Synthesis and in vitro evaluation of West Nile virus protease inhibitors based on the 1,3,4,5-tetrasubstituted 1H-pyrrol-2(5H)-one scaffold.

West Nile virus (WNV), a member of the Flaviviridae family, is a mosquito-borne pathogen that causes a large number of human infections each year. There are currently no vaccines or antiviral therapies available for human use against WNV. Therefore, efforts to develop new chemotherapeutics against this virus are highly desired. In this study, a WNV NS2B-NS3 protease inhibitor with a 1,3,4,5-tetrasubstituted 1H-pyrrol-2(5H)-one scaffold was identified by screening a small library of nonpeptidic compounds. Optimization of this initial hit by the synthesis and screening of a focused library of compounds with this scaffold led to the identification of a novel uncompetitive inhibitor ((-)-1a16, IC50 =2.2±0.7 µM) of the WNV NS2B-NS3 protease. Molecular docking of the chiral compound onto the WNV protease indicates that the R enantiomer of 1a16 interferes with the productive interactions between the NS2B cofactor and the NS3 protease domain and is thus the preferred isomer for inhibition of the WNV NS2B-NS3 protease.

Amine-catalyzed [3+2] Huisgen cycloaddition strategy for the efficient assembly of highly substituted 1,2,3-triazoles.

An enamine-catalyzed strategy has been utilized to fully promote the Huisgen [3+2] cycloaddition with a broad spectrum of carbonyl compounds and azides, thereby permitting the efficient assembly of a vast pool of highly substituted 1,2,3-triazoles. In particular, the employment of commonly used and commercially available carbonyl compounds has resulted in the introduction of a diverse set of functional groups, such as alkyl, aryl, nitrile, ester, and ketone groups, at the 1-, 4-, or 5-positions of the 1,2,3-triazole scaffold. This approach might be manipulated to access more useful and sophisticated heterocyclic compounds. Most significantly, the reaction process exhibits complete regioselectivity, with the formation of only one regioisomer.

Expeditious diastereoselective construction of a thiochroman skeleton via a cinchona alkaloid-derived catalyst.

An example of diastereoselective and enantioselective synthesis of thiochroman derivatives through a sulfa-Michael-Michael cascade sequence is disclosed. This is a significant complement of the quinine-thiourea catalyzed sulfa-Michael-Michael cascade reaction. The densely functionalized target thiochromans were obtained in high diastereoselectivities, and with high to excellent enantioselectivities.

Chiral indane skeleton based thiourea catalyzed highly stereoselective cascade Michael-enolation-cyclization reaction.

An efficient asymmetric cascade reaction catalyzed by a chiral bifunctional indane amine-thiourea catalyst has been developed. From a broad substrate scope, chiral dihydro-2H-pyran complexes that contained two stereogenic centers were obtained in a one-pot manner in good to excellent yields (72-97%) and high to excellent stereoselectivities (92-97% ee).

Asymmetric organocatalytic cascade Michael/hemiketalization/retro-Henry reaction of ß,γ-unsaturated ketoesters with α-nitroketones.

An unprecedented enantioselective organocatalytic Michael/hemiketalization/retro-Henry cascade sequence is described, which catalyzed by a simple bifunctional indane amine-thiourea catalyst. This process provides a new route to the enantioselective synthesis of 5-nitro-pent-2-enoates, a precursor to α-ketolactam.

Enantioselective organocatalytic Michael-hemiketalization catalyzed by a trans-bifunctional indane thiourea catalyst.

An efficient, convenient and enantioselective Michael-hemiketalization reaction has been developed for the synthesis of naphthoquinones. In this work, a novel trans-bifunctional indane thiourea catalyst has been reported to promote this process to afford high yields (up to 99%) and high to excellent enantiomeric excesses (90-98% ee).

Enantioselective heterocyclic synthesis of spiro chromanone-thiochroman complexes catalyzed by a bifunctional indane catalyst.

Novel asymmetric domino reactions of benzylidenechroman-4-ones and 2-mercaptobenzaldehydes for efficient construction of spiro chromanone-thiochroman complexes were accomplished with high yields and excellent selectivities via a novel bifunctional indane catalyst.

Synthesis and disulfide bond connectivity-activity studies of a kalata B1-inspired cyclopeptide against dengue NS2B-NS3 protease.

Kalata B1 is a plant protein with remarkable thermal, chemical and enzymatic stability. Its potential applications could be centered on the possibility of using its cyclic structure and cystine knot motif as a scaffold for the design of stable pharmaceuticals. To discover potent dengue NS2B-NS3 protease inhibitors, we have prepared various kalata B1 analogues by varying the amino acid sequence. Mass spectrometric and biochemical investigations of these analogues revealed a cyclopeptide whose two fully oxidized forms are substrate-competitive inhibitors of the dengue viral NS2B-NS3 protease. Both oxidized forms showed potent inhibition with K(i) of 1.39+/-0.35 and 3.03+/-0.75 microM, respectively.

Synthesis of pyrazolo[5,1-d][1,2,3,5]tetrazine-4(3H)-ones.

A solid-phase synthesis of 5-aminopyrazole has been developed and applied to the preparation of pyrazolo[5,1-d][1,2,3,5]tetrazine-4(3H)-ones. In this strategy, a one-pot reaction from 5-aminopyrazoles to the pyrazolo[5,1-d][1,2,3,5]tetrazine-4(3H)-ones which provided the compounds in good yields was demonstrated. Using this synthetic strategy, we prepared a representative set of 16 pyrazolo[5,1-d][1,2,3,5]tetrazine-4(3H)-ones.

Hydrogen peroxide enhances enterokinase-catalysed proteolytic cleavage of fusion protein.

The effects of hydrogen peroxide on enterokinase catalysis were studied using several fusion proteins recombinantly produced from E. coli. It was demonstrated that hydrogen peroxide enhanced the rate of enterokinase cleavage reaction, leading to a faster release of the target peptide as discussed in patent WO07149053. Among the conditions tested, we observed that hydrogen peroxide could exert its effect on the cleavage of fusion proteins over a wide range of pH and temperature. This finding might provide a simple solution for the accelerated enterokinase cleavage of thermolabile fusion proteins at low temperature.

Efficient preparation of an acyclic permutant of kalata B1 from a recombinant fusion protein with thioredoxin.

A new approach to prepare an acyclic permutant of kalata B1, a cysteine-rich plant cyclopeptide with uterotonic activity, is described. The synthetic codon-optimized cDNA sequence encoding this 29-residue peptide was cloned and fused in-frame to the His(6)-tagged thioredoxin gene in the bacterial expression vector pET-32a. The fusion protein was overexpressed in the bacterial host, Escherichia coli strain BL21 (DE3), and isolated by affinity chromatography on a metal-chelating Sepharose column. An enterokinase recognition sequence incorporated immediately upstream of the target peptide allowed the 29-residue peptide to be released without any unwanted residues upon treatment with enterokinase. This peptide was subsequently separated from the larger thioredoxin moiety by ultracentrifugation through a semipermeable membrane. Further purification was achieved using reversed-phase HPLC. Hydrogen peroxide was found to enhance the rate of enterokinase cleavage in a concentration-dependent manner. Thermal stability studies demonstrated that the recombinant acyclic kalata B1 (ac kalata) was exceptionally stable against thermal denaturation. Mass spectrometric analysis revealed that the recombinant ac kalata was obtained in a fully oxidized form, indicating a high reducing potential and a strong tendency of the 29-residue peptide to form a tightly folded structure.