Palladium-Catalyzed Arylation of Cyclopropanes via Directing Group-Mediated C(sp³)–H Bond Activation To Construct Quaternary Carbon Centers: Synthesis of <i>cis</i>- and <i>trans</i>-1,1,2-Trisubstituted Chiral Cyclopropanes

Pd(II)-catalyzed tertiary C(sp³)–H arylation of cyclopropanes via directing group-mediated C–H activation for the construction of a chiral quaternary carbon center on cyclopropanes using aryl iodides as a coupling partner is reported. The arylation had a wide substrate scope and good functional group tolerance, including heteroaryl iodides, to provide various chiral arylcyclopropanes with the <i>cis</i>- and <i>trans</i>-1,1,2-trisubstituted structures

<i>In cellulo</i> mizoribine capping inhibition is rescued by HCE over-expression.

<p>(A) Timeline of the experiments. (B) Cells over-expressing HCE-WT-HA, HCE-K294A-HA, GFP, or control cells were pre-treated with mizoribine for 42 h and transfected with the luciferase encoding vector PGL3. The RNA pol II transcribe reporter gene was allowed to express for 30 h prior to luciferase assay. Luciferase intensity, as a mesure of efficient capping of the reporter protein mRNA, was normalized for untreated cells and plotted against mizoribine concentration. For both mizoribine concentrations tested, the luciferase activity was significantly higher in cells over-expressing HCE-WT-HA as compared to control cells (cells over-expressing the GTase defective HCE-K294A-HA mutant, over-expressing GFP, or transfected with the control Vector). Shown is the mean of 18 experiments for each condition. (C) Western blots using anti-HA, anti-GFP and anti-actin antibody were performed on protein extracts from each cell line. (**P<0.01, ***P<0.001).</p

RNA guanylyltransferase mechanistic pathway and associated structures.

<p>The phosphoryltransfer catalysis requires conformational changes between the open (blue) and close (red) form of the RNA guanylyltransferase enzyme. The apo-enzyme (structure 1) first binds GTP (grey sphere, structure 2) which promotes the closure of the OB fold domain toward the NT domain (structure 3). In the catalytically active close conformation, the enzyme hydrolyzes the GTP to form the hallmark enzyme-GMP covalent intermediate complex (black sphere, structure 4). The lost of interactions between the bound guanylate and the OB fold domain, upon GTP hydrolysis, destabilizes the close conformation of the enzyme and leads to its reopening (structure 5) concomitant with the release of the pyrophosphate product. This exposes the RNA-binding site of the enzyme (exact location unknown), thereby allowing 5′-diphosphate RNA binding (structure 6) and subsequent GMP moiety transfer onto the acceptor RNA (structure 7). The capped RNA is then released and the apo-enzyme (structure 1) is regenerated allowing reinitiation of the pathway. (PDB: 1CKN).</p

Inhibition of capping by MZP.

<p>(A) Structure of mizoribine 5-monophosphate (MZP) and guanosine-5′-triphosphate (GTP). (B) An aliquot of the purified HCE protein (P) was analyzed by electrophoresis through a 12.5% polyacrylamide gel containing 0.1% SDS and visualized by staining with Coomassie blue dye. The position and size (in kDa) of the molecular weight marker (M) are indicated on the left. (C) RNA capping assay by the sequential RTase and GTase activity of HCE. A 5′-triphosphate RNA and [α-³²P]GTP were incubated in presence of HCE in a buffer containing 5 mM MgCl₂. As a control, EDTA was added to a final concentration of 50 mM to prevent RNA capping. Radiolabeled RNA products were analyzed by SDS-PAGE, an autoradiogram of the gel is shown. (D and E) Capping assays were performed in the presence of increasing concentration of MZP or GMP and the capped RNA products were analyzed by SDS-PAGE and quantified by autoradiography.</p

Specificity of MZP inhibition toward RNA guanylyltransferase.

<p>(A) Complete GTase reaction. 5′-diphosphate RNA was capped with HCE, HCE-G_229–597, D1R, A103R or Ceg1 GTases in presence of increasing concentration of MZP. (B) Formation of EpG complex. The GTases from human, vaccinia virus, <i>Chlorella</i> virus or <i>S. cerevisiae</i> were allowed to form enzyme-GMP covalent complex in presence of increasing concentration of MZP. (C) Amino acids alignment of the human (HCE), <i>S. cerevisiae</i> (Ceg1), <i>C. albicans</i> and <i>Chlorella</i> virus (A103R) GTases highlighting the sequence conservation. Representation of the six conserved GTase signature motifs (I, III, IIIa, IV, V, VI) and their associated secondary structures (above). Numbering is based on the HCE amino acids. Amino acids predicted to coordinate MZP low affinity binding site are highlighted by an asterisk. (D) GTases harbor a similar three-dimensional architecture. Ribbon diagram of HCE GTase domain in close conformation (PDB: 3S24), in open conformation (homology model base on PDB: 1P16), <i>Chlorella</i> virus GTase (PDB: 1CKN), <i>S. cerevisiae</i> GTase (PDB: 3KYH), <i>C. albicans</i> GTase (PDB: 1P16). Active site color code is representative of the GTase signature motif as displayed in panel C.</p

MZP mechanism of inhibition.

<p>(A) MZP does not inhibits the RTase activity. An RNA substrate radiolabeled at its 5′-terminal γ-phosphate was incubated with HCE and increasing concentration of MZP. ³²Pi product was separated from RNA substrate by polyethyleneimine-cellulose TLC plate and quantified by autoradiography. (B) The GTase activity is inhibited by MZP. A 5′-diphosphate RNA was incubated with [α-³²P]GTP, HCE and increasing concentration of MZP. Radiolabeled RNA products were analyzed on denaturing polyacrylamide gel and quantified by autoradiography. (C) The formation of HCE-GMP complex, the first step of GTase activity, is inhibited to a lesser extent then the complete GTase reaction by MZP. HCE was incubated with [α-³²P]GTP and increasing concentration of MZP. Radiolabeled EpG complex were analyzed by SDS-PAGE and quantified by autoradiography. (D) MZP does not significantly inhibit the transfer of a GMP on an acceptor RNA; the second step of GTase activity. HCE was preincubated with [α-³²P]GTP to allow EpG formation, a 5′-diphosphate RNA and increasing concentration of MZP were next added to the reaction. Formation of radiolabeled RNA products was analyzed on denaturing polyacrylamide gel and quantified by autoradiography. (E) Lineweaver-Burk representation of the enzyme-GMP covalent complex formation (GTase first step) as a function of the substrate concentration in presence of various MZP concentration. The maximal velocity of EpG formation is not affected by various MZP concentration (curves crossing on the Y axis). (F) Lineweaver-Burk representation of the complete GTase reaction velocity as a function of the substrate concentration in presence of various MZP concentration. HCE K_m for GTP is independent from the MZP concentration (curves crossing on the X axis) for the complete reaction. MZP concentration : ✯ 0 µM, □ 50 µM, ▵ 200 µM, ▿ 500 µM, ✵ 1 mM, ★ 2 mM, ▪ 5 mM, ▴ 10 mM, ▾ 20 mM.</p

Identification of 8‑Aminoadenosine Derivatives as a New Class of Human Concentrative Nucleoside Transporter 2 Inhibitors

Purine-rich foods have long been suspected as a major cause of hyperuricemia. We hypothesized that inhibition of human concentrative nucleoside transporter 2 (hCNT2) would suppress increases in serum urate levels derived from dietary purines. To test this hypothesis, the development of potent hCNT2 inhibitors was required. By modifying adenosine, an hCNT2 substrate, we successfully identified 8-aminoadenosine derivatives as a new class of hCNT2 inhibitors. Compound <b>12</b> moderately inhibited hCNT2 (IC₅₀ = 52 ± 3.8 μM), and subsequent structure–activity relationship studies led to the discovery of compound <b>48</b> (IC₅₀ = 0.64 ± 0.19 μM). Here we describe significant findings about structural requirements of 8-aminoadenosine derivatives for exhibiting potent hCNT2 inhibitory activity

Synthesis of 7‑Deaza-cyclic Adenosine-5′-diphosphate-carbocyclic-ribose and Its 7‑Bromo Derivative as Intracellular Ca²⁺-Mobilizing Agents

Cyclic ADP-carbocyclic-ribose (cADPcR, <b>3</b>) is a biologically and chemically stable equivalent of cyclic ADP-ribose (cADPR, <b>1</b>), a Ca²⁺-mobilizing second messenger. We became interested in the biological activity of the 7-deaza analogues of cADPcR, i.e., 7-deaza-cADPcR (<b>7</b>) and its 7-bromo derivative, i.e., 7-deaza-7-Br-cADPcR (<b>8</b>), because 7-deazaadenosine is an efficient bioisostere of adenosine. The synthesis of <b>7</b> and <b>8</b> required us to construct the key <i>N</i>1-carbocyclic-ribosyl-7-deazaadenosine structure. Therefore, we developed a general method for preparing N1-substituted 7-deazaadenosines by condensing a 2,3-disubstituted pyrrole nucleoside with amines. Using this method, we prepared the <i>N</i>1-carbocyclic ribosyl 7-deazaadenosine derivative <b>10a</b>, from which we then synthesized the target 7-deaza-cADPcR (<b>7</b>) via an Ag⁺-promoted intramolecular condensation to construct the 18-membered pyrophosphate ring structure. The corresponding 7-bromo derivative <b>8</b>, which was the first analogue of cADPR with a substitution at the 7-position, was similarly synthesized. Biological evaluation for Ca²⁺-mobilizing activity in the sea urchin egg homogenate system indicated that 7-deaza-cADPcR (<b>7</b>) and 7-deaza-7-Br-cADPcR (<b>8</b>) acted as a full agonist and a partial agonist, respectively

Ligand–Phospholipid Conjugation: A Versatile Strategy for Developing Long-Acting Ligands That Bind to Membrane Proteins by Restricting the Subcellular Localization of the Ligand

We hypothesized that if drug localization can be restricted to a particular subcellular domain where their target proteins reside, the drugs could bind to their target proteins without being metabolized and/or excreted, which would significantly extend the half-life of the corresponding drug–target complex. Thus, we designed ligand–phospholipid conjugates in which the ligand is conjugated with a phospholipid through a polyethylene glycol linker to restrict the subcellular localization of the ligand in the vicinity of the lipid bilayer. Here, we present the design, synthesis, pharmacological activity, and binding mode analysis of ligand–phospholipid conjugates with muscarinic acetylcholine receptors as the target proteins. These results demonstrate that ligand–phospholipid conjugation can be a versatile strategy for developing long-acting ligands that bind to membrane proteins in drug discovery

Entry to Chiral 1,1,2,3-Tetrasubstituted Arylcyclopropanes by Pd(II)-Catalyzed Arylation via Directing Group-Mediated C(sp³)‑H Activation

Here we report the construction of highly functionalized chiral 1,1,2,3-tetrasubstituted arylcyclopropanes of medicinal chemical importance using Pd­(II)-catalyzed arylation via directing group-mediated C­(sp³)-H activation. The key aspect for the effective arylation was control of the substrate conformation based on the characteristic steric and stereoelectronic features of cyclopropane by manipulating the protecting group at the hydroxyl. The arylation with good functional group tolerance is pivotal as the first entry to chiral 1,1,2,3-tetrasubstituted arylcyclopropanes with wide variety of aryl groups, including heteroaryl groups