Gold-Catalyzed Unexpected Ring Transformation of Pyrimidodiazepine Derivatives

Pyrimidodiazepine derivatives underwent an unexpected gold-catalyzed retro-Mannich-type carbon–carbon bond cleavage and intramolecular nucleophilic cyclization. The pyrimidodiazepines bearing an alkyne moiety showed novel orthogonal reactivity in the presence of a gold catalyst, as opposed to the alkynophilicity that is commonly observed with gold catalysts. The ring transformation reaction of pyrimidodiazepines probably proceeds through an acyclic iminium intermediate. The potential of this synthetic method for the skeletal diversification of pyrimidine-containing macrocycles was also demonstrated

Privileged Substructure-Based Diversity-Oriented Synthesis Pathway for Diverse Pyrimidine-Embedded Polyheterocycles

A new diversity-oriented synthesis pathway for the fabrication of a pyrimidine-embedded polyheterocycles library was developed for potential interactions with diverse biopolymers. Five different pyrimidine-embedded core skeletons were synthesized from <i>ortho</i>-alkynylpyrimidine carbaldehydes by a silver- or iodine-mediated tandem cyclization strategy. The resulting polyheterocycles possess diverse fused ring sizes and positions with potential functionalities for further modification

Construction of Polyheterocyclic Benzopyran Library with Diverse Core Skeletons through Diversity-Oriented Synthesis Pathway: Part II

As a continuation of our previous report (<i>J. Comb. Chem.</i> <b>2010</b>, <i>12</i>, 548–558), we accomplished the diversity-oriented synthesis of polyheterocyclic small-molecule library with privileged benzopyran substructure. To ensure the synthetic efficiency, we utilized the solid-phase parallel platform and the fluorous-tag-based solution-phase parallel platform to construct a 284-member polyheterocyclic library with six distinct core skeletons with an average purity of 87% on a scale of 5–10 mg. This library was designed to maximize the skeletal diversity with discrete core skeletons in three-dimensional space and the combinatorial diversity with four different benzopyranyl starting materials and various building blocks. Together with our reported benzopyranyl library, we completed the construction of polyheterocyclic benzopyran library with 11 unique scaffolds and their molecular diversity was visualized in chemical space using principle component analysis (PCA)

Suppression of type I IFN induction by HCV F/ARFP.

<p>(A) Huh7 cells transfected with pEF (empty vector), pHA-F, pFLAG-NS3/4A, or pHA-F plus pFLAG-NS3/4A were analyzed by Western blots using anti-HA, FLAG, and actin antibodies. (B—D) Huh7 cells were transfected with indicated plasmids and, after 48 hrs, transfected with 5 μg of HCV RNA PAMP corresponding to the UTR or poly(IC). Samples were analyzed for the indicated mRNAs, 24 hrs after PAMP stimulation by qRT-PCR. For (B–D), star indicates statistically significant difference (P < 0.05) from respective minus PAMP controls. Letter “a” indicates statistically significant difference (P < 0.05) from the corresponding pEF control for each -PAMP or +PAMP group. Lines with P values also indicate statistically significant difference between those samples. All mRNA data were normalized by GAPDH mRNA and shown as percentage of controls.</p

Role of RIG-I in the modulation of IFNβ1 by HCV -2/+1 frame mutants.

<p>(A) Huh7 and Huh7.5 cells were transfected with JFH1wt, JFH1Δ, and JFH1Δ4 RNAs or mock-transfected and analyzed for IFNβ1 mRNA and HCV RNA by qRT-PCR after 72 hrs. (B) Huh7 cells were transfected with siRNAs and then transfected with JFH1wt and JFH1Δ4 RNAs or mock-transfected 48–72 hrs later. Cells were then analyzed for IFNβ1 and RIG-I mRNAs by qRT-PCR. Data were normalized by GAPDH mRNA. (C) Huh7 cells supporting JFH1wt, JFH1Δ, and JFH1Δ4 were incubated with 0, 100, or 250 U/ml of exogenous IFNα (NIAID Reference Reagent Repository and Sigma Aldrich) with change of cell culture medium daily for 72 hrs. Then, samples were collected and analyzed by qRT-PCR. Data are shown as percentage of respective 0 U/ml IFNα controls. Star indicates statistically significant difference (P < 0.05) compared to controls. Lines with P values also indicate statistically significant difference between those samples.</p

Regioselective Construction and Screening of 1,3-Disubstituted Tetrahydroindazolones in Enantiomerically Pure Pairs

In this paper, we describe a regioselective synthetic pathway for enantiopure 1,3-disubstituted tetrahydroindazolone derivatives via the condensation of 2-acylcyclohexane-1,3-dione with various alkyl- and arylhydrazines using the steric effects of a Boc-protected pyrrolidine ring. This synthetic method has a broad scope for substrate generality for various hydrazines with excellent regioselectivity. To maximize the molecular diversity, further diversifications of 1,3-disubstituted tetrahydroindazolones were pursued by systematic <i>N</i>-modification of the secondary amine of the pyrrolidine ring using solution-phase parallel synthesis with polymer-supported reagents. A library containing a total of 272 drug-like tetrahydroindazolones, including 85 enantiomeric pairs, was constructed; the average purity, without further purification, was 95%

Hepatitis C Virus Frameshift/Alternate Reading Frame Protein Suppresses Interferon Responses Mediated by Pattern Recognition Receptor Retinoic-Acid-Inducible Gene-I

<div><p>Hepatitis C virus (HCV) actively evades host interferon (IFN) responses but the mechanisms of how it does so are not completely understood. In this study, we present evidence for an HCV factor that contributes to the suppression of retinoic-acid-inducible gene-I (RIG-I)-mediated IFN induction. Expression of <u>f</u>rameshift/alternate reading frame protein (F/ARFP) from HCV -2/+1 frame in Huh7 hepatoma cells suppressed type I IFN responses stimulated by HCV RNA pathogen-associated molecular pattern (PAMP) and poly(IC). The suppression occurred independently of other HCV factors; and activation of interferon stimulated genes, TNFα, IFN-λ1, and IFN-λ2/3 was likewise suppressed by HCV F/ARFP. Point mutations in the full-length HCV sequence (JFH1 genotype 2a strain) were made to introduce premature termination codons in the -2/+1 reading frame coding for F/ARFP while preserving the original reading frame, which enhanced IFNα and IFNβ induction by HCV. The potentiation of IFN response by the F/ARFP mutations was diminished in Huh7.5 cells, which already have a defective RIG-I, and by decreasing RIG-I expression in Huh7 cells. Furthermore, adding F/ARFP back <i>via trans</i>-complementation suppressed IFN induction in the F/ARFP mutant. The F/ARFP mutants, on the other hand, were not resistant to exogenous IFNα. Finally, HCV-infected human liver samples showed significant F/ARFP antibody reactivity, compared to HCV-uninfected control livers. Therefore, HCV F/ARFP likely cooperates with other viral factors to suppress type I and III IFN induction occurring through the RIG-I signaling pathway. This study identifies a novel mechanism of pattern recognition receptor modulation by HCV and suggests a biological function of the HCV alternate reading frame in the modulation of host innate immunity.</p></div

Effects of HCV core versus F/ARFP on IFNβ1 mRNA elevation by JFH1Δ4.

<p>(A) Huh7 cells were transfected with pCoreΔ or pF as well as JFH1wt or JFH1Δ4 RNA and analyzed for IFNβ1 mRNA by qRT-PCR. Data are normalized by JFH1 RNA and GAPDH mRNA levels and expressed as percentage of pCoreΔ/JFH1wt transfected control. Star indicates statistically significant difference (P < 0.05) from JFH1wt for each plasmid group; letter “a” indicates statistically significant difference (P < 0.05) from pCoreΔ. (B) Huh7 cells transfected with pCoreΔ4, pHA-F, pF, and pFLAG-NS3/4A were analyzed for IFNβ1 mRNA by qRT-PCR. Data were normalized by GAPDH mRNA. Star indicates statistically significant difference (P < 0.05) from respective minus PAMP controls. Letter “a” indicates statistically significant difference (P value less than 0.05) from pCoreΔ4.</p

HCV F/ARFP suppresses ISGs, pro-inflammatory cytokines, and type III IFN responses in Huh7 cells.

<p>(A—B) Huh7 cells were transfected with indicated plasmids and, after 48 hrs, transfected with 5 μg of HCV RNA PAMP corresponding to the UTR or poly(IC). Samples were analyzed for the indicated mRNAs 24 hrs after stimulation with PAMP by qRT-PCR. Data were normalized by GAPDH mRNA and shown as percentage of controls. Star indicates statistically significant difference (P < 0.05) from respective minus PAMP controls. Letter “a” indicates statistically significant difference (P < 0.05) from the corresponding pEF controls for each -PAMP or +PAMP group. Lines with P values also indicate statistically significant difference between those samples.</p

List of qRT-PCR primers.

<p>List of qRT-PCR primers.</p