ring closure of 3,3-difluoro-4-pentenyl carbon radicals and synthesis of a gem-difluorocyclobutane nucleoside.

4-exo-trig Cyclization reaction of a 4-pentenyl carbon radical containing the gem-difluoromethylene moiety adjacent to a radical accepting α,ß-unsaturated ester was found to proceed efficiently to furnish a novel gem-difluorocyclobutane derivative. The cyclized product could be transformed into a gem-difluoromethylene analogue of oxetanocin T.

Synthesis of 4'-substituted cordycepins via benzenesulfenylation at the 4'-position as a key step.

With an aim to synthesize 4'-substituted cordycepins, the 4'-phenylthio precursor 4 was prepared from adenosine through an electrophilic addition to the 3',4'-unsaturated derivative 2 by using NIS/PhSH system. Nucleophilic substitution of 4 with a series of alcohols in the presence of NBS gave the respective 4'-alpha-alkoxy cordycepins 6 as the major stereoisomer. Use of DAST, in stead of alcohol in this reaction, gave the 4'-fluoro analogue 7. The 4'-sulfone derivative 8 obtained by m-CPBA oxidation of 4 was employed for the reaction with organoaluminum reagents. These reactions furnished various types of the 4'-carbon-substituted cordycepins 9.

Synthesis of 5'-fluoro-2'-beta-methylneplanocin analogues.

Synthesis of 5'-fluoro-2'-beta-methylneplanocin analogues (5) was carried out. The cyclopentenone 16 was prepared from methyl mannopyranoside by using ring closing metathesis, stereoselective introduction of methyl group, and seleno cyclization as representative steps. Introduction of a fluorine atom was conducted by electrophilic fluorination. Antiviral activity of 5 will also be presented.

Synthesis and antiviral evaluation of (+/-)-4'-ethynyl-5'-difluorocarbocyclic-d4T analogue.

Synthesis of (+/-)-4'-ethynyl-5'-difluorocarbocyclic-d4T analogue 8, in which the furanose ring oxygen of usual nucleosides is replaced with a geminal-difluoromethylidene group, was carried out. Electrophilic fluorination with Selectfluor was applied to construct a gem-difluorocyclopentenone system to give 12. Regioselective introduction of thymine base was performed under the Mitsunobu conditions by employing the 4-methoxycarbonyl derivative 13. Antiviral evaluation of 8 was also examined.

Looking for new pyrimidine acyclic nucleotide analogues designed for phosphorylation by human UMP-CMP kinase.

Human UMP-CMP kinase is involved in the phosphorylation of nucleic acid precursors and also in the activation of antiviral analogues including cidofovir, an acyclic phosphonate compound that mimicks dCMP and shows a broad antiviral spectrum. The binding of ligands to the enzyme was here investigated using a fluorescent probe and a competitive titration assay. At the acceptor site, the enzyme was found to accommodate any base, purine and pyrimidine, including thymidine. A method for screening analogues based on their affinity for the UMP binding site was developed. The affinities of uracil vinylphosphonate derivatives modified in the 5 position were found similar to (d)UMP and (d)CMP and improved when compared to cidofovir.

Cross-metathesis mediated synthesis of new acyclic nucleoside phosphonates.

With the commercial availability of well-defined ruthenium metathesis catalysts which combine high stability and broad functional group compatibility, olefin metathesis is now routinely integrated in various syntheses. We will report here the overwhelming power and scope of cross-metathesis in the area of new acyclic nucleoside phosphonates. Scope and limitations of this approach, and especially the E/Z stereocontrol, are discussed on selected examples from our drug discovery group.

Synthesis of novel 4'-modified neplanocin A analogues and their inhibitory activity against S-adenosyl-L-l-homocysteine hydrolase.

A new approach was developed for the synthesis of 4'-modified neplanocin A analogues, as potential inhibitors against S-adenosyl-L-homocysteine hydrolase. The vinylstannane 13, a key intermediate in the present approach, was prepared by radical-mediated sulfur-extrusive stannylation.