Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing.

CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo.

Efficient Liver Targeting by Polyvalent Display of a Compact Ligand for the Asialoglycoprotein Receptor.

A compact and stable bicyclic bridged ketal was developed as a ligand for the asialoglycoprotein receptor (ASGPR). This compound showed excellent ligand efficiency, and the molecular details of binding were revealed by the first X-ray crystal structures of ligand-bound ASGPR. This analogue was used to make potent di- and trivalent binders of ASGPR. Extensive characterization of the function of these compounds showed rapid ASGPR-dependent cellular uptake in vitro and high levels of liver/plasma selectivity in vivo. Assessment of the biodistribution in rodents of a prototypical Alexa647-labeled trivalent conjugate showed selective hepatocyte targeting with no detectable distribution in nonparenchymal cells. This molecule also exhibited increased ASGPR-directed hepatocellular uptake and prolonged retention compared to a similar GalNAc derived trimer conjugate. Selective release in the liver of a passively permeable small-molecule cargo was achieved by retro-Diels-Alder cleavage of an oxanorbornadiene linkage, presumably upon encountering intracellular thiol. Therefore, the multicomponent construct described here represents a highly efficient delivery vehicle to hepatocytes.

C2-Selective Branched Alkylation of Benzimidazoles by Rhodium(I)-Catalyzed C-H Activation.

Herein, we report a Rh(I)/bisphosphine/K3PO4 catalytic system allowing for the first time the selective branched C-H alkylation of benzimidazoles with Michael acceptors. Branched alkylation with N,N-dimethyl acrylamide was successfully applied to the alkylation of a broad range of benzimidazoles incorporating a variety of N-substituents and with both electron-rich and -poor functionality displayed at different sites of the arene. Moreover, the introduction of a quaternary carbon was achieved by alkylation with ethyl methacrylate. The method was also shown to be applicable to the C2-selective branched alkylation of azabenzimidazoles.

Base-Controlled Completely Selective Linear or Branched Rhodium(I)-Catalyzed C-H ortho-Alkylation of Azines without Preactivation.

A [RhI ]/bisphosphine/base catalytic system for the ortho-selective C-H alkylation of azines by acrylates and acrylamides is reported. This catalytic system features an unprecedented complete linear or branched selectivity that is solely dependent on the catalytic base that is used. Complete branched selectivity is even achieved for ethyl methacrylate, which enables the introduction of a quaternary carbon center. Excellent functional group compatibility is demonstrated for both linear and branched alkylations. The operational simplicity and broad scope of this transformation allow for rapid access to functionalized azines of direct pharmaceutical and agrochemical relevance.

Fluorodecarboxylation for the Synthesis of Trifluoromethyl Aryl Ethers.

The synthesis of mono-, di-, and trifluoromethyl aryl ethers by fluorodecarboxylation of the corresponding carboxylic acids is reported. AgF2 induces decarboxylation of aryloxydifluoroacetic acids, and AgF, either generated in situ or added separately, serves as a source of fluorine to generate the fluorodecarboxylation products. The addition of 2,6-difluoropyridine increased the reactivity of AgF2 , thereby increasing the range of functional groups and electronic properties of the aryl groups that are tolerated. The reaction conditions used for the formation of trifluoromethyl aryl ethers also served to form difluoromethyl and monofluoromethyl aryl ethers.

Palladium-Catalyzed Synthesis of (Hetero)Aryl Alkyl Sulfones from (Hetero)Aryl Boronic Acids, Unactivated Alkyl Halides, and Potassium Metabisulfite.

A palladium-catalyzed one-step synthesis of (hetero)aryl alkyl sulfones from (hetero)arylboronic acids, potassium metabisulfite, and unactivated or activated alkylhalides is described. This transformation is of broad scope, occurs under mild conditions, and employs readily available reactants. A stoichiometric experiment has led to the isolation of a catalytically active dimeric palladium sulfinate complex, which was characterized by X-ray diffraction analysis.

Application of fundamental organometallic chemistry to the development of a gold-catalyzed synthesis of sulfinate derivatives.

The development of a gold(I)-catalyzed sulfination of aryl boronic acids is described. This transformation proceeds through an unprecedented mechanism which exploits the reactivity of gold(I)-heteroatom bonds to form sulfinate anions. Further in situ elaboration of the sulfinate intermediates leads to the corresponding sulfones and sulfonamides, two pharmacophores routinely encountered in drug discovery.

Pharmacokinetics, metabolism, and excretion of the antidiabetic agent ertugliflozin (PF-04971729) in healthy male subjects.

The disposition of ertugliflozin (PF-04971729), an orally active selective inhibitor of the sodium-dependent glucose cotransporter 2, was studied after a single 25-mg oral dose of [(14)C]-ertugliflozin to healthy human subjects. Mass balance was achieved with approximately 91% of the administered dose recovered in urine and feces. The total administered radioactivity excreted in feces and urine was 40.9% and 50.2%, respectively. The absorption of ertugliflozin in humans was rapid with a T(max) at ∼1.0 hour. Of the total radioactivity excreted in feces and urine, unchanged ertugliflozin collectively accounted for ∼35.3% of the dose, suggestive of moderate metabolic elimination in humans. The principal biotransformation pathway involved glucuronidation of the glycoside hydroxyl groups to yield three regioisomeric metabolites, M4a, M4b, and M4c (∼39.3% of the dose in urine), of which M4c was the major regioisomer (∼31.7% of the dose). The structure of M4a and M4c were confirmed to be ertugliflozin -4-O-ß- and -3-O-ß-glucuronide, respectively, via comparison of the HPLC retention time and mass spectra with authentic standards. A minor metabolic fate involved oxidation by cytochrome P450 to yield monohydroxylated metabolites M1 and M3 and des-ethyl ertugliflozin (M2), which accounted for ∼5.2% of the dose in excreta. In plasma, unchanged ertugliflozin and the corresponding 4-O-ß- (M4a) and 3-O-ß- (M4c) glucuronides were the principal components, which accounted for 49.9, 12.2, and 24.1% of the circulating radioactivity. Overall, these data suggest that ertugliflozin is well absorbed in humans, and eliminated largely via glucuronidation.

From partial to full agonism: identification of a novel 2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole as a full agonist of the human GPR119 receptor.

A novel GPR119 agonist based on the 2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole scaffold was designed through lead optimization starting from pyrazole-based GPR119 agonist 1. The design is centered on the conformational restriction of the core scaffold, while minimizing the change in spatial relationships of two key pharmacophoric elements (piperidine-carbamate and aryl sulfone).

Glycomimetic ligands for the human asialoglycoprotein receptor.

The asialoglycoprotein receptor (ASGPR) is a high-capacity galactose-binding receptor expressed on hepatocytes that binds its native substrates with low affinity. More potent ligands are of interest for hepatic delivery of therapeutic agents. We report several classes of galactosyl analogues with varied substitution at the anomeric, C2-, C5-, and C6-positions. Significant increases in binding affinity were noted for several trifluoromethylacetamide derivatives without covalent attachment to the protein. A variety of new ligands were obtained with affinity for ASGPR as good as or better than that of the parent N-acetylgalactosamine, showing that modification on either side of the key C3,C4-diol moiety is well tolerated, consistent with previous models of a shallow binding pocket. The galactosyl pyranose motif therefore offers many opportunities for the attachment of other functional units or payloads while retaining low-micromolar or better affinity for the ASGPR.

Preclinical species and human disposition of PF-04971729, a selective inhibitor of the sodium-dependent glucose cotransporter 2 and clinical candidate for the treatment of type 2 diabetes mellitus.

(1S,2S,3S,4R,5S)-5-[4-Chloro-3-(4-ethoxybenzyl)phenyl]-1-hydroxymethyl-6,8-dioxabicyclo[3.2.1]octane-2,3,4-triol (PF-04971729), a potent and selective inhibitor of the sodium-dependent glucose cotransporter 2, is currently in phase 2 trials for the treatment of diabetes mellitus. This article describes the preclinical species and in vitro human disposition characteristics of PF-04971729 that were used in experiments performed to support the first-in-human study. Plasma clearance was low in rats (4.04 ml · min(-1) · kg(-1)) and dogs (1.64 ml · min(-1) · kg(-1)), resulting in half-lives of 4.10 and 7.63 h, respectively. Moderate to good bioavailability in rats (69%) and dogs (94%) was observed after oral dosing. The in vitro biotransformation profile of PF-04971729 in liver microsomes and cryopreserved hepatocytes from rat, dog, and human was qualitatively similar; prominent metabolic pathways included monohydroxylation, O-deethylation, and glucuronidation. No human-specific metabolites of PF-04971729 were detected in in vitro studies. Reaction phenotyping studies using recombinant enzymes indicated a role of CYP3A4/3A5, CYP2D6, and UGT1A9/2B7 in the metabolism of PF-04971729. No competitive or time-dependent inhibition of the major human cytochrome P450 enzymes was discerned with PF-04971729. Inhibitory effects against the organic cation transporter 2-mediated uptake of [(14)C]metformin by PF-04971729 also were very weak (IC(50) = ∼900 µM). Single-species allometric scaling of rat pharmacokinetics of PF-04971729 was used to predict human clearance, distribution volume, and oral bioavailability. Human pharmacokinetic predictions were consistent with the potential for a low daily dose. First-in-human studies after oral administration indicated that the human pharmacokinetics/dose predictions for PF-04971729 were in the range that is likely to yield a favorable pharmacodynamic response.

Intrinsic electrophilicity of a 4-substituted-5-cyano-6-(2-methylpyridin-3-yloxy)pyrimidine derivative: structural characterization of glutathione conjugates in vitro.

Isopropyl 9-anti-[5-cyano-6-(2-methyl-pyridin-3-yloxy)-pyrimidin-4-yloxy]-3-oxa-7-aza-bicyclo[3.3.1]nonane-7-carboxylate (1) represents a prototypic compound from a lead chemical series of G protein-coupled receptor 119 agonists, intended for treatment of type 2 diabetes. When compound 1 was incubated with NADPH-supplemented human liver microsomes in the presence of glutathione, two thioether conjugates M4-1 and M5-1 were observed. Omission of NADPH from the microsomal incubations prevented the formation of M5-1 but not M4-1. The formation of M4-1 was also discerned in incubations of 1 and glutathione with human liver cytosol, partially purified glutathione transferase, and in phosphate buffer at pH 7.4. M4-1 was isolated, and its structure ascertained from LC-MS/MS and NMR analysis. The mass spectral and NMR data suggested that M4-1 was obtained from a nucleophilic displacement of the 6-(2-methylpyridin-3-yloxy) group in 1 by glutathione. In addition, mass spectral studies revealed that M5-1 was derived from an analogous displacement reaction on a monohydroxylated metabolite of 1; the regiochemistry of hydroxylation was established to be on the isopropyl group. Of great interest were the findings that replacement of the 5-cyano group in 1 with a 5-methyl group resulted in 2, which was practically inert toward reaction with glutathione. This observation suggests that the electron-withdrawing potential of the C5 cyano group serves to increase the electrophilicity of the C6 carbon (via stabilization of the transition state) and favors reaction with the nucleophilic thiol. The mechanistic insights gained from these studies should assist medicinal chemistry efforts toward the design of analogs that retain primary pharmacology but are latent toward reaction with biological nucleophiles, thus mitigating the potential for toxicological outcome due to adduction with glutathione or proteins.

Design and evaluation of a 2-(2,3,6-trifluorophenyl)acetamide derivative as an agonist of the GPR119 receptor.

The design and synthesis of a GPR119 agonist bearing a 2-(2,3,6-trifluorophenyl)acetamide group is described. The design capitalized on the conformational restriction found in N-ß-fluoroethylamide derivatives to help maintain good levels of potency while driving down both lipophilicity and oxidative metabolism in human liver microsomes. The chemical stability and bioactivation potential are discussed.

C-Aryl glycoside inhibitors of SGLT2: Exploration of sugar modifications including C-5 spirocyclization.

Modifications to the sugar portion of C-aryl glycoside sodium glucose transporter 2 (SGLT2) inhibitors were explored, including systematic deletion and modification of each of the glycoside hydroxyl groups. Based on results showing activity to be quite tolerant of structural change at the C-5 position, a series of novel C-5 spiro analogues was prepared. Some of these analogues exhibit low nanomolar potency versus SGLT2 and promote urinary glucose excretion (UGE) in rats. However, due to sub-optimal pharmacokinetic parameters (in particular half-life), predicted human doses did not meet criteria for further advancement.

2-[4-Chloro-3-(4-ethoxy-benz-yl)phen-yl]-1,3-dithiane.

In the title compound, C(19)H(21)ClOS(2), the dithiane ring adopts a chair conformation. The dihedral angle between the benzene rings is 87.88 (4)°. In the crystal, inversion dimmers linked by pairs of C-H⋯O inter-actions occur.

Optimizing the Benefit/Risk of Acetyl-CoA Carboxylase Inhibitors through Liver Targeting.

Preclinical and clinical data suggest that acetyl-CoA carboxylase (ACC) inhibitors have the potential to rebalance disordered lipid metabolism, leading to improvements in nonalcoholic steatohepatitis (NASH). Consistent with these observations, first-in-human clinical trials with our ACC inhibitor PF-05175157 led to robust reduction of de novo lipogenesis (DNL), albeit with concomitant reductions in platelet count, which were attributed to the inhibition of fatty acid synthesis within bone marrow. Herein, we describe the design, synthesis, and evaluation of carboxylic acid-based ACC inhibitors with organic anion transporting polypeptide (OATP) substrate properties, which facilitated selective distribution of the compounds at the therapeutic site of action (liver) relative to the periphery. These efforts led to the discovery of clinical candidate PF-05221304 (12), which selectively inhibits liver DNL in animals, while demonstrating considerable safety margins against platelet reduction in a nonhuman primate model.

Pyridine sulfinates as general nucleophilic coupling partners in palladium-catalyzed cross-coupling reactions with aryl halides.

Pyridine rings are ubiquitous in drug molecules; however, the pre-eminent reaction used to form carbon-carbon bonds in the pharmaceutical industry, the Suzuki-Miyaura cross-coupling reaction, often fails when applied to these structures. This phenomenon is most pronounced in 2-substituted pyridines, and results from the difficulty in preparing, the poor stability of, and low efficiency in reactions of pyridine-2-boronates. We demonstrate that by replacing these boronates with pyridine-2-sulfinates, a cross-coupling process of unrivalled scope and utility is realized. The corresponding 3- and 4-substituted pyridine variants are also efficient coupling partners. In addition, we apply these sulfinates in a library format to the preparation of medicinally relevant derivatives of the drugs varenicline (Chantix) and mepyramine (Anthisan).

Catalyst Selection Facilitates the Use of Heterocyclic Sulfinates as General Nucleophilic Coupling Partners in Palladium-Catalyzed Coupling Reactions.

A range of 5- and 6-membered heterocycle-derived sulfinates are shown to be effective nucleophilic coupling partners with aryl chlorides and bromides using Pd(0) catalysis. The use of optimal reaction conditions, specifically incorporating a P(t-Bu)2Me-derived Pd catalyst, allowed reactions to be performed at moderate temperatures and enabled the inclusion of a variety of sensitive functional groups. Challenging heterocyclic sulfinates, including pyrazine, pyridazine, pyrimidine, pyrazole, and imidazole, were all shown to perform well.

One-Step Synthesis of Sulfonamides from N-Tosylhydrazones.

The first described reaction between N-tosylhydrazone and SO2 is reported to provide alkyl sulfonamides in the presence of various amines. In this procedurally simple method, hydrazones of both unsaturated aldehydes and ketones proceed in moderate to excellent yields. Primary and secondary aliphatic amines are accommodated in this reaction, which provides a novel route to sulfonamides.

Short Hydrophobic Peptides with Cyclic Constraints Are Potent Glucagon-like Peptide-1 Receptor (GLP-1R) Agonists.

Cyclic constraints are incorporated into an 11-residue analogue of the N-terminus of glucagon-like peptide-1 (GLP-1) to investigate effects of structure on agonist activity. Cyclization through linking side chains of residues 2 and 5 or 5 and 9 produced agonists at nM concentrations in a cAMP assay. 2D NMR and CD spectra revealed an N-terminal ß-turn and a C-terminal helix that differentially influenced affinity and agonist potency. These structures can inform development of small molecule agonists of the GLP-1 receptor to treat type 2 diabetes.