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).

Long-chain acyl-CoA synthetase 4 participates in the formation of highly unsaturated fatty acid-containing phospholipids in murine macrophages.

Long-chain acyl-coenzyme A synthetases (ACSLs) are a family of enzymes that convert free long-chain fatty acids into their acyl-coenzyme A (CoA) forms. ACSL4, belonging to the ACSL family, shows a preferential use of arachidonic acid (AA) as its substrate and plays a role in the remodeling of AA-containing phospholipids by incorporating free AA. However, little is known about the roles of ACSL4 in inflammatory responses. Here, we assessed the roles of ACSL4 on the effector functions of bone marrow-derived macrophages (BMDMs) obtained from mice lacking ACSL4. Liquid chromatography-tandem mass spectrometry analysis revealed that various highly unsaturated fatty acid (HUFA)-derived fatty acyl-CoA species were markedly decreased in the BMDMs obtained from ACSL4-deficient mice compared with those in the BMDMs obtained from wild-type mice. BMDMs from ACSL4-deficient mice also showed a reduced incorporation of HUFA into phosphatidylcholines. The stimulation of BMDMs with lipopolysaccharide (LPS) elicited the release of prostaglandins (PGs), such as PGE2, PGD2 and PGF2α, and the production of these mediators was significantly enhanced by ACSL4 deficiency. In contrast, neither the LPS-induced release of cytokines, such as IL-6 and IL-10, nor the endocytosis of zymosan or dextran was affected by ACSL4 deficiency. These results suggest that ACSL4 has a crucial role in the maintenance of HUFA composition of certain phospholipid species and in the incorporation of free AA into the phospholipids in LPS-stimulated macrophages. ACSL4 dysfunction may facilitate inflammatory responses by an enhanced eicosanoid storm.

Analysis on the Substrate Specificity of Recombinant Human Acyl-CoA Synthetase ACSL4 Variants.

Acyl-CoA synthetase long-chain family members (ACSLs) are a family of enzymes that convert long-chain free fatty acids into their acyl-CoAs. ACSL4 is an ACSL isozyme with a strong preference for arachidonic acid (AA) and has been hypothesized to modulate the metabolic fates of AA. There are two ACSL4 splice variants: ACSL4V1, which is the more abundant transcript, and ACSL4V2, which is believed to be restricted to the brain. In the present study, we expressed recombinant human ACSL4V1 and V2 in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus expression system and then partially purified both variants by cobalt affinity column chromatography. We then established a novel ACSL assay system with LC-MS/MS, which is highly sensitive and applicable to various kinds of fatty acids, and used it to investigate the substrate specificity of recombinant human ACSL4V1 and V2. The results showed that both ACSL4 variants preferred various kinds of highly unsaturated fatty acids (HUFAs), including docosahexaenoic acid (DHA), adrenic acid (docosatetraenoic acid) and eicosapentaenoic acid (EPA), as well as AA as a substrate. Moreover, our kinetic studies revealed that the two variants had similar relative affinities for AA, EPA and DHA but different reaction rates for each HUFA. These results confirmed the importance of both of ACSL4 variants in the maintenance of membrane phospholipids bearing HUFAs. Structural analysis of these variants might reveal the molecular mechanism by which they maintain membrane phospholipids bearing HUFAs.

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.