Structure-based Design of Novel Hepatitis B Virus Capsid Assembly Modulators.

Small-molecule capsid assembly modulators (CAMs) have been recently recognized as promising antiviral agents for curing chronic hepatitis B virus (HBV) infection. A target-based in silico screening study is described, aimed towards the discovery of novel HBV CAMs. Initial optimization of four weakly active screening hits was performed via focused library synthesis. Lead compound 42 and close analogues 56 and 57 exhibited in vitro potency in the sub- and micromolar range along with good physico-chemical properties and were further evaluated in molecular docking and mechanism of action studies.

An automated microfluidic platform for the screening and characterization of novel hepatitis B virus capsid assembly modulators.

To date, hepatitis B virus (HBV) capsid assembly modulators (CAMs), which target the viral core protein and induce the formation of non-functional viral capsids, have been identified and characterized in microtiter plate-based biochemical or cell-based in vitro assays. In this work, we developed an automated microfluidic screening assay, which uses convection-dominated Taylor-Aris dispersion to generate high-resolution dose-response curves, enabling the measurements of compound EC50 values at very short incubation times. The measurement of early kinetics down to 7.7 seconds in the microfluidic format was utilized to discriminate between the two different classes of CAMs known so far. The CAM (-N), leading to the formation of morphologically normal capsids and the CAM (-A), leading to aberrant HBV capsid structures. CAM-A compounds like BAY 41-4109 and GLS4 showed rapid kinetics, with assembly rates above 80% of the core protein after only a 7 second exposure to the compound, whereas CAM-N compounds like ABI-H0731 and JNJ-56136379 showed significantly slower kinetics. Using our microfluidic system, we characterized two of our in-house screening compounds. Interestingly, one compound showed a CAM-N/A intermediate behavior, which was verified with two standard methods for CAM classification, size exclusion chromatography, and anti-HBc immunofluorescence microscopy. With this proof-of-concept study, we believe that this microfluidic system is a robust primary screening tool for HBV CAM drug discovery, especially for the hit finding and hit-to-lead optimization phases. In addition to EC50 values, this system gives valuable first information about the mode of action of novel CAM screening compounds.

Synthesis of a novel polycyclic ring scaffold with antimitotic properties via a selective domino Heck-Suzuki reaction.

The synthesis of a previously undescribed sp3-rich 6-5-5-6 tetracyclic ring scaffold using a palladium catalysed domino Heck-Suzuki reaction is reported. This reaction is high-yielding, selective for the domino process over the direct Suzuki reaction and tolerant towards a variety of boronic acids. The novel scaffold can also be accessed via domino Heck-Stille and radical cyclisations. Compounds based around this scaffold were found to be effective antimitotic agents in a human cancer cell line. Detailed phenotypic profiling showed that the compounds affected the congression of chromosomes to give mitotic arrest and apoptotic cell death. Thus, a novel structural class of antimitotic agents that does not disrupt the tubulin network has been identified.

Diversity-oriented synthesis as a tool for identifying new modulators of mitosis.

The synthesis of diverse three-dimensional libraries has become of paramount importance for obtaining better leads for drug discovery. Such libraries are predicted to fare better than traditional compound collections in phenotypic screens and against difficult targets. Herein we report the diversity-oriented synthesis of a compound library using rhodium carbenoid chemistry to access structurally diverse three-dimensional molecules and show that they access biologically relevant areas of chemical space using cheminformatic analysis. High-content screening of this library for antimitotic activity followed by chemical modification identified 'Dosabulin', which causes mitotic arrest and cancer cell death by apoptosis. Its mechanism of action is determined to be microtubule depolymerization, and the compound is shown to not significantly affect vinblastine binding to tubulin; however, experiments suggest binding to a site vicinal or allosteric to Colchicine. This work validates the combination of diversity-oriented synthesis and phenotypic screening as a strategy for the discovery of biologically relevant chemical entities.

Polystyrene-supported diarylprolinol ethers as highly efficient organocatalysts for Michael-type reactions.

α,α-Diphenylprolinol methyl- and trimethylsilyl ethers anchored onto a polystyrene resin have been prepared by a copper-catalyzed azide-alkyne cycloadditions (CuAAC). The catalytic activity and enantioselectivity displayed by the O-trimethylsilyl derivative are comparable to those exhibited by the best known homogeneous catalysts for the addition of aldehydes to nitroolefins and of malonates or nitromethane to α,ß-unsaturated aldehydes. The combination of the catalytic unit, the triazole linker, and the polymeric matrix provides unprecedented substrate selectivity, in favor of linear, short-chain aldehydes, when the organocatalyzed reaction proceeds by an enamine mechanism. High versatility is noted in reactions that proceed via an iminium ion intermediate. The catalytic behavior of polystyrene-supported α,α-diphenylprolinol methyl ether was also evaluated in asymmetric Michael addition reactions. As a general trend, the CuAAC immobilization of diarylprolinol ethers onto insoluble polystyrene resins offers important operational advantages, such as high catalytic activity, easy recovery from the reaction mixture by simple filtration, and the possibility of extended reuse.

Continuous-flow enantioselective α-aminoxylation of aldehydes catalyzed by a polystyrene-immobilized hydroxyproline.

The application of polystyrene-immobilized proline-based catalysts in packed-bed reactors for the continuous-flow, direct, enantioselective α-aminoxylation of aldehydes is described. The system allows the easy preparation of a series of ß-aminoxy alcohols (after a reductive workup) with excellent optical purity and with an effective catalyst loading of ca. 2.5% (four-fold reduction compared to the batch process) working at residence times of ca. 5 min.

A solid-supported organocatalyst for highly stereoselective, batch, and continuous-flow Mannich reactions.

The fast and highly stereoselective Mannich reaction of aldehydes and ketones with the N-(p-methoxyphenyl) ethyl glyoxylate imine catalyzed by polystyrene resins functionalized with (2S,4R)-hydroxyproline is reported. The effect of the nature of the linker connecting proline with the polymeric backbone has been studied, and a 1,2,3-triazole linker constructed from azidomethyl polystyrene and O-propargyl hydroxyproline turns out to be optimal for catalytic activity and enantioselectivity. With aldehyde donors, fast reactions leading to complete conversion in 1-3 h are recorded in DMF. With ketone donors, the reactions tend to be slower, but can be efficiently accelerated (six-membered ring cycloalkanones) by low-power microwave irradiation. This approach, which greatly facilitates product isolation since the catalyst is removed by simple filtration, has allowed the implementation of the reactions of aldehyde substrates in a continuous-flow, single-pass system. In this manner, the continuous synthesis of the enantiomerically and diastereomerically pure adducts (syn/anti>97:3; ee>99 %) has been achieved at room temperature with residence times of 6.0 min. This methodology has allowed for the preparation of up to 7.8 mmol of the desired Mannich adduct through the use of 0.46 mmol of catalytic resin (5.9 mol %), in a greatly simplified experimental protocol that avoids purification steps.

Highly enantioselective Michael additions in water catalyzed by a PS-supported pyrrolidine.

The development of a highly efficient, polymer-supported organocatalyst for the Michael addition of ketones to nitroolefins is described. A 1,2,3-triazole ring, constructed through a click 1,3-cycloaddition, plays the double role of grafting the chiral pyrrolidine monomer onto the polystyrene backbone and of providing a structural element, complementary to pyrrolidine, key to high catalytic activity and enantioselectivity. Optimal operation in water and full recyclability make the triazole linker attractive for the immobilization of organocatalysts.