Chiral Amines via Enantioselective π-Allyliridium-C,O-Benzoate-Catalyzed Allylic Alkylation: Student Training via Industrial-Academic Collaboration.

Cyclometalated π-allyliridium-C,O-benzoate complexes discovered in the Krische laboratory display unique amphiphilic properties, catalyzing both nucleophilic carbonyl allylation and electrophilic allylation of diverse amines as well as nitronates. Given the importance of chiral amines in FDA-approved small-molecule drugs, a collaboration with medicinal chemists at Genentech that included on-site graduate student internships was undertaken to explore and expand the scope of π-allyliridium-C,O-benzoate-catalyzed allylic amination and related processes. As described in this Account, our collective experimental studies have unlocked asymmetric allylic aminations of exceptionally broad utility and scope. Specifically, using racemic branched alkyl-substituted allylic acetate proelectrophiles, primary and secondary aliphatic or aromatic amines, including indoles, engage in highly regio- and enantioselective allylic amination. Additionally, unactivated nitronates were found to be competent nucleophilic partners for regio- and enantioselective allylic alkylation, enabling entry to ß-stereogenic α-quaternary primary amines. Notably, these π-allyliridium-C,O-benzoate-catalyzed allylic substitutions, which display complete branched regioselectivity in reactions of alkyl-substituted allyl electrophiles, complement the scope of corresponding iridium phosphoramidite-catalyzed allylic aminations, which require aryl-substituted allyl electrophiles to promote high levels of branched regioselectivity. Computational, kinetic, ESI-CID-MS, and isotopic labeling studies were undertaken to understand the mechanism of these processes, including the origins of regio- and enantioselectivity. Isotopic labeling studies suggest that C-N bond formation occurs through outer-sphere addition to the π-allyl. DFT calculations corroborate C-N bond formation via outer-sphere addition and suggest that early transition states and distinct trans effects of diastereomeric chiral-at-iridium π-allyl complexes render the reaction less sensitive to steric effects, accounting for complete levels of branched regioselectivity in reactions of hindered amine and nitronate nucleophiles. Reaction progress kinetic analysis (RPKA) reveals a zero-order dependence on allyl acetate, a first-order dependence on the catalyst, and a fractional-order dependence on the amine. As corroborated by ESI-CID-MS analysis, the 0.4 kinetic order dependence on the amine may reflect the intervention of cesium-bridged amine dimers, which dissociate to form monomeric cesium amide nucleophiles. Hence, the requirement of cesium carbonate (vs lower alkali metal carbonates) in these processes may reside in cesium's capacity for Lewis acid-enhanced Brønsted acidification of the amine pronucleophile. Beyond the development of catalytic processes for the synthesis of novel chiral amines, the present research was conducted by graduate students who benefited from career development experiences associated with training in both academic and industrial laboratories.

Lipid droplets can promote drug accumulation and activation.

Genetic screens in cultured human cells represent a powerful unbiased strategy to identify cellular pathways that determine drug efficacy, providing critical information for clinical development. We used insertional mutagenesis-based screens in haploid cells to identify genes required for the sensitivity to lasonolide A (LasA), a macrolide derived from a marine sponge that kills certain types of cancer cells at low nanomolar concentrations. Our screens converged on a single gene, LDAH, encoding a member of the metabolite serine hydrolase family that is localized on the surface of lipid droplets. Mechanistic studies revealed that LasA accumulates in lipid droplets, where it is cleaved into a toxic metabolite by LDAH. We suggest that selective partitioning of hydrophobic drugs into the oil phase of lipid droplets can influence their activation and eventual toxicity to cells.

MCC950/CRID3 potently targets the NACHT domain of wild-type NLRP3 but not disease-associated mutants for inflammasome inhibition.

The nucleotide-binding-domain (NBD)-and leucine-rich repeat (LRR)-containing (NLR) family, pyrin-domain-containing 3 (NLRP3) inflammasome drives pathological inflammation in a suite of autoimmune, metabolic, malignant, and neurodegenerative diseases. Additionally, NLRP3 gain-of-function point mutations cause systemic periodic fever syndromes that are collectively known as cryopyrin-associated periodic syndrome (CAPS). There is significant interest in the discovery and development of diarylsulfonylurea Cytokine Release Inhibitory Drugs (CRIDs) such as MCC950/CRID3, a potent and selective inhibitor of the NLRP3 inflammasome pathway, for the treatment of CAPS and other diseases. However, drug discovery efforts have been constrained by the lack of insight into the molecular target and mechanism by which these CRIDs inhibit the NLRP3 inflammasome pathway. Here, we show that the NAIP, CIITA, HET-E, and TP1 (NACHT) domain of NLRP3 is the molecular target of diarylsulfonylurea inhibitors. Interestingly, we find photoaffinity labeling (PAL) of the NACHT domain requires an intact (d)ATP-binding pocket and is substantially reduced for most CAPS-associated NLRP3 mutants. In concordance with this finding, MCC950/CRID3 failed to inhibit NLRP3-driven inflammatory pathology in two mouse models of CAPS. Moreover, it abolished circulating levels of interleukin (IL)-1ß and IL-18 in lipopolysaccharide (LPS)-challenged wild-type mice but not in Nlrp3L351P knock-in mice and ex vivo-stimulated mutant macrophages. These results identify wild-type NLRP3 as the molecular target of MCC950/CRID3 and show that CAPS-related NLRP3 mutants escape efficient MCC950/CRID3 inhibition. Collectively, this work suggests that MCC950/CRID3-based therapies may effectively treat inflammation driven by wild-type NLRP3 but not CAPS-associated mutants.

Correction: MCC950/CRID3 potently targets the NACHT domain of wild-type NLRP3 but not disease-associated mutants for inflammasome inhibition.

[This corrects the article DOI: 10.1371/journal.pbio.3000354.].

Enantioselective Iridium-Catalyzed Allylation of Nitroalkanes: Entry to ß-Stereogenic α-Quaternary Primary Amines.

The first systematic study of simple nitronate nucleophiles in iridium-catalyzed allylic alkylation is described. Using a tol-BINAP-modified π-allyliridium C,O-benzoate catalyst, α,α-disubstituted nitronates substitute racemic branched alkyl-substituted allylic acetates, thus providing entry to ß-stereogenic α-quaternary primary amines. DFT calculations reveal early transition states that render the reaction less sensitive to steric effects and distinct trans-effects of diastereomeric chiral-at-iridium π-allyl complexes that facilitate formation of congested tertiary-quaternary C-C bonds.

Regio- and Enantioselective Iridium-Catalyzed Amination of Racemic Branched Alkyl-Substituted Allylic Acetates with Primary and Secondary Aromatic and Heteroaromatic Amines.

The air- and water-stable π-allyliridium C,O-benzoate modified by ( S)-tol-BINAP, ( S)-Ir-II, catalyzes highly regio- and enantioselective Tsuji-Trost-type aminations of racemic branched alkyl-substituted allylic acetates using primary or secondary (hetero)aromatic amines. Specifically, in the presence of ( S)-Ir-II (5 mol%) in DME solvent at 60-70 °C, α-methyl allyl acetate 1a (100 mol%) reacts with primary (hetero)aromatic amines 2a-2l (200 mol%) or secondary (hetero)aromatic amines 3a-3l (200 mol%) to form the branched products of allylic amination 4a-4l and 5a-5l, respectively, as single regioisomers in good to excellent yield with uniformly high levels of enantioselectivity. As illustrated by the conversion of heteroaromatic amine 3m to adducts 6a-6g, excellent levels of regio- and enantioselectivity are retained across diverse branched allylic acetates bearing normal alkyl or secondary alkyl substituents. For reactants 3n-3p, which incorporate both primary and secondary aryl amine moieties, regio- and enantioselective amination occurs with complete site-selectivity to furnish adducts 7a-7c. Mechanistic studies involving amination of the enantiomerically enriched, deuterium-labeled acetate 1h corroborate C-N bond formation via outer-sphere addition.

Regio- and Enantioselective Iridium-Catalyzed N-Allylation of Indoles and Related Azoles with Racemic Branched Alkyl-Substituted Allylic Acetates.

Cyclometallated π-allyliridium C,O-benzoates modified with (S)-tol-BINAP, which are stable to air, water, and SiO2 , catalyze highly enantioselective N-allylations of indoles and related azoles. This reaction complements previously reported metal-catalyzed indole allylations in that complete levels of N versus C3 and branched versus linear regioselectivity are observed.

Hydroamination versus Allylic Amination in Iridium-Catalyzed Reactions of Allylic Acetates with Amines: 1,3-Aminoalcohols via Ester-Directed Regioselectivity.

In the presence of a neutral dppf-modified iridium catalyst and Cs2CO3, linear allylic acetates react with primary amines to form products of hydroamination with complete 1,3-regioselectivity. The collective data, including deuterium labeling studies, corroborate a catalytic mechanism involving rapid, reversible acetate-directed aminoiridation with inner-sphere/outer-sphere crossover followed by turnover-limiting proto-demetalation mediated by amine.

Amphiphilic π-Allyliridium C,O-Benzoates Enable Regio- and Enantioselective Amination of Branched Allylic Acetates Bearing Linear Alkyl Groups.

The first examples of amphiphilic reactivity in the context of enantioselective catalysis are described. Commercially available π-allyliridium C,O-benzoates, which are stable to air, water and SiO2 chromatography, and are well-known to catalyze allyl acetate-mediated carbonyl allylation, are now shown to catalyze highly chemo-, regio- and enantioselective substitutions of branched allylic acetates bearing linear alkyl groups with primary amines.

Enantioselective Synthesis of des-Epoxy-Amphidinolide N.

The synthesis of des-epoxy-amphidinolide N was achieved in 22 longest linear and 33 total steps. Three generations of synthetic endeavors are reported herein. During the first generation, our key stitching strategy that highlighted an intramolecular Ru-catalyzed alkene-alkyne (Ru AA) coupling and a late-stage epoxidation proved successful, but the installation of the α,α'-dihydroxyl ketone motif employing a dihydroxylation method was problematic. Our second generation of synthetic efforts addressed the scalability problem of the southern fragment synthesis and significantly improved the efficiency of the atom-economical Ru AA coupling, but suffered from several protecting group-based issues that proved insurmountable. Finally, relying on a judicious protecting group strategy together with concise fragment preparation, des-epoxy-amphidinolide N was achieved in a convergent fashion. Calculations disclose a hydrogen-bonding bridge within amphidinolide N. Comparisons of 13C NMR chemical shift differences using our synthetic des-epoxy-amphidinolide N suggest that amphidinolide N and carbenolide I are probably identical.

Lithium Enolates in the Enantioselective Construction of Tetrasubstituted Carbon Centers with Chiral Lithium Amides as Noncovalent Stereodirecting Auxiliaries.

Lithium enolates derived from carboxylic acids are ubiquitous intermediates in organic synthesis. Asymmetric transformations with these intermediates, a central goal of organic synthesis, are typically carried out with covalently attached chiral auxiliaries. An alternative approach is to utilize chiral reagents that form discrete, well-defined aggregates with lithium enolates, providing a chiral environment conducive of asymmetric bond formation. These reagents effectively act as noncovalent, or traceless, chiral auxiliaries. Lithium amides are an obvious choice for such reagents as they are known to form mixed aggregates with lithium enolates. We demonstrate here that mixed aggregates can effect highly enantioselective transformations of lithium enolates in several classes of reactions, most notably in transformations forming tetrasubstituted and quaternary carbon centers. Easy recovery of the chiral reagent by aqueous extraction is another practical advantage of this one-step protocol. Crystallographic, spectroscopic, and computational studies of the central reactive aggregate, which provide insight into the origins of selectivity, are also reported.

Total Synthesis of (-)-Lasonolide A.

The lasonolides are novel polyketides that have displayed remarkable biological activity in vitro against a variety of cancer cell lines. Herein we describe our first-generation approach to the formal synthesis of lasonolide A. The key findings from these studies ultimately allowed us to go on and complete a total synthesis of lasonolide A. The convergent approach unites two highly complex fragments utilizing a Ru-catalyzed alkene-alkyne coupling. This type of coupling typically generates branched products; however, through a detailed investigation, we are now able to demonstrate that subtle structural changes to the substrates can alter the selectivity to favor the formation of the linear product. The synthesis of the fragments features a number of atom-economical transformations which are highlighted by the discovery of an engineered enzyme to perform a dynamic kinetic reduction of a ß-ketoester to establish the absolute stereochemistry of the southern tetrahydropyran ring with high levels of enantioselectivity.

Synthesis and biology of cyclic imine toxins, an emerging class of potent, globally distributed marine toxins.

From a small group of exotic compounds isolated only two decades ago, Cyclic Imine (CI) toxins have become a major class of marine toxins with global distribution. Their distinct chemical structure, biological mechanism of action, and intricate chemistry ensures that CI toxins will continue to be the subject of fascinating fundamental studies in the broad fields of chemistry, chemical biology, and toxicology. The worldwide occurrence of potent CI toxins in marine environments, their accumulation in shellfish, and chemical stability are important considerations in assessing risk factors for human health. This review article aims to provide an account of chemistry, biology, and toxicology of CI toxins from their discovery to the present day.

A concise synthesis of (-)-lasonolide A.

Lasonolide A is a novel polyketide displaying potent anticancer activity across a broad range of cancer cell lines. Here, an enantioselective convergent total synthesis of the (-)-lasonolide A in 16 longest linear and 34 total steps is described. This approach significantly reduces the step count compared to other known syntheses. The synthetic strategy utilizes alkyne-bearing substrates as core building blocks and is highlighted by stitching together two similarly complex halves via a key Ru-catalyzed alkene-alkyne coupling and macrolactionization.

Enediolate-dilithium amide mixed aggregates in the enantioselective alkylation of arylacetic acids: structural studies and a stereochemical model.

A combination of X-ray crystallography, (6)Li, (15)N, and (13)C NMR spectroscopies, and density functional theory computations affords insight into the structures and reactivities of intervening aggregates underlying highly selective asymmetric alkylations of carboxylic acid dianions (enediolates) mediated by the dilithium salt of a C2-symmetric chiral tetraamine. Crystallography shows a trilithiated n-butyllithium-dilithiated amide that has dimerized to a hexalithiated form. Spectroscopic studies implicate the non-dimerized trilithiated mixed aggregate. Reaction of the dilithiated amide with the dilithium enediolate derived from phenylacetic acid affords a tetralithio aggregate comprised of the two dianions in solution and the dimerized octalithio form in the solid state. Computational studies shed light on the details of the solution structures and afford a highly predictive stereochemical model.

Stability of cyclic imine toxins: interconversion of pinnatoxin amino ketone and pinnatoxin A in aqueous media.

Pinnatoxins belong to the cyclic imine (CI) group of marine toxins with a unique toxicological profile. The need for structural integrity of the aliphatic 7-membered cyclic imine for the potent bioactivity of pinnatoxins has been experimentally demonstrated. In this study, we probe interconversion of the natural cyclic imine and its open form, pinnatoxin A amino ketone (PnTX AK), under physiologically relevant aqueous conditions. Our studies demonstrate the high stability of PnTX A. The unusual stability of the imine ring in PnTX A has implications for its oral toxicity and detoxification. These studies, as well the access to PnTX amino ketone, were enabled by the total synthesis of (+)-pinnatoxin A completed previously in our laboratory.

Kinetic, ESI-CID-MS and Computational Studies of π-Allyliridium C,O-Benzoate-Catalyzed Allylic Amination: Understanding the Effect of Cesium Ion.

The mechanism of π-allyliridium C,O-benzoate-catalyzed allylic amination was studied by (a) reaction progress kinetic analysis (RPKA), (b) tandem ESI-MS analysis, and (c) computational studies involving density functional theory (DFT) calculations. Reaction progress kinetic analysis (RPKA) reveals a zero-order dependence on allyl acetate, first-order dependence on catalyst and fractional-order dependence on amine. These data corroborate rapid ionization of the allylic acetate followed by turnover limiting C-N bond formation. To illuminate the origins of the 0.4 kinetic order dependence on amine, ESI-MS analyses of quaternary ammonium-labelled piperazine with multistage collision induced dissociation (CID) were conducted that corroborate intervention of cesium-bridged amine dimers that dissociate to form monomeric cesium amide nucleophiles. Computational data align with RPKA and ESI-CID-MS analyses and suggest early transition states mitigate the impact of steric factors, thus enabling formation of highly substituted C-N bonds with complete levels of branched regioselectivity. Specifically, trans-effects of the iridium complex facilitate nucleophilic attack at the more substituted allyl terminus trans to phosphorus with enantioselectivity governed by steric repulsions between the chiral bisphosphine ligand and the π-allyl of a dominant diastereomer of the stereogenic-at-metal complex. Beyond defining aspects of the mechanism of π-allyliridium C,O-benzoate-catalyzed allylic amination, these data reveal that a key feature of cesium carbonate not only lies in its enhanced basicity, but also its capacity for Lewis-acid enhanced Brønsted acidification of amines.

Regio- and Enantioselective Iridium-Catalyzed Amination of Alkyl-Substituted Allylic Acetates with Secondary Amines.

Robust air-stable cyclometalated π-allyliridium C,O-benzoates modified by (S)-tol-BINAP catalyze the reaction of secondary aliphatic amines with racemic alkyl-substituted allylic acetates to furnish products of allylic amination with high levels of enantioselectivity. Complete branched regioselectivities were observed despite the formation of more highly substituted C-N bonds.

Heterobivalent Inhibitors of Acetyl-CoA Carboxylase: Drug Target Residence Time and Time-Dependent Antibacterial Activity.

The relationship between drug-target residence time and the post-antibiotic effect (PAE) provides insights into target vulnerability. To probe the vulnerability of bacterial acetyl-CoA carboxylase (ACC), a series of heterobivalent inhibitors were synthesized based on pyridopyrimidine 1 and moiramide B (3) which bind to the biotin carboxylase and carboxyltransferase ACC active sites, respectively. The heterobivalent compound 17, which has a linker of 50 Å, was a tight binding inhibitor of Escherichia coli ACC (Kiapp 0.2 nM) and could be displaced from ACC by a combination of both 1 and 3 but not just by 1. In agreement with the prolonged occupancy of ACC resulting from forced proximity binding, the heterobivalent inhibitors produced a PAE in E. coli of 1-4 h in contrast to 1 and 3 in combination or alone, indicating that ACC is a vulnerable target and highlighting the utility of kinetic, time-dependent effects in the drug mechanism of action.

Overcoming Preclinical Safety Obstacles to Discover (S)-N-((1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)carbamoyl)-6-(methylamino)-6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazine-3-sulfonamide (GDC-2394): A Potent and Selective NLRP3 Inhibitor.

Inappropriate activation of the NLRP3 inflammasome has been implicated in multiple inflammatory and autoimmune diseases. Herein, we aimed to develop novel NLRP3 inhibitors that could minimize the risk of drug-induced liver injury. Lipophilic ligand efficiency was used as a guiding metric to identify a series of 6,7-dihydro-5H-pyrazolo[5,1-b][1,3]oxazinesulfonylureas. A leading compound from this series was advanced into safety studies in cynomolgus monkeys, and renal toxicity, due to compound precipitation, was observed. To overcome this obstacle, we focused on improving the solubility of our compounds, specifically by introducing basic amine substituents into the scaffold. This led to the identification of GDC-2394, a potent and selective NLRP3 inhibitor, with an in vitro and in vivo safety profile suitable for advancement into human clinical trials.