Design of Potent and Selective Covalent Inhibitors of Bruton's Tyrosine Kinase Targeting an Inactive Conformation.

Bruton's tyrosine kinase (BTK) is a member of the TEC kinase family and is selectively expressed in a subset of immune cells. It is a key regulator of antigen receptor signaling in B cells and of Fc receptor signaling in mast cells and macrophages. A BTK inhibitor will likely have a positive impact on autoimmune diseases which are caused by autoreactive B cells and immune-complex driven inflammation. We report the design, optimization, and characterization of potent and selective covalent BTK inhibitors. Starting from the selective reversible inhibitor 3 binding to an inactive conformation of BTK, we designed covalent irreversible compounds by attaching an electrophilic warhead to reach Cys481. The first prototype 4 covalently modified BTK and showed an excellent kinase selectivity including several Cys-containing kinases, validating the design concept. In addition, this compound blocked FcγR-mediated hypersensitivity in vivo. Optimization of whole blood potency and metabolic stability resulted in compounds such as 8, which maintained the excellent kinase selectivity and showed improved BTK occupancy in vivo.

An allosteric MALT1 inhibitor is a molecular corrector rescuing function in an immunodeficient patient.

MALT1 paracaspase is central for lymphocyte antigen-dependent responses including NF-κB activation. We discovered nanomolar, selective allosteric inhibitors of MALT1 that bind by displacing the side chain of Trp580, locking the protease in an inactive conformation. Interestingly, we had previously identified a patient homozygous for a MALT1 Trp580-to-serine mutation who suffered from combined immunodeficiency. We show that the loss of tryptophan weakened interactions between the paracaspase and C-terminal immunoglobulin MALT1 domains resulting in protein instability, reduced protein levels and functions. Upon binding of allosteric inhibitors of increasing potency, we found proportionate increased stabilization of MALT1-W580S to reach that of wild-type MALT1. With restored levels of stable MALT1 protein, the most potent of the allosteric inhibitors rescued NF-κB and JNK signaling in patient lymphocytes. Following compound washout, MALT1 substrate cleavage was partly recovered. Thus, a molecular corrector rescues an enzyme deficiency by substituting for the mutated residue, inspiring new potential precision therapies to increase mutant enzyme activity in other deficiencies.

Modulating ADME Properties by Fluorination: MK2 Inhibitors with Improved Oral Exposure.

MAP-activated protein kinase 2 (MK2) plays an important role in the regulation of innate immune response as well as in cell survival upon DNA damage. Despite its potential for the treatment of inflammation and cancer, to date no MK2 low molecular weight inhibitors have reached the clinic, mainly due to inadequate absorption, distribution, metabolism, and excretion (ADME) properties. We describe here an approach based on specifically placed fluorine within a recently described pyrrole-based MK2 inhibitor scaffold for manipulation of its physicochemical and ADME properties. While preserving target potency, the novel fluoro-derivatives showed greatly improved permeability as well as enhanced solubility and reduced in vivo clearance leading to significantly increased oral exposure.

Pathophysiological Consequences of a Break in S1P1-Dependent Homeostasis of Vascular Permeability Revealed by S1P1 Competitive Antagonism.

RATIONAL: Homeostasis of vascular barriers depends upon sphingosine 1-phosphate (S1P) signaling via the S1P1 receptor. Accordingly, S1P1 competitive antagonism is known to reduce vascular barrier integrity with still unclear pathophysiological consequences. This was explored in the present study using NIBR-0213, a potent and selective S1P1 competitive antagonist. RESULTS: NIBR-0213 was tolerated at the efficacious oral dose of 30 mg/kg BID in the rat adjuvant-induced arthritis (AiA) model, with no sign of labored breathing. However, it induced dose-dependent acute vascular pulmonary leakage and pleural effusion that fully resolved within 3-4 days, as evidenced by MRI monitoring. At the supra-maximal oral dose of 300 mg/kg QD, NIBR-0213 impaired lung function (with increased breathing rate and reduced tidal volume) within the first 24 hrs. Two weeks of NIBR-0213 oral dosing at 30, 100 and 300 mg/kg QD induced moderate pulmonary changes, characterized by alveolar wall thickening, macrophage accumulation, fibrosis, micro-hemorrhage, edema and necrosis. In addition to this picture of chronic inflammation, perivascular edema and myofiber degeneration observed in the heart were also indicative of vascular leakage and its consequences. CONCLUSIONS: Overall, these observations suggest that, in the rat, the lung is the main target organ for the S1P1 competitive antagonism-induced acute vascular leakage, which appears first as transient and asymptomatic but could lead, upon chronic dosing, to lung remodeling with functional impairments. Hence, this not only raises the question of organ specificity in the homeostasis of vascular barriers, but also provides insight into the pre-clinical evaluation of a potential safety window for S1P1 competitive antagonists as drug candidates.

An oral sphingosine 1-phosphate receptor 1 (S1P(1)) antagonist prodrug with efficacy in vivo: discovery, synthesis, and evaluation.

A prodrug approach to optimize the oral exposure of a series of sphingosine 1-phosphate receptor 1 (S1P(1)) antagonists for chronic efficacy studies led to the discovery of (S)-2-{[3'-(4-chloro-2,5-dimethylphenylsulfonylamino)-3,5-dimethylbiphenyl-4-carbonyl]methylamino}-4-dimethylaminobutyric acid methyl ester 14. Methyl ester prodrug 14 is hydrolyzed in vivo to the corresponding carboxylic acid 15, a potent and selective S1P(1) antagonist. Oral administration of the prodrug 14 induces sustained peripheral blood lymphocyte reduction in rats. In a rat cardiac transplantation model coadministration of a nonefficacious dose of prodrug 14 with a nonefficacious dose of sotrastaurin (19), a protein kinase C inhibitor, or everolimus (20), an mTOR inhibitor, effectively prolonged the survival time of rat cardiac allografts. This demonstrates that clinically useful immunomodulation mediated by the S1P(1) receptor can be achieved with an S1P(1) antagonist generated in vivo after oral administration of its prodrug.

A potent and selective S1P(1) antagonist with efficacy in experimental autoimmune encephalomyelitis.

Lymphocyte trafficking is critically regulated by the Sphingosine 1-phosphate receptor-1 (S1P(1)), a G protein-coupled receptor that has been highlighted as a promising therapeutic target in autoimmunity. Fingolimod (FTY720, Gilenya) is a S1P(1) receptor agonist that has recently been approved for the treatment of multiple sclerosis (MS). Here, we report the discovery of NIBR-0213, a potent and selective S1P(1) antagonist that induces long-lasting reduction of peripheral blood lymphocyte counts after oral dosing. NIBR-0213 showed comparable therapeutic efficacy to fingolimod in experimental autoimmune encephalomyelitis (EAE), a model of human MS. These data provide convincing evidence that S1P(1) antagonists are effective in EAE. In addition, the profile of NIBR-0213 makes it an attractive candidate to further study the consequences of S1P(1) receptor antagonism and to differentiate the effects from those of S1P(1) agonists.

Design and preparation of 2-benzamido-pyrimidines as inhibitors of IKK.

The design, synthesis, and the biological evaluation of 2-benzamido-pyrimidines as novel IKK inhibitors are described. By optimization of the lead compound 3, compounds 16 and 24 are identified as good inhibitors of IKK2 with IC(50) values of 40 and 25 nM, respectively. Compound 16 also demonstrated significant in vivo activity in an acute model of cytokine release.

A locally active antiinflammatory macrolide (MLD987) for inhalation therapy of asthma.

One of the characteristic features of asthma is a persistent pulmonary inflammation, with increased numbers of eosinophils and activated T-lymphocytes in the airways. T-helper cells of the Th2 phenotype play a pivotal role in the pathogenesis of asthma, and they are believed to orchestrate the asthmatic response by releasing a wide repertoire of cytokines. Herein, we describe the design, synthesis, and evaluation in models of allergic asthma of a locally active T-cell modulator, MLD987 (1). Compound 1 is a potent immunosuppressant that inhibits the activation, proliferation, and release of cytokines from T-cells with IC(50) values in the low nanomolar range. In a Brown-Norway rat model of allergic asthma, 1, when given into the airways by intratracheal administration (ED(50) = 1 mg/kg) or by inhalation (ED(50) = 0.4 mg/kg), potently reduced the influx of leukocytes into bronchoalveolar lavage fluid samples obtained from antigen-challenged animals. In contrast, 1 had an appreciably weaker activity in this model when given orally or intravenously. Pharmacokinetic evaluation in rat and rhesus monkey showed that 1 had both a low oral (2-4%) and a low pulmonary (7%, monkey) bioavailability. These findings are consistent with a local site of action of the compound and rule out that its antiinflammatory activity in the lung was caused by systemically absorbed material, which had been swallowed during inhalation or which had entered the circulation via the airways. Local administration and the metabolically soft structure of 1, which favors rapid systemic metabolism to less immunosuppressive metabolite 2, are the main reasons for the low exposure and weak systemic activity of the compound. Administration of a locally active compound such as 1, by inhalation, should reduce systemic side effects. Our results indicate that 1 has the potential to serve as an alternative to inhaled glucocorticosteroids for the long-term therapy of asthma of all grades of severity.

Pharmacological profile of a novel phosphodiesterase 4 inhibitor, 4-(8-benzo[1,2,5]oxadiazol-5-yl-[1,7]naphthyridin-6-yl)-benzoic acid (NVP-ABE171), a 1,7-naphthyridine derivative, with anti-inflammatory activities.

We investigated the pharmacology of a new class of phosphodiesterase 4 (PDE4) inhibitor, 6,8-disubstituted 1,7-naphthyridines, by using 4-(8-benzo[1,2,5]oxadiazol-5-yl-[1,7]naphthyridin-6-yl)-benzoic acid (NVP-ABE171) as a representative compound and compared its potency with the most advanced PDE4 inhibitor, undergoing clinical trials, Ariflo [cis-4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl-r-1-cyclohexanecarboxylic acid)]. NVP-ABE171 inhibited the activity of phosphodiesterase 4A, 4B, 4C, and 4D with respective IC(50) values of 602, 34, 1230, and 1.5 nM. Ariflo was about 40 times less potent. In human cells, NVP-ABE171 inhibited the eosinophil and neutrophil oxidative burst, the release of cytokines by T cells, and the tumor necrosis factor-alpha release from monocytes, in the nanomolar range. Ariflo presented a similar inhibition profile but was 7 to 50 times less potent. In BALB/c mice challenged with lipopolysaccharide, NVP-ABE171 inhibited the airway neutrophil influx and activation with an ED(50) in the range of 3 mg/kg. Ariflo was inactive up to a dose of 10 mg/kg. In ovalbumin sensitized Brown Norway rats, NVP-ABE171 inhibited the lipopolysaccharide-induced airway neutrophil influx and activation (ED(50) of 0.2 mg/kg) and the ovalbumin-induced airway eosinophil influx and activation (ED(50) of 0.1 mg/kg). Ariflo was about 100 times less potent in both models. In the ovalbumin model, NVP-ABE171 had a duration of action of more than 24 h. NVP-ABE171 is a novel PDE4 inhibitor that shows activity both in vitro on human inflammatory cells and in vivo in animal models of lung inflammation. This compound class may have potential for the treatment of airway inflammatory conditions such as asthma and chronic obstructive pulmonary diseases.

Synthesis of 4-(8-benzo[1,2,5]oxadiazol-5-yl-[1,7]naphthyridine-6-yl)-benzoic acid: a potent and selective phosphodiesterase type 4D inhibitor.

The synthesis of a 6,8-disubstituted 1,7-naphthyridine 1 and its characterization as a potent and selective phosphodiesterase type 4D inhibitor (IC(50)=1.5nM) are described. The compound inhibited TNFalpha-release from human peripheral blood mononuclear cells and was orally active in a model of adjuvant-induced arthritis in rats.