Enyne Amides to Fused Pyridones: Scope and Limitations.

Herein we present an investigation into the scope and mechanism for the synthesis of cyclopentyl and heterocyclic fused pyridones from the corresponding enyne amides. In the presence of a secondary amine, cyclization proceeds smoothly to form 5,6-bicyclic pyridones in 12-90% yield. The cyclization fails with enyne amides of six-membered and larger ring systems. The ring closure reaction is catalytic in nature with respect to the secondary amine and proceeds via sequential 1,6-addition of the amine, 6-exo-trig ring closure of the iminium intermediate, and subsequent elimination of the secondary amine. Computations show reduced conjugation between the enyne and amide for six-membered and larger systems, thereby providing an explanation for the inability of such enyne amides to form fused pyridones.

Novel Homodimer Metabolites of GDC-0994 via Cytochrome P450-Catalyzed Radical Coupling.

Two novel homodimer metabolites were identified in rat samples collected during the in vivo study of GDC-0994. In this study, we investigated the mechanism of the formation of these metabolites. We generated and isolated the dimer metabolites using a biomimetic oxidation system for NMR structure elucidation to identify a symmetric dimer formed via carbon-carbon bond between two pyrazoles and an asymmetric dimer formed via an aminopyrazole-nitrogen to pyrazole-carbon bond. In vitro experiments demonstrated formation of these dimers was catalyzed by cytochrome P450 enzymes (P450s) with CYP3A4/5 being the most efficient. Using density functional theory, we determined these metabolites share a mechanism of formation, initiated by an N-H hydrogen atom abstraction by the catalytically active iron-oxo of P450s. Molecular modeling studies also show these dimer metabolites fit in the CYP3A4 binding site in low energy conformations with minimal protein rearrangement. Collectively, the results of these experiments suggest that formation of these two homodimer metabolites is mediated by CYP3A, likely involving activation of two GDC-0994 molecules by a single P450 enzyme and proceeding through a radical coupling mechanism. SIGNIFICANCE STATEMENT: These studies identified structures and enzymology for two distinct homodimer metabolites and indicate a novel biotransformation reaction mediated by CYP3A. In it, two molecules may bind within the active site and combine through radical coupling. The mechanism of dimerization was elucidated using density functional theory computations and supported by molecular modeling.

cis-Selective synthesis of 1,3-disubstituted tetrahydro-ß-carbolines from N-sulfonyl N,S-acetals.

Nucleophilic addition of Grignard reagents to tetrahydro-ß-carboline (THC) N-sulfonyl N,S-acetal generates exclusively cis-1,3-disubstituted THCs with a unique 1,3-diaxial conformation. The stereochemical relationship of the 1,3-substituents was confirmed by 2-dimensional NMR spectroscopy and X-ray crystallography. The mechanism of the reaction is proposed based on crystal structures and molecular orbital calculations.

Eicosanoids: Emerging contributors in stem cell-mediated wound healing.

Eicosanoids are bioactive lipid products primarily derived from the oxidation of arachidonic acid (AA). The individual contributions of eicosanoids and stem cells to wound healing have been of great interest. This review focuses on how stem cells work in concert with eicosanoids to create a beneficial environment in the wound bed and in the promotion of wound healing. Stem cells contribute to wound healing through modulating inflammation, differentiating into skin cells or endothelial cells, and exerting paracrine effects by releasing various potent growth factors. Eicosanoids have been shown to stimulate proliferation, migration, homing, and differentiation of stem cells, all of which contribute to the process of wound healing. Increasing evidence has shown that eicosanoids improve wound healing through increasing stem cell densities, stimulating differentiation, and enhancing the angiogenic properties of stem cells. Chronic wounds have become a major problem in health care. Therefore, research regarding the effects of stem cells and eicosanoids in the promotion wound healing is of great importance.

Targeted drug delivery through the traceless release of tertiary and heteroaryl amines from antibody-drug conjugates.

The reversible attachment of a small-molecule drug to a carrier for targeted delivery can improve pharmacokinetics and the therapeutic index. Previous studies have reported the delivery of molecules that contain primary and secondary amines via an amide or carbamate bond; however, the ability to employ tertiary-amine-containing bioactive molecules has been elusive. Here we describe a bioreversible linkage based on a quaternary ammonium that can be used to connect a broad array of tertiary and heteroaryl amines to a carrier protein. Using a concise, protecting-group-free synthesis we demonstrate the chemoselective modification of 12 complex molecules that contain a range of reactive functional groups. We also show the utility of this connection with both protease-cleavable and reductively cleavable antibody-drug conjugates that were effective and stable in vitro and in vivo. Studies with a tertiary-amine-containing antibiotic show that the resulting antibody-antibiotic conjugate provided appropriate stability and release characteristics and led to an unexpected improvement in activity over the conjugates previously connected via a carbamate.

Synthesis of Fused Imidazole-Containing Ring Systems via Dual Oxidative Amination of C(sp(3))-H Bonds.

A general and efficient method for a metal-free one-pot synthesis of highly substituted fused imidazole-containing 5,5- and 5,6-fused bicyclic heterocycles is described. Starting from commercially available substrates and reagents, the reaction proceeds through two C-N bond formations and an oxidative dehydrogenation to form highly substituted products in good to excellent yield.

Structure and Optical Thermometry Characterization of Er3+/Yb3+ Co-Doped BaGd2CuO5.

Er3+/Yb3+ co-doped BaGd2CuO5 upconversion luminescent materials are obtained by solid phase method. Rietveld refinement on X-ray diffraction data indicates that Er3+/Yb3+ ions are inclined to occupy the Gd(1) site in the structure of BaGd2CuO5 (green phase). Two green emission peaks located at 523 nm and 547 nm have been produced by the excitation of 971 nm LD. The fluorescence intensity ratio (FIR) of the two green emission peaks have been investigated in the temperature range of 290 K-594 K. The maximum sensitivity derived from the FIR technique of the green upconversion emission is approximately 0.0038 K-1, and it has a high transmission power at low excitation density. This result implies that the Er3+/Yb3+ co-doped BaGd2CuO5 phosphors can play an important role in temperature measurements with a better sensitivity.

Synthesis and Luminescence Properties of La2W2O9:Eu3+ Micron-Crystals.

La2W2O9:2%Eu3+ phosphors were synthesized by a typical hydrothermal procedure. The samples were characterized by X-ray diffraction and scanning electron microscope (SEM). X-ray diffraction analysis showed that a stock solution pH value equal to 9 is the ideal value, while the crystallization of the hydroxyl sodium yttrium tungstate crystal is improved by increasing the PH values of stock solution within limits. Meanwhile, SEMs of different pH values were recorded. Additionally, photo-luminescence excitation (PLE) and emission (PL) spectra were measured. It was found that this phosphor can be effectively excited by C-T band (266 nm) and ultraviolet light 342 nm. The wave-lengths at 342 nm fit in nicely with the whole visible region, thus the La2W2O9:2%Eu3+ phosphors emit white light. Furthermore, the annealing temperature's impact on PLE and PL spectra was also studied. The Eu3+-doped La2W2O9 phosphor may be a better candidate than current method for solid-state lighting applications.

Discovery of GluN2A-Selective NMDA Receptor Positive Allosteric Modulators (PAMs): Tuning Deactivation Kinetics via Structure-Based Design.

The N-methyl-D-aspartate receptor (NMDAR) is a Na(+) and Ca(2+) permeable ionotropic glutamate receptor that is activated by the coagonists glycine and glutamate. NMDARs are critical to synaptic signaling and plasticity, and their dysfunction has been implicated in a number of neurological disorders, including schizophrenia, depression, and Alzheimer's disease. Herein we describe the discovery of potent GluN2A-selective NMDAR positive allosteric modulators (PAMs) starting from a high-throughput screening hit. Using structure-based design, we sought to increase potency at the GluN2A subtype, while improving selectivity against related α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). The structure-activity relationship of channel deactivation kinetics was studied using a combination of electrophysiology and protein crystallography. Effective incorporation of these strategies resulted in the discovery of GNE-0723 (46), a highly potent and brain penetrant GluN2A-selective NMDAR PAM suitable for in vivo characterization.

Scaffold-Hopping and Structure-Based Discovery of Potent, Selective, And Brain Penetrant N-(1H-Pyrazol-3-yl)pyridin-2-amine Inhibitors of Dual Leucine Zipper Kinase (DLK, MAP3K12).

Recent data suggest that inhibition of dual leucine zipper kinase (DLK, MAP3K12) has therapeutic potential for treatment of a number of indications ranging from acute neuronal injury to chronic neurodegenerative disease. Thus, high demand exists for selective small molecule DLK inhibitors with favorable drug-like properties and good CNS penetration. Herein we describe a shape-based scaffold hopping approach to convert pyrimidine 1 to a pyrazole core with improved physicochemical properties. We also present the first crystal structures of DLK. By utilizing a combination of property and structure-based design, we identified inhibitor 11, a potent, selective, and brain-penetrant inhibitor of DLK with activity in an in vivo nerve injury model.

Structure-Guided Design of Group I Selective p21-Activated Kinase Inhibitors.

The p21-activated kinases (PAKs) play important roles in cytoskeletal organization, cellular morphogenesis, and survival and have generated significant attention as potential therapeutic targets for cancer. Following a high-throughput screen, we identified an aminopyrazole scaffold-based series that was optimized to yield group I selective PAK inhibitors. A structure-based design effort aimed at targeting the ribose pocket for both potency and selectivity led to much-improved group I vs II selectivity. Early lead compounds contained a basic primary amine, which was found to be a major metabolic soft spot with in vivo clearance proceeding predominantly via N-acetylation. We succeeded in identifying replacements with improved metabolic stability, leading to compounds with lower in vivo rodent clearance and excellent group I PAK selectivity.

Leveraging the Pre-DFG Residue Thr-406 To Obtain High Kinase Selectivity in an Aminopyrazole-Type PAK1 Inhibitor Series.

To increase kinase selectivity in an aminopyrazole-based PAK1 inhibitor series, analogues were designed to interact with the PAK1 deep-front pocket pre-DFG residue Thr-406, a residue that is hydrophobic in most kinases. This goal was achieved by installing lactam head groups to the aminopyrazole hinge binding moiety. The corresponding analogues represent the most kinase selective ATP-competitive Group I PAK inhibitors described to date. Hydrogen bonding with the Thr-406 side chain was demonstrated by X-ray crystallography, and inhibitory activities, particularly against kinases with hydrophobic pre-DFG residues, were mitigated. Leveraging hydrogen bonding side chain interactions with polar pre-DFG residues is unprecedented, and similar strategies should be applicable to other appropriate kinases.

[Er3+:Yb3+ co-doped nanocrystals BaGd2ZnO5 of up-conversion optical temperature sensing].

By far, the most efficient upconversion nanocrystals luminescence materials BaGd2ZnO5: 4%Yb3+ , 1%Er3+, with stable chemical performance, were prepared by using Sol-gel method. XRD pattern shows that the sample is pure phase, belongs to the orthogonal crystals, and space group is Pbnm; SEM micrograph shows that the prepared sample of the morphology sized around 150 nm is evenly distributed. Samples with 971 nm semiconductor laser excitation produce a strong green emission, visible to the naked eye, and uponversion strength and pump energy relation n = 1.22 is two-photon for the realization of the upconversion emission. They originated from Er3+ ions 2H(11/2)--2H(11/2)-->4I(15/2) and 4S(3/2)-->4I(15/2) transition emission, Er3+ ions main excited state absorption (ESA) process is: 4I(15/2)-->4I(11/2)-->2F(7/2)-->2H(11/2), 4S(3/2), Yb3+ was added because of its large absorption cross section (10(4) cm(-1)) so that it is easy to transfer excitation energy to the E3+ ions which enhance the layout particles number and the energy state of the 1F7/2, thereby enhancing the intensity of the peaks of the spectrum. Fluorescence intensity ratio (FIR) technique based on the green upconversion emission of the sample has been studied because the Er3+ ions 2H(11/2) and 4S(3/2) energy level spacing is small. The electrons at the two levels conform to the Boltzmann distribution which is a function of temperature, and thus the fluorescence intensity ratio of two levels can be used to measure the temperature of the substrate material. This method does not interfere with temperature field of the measured object, and can eliminate the uncertainty of the accuracy; the test has a wide temperature range and reasonable temperature resolution, the pump source used is simple, convenient and inexpensive, and has more commercial values. The temperature range of the samples is from 350 to 800 K, and the highest temperature measuring sensitivity can reach 0.0031 K(1). At the same time, under low excitation density, it can produce higher conversion transmission power, making it become ideal material for distance non-contact temperature measurement.

White up-conversion luminescence power and efficiency in Yb(3+)-, Er(3+)- and Tm(3+)-doped BaIn6Y2O13.

A series of BaIn6Y2O13:Yb(3+), Er(3+), Tm(3+) phosphors with different dopant concentrations have been successfully synthesized by a sol-gel method. In order to obtain efficient white light emitting samples, tri-doped and biphasic samples were prepared. Afterwards, we carried out a systematic study on the up-conversion luminescence (UCL) properties of the samples as functions of doping concentrations and excitation densities. The up-conversion (UC) white light emission powers and efficiencies of samples with different dopant concentrations at different excitation power densities were obtained. The highest white UCL efficiency of up to 0.38% was achieved at excitation densities down to 17.5 W cm(-2) in the biphasic samples. More importantly, we demonstrated that the UC materials with higher efficiency do not necessarily have stronger emission power, and the emission power is the most important parameter. A maximum emission power of up to 1.23 mW was obtained. Furthermore, the impact of temperature on the white UCL was studied, and transparent polymer composites that can emit white light were synthesized by doping BaIn6Y2O13 samples into polymethylmethacrylate (PMMA).

Ultraviolet C upconversion fluorescence of trivalent erbium in BaGd2ZnO5 phosphor excited by a visible commercial light-emitting diode.

Multiple ultraviolet (UV) emission bands have been obtained in Er3+ doped BaGd2ZnO5 phosphor under the excitation of a 532 nm solid-state laser, and the emission peaks at 217, 254, 278, 296, 314, 348, 374 and 394 nm were determined to stem from the high-energy states 4D(1/2), 4D(7/2), 2H(9/2), 2P(1/2), 2P(3/2), 4G(7/2), 4G(11/2), 4H(9/2) of trivalent erbium, respectively. Some UV emission bands in the UVC region can be observed when the sample was excited by commercial green (529 nm) and blue (460 nm) LED. In view of the small size, low-drive voltage and price of LED, UVC upconversion phosphor BaGd2ZnO5:Er3+ excited by visible LED has potential application in environmental sciences.

Lead optimization of a 4-aminopyridine benzamide scaffold to identify potent, selective, and orally bioavailable TYK2 inhibitors.

Herein we report our lead optimization effort to identify potent, selective, and orally bioavailable TYK2 inhibitors, starting with lead molecule 3. We used structure-based design to discover 2,6-dichloro-4-cyanophenyl and (1R,2R)-2-fluorocyclopropylamide modifications, each of which exhibited improved TYK2 potency and JAK1 and JAK2 selectivity relative to 3. Further optimization eventually led to compound 37 that showed good TYK2 enzyme and interleukin-12 (IL-12) cell potency, as well as acceptable cellular JAK1 and JAK2 selectivity and excellent oral exposure in mice. When tested in a mouse IL-12 PK/PD model, compound 37 showed statistically significant knockdown of cytokine interferon-γ (IFNγ), suggesting that selective inhibition of TYK2 kinase activity might be sufficient to block the IL-12 pathway in vivo.

Step-economic synthesis of (+)-crocacin C: a concise crotylboronation/[3,3]-sigmatropic rearrangement approach.

The step-economic total synthesis of (+)-crocacin C has been achieved in 20% yield from commercially available starting materials. This approach requires the isolation of only 8 intermediates and can provide a reliable supply of (+)-crocacin C for the development of new antifungal and crop protection agents.

Isolation and identification of ester impurities in RG7128, an HCV polymerase inhibitor.

RG7128 is a di-ester prodrug of a cytidine analog for the treatment of hepatitis C virus (HCV) infection. The structures of nine low level impurities (0.05-0.10%) in RG7128 drug substance were elucidated. The majority of the impurities were formed during the synthesis of the prodrug from the parent drug. Structural elucidations of the impurities were achieved either by enrichment of the impurities using preparative chromatography followed by spectroscopic techniques or by confirmation with a reference sample. Heart-cut and recycle chromatographic techniques were applied to purify closely eluting isomers of RG7128.

An integrative genomic analysis identifies Bhmt2 as a diet-dependent genetic factor protecting against acetaminophen-induced liver toxicity.

Acetaminophen-induced liver toxicity is the most frequent precipitating cause of acute liver failure and liver transplant, but contemporary medical practice has mainly focused on patient management after a liver injury has been induced. An integrative genetic, transcriptional, and two-dimensional NMR-based metabolomic analysis performed using multiple inbred mouse strains, along with knowledge-based filtering of these data, identified betaine-homocysteine methyltransferase 2 (Bhmt2) as a diet-dependent genetic factor that affected susceptibility to acetaminophen-induced liver toxicity in mice. Through an effect on methionine and glutathione biosynthesis, Bhmt2 could utilize its substrate (S-methylmethionine [SMM]) to confer protection against acetaminophen-induced injury in vivo. Since SMM is only synthesized in plants, Bhmt2 exerts its beneficial effect in a diet-dependent manner. Identification of Bhmt2 and the affected biosynthetic pathway demonstrates how a novel method of integrative genomic analysis in mice can provide a unique and clinically applicable approach to a major public health problem.

2D NMR metabonomic analysis: a novel method for automated peak alignment.

MOTIVATION: Comparative metabolic profiling by nuclear magnetic resonance (NMR) is showing increasing promise for identifying inter-individual differences to drug response. Two dimensional (2D) (1)H (13)C NMR can reduce spectral overlap, a common problem of 1D (1)H NMR. However, the peak alignment tools for 1D NMR spectra are not well suited for 2D NMR. An automated and statistically robust method for aligning 2D NMR peaks is required to enable comparative metabonomic analysis using 2D NMR. RESULTS: A novel statistical method was developed to align NMR peaks that represent the same chemical groups across multiple 2D NMR spectra. The degree of local pattern match among peaks in different spectra is assessed using a similarity measure, and a heuristic algorithm maximizes the similarity measure for peaks across the whole spectrum. This peak alignment method was used to align peaks in 2D NMR spectra of endogenous metabolites in liver extracts obtained from four inbred mouse strains in the study of acetaminophen-induced liver toxicity. This automated alignment method was validated by manual examination of the top 50 peaks as ranked by signal intensity. Manual inspection of 1872 peaks in 39 different spectra demonstrated that the automated algorithm correctly aligned 1810 (96.7%) peaks. AVAILABILITY: Algorithm is available upon request.