Discovery of Covalent Bruton's Tyrosine Kinase Inhibitors with Decreased CYP2C8 Inhibitory Activity.

Bruton's tyrosine kinase (BTK) is a member of the Tec kinase family that is expressed in cells of hematopoietic lineage. Evidence has shown that inhibition of BTK has clinical benefit for the treatment of a wide array of autoimmune and inflammatory diseases. Previously we reported the discovery of a novel nicotinamide selectivity pocket (SP) series of potent and selective covalent irreversible BTK inhibitors. The top molecule 1 of that series strongly inhibited CYP2C8 (IC50 =100 nM), which was attributed to the bridged linker group. However, our effort on the linker replacement turned out to be fruitless. With the study of the X-ray crystal structure of compound 1, we envisioned the opportunity of removal of this liability via transposition of the linker moiety in 1 from C6 to C5 position of the pyridine core. With this strategy, our optimization led to the discovery of a novel series, in which the top molecule 18 A displayed reduced CYP inhibitory activity and good potency. To further explore this new series, different warheads besides acrylamide, for example cyanamide, were also tested. However, this effort didn't lead to the discovery of molecules with better potency than 18 A. The loss of potency in those molecules could be related to the reduced reactivity of the warhead or reversible binding mode. Further profiling of 18 A disclosed that it had a strong hERG (human Ether-a-go-go Related Gene) inhibition, which could be related to the phenoxyphenyl group.

Discovery of 4-aminopyrimidine analogs as highly potent dual P70S6K/Akt inhibitors.

Activation of the PI3K/Akt/mTOR kinase pathway is associated with human cancers. A dual p70S6K/Akt inhibitor is sufficient to inhibit strong tumor growth and to block negative impact of the compensatory Akt feedback loop activation. A scaffold docking strategy based on an existing quinazoline carboxamide series identified 4-aminopyrimidine analog 6, which showed a single-digit nanomolar and a micromolar potencies in p70S6K and Akt enzymatic assays. SAR optimization improved Akt enzymatic and p70S6K cellular potencies, reduced hERG liability, and ultimately discovered the promising candidate 37, which exhibited with a single digit nanomolar value in both p70S6K and Akt biochemical assays, and hERG activities (IC50 = 17.4 µM). This agent demonstrated dose-dependent efficacy in inhibiting mice breast cancer tumor growth and covered more than 90% pS6 inhibition up to 24 h at a dose of 200 mg/kg po.

Discovery of potent and selective reversible Bruton's tyrosine kinase inhibitors.

Bruton's tyrosine kinase (BTK) is a cytoplasmic, non-receptor tyrosine kinase member of the TEC family of tyrosine kinases. Pre-clinical and clinical data have shown that targeting BTK can be used for the treatment for B-cell disorders. Here we disclose the discovery of a novel imidazo[4,5-b]pyridine series of potent, selective reversible BTK inhibitors through a rational design approach. From a starting hit molecule 1, medicinal chemistry optimization led to the development of a lead compound 30, which exhibited 58 nM BTK inhibitory potency in human whole blood and high kinome selectivity. Additionally, the compound demonstrated favorable pharmacokinetics (PK), and showed potent dose-dependent efficacy in a rat CIA model.

Discovery of Evobrutinib: An Oral, Potent, and Highly Selective, Covalent Bruton's Tyrosine Kinase (BTK) Inhibitor for the Treatment of Immunological Diseases.

Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib hold a prominent role in the treatment of B cell malignancies. However, further refinement is needed to this class of agents, particularly in terms of adverse events (potentially driven by kinase promiscuity), which preclude their evaluation in nononcology indications. Here, we report the discovery and preclinical characterization of evobrutinib, a potent, obligate covalent inhibitor with high kinase selectivity. Evobrutinib displayed sufficient preclinical pharmacokinetic and pharmacodynamic characteristics which allowed for in vivo evaluation in efficacy models. Moreover, the high selectivity of evobrutinib for BTK over epidermal growth factor receptor and other Tec family kinases suggested a low potential for off-target related adverse effects. Clinical investigation of evobrutinib is ongoing in several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.

Efficacy and Pharmacodynamic Modeling of the BTK Inhibitor Evobrutinib in Autoimmune Disease Models.

Because of its role in mediating both B cell and Fc receptor signaling, Bruton's tyrosine kinase (BTK) is a promising target for the treatment of autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Evobrutinib is a novel, highly selective, irreversible BTK inhibitor that potently inhibits BCR- and Fc receptor-mediated signaling and, thus, subsequent activation and function of human B cells and innate immune cells such as monocytes and basophils. We evaluated evobrutinib in preclinical models of RA and SLE and characterized the relationship between BTK occupancy and inhibition of disease activity. In mouse models of RA and SLE, orally administered evobrutinib displayed robust efficacy, as demonstrated by reduction of disease severity and histological damage. In the SLE model, evobrutinib inhibited B cell activation, reduced autoantibody production and plasma cell numbers, and normalized B and T cell subsets. In the RA model, efficacy was achieved despite failure to reduce autoantibodies. Pharmacokinetic/pharmacodynamic modeling showed that mean BTK occupancy in blood cells of 80% was linked to near-complete disease inhibition in both RA and SLE mouse models. In addition, evobrutinib inhibited mast cell activation in a passive cutaneous anaphylaxis model. Thus, evobrutinib achieves efficacy by acting both on B cells and innate immune cells. Taken together, our data show that evobrutinib is a promising molecule for the chronic treatment of B cell-driven autoimmune disorders.

Discovery of Affinity-Based Probes for Btk Occupancy Assays.

Bruton's tyrosine kinase (Btk) is an attractive target for the treatment of a wide array of B-cell malignancies and autoimmune diseases. Small-molecule covalent irreversible Btk inhibitors targeting Cys481 have been developed for the treatment of such diseases. In clinical trials, probe molecules are required in occupancy studies to measure the level of engagement of the protein by these covalent irreversible inhibitors. The result of this pharmacodynamic (PD) activity provides guidance for appropriate dosage selection to optimize inhibition of the drug target and correlation of target inhibition with disease treatment efficacy. This information is crucial for successful evaluation of drug candidates in clinical trials. Based on the pyridine carboxamide scaffold of a novel solvent-accessible pocket (SAP) series of covalent irreversible Btk inhibitors, we successfully developed a potent and selective affinity-based biotinylated probe 12 (2-[(4-{4-[5-(1-{5-[(3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanamido}-3,6,9,12-tetraoxapentadecan-15-amido)pentanoyl]piperazine-1-carbonyl}phenyl)amino]-6-[1-(prop-2-enoyl)piperidin-4-yl]pyridine-3-carboxamide). Compound 12 has been used in Btk occupancy assays for preclinical studies to determine the therapeutic efficacy of Btk inhibition in two mouse lupus models driven by TLR7 activation and type I interferon.

Discovery of a novel series of pyridine and pyrimidine carboxamides as potent and selective covalent inhibitors of Btk.

Btk is an attractive target for the treatment of a range of Bcell malignancies as well as several autoimmune diseases such as murine lupus and rheumatoid arthritis. Several covalent irreversible inhibitors of Btk are currently in development including ibrutinib which was approved for treatment of B-cell malignancies. Herein, we describe our efforts using X-ray guided structure based design (SBD) to identify a novel chemical series of covalent Btk inhibitors. The resulting pyridine carboxamides were potent and selective inhibitors of Btk having excellent enzymatic and cellular inhibitory activity.

Optimization of the efflux ratio and permeability of covalent irreversible BTK inhibitors.

Bruton's tyrosine kinase (Btk) is a member of the Tec kinase family that is expressed in cells of hematopoietic lineage (e.g. B cells, macrophages, monocytes, and mast cells). Small molecule covalent irreversible Btk inhibitors targeting Cys481 within the ATP-binding pocket have been applied in the treatment of B-cell malignancies. Starting from a fragment, we discovered a novel series of potent covalent irreversible Btk inhibitors that bear N-linked groups occupying the solvent accessible pocket (SAP) of the active site of the Btk kinase domain. The hit molecules, however, displayed high P-gp mediated efflux ratio (ER) and poor A-B permeability in Caco-2 assay. By decreasing tPSA, installing steric hindrance and adjusting clogP, one top molecule 9 was discovered, which showed a 99% decrease in efflux ratio and a 90-fold increase in A-B permeability compared to hit molecule 1.

Discovery of potent, highly selective covalent irreversible BTK inhibitors from a fragment hit.

Bruton's Tyrosine Kinase (BTK) is a member of the TEC kinase family that is expressed in cells of hematopoietic lineage (e.g., in B cells, macrophages, monocytes, and mast cells). Small molecule covalent irreversible BTK inhibitor targeting Cys481 within the ATP-binding pocket, for example ibrutinib, has been applied in the treatment of B-cell malignancies. Starting from a fragment hit, we discovered a novel series of potent covalent irreversible BTK inhibitors that occupy selectivity pocket of the active site of the BTK kinase domain. Guided by X-ray structures and a fragment-based drug design (FBDD) approach, we generated molecules showing comparable cellular potency to ibrutinib and higher kinome selectivity against undesirable off-targets like EGFR.

Ability of Bruton's Tyrosine Kinase Inhibitors to Sequester Y551 and Prevent Phosphorylation Determines Potency for Inhibition of Fc Receptor but not B-Cell Receptor Signaling.

Bruton's tyrosine kinase (Btk) is expressed in a variety of hematopoietic cells. Btk has been demonstrated to regulate signaling downstream of the B-cell receptor (BCR), Fc receptors (FcRs), and toll-like receptors. It has become an attractive drug target because its inhibition may provide significant efficacy by simultaneously blocking multiple disease mechanisms. Consequently, a large number of Btk inhibitors have been developed. These compounds have diverse binding modes, and both reversible and irreversible inhibitors have been developed. Reported herein, we have tested nine Btk inhibitors and characterized on a molecular level how their interactions with Btk define their ability to block different signaling pathways. By solving the crystal structures of Btk inhibitors bound to the enzyme, we discovered that the compounds can be classified by their ability to trigger sequestration of Btk residue Y551. In cells, we found that sequestration of Y551 renders it inaccessible for phosphorylation. The ability to sequester Y551 was an important determinant of potency against FcεR signaling as Y551 sequestering compounds were more potent for inhibiting basophils and mast cells. This result was true for the inhibition of FcγR signaling as well. In contrast, Y551 sequestration was less a factor in determining potency against BCR signaling. We also found that Btk activity is regulated differentially in basophils and B cells. These results elucidate important determinants for Btk inhibitor potency against different signaling pathways and provide insight for designing new compounds with a broader inhibitory profile that will likely result in greater efficacy.

Btk inhibition treats TLR7/IFN driven murine lupus.

Bruton's tyrosine kinase (Btk) is expressed in a variety of immune cells and previous work has demonstrated that blocking Btk is a promising strategy for treating autoimmune diseases. Herein, we utilized a tool Btk inhibitor, M7583, to determine the therapeutic efficacy of Btk inhibition in two mouse lupus models driven by TLR7 activation and type I interferon. In BXSB-Yaa lupus mice, Btk inhibition reduced autoantibodies, nephritis, and mortality. In the pristane-induced DBA/1 lupus model, Btk inhibition suppressed arthritis, but autoantibodies and the IFN gene signature were not significantly affected; suggesting efficacy was mediated through inhibition of Fc receptors. In vitro studies using primary human macrophages revealed that Btk inhibition can block activation by immune complexes and TLR7 which contributes to tissue damage in SLE. Overall, our results provide translational insight into how Btk inhibition may provide benefit to a variety of SLE patients by affecting both BCR and FcR signaling.

Guanfacine ER for the treatment of adolescent attention-deficit/hyperactivity disorder.

INTRODUCTION: Guanfacine extended release (GXR) is an alpha 1A noradrenergic agonist that has been approved by the FDA for the treatment of Attention-Deficit/Hyperactivity Disorder (ADHD) as a monotherapy, and as an adjunctive therapy to stimulants for the treatment of ADHD in children and adolescents age 6 - 17. AREAS COVERED: PubMed, the Ovid Medline database, and the PsycInfo database were searched using the term 'guanfacine'. Results were then limited to criteria such as English and human, from 1990 through December 2011. The resulting yield from the comprehensive literature search was 4391 articles. The titles and abstracts of all articles were reviewed. Studies were selected for full-text review based upon their place in the hierarchy of evidence (e.g., randomized controlled trials), relevance and quality of individual studies, and generalizability to clinical practice. The search was augmented by further search of article reference lists. A total of 15 articles were selected for full-text examination. EXPERT OPINION: Due to the absence of positive evidence for the efficacy of GXR for monotherapy in adolescents, clinicians should be guarded in the use of GXR for monotherapy in adolescents with ADHD. The use of GXR has considerable promise as an adjunct to stimulants for other behavioral conditions associated with ADHD.