Efficient Inhibition of Hepatitis B Virus (HBV) Replication and cccDNA Formation by HBV Ribonuclease H Inhibitors during Infection.

The hepatitis B virus (HBV) ribonuclease H (RNase H) is an attractive but unexploited drug target. Here, we addressed three limitations to the current state of RNase H inhibitor development: (a) Efficacy has been assessed only in transfected cell lines. (b) Cytotoxicity data are from transformed cell lines rather than primary cells. (c) It is unknown how the compounds work against nucleos(t)ide analog resistant HBV strains. Three RNase H inhibitors from different chemotypes, 110 (α-hydroxytropolone), 1133 (N-hydroxypyridinedione), and 1073 (N-hydroxynapthyridinone), were tested in HBV-infected HepG2-NTCP cells for inhibition of cccDNA accumulation and HBV product formation. 50% effective concentrations (EC50s) were 0.049-0.078 µM in the infection studies compared to 0.29-1.6 µM in transfected cells. All compounds suppressed cccDNA formation by >98% at 5 µM when added shortly after infection. HBV RNA, intracellular and extracellular DNA, and HBsAg secretion were all robustly suppressed. The greater efficacy of the inhibitors when added shortly after infection is presumably due to blocking amplification of the HBV cccDNA, which suppresses events downstream of cccDNA formation. The compounds had 50% cytotoxic concentrations (CC50s) of 16-100 µM in HepG2-derived cell lines but were nontoxic in primary human hepatocytes, possibly due to the quiescent state of the hepatocytes. The compounds had similar EC50s against replication of wild-type, lamivudine-resistant, and adefovir/lamivudine-resistant HBV, as expected because the RNase H inhibitors do not target the viral reverse transcriptase active site. These studies expand confidence in inhibiting the HBV RNase H as a drug strategy and support inclusion of RNase H inhibitors in novel curative drug combinations for HBV.

Synthesis of Imide and Amine Derivatives via Deoxyamination of Alcohols Using N-Haloimides and Triphenylphosphine.

A deoxyamination methodology of activated and unactivated alcohols is presented. The reaction is mediated by phosphonium intermediates generated in situ from N-haloimides and triphenylphosphine. The protocol allows for the synthesis of phthalimide and amine derivatives in moderate to good yields at room temperature. A series of NMR experiments have provided insight into the reactive intermediates involved and the mechanism of this deoxyamination reaction.

Superelectrophilic carbocations: preparation and reactions of a substrate with six ionizable groups.

A substrate has been prepared having two triarylmethanol centers and four pyridine-type substituent groups. Upon ionization in the Brønsted superacid CF3SO3H, the substrate undergoes two types of reactions. In the presence of only the superacid, the highly ionized intermediate(s) provide a double cyclization product having two pyrido[1,2-a]indole rings. With added benzene, an arylation product is obtained. A mechanism is proposed involving tetra-, penta-, or hexacationic species.

Regioselective α-Amination of Ethers Using Stable N-Chloroimides and Lithium tert-Butoxide.

Herein we describe a metal-free regioselective α-amination of ethers mediated by N-chloroimides in ethereal solvents in the presence of lithium tert-butoxide. This reactivity of N-chloroimides leads to the synthesis of hemiaminal ethers in good to excellent yields at room temperature. This C-H functionalization is achieved without the use of a light, heat source, or external radical initiators. Initial mechanistic work indicates that the reaction proceeds through a radical pathway.

Synthesis of Heterocycle-Containing 9,9-Diarylfluorenes Using Superelectrophiles.

A superacid-promoted method for the synthesis of 9,9-diarylfluorenes is described. The chemistry involves cyclizations and arylations with biphenyl-substituted heterocyclic ketones and a mechanism is proposed involving superelectrophilic intermediates. The key reactive intermediates-dicationic and trication fluorenyl cations have been observed by low-temperature NMR and the mechanism has been further studied using DFT calculations.

Use of Charge-Charge Repulsion to Enhance π-Electron Delocalization into Anti-Aromatic and Aromatic Systems.

A series of 9-fluorenyl cations has been studied and it is shown that increasing charge on a heterocyclic substituent group enhances the anti-aromatic character of the carbocation system. Similarly, a series of dibenzosuberenyl cations has been studied and increasing charge on a substituent group is shown to enhance aromatic character in the carbocation system. These studies include the direct observations of dicationic and tricationic species using stable-ion conditions and low temperature NMR. The structures of these ions were further characterized using DFT calculations, confirming that highly charged organic ions may exhibit unusual distributions of π-electrons and delocalization of electrons in 4n or 4n+2 π-systems.

Tetra- and Pentacationic Electrophiles and Their Chemistry.

A tetracationic electrophile has been generated in superacid and shown to undergo an arylation reaction with benzene. A cyclization product is also obtained in the absence of benzene, presumably from a tricationic intermediate. Using low-temperature NMR, the tetracationic species is directly observed from a FSO3H-SbF5-SO2ClF solution. Similar chemistry is described with a system involving penta- and tetracationic intermediates. These highly ionized structures and their chemistry were also examined by DFT calculation.

Intramolecular Conjugate Additions with Heterocyclic Olefins.

The intramolecular reactions of olefinic N-heterocycles have been studied. In triflic acid-promoted reactions, conjugate addition is observed with pyrazine-, 2-pyrimidine-, and 2-quinoxaline-based olefins and a phenyl group nucleophile. Markovnikov addition is observed with pyridine and 5-quinoxaline-based olefins. These results are in accordance with previous observations relating the type of addition-conjugate or Markovnikov-to the positions of olefinic substituents of the N-heterocycle.