Triazinone derivatives as NLRP3 inhibitors

Abstract

The present invention relates to novel compounds having the formula 6-[[(3R)-1-ethyl-3-piperidyl]amino]-3-(4-hydroxyindan-5-yl)-4-methyl-1,2,4-triazin-5-one, or 6-[[(3R)-1-ethyl-3-piperidyl]amino]-3-(2-hydroxy-3-bicyclo[4.2.0]octa-1,3,5-trienyl)-4-methyl-1,2,4-triazin-5-one, and pharmaceutically acceptable salts thereof, compositions containing the compounds, and methods of using the compounds.

Description

[Technical Field]

[0001] The present invention relates to organic compounds useful for therapy and / or prophylaxis in mammals, in particular compounds that modulate NLRP3 inhibition.

[0002] The present invention provides 6-[[(3R)-1-ethyl-3-piperidyl]amino]-3-(4-hydroxyindan-5-yl)-4-methyl-1,2,4-triazin-5-one; and 6-[[(3R)-1-ethyl-3-piperidyl]amino]-3-(2-hydroxy-3-bicyclo[4.2.0]octa-1,3,5-trienyl)-4-methyl-1,2,4-triazin-5-one; 6-[[(3R)-1-ethyl-3-piperidyl]-methyl-amino]-3-(4-hydroxyindan-5-yl)-4-methyl-1,2,4 triazin-5-one; or a pharmaceutically acceptable salt thereof. [Background technology]

[0003] The NOD-like receptor (NLR) family pyrin domain-containing protein 3 (NLRP3) inflammasome is a component of inflammatory processes, and its aberrant activity is pathogenic in genetic disorders such as cryopyrin-associated periodic syndromes (CAPS), and complex diseases such as multiple sclerosis, type 2 diabetes, Alzheimer's disease, and atherosclerosis.

[0004] NLRP3 is an intracellular signaling molecule that senses many pathogen-, environmental, and host-derived factors. Upon activation, NLRP3 binds to apoptosis-associated speck-like proteins containing a caspase activation and recruitment domain (ASC). ASC subsequently polymerizes to form large aggregates known as ASC specks. Polymerized ASC then interacts with the cysteine ​​protease caspase-1 to form a complex called the inflammasome. This leads to the activation of caspase-1, which cleaves the precursor forms of the pro-inflammatory cytokines IL-1β and IL-18 (pro-IL-1β and pro-IL-18, respectively), thereby activating these cytokines. Caspase-1 also mediates a form of inflammatory cell death known as pyroptosis. ASC specks can also recruit and activate caspase-8, which can process pro-IL-1β and pro-IL-18, leading to apoptotic cell death.

[0005] Caspase-1 cleaves pro-IL-1β and pro-IL-18 to their active forms, which are secreted from cells. Active caspase-1 also cleaves gasdermin-D, causing pyroptosis. Through its control of the pyroptotic cell death pathway, caspase-1 also mediates the release of alarmin molecules such as IL-33 and high-mobility group box 1 protein (HMGB1). Caspase-1 also cleaves intracellular IL-1R2, leading to its degradation and allowing IL-1α to be released. In human cells, caspase-1 can also regulate the processing and secretion of IL-37. Several other caspase-1 substrates, such as components of the cytoskeleton and glycolytic pathways, may contribute to caspase-1-dependent inflammation.

[0006] NLRP3-dependent ASC specks are released into the extracellular milieu where they can activate caspase-1, induce the processing of caspase-1 substrates, and propagate inflammation.

[0007] Active cytokines derived from NLRP3 inflammasome activation are key drivers of inflammation and interact with other cytokine pathways to shape immune responses to infection and injury. For example, IL-1β signaling induces the secretion of the pro-inflammatory cytokines IL-6 and TNF. IL-1β and IL-18 synergize with IL-23 to induce IL-17 production by memory CD4 Th17 cells and γδ T cells in the absence of T cell receptor engagement. IL-18 and IL-12 also act synergistically to induce IFN-γ production from memory T cells and NK cells, driving Th1 responses.

[0008] The inherited CAPS disorders Muckle-Wells syndrome (MWS), familial common cold autoinflammatory syndrome (FCAS), and neonatal-onset multisystem inflammatory disease (NOMID) are caused by gain-of-function mutations in NLRP3, defining it as a key component of the inflammatory process. NLRP3 is also involved in the pathogenesis of several complex diseases, including metabolic disorders such as type 2 diabetes, atherosclerosis, obesity, and gout, among others.

[0009] The role of NLRP3 in central nervous system diseases is becoming clearer, and lung diseases have also been shown to be affected by NLRP3. NLRP3 has also been implicated in many central nervous system conditions, including Parkinson's disease (PD), Alzheimer's disease (AD), dementia, Huntington's disease, cerebral malaria, and brain damage due to pneumococcal meningitis (Walsh et al., Nature Reviews, 15:84-97, 2014 and Dempsey et al., Brain. Behav. Immun. 201761:306-316). NLRP3 has also been shown to be involved in many lung diseases, such as chronic obstructive pulmonary disorder (COPD), asthma (including steroid-resistant asthma), asbestosis, and silicosis (De Nardo et al., Am. J. Pathol., 184:42-54, 2014 and Kim et al., Am J Respir Crit Care Med. 2017 196(3):283-97). Furthermore, NLRP3 is involved in the development of liver disease, kidney disease, and aging. Many of these associations are due to the involvement of NLRP3. - / - Although defined in mice, insights into the specific activation of NLRP3 in these diseases also exist. In type 2 diabetes (T2D), deposition of islet amyloid polypeptide in the pancreas activates NLRP3 and IL-1β signaling, leading to cell death and inflammation.

[0010] Several small molecules have been shown to inhibit the NLRP3 inflammasome. Glyburide inhibits IL-1β production at micromolar concentrations in response to NLRP3 activation, but not in response to NLRC4 or NLRP1 activation. Other previously characterized weak NLRP3 inhibitors include parthenolide, 3,4-methylenedioxy-β-nitrostyrene, and dimethyl sulfoxide (DMSO), but these agents have limited efficacy and are nonspecific.

[0011] Current treatments for NLRP3-related diseases include biologic agents that target IL-1. These include the recombinant IL-1 receptor antagonist anakinra, the neutralizing IL-1β antibody canakinumab, and the soluble decoy IL-1 receptor rilonacept. These approaches have proven successful in treating CAPS, and these biologic agents are being used in clinical trials for other IL-1β-related diseases.

[0012] There is a need to provide compounds that have improved pharmacological and / or physiological and / or physicochemical properties and / or that offer useful alternatives to known compounds, in particular compounds that have good activity and improved in vitro stability. Summary of the Invention

[0013] The present invention provides 6-[[(3R)-1-ethyl-3-piperidyl]amino]-3-(4-hydroxyindan-5-yl)-4-methyl-1,2,4-triazin-5-one; and 6-[[(3R)-1-ethyl-3-piperidyl]amino]-3-(2-hydroxy-3-bicyclo[4.2.0]octa-1,3,5-trienyl)-4-methyl-1,2,4-triazin-5-one; Novel compounds selected from 6-[[(3R)-1-ethyl-3-piperidyl]-methyl-amino]-3-(4-hydroxyindan-5-yl)-4-methyl-1,2,4 triazin-5-one; and Pharmaceutically acceptable salts thereof are provided. [Brief explanation of the drawings]

[0014] [Figure 1] 1 is a graph showing the voltage patterns used in the hERG screening assay.

[0015] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the free base or free acid, and is not biologically or otherwise undesirable.Salts are formed with inorganic acids, such as trifluoroacetic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, especially hydrochloric acid, and organic acids, such as formic acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and N-acetylcysteine.In addition, these salts can be prepared by adding inorganic or organic bases to free acids.Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, and polyamine resins. The compounds of formula (I) may also exist in zwitterionic form. Particularly preferred pharmaceutically acceptable salts of the compounds of formula (I) are salts formed with formic acid and with hydrochloric acid, resulting in the hydrochloride, dihydrochloride, or trihydrochloride salts.

[0016] The abbreviation uM means micromolar and is equivalent to the symbol μM.

[0017] The abbreviation uL means microliter and is equivalent to the symbol μL.

[0018] The abbreviation ug stands for microgram and is equivalent to the symbol μg.

[0019] Also, one embodiment of the present invention provides compounds as described herein and pharmaceutically acceptable salts or esters thereof, particularly compounds as described herein and pharmaceutically acceptable salts thereof, more particularly compounds as described herein.

[0020] Specific examples of the compounds described herein are: 6-[[(3R)-1-ethyl-3-piperidyl]amino]-3-(4-hydroxyindan-5-yl)-4-methyl-1,2,4-triazin-5-one; 6-[[(3R)-1-ethyl-3-piperidyl]amino]-3-(2-hydroxy-3-bicyclo[4.2.0]octa-1,3,5-trienyl)-4-methyl-1,2,4-triazin-5-one; and pharmaceutically acceptable salts thereof.

[0021] Another specific example of a compound described herein is 6-[[(3R)-1-ethyl-3-piperidyl]-methyl-amino]-3-(4-hydroxyindan-5-yl)-4-methyl-1,2,4-triazin-5-one, or a pharmaceutically acceptable salt thereof.

[0022] One embodiment of the present invention provides a compound of the formula 6-[[(3R)-1-ethyl-3-piperidyl]amino]-3-(4-hydroxyindan-5-yl)-4-methyl-1,2,4-triazin-5-one, or a pharmaceutically acceptable salt thereof.

[0023] One embodiment of the present invention provides a compound of the formula 6-[[(3R)-1-ethyl-3-piperidyl]amino]-3-(2-hydroxy-3-bicyclo[4.2.0]octa-1,3,5-trienyl)-4-methyl-1,2,4-triazin-5-one, or a pharmaceutically acceptable salt thereof.

[0024] Another embodiment of the present invention provides pharmaceutical compositions or medicaments containing a compound of the present invention and a therapeutically inert carrier, diluent, or excipient, as well as methods for preparing such compositions and medicaments using the compound of the present invention. In one example, the compound can be formulated at an appropriate pH and desired purity by mixing with a physiologically acceptable carrier, i.e., a carrier that is non-toxic to recipients, at the volume and concentration used in galenic formulations, at room temperature. The pH of the formulation will depend primarily on the specific application and compound concentration, but is preferably somewhere in the range of about 3 to about 8. In one example, the compound of formula is formulated in an acetate buffer at pH 5. In another embodiment, the compound of formula is sterile. The compound can be stored, for example, as a solid or amorphous composition, as a lyophilized formulation, or as an aqueous solution.

[0025] The compositions are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the administration schedule, and other factors known to medical practitioners.

[0026] The compounds of the present invention can be administered by any suitable means, including oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal, and epidural and intranasal, and, if desired for local treatment, intralesional administration. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0027] The compounds of the present invention may be administered in any convenient dosage form, for example, tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc. Such compositions may contain conventional components of pharmaceutical formulations, such as diluents, carriers, pH adjusters, sweeteners, fillers, and additional active agents.

[0028] Typical preparation is prepared by mixing the compound of the present invention with carrier or excipient.Suitable carrier and excipient are well known to those skilled in the art, and are described in detail in, for example, Ansel, Howard C. et al., "Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems", Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R. et al., "Remington: The Science and Practice of Pharmacy", Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C., "Handbook of Pharmaceutical Excipients", Chicago, Pharmaceutical Press, 2005. The formulation may also include one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, fragrances, flavorings, diluents, and other known additives to provide a superior presentation of the drug (i.e., the compound of the present invention or a pharmaceutical composition thereof) or to aid in the manufacture of a pharmaceutical product (i.e., a medicament).

[0029] The compounds and their pharmaceutically acceptable salts may be processed with pharmaceutically inert inorganic or organic adjuvants for the manufacture of tablets, coated tablets, dragees, hard gelatin capsules, injection solutions or topical preparations, and lactose, corn starch or its derivatives, talc, stearic acid or its salts, etc. may be used, for example, as such adjuvants for tablets, dragees and hard gelatin capsules.

[0030] Suitable adjuvants for soft gelatin capsules include, for example, vegetable oils, waxes, fats, semisolids, liquid polyols, and the like.

[0031] Suitable adjuvants for the production of solutions and syrups are, for example, water, polyols, saccharose, invert sugar, glucose etc.

[0032] Suitable adjuvants for injection solutions are, for example, water, alcohols, polyols, glycerol, vegetable oils and the like.

[0033] Suitable adjuvants for suppositories are, for example, natural or hardened oils, waxes, fats, semi-solid or liquid polyols etc.

[0034] Suitable adjuvants for topical ophthalmic formulations are, by way of example, cyclodextrin, mannitol, or many other carriers and excipients known in the art.

[0035] In addition, pharmaceutical preparations may contain preservatives, solubilizers, viscosity-increasing substances, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, salts for varying osmotic pressure, buffers, masking agents, or antioxidants. They may also contain still other therapeutically valuable substances.

[0036] Dosages can vary widely and will, of course, be tailored to the individual requirements of each particular case. Generally, for oral administration, the daily dose is about 0.1 mg to 20 mg per kg of body weight, preferably about 0.5 mg to 4 mg per kg of body weight (e.g., about 300 mg per person), preferably administered individually in 1 to 3 doses, which may, if appropriate, be composed of equal amounts. For topical administration, the formulation may contain 0.001% to 15% by weight of the pharmaceutical agent, and the required dose, which may be between 0.1 and 25 mg, may be administered either as a single dose per day or per week, as multiple doses (2 to 4 times per day), or as multiple doses per week. However, it will be apparent that the upper or lower limits given herein may be exceeded where indicated.

[0037] One embodiment of the present invention is a compound according to the invention as described herein for use as a therapeutically active substance.

[0038] One embodiment of the present invention is a compound according to the invention as described herein for use in the treatment or prevention of a disease, disorder or condition, wherein the disease, disorder or condition is responsive to NLRP3 inhibition.

[0039] One embodiment of the present invention is a compound according to the invention as described herein for the treatment or prevention of a disease, disorder or condition, wherein the disease or condition is responsive to NLRP3 inhibition.

[0040] As used herein, the term "NLRP3 inhibition" refers to a complete or partial reduction in the activity level of NLRP3, including, for example, inhibition of active NLRP3 and / or inhibition of NLRP3 activation.

[0041] There is evidence for a role for NLRP3-induced IL-1 and IL-18 in the inflammatory responses associated with or resulting from a number of different disorders (Menu et al., Clinical and Experimental Immunology, 166:1-15, 2011; Strowig et al., Nature, 481:278-286, 2012).

[0042] In one embodiment, the disease, disorder or condition is selected from the following: (i) Inflammation; (ii) autoimmune diseases; (iii) cancer; (iv) infectious diseases; (v) central nervous system disorders; (vi) metabolic diseases; (vii) cardiovascular disease; (viii) respiratory diseases; (ix) liver disease; (x) Kidney disease; (xi) eye diseases; (xii) skin diseases; (xiii) lymphatic symptoms; (xiv) mental disorder; (xv) graft-versus-host disease; (xvi) allodynia; (xvii) symptoms related to diabetes; and (xviii) Any disease in which an individual is determined to have a germline or somatic non-silent mutation in NLRP3.

[0043] In another embodiment, the disease, disorder or condition is selected from the following: (i) Cancer; (ii) infectious diseases; (iii) central nervous system disorders; (iv) cardiovascular disease; (v) liver disease; (vi) eye disease; or (vii) Skin diseases.

[0044] In a further exemplary embodiment of the invention, the disease, disorder or condition is inflammation. Examples of inflammation that may be treated or prevented include inflammatory responses associated with or resulting from: (i) Skin conditions such as contact sensitivity, bullous pemphigoid, sunburn, psoriasis, atopic dermatitis, contact dermatitis, allergic contact dermatitis, seborrheic dermatitis, lichen planus, scleroderma, pemphigus, epidermolysis bullosa, urticaria, erythema, or alopecia; (ii) articular conditions such as osteoarthritis, systemic juvenile idiopathic arthritis, adult-onset Still's disease, relapsing polychondritis, rheumatoid arthritis, juvenile chronic arthritis, gout, or seronegative spondyloarthropathy (e.g., ankylosing spondylitis, psoriatic arthritis, or Reiter's disease); (iii) muscle conditions such as polymyositis or myasthenia gravis; (iv) gastrointestinal conditions such as inflammatory bowel disease (including Crohn's disease and ulcerative colitis), colitis, gastric ulcer, celiac disease, proctitis, pancreatitis, eosinophilic gastroenteritis, mastocytosis, antiphospholipid syndrome, or food-related allergies that may have effects remote from the gut (e.g., migraine, rhinitis, or eczema); (v) respiratory conditions such as chronic obstructive pulmonary disease (COPD), asthma (including eosinophilic, bronchial, allergic, intrinsic, extrinsic or dust-induced asthma, especially chronic or refractory asthma, e.g., late-stage asthma and airway hyperresponsiveness), bronchitis, rhinitis (including acute rhinitis, allergic rhinitis, atrophic rhinitis, chronic rhinitis, caseous rhinitis, hypertrophic rhinitis, panlentic rhinitis, rhinitis sicca, rhinitis medicamentosa, membranous rhinitis, seasonal rhinitis, e.g., hay fever and vasomotor rhinitis), sinusitis, idiopathic pulmonary fibrosis (IPF), sarcoidosis, farmer's lung, silicosis, asbestosis, volcanic ash-induced inflammation, adult respiratory distress syndrome, hypersensitivity pneumonitis, or idiopathic interstitial pneumonia; (vi) vascular conditions such as atherosclerosis, Behçet's disease, vasculitis, or Wegener's granulomatosis; (vii) autoimmune conditions such as systemic lupus erythematosus, Sjögren's syndrome, systemic sclerosis, Hashimoto's thyroiditis, type I diabetes, idiopathic thrombocytopenic purpura, or Graves' disease; (viii) ocular conditions such as uveitis, allergic conjunctivitis, or vernal conjunctivitis; (ix) neurological conditions such as multiple sclerosis or encephalomyelitis; (x) an infection or infection-related condition such as acquired immunodeficiency syndrome (AIDS), acute or chronic bacterial infection, acute or chronic parasitic infection, acute or chronic viral infection, acute or chronic fungal infection, meningitis, hepatitis (A, B, or C, or other viral hepatitis), peritonitis, pneumonia, epiglottitis, malaria, hemorrhagic dengue fever, leishmaniasis, streptococcal myositis, Mycobacterium tuberculosis (including Mycobacterium tuberculosis and HIV co-infection), Mycobacterium avium intracellulare, Pneumocystis carinii pneumonia, orchitis / epidimitis, Legionella, Lyme disease, influenza A, Epstein-Barr virus infection, viral encephalitis / aseptic meningitis, or pelvic inflammatory disease; (xi) renal conditions such as mesangial proliferative glomerulonephritis, renal syndrome, nephritis, glomerulonephritis, obesity-related glomerulopathy, acute renal failure, acute kidney injury, uremia, nephritic syndrome, renal fibrosis including chronic crystalline nephropathy, or renal hypertension; (xii) lymphatic conditions such as Castleman's disease; (xiii) conditions of or involving the immune system, such as hyper-IgE syndrome, lepromatous leprosy, familial hemophagocytic lymphohistiocytosis, or graft-versus-host disease; (xiv) hepatic conditions such as chronic active hepatitis, nonalcoholic steatohepatitis (NASH), alcohol-induced hepatitis, nonalcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease (AFLD), alcoholic steatohepatitis (ASH), primary biliary cirrhosis, fulminant hepatitis, hepatic fibrosis, or liver failure; (xv) Cancer, including those mentioned above; (xvi) burns, wounds, trauma, hemorrhage, or stroke; (xvii) radiation exposure; (xviii) metabolic diseases such as type 2 diabetes (T2D), atherosclerosis, obesity, gout, or pseudogout; and / or (xix) Pain such as inflammatory hyperalgesia, pelvic pain, allodynia, neuropathic pain, or cancer-induced bone pain.

[0045] One embodiment of the present invention is a compound according to the invention as described herein for the treatment or prevention of a disease, disorder or condition selected from: (i) Inflammation; (ii) autoimmune diseases; (iii) cancer; (iv) infectious diseases; (v) central nervous system disorders; (vi) metabolic diseases; (vii) cardiovascular disease; (viii) respiratory diseases; (ix) liver disease; (x) Kidney disease; (xi) eye diseases; (xii) skin diseases; (xiii) lymphatic symptoms; (xiv) mental disorder; (xv) graft-versus-host disease; (xvi) allodynia; (xvii) symptoms related to diabetes; and (xviii) Any disease in which an individual is determined to have a germline or somatic non-silent mutation in NLRP3.

[0046] One embodiment of the present invention is the use of a compound according to the invention as described herein in the treatment or prevention of a disease, disorder or condition, wherein the disease, disorder or condition is responsive to NLRP3 inhibition.

[0047] One embodiment of the present invention is the use of a compound according to the invention as described herein in the treatment or prevention of a disease, disorder or condition selected from Alzheimer's disease and Parkinson's disease.

[0048] One embodiment of the present invention is the use of a compound according to the invention as described herein for use in the treatment or prevention of a disease, disorder or condition selected from asthma or COPD.

[0049] One embodiment of the present invention is a compound according to the invention as described herein for the treatment or prevention of a disease, disorder or condition selected from Alzheimer's disease and Parkinson's disease.

[0050] One embodiment of the present invention is a compound according to the invention as described herein for the treatment or prevention of a disease, disorder or condition selected from asthma or COPD.

[0051] One embodiment of the present invention is the use of a compound according to the invention as described herein for the preparation of a medicament for the treatment or prevention of a disease, disorder or condition selected from Alzheimer's disease and Parkinson's disease.

[0052] One embodiment of the present invention is the use of a compound according to the invention as described herein for the preparation of a medicament for the treatment or prevention of a disease, disorder or condition selected from asthma or COPD.

[0053] One embodiment of the present invention is a method for treating or preventing a disease, disorder, or condition selected from Alzheimer's disease and Parkinson's disease, comprising administering an effective amount of a compound according to the invention described herein.

[0054] One embodiment of the present invention is a method of treating or preventing a disease, disorder, or condition selected from asthma or COPD, comprising administering an effective amount of a compound according to the invention as described herein.

[0055] One embodiment of the present invention relates to a method of inhibiting NLRP3, comprising administering an effective amount of a compound according to the invention described herein.

[0056] Also, one embodiment of the present invention is a compound of the formula described herein when prepared according to any one of the methods described.

[0057] One embodiment of the present invention is a pharmaceutical composition comprising a compound according to the invention as described herein and a therapeutically inert carrier.

[0058] Assay procedure NLRP3 and pyroptosis It is well established that activation of NLRP3 leads to cell pyroptosis, a hallmark of which plays a key role in the development of clinical disease (Yan-gang Liu et al., Cell Death & Disease, 2017, 8(2), e2579; Alexander Wree et al., Hepatology, 2014, 59(3), 898-910; Alex Baldwin et al., Journal of Medicinal Chemistry, 2016, 59(5), 1691-1710; Ema Ozaki et al., Journal of Inflammation Research, 2015, 8, 15-27; Zhen Xie & Gang Zhao, Neuroimmunology Neuroinflammation, 2014, 1(2), 60-65; Mattia Cocco et al., Journal of Medicinal Chemistry, 2014, 57(24), 10366-10382; T. Satoh et al., Cell Death & Disease, 2013, 4, e644). Therefore, inhibitors of NLRP3 are expected to block pyroptosis and the release of pro-inflammatory cytokines (e.g., IL-1β) from cells.

[0059] THP-1 cells: culture and preparation THP-1 cells (ATCC No. TIB-202) were grown in RPMI containing L-glutamine (Gibco No. 11835) supplemented with 1 mM sodium pyruvate (Sigma No. S8636) and penicillin (100 units / ml) / streptomycin (0.1 mg / ml) (Sigma No. P4333) in 10% fetal bovine serum (FBS) (Sigma No. F0804). Cells were passaged periodically until confluent (approximately 10 6The THP-1 cells were grown to a concentration of 625,000 cells / ml. On the day of the experiment, the cells were harvested and resuspended in RPMI medium (without FBS). The cells were then counted, and viability (>90%) was confirmed using trypan blue (Sigma #T8154). Appropriate dilutions were made to obtain a concentration of 625,000 cells / ml. LPS (Sigma #L4524) was added to this diluted cell solution to obtain a final assay concentration (FAC) of 1 μg / ml. 40 μl of the final preparation was dispensed into each well of a 96-well plate. The plates prepared in this manner were used for compound screening.

[0060] THP-1 cell pyroptosis assay For compound screening, the following method stepwise assay was followed.

[0061] 1. Seed THP-1 cells (25,000 cells / well) in 40 μl of RPMI medium (without FBS) containing 1.0 μg / ml LPS in a 96-well black-walled, clear-bottom cell culture plate coated with poly-D-lysine (VWR No. 734-0317). Add 2.5 μl of compound (8-point half-log dilutions with the highest dose at 10 μM) or vehicle (DMSO 0.1% FAC) to the appropriate wells. 3. Incubate at 37°C, 5% CO2 for 3 hours. Add 4.5 μl of nigericin (Sigma No. N7143) (FAC 5 μM) to all wells 5. Incubate at 37°C, 5% CO2 for 1 hour. 6. At the end of the incubation period, spin the plate at 300 x g for 3 minutes and remove the supernatant. 7. 50 μl of resazurin (Sigma #R7017) (100 μM resazurin in RPMI medium without FBS) is then added and the plate is incubated at 37°C and 5% CO for a further 1-2 hours. 8. Plates were read on an Envision reader at Ex 560nm and Em 590nm 9.IC 50Fit the data to a nonlinear regression equation (log inhibitor vs. response variable slope, 4 parameters)

[0062] The results of the pyroptosis assay were compared with those of THP IC 50 These are summarized in Table 1 below.

[0063] Human whole blood IL-1β release assay For systemic delivery, the ability of a compound to inhibit NLRP3 when present in the bloodstream is crucial. Therefore, we investigated the NLRP3 inhibitory activity of several compounds in human whole blood according to the following protocol.

[0064] Human whole blood in Li-heparin tubes was obtained from healthy donors from a volunteer donor panel.

[0065] 1. Plate out 80 μl of whole blood containing 1 μg / ml LPS into a 96-well clear-bottom cell culture plate (Corning #3585). 2. Add 10 μl of compound (8-point half-log dilutions at the highest dose of 10 μM) or vehicle (FAC in DMSO 0.1%) to the appropriate wells. 3. Incubate at 37°C, 5% CO2 for 3 hours. 4. Add 10 μl of nigericin (Sigma #N7143) (10 μM FAC) to all wells 5. Incubate at 37°C, 5% CO2 for 1 hour. 6. At the end of the incubation period, spin the plate at 300 x g for 5 minutes to pellet the cells, remove 20 μl of the supernatant and add to a 96-well v-bottom plate for IL-1β analysis (Note: These plates containing supernatant may be stored at -80°C for analysis at a later date). 7. IL-1β was measured according to the manufacturer's protocol (Perkin Elmer-AlphaLisa IL-1 Kit AL220F-5000). 8.IC 50 Fit the data to a nonlinear regression equation (log inhibitor vs. response variable slope, 4 parameters)

[0066] Human whole blood assay results HWB IC 50 These are summarized in Table 1 below.

[0067] hERG screening assay Cardiac arrhythmias are one of the most common side effects leading to drug failure during small molecule drug development. Such failures are often related to the drug's ability to inhibit the human ether-a-go-go-related gene (hERG) cardiac potassium channel. Therefore, no or low inhibition of the hERG cardiac potassium channel is considered beneficial.

[0068] cell The CHO-crelox hERG cell line (ATCC reference number PTA-6812, female Chinese hamster cells) was generated and validated at Roche. Ready-to-use frozen instant CHO-hERG cells were cryopreserved at Evotec (Germany) and used directly in the experiments.

[0069] Experimental solution The extracellular solution contained (in mM): NaCl 150; KCl 4; CaCl 1; MgCl 1; HEPES 10; pH 7.2-7.4 with NaOH, osmolality 290-330 mOsm. The internal solution contained (in mM): KCl 10; KF 100; NaCl 10; HEPES 10; EGTA 20; pH 7.0-7.4 with KOH, osmolality 260-300 mOsm.

[0070] electrophysiology The effects of compounds on hERG K+-current parameters will be evaluated at two concentrations in at least four cells.

[0071] hERG studies are performed using the automated patch clamp system SynchroPatch® 384 (Nanion Technologies GmbH, Germany). K+ currents are measured using the patch voltage clamp technique in the whole-cell configuration at 35-37°C.

[0072] Cells were held at a resting voltage of -80 mV and stimulated with the voltage pattern shown in Figure 1 (outward K at 35–37 °C). + The pulse pattern used to elicit the current) activated hERG channels at a stimulation frequency of 0.1 Hz (6 bpm) to conduct outward IK hERG currents.

[0073] Data analysis The amplitude of the IKhERG was recorded at each drug concentration and compared to vehicle control values ​​(set at 100%) to define fractional blocks. The concentration-response data were fitted with the following relationship: [Table 1]

[0074] Concentration-response curves were fitted by nonlinear regression analysis using the EworkBook suite (ID Business Solutions Ltd, UK). Data were fitted using a four-parameter logistic model (fit = (A + (B / (1 + ((x / C)^D)))), where A = 0 and B = 100).

[0075] hERG assay results are compared with hERG IC 20 The results are summarized in Table 2 below.

[0076] Intracellular P-gp assay: A typical assay uses transfected LLC-PK1 cells (porcine kidney epithelial cells) overexpressing human P-gp or mouse P-gp cultured on 96-well semi-permeable filter membrane plates, where these cells form a polarized monolayer with tight junctions, which act as a barrier between the apical and basolateral compartments.

[0077] P-gp is expressed in the apical membrane of the monolayer.

[0078] The adhesion of the cell monolayer and the functional activity of P-gp are confirmed by the addition of the cell-impermeable marker Lucifer Yellow and the reference P-gp substrate edoxaban, respectively.

[0079] PAMPA: PAMPA (Parallel Artificial Membrane Permeability Assay) is a first-order selective permeability screen for drug candidates. The PAMPA assay uses artificial phospholipid membranes to mimic intracellular absorption conditions. This assay determines permeability values ​​that can be used for compound optimization and ranking purposes and as input parameters for in silico models predicting intestinal absorption.

[0080] The donor concentration is measured at t-start (baseline) and compared to the donor and acceptor concentrations after a certain time period (t-end), and the extent of compound crossing the membrane is calculated.

[0081] Microsomal stability: Incubations with 1 μM test compound in microsomes (0.5 mg / mL) and the cofactor NADPH are performed in 96-well plates at 37°C using a TECAN (Tecan Group Ltd, Switzerland) automated liquid handling system. After a 10-minute preincubation step between microsomes and test compound, the enzymatic reaction is initiated by the addition of the cofactor. Aliquots of the incubation are removed at 1, 3, 6, 9, 15, 25, 35, and 45 minutes and quenched with 1:3 (v / v) acetonitrile containing an internal standard. Samples are then cooled and centrifuged, after which the supernatant is analyzed by LC-MS / MS2.

[0082] Metabolic stability in hepatocytes: Assay Description: Biological materials. Obtain cryopreserved hepatocytes [mouse, rat, rabbit, monkey, and human (mixed sex)]. Post-reconstitution hepatocyte viability is at least 80% throughout the study. Obtain ready-to-use rat / human HepatoPac® cultures [long-term hepatocyte co-cultures; pooled (human male n=5, female n=5)], stromal mouse fibroblasts (negative control; pooled), culture plates, application medium, and maintenance medium.

[0083] Metabolism by Suspension Hepatocytes. Initially, pooled cryopreserved hepatocytes were reconstituted with prewarmed William's E medium containing 10% FCS, 0.05 mg / mL streptomycin, 50 U / mL penicillin, and 0.4 mM L-glutamine, as well as 0.01 mg / mL gentamicin, 0.048 mg / mL hydrocortisone, and 0.004 mg / mL insulin to a final suspension density of 1 × 10 cells / mL. Incubations were performed automatically using a Liquid Handling System (Tecan) equipped with a CO2 incubator with an orbital shaker. After adding test compounds (e.g., 1 μM) to wells (1 × 10 cells / well), the 96-well hepatocyte suspension culture plate was incubated at 37 °C with 5% CO2. Samples were quenched at the designated time points by adding acetonitrile (containing the internal standard) to the incubation wells for up to 2 hours.

[0084] Incubations of test substances (e.g., 1 μM, 0.1% v / v DMSO) performed in metabolic suspension assays using HepatoPac® are performed in 96-well plates containing either cocultures of adherent hepatocytes with mouse fibroblast control cells or control cells alone (5% CO2 atmosphere, 37°C). The incubation medium for human HepatoPac® is identical to that used in suspension stem cells. At designated time points (2, 18, 26, 48, 72, and 96 hours), all wells are quenched with ice-cold acetonitrile containing an internal standard.

[0085] The samples are then appropriately centrifuged and the supernatants are analyzed by LC-MS / MS. n=1 or 2 incubations are performed.

[0086] Pharmacokinetic profile of test article in minipigs: The pharmacokinetics of the test substance was measured in minipigs after intravenous and oral administration. The experimental design consisted of three male minipigs, each of which received a single intravenous bolus dose and a single oral dose with the test substance. The intravenous dose was administered at a nominal volume of 1 mL / kg. The oral dose was administered by gavage at a nominal volume of 5 mL / kg. There was a washout period of at least 7 days between the last sampling and the next dose in the same animal. The content of all formulations was within the desired range of 85-115% of the nominal content. After administration, blood samples (1 mL) were collected from the saphenous vein (via cannula) or jugular vein of each animal pre-dose, 5, 15, and 30 minutes after IV administration, 1, 2, 4, 8, and 24 hours after administration, and 15, 30 minutes, 1, 2, 4, 6, 8, 24, and 48 hours after oral administration. Hematocrit was measured at all time points. Blood:plasma partition coefficients were determined at 2 and 4 hours, and urine was collected as a single sample over 24 hours post-dose. Blood samples (nominal 1 mL) were collected from each animal's saphenous vein (via cannula) or jugular vein into polypropylene tubes containing K2EDTA anticoagulant and centrifuged (1500 g, 10 minutes, 4°C) to prepare plasma for analysis. Residual blood cells were discarded. Plasma vials were capped and stored on wet ice for no more than 60 minutes before being transferred to storage below -50°C (nominal -80°C) before analysis by specific LC-MS methods.

[0087] Toxicity evaluation of test substances in minipigs The maximum tolerated dose (MTD) of the test article is determined after a single daily oral (gavage) administration to minipigs. Toxicity is then assessed following repeated daily administration for 14 days. Furthermore, the toxicokinetic profile of the test article is characterized. Purpose-bred Göttingen minipigs are obtained from Ellegaard Göttingen, Dalmoos, Denmark (animals: age range: 2-3 months, body weight range: 4-6 kg). At the start of dosing, animals are 4-5 months old and weigh between 6-9.5 kg. A dose volume of 10 mL / kg is used. Individual doses are calculated from each animal's most recent body weight up to 30, 100, and 300 mg / kg / day or other target dose levels depending on non-MTD results. Blood samples are collected on days 1 and 14 to determine plasma drug concentrations and derived toxicokinetic parameters. Animals are not fed on the day of the scheduled necropsy. Each animal was anesthetized by intramuscular injection of the Zoletil mixture and then sacrificed by exsanguination. All tissues were preserved in appropriate fixatives. Further analysis included food consumption, body weight, clinical pathology, and complete histopathological examination of target organs. [Table 2] [Table 3] [Table 4]

[0088] The invention will now be illustrated by the following examples, which have no limiting character.

[0089] Where preparations are obtained as mixtures of enantiomers or diastereomers, the pure enantiomers or diastereomers can be obtained by the methods described herein or by methods known to those skilled in the art, such as, for example, chiral chromatography or crystallization.

[0090] Experimental Method [Table 5] [Example]

[0091] Unless otherwise specified, all examples and intermediates were prepared under a nitrogen atmosphere.

[0092] Intermediates Intermediate 1: 3-chloro-6-[[(3R)-1-ethyl-3-piperidyl]amino]-4-methyl-1,2,4-triazin-5-one [ka] Step A: 6-bromo-2-[(4-methoxyphenyl)methyl]-4-methyl-1,2,4-triazine-3,5-dione 6-Bromo-4-methyl-2H-1,2,4-triazine-3,5-dione (CAS number 15870-75-4, 13.8 g, 63.1 mmol, 1.0 equiv.) and potassium carbonate (4.84 g, 31.5 mmol, 0.50 equiv.) were suspended in dry DMF (125 mL) and 4-methoxybenzyl chloride (10.3 mL, 75.7 mmol, 1.2 equiv.) was added. The reaction mixture was stirred at room temperature for 24 h. The reaction mixture was diluted with EtOAc (50 mL), washed with 10 wt% aqueous LiCl solution (2 × 30 mL), dried using a phase separator, and concentrated in vacuo. The resulting residue was purified by silica gel chromatography (0–50% EtOAc / isohexane) to afford the title compound (15.9 g, 77% yield) as a white solid. 1 H NMR(500 MHz,DMSO-d6)[ppm]:δ 7.33-7.25(m,2H),6.97-6.89(m,2H),5.00(s,2H),3.74(s,3H),3.20(s,3H).

[0093] Step B: 6-[[(3R)-1-ethyl-3-piperidyl]amino]-2-[(4-methoxyphenyl)methyl]-4-methyl-1,2,4-triazine-3,5-dione (3R)-1-Ethylpiperidin-3-amine (6.0 g, 46.9 mmol, 1.53 equiv.) and the above 6-bromo-2-[(4-methoxyphenyl)methyl]-4-methyl-1,2,4-triazine-3,5-dione (10.0 g, 30.7 mmol, 1.0 equiv.) and cesium carbonate (20 g, 61.3 mmol, 2.0 equiv.) were dissolved in DMSO (125 mL), and the mixture was degassed (N ) for 5 minutes. The reaction vessel was evacuated and refilled with N ) three times, then (rac)-BINAP Pd G ) (1 g, 1.01 mmol, 0.030 equiv.) was added, and the reaction mixture was placed under N , then stirred at 95 °C for 24 hours. The reaction mixture was partitioned with EtOAc (500 mL) and water (500 mL). The organic phase was isolated, washed with brine (3 x 300 mL), dried using a phase separator, and concentrated in vacuo. The residue was purified by silica gel chromatography (0-7% (0.7N ammonia in MeOH) in DCM) to give the title compound (10.4 g, 86% yield) as an orange oil. LCMS m / z 374.2 [M+H] + ,ESI pos.

[0094] Step C: 6-[[(3R)-1-ethyl-3-piperidyl]amino]-4-methyl-2H-1,2,4-triazine-3,5-dione; trifluoromethanesulfonate The aforementioned 6-[[(3R)-1-ethyl-3-piperidyl]amino]-2-[(4-methoxyphenyl)methyl]-4-methyl-1,2,4-triazine-3,5-dione (10.4 g, 25.1 mmol, 1.0 equiv.) was dissolved in DCM (75 mL). Trifluoromethanesulfonic acid (3.33 mL, 37.7 mmol, 1.5 equiv.) was added to the reaction. The resulting solution was stirred at room temperature for 24 h. Additional trifluoromethanesulfonic acid (3.33 mL, 37.7 mmol, 1.5 equiv.) was added, and the reaction mixture was stirred for an additional 3 h. The reaction mixture was concentrated in vacuo, and the resulting residue was purified by silica gel chromatography (0-10% (0.7 N ammonia in MeOH / DCM)) to afford the title compound (17.04 g, 84% yield) as a yellow oil. LCMS m / z 254.5 [M+H]+ ,ESI pos.

[0095] Step D: 3-chloro-6-[[(3R)-1-ethyl-3-piperidyl]amino]-4-methyl-1,2,4-triazin-5-one The above 6-[[(3R)-1-ethyl-3-piperidyl]amino]-4-methyl-2H-1,2,4-triazine-3,5-dione; trifluoromethanesulfonate salt (17.04 g, 21.1 mmol, 1.0 equiv.) was dissolved in phosphorus oxychloride (75.0 mL, 804.6 mmol, 38.1 equiv.). The reaction was stirred at 120 °C for 72 h. A 15 mL aliquot of the reaction mixture was concentrated in vacuo, and the resulting residue was diluted with EtOAc (200 mL) and washed with 1:1 brine:saturated aqueous NaHCO3 (200 mL). The organic phase was isolated, and the aqueous phase was back-extracted with EtOAc (200 mL). The combined organic extracts were dried (MgSO4) and concentrated in vacuo to give the title compound (1.03 g, 17% yield) as a brown oil. The remaining reaction mixture was subjected to the same work-up conditions and scaled proportionally to give the title compound (5.06 g, 79% yield) as a brown oil. LCMS m / z 274.4 ([37Cl]M+H). + ,ESI pos.

[0096] Intermediate 2: 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indan-4-ol [ka] A mixture of commercially available 5-bromoindan-4-ol (CAS no. 575504-23-3) (950 mg, 4.46 mmol, 1.0 equiv.), bis(pinacolato)diboron (5.66 g, 22.3 mmol, 5.0 equiv.), Pd(dppf)Cl₂·DCM complex (364.1 mg, 0.45 mmol, 0.1 equiv.), and potassium acetate (1.31 g, 13.4 mmol, 3.0 equiv.) in MeCN (40 mL) was degassed with nitrogen for 5 min. The reaction mixture was then heated to 80 °C and stirred at this temperature for 16 h. The reaction mixture was cooled and concentrated in vacuo. The residue was purified by silica gel chromatography (0–100% EtOAc / isohexane) to afford the title compound (211.8 mg, 17%) as a colorless oil. LCMS: No ionization. 1 H NMR (500 MHz, CDCl3) [ppm]: δ 7.91(s,1H),7.43(d,1H),6.81(d,1H),2.94-2.86(m,4H),2.07(d,2H),1.35(s,12H).

[0097] Intermediate 3: 3-Bromobicyclo[4.2.0]octa-1(6), 2,4-trien-2-ol [ka] Step A: 5-benzyloxybicyclo[4.2.0]octa-1(6), 2,4-trien-7-ol A 2.5 M solution of butyllithium in hexanes (38.0 mL, 95.01 mmol, 5.0 equiv.) was added dropwise to stirred THF (100 mL) under N2 at 0 °C. The reaction was allowed to warm to room temperature and then stirred for approximately 16 hours. In a separate flask, a 2.5 M solution of butyllithium in hexanes (15.2 mL, 38.0 mmol, 2.0 equiv.) was added dropwise to a solution of 2,2,6,6-tetramethylpiperidine (6.41 mL, 38.0 mmol, 2.0 equiv.) in THF (60 mL) at 0 °C, and the reaction mixture was stirred for 30 minutes. The first flask was then cooled to -78 °C, and a solution of 3-benzyloxybromobenzene (5000 mg, 19.0 mmol, 1.0 equiv.; CAS No. 53087-13-1) in THF (25 mL) was added. The lithium 2,2,6,6-tetramethylpiperidine solution was then added dropwise via cannula to the reaction mixture, and the reaction was stirred for 1 hour. The reaction was quenched by the addition of NH4Cl (200 mL, saturated aqueous solution) and allowed to warm to room temperature. The reaction was diluted with water (100 mL) and extracted with EtOAc (3 x 150 mL). The combined organic layers were dried over MgSO4, concentrated in vacuo, and purified by silica gel column chromatography (0-10% EtOAc / isohexane) to give the title compound (3.22 g, 74% yield) as a white solid. LCMS: m / z = 225.3 [M−H] - ,ESI neg.

[0098] Step B: 2-Benzyloxy-8-bromo-bicyclo[4.2.0]octa-1(6),2,4-triene Carbon tetrabromide (1.76 g, 5.3 mmol, 1.2 equiv.) and triphenylphosphine (2.09 g, 7.96 mmol, 1.8 equiv.) were added to a stirred solution of the aforementioned 5-benzyloxybicyclo[4.2.0]octa-1(6),2,4-trien-7-ol (1.0 g, 4.42 mmol, 1.0 equiv.) in EtO (40 mL) at room temperature, and the reaction was stirred for 2 days. The solution was filtered, the solid was washed with ether (40 mL), and the filtrate was concentrated in vacuo. The crude material was purified by silica gel column chromatography (0–20% EtOAc / isohexane) to afford the title compound (1186 mg, 4.1 mmol, 89% yield) as a colorless oil. LCMS: m / z not observed. 1 H NMR(500 MHz,DMSO-d6)[ppm]:δ 7.48-7.43(m,2H),7.43-7.38(m,2H),7.37-7.28(m,2H),6.87(d,1H),6.77(d,1H) ),5.74(dd,1H),5.32(d,1H),5.27(d,1H),3.94-3.81(m,1H),3.40-3.34(m,1H).

[0099] Step C: Bicyclo[4.2.0]octa-1(6),2,4-trien-2-ol A mixture of the aforementioned 2-benzyloxy-8-bromo-bicyclo[4.2.0]octa-1(6),2,4-triene (1180.0 mg, 4.08 mmol, 1.0 equiv.) and 5 wt.% palladium on carbon, R434 form, 50 wt.% water (173.7 mg, 0.04 mmol, 0.01 equiv.) in ethanol (25 mL) was stirred under H2 (5 bar) for 3 h. The reaction was filtered, diluted with DCM (200 mL), and then washed with sodium thiosulfate (1 × 50 mL, 10% aqueous solution), sodium bicarbonate (1 × 50 mL, saturated aqueous solution), and brine (1 × 50 mL). The organic phase was dried over MgSO4, concentrated in vacuo, and purified by silica gel column chromatography (0–10% EtOAc / isohexane) to give the title compound (471.7 mg, 3.93 mmol, 91% yield) as an off-white crystalline solid. LCMS: m / z=121.1 [M+H] + ,ESI pos.

[0100] Step D: 3-Bromobicyclo[4.2.0]octa-1(6),2,4-trien-2-ol N-Bromosuccinimide (628.34 mg, 3.53 mmol, 0.95 equiv) in DCM (10 mL) was added portionwise to a stirred solution of the aforementioned bicyclo[4.2.0]octa-1(6),2,4-trien-2-ol (470.0 mg, 3.72 mmol, 1.0 equiv) and diisopropylamine (52.08 μL, 0.37 mmol, 0.1 equiv) in DCM (40 mL) at 0 °C, and the reaction was stirred for 1 h. The reaction was concentrated in vacuo and purified by silica gel column chromatography (0-10% EtOAc / isohexane) to afford the title compound (522.4 mg, 2.62 mmol, 67% yield) as a white solid. LCMS: m / z not observed. 1 H NMR(500 MHz,CDCl3)[ppm]:δ 7.32(d,1H),6.55(d,1H),5.43(s,1H),3.20-3.13(m,2H),3.13-3.06(m,2H).

[0101] Working Example: Example 1: 6-[[(3R)-1-ethyl-3-piperidyl]amino]-3-(4-hydroxyindan-5-yl)-4-methyl-1,2,4 triazin-5-one [ka] A mixture of the above 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indan-4-intermediate 2 (60.0 mg, 0.23 mmol, 1.25 equiv.), 3-chloro-6-[[(3R)-1-ethyl-3-piperidyl]amino]-4-methyl-1,2,4-triazin-5-one intermediate 1 (50.0 mg, 0.18 mmol, 1.0 equiv.), meCgPPhPdG3 (CAS No. 2230788-58-4) (12.2 mg, 0.02 mmol, 0.1 equiv.), and potassium carbonate (76.29 mg, 0.55 mmol, 3.0 equiv.) in 1,4-dioxane (2 mL) and water (0.5 mL) was degassed with nitrogen for 5 minutes, and the reaction was then heated to 90 °C for 2 hours. The reaction mixture was cooled and concentrated in vacuo. The residue was purified by silica gel chromatography (24 g cartridge, 0-10% (0.7 M NH3) MeOH / DCM, then 12 g cartridge, 0-7% (0.7 M NH3) MeOH / DCM) to give the title compound (19.6 mg, 28%) as an off-white solid. LCMS m / z=370.2 [M+H] + ,ESI pos.

[0102] Example 2: 6-[[(3R)-1-ethyl-3-piperidyl]amino]-3-(2-hydroxy-3-bicyclo[4.2.0]octa-1,3,5-trienyl)-4-methyl-1,2,4-triazin-5-one [ka] A mixture of 3-bromobicyclo[4.2.0]octa-1(6),2,4-trien-2-ol intermediate 3 (100 mg, 0.50 mmol, 1.0 equiv.), bis(pinacolato)diboron (640.0 mg, 2.52 mmol, 5.0 equiv.), Pd(dppf)Cl·DCM complex (50.0 mg, 0.06 mmol, 0.12 equiv.; CAS No. 95464-05-4), and potassium acetate (150.0 mg, 1.53 mmol, 3.0 equiv.) in 1,4-dioxane (5 mL) was degassed with N for 5 min, then heated to 90 °C and stirred for 6 h. The reaction was cooled to room temperature, and then KPO (2 mL, 10% aqueous solution) was added, and the reaction was stirred for 30 min. The reaction mixture was diluted with water (30 mL) and extracted with DCM (30 mL). The aqueous layer was acidified to approximately pH 3–4 by dropwise addition of HCl (1 M aqueous solution) and then extracted again with DCM (5 × 30 mL). The combined organic extracts were dried over MgSO and concentrated in vacuo. The resulting residue was dissolved in 1,4-dioxane (4 mL), and 3-chloro-6-[[(3R)-1-ethyl-3-piperidyl]amino]-4-methyl-1,2,4-triazin-5-one intermediate 1 (140.0 mg, 0.36 mmol, 0.72 equiv.), XphosPdG3 (22.0 mg, 0.03 mmol, 0.05 equiv.), potassium carbonate (210.0 mg, 1.52 mmol, 3.02 equiv.), and water (1 mL) were added. The resulting mixture was degassed with N2 for 5 min and then heated to 90 °C for 4 h. The reaction was cooled to room temperature, concentrated in vacuo, and purified by silica gel column chromatography (0-10% (0.7M NH3) MeOH / DCM) to give 58 mg of the desired compound in approximately 93% purity. The material was purified again by reverse phase chromatography (0.1% ammonium hydroxide, 5-35% MeCN / water) to give the title compound (33.8 mg, 18% yield) as a white solid. LCMS: m / z=356.2 [M+H] + ,ESI pos.

[0103] Example 3: 6-[[(3R)-1-Ethyl-3-piperidyl]-methyl-amino]-3-(4-hydroxyindan-5-yl)-4-methyl-1,2,4 triazin-5-one [ka] Step A: 3-(4-benzyloxyindan-5-yl)-6-[[(3R)-1-ethyl-3-piperidyl]amino]-4-methyl-1,2,4 triazin-5-one 2-(4-Benzyloxyindan-5-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (737.0 mg, 2.1 mmol, 1.0 equiv., CAS No. 2878443-82-2), 3-chloro-6-[[(3R)-1-ethyl-3-piperidyl]amino]-4-methyl-1,2,4-triazin-5-one intermediate 1 (603.57 mg, 2.0 mmol, 0.95 equiv.), XPhosPdG3 (178.32 mg, 0.21 mmol, 0.1 equiv.), and saturated aqueous sodium carbonate (3.0 mL, 2.1 mmol, 1.0 equiv.) were dissolved in MeCN (15 mL), the mixture was degassed with nitrogen, and then stirred at 80 °C for 20 h. The mixture was diluted with EtOAc (100 mL) and washed with brine (100 mL). The organic phase was separated, dried using a phase separator, and concentrated in vacuo. The resulting residue was purified by flash chromatography on silica gel (24 g column, 0-10% (0.7 N ammonia in MeOH) / DCM) to afford the title compound (675.0 mg, 1.47 mmol, 67% yield) as a yellow solid. LCMS: m / z=460.3 [M+H] + ,ESI pos.

[0104] Step B: 3-(4-benzyloxyindan-5-yl)-6-[[(3R)-1-ethyl-3-piperidyl]-methyl-amino]-4-methyl-1,2,4 triazin-5-one 3-(4-benzyloxyindan-5-yl)-6-[[(3R)-1-ethyl-3-piperidyl]amino]-4-methyl-1,2,4-triazin-5-one (250.0 mg, 0.54 mmol, 1.0 equiv.) was dissolved in NMP (2 mL) and sodium hydride (60% in mineral oil, 108.79 mg, 2.72 mmol, 5.0 equiv.) was added. The mixture was stirred for 15 minutes, and then iodomethane (0.04 mL, 0.65 mmol, 1.2 equiv.) was added. The mixture was stirred for 18 hours, and then additional sodium hydride (60% in mineral oil, 108.79 mg, 2.72 mmol, 5.0 equiv.) and iodomethane (0.04 mL, 0.65 mmol, 1.2 equiv.) were added, and the mixture was stirred for an additional 2 hours. The mixture was quenched with MeOH (5 mL), followed by water (10 mL), and diluted with EtOAc (50 mL). The organic phase was washed with brine (3 × 30 mL), dried using a phase separator, and concentrated in vacuo. The resulting residue was purified by flash chromatography on silica gel (4 g column, 0–7% (0.7 N NH3 in MeOH) / DCM) to afford the title compound (13.0 mg, 4% yield) as a pale yellow solid. LCMS: m / z = 474.5 [M+H] + ,ESI pos.

[0105] Step B: 6-[[(3R)-1-ethyl-3-piperidyl]-methyl-amino]-3-(4-hydroxyindan-5-yl)-4-methyl-1,2,4-triazin-5-one 3-(4-benzyloxyindan-5-yl)-6-[[(3R)-1-ethyl-3-piperidyl]-methyl-amino]-4-methyl-1,2,4-triazin-5-one (13.0 mg, 0.02 mmol, 1.0 equiv.) was dissolved in EtOH (1 mL) and Pd(OH) (10 wt.% on carbon, 7.53 mg, 0.0 mmol, 0.2 equiv.) was added. The mixture was stirred under H (2 bar) for 24 h. The reaction mixture was then filtered through a Celite plug and the filtrate was concentrated in vacuo. The resulting residue was purified by flash chromatography on silica gel (4 g column, 0–10% (0.7 N NH in MeOH / DCM) to afford the title compound (7.0 mg, 73% yield) as an off-white solid. LCMS: m / z = 384.3 [M+H] + ,ESI pos.

[0106] Example A The compound of the formula can be used in a manner known per se as the active ingredient for producing tablets of the following composition: Per tablet Active ingredient 200mg Microcrystalline cellulose 155mg Cornstarch 25mg Talc 25mg Hydroxypropyl methylcellulose 20mg 425mg

[0107] Example B The compound of the formula can be used in a manner known per se as the active ingredient for producing capsules of the following composition: Per capsule Active ingredient 100.0mg Cornstarch 20.0mg Lactose 95.0mg Talc 4.5mg Magnesium stearate 0.5mg 220.0mg

Claims

1. 6-[[(3R)-1-ethyl-3-piperidyl]amino]-3-(4-hydroxyindan-5-yl)-4-methyl-1,2,4-triazin-5-one; 6-[[(3R)-1-ethyl-3-piperidyl]amino]-3-(2-hydroxy-3-bicyclo[4.2.0]octa-1,3,5-trienyl)-4-methyl-1,2,4-triazin-5-one; A compound selected from 6-[[(3R)-1-ethyl-3-piperidyl]-methyl-amino]-3-(4-hydroxyindan-5-yl)-4-methyl-1,2,4-triazin-5-one; or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, which is 6-[[(3R)-1-ethyl-3-piperidyl]amino]-3-(4-hydroxyindan-5-yl)-4-methyl-1,2,4-triazin-5-one, or a pharmaceutically acceptable salt thereof.

3. 2. The compound of claim 1, which is 6-[[(3R)-1-ethyl-3-piperidyl]amino]-3-(2-hydroxy-3-bicyclo[4.2.0]octa-1,3,5-trienyl)-4-methyl-1,2,4-triazin-5-one, or a pharmaceutically acceptable salt thereof.

4. 2. The compound of claim 1, which is 6-[[(3R)-1-ethyl-3-piperidyl]-methyl-amino]-3-(4-hydroxyindan-5-yl)-4-methyl-1,2,4-triazin-5-one, or a pharmaceutically acceptable salt thereof.

5. A compound according to any one of claims 1 to 4 for use as a therapeutically active substance.

6. 10. A compound according to any one of claims 1 to 4 for use in the treatment or prevention of a disease, disorder or condition, wherein the disease, disorder or condition is responsive to NLRP3 inhibition.

7. A pharmaceutical composition comprising a compound according to any one of claims 1 to 4 and a therapeutically inert carrier.

8. 10. Use of a compound according to any one of claims 1 to 4 for the treatment or prevention of a disease, disorder or condition, wherein said disease, disorder or condition is responsive to NLRP3 inhibition.

9. A compound according to any one of claims 1 to 4 for the treatment or prevention of a disease, disorder or condition selected from asthma or COPD.

10. A compound according to any one of claims 1 to 4 for the treatment or prevention of a disease, disorder or condition selected from Alzheimer's disease and Parkinson's disease.

11. 10. Use of a compound according to any one of claims 1 to 4 in the treatment or prevention of a disease, disorder or condition selected from asthma or COPD.

12. 10. Use of a compound according to any one of claims 1 to 4 in the treatment or prevention of a disease, disorder or condition selected from Parkinson's disease or Alzheimer's disease.

13. Use of a compound according to any one of claims 1 to 4 for the preparation of a medicament for the treatment or prevention of a disease, disorder or condition selected from asthma or COPD.

14. Use of a compound according to any one of claims 1 to 4 for the preparation of a medicament for the treatment or prevention of a disease, disorder or condition selected from Parkinson's disease or Alzheimer's disease.

15. A method of inhibiting NLRP3, comprising administering an effective amount of a compound according to any one of claims 1 to 4 to inhibit NLRP3.

16. 10. A method for the treatment or prevention of a disease, disorder or condition, comprising administering an effective amount of a compound according to any one of claims 1 to 4, wherein the disease, disorder or condition is selected from asthma or COPD.

17. 10. A method for the treatment or prevention of a disease, disorder or condition, comprising administering an effective amount of a compound according to any one of claims 1 to 4, wherein the disease, disorder or condition is selected from Parkinson's disease or Alzheimer's disease.

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