A pyridopyrimidine compound and use thereof

Abstract

The present application provides a kind of pyridopyrimidine compound prodrug, oxide, salt, metal complex or stereochemical isomer, and its preparation method, and the pharmaceutical composition containing it, and in the preparation for treating or preventing the drug use related to the regulation miRNA124 level, including the use for treating inflammatory diseases.The compound provided by the present application has the advantages of high metabolic stability, better bioavailability, low brain distribution amount, good up-regulation miRNA-124 level activity, low cardiotoxicity, no potential central nervous side effects such as headache.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry, specifically relating to a pyridopyrimidine compound or a pharmaceutically acceptable salt thereof, a pharmaceutical composition containing the same, and its use in the preparation of a medicament for upregulating miRNA-124 levels, including its use in the preparation of a medicament for the prevention or treatment of inflammatory diseases. Background Technology

[0002] Inflammation is the immune system's protective response to tissue damage and infection. However, in some cases, the inflammatory response can damage the body. In the acute phase, inflammation manifests as pain, fever, redness, swelling, and loss of function. Many inflammatory conditions exist, affecting millions of people worldwide. Inflammatory diseases include those related to autoimmune disorders, inflammatory diseases of the central nervous system (CNS), arthritis, inflammatory digestive tract diseases, inflammatory skin conditions, and cancer-related inflammation.

[0003] Overexpression of miRNA-124 initiates an anti-inflammatory cascade. miRNA-124 targets signal transducers and activator of transcription 3 (STAT3) to regulate macrophage cytokine production, reducing the secretion of IL-1β, IL-6, and TNF-α, thereby decreasing Th17 proliferation. Upregulation of miRNA-124 in macrophages also reduces MCP1 production, thereby limiting neutrophil recruitment, activating macrophage transdifferentiation from M1 to M2, inactivating inflammatory macrophages, and transforming them into microglia-like cells.

[0004] Prior art has been disclosed that quinoline derivatives can be used to treat inflammatory diseases by modulating miRNA-124. WO2010 / 143170 teaches the use of compounds for treating conditions associated with premature aging. WO2010 / 143169 and WO2012 / 080953 teach the use of compounds for treating AIDS. WO2010 / 143168 teaches the use of compounds for treating a range of cancers. These compounds have been shown to correct defects in alternative splicing. Abivax's miRNA-124 modulator ABX464, with a novel anti-inflammatory mechanism, is being developed for a variety of inflammatory diseases.

[0005]

[0006] However, these drugs suffer from high concentrations in brain tissue, leading to central nervous system side effects such as headaches and gastrointestinal side effects, which limits their clinical application. Therefore, there is an urgent clinical need to develop drugs that effectively upregulate miRNA-124 activity, have low brain distribution, and minimal central nervous system side effects. Summary of the Invention

[0007] To address the aforementioned technical problems, the present invention provides a pyridopyrimidine compound, hydrate, solvate, polymorph, active metabolite, pharmaceutically acceptable salt thereof, or prodrug as shown in formula (I):

[0008] in: Z is either C or N; R 1 Independently selected from hydrogen, deuterium, halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C3-C6)heterocyclic alkyl, hydroxyl, nitro, cyano, morpholino, N-methylpiperazinyl, (C1-C3)haloalkyl, (C1-C3)haloalkoxy, (C3-C6)halocycloalkyl, (C3-C6)haloheterocyclic alkyl; R 2 Selected from hydrogen, glucuronidyl alkyl, (C1-C3)alkyl, (C3-C6)cycloalkyl, (C3-C6) heterocycloalkyl, (C1-C3) fluoroalkyl, (C3-C6) halocycloalkyl, (C3-C6) haloheteroalkyl, (C1-C3) deuterated alkyl; R 3 Independently selected from hydrogen, deuterium, halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C3-C6)heterocycloalkyl, hydroxyl, nitro, cyano, vinyl, (C1-C3)haloalkyl, (C1-C3)haloalkoxy, (C3-C6) substituted cycloalkyl, (C3-C6) halocycloalkyl, difluoromethylenedioxy, (C3-C6) haloheterocycloalkyl, fluorovinyl, pentafluorothio, trifluoromethylthio, -CF2OCH3, -CF2OCH2CH3; The heterocyclic alkyl group contains one to two heteroatoms selected from N and O; n is 1, 2, 3 or 4; m can be 1, 2, 3, 4 or 5.

[0009] As shown in formula (I) above, pyridopyrimidine compounds, hydrates, solvates, polymorphs, active metabolites, pharmaceutically acceptable salts thereof, or prodrugs thereof, wherein R 1 It is independently selected from hydrogen, deuterium, chlorine, fluorine, cyano, cyclopropyl, morpholino, (C1-C3)alkyl, (C1-C3)fluoroalkyl, (C1-C3)alkoxy, and (C1-C3)fluoroalkoxy.

[0010] As shown in formula (I) above, pyridopyrimidine compounds, hydrates, solvates, polymorphs, active metabolites, pharmaceutically acceptable salts thereof, or prodrugs thereof, wherein R 2Selected from hydrogen, glucuronidyl, methyl, ethyl, cyclopropyl, trifluoromethyl, and trifluoroethyl.

[0011] As shown in formula (I) above, pyridopyrimidine compounds, hydrates, solvates, polymorphs, active metabolites, pharmaceutically acceptable salts thereof, or prodrugs thereof, wherein R 3 Independently selected from hydrogen, deuterium, chlorine, fluorine, cyano, cyclopropyl, morpholino, (C1-C3)alkyl, (C1-C3)fluoroalkyl, (C1-C3)alkoxy, (C1-C3)fluoroalkoxy, difluoromethylenedioxy, fluorovinyl, pentafluorothio, trifluoromethylthio, -CF2OCH3, -CF2OCH2CH3 .

[0012] n is 1, 2, or 3; m can be 1, 2, 3 or 4.

[0013] The pyridopyrimidine compounds of formula (I) preferably have the structure shown in any of the following structural formulas:

[0014] The compounds of the present invention are generally used in the form of free acids or free bases. Alternatively, the compounds of the present invention may be used in the form of acid or base salts. Acid adducts of the free amino compounds of the present invention can be prepared using methods known in the art, and can be prepared from organic and inorganic acids. Suitable organic acids include maleic acid, fumaric acid, benzoic acid, ascorbic acid, succinic acid, methanesulfonic acid, acetic acid, trifluoroacetic acid, oxalic acid, propionic acid, tartaric acid, salicylic acid, citric acid, gluconic acid, lactic acid, mandelic acid, phenylacetic acid, aspartic acid, stearic acid, palmitic acid, ethylene glycol, glutamic acid, and benzenesulfonic acid. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-heptate, glyceryl phosphate, gluconate, hemisulfate, heptaate, hydroiodate, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Basic salts include salts formed with carboxylate anions, and also include salts formed with organic and inorganic cations such as those selected from alkali metal ions, alkaline earth metal ions (e.g., lithium, sodium, potassium, magnesium, barium, calcium), and ammonium ions, as well as their substituted derivatives (e.g., dibenzylamine, benzylamine, 2-hydroxyethylamine, etc.). Therefore, the term "pharmaceutical-acceptable salt" of general formula (I) should include all acceptable salt forms.

[0015] Furthermore, prodrugs are also included within the scope of this invention. A prodrug is any covalently bound carrier that, when administered to a patient, releases in vivo a compound accepting general formula (I). Prodrugs are typically prepared by modifying functional groups in a way that allows the modification to be resolved in vivo via conventional exchange or degradation to yield the parent compound. Prodrugs include compounds of this invention, for example, hydroxyl, amino, or thiol groups bound to any group, wherein, when administered to a patient, the group is detached to yield a hydroxyl, amino, or thiol group.

[0016] Therefore, representative examples of prodrugs include (but are not limited to) acetates (esters), formates (esters), and benzoate derivatives of compounds with alcohol and amine functional groups of general formula (I). Furthermore, in the case of carboxylic acids (-COOH), esters such as methyl esters and ethyl esters may be included. In the case of hydroxyl groups, mixed acid anhydrides such as methoxy, ethoxy, propoxy, and tert-butoxy may be included.

[0017] For stereoisomers, compounds of general formula (I) may have a chiral center and may exist as racemates, racemic mixtures, and individual enantiomers or diastereomers. All isomeric forms are included within the scope of this invention, including mixtures thereof. Furthermore, certain crystalline forms of compounds of general formula (I) may exist in polymorphic forms, which are also included in this invention. In addition, some compounds of said general formula (I) may also form solvates with water, ethanol, or other organic solvents. Such solvates are similarly included within the scope of this invention.

[0018] Those skilled in the art will understand that any compound may contain atomic isotopes in non-natural proportions on one or more of the atoms constituting the compound. As used in this invention, in the specification and claims, H refers to hydrogen and includes any stable isotope of hydrogen, i.e. 1 H and D. In embodiments where the atom is designated as H, no work was performed to enrich the specific isotope of hydrogen, and therefore those skilled in the art will understand that such hydrogen atoms may be present at approximately the natural abundance concentration of hydrogen. In the pyridopyrimidine compounds involved in this invention, deuteration means that the atom at the relevant site of the compound contains deuterium atoms in a proportion exceeding the natural proportion (i.e., exceeding the natural abundance of deuterium). Therefore, any pyridopyrimidine compound of formula (I) containing deuterium atoms at the relevant site in a proportion exceeding the natural abundance of deuterium is within the scope of protection of this invention. For example, it can be understood that corresponding pyridopyrimidine compounds of formula (I) with corresponding deuteration rates or deuterium contents obtained by introducing deuterium atoms using commercially available deuterating agents through the same or similar chemical synthesis methods shown in the embodiments of this invention are within the scope of protection of this invention. The chemical synthesis methods and deuterated reagents mentioned herein are not limited to those exemplified in the examples, but should be understood as all synthetic methods or routes that can be used in the art to obtain the compounds of the present invention, and all deuterated reagents that can be used in conjunction with the aforementioned synthetic methods or routes to introduce deuterium atoms into the target molecule.

[0019] Isotope-labeled compounds and salts can be used in a variety of advantageous ways, including as pharmaceuticals. In some embodiments, the isotope-labeled compounds and salts are deuterium (D)-labeled. Deuterium (D)-labeled compounds and salts are therapeutically useful, possessing potential therapeutic advantages over unlabeled compounds. Generally, due to the kinetic isotope effect described below, deuterium (D)-labeled compounds and salts can exhibit higher metabolic stability compared to unlabeled compounds and salts. Higher metabolic stability directly translates to prolonged in vivo half-life or reduced dose, which in most cases will represent preferred embodiments of the invention. Isotope-labeled compounds and salts can generally be prepared by performing the procedures disclosed in the synthetic schemes, examples, and related descriptions, replacing the unlabeled reactants with readily available isotope-labeled reactants. Deuterium (D)-labeled compounds and salts can manipulate the oxidative metabolic rate of said compounds through primary kinetic isotope effects. Primary kinetic isotope effects are rate changes in chemical reactions resulting from isotopic nuclear exchange, which in turn is caused by changes in the ground-state energy of the covalent bonds involved in the reaction. Exchange with heavier isotopes typically lowers the ground-state energy of chemical bonds and thus reduces the rate-limiting bond breaking. If bond breaking occurs in or near a saddle point region along the coordinates of a multi-product reaction, it can substantially alter the product distribution ratio.

[0020] Based on the specific embodiments disclosed below according to the present invention, those skilled in the art can use the same or similar principles and methods to prepare the specific compounds involved in the pyridopyrimidine compounds of the present invention as shown in formula (I).

[0021] The present invention further provides the use of a pyridopyrimidine compound of formula (I), its stereoisomers, hydrates, solvates, polymorphs, active metabolites, pharmaceutically acceptable salts thereof, or prodrugs thereof in the preparation of a medicament for regulating miRNA-124-related diseases. The miRNA-124-related diseases include autoimmune disorders and inflammatory disorders; neurological disorders and neurodegenerative disorders; pain disorders and nociceptive disorders; cardiovascular disorders; metabolic disorders; eye disorders; and tumor disorders.

[0022] Inflammatory and autoimmune disorders include systemic autoimmune disorders, autoimmune endocrine disorders, and organ-specific autoimmune disorders. Systemic autoimmune disorders include systemic lupus erythematosus (SLE), psoriasis, psoriatic arthritis, vasculitis, inflammatory myopathies (including polymyositis, dermatomyositis, and inclusion body myositis), scleroderma, multiple sclerosis, systemic sclerosis, ankylosing spondylitis, rheumatoid arthritis, nonspecific inflammatory arthritis, juvenile inflammatory arthritis, juvenile idiopathic arthritis (including its oligoarticular and polyarticular forms), anemia of chronic disease (ACD), Still's disease (juvenile and / or adult-onset), Behcet's disease, and Sjögren's syndrome. Autoimmune endocrine disorders include thyroiditis. Organ-specific autoimmune disorders include Addison's disease, hemolytic or pernicious anemia, acute kidney injury (AKI; including cisplatin-induced AKI), diabetic nephropathy (DN), obstructive urinary tract disease (including cisplatin-induced obstructive urinary tract disease), glomerulonephritis (including Goodpassuia syndrome, immune complex-mediated glomerulonephritis, and anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis), lupus nephritis (LN), minimally invasive nephropathy, Graves' disease, idiopathic thrombocytopenic purpura, inflammatory bowel disease (including Crohn's disease, ulcerative colitis, indeterminate colitis, and cryptitis), pemphigus, atopic dermatitis, and autoimmune diseases. Hepatitis B, primary biliary cirrhosis, autoimmune pneumonia, autoimmune carditis, myasthenia gravis, spontaneous infertility, osteoporosis, osteopenia, erosive bone disease, osteitis, cartilage degeneration and / or destruction, fibrotic disorders (including various forms of liver and lung fibrosis), asthma, rhinitis, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome, sepsis, fever, muscular dystrophy (including Duchenne muscular dystrophy), organ transplant rejection (including kidney allogeneic graft rejection), scleritis (including giant cell arteritis scleritis), aortitis, hidradenitis suppurativa, pyoderma gangrenosa, sarcoidosis, polymyalgia rheumatica, and axial spondyloarthritis.

[0023] Neurological disorders and neurodegenerative disorders include Alzheimer's disease, Parkinson's disease, Huntington's disease, ischemia, stroke, amyotrophic lateral sclerosis, spinal-hip injury, head injury, seizures, and epilepsy.

[0024] Cardiovascular disorders include thrombosis, cardiac hypertrophy, hypertension, irregular heart contractions (such as during heart failure), and sexual disorders (including erectile dysfunction and female sexual dysfunction).

[0025] Metabolic disorders include diabetes (including insulin-dependent diabetes and juvenile diabetes), dyslipidemia, and metabolic syndrome.

[0026] Eye disorders include retinopathy (including diabetic retinopathy, proliferative retinopathy, nonproliferative retinopathy, and retinopathy of prematurity), macular edema (including diabetic macular edema), age-related macular degeneration (ARMD), vascularization (including corneal vascularization and neovascularization), retinal vein occlusion, and various forms of uveitis (including iritis) and keratitis.

[0027] Cancer disorders (which can be acute or chronic) include proliferative disorders, particularly cancer and cancer-related complications (including bone complications, cachexia, and anemia). Specific types of cancer include hematologic malignancies (including leukemia and lymphoma) and non-hematologic malignancies (including solid tumors, sarcomas, meningiomas, glioblastoma multiforme, neuroblastoma, melanoma, gastric cancer, and renal cell carcinoma). Chronic leukemia can be myelopathic or lymphoid. Leukemia includes types such as lymphoblastic T-cell leukemia, chronic myeloid leukemia (CML), chronic lymphocytic / lymphoid leukemia (CLL), hairy cell leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), myelodysplastic syndromes, chronic neutrophilic leukemia, acute lymphoblastic T-cell leukemia, plasmacytoma, immunoblastic large cell leukemia, mantle cell leukemia, multiple myeloma, acute megakaryoblastic leukemia, acute megakaryocytic leukemia, promyelocytic leukemia, and erythroleukemia. Lymphoma includes types such as malignant lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, lymphoblastic T-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, MALT1 lymphoma, and marginal zone lymphoma. Non-hematologic malignancies include cancers of the prostate, lungs, breast, rectum, colon, lymph nodes, bladder, kidneys, pancreas, liver, ovaries, uterus, cervix, brain, skin, bone, stomach, and muscles.

[0028] The present invention further provides a pyridopyrimidine compound of formula (I), its stereoisomers, hydrates, solvates, polymorphs, active metabolites, pharmaceutically acceptable salts thereof, or prodrugs thereof, for the preparation of a drug for the treatment / prevention of miRNA-124-related inflammatory diseases. The miRNA-124-related inflammatory diseases include: inflammatory bowel disease, rheumatoid arthritis, Crohn's disease, ulcerative colitis, multiple sclerosis, Alzheimer's disease, Parkinson's disease, osteoarthritis, atherosclerosis, ankylosing spondylitis, psoriasis, dermatitis, Sjogren's syndrome, bronchitis, asthma, or inflammation associated with colon cancer.

[0029] The present invention further provides a pharmaceutical composition comprising a therapeutically effective amount of a pyridopyrimidine compound of formula (I), its stereoisomer, hydrate, solvate, polymorph, active metabolite, pharmaceutically acceptable salt thereof or a prodrug thereof, and a pharmaceutically acceptable carrier.

[0030] The present invention further provides a pharmaceutical composition comprising, in addition to a therapeutically effective amount of a pyridopyrimidine compound of formula (I), its stereoisomers, hydrates, solvates, polymorphs, active metabolites, pharmaceutically acceptable salts thereof, or a prodrug thereof, and a pharmaceutically acceptable carrier, one or more other pharmaceutically active ingredients. The other pharmaceutically active ingredients are selected from the group consisting of: vetuzumab, PF-06835375, eculizumab, mirtuzumab, SM-06, SM-03, BT-063, QX-006-N, BOS-161721, AK-101, TNX-1500, cilalizumab, dastardilmab, TAK-079, frizzetuzumab, iributumab, and aniluzumab. Iscalimumab, pegylated dapirozumab, lanalitumab, LY-3361237, JNJ-55920839, UBP-1213, DS7011, PFI-102, BIIB-059, obbecilimab, tatoluzumab, vobalizumab, TE-2324, PRV-3279, chloroquine, hydroxychloroquine, hydroxychloroquine sulfate, COV-08-0064;GNKS-356, AVO-101, Rozibafusp ALFA), VRN-02, annexuzlimab, ALPN-101, bendamustine hydrochloride, BMS-986256, NKTR35, acecept, telitacept, M-5049, KZR-616, KPG-818, verdinexor, ALPN-303, valacyclocept, LA-1, sinemod, prednisone, adrenocorticotropic hormone, deuterocelexitinib, CPL409116, CS-12192, tofacitinib citrate, ISB-830, DV-1079, julemic acid, ibelidomide, TAM-01, BML-258, bupraxitinib, SDC-1801, SDC-1802, ICP-330, NTR-441, dabra Zasatide, GSK-2646264, SKI-O-703, Lanlapunib (GS-9876), GNS-1653, HMPL-523, RSLV-132, Interleukin-2 follow-up biologics, Interleukin-2, Anteluke, Interking recombinant human interleukin-2, ILT-101, CUG-252, DZ-2002, PEGylated HLA-x (SLE), AC-0058, Fenibutinib, XNW-1011, Tiralutinib hydrochloride, Brimbutinib, Elbutinib, Orelabrutinib, DWP-213388, INV-103, R-salbutamol sulfate, Anchoring protein, NIK-SMI1, X-6, INV-17, Oshadi D (Oshadi D) Baricitinib, Upatinib, Figotinib, Itatinib, INCB-54707, Digotinib, DWP-212525, CKD-971, Mometasone, Betamethasone, Furemod, Arachidonic acid ethanolamine, DCB SLE1, Arsenic trioxide, Tairuimide, TV-4710 (Eridinide), Allogeneic human umbilical cord mesenchymal stem cell therapy (hUC-MSC), LC-200, BI-705564, SM-934, GX-101, TXR-712, TXR-711, CIT-013, MHV-370, TPX-6001, TPX-7001, Dihydroartemisinin and AMG-592, or any pharmaceutically acceptable salt of the foregoing substances or any combination thereof.

[0031] Furthermore, combinations of other anti-inflammatory agents and pyridopyrimidine compounds as shown in formula (I) can be used as pharmaceutical preparations. Therefore, the present invention also relates to products containing (a) a pyridopyrimidine compound as shown in formula (I) and (b) other anti-inflammatory compounds, as combination formulations for simultaneous, separate, or sequential anti-inflammatory treatment. Different pharmaceutical preparations can be co-combined with pharmaceutically acceptable carriers in a single formulation.

[0032] The compounds of the present invention, or their pharmaceutically acceptable salts, in pure form or suitable pharmaceutical compositions, may be administered in any acceptable or similar mode of administration. The pharmaceutical compositions of the present invention may be prepared by combining the compounds of the present invention with suitable pharmaceutically acceptable carriers, diluents, or excipients, and may be formulated into solid, semi-solid, liquid, or gaseous formulations, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalers, gels, microspheres, and aerosols. Typical routes of administration of the pharmaceutical compositions include (but are not limited to) oral, topical, transdermal, inhalation, non-enteric, sublingual, buccal, rectal, vaginal, and intranasal administration. As used herein, the term non-enteric includes subcutaneous injection, intravenous, intramuscular, intrasternal injection, or infusion techniques. The pharmaceutical compositions of the present invention are formulated to allow the active ingredient contained therein to be bioavailable after administration to a patient. The composition to be administered to an individual or patient may be in the form of one or more dose units, wherein, for example, a tablet may be a single dose unit, while a container containing the compound of the invention in aerosol form may hold multiple dose units. The actual methods for preparing the dosage form are known to, or will be known to, those skilled in the art. The composition to be administered will in any case contain a therapeutically effective amount of the compound of the invention or a pharmaceutically acceptable salt thereof, in order to treat the disease or condition of interest according to the teachings of the invention.

[0033] The pharmaceutical compositions of the present invention can be in solid or liquid form. On one hand, the carrier is a microparticle, so that the composition is in the form of, for example, tablets or powders. The carrier can be a liquid, and the composition is, for example, an oral syrup, an injectable liquid, or an aerosol suitable for, for example, inhalation. When intended for oral administration, the pharmaceutical composition is preferably in solid or liquid form, wherein semi-solid, semi-liquid, suspension, and gel forms are included in forms considered solid or liquid herein. For oral solid compositions, the pharmaceutical composition can be formulated into powders, granules, compressed tablets, pills, capsules, chewable tablets, powder tablets, etc. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more of the following substances may be present: binders, such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, xanthan gum, or gelatin; excipients, such as starch, lactose, or dextrin; disintegrants, such as alginate, sodium alginate, Primogel, corn starch, etc.; lubricants, such as magnesium stearate or hydrogenated vegetable oil (Sterotex); flow aids, such as colloidal silica; sweeteners, such as sucrose or saccharin; flavoring agents, such as peppermint, methyl salicylate, or sweet orange flavoring; and coloring agents.

[0034] In preparing compositions for oral administration, any common pharmaceutical medium can be used, such as water, glycols, oils, alcohols, etc., in the case of oral liquid compositions (e.g., suspensions, syrups, free radicals, emulsions, and solutions); or solid carriers such as starch, sugar, kaolin, lubricants, binders, disintegrants, etc., in the case of solid compositions. For parenteral compositions, the carrier will typically consist of at least predominantly sterile water, but other components such as solubilizers, emulsifiers, or additional adjuvants may be added. Injectable solutions can be prepared in which the carrier includes physiological saline, glucose solution, or a mixture of both. Injectable suspensions can also be prepared, in which case appropriate liquid carriers, suspending agents, etc., can be used. Solid formulations intended to be converted into liquid form shortly before use are also included, such as powders for reconstitution.

[0035] When the pharmaceutical composition is in capsule form, such as a gelatin capsule, it may contain a liquid carrier, such as polyethylene glycol or oil, in addition to the substances described above. The pharmaceutical composition may be in liquid form, such as a tincture, syrup, solution, emulsion, or suspension. This liquid may be administered orally or by injection, as two examples. When intended for oral administration, the composition preferably contains one or more of the following in addition to the compounds of the present invention: a sweetener, a preservative, a dye / coloring agent, and a flavor enhancer. In compositions intended for injection, one or more of the following may be included: a surfactant, a preservative, a wetting agent, a dispersant, a suspending agent, a buffer, a stabilizer, and an isotonic agent.

[0036] Regardless of whether it is a solution, suspension, or other similar form, the liquid pharmaceutical composition of the present invention may include one or more of the following adjuvants: a sterile diluent, such as water for injection, physiological saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, non-volatile oils (e.g., synthetic mono- or diglycerides, which can be used as solvents or suspension media), polyethylene glycol, glycerol, propylene glycol, etc.; an antibacterial agent, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate, or phosphate; and a tonic agent, such as sodium chloride or dextran. Enteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0037] Liquid pharmaceutical compositions of the present invention intended for oral or intestinal administration should contain an amount of the compounds of the present invention to achieve a suitable dosage. Pharmaceutical compositions of the present invention may be intended for topical administration, in which case the carrier preferably comprises a solution, emulsion, ointment, or gel matrix. For example, this matrix may comprise one or more of the following: paraffin oil, lanolin, polyethylene glycol, beeswax, mineral oil, diluents (e.g., water and alcohol), and emulsifiers and stabilizers. Thickeners may be present in pharmaceutical compositions intended for topical administration. If intended for transdermal administration, the composition may comprise a transdermal patch or an iontophoresis device.

[0038] The pharmaceutical compositions of this invention can be administered rectally, for example in suppository form, where they melt in the rectum and release the drug. The compositions for rectal administration may contain an oily matrix as a suitable non-irritating excipient. The matrix includes (but is not limited to) lanolin, cocoa butter, and polyethylene glycol.

[0039] The pharmaceutical compositions of the present invention may include various substances in physical form that alter the dosage units of solids or liquids. For example, the composition may include a substance that forms a coating around the active ingredient. The substance forming the coating is generally inert and may be selected from, for example, sugars, shellac, and other enteric coatings. Alternatively, the active ingredient may be encapsulated in a gelatin capsule.

[0040] Pharmaceutical compositions of the present invention in solid or liquid form may include an agent that binds to and thereby facilitates the delivery of the compound. Suitable agents with this capability include monoclonal or polyclonal antibodies, proteins, or liposomes.

[0041] The pharmaceutical compositions of the present invention can consist of dosage units that can be administered in aerosol form. The term aerosol is used to refer to a variety of systems, ranging from gel-like forms to systems consisting of pressurized packaging. Delivery can be carried out by liquefied or compressed gas, or by a suitable pump system dispensing the active ingredient. The aerosols of the compounds of the present invention can be delivered in a single-phase, two-phase, or three-phase system to deliver the active ingredient. Aerosol delivery includes necessary containers, initiators, valves, sub-containers, etc., which together form a kit. Preferred aerosols can be determined by those skilled in the art without extensive experimentation.

[0042] The pharmaceutical compositions of the present invention can be prepared using methods well-known in the pharmaceutical industry. For example, a pharmaceutical composition intended for injection can be prepared by combining the compounds of the present invention with sterile distilled water to form a solution. Surfactants may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the compounds of the present invention, thereby promoting the dissolution or homogeneous suspension of the compounds in an aqueous delivery system.

[0043] The effective amount of the compound of the present invention can be mixed with various pharmaceutical additives such as excipients, binders, disintegrants, and lubricants suitable for its dosage form, as needed, to form a pharmaceutical composition. Furthermore, by appropriately changing the effective amount, dosage form, and / or various pharmaceutical additives of the compound of the present invention, this pharmaceutical composition can be formulated for pediatric use, elderly use, critically ill patients, or surgical use. For example, the pediatric pharmaceutical composition can be administered to newborns (under 4 weeks old), infants (4 weeks to under 1 year old), toddlers (1 year to under 7 years old), children (7 years to under 15 years old), or patients aged 15 to 18 years. For example, the elderly pharmaceutical composition can be administered to patients aged 65 years and older.

[0044] The dosage of the pharmaceutical composition of the present invention is preferably determined taking into account the patient's age, weight, type or severity of disease, route of administration, etc. For oral administration, the dosage is typically from 0.05 to 100 mg / kg / day, preferably from 0.1 to 10 mg / kg / day. For non-oral administration, the dosage varies considerably depending on the route of administration, but is typically from 0.005 to 10 mg / kg / day, preferably from 0.01 to 1 mg / kg / day. This can be administered once daily or divided into several doses.

[0045] The dosage of the concurrent drug can be appropriately selected based on clinically used dosages. Furthermore, the mixing ratio of the compound of the present invention with the concurrent drug can be appropriately selected according to the target population, route of administration, target disease, symptoms, and combination. For example, when the target population is humans, the concurrent drug can be used in quantities of 0.01 to 100 parts by weight relative to 1 part by weight of the compound of the present invention.

[0046] This invention relates to pyridopyrimidine compounds as shown in formula (I), pharmaceutical compositions containing the same, and the use of such compositions for the treatment / prevention of miRNA-124-related inflammatory diseases. The miRNA-124-related inflammatory diseases include: inflammatory bowel disease, rheumatoid arthritis, Crohn's disease, ulcerative colitis, multiple sclerosis, Alzheimer's disease, Parkinson's disease, osteoarthritis, atherosclerosis, ankylosing spondylitis, psoriasis, dermatitis, Sjogren's syndrome, bronchitis, asthma, or inflammation associated with colon cancer.

[0047] The compounds provided by this invention possess advantages such as high metabolic stability, superior bioavailability, low brain distribution, higher upregulation of miRNA-124 levels, excellent anti-inflammatory activity, low cardiotoxicity, and the potential to reduce potential central nervous system side effects such as headaches. Therefore, the compounds of this invention have the potential to have better therapeutic effects on inflammatory diseases and exhibit better pharmaceutical compatibility.

[0048] Unless otherwise specified, all reagents and raw materials used in this invention are commercially available. Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention. Detailed Implementation

[0049] The meanings of all terms used in this instruction manual are as follows: unless otherwise stated, each term has the same meaning when used alone or in combination with other terms.

[0050] "Halogen" contains fluorine, chlorine, bromine, and iodine atoms. Fluorine and chlorine atoms are particularly preferred.

[0051] The term "alkyl" comprises a straight-chain or branched hydrocarbon group having 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, and even more preferably 1 to 4 carbon atoms. Examples include: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, isooctyl, n-nonyl, n-decyl, etc. Preferred embodiments of "alkyl" include: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl. More preferred embodiments include: methyl, ethyl, n-propyl, isopropyl, tert-butyl.

[0052] "Cycloalkyl" refers to a cyclic hydrocarbon group having 3 to 10 carbon atoms, preferably 3 to 8 carbon atoms, more preferably 3 to 6 carbon atoms, and even more preferably 3 to 5 carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0053] "Alkoxy" refers to an alkyl radical attached to an oxygen atom and is represented by -O-alkyl. For example, "(C1-C6)alkoxy" includes methoxy, ethoxy, propoxy, and butoxy.

[0054] When used alone or as part of a larger group, "heterocyclic alkyl" means a saturated or unsaturated 3-6 membered cyclic radical, optionally containing one or more double bonds, and generally having 3-6 cyclic atoms selected from carbon and at least one (generally 1 to 4, more generally 1 or 2) heteroatoms (e.g., oxygen, nitrogen).

[0055] "Halogenated alkyl" refers to a (C1-C3) alkyl group that is substituted with one or more fluorine atoms, such as trifluoromethyl, difluoromethyl, and 2,2,2-trifluoroethyl; "halogenated alkoxy" refers to a group formed by the connection of a haloalkyl group to the parent nucleus through an oxygen atom, such as trifluoromethoxy.

[0056] "Fluorovinyl" refers to a vinyl group substituted with one or two fluorine atoms, including 1-fluorovinyl, 2-fluorovinyl, 1,1-difluorovinyl, 1,2-difluorovinyl and 2,2-difluorovinyl; "pentafluoride sulfide" refers to the -SF5 group; "glucuronide glycoside" refers to the glucuronic acid group linked to the compound of the present invention via an oxyglycosidic bond.

[0057] Certain preferred embodiments of the present invention are illustrated in the following non-limiting examples. Experimental methods not specifically described in the following examples are performed according to conventional methods and conditions, or as selected according to the trade instructions. Raw materials may be commercially available, or prepared by methods known in the art, or according to the methods described herein.

[0058] Example 1: Synthesis of Compound 1 The synthesis route is as follows:

[0059] Preparation of Compound 1 Intermediate 3 (11.3 g, 51.7 mmol, 1.50 eq) and DIEA (8.91 g, 68.9 mmol, 12.0 mL, 2.00 eq) were added to a DMA (55 mL) solution of intermediate 2 (5.50 g, 34.4 mmol, 1.00 eq). The reaction mixture was stirred at 135°C for 3 hours. LC-MS showed that the reaction was complete. The reaction mixture was diluted with water (100 mL), washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by rapid silica gel column chromatography (40 g SepaFlash® silica gel column; eluent 0–40%, ethyl acetate / petroleum ether). The purified product was further purified by preparative chromatography (column: Welch Xtimate C18 150×50 mm×5 μm; mobile phase: [water (10 mM NH4HCO3)-ACN]; gradient: 46%–76% acetonitrile over 20.0 min) to give a yellow solid compound 1 (yield 55.6%, purity 96.8%). 1 H NMR (400MHz, DMSO- d 6): δ = 10.82 (br s, 1 H), 9.55 (s, 1 H), 9.14 (s, 1 H), 8.84 (s, 1 H), 8.21 (br s, 2 H), 7.40 (br d, J =8.6 Hz, 2 H) ppm. LCMS: RT = 0.547 min, MS (ESI) m / z: 341.0 [M+H] + . HPLC: RT = 2.955 min, purity: 96.8%.

[0060] Solution 1: Intermediate 1 (25.0 g, 190 mmol, 1.00 eq) dissolved in 500.0 mL THF. Solution 2: LDA (2 M, 99.7 mL, 1.05 eq). Solution 3: Ethyl formate (42.2 g, 570 mmol, 45.8 mL, 3.00 eq) dissolved in 500 mL THF. Solutions 1 and 2 were mixed in a T-mixer at -60.0°C and then fed into a perfluoroalkoxy (PFA) coil reactor (outer diameter: 3.175 mm (1 / 8 inch); internal volume: 30.0 mL) at -60.0°C at flow rates of 6.37 mL / min and 3.63 mL / min, respectively. The effluent from the reactor was immediately mixed with solution 3 (delivered at a flow rate of 6.37 mL / min) in a second T-mixer at -60.0°C, and the combined stream entered a second perfluoroalkoxy (PFA) coil reactor (outer diameter: 3.175 mm (1 / 8 inch); internal volume: 60.0 mL) at -60.0°C. The residence times in the first and second reactors were 3.0 min and 3.67 min, respectively. The reactor effluent was collected in a flat-bottomed container (quenched at 0°C with 1200.0 mL NH4Cl(aq) / MeOH = 2:1). Flow reaction time: 1.4 h. TLC (PE:EtOAc = 3:1) showed complete consumption of the starting material. The reaction solution was extracted with EA (3 × 200 mL). The organic layer was washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by rapid silica gel column chromatography (400 g SepaFlash® silica gel column; eluent 0-10%, ethyl acetate / petroleum ether). A yellow oily intermediate 2 (5.50 g, 34.4 mmol, yield 18.1%) was obtained. 1 H NMR (400 MHz, DMSO- d 6): δ = 10.20(s, 1 H), 9.03 (d, J =8.8 Hz, 1 H), 8.95 (d, J =8.2 Hz, 1 H) ppm.

[0061] Preparation of intermediate 3 Concentrated HCl (22.1 g, 218 mmol, 21.7 mL, 36% purity, 1.31 eq) was added to a solution of cyanamide (40.3 g, 479 mmol, 40.3 mL, 2.86 eq) and intermediate 3A (30.0 g, 167 mmol, 22.9 mL, 1.00 eq) in 90 mL of EtOH. The mixture was stirred at 108°C for 20 hours. LC-MS showed that the reaction was complete. The reaction solution was cooled to room temperature, concentrated, and diluted with 100 mL of water. The pH was adjusted to 7 with 1 N NaOH, extracted with ethyl acetate (100 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by rapid silica gel column chromatography (100 g SepaFlash® silica gel column; eluent 0–20%, MeOH / DCM). A colorless oily intermediate 3 was obtained (26.0 g, 106 mmol, yield 63.6%, purity 90%). LCMS: RT = 0.413 min, MS (ESI) m / z: 220.3 [M+H] + .

[0062] Example 2, Synthesis of Compound 2 The synthesis route is as follows:

[0063] Preparation of compound 2 Intermediate 3 (5.60 g, 25.5 mmol, 1.50 eq) and DIEA (4.40 g, 34.0 mmol, 5.93 mL, 2.00 eq) were added to a DMA (25 mL) solution of intermediate 2A (2.72 g, 17.0 mmol, 1.00 eq). The reaction mixture was stirred at 180°C for 3 hours, and LC-MS showed that the reaction was complete. The organic layer was diluted with water (5 mL), washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by rapid silica gel column chromatography (20 g SepaFlash® silica gel column; eluent 0–40%, ethyl acetate / petroleum ether). The purified product was further purified by preparative chromatography (column: PrePulite XPtC18 250 × 50 mm × 7 μm; mobile phase: [water (0.05% HCl)-ACN]; gradient: 28%–68% acetonitrile over 20.0 min). A yellow solid compound 2 was obtained (yield 53.1%, purity 96.7%). 1 H NMR (400 MHz, DMSO- d 6): δ = 10.67(s, 1 H), 9.52 (s, 1 H), 9.07 (s, 1 H), 8.07 (br d, J=7.8 Hz, 2 H), 7.66 (s,1 H), 7.38 (br d, J =8.6 Hz, 2 H) ppm. LCMS: RT = 0.599 min, MS (ESI) m / z =341.1 [M+H] + , HPLC: RT = 3.488 min, purity: 96.7%. Example 3: Synthesis of Compound 12 The synthesis route is as follows:

[0064] Preparation of compound 12 Hydrogen peroxide (30%, 20.8 mL) was added to a stirred aqueous suspension of lithium hydroxide monohydrate (9.46 g, 224 mmol) in 85 mL, and stirred for 15 minutes. The mixture was then added to a stirred intermediate 6 (6.8 g, 10.4 mmol) in 280 mL of THF. A precipitate formed within 20 minutes, and the reaction was quenched by stirring for 2 hours with sodium thiosulfate. The reaction was confirmed by TLC. The reaction mixture was acidified to pH 2.5 with 1 M hydrochloric acid, and the mixture was extracted three times with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. The crude product was further purified by preparative chromatography (column: PrePulite XP tC18 250 × 50 mm × 7 μm; mobile phase: [water (0.05% HCl) - ACN]; gradient: 20%–50% acetonitrile over 20.0 min). A yellow solid compound 12 was obtained (yield 58.1%, purity 95.5%). 1 H NMR (400 MHz, DMSO- d 6): δ = 9.54(s, 1 H), 9.12 (s, 1 H), 8.83(s, 1 H), 8.20 (br s, 2 H), 7.39 (br, 2 H), 5.18(br, 1H), 3.79 (m, 1H), 3.13-3.50(m, 4H) ppm. MS (ESI) m / z: 517.1 [M+H] + .

[0065] Preparation of intermediate 5 Under a nitrogen atmosphere at 0°C, 45% HBr / acetic acid (38 mL) was added dropwise to intermediate 4 (11.6 g, 30.81 mmol). The round-bottom flask was placed in a desiccator and stirred at 5°C for 48 hours. The reaction solution was diluted with ethyl acetate (200 mL) and poured onto ice (200 g). The solution was washed with saturated aq. NaHCO3 (150 mL), brine (200 mL), and water (200 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and the solvent was removed under reduced pressure. The product was purified by rapid silica gel column chromatography (2:1 petroleum ether / ethyl acetate) to give intermediate 5 (39% yield) as a pink gel.

[0066] Preparation of intermediate 6 Cadmium carbonate (5.2 g, 30.24 mmol) was added to an anhydrous toluene (600 mL) solution of compound 1 (17.5 g, 51.44 mmol), and the mixture was refluxed for 12 hours using a Dean Stark apparatus. After cooling to room temperature, intermediate 5 (24.3 g, 61.3 mmol) was added, and the reaction mixture was refluxed again for 24 hours. The precipitate was removed by filtration, and the mixture was washed with a DCM / MeOH: 95:5 (v / v) mixed solvent. The filtrates and washings were combined and concentrated under reduced pressure to obtain the product. The product was purified by rapid silica gel column chromatography (95:5 DCM / MeOH) to give intermediate 6 (37% yield) as a white frothy substance.

[0067] Referring to the synthesis schemes of the above embodiments, the compounds shown in Table 1 below were prepared.

[0068] Table 1. Compound Structures and Mass Spectrometry Data

[0069]

[0070] Example 1: Upregulation of miRNA-124 by the compound

[0071] Human peripheral blood mononuclear cells (PBMCs) were resuscitated and incubated at 37°C and 5% CO2 for 30 min. 3.0 × 10⁻⁶ cells were then... 6 Personal PBMCs were seeded at 4 mL per well in 6-well plates, with cell culture medium containing PMA (10 ng / mL) and ionomycin (200 ng / mL). Cells were incubated at 37°C and 5% CO2 for 48 h. The test compound (40 nM) was added to each well, and the cells were incubated at 37°C and 5% CO2 for 72 h. Cells were then centrifuged at 400 × g for 10 min and collected into 1.5 mL centrifuge tubes, which were stored at -80°C. miRNA-124 was subsequently detected using qPCR.

[0072] Collect cells by centrifugation, and add 1 mL of RNA isolater to the cell sample. Vortex or aspirate the cell pellet and incubate at room temperature for 2-3 minutes to allow for complete lysis.

[0073] RNA purification was performed according to the MiPure Cell / Tissue miRNA Kit instructions. Add 200 μL of chloroform to the lysis buffer, vortex vigorously for 15 seconds, and incubate at room temperature for 3 minutes. Centrifuge at 12,000 rpm for 15 minutes at 4°C, and transfer 500 μL of supernatant to a new 1.5 mL centrifuge tube. Add 160 μL of anhydrous ethanol to the supernatant and vortex to mix. Place the MiPure RNA column in a 2 mL collection tube, transfer the mixture to the RNA column, and centrifuge at 12,000 rpm. Add 0.9 volumes of anhydrous ethanol to the filtrate and pipette 3-5 times. Place the miRNA column (MiPure miRNA Column) in a 2 mL collection tube, transfer half the volume of the mixture to the miRNA column, and centrifuge at 12,000 rpm. Discard the filtrate, return the miRNA column to the collection tube, transfer the remaining mixture to the miRNA column, and centrifuge at 12,000 rpm. Discard the filtrate, return the miRNA column to the collection tube, add 500 μL of Buffer miRW1 (with added ethanol), incubate at room temperature for 1 min, and centrifuge at 12,000 rpm for 30 s. Discard the filtrate, return the miRNA column to the collection tube, add 500 μL of Buffer miRW2 (with added ethanol), incubate at room temperature for 1 min, and centrifuge at 12,000 rpm. Discard the filtrate, return the miRNA column to the collection tube, add 500 μL of 80% ethanol (freshly prepared with RNase-free ddH2O), incubate at room temperature for 1 min, and centrifuge at 12,000 rpm (13,400 × g) for 30 s. Discard the filtrate, return the miRNA column to the collection tube, centrifuge the empty column at 12,000 rpm (13,400 × g) for 2 min, and dry the miRNA column matrix. This step completely removes residual ethanol from the miRNA column. Transfer the miRNA column to a new 1.5 mL centrifuge tube and allow it to dry at room temperature for 2-5 min. Add 30-50 μL of RNase-free ddH2O to the center of the miRNA column membrane, incubate at room temperature for 2 min, and centrifuge at 12,000 rpm (13,400 × g) for 1 min. Collect the filtrate. Detect the concentration and amount of collected RNA using a Thermo NanoDrop 1000 spectrophotometer.

[0074] Prepare the genomic DNA elimination reaction solution on ice. Prepare a premix of all components except the RNA sample, with a volume sufficient for the reaction number and a slight excess. Dispense an appropriate volume of the premix (total volume minus total RNA volume) into microcentrifuge tubes, and then add the RNA sample.

[0075] Prepare the reverse transcription reaction mixture on ice. Prepare a sufficient number of reactions with an appropriate excess of premix. The premix should contain all components except for the genomic DNA elimination reaction mixture. Add 10 μL of the premix to the reaction mixture, mix gently, and immediately perform the reverse transcription reaction. Store the reverse transcription product at -20°C for real-time PCR analysis.

[0076] Prepare the PCR mixture. To compensate for pipetting errors, prepare a premix at least 10% larger than the total reaction volume. The mixture contains: 5 μL 2×miRNA Unimodal SYBR qPCR Master Mix, 0.2 μL Specific Primer (10 μM), 0.2 μL mQPrimer R (10 μM), 1 μL template cDNA, and 3.6 μL ddH2O. Place the reaction mixture in a real-time PCR instrument for detection.

[0077] Ct values ​​were automatically calculated by Quant Studio 7 software using default settings. The Ct values ​​were exported as an Excel file. The relative gene expression levels were calculated using the following formula: ΔCt = Ct(target gene) − Ct(internal reference gene) ΔCt = ΔCt(treatment group) − ΔCt(control group) The relative expression level = 2^(−ΔΔCt), and the results are shown in Table 2.

[0078] Table 2: Experimental results of compounds regulating miRNA-124 levels

[0079] Experimental results show that, except for compound 18, all the compounds in the examples, as miRNA regulators, exhibited better upregulation activity against miRNA-124 than the control compound ABX464. In particular, compounds 1 and 3 in the examples showed upregulation of miRNA-124 by more than two times that of ABX464. These experimental results indicate that the compounds described in this application may have clinical advantages such as lower dosage and fewer gastrointestinal and neurological side effects.

[0080] To assess the risk of QT interval prolongation on electrocardiogram, HEK293 cells expressing human ether-a-go-go-related gene (hERG) channels were used to investigate the role of delayed rectified K+ current (IKr), which plays an important role in ventricular repolarization.

[0081] Using a fully automated patch-clamp system, the IKr induced by maintaining cells at a membrane potential of -80 mV, followed by a 2-second depolarization stimulus of +50 mV and a further 2-second repolarization stimulus of -50 mV was recorded via whole-cell patch-clamp technique. Drug concentrations ranged from 10 to 50 μmol / L. After the generated current stabilized, extracellular fluid containing the target concentration of the test substance (NaCl: 137 mmol / L, KCl: 4 mmol / L, CaCl2: 1.8 mmol / L, MgCl2-6H2O: 1 mmol / L, glucose: 10 mmol / L, HEPES: 10 mmol / L, pH 7.4) was applied to the cells at room temperature for 10 minutes. The obtained IKr was analyzed using software to measure the absolute value of the maximum tail current, based on the current value at resting membrane potential. Furthermore, the suppression rate of the maximum tail current relative to the test substance before application was calculated and compared with the medium application group (0.1% DMSO solution) to evaluate the effect of the test substance on IKr.

[0082] Table 3. Inhibition rate of the compounds in the examples on hERG potassium channels

[0083] As shown in Table 3, under the same experimental conditions, the inhibition rate of the control compound ABX464 on the hERG potassium channel was 2.4 times that of compound 1 in the examples. This indicates that the compound in the examples has a smaller effect on C-QTc than ABX464, i.e., lower cardiotoxicity. The experimental results suggest that compound 1 of this patent may have broader clinical application value in the elderly and patients with heart failure.

[0084] Using SD rats as test animals, the plasma (CP) and cerebrospinal fluid (CSF) concentrations of the test compounds at different time points after gavage administration in Sprague-Dawley (SD) rats were determined by LC / MS / MS. The pharmacokinetic behavior of the test compounds in SD rats was investigated to evaluate their pharmacokinetic and tissue distribution characteristics.

[0085] 1) Test drugs Examples of compounds 1, 18 and ABX464.

[0086] 2) Experimental animals Seventy-two Sprague-Dawley (SD) rats, divided into groups of 24 (half male and half female), were administered the drug via gavage after being fasted for one night but allowed free access to water.

[0087] 3) Drug preparation Weigh out a certain amount of the test compound and add the drug solvent: 20% PEG400 / 10% Solutol / 70% 50mM citric acid buffer (pH 6.0) to prepare a homogeneous solution of 3 mg / mL.

[0088] 4) Administration The dose administered by gavage was 30 mg / kg, and the volume of administration was 10.0 mL / kg.

[0089] 5) Operation Blood samples of 0.2 mL were collected from the orbital cavity before administration and at 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, and 12.0 hours after administration. The samples were placed in EDTA-K2 anticoagulant tubes and centrifuged at 10,000 rpm for 1 minute (4°C). Plasma was separated within 1 hour and stored at -20°C for analysis. The entire process from blood collection to centrifugation was performed under ice bath conditions. Cerebrospinal fluid (CSF) samples were collected from the animals and stored on dry ice at -60°C or lower for analysis. The animals were fed 2 hours after administration.

[0090] Determination of the content of analyte compounds in plasma and cerebrospinal fluid of SD rats after administration of different drugs: CP and CSF samples from SD rats at various time points after drug administration were diluted 10-fold with acetonitrile solution containing internal standard, vortexed, and centrifuged at 3700 rpm for 10 minutes. The supernatant was mixed with water at a 1:1 ratio, and 0.5 μL of the supernatant was analyzed by LC / MS / MS.

[0091] 6) Pharmacokinetic parameters and tissue distribution results Table 4: Pharmacokinetic parameters and tissue distribution of the compounds in male and female SD rats.

[0092] Experimental results showed that compound 1 had a rat plasma AUC 0-t Compound 1's AUC ratio was twice that of ABX464, and only 0.2 times that of ABX464, indicating that compound 1 maintained high systemic exposure while having low cerebrospinal fluid exposure. This result preliminarily suggests that compound 1 had low brain distribution, indicating very low drug concentrations in brain tissue and less impact on brain nerves. Compound 18's plasma AUC (1965±160 ng•h / mL) was lower than ABX464, and its cerebrospinal fluid / plasma ratio (0.21) was comparable to ABX464 (0.23), showing no similar distribution characteristics to compound 1.

[0093] The above experimental results show that the compound of the present invention has a low brain distribution while maintaining high systemic exposure, which is expected to reduce the risk of potential central nervous system side effects such as headache. It has a wider range of clinical users, a wider drug safety window, and higher drug safety.

[0094] A mouse model of acute enteritis was induced using sodium dextran sulfate (DSS) to evaluate the in vivo anti-inflammatory efficacy of the test compound and simultaneously detect the levels of intestinal inflammatory factors.

[0095] 4.1 Animal Information and Grouping Male C57BL / 6J mice, 6-8 weeks old, weighing approximately 25g; Groups 1, 2, 3, 4, 5, 6, 7, with N=6 per group, 3 mice per cage. Feeding regimen: Standard diet, free access to food and water.

[0096] 4.2 Drug Preparation Weigh out a certain amount of the test compound and add the drug solvent: 20% PEG400 / 10% Solutol / 70% 50mM citric acid buffer (pH 6.0) to prepare homogeneous solutions of 2.5 mg / mL and 5 mg / mL.

[0097] 4.3 Modeling and Dosing Regimen From day 0 onwards, all groups were given 2.5% DSS in drinking water, with fresh DSS solution changed daily. Modeling was performed using 2.5% DSS from day 0 to day 6. Administration method: oral gavage (po), with administration from day 0 to day 7. See Table 5 for the protocol.

[0098] Table 5: Modeling and Dosing Protocols

[0099] 4.4 Sample Collection and Processing 4.4.1. Weight Records: Fixed weighing records at 8 time points: D0, D1, D2, D3, D4, D5, D6, and D7.

[0100] 4.4.2. Survival observation: Daily clinical condition observation, record the animal's health status and daily water intake.

[0101] 4.4.3. Stool score (daily testing from D0 to D7): Stool morphology is observed daily, and bleeding is detected using a stool occult blood test kit (o-toluidine method). Scoring is performed according to a unified standard, and the DAI score is calculated comprehensively.

[0102] The criteria for judging fecal occult blood are as follows: it turns blue-black immediately after the reagent is added (4+), turns blue-brown immediately after the reagent is added and gradually turns blackish-brown (3+), initially appears light green after the reagent is added and gradually turns obvious blackish-brown (2+), gradually changes from light green to green after 10 seconds (1+), and no color development within 2 minutes is negative.

[0103] DAI Comprehensive Scoring Criteria: Weight loss, stool characteristics, and bleeding are shown in Table 6. The total DAI score = weight loss score + stool characteristics score + bleeding score.

[0104] Table 6: DAI Comprehensive Scoring Criteria

[0105] 4.4.4. Colon sample processing: After euthanasia, the colon was completely dissected, feces were removed, and the colon was photographed, its length was measured and it was weighed. The colon tissue was ground to prepare a homogenate supernatant for CBA multifactor detection of TNF-α and IL-17A.

[0106] 4.4.5. Flow cytometry multifactor detection of CBA in colon supernatant (TNF-α, IL-17A): ① Prepare standard curves by serially diluting the standards by two-fold after reconstitution, and set up blank controls. Both samples and standards are replicated. ② Premix capture microspheres, add microspheres and test samples to each well, and incubate at room temperature in the dark for 1 h. ③ Add PE fluorescent detection working solution and incubate in the dark for 2 h. ④ After washing and resuspending, perform flow cytometry detection, fit the standard curve with software, and calculate the cytokine concentrations in the samples.

[0107] 4.5 Results of the trial on prevention and treatment of ulcerative colitis (UC), the results are shown in Table 7.

[0108] D7 relative change in body weight %: The rate of change in body weight relative to D0 at the experimental endpoint in mice; D7 DAI: Total score of DAI at the endpoint of mouse experiments; D7 relative colon length %: The endpoint of mouse experiments relative to the control group's relative colon length.

[0109] Table 7: Results of the trial on the prevention and treatment of ulcerative colitis (UC)

[0110] ### indicates P<0.001 compared with the control group; * indicates P<0.05 compared with the model group; ** indicates P<0.001 compared with the model group; *** indicates P<0.0001 compared with the model group.

[0111] The results of the weight change experiment showed that the relative weight change rate of the DSS model group was significantly reduced at the experimental endpoint D7 (P<0.001). The weight reduction of compounds 1, 2 and ABX464 was less than that of the DSS model group. In particular, the relative weight reduction rate of the 50mpk compound 1 and compound 2 experimental groups was less than that of the 50mpk ABX464 group.

[0112] The DAI comprehensive score results showed that the DSS model group had a significantly higher DAI comprehensive score at the experimental endpoint D7 (P<0.001). The DAI comprehensive scores of compound 1, compound 2 and ABX464 were lower than those of the DSS model group, especially the DAI comprehensive scores of compound 1 and compound 2 at 50 mpk were lower than those of ABX464 at 50 mpk.

[0113] The results of the relative colon length experiment showed that the relative colon length of the DSS model group decreased significantly at the experimental endpoint (P<0.001). The relative colon lengths of compounds 1, 2 and ABX464 were greater than those of the DSS model group. In particular, the relative colon lengths of the 50 mpk compound 1 and compound 2 experimental groups were greater than those of the 50 mpk ABX464 group. The relative colon lengths of the low-dose example compound groups were roughly equivalent to those of the 50 mpk ABX464 group.

[0114] The results of the cytokine detection experiment showed that the DSS model group had significantly increased levels of cytokines mTNF-α and mIL-17A at the experimental endpoint (P<0.001). The levels of cytokines mTNF-α and mIL-17A in compounds 1, 2, and ABX464 were significantly lower than those in the DSS model group. Furthermore, the levels of cytokines mTNF-α in the low- and high-dose compound 1 and compound 2 experimental groups were lower than those in the 50 mpk ABX464 group; and the levels of cytokines mIL-17A in the 50 mpk compound 1 and compound 2 experimental groups were lower than those in the 50 mpk ABX464 group.

[0115] In summary, the compounds of this invention have a good restorative effect on weight loss in mice with DSS-induced ulcerative colitis; a good therapeutic effect on colonic shortening in mice with ulcerative colitis; and a significant reduction in cytokines in mice with ulcerative colitis. At a dose of 50 mg / kg, compounds 1 and 2 generally showed better improvement than ABX464 in the above indicators; at a dose of 25 mg / kg, their improvement effect was generally comparable to that of ABX464 at 50 mg / kg, with some indicators being even better. That is, compounds 1 and 2 in the examples are more effective than ABX464 in treating ulcerative colitis.

[0116] The preferred embodiments of the present invention have been described above. Those skilled in the art should understand that various modifications and combinations can be made to the above embodiments without departing from the spirit of the present invention, and all such modifications fall within the protection scope defined by the claims of the present invention.

Claims

1. Pyridopyrimidine compounds of formula (I) or pharmaceutically acceptable salts thereof: in: Z is either C or N; R 1 Independently selected from hydrogen, deuterium, halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C3-C6)heterocyclic alkyl, hydroxyl, nitro, cyano, morpholino, N-methylpiperazinyl, (C1-C3)haloalkyl, (C1-C3)haloalkoxy, (C3-C6)halocycloalkyl, (C3-C6)haloheterocyclic alkyl; R 2 Selected from hydrogen, glucuronidyl alkyl, (C1-C3)alkyl, (C3-C6)cycloalkyl, (C3-C6) heterocycloalkyl, (C1-C3) fluoroalkyl, (C3-C6) halocycloalkyl, (C3-C6) haloheteroalkyl, (C1-C3) deuterated alkyl; R 3 Independently selected from hydrogen, deuterium, halogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C3-C6)heterocycloalkyl, hydroxyl, nitro, cyano, vinyl, (C1-C3)haloalkyl, (C1-C3)haloalkoxy, (C3-C6) substituted cycloalkyl, (C3-C6) halocycloalkyl, difluoromethylenedioxy, (C3-C6) haloheterocycloalkyl, fluorovinyl, pentafluorothio, trifluoromethylthio, -CF2OCH3, -CF2OCH2CH3; The heterocyclic alkyl group contains one to two heteroatoms selected from N and O; n is 1, 2, 3 or 4; m can be 1, 2, 3, 4 or 5.

2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein: Z is either C or N; R 1 It is independently selected from hydrogen, deuterium, chlorine, fluorine, cyano, cyclopropyl, morpholino, (C1-C3)alkyl, (C1-C3)fluoroalkyl, (C1-C3)alkoxy, and (C1-C3)fluoroalkoxy. R 2 Selected from hydrogen, glucuronidyl, methyl, ethyl, cyclopropyl, trifluoromethyl, and trifluoroethyl; R 3 Independently selected from hydrogen, deuterium, chlorine, fluorine, cyano, cyclopropyl, morpholino, (C1-C3)alkyl, (C1-C3)fluoroalkyl, (C1-C3)alkoxy, (C1-C3)fluoroalkoxy, difluoromethylenedioxy, fluorovinyl, pentafluorothio, trifluoromethylthio, -CF2OCH3, -CF2OCH2CH3 ; n is 1, 2, or 3; m can be 1, 2, 3 or 4.

3. The compound of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula: 。 4. A pharmaceutical composition comprising a therapeutically effective amount of the compound as described in any one of claims 1-3 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

5. Use of the compound of any one of claims 1-3 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for upregulating miRNA-124 levels.

6. Use of the compound of any one of claims 1-3 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of inflammatory diseases.

7. The use according to claim 6, characterized in that, The inflammatory disease is selected from inflammatory bowel disease, rheumatoid arthritis, Crohn's disease, ulcerative colitis, multiple sclerosis, Alzheimer's disease, Parkinson's disease, osteoarthritis, atherosclerosis, ankylosing spondylitis, psoriasis, dermatitis, Sjögren's syndrome, bronchitis, asthma, or inflammation related to colon cancer.

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