Noriyuki Ashida

Mechanism of inhibition of HIV-1 reverse transcriptase by 4′-ethynyl-2-fluoro-2′-deoxyadenosine triphosphate, a translocation defective reverse transcriptase inhibitor

Nucleoside reverse transcriptase inhibitors (NRTIs) are employed in first line therapies for the treatment of human immunodeficiency virus (HIV) infection. They generally lack a 3′-hydroxyl group, and thus when incorporated into the nascent DNA they prevent further elongation. In this report we show that 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA), a nucleoside analog that retains a 3′-hydroxyl moiety, inhibited HIV-1 replication in activated peripheral blood mononuclear cells with an EC50 of 0.05 nm, a potency several orders of magnitude better than any of the current clinically used NRTIs. This exceptional antiviral activity stems in part from a mechanism of action that is different from approved NRTIs. Reverse transcriptase (RT) can use EFdA-5′-triphosphate (EFdA-TP) as a substrate more efficiently than the natural substrate, dATP. Importantly, despite the presence of a 3′-hydroxyl, the incorporated EFdA monophosphate (EFdA-MP) acted mainly as a de facto terminator of further RT-catalyzed DNA synthesis because of the difficulty of RT translocation on the nucleic acid primer possessing 3′-terminal EFdA-MP. EFdA-TP is thus a translocation-defective RT inhibitor (TDRTI). This diminished translocation kept the primer 3′-terminal EFdA-MP ideally located to undergo phosphorolytic excision. However, net phosphorolysis was not substantially increased, because of the apparently facile reincorporation of the newly excised EFdA-TP. Our molecular modeling studies suggest that the 4′-ethynyl fits into a hydrophobic pocket defined by RT residues Ala-114, Tyr-115, Phe-160, and Met-184 and the aliphatic chain of Asp-185. These interactions, which contribute to both enhanced RT utilization of EFdA-TP and difficulty in the translocation of 3′-terminal EFdA-MP primers, underlie the mechanism of action of this potent antiviral nucleoside.

Potential of 4'-C-substituted nucleosides for the treatment of HIV-1.

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.

Potent Activity of a Nucleoside Reverse Transcriptase Inhibitor, 4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine, against Human Immunodeficiency Virus Type 1 Infection in a Model Using Human Peripheral Blood Mononuclear Cell-Transplanted NOD/SCID Janus Kinase 3 Knockout Mice

ABSTRACT 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 μM). 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 × 102 copies/ml [range, 8.1 × 102 to 1.1 × 103 copies/ml] versus median of 9.9 × 104 copies/ml [range, 8.1 × 102 to 1.1 × 103 copies/ml]; P < 0.001), the p24 level in plasma (2.5 × 103 pg/ml [range, 8.2 × 102 to 5.6 × 103 pg/ml] versus 2.8 × 102 pg/ml [range, 8.2 × 101 to 6.3 × 102 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.

Design, efficient synthesis, and anti-HIV activity of 4'-C-cyano- and 4'-C-ethynyl-2'-deoxy purine nucleosides.

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.

Synthesis and Biological Evaluation of 1′-C-Cyano-Pyrimidine Nucleosides †

Abstract 2′-Deoxy-, 2′-bromo-, and arabino-1′-C-cyano-pyrimidine nucleosides were synthesized from O2 ,2′-cyclouridine. Incorporation of cyano group at the anomeric position was achieved by treatment of 1′,2′-unsaturated uridine with NBS in the presence of pivalic acid followed by TMS-cyanide and stannic chloride. Antineoplastic and antiviral activities of those compounds are also discussed. †This paper is dedicated to Dr. Yoshihisa Mizuno on the occasion of his 75th birthday.

In vitro anti-herpesvirus activities of 5-substituted 2'-deoxy-2'-methylidene pyrimidine nucleosides

New pyrimidine deoxyribonucleoside analogues, 2′-deoxy-2′-methylideneuridine (DMDU), 2′-deoxy-2′-methylidenecytidine (DMDC), and their 5-substituted derivatives were tested for the anti-herpesvirus activities and anti-proliferative activity. E-5-(2-Bromovinyl)uracil derivative (BV-DMDU) and its cytosine congener were synthesized from 1-β-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU). 5-Bromo, 5-iodo, 5-methyl, and 5-ethyl derivatives of DMDU and BV-DMDU showed activities against herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV). The corresponding DMDC derivatives had no or only weak antiviral activity. Among the 2′-deoxy-2′-methylidene pyrimidine nucleosides, BV-DMDU showed the most potent and selective anti-VZV activity. BV-DMDU was more potent than acyclovir, but less active than BV-araU. BV-DMDU was inactive against human diploid and tumour cells. DMDC and F-DMDC (5-fluoro derivative) were potent inhibitors of HSV-1, herpes simplex virus type 2, VZV, and human cytomegalovirus (HCMV) and also had significant anti-proliferative activity. Their potency against HCMV was better than that of ganciclovir and araC. Some DMDU derivatives also showed anti-HCMV activity, but they had anti-proliferative activity. The anti-HCMV activity of these DMDC and DMDU compounds was generally more potent than those against HSV-1 and VZV thereof, suggesting the participation of cellular kinase in their antiviral action.

Facile Synthesis and Evaluation of Antitumor and Antiviral Activities of [1,2,5]Thiadiazolo[3,4-d]pyrimidines (8-Thiapurines) and 4-b-D-Ribofuranosyl-[1,2,5]thiadiazolo[3,4-d]pyrimidines

Synthesis of 5-amino-[1,2,5]thiadiazolo[3,4-d]pyrimidin-7(6H)-ones (8-thiaguanine) (4a-m), 7-amino- l,2,5|thiadiazolo|3,4-c?]pyrimidin-5(4//)-ones (8-thiaisoguanine) (6a-k), 5,7-diamino-[1,2,5]thiadiazolo]3,4-d]pyrimidines (8a-d) and some other thiadiazolopyrimidine derivatives were prepared by treating 6-amino-5-nitrosopyrimidine derivatives with sodium thiosulfate in aqueous acid media. 4-β-D-Ribofuranosyl-[1,2,5]thiadiazolo[3,4-d]pyrimidine-5,7(4H,6H)-dione (8-thiaxanthosine) (18) was also prepared by the reliable method. Moderate antitumor and antiviral activities of the synthesized compounds have been evaluated in vitro.

Antitumor studies. Part 5: Synthesis, antitumor activity, and molecular docking study of 5-(monosubstituted amino)-2-deoxo-2-phenyl-5-deazaflavins

Various novel 5-(monosubstituted amino)-2-deoxo-2-phenyl-5-deazaflavins derivatives have been synthesized by direct coupling of 5-deazaflavins and N-alkyl or aryl amines. The antitumor activities against human tumor cell lines CCRF-HSB-2 and KB cells have been investigated in vitro and many compounds showed promising potential antitumor activities with less cytotoxicities. AutoDock molecular docking into PTK (PDB code: 1t46) has been done for lead optimization of these compounds as potential PTK inhibitors. Some of the synthesized 5-(monosubstituted amino)-2-deoxo-2-phenyl-5-deazaflavins at the 5-position exhibited reasonable binding affinities into PTK with the hydrogen bond through their C(5)-NH moiety.

Synthesis and Biological Activities of 2′-Modified 4′-Thionucleosides

Abstract We have synthesized 4′-thioDMDC, 4′-thiogemcitabine, and 4′-thioarabinonucleosides, as potential antitumor and antiviral agents, originated from D-glucose. Biological activities of these compounds are also described.

Synthesis of (2′S)-1-(2-C-Azidomethyl-2-deoxy and 2-C-Cyanomethyl-2-deoxy-β-D-arabinofuranosyl)cytosines

Abstract 2′-C-Cyanomethyl-2′-deoxy-arabinosylcytosine 3 and 2′-C-azidomethyl-2′-deoxy-arabinosylcytosine 4 were synthesized from uridine. The antineoplastic activities of these compounds were evaluated.