Identification of potent, selective and orally bioavailable phenyl ((R)-3-phenylpyrrolidin-3-yl)sulfone analogues as RORγt inverse agonists.

An X-ray crystal structure of one of our previously discovered RORγt inverse agonists bound to the RORγt ligand binding domain revealed that the cyclohexane carboxylic acid group of compound 2 plays a significant role in RORγt binding, forming four hydrogen bonding and ionic interactions with RORγt. SAR studies centered around the cyclohexane carboxylic acid group led to identification of several structurally diverse and more potent compounds, including new carboxylic acid analogues 7 and 20, and cyclic sulfone analogues 34 and 37. Notably, compounds 7 and 20 were found to maintain the desirable pharmacokinetic profile of 2.

PK/PD Disconnect Observed with a Reversible Endothelial Lipase Inhibitor.

Screening of a small set of nonselective lipase inhibitors against endothelial lipase (EL) identified a potent and reversible inhibitor, N-(3-(3,4-dichlorophenyl)propyl)-3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamide (5; EL IC50 = 61 nM, ELHDL IC50 = 454 nM). Deck mining identified a related hit, N-(3-(3,4-dichlorophenyl)propyl)-4-hydroxy-1-methyl-5-oxo-2,5-dihydro-1H-pyrrole-3-carboxamide (6a; EL IC50 = 41 nM, ELHDL IC50 = 1760 nM). Both compounds were selective against lipoprotein lipase (LPL) but nonselective versus hepatic lipase (HL). Optimization of compound 6a for EL inhibition using HDL as substrate led to N-(4-(3,4-dichlorophenyl)butan-2-yl)-1-ethyl-4-hydroxy-5-oxo-2,5-dihydro-1H-pyrrole-3-carboxamide (7c; EL IC50 = 148 nM, ELHDL IC50 = 218 nM) having improved PK over compound 6a, providing a tool molecule to test for the ability to increase HDL-cholesterol (HDL-C) levels in vivo using a reversible EL inhibitor. Compound 7c did not increase HDL-C in vivo despite achieving plasma exposures targeted on the basis of enzyme activity and protein binding demonstrating the need to develop more physiologically relevant in vitro assays to guide compound progression for in vivo evaluation.

Identification of a Potent, Selective, and Efficacious Phosphatidylinositol 3-Kinase δ (PI3Kδ) Inhibitor for the Treatment of Immunological Disorders.

PI3Kδ plays an important role controlling immune cell function and has therefore been identified as a potential target for the treatment of immunological disorders. This article highlights our work toward the identification of a potent, selective, and efficacious PI3Kδ inhibitor. Through careful SAR, the successful replacement of a polar pyrazole group by a simple chloro or trifluoromethyl group led to improved Caco-2 permeability, reduced Caco-2 efflux, reduced hERG PC activity, and increased selectivity profile while maintaining potency in the CD69 hWB assay. The optimization of the aryl substitution then identified a 4'-CN group that improved the human/rodent correlation in microsomal metabolic stability. Our lead molecule is very potent in PK/PD assays and highly efficacious in a mouse collagen-induced arthritis model.

Discovery of 7-(3-(piperazin-1-yl)phenyl)pyrrolo[2,1-f][1,2,4]triazin-4-amine derivatives as highly potent and selective PI3Kδ inhibitors.

As demonstrated in preclinical animal models, the disruption of PI3Kδ expression or its activity leads to a decrease in inflammatory and immune responses. Therefore, inhibition of PI3Kδ may provide an alternative treatment for autoimmune diseases, such as RA, SLE, and respiratory ailments. Herein, we disclose the identification of 7-(3-(piperazin-1-yl)phenyl)pyrrolo[2,1-f][1,2,4]triazin-4-amine derivatives as highly potent, selective and orally bioavailable PI3Kδ inhibitors. The lead compound demonstrated efficacy in an in vivo mouse KLH model.

Discovery of 5-Phenyl-N-(pyridin-2-ylmethyl)-2-(pyrimidin-5-yl)quinazolin-4-amine as a Potent I Kur Inhibitor.

A new series of phenylquinazoline inhibitors of Kv 1.5 is disclosed. The series was optimized for Kv 1.5 potency, selectivity versus hERG, pharmacokinetic exposure, and pharmacodynamic potency. 5-Phenyl-N-(pyridin-2-ylmethyl)-2-(pyrimidin-5-yl)quinazolin-4-amine (13k) was identified as a potent and ion channel selective inhibitor with robust efficacy in the preclinical rat ventricular effective refractory period (VERP) model and the rabbit atrial effective refractory period (AERP) model.

Discovery and SAR of pyrrolo[2,1-f][1,2,4]triazin-4-amines as potent and selective PI3Kδ inhibitors.

Aberrant Class I PI3K signaling is a key factor contributing to many immunological disorders and cancers. We have identified 4-amino pyrrolotriazine as a novel chemotype that selectively inhibits PI3Kδ signaling despite not binding to the specificity pocket of PI3Kδ isoform. Structure activity relationship (SAR) led to the identification of compound 30 that demonstrated efficacy in mouse Keyhole Limpet Hemocyanin (KLH) and collagen induced arthritis (CIA) models.

Pseudosaccharin amines as potent and selective KV1.5 blockers.

Phenethyl aminoheterocycles like compound 1 were known to be potent I(Kur) blockers although they lacked potency in vivo. Modification of the heterocycle led to the design and synthesis of pseudosaccharin amines. Compounds such as 14, 17d and 21c were found to be potent K(V)1.5 blockers and selective over other cardiac ion channels. These compounds had potent pharmacodynamic activity, however, they also showed off-target activities such as hemodynamic effects.

Chemical and enzymatic reductive activation of acylfulvene to isomeric cytotoxic reactive intermediates.

Acylfulvenes (AFs), a class of semisynthetic analogues of the sesquiterpene natural product illudin S, are cytotoxic toward cancer cells. The minor structural changes between illudin S and AFs translate to an improved therapeutic window in preclinical cell-based assays and xenograft models. AFs are, therefore, unique tools for addressing the chemical and biochemical basis of cytotoxic selectivity. AFs elicit cytotoxic responses by alkylation of biological targets, including DNA. While AFs are capable of direct alkylation, cytosolic reductive bioactivation to an electrophilic intermediate is correlated with enhanced cytotoxicity. Data obtained in this study illustrate chemical aspects of the process of AF activation. By tracking reaction mechanisms with stable isotope-labeled reagents, enzymatic versus chemical activation pathways for AF were compared for reactions involving the NADPH-dependent enzyme prostaglandin reductase 1 (PTGR1) or sodium borohydride, respectively. These two processes resulted in isomeric products that appear to give rise to similar patterns of DNA modification. The chemically activated isomer has been newly isolated and chemically characterized in this study, including an assessment of its relative stereochemistry and stability at varying pH and under bioassay conditions. In mammalian cancer cells, this chemically activated analogue was shown to not rely on further cellular activation to significantly enhance cytotoxic potency, in contrast to the requirements of AF. On the basis of this study, we anticipate that the chemically activated form of AF will serve as a useful chemical probe for evaluating biomolecular interactions independent of enzyme-mediated activation.

Quantitative correlation of drug bioactivation and deoxyadenosine alkylation by acylfulvene.

Acylfulvenes (AFs) are a class of antitumor agents that exert their cytotoxic effects by forming covalent adducts with biomolecules, including DNA and proteins; clinical trials are ongoing for (-)-(hydroxymethyl)AF. Recently, depurinating DNA adducts N3-AF-deoxyadenosine (dAdo) and N7-AF-deoxyguanosine (dGuo) were identified from reactions of the parent compound, AF, with calf thymus DNA in the presence of the reductase enzyme alkenal/one oxidoreductase (AOR) and cofactor NADPH. We report here the development of a structure-specific quantitative analytical method for evaluating levels of the major base adduct N3-AF-adenine (Ade), which results from depurination of N3-AF-dAdo, and its utilization to further probe the relationship between AOR-mediated bioactivation and adduct formation in a cell-free system. As an internal standard, the isotopomer N3-AF-Ade-d3 was synthesized, and electrospray-ionization mass spectrometry coupled with high-performance liquid chromatography (HPLC-ESI-MS/MS) was used to detect and quantitate the adduct. This method was validated and found to be accurate (R2>or=0.99) and precise (relative standard deviation 5.8-6.4%), with a limit of detection of 2 fmol. DNA samples, to which the stable-isotope-labeled internal standard was added, were subjected to neutral thermal hydrolysis yielding N3-AF-Ade. Adducts were isolated by a simple solid-liquid methanol extraction procedure, and adduct formation was examined in the presence of either high (1-3 micromol) or low (15 nmol) levels of DNA. Absolute amounts of N3-AF-Ade were measured in cell-free reaction mixtures containing varying levels of AOR as the only drug-activating enzyme. The increase in adduct formation (5-100 adducts per 10(5) DNA bases) over a range of enzyme concentrations (1-24 nM of AOR) showed saturation type behavior. This study reports a sensitive HPLC-ESI-MS/MS method for quantitation of the major DNA adduct induced by AF and illustrates a correlation between N3-AF-Ade formation and AOR-mediated enzymatic activation in a cell-free system, thus providing a template for further studies of drug toxicity in cells and in vivo.

Investigating the role of stereochemistry in the activity of anticancer acylfulvenes: synthesis, reductase-mediated bioactivation, and cellular toxicity.

Acylfulvenes comprise a family of semisynthetic natural product derivatives with potent antitumor activities. Previous studies indicated that acylfulvenes are bioactivated by NADPH-dependent alkenal/one reductase (AOR), presumably generating intermediates with the capacity to alkylate cellular targets, such as DNA, proteins, and glutathione. This process is thought to induce apoptosis, and the chemical and biochemical pathways involved are topics of current investigation. In this study, four acylfulvene analogues were synthesized: (-)-acylfulvene, (+)-acylfulvene, (-)-(hydroxymethyl)acylfulvene, and (+)-(hydroxymethyl)acylfulvene. These compounds were synthesized by a chiral-resolution method, described for the first time in this report, and by asymmetric synthesis using a method formally demonstrated previously. Cell toxicity studies indicate a positive correlation between AOR level and acylfulvene sensitivity. The absolute configuration of acylfulvene analogues has a significant influence on cytotoxicity. (-)-(Hydroxymethyl)acylfulvene is 25 times more potent than (+)-(hydroxymethyl)acylfulvene in cells transfected with an AOR overexpression vector. Based on kinetic parameters, the rates of AOR-mediated activation are more strongly dependent on acylfulvene substitution than on absolute stereochemistry. These data support the role of AOR-mediated metabolism and indicate the involvement of other stereochemically dictated pathways, such as transport and biomolecule binding, in contributing to the cytotoxicity of acylfulvenes.