Timothy D. Owens

Prolonged and tunable residence time using reversible covalent kinase inhibitors

Drugs with prolonged, on-target residence time often show superior efficacy, yet general strategies for optimizing drug-target residence time are lacking. Here, we demonstrate progress toward this elusive goal by targeting a noncatalytic cysteine in Brutons tyrosine kinase (BTK) with reversible covalent inhibitors. Utilizing an inverted orientation of the cysteine-reactive cyanoacrylamide electrophile, we identified potent and selective BTK inhibitors that demonstrate biochemical residence times spanning from minutes to 7 days. An inverted cyanoacrylamide with prolonged residence time in vivo remained bound to BTK more than 18 hours after clearance from the circulation. The inverted cyanoacrylamide strategy was further utilized to discover fibroblast growth factor receptor (FGFR) kinase inhibitors with residence times of several days, demonstrating generalizability of the approach. Targeting noncatalytic cysteines with inverted cyanoacrylamides may serve as a broadly applicable platform that facilitates “residence time by design”, the ability to modulate and improve the duration of target engagement in vivo.

RN486, a Selective Bruton's Tyrosine Kinase Inhibitor, Abrogates Immune Hypersensitivity Responses and Arthritis in Rodents

Genetic mutation and pharmacological inhibition of Brutons tyrosine kinase (Btk) both have been shown to prevent the development of collagen-induced arthritis (CIA) in mice, providing a rationale for the development of Btk inhibitors for treating rheumatoid arthritis (RA). In the present study, we characterized a novel Btk inhibitor, 6-cyclopropyl-8-fluoro-2-(2-hydroxymethyl-3-{1-methyl-5-[5-(4-methyl-piperazin-1-yl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-2H-isoquinolin-1-one (RN486), in vitro and in rodent models of immune hypersensitivity and arthritis. We demonstrated that RN486 not only potently and selectively inhibited the Btk enzyme, but also displayed functional activities in human cell-based assays in multiple cell types, blocking Fcε receptor cross-linking-induced degranulation in mast cells (IC50 = 2.9 nM), Fcγ receptor engagement-mediated tumor necrosis factor α production in monocytes (IC50 = 7.0 nM), and B cell antigen receptor-induced expression of an activation marker, CD69, in B cells in whole blood (IC50 = 21.0 nM). RN486 displayed similar functional activities in rodent models, effectively preventing type I and type III hypersensitivity responses. More importantly, RN486 produced robust anti-inflammatory and bone-protective effects in mouse CIA and rat adjuvant-induced arthritis (AIA) models. In the AIA model, RN486 inhibited both joint and systemic inflammation either alone or in combination with methotrexate, reducing both paw swelling and inflammatory markers in the blood. Together, our findings not only demonstrate that Btk plays an essential and conserved role in regulating immunoreceptor-mediated immune responses in both humans and rodents, but also provide evidence and mechanistic insights to support the development of selective Btk inhibitors as small-molecule disease-modifying drugs for RA and potentially other autoimmune diseases.

Bruton’s Tyrosine Kinase Inhibitors: Approaches to Potent and Selective Inhibition, Preclinical and Clinical Evaluation for Inflammatory Diseases and B Cell Malignancies

■ INTRODUCTION Bruton’s tyrosine kinase (BTK) is a member of the Tec tyrosine kinase family. BTK is expressed in most hematopoietic cells such as B cells, mast cells, and macrophages but not in T cells, natural killer cells, and plasma cells. BTK plays key roles in multiple cell signaling pathways including BCR and FcR signaling cascades. Mutations in the human BTK gene cause the inherited disease X-linked agammaglobulinemia (XLA), with lack of peripheral B cells and low levels of serum Ig. In XLA, the primary immune deficit is B cell specific. In fact Rituxan, a CD20 antibody, has impacted B cells on the pathogenesis of many autoimmune diseases, such as RA, SLE, and MS. This has fueled interest by multiple pharmaceutical companies in pursuing small molecule BTK inhibitors in the treatment of autoimmune diseases. Likewise, there is also interest in the development of BTK inhibitors for the treatment of hematological malignancies, as aberrant activating BTK has been implicated in the pathogenesis of B cell lymphoma. Detailed reviews and articles on BTK biology and its therapeutic potentials have been reported. Kinase selectivity is a central issue in discovering efficacious and safe small molecule inhibitors for kinase targets, especially for non-life-threatening diseases such as RA. To prevent adverse toxicological events caused by immunological responses, the pharmaceutical industry has largely focused its small molecule drug discovery efforts on agents that interact noncovalently with their target proteins. This strategy has been followed despite numerous examples of marketed drugs with target-specific covalent mode-of-action. In the case of protein kinase targets for which selectivity and efficacy pose major challenges for noncovalent inhibitors, targeted covalent inhibition has provided an attractive alternative. Both approaches, noncovalent and covalent inhibition of protein kinases, benefit tremendously from high resolution structural information from protein crystal structures. This article will review BTK structural biology with a focus on design features for selective BTK inhibitors. We first summarize the publically available structural information on the BTK kinase domain. Then we will provide a brief summary and analysis with key SAR information for the most potent inhibitors reported for the chemical classes that have been disclosed in patents and publications. When available, preclinical and clinical data for advanced compounds will be summarized.

Concise total synthesis of the prolyl endopeptidase inhibitor eurystatin A via a novel Passerini reaction-deprotection-acyl migration strategy †

Abstract The Passerini reaction between suitably protected alaninal, leucine isonitrile, and ornithine components delivered adducts 10a , b in high yield. Orthogonal N -deprotection of 10a led, via a smooth O - to N -acyl migration, to 11 , which constitutes the entire skeleton of the eurystatins. Subsequent deprotection, macrocyclization, elaboration, and final oxidation steps efficiently afforded eurystatin A 1a in high overall yield.

Structure-Based Drug Design of RN486, a Potent and Selective Bruton's Tyrosine Kinase (BTK) Inhibitor, for the Treatment of Rheumatoid Arthritis.

Structure-based drug design was used to guide the optimization of a series of selective BTK inhibitors as potential treatments for Rheumatoid arthritis. Highlights include the introduction of a benzyl alcohol group and a fluorine substitution, each of which resulted in over 10-fold increase in activity. Concurrent optimization of drug-like properties led to compound 1 (RN486) ( J. Pharmacol. Exp. Ther. 2012 , 341 , 90 ), which was selected for advanced preclinical characterization based on its favorable properties.

Pyrrolopyrazines as selective spleen tyrosine kinase inhibitors.

We describe the discovery of several pyrrolopyrazines as potent and selective Syk inhibitors and the efforts that eventually led to the desired improvements in physicochemical properties and human whole blood potencies. Ultimately, our mouse model revealed unexpected toxicity that precluded us from further advancing this series.

Finding the perfect spot for fluorine: improving potency up to 40-fold during a rational fluorine scan of a Bruton's Tyrosine Kinase (BTK) inhibitor scaffold.

A rational fluorine scan based on co-crystal structures was explored to increase the potency of a series of selective BTK inhibitors. While fluorine substitution on a saturated bicyclic ring system yields no apparent benefit, the same operation on an unsaturated bicyclic ring can increase HWB activity by up to 40-fold. Comparison of co-crystal structures of parent molecules and fluorinated counterparts revealed the importance of placing fluorine at the optimal position to achieve favorable interactions with protein side chains.

Potent and selective thrombin inhibitors featuring hydrophobic, basic P3P4-aminoalkyllactam moieties

Abstract Crystal structure and evolving SAR considerations of potent, selective benzylsulfonamide lactam thrombin inhibitors and related serine protease inhibitors have led to the design of novel thrombin inhibitors 1a-g , featuring hydrophobic, basic, P 4 -alkylaminolactam scaffolds that serve as novel types of P 3 P 4 dipeptide mimics. The design, synthesis, and biological activity of these targets is presented.

Novel, potent and selective chimeric FXa inhibitors featuring hydrophobic P1-ketoamide moieties.

Judicious combination of P-region sequences of highly potent anticoagulant proteins including NAP5, NAP6, Ecotin, and Antistasin with SAR from small molecule FXa inhibitors led to a series of chimeric inhibitors of formula 1a-j. We report herein the design, synthesis, and biological activity of this novel family of FXa inhibitors that express both high in vitro potency and superb selectivity against related serine proteases.

Alkoxide-catalyzed ring-opening of a novel homosaccharin derivative: synthesis of potent, selective P3-lactam thrombin inhibitors containing P4-o-alkoxycarbonylbenzylsulfonamide residues

A series of lactam derivatives 1b-g featuring P4-o-alkoxycarbonylbenzylsulfonamide residues along with the potential P4-homosaccharin prodrug candidate 1h was prepared in order to probe the thrombin S3 specificity pocket. The synthesis and alkoxide-catalyzed ring opening of the novel homosaccharin intermediate 7 followed by subsequent elaboration delivered the targets 1b-h which were potent and selective thrombin inhibitors. The design, synthesis, and biological activity of these targets will be presented.