Hpki kinase inhibitor compounds

Abstract

The present application provides a kind of compound with inhibiting HPK1 kinase activity of structure (I) and pharmaceutical composition comprising the compound.The present application also provides the use of the compound in preventing and / or treating cancer, tumor, inflammatory disease, autoimmune disease or immune-mediated disease.

Description

[0001] This application claims priority to Chinese Patent Application No. 202110308954.9, filed on March 23, 2021, entitled “HPK1 Kinase Inhibitor Compound,” the contents of which are incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application relates to a heterocyclic compound, in particular to a highly active HPK1 kinase inhibitor and its use. BACKGROUND

[0003] HPK1 is one of the members of the MAP4K family, mainly expressed in hematopoietic system cells, and acts as an intracellular negative regulator of T cell proliferation and signaling. After antigen stimulation of T cells, the linker protein SLP-76 in the cytoplasm is recruited to the lipid membrane TCR complex, providing a binding site for signal transduction-related kinases to achieve TCR-mediated signal transmission and induce T cell activation. In this process, HPK1 is activated by tyrosine kinases Lck and Zap70 phosphorylation, which participates in the regulation of T cell receptor protein interaction. HPK1 phosphorylates the Ser376 site of the linker protein SLP-76, which binds to the scaffold protein 14-3-3 epsilon and is then degraded by the proteasome, and this effect reduces the binding of SLP-76 to signal transduction-related kinases, thereby blocking TCR signal transduction, and then inhibiting T cell activation and proliferation. On the other hand, HPK1 is also involved in the regulation of dendritic cell (DC) maturation and activation, especially the inhibition of the expression of proteins related to T cell activation in DC cells, such as CD80, CD86, and MHC complex, thereby affecting the role of DC in regulating T cell activation; the presentation of tumor antigens by activated DC and the cooperation between DC and T cells is one of the most important links in the anti-tumor immune system. In addition, there are a large number of immunosuppressive molecules in the tumor microenvironment, such as PGE2 and TGF-β, and the immunosuppressive effect mediated by these factors is also closely related to HPK1. In general, specific small molecule compounds that target and inhibit HPK1 can improve T cell function, enhance DC cell function, and at the same time reverse the tumor immunosuppressive microenvironment, thereby playing a role in enhancing anti-tumor immune effects through multiple pathways, thereby achieving the effect of inhibiting tumor growth. However, there is currently a lack of effective HPK1 kinase activity inhibitors.

[0004] Therefore, there is still an urgent need in the prior art for effective HPK1 kinase activity inhibitors in order to provide more effective options for anti-tumor. SUMMARY

[0005] The present invention has unexpectedly discovered a compound of formula (I) having inhibitory activity against HPK1 kinase, as well as its pharmaceutically acceptable salts, isotope derivatives, or stereoisomers. Therefore, in a first aspect, the present invention provides a compound having the structure of formula (I) or a pharmaceutically acceptable salt, isotope derivative, or stereoisomer:

[0006]

[0007] in

[0008] R1 represents hydrogen, (C1-C6)alkyl, or (C3-C8)cycloalkyl;

[0009] R2 represents hydrogen, (C1-C6) alkyl, halogen, cyano, -OR a Or (C1-C6) haloalkyl;

[0010] R3 represents hydrogen, halogen, -OR a (C1-C6)alkyl, (C1-C6)haloalkyl, hydroxy(C1-C6)alkyl, (C2-C6)alkenyl, -(C0-C6 alkylene)(C3-C8)cycloalkyl, -(C0-C6 alkylene)(4-8-membered)heterocyclic alkyl, -(C0-C6 alkylene)(C3-C8)cycloalkyloxy, -(C0-C6 alkylene)(4-8-membered)heterocyclic alkyloxy;

[0011] A represents

[0012] B represents -(C0-C6)alkylene-, -O-(C1-C6)alkyl-, -S-(C1-C6)alkyl-, and -S(O)-(C1-C6)alkyl-.

[0013] R4 and R4' each independently represent hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, or halogen;

[0014] Or R4 and R 4’ Together with the carbon atoms attached thereto, they form a 3-6 membered ring, which may also contain 0, 1 or 2 heteroatoms selected from N, O or S.

[0015] R5 represents hydrogen, C1-C6 alkyl, halo(C1-C6)alkyl, (C3-C6)alkenyl, (C3-C8)cycloalkyl, halo(C3-C8)cycloalkyl, (4-8-membered) heterocyclic alkyl, halo(4-8-membered) heterocyclic alkyl, -(C0-C6)alkylene-OR a -(C0-C6)alkylene-COOR a Or -(C0-C6)alkylene-C(O)NR a Ra

[0016] R6and R 6’ each independently represent hydrogen, C1-C6alkyl, (C1-C6)haloalkyl, (C2-C6)alkenyl, halogen or -(C0-C6)alkylene-OR a ;

[0017] or R6and R 6’ together with the carbon atom to which they are attached form a 3-6 membered ring, which can optionally further contain 0, 1 or 2 heteroatoms selected from N, O, S;

[0018] X1represents N or CR7;

[0019] wherein R7represents hydrogen, halogen, (C1-C6)alkyl, (C1-C6)haloalkyl, (C2-C6)alkenyl, -(C0-C6alkylene)(C3-C8)cycloalkyl, -(C0-C6alkylene)(4-10 membered)heterocycloalkyl, -(C0-C6alkylene)(C6-C 10 )aryl, -(C0-C6alkylene)(5-10 membered)heteroaryl,

[0020] or, when X1represents CR7, R7may form together with the adjacent R3a (5-10 membered)cycloalkyl or (5-10 membered)heterocycloalkyl, optionally substituted with halogen;

[0021] R M and R N each independently represent hydrogen, (C1-C6)alkyl, (C1-C6)haloalkyl, or R M and R N together with the carbon atom to which they are attached form a 3-6 membered ring, which can optionally further contain 0, 1 or 2 heteroatoms selected from O, N, S, optionally substituted with halogen; further, the ring can be optionally substituted with 0, 1 or 2 substituents selected from halogen, OR a ; and R M and R N are not simultaneously hydrogen;

[0022] wherein R a , R b represent hydrogen or (C1-C6)alkyl;

[0023] m, n represent 0, 1, 2, 3.

[0024] In a preferred technical solution of the present application, A represents wherein R a represents hydrogen or (C1-C6)alkyl.

[0025] ​In a preferred embodiment of the application, R1 represents (C1-C6)alkyl.

[0026] In a preferred embodiment of the application, R2 represents hydrogen, halogen or (C1-C6)alkyl or halo(C1-C6)alkyl.

[0027] In a preferred embodiment of the application, X1 represents N or CH.

[0028] In a preferred embodiment of the application, R3 represents -OR a (C1-C6)alkyl or (C3-C8)cycloalkyl.

[0029] In a preferred embodiment of the application, R4 and R 4’ each independently represent hydrogen.

[0030] In a preferred embodiment of the application, R5 represents hydrogen, (C1-C6)alkyl or (C3-C8)cycloalkyl.

[0031] In a preferred embodiment of the application, R6 and R 6’ each independently represent hydrogen, halogen, (C1-C6)alkyl or (C1-C6)haloalkyl.

[0032] In a preferred embodiment of the application, R7 represents hydrogen, halogen, (C1-C6)alkyl, (C1-C6)haloalkyl, -(C0-C6alkylene)(C3-C8)cycloalkyl or -(C0-C6alkylene)(4-10 membered)heterocycloalkyl.

[0033] In a preferred embodiment of the application, m represents 1 or 2.

[0034] In a preferred embodiment of the application, n represents 1 or 2.

[0035] In a preferred embodiment of the application, m represents 1 and n represents 2.

[0036] In particular, the application provides the following specific compounds:

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044] In another aspect, the present application also provides a pharmaceutical composition comprising a compound of the present application and a pharmaceutically acceptable carrier.

[0045] In yet another aspect, the present application also provides use of a compound or a pharmaceutical composition of the present application in the manufacture of a medicament for preventing and / or treating cancer, tumor, inflammatory disease, autoimmune disease or immune-mediated disease.

[0046] It is particularly noted that herein, when referring to a "compound" having a specific structural formula, it generally also encompasses its stereoisomers, diastereomers, enantiomers, racemic mixtures and isotopic derivatives.

[0047] It is well known to those skilled in the art that a salt, solvate, hydrate of a compound is an alternative form of existence of the compound, which can be converted into the compound under certain conditions, therefore, it is particularly noted that herein, when referring to a compound, it generally also includes its pharmaceutically acceptable salt, and further includes its solvate and hydrate.

[0048] Similarly, herein, when referring to a compound, it generally also includes its prodrugs, metabolites and nitroxides.

[0049] The pharmaceutically acceptable salts of the present application can be formed using, for example, inorganic or organic acids, as follows: "Pharmaceutically acceptable salt" refers to salts of a compound of the present application which are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio. The salts can be prepared in situ during the final isolation and purification of the compounds of the application, or separately by reacting the free base or free acid with a suitable reagent, as outlined below. For example, the free base functionality can be reacted with the appropriate acid. Also, where the compound of the present application carries an acidic moiety, suitable pharmaceutically acceptable salts thereof can include metal salts, such as alkali metal salts, e.g., sodium or potassium salts; and alkaline earth metal salts, e.g., calcium or magnesium salts. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, and perchloric acid, or with organic acids such as acetic, oxalic, maleic, tartaric, citric, succinic, or malonic acid, or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hernisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium salts, and the like. Other pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.

[0050] The pharmaceutically acceptable salts of the present application can be prepared by conventional methods, e.g., by dissolving the compound of the present application in an organic solvent which is miscible with water, such as acetone, methanol, ethanol, and acetonitrile, adding thereto an excess of aqueous solution of an organic or inorganic acid, so that the salt precipitates from the resulting mixture, removing the solvent and the remaining free acid, and isolating the precipitated salt.

[0051] The precursors or metabolites described in this invention can be precursors or metabolites known in the art, as long as they can be metabolized and transformed in vivo to form the target compound. For example, "prodrug" refers to those prodrugs of the compounds of this invention that, within a reasonable medical judgment, are suitable for contact with human and lower animal tissues without undue toxicity, irritation, allergic reactions, etc., and have a reasonable benefit / risk ratio and are effective for their intended use. The term "prodrug" refers to a compound that is rapidly transformed in vivo to produce the parent compound of the above formula, for example, through in vivo metabolism, or through N-demethylation of the compounds of this invention.

[0052] The term "solvate" as used in this invention refers to the physical association of the compound of this invention with one or more solvent molecules (organic or inorganic). This physical association includes hydrogen bonding. In some cases, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate can be separated. The solvent molecules in the solvate may be present in a regular and / or disordered arrangement. The solvate may contain stoichiometric or non-stoichiometric solvent molecules. "Solvate" encompasses both solution phases and separable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.

[0053] The "stereoisomerism" described in this invention is divided into conformational isomerism and configurational isomerism. Configurational isomerism can be further divided into cis-trans isomerism and optical isomerism. Conformational isomerism refers to the phenomenon where organic molecules with a certain configuration undergo different spatial arrangements of atoms or groups of atoms due to the rotation or twisting of carbon or carbon single bonds. Common examples include the structures of alkanes and cycloalkanes, such as the chair conformation and boat conformation in the cyclohexane structure. "Stereoisomers" refer to compounds of this invention containing one or more asymmetric centers, thus allowing them to exist as racemic mixtures and racemic mixtures, single enantiomers, diastereomer mixtures, and single diastereomers. The compounds of this invention have asymmetric centers, each of which produces two optical isomers. The scope of this invention includes all possible optical isomers and diastereomer mixtures, as well as pure or partially pure compounds. The compounds of this invention can exist as tautomers, which have different hydrogen bonding sites through one or more double bond shifts. For example, ketones and their enol forms are ketone-enol tautomers. All tautomers and mixtures thereof are included in the compounds of this invention. All enantiomers, diastereomers, racemates, mesomates, cis-trans isomers, tautomers, geometric isomers, epimers, and mixtures thereof of all compounds of formula (I) are included within the scope of this invention.

[0054] An "isotopically-labeled" compound of the application is a molecule of the application in which one or more atoms are replaced by an isotope of the atom. Typically, isotopes of hydrogen, carbon, nitrogen, oxygen and sulfur are preferred isotopes for inclusion in the compounds of the application. The inclusion of isotopic atoms in compounds of the application can afford insight into a compound's distribution, transport, and / or metabolism in a tissue of interest. In particular, deuterium 2 H and 3 H; isotopes of carbon: 11 C, 13 C and 14 C; isotopes of chlorine: 35 Cl and 37 Cl; isotopes of fluorine: 18 F; isotopes of iodine: 123 I and 125 I; isotopes of nitrogen: 13 N and 15 N; isotopes of oxygen: 15 O, 17 O and 18 O and isotopes of sulfur 35 S. These isotopically-labeled compounds are useful in metabolic studies, as diagnostic tools, as probes in biological assays, and the like. In particular, deuterium 3 H and carbon 13 C are useful because of their ease of incorporation, relative safety, and non- interfering mass. Certain heavier isotopes, such as hydrogen 2 H), can afford enhanced metabolic stability, which in turn allows for

[0055] The present application also provides the use of a compound of the present application for the manufacture of a medicament for the prevention and / or treatment of cancer, a tumor, an inflammatory disease, an autoimmune disease, or an immune-mediated disease.

[0056] In addition, the present application provides a pharmaceutical composition for the prevention and / or treatment of cancer, a tumor, an inflammatory disease, an autoimmune disease, a neurodegenerative disease, an attention-related disease, or an immune-mediated disease, which comprises a compound of the present application as an active ingredient.

[0057] In addition, the present application provides a method for the prevention and / or treatment of cancer, a tumor, an inflammatory disease, an autoimmune disease, a neurodegenerative disease, an attention-related disease, or an immune-mediated disease, which comprises administering a compound of the present application to a mammal in need thereof.

[0058] Representative examples of inflammatory diseases, autoimmune diseases, and immune-mediated diseases can include, but are not limited to, arthritis, rheumatoid arthritis, spondyloarthritides, gouty arthritis, osteoarthritis, juvenile arthritis, other arthritic conditions, lupus, systemic lupus erythematosus (SLE), skin-related diseases, psoriasis, eczema, dermatitis, allergic dermatitis, pain, lung diseases, lung inflammation, adult respiratory distress syndrome (ARDS), pulmonary sarcoidosis, chronic lung inflammatory disease, chronic obstructive pulmonary disease (COPD), cardiovascular disease, atherosclerosis, myocardial infarction, congestive heart failure, myocardial ischemia reperfusion injury, inflammatory bowel disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, asthma, Sjogren's syndrome, autoimmune thyroid disease, urticaria (hives), multiple sclerosis, scleroderma, organ transplant rejection, xenotransplantation, idiopathic thrombocytopenic purpura (ITP), Parkinson's disease, Alzheimer's disease, diabetes-related diseases, inflammation, pelvic inflammatory disease, allergic rhinitis, allergic bronchitis, allergic rhinosinusitis, leukemia, lymphoma, B-cell lymphoma, T-cell lymphoma, myeloma, acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), hairy cell leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), diffuse large B-cell lymphoma, and follicular lymphoma.

[0059] Representative examples of cancers or tumors can include, but are not limited to, skin cancer, bladder cancer, ovarian cancer, breast cancer, stomach cancer, pancreatic cancer, prostate cancer, colon cancer, lung cancer, bone cancer, brain cancer, neuroblastoma, rectal cancer, colon cancer, familial adenomatous polyposis cancer, hereditary nonpolyposis colorectal cancer, esophageal cancer, lip cancer, larynx cancer, hypopharynx cancer, tongue cancer, salivary gland cancer, stomach cancer, adenocarcinoma, medullary thyroid cancer, papillary thyroid cancer, kidney cancer, renal parenchymal cancer, ovarian cancer, cervical cancer, uterine body cancer, endometrial cancer, choriocarcinoma, pancreatic cancer, prostate cancer, testicular cancer, urological cancer, melanoma, brain tumors such as glioblastoma, astrocytoma, meningioma, medulloblastoma, and peripheral neuroectodermal tumor, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), adult T-cell leukemia lymphoma, diffuse large B-cell lymphoma (DLBCL), hepatocellular carcinoma, gallbladder cancer, bronchial cancer, small cell lung cancer, non-small cell lung cancer, multiple myeloma, basal cell carcinoma, teratocarcinoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, craniopharyngioma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing's sarcoma, or plasmacytoma.

[0060] When the compound of the present application or a pharmaceutically acceptable salt thereof is administered in combination with another anticancer agent or immune checkpoint inhibitor for the treatment of cancer or tumor, the compound of the present application or a pharmaceutically acceptable salt thereof can provide an enhanced anticancer effect.

[0061] Representative examples of the anticancer agent for the treatment of cancer or tumor can include, but are not limited to, cell signal transduction inhibitors, chlorambucil, melphalan, cyclophosphamide, ifosfamide, busulfan, carmustine, lomustine, streptozocin, cisplatin, carboplatin, oxaliplatin, dacarbazine, temozolomide, procarbazine, methotrexate, fluorouracil, cytarabine, gemcitabine, mercaptopurine, fludarabine, vinblastine, vincristine, vinorelbine, paclitaxel, docetaxel, topotecan, irinotecan, etoposide, trabectedin, dactinomycin, doxorubicin, epirubicin, daunorubicin, mitoxantrone, bleomycin, mitomycin C, ixabepilone, tamoxifen, flutamide, goserelin analogs, megestrol, prednisone, dexamethasone, methylprednisolone, thalidomide, interferon alpha, leucovorin, sirolimus, sirolimus lipidate, everolimus, afatinib, alisertib, amuvatinib, apatinib, axitinib, bortezomib, bosutinib, brivanib, cabozantinib, cediranib, crenolanib, crotetuzumab, dabrafenib, dacotuzumab, danusertib, dasatinib, dovitinib, erlotinib, foretinib, ganetespib, gefitinib, ibrutinib, icotinib, imatinib, iniparib, lapatinib, lenvatinib, linifanib, linsitinib, masitinib, momelotinib, motesanib, neratinib, nilotinib, niraparib, oprozomib, olaparib, pazopanib, pictilisib, ponatinib, quizartinib, regorafenib, rigosertib, rucaparib, ruxolitinib, seribantumab, sorafenib, sunitinib, tivantinib, tivozanib, tofacitinib, trametinib, vandetanib, velpatasarin, vemurafenib, vismodegib, volasertib, alemtuzumab, bevacizumab, brentuximab vedotin, catumaxomab, cetuximab, denosumab, gemtuzumab ozogamicin, ipilimumab, nimotuzumab, ofatumumab, panitumumab, rituximab, tositumomab, trastuzumab, PI3K inhibitors, CSF1R inhibitors, A2A and / or A2B receptor antagonists, IDO inhibitors, anti-PD-1 antibodies, anti-PD-L1 antibodies, LAG3 antibodies, TIM-3 antibodies, and anti-CTLA-4 antibodies, or any combination thereof.

[0062] When the compound of the present application or a pharmaceutically acceptable salt thereof is administered in combination with another therapeutic agent for the treatment of inflammatory diseases, autoimmune diseases and immune-mediated diseases, the compound of the present application or a pharmaceutically acceptable salt thereof can provide an enhanced therapeutic effect.

[0063] Representative examples of therapeutic agents for the treatment of inflammatory diseases, autoimmune diseases and immune-mediated diseases can include, but are not limited to, steroidal drugs (e.g., prednisone, prednisolone, methylprednisolone, cortisone, hydrocortisone, betamethasone, dexamethasone, etc.), methotrexate, leflunomide, anti-TNFa agents (e.g., etanercept, infliximab, adalimumab, etc.), calcineurin inhibitors (e.g., tacrolimus, pimecrolimus, etc.), and antihistamines (e.g., diphenhydramine, hydroxyzine, loratadine, ebastine, ketotifen, cetirizine, levocetirizine, fexofenadine, etc.), and at least one therapeutic agent selected from among them can be included in the pharmaceutical composition of the present application.

[0064] The compound of the present application or a pharmaceutically acceptable salt thereof can be orally or parenterally administered as an active ingredient in an effective amount ranging from 0.1 to 2000 mg / kg body weight / day, preferably 1 to 1000 mg / kg body weight / day, in the case of a mammal including a human (body weight about 70 kg), and administered in a single or 4 divided doses per day, or in compliance with / incompliance with a predetermined time. The dose of the active ingredient can be adjusted according to various relevant factors (e.g., the condition of the subject to be treated, the type and severity of the disease, the rate of administration, and the physician's opinion). In some cases, an amount less than the above dose can be appropriate. An amount greater than the above dose can be used if it does not cause harmful side effects and can be administered in divided doses per day.

[0065] In addition, the present application provides a method for preventing and / or treating a tumor, cancer, viral infection, organ transplant rejection, neurodegenerative disease, attention-related disease, or autoimmune disease, which comprises administering to a mammal in need thereof a compound of the present application or a pharmaceutical composition of the present application.

[0066] The pharmaceutical composition of the present application can be formulated into a dosage form for oral administration or parenteral administration (including intramuscular, intravenous, and subcutaneous routes, intratumoral injection) such as a tablet, granule, powder, capsule, syrup, emulsion, microemulsion, solution, or suspension according to any one of the conventional methods.

[0067] The pharmaceutical compositions of the present application for oral administration can be prepared by mixing the active ingredient with a carrier, for example, a cellulose, calcium silicate, corn starch, lactose, sucrose, dextrose, calcium phosphate, stearic acid, magnesium stearate, calcium stearate, gelatin, talc, a surface active agent, a suspending agent, an emulsifying agent, and a diluent. Examples of the carriers employed in the injection compositions of the present application are water, a salt solution, a glucose solution, a glucose-like solution, an alcohol, a glycol, an ether (e.g., polyethylene glycol 400), an oil, a fatty acid, a fatty acid ester, a glyceride, a surface active agent, a suspending agent, and an emulsifying agent.

[0068] Other features of the application will be apparent from consideration of the specification and the examples, given by way of illustration of the application and not intended to be limiting thereof, which follow.

[0069] The compounds of the present application can be prepared in a number of ways known to one skilled in the art of organic synthesis, using the methods described below, together with synthetic methods known in the art of synthetic organic chemistry, or by modifications thereof, as appreciated by those skilled in the art. Preferred methods include, but are not limited to, those described below. The reactions are performed in solvents appropriate to the reagents and materials employed and suitable for the transformations being effected. It will be understood by those skilled in the art of organic synthesis that the functionality present on the molecule should be consistent with the chemistry that is being effected and that the functionality can be modified as appreciated by one skilled in the art to give the desired compound. The order of carrying out the various processes disclosed herein will be determined, in part, by the specific reagents and materials used in the synthesis. DETAILED DESCRIPTION

[0070] TERMINOLOGY

[0071] If not otherwise specified, the terms used in the present application, including the specification and claims, are defined as follows. It must be noted that, as used in the specification and the appended claims, the singular form "a" includes plural referents unless the context clearly dictates otherwise. If not otherwise specified, conventional methods of mass spectrometry, nuclear magnetic resonance, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology are used. In the present application, "or" or "and" are used to mean "and / or" unless otherwise indicated.

[0072] In the specification and claims, a given chemical formula or name shall cover all stereoisomers and optical isomers and racemates in which such isomers are present. Unless otherwise indicated, all chiral (enantiomers and diastereomers) and racemic forms are within the scope of the present invention. Many geometric isomers of C═C double bonds, C═N double bonds, ring systems, etc. may also be present in the compounds, and all such stable isomers are covered by the present invention. The present invention describes the cis- and trans- (or E- and Z-) geometric isomers of the compounds of the present invention, and they can be separated into mixtures of isomers or separate isomer forms. The compounds of the present invention can be separated in optically active or racemic forms. All methods for preparing the compounds of the present invention and the intermediates prepared therein are considered to be part of the present invention. When preparing enantiomeric or diastereomeric products, they can be separated by conventional methods (such as by chromatography or fractional crystallization). Depending on the method conditions, the final products of the present invention are obtained in free (neutral) or salt form. Both the free form and the salts of these final products are within the scope of the present invention. If desired, one form of the compound can be converted into another form. The free base or acid can be converted into a salt; the salt can be converted into the free compound or another salt; a mixture of isomeric compounds of the present invention can be separated into individual isomers. The compounds of the present invention, their free forms and salts can exist in a variety of tautomeric forms, in which a hydrogen atom is transposed to other parts of the molecule and the chemical bonds between the atoms of the molecule are rearranged. It should be understood that all tautomeric forms that can exist are included in the present invention.

[0073] Unless otherwise defined, the definitions of the substituents of the present invention are each independent and not interrelated, for example for the substituent R a (or R a ’), which is independent in the definitions of different substituents. Specifically, for R a (or R a ’) when selecting one definition in one substituent, it does not mean that this R a (or R a ’) has the same definition in other substituents. More specifically, for example (only listing non-exhaustively) for NR a R a ’, when the definition of R a (or R a ’) is selected from hydrogen, it does not mean that in -C(O)-NR a R a ’, R a (or R a ’) must necessarily be hydrogen.

[0074] Unless otherwise defined, when a substituent group is designated as "optionally substituted" the substituent group is selected from, for example, alkyl, cycloalkyl, aryl, heterocyclyl, halo, hydroxy, alkoxy, oxo, alkanoyl, aryloxy, alkanoyloxy, amino, alkylamino, arylamino, arylalkylamino, disubstituted amine groups wherein the 2 amino substituents are selected from alkyl, aryl or arylalkyl, alkanoylamino, aroylamino, aralkanoylamino, substituted alkanoylamino, substituted arylamino, substituted aralkanoylamino, thio, alkylthio, arylthio, arylalkylthio, arylthiocarbonyl, arylalkylthiocarbonyl, alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, sulfonamido such as -SO2NH2, substituted sulfonamido, nitro, cyano, carboxy, carbamoyl such as -CONH2, substituted carbamoyl such as -CONHalkyl, -CONHaryl, -CONHarylalkyl or where the nitrogen has two substituents selected from alkyl, aryl or arylalkyl, alkoxycarbonyl, aryl, substituted aryl, guanidino, heterocyclyl such as indolyl, imidazolyl, furanyl, thienyl, thiazolyl, pyrrolidinyl, pyridyl, pyrimidinyl, pyrrolidinyl, piperidyl, morpholinyl, piperazinyl, homopiperazinyl and substituted heterocyclyl.

[0075] The term "alkyl" or "alkylene" as used herein is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For example, "Ci-C6alkyl" denotes alkyl groups having from 1 to 6 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl) and pentyl (e.g., n-pentyl, isopentyl, neopentyl). Preferred alkyl groups are Ci-C6alkyl. Preferred alkylene groups are Co-C6alkylene or Ci-C6alkylene.

[0076] The term "alkenyl" denotes straight or branched-chain hydrocarbon groups containing one or more double bonds and typically having a length of from 2 to 20 carbon atoms. For example, "C2-C6alkenyl" contains from two to six carbon atoms. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl and the like. Preferred alkenyl groups are (C3-C6)alkenyl.

[0077] The term "alkynyl" denotes straight or branched-chain hydrocarbon groups containing one or more triple bonds and typically having a length of from 2 to 20 carbon atoms. For example, "C2-C6alkynyl" contains from two to six carbon atoms. Representative alkynyl groups include, but are not limited to, for example, ethynyl, 1-propynyl, 1-butynyl and the like.

[0078] The term "alkoxy" or "alkyloxy" refers to -O-alkyl. "C1-C6alkoxy" (or alkyloxy) is intended to include C1, C2, C3, C4, C5, C6alkoxy. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propyloxy (e.g., n- propyloxy and isopropyloxy), and t-butyloxy. Similarly, "alkylthio" or "thioalkoxy" denotes an alkyl group as defined above attached through a sulfur bridge; for example, methyl-S- and ethyl-S-. A preferred alkoxy group is C1-C6alkoxy.

[0079] The term "carbonyl" refers to the organic functional group (C=0) connected by a double bond between a carbon and an oxygen atom.

[0080] The term "aryl," by itself or as part of another substituent such as "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to a monocyclic, bicyclic or tricyclic ring system having from 5 to 12 ring members, wherein at least one ring is aromatic and wherein each ring in the system contains from 3 to 7 ring members. In certain embodiments of the application, "aryl" refers to aromatic ring systems including, but not limited to, phenyl, biphenyl, indanyl, 1-naphthyl, 2-naphthyl, and tetrahydronaphthyl. The term "aralkyl" or "arylalkyl" refers to an alkyl radical attached to an aryl ring. Non-limiting examples include benzyl, phenethyl, and the like. A fused aryl group can be attached to another group at a suitable position on either the cycloalkyl ring or the aromatic ring. The dashed line drawn from the ring system indicates that the bond can be attached to any suitable ring atom. A preferred aryl group is C6-C10aryl. 10 aryl.

[0081] The term "cycloalkyl" refers to a monocyclic or bicyclic ring system of cyclic alkyl groups, preferably having from 3 to 8 ring members. Monocyclic ring systems of cycloalkyl groups refer to C3-C8cycloalkyl groups, including, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and norbornyl. Branched cycloalkyl groups such as 1-methylcyclopropyl and 2-methylcyclopropyl are included in the definition of "cycloalkyl." Bicyclic ring systems of cycloalkyl groups include bridged, spiro, or fused ring cycloalkyl groups.

[0082] The term "cycloalkenyl" refers to a monocyclic or bicyclic ring system of cyclic alkenyl groups, preferably having from 3 to 8 ring members. Monocyclic ring systems of cycloalkenyl groups refer to C3-C8cycloalkenyl groups, including, but not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, and norbornenyl. Branched cycloalkenyl groups such as 1-methylcyclopropenyl and 2-methylcyclopropenyl are included in the definition of "cycloalkenyl." Bicyclic ring systems of cycloalkenyl groups include bridged, spiro, or fused ring cycloalkenyl groups.

[0083] "Halo" or "halogen" includes fluoro, chloro, bromo, and iodo. "Haloalkyl" is intended to include both branched and straight chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms and substituted with one or more halogens (preferably 1, 2, or 3 halogens). Examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, trichloromethyl, pentafluoroethyl, pentachloroethyl, 2,2,2-trifluoroethyl, heptafluoropropyl, and heptafluoropropyl. Examples of haloalkyl also include "fluoroalkyl" intended to include both branched and straight chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms (preferably 1 to 6 carbon atoms) and substituted with one or more fluorine atoms.

[0084] "Haloalkoxy" or "haloalkyloxy" denotes an oxygen-bridged haloalkyl group as defined above having the specified number of carbon atoms (preferably 1 to 6 carbon atoms). For example, "halo C1-C6alkoxy" is intended to include C1, C2, C3, C4, C5, C6haloalkoxy groups. Examples of haloalkoxy include, but are not limited to, trifluoromethoxy, 2,2,2-trifluoroethoxy, and pentafluoroethoxy. Similarly, "haloalkylthio" or "thiohaloalkoxy" denotes a sulfur-bridged haloalkyl group as defined above having the specified number of carbon atoms (preferably 1 to 6 carbon atoms); for example, trifluoromethyl-S- and pentafluoroethyl-S-.

[0085] In the present disclosure, when referring to some substituent groups, the expression C x1 -C x2 denotes that the number of carbon atoms in the substituent group can be from x1 to x2. For example, C0-C8denotes that the group contains 0, 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms, C1-C8denotes that the group contains 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms, C2-C8denotes that the group contains 2, 3, 4, 5, 6, 7, or 8 carbon atoms, C3-C8denotes that the group contains 3, 4, 5, 6, 7, or 8 carbon atoms, C4-C8denotes that the group contains 4, 5, 6, 7, or 8 carbon atoms, C0-C6denotes that the group contains 0, 1, 2, 3, 4, 5, or 6 carbon atoms, C1-C6denotes that the group contains 1, 2, 3, 4, 5, or 6 carbon atoms, C2-C6denotes that the group contains 2, 3, 4, 5, or 6 carbon atoms, C3-C6denotes that the group contains 3, 4, 5, or 6 carbon atoms.

[0086] In the present disclosure, when referring to cyclic groups (e.g., aryl, heteroaryl, cycloalkyl, and heterocycloalkyl), the expression "x1-x2-membered ring" is used, which means that the number of ring atoms of the group can be from x1 to x2. For example, the 3-12 membered cyclic group can be a 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, or 12-membered ring, which can have from 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 ring atoms; 3-6 membered ring means that the cyclic group can be a 3-, 4-, 5-, or 6-membered ring, which can have from 3, 4, 5, or 6 ring atoms; 3-8 membered ring means that the cyclic group can be a 3-, 4-, 5-, 6-, 7-, or 8-membered ring, which can have from 3, 4, 5, 6, 7, or 8 ring atoms; 3-9 membered ring means that the cyclic group can be a 3-, 4-, 5-, 6-, 7-, 8-, or 9-membered ring, which can have from 3, 4, 5, 6, 7, 8, or 9 ring atoms; 4-7 membered ring means that the cyclic group can be a 4-, 5-, 6-, or 7-membered ring, which can have from 4, 5, 6, or 7 ring atoms; 5-8 membered ring means that the cyclic group can be a 5-, 6-, 7-, or 8-membered ring, which can have from 5, 6, 7, or 8 ring atoms; 5-12 membered ring means that the cyclic group can be a 5-, 6-, 7-, 8-, 9-, 10-, 11-, or 12-membered ring, which can have from 5, 6, 7, 8, 9, 10, 11, or 12 ring atoms; 6-12 membered ring means that the cyclic group can be a 6-, 7-, 8-, 9-, 10-, 11-, or 12-membered ring, which can have from 6, 7, 8, 9, 10, 11, or 12 ring atoms. The ring atoms can be carbon atoms or heteroatoms, e.g., heteroatoms selected from N, O, and S. When the ring is a heterocycle, the heterocycle can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more ring heteroatoms, e.g., heteroatoms selected from N, O, and S.

[0087] In the present disclosure, one or more halogens can each be independently selected from fluorine, chlorine, bromine, and iodine.

[0088] The term "heteroaryl" means a stable 3-, 4-, 5-, 6-, or 7-membered aromatic monocyclic or aromatic bicyclic or 7-, 8-, 9-, 10-, 11-, 12-membered polyaromatic heterocyclic ring which is fully unsaturated, partially unsaturated, and which contains carbon atoms and 1, 2, 3, or 4 heteroatoms independently selected from N, O, and S; and includes any of the following polycyclic groups wherein any of the heterocyclic rings defined above are fused to a benzene ring. The nitrogen and sulfur heteroatoms can optionally be oxidized. The nitrogen atoms are substituted or unsubstituted (i.e., N or NR, where R is H or, if defined, another substituent). The heterocyclic rings can be attached to their side groups at any heteroatom or carbon atom that results in a stable structure. The heterocyclyl groups described herein can be substituted on a carbon or a nitrogen atom if the resulting compound is stable. The nitrogens in the heterocyclic ring can optionally be quaternized. Preferably, when the total number of S and O atoms in the heterocyclic ring exceeds one, then these heteroatoms are not adjacent to one another. Preferably, the total number of S and O atoms in the heterocyclic ring is not more than one. When the term "heterocycle" is used, it is intended to include heteroaryl.Examples of heteroaryl groups include, but are not limited to, acridinyl, azetidinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, imidazopyridinyl, indoleninyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isochromanyl, isochromenyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isothiazolopyridinyl, isoxazolyl, isoxazolopyridinyl, methylenedioxyphenyl, morpholinyl, naphthizidinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolopyridinyl, oxazolidinyl, perimidinyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolopyridinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2-pyrrolidonyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrazolyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 6H-1,2,5-thiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienopyridinyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, and xanthenyl, quinolinyl, isoquinolinyl, phthalazinyl, quinazolinyl, indolyl, isoindolyl, indolinyl, 1H-indazolyl, benzimidazolyl, 1,2,3,4-tetrahydroquinolinyl, 1,2,3,4-tetrahydroisoquinolinyl, 5,6,7,8-tetrahydro-quinolinyl, 2,3-dihydro-benzofuranyl, chromanyl, 1,2,3,4-tetrahydro- quinoxalinyl and 1,2,3,4-tetrahydro-quinazolinyl.The term "heteroaryl" can also include biaryl structures formed from the above defined "aryl" groups with monocyclic "heteroaryl" groups, such as, but not limited to, "-phenylbipyridyl-", "-phenylbipyrimidyl-", "-pyridylbiphenyl-", "-pyridylbipyrimidyl-", "-pyrimidylbiphenyl-"; wherein the present application also includes fused ring and spiro compounds containing, for example, the above heterocycles.

[0089] The term "heterocycloalkyl" as used herein refers to a monocyclic heterocycloalkyl ring system, or to a bicyclic heterocycloalkyl ring system, and also includes spiro or bridged heterocycloalkyl groups. Monocyclic heterocycloalkyl refers to a 3-8 membered or 4-8 membered, saturated or unsaturated, but not aromatic, cyclic alkyl ring system containing at least one ring member selected from O, N, S, P. Bicyclic heterocycloalkyl ring systems refer to a heterocycloalkyl fused to a phenyl, or a cycloalkyl, or a cycloalkenyl, or a heterocycloalkyl, or a heteroaryl.

[0090] The term "bridged cycloalkyl" as used herein refers to polycyclic compounds sharing two or more carbon atoms. It can be divided into bicyclic bridged cycloalkanes and polycyclic bridged cycloalkanes. The former consists of two alicyclic rings sharing two or more carbon atoms; the latter is a bridged cycloalkane consisting of three or more rings.

[0091] The term "spirocycloalkyl" as used herein refers to polycyclic hydrocarbons sharing one carbon atom (called spiro atom) between single rings.

[0092] The term "bridged heterocyclyl" as used herein refers to polycyclic compounds sharing two or more carbon atoms, at least one of which is selected from O, N, S. It can be divided into bicyclic bridged heterocycles and polycyclic bridged heterocycles.

[0093] The term "spiroheterocyclyl" as used herein refers to polycyclic hydrocarbons sharing one carbon atom (called spiro atom) between single rings, at least one of which is selected from O, N, S.

[0094] The term "substituted" as used herein means that at least one hydrogen atom has been replaced by a non-hydrogen group, provided that a stable compound results. A ring double bond as used herein is a double bond between two adjacent ring atoms (e.g., C=C, C=N, or N=N).

[0095] In cases where there are nitrogen atoms (e.g., amines) on the compounds of the present application, these nitrogen atoms can be converted to N-oxides by treatment with an oxidizing agent (e.g., mCPBA and / or hydrogen peroxide) to afford additional compounds of the present application. Thus, shown and claimed nitrogen atoms are considered to cover both the shown nitrogen and its N-oxide to afford derivatives of the present application.

[0096] When any variable occurs more than one time in any constituent or formula, its definition in each occurrence is independent of its definition in every other case. Thus, for example, if a group is shown to be substituted with 0 to 3 R groups, said group can optionally be substituted with up to three R groups, and at each occurrence R is independently selected from the definition of R. Also, combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0097] The term "patient" as used herein refers to an organism to be treated by the methods of the present application. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians / monkeys, equines, bovines, porcines, canines, felines, etc.) and most preferably refers to humans.

[0098] The term "effective amount" as used herein means that amount of a drug or pharmaceutical agent (i.e., a compound of the present application), which will elicit the biological or medical response of a tissue, system, animal, or human that is being sought, for instance, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means an amount of a compound effective to improve the treatment, cure, prevent, or reduce the symptoms of a disease, condition, or disorder, or to reduce the speed of progression of a disease or condition, as compared to an untreated subject. An effective amount can be given in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or administration route. The term also includes within its scope amounts effective to enhance normal physiological function.

[0099] The term "treatment" as used herein includes any effect that relieves, reduces, modulates, ameliorates, or eliminates a condition, disease, disorder, etc., or its symptoms.

[0100] The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, and / or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0101] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium stearate or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as glucose or beta-lactose;

[0102] The term "pharmaceutical composition" means a composition comprising a compound of the present application in combination with at least one other pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" means a medium generally accepted in the art for the delivery of a biologically active agent to an animal, particularly a mammal, and includes, i.e., an adjuvant, excipient, or vehicle such as diluent, preservative, filler, flow regulator, disintegrating agent, wetting agent, emulsifying agent, suspending agent, sweetening agent, flavoring agent, perfuming agent, antibacterial agent, antifungal agent, lubricating agent, and dispersing agent, depending on the nature of the mode of administration and dosage form.

[0103] Specific Pharmaceutical and Medical Terms

[0104] The term "acceptable", as used herein, means no undue harmful effect on the general health of the subject of treatment of the active ingredients of the prescription.

[0105] The term "cancer", as used herein, means an uncontrolled abnormal growth of cells and, under certain conditions, the ability to metastasize (spread). This type of cancer includes, but is not limited to, solid tumors (e.g., bladder, bowel, brain, breast, uterine, cardiac, kidney, lung, lymphatic tissue (lymphoma), ovarian, pancreatic or other endocrine organ (e.g., thyroid), prostate, skin (melanoma), or blood tumors (e.g., non-leukemic leukemia).

[0106] The term "co-administration" or its grammatical equivalents, as used herein, means the administration of two or more selected therapeutic agents to a single patient in close enough time proximity as to provide a desired therapeutic effect.

[0107] The term "enhance" or "enhancing", as used herein, means an intended result that can be an increase or prolongation in potency or duration. Thus, in the context of enhancing the therapeutic effect of a drug, the term "enhancing" means the ability of a drug to increase or prolong the potency or duration of action in a system. "Enhancing value", as used herein, means the ability to maximize the enhancement of another therapeutic agent in a desired system.

[0108] The term "immune disease" means a disease or condition resulting from an adverse or deleterious reaction to an endogenous or exogenous antigen. The result is usually a dysfunction of cells, or destruction and dysfunction as a result, or destruction of organs or tissues that can produce the immune condition.

[0109] The terms "kit" and "product package" are synonymous.

[0110] The term "subject" or "patient" includes mammals and non-mammals. Mammals include, but are not limited to, mammals: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and house cats; laboratory animals such as rats, mice, and guinea pigs; and the like. Non-mammalian animals include, but are not limited to, birds, fish, and the like. In a preferred embodiment, the mammal is a human.

[0111] The terms "treatment," "treatment regime," or "therapy," as used herein, include alleviating, inhibiting or ameliorating a disease symptom or condition; inhibiting the onset of a complication; ameliorating or preventing an underlying metabolic syndrome; inhibiting the development of a disease or symptom, such as controlling the progression of a disease or condition; abating a disease or symptom; causing regression of a disease or symptom; relieving a complication caused by a disease or symptom, or preventing and / or treating an indication caused by a disease or symptom.

[0112] As used herein, an improvement in a disease, symptom, or condition, with respect to a compound or pharmaceutical composition, means, inter alia, that the severity of the disease, symptom, or condition is improved, the onset of the disease, symptom, or condition is delayed, the progression of the disease, symptom, or condition is slowed, or the duration of the disease, symptom, or condition is lessened. The improvement can be attributed to or associated with the administration, whether fixed or contingent, whether continuous or intermittent.

[0113] Routes of administration

[0114] Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ocular, pulmonary, transdermal, vaginal, aural, nasal, and topical. In addition, parenteral administration includes, by way of illustration, intramuscular, subcutaneous, intravenous, intramedullary, intraventricular, intraperitoneal, intralymphatic, and intranasal.

[0115] In one aspect, the compounds described herein are administered in a manner that is local rather than systemic. In a particular embodiment, the long-acting formulation is administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. In addition, in another embodiment, the drug is administered by a targeted drug delivery system. For example, a liposome encapsulated with an organ-specific antibody. In this embodiment, the liposome is selectively directed to a particular organ and is taken up.

[0116] Pharmaceutical compositions and dosages

[0117] The present application also provides pharmaceutical compositions comprising a therapeutically effective amount of one or more compounds of the present application formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents and, optionally, one or more other therapeutic agents as described above. The compounds of the present application can be administered by any suitable means, for any of the above-mentioned uses, e.g., orally, such as tablets, pills, powders, granules, elixirs, tinctures, suspensions (including nano-, micro-, spray-dried dispersions), syrups, and emulsions; sublingually; buccally; parenterally, such as by subcutaneous, intravenous, intramuscular, or intrasternal injection or infusion techniques (e.g., as sterile injectable aqueous or nonaqueous solutions or suspensions); nasally, including to the nasal membranes, such as by inhalable sprays; topically, such as in creams or ointments; or rectally, such as in suppositories; or intratumorally. They can be administered alone, but generally are administered in admixture with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

[0118] Pharmaceutically acceptable carriers are formulated in accordance with a number of factors well within the purview of those skilled in the art. These factors include, but are not limited to: the type and nature of the active agent being formulated; the subject to which the composition containing the active agent is to be administered; the intended route of administration of the composition; and the therapeutic indication being targeted. Pharmaceutically acceptable carriers include aqueous and nonaqueous liquid media and various solid and semi-solid dosage forms.

[0119] The above-mentioned carriers can include a wide variety of different components and additives, in addition to the active agent, which are included in the formulation for a variety of reasons, e.g., to stabilize the active agent, to bind the agents, etc. Descriptions of suitable pharmaceutically acceptable carriers and the factors involved in selection of carriers are well-documented, e.g., in Allen L.V. Jr. et al. Remington: The Science and Practice of Pharmacy (2 Volumes), 22nd Edition (2012), Pharmaceutical Press.

[0120] The dosage regimen for compounds of the present application will, of course, depend on known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition, and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment(s) and the frequency and route of administration, the renal and hepatic function of the patient, and the desired effect. In general, a daily oral dosage of each active ingredient for use in humans will range from about 0.001 mg / day to about 10-5000 mg / day, preferably from about 0.01 mg / day to about 1000 mg / day, and most preferably from about 0.1 mg / day to about 250 mg / day, when employed for the indicated effects. Intravenous dosages during a constant rate infusion will preferably be about 0.01 mg / kg / min to about 10 mg / kg / min. Compounds of the present application can be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three or four times daily.

[0121] The compounds are generally administered in the form of a pharmaceutical composition in admixture with a suitable pharmaceutical diluent, excipient, or carrier (collectively referred to as a "pharmaceutical carrier") selected with due regard to the intended form of administration (e.g., oral tablets, capsules, elixirs, and syrups) and consistent with conventional pharmaceutical practice.

[0122] Dosage forms (pharmaceutical compositions) suitable for administration include about 1 milligram to about 2000 milligrams of active ingredient per dosage unit.

[0123] A typical capsule for oral administration contains at least one compound of the present application (250 mg), lactose (75 mg) and magnesium stearate (15 mg). The mixture is passed through a 60 mesh sieve and packed into a No. 1 gelatin capsule.

[0124] A typical injectable formulation can be prepared by aseptically placing at least one compound of the present application (250 mg) into a bottle, freeze-drying and sealing. For use, the contents of the bottle are mixed with 2 mL of normal saline to produce an injectable formulation.

[0125] The scope of the present application includes pharmaceutical compositions comprising a therapeutically effective amount of at least one compound of the present application as an active ingredient, alone or in combination with a pharmaceutical carrier. Optionally, a compound of the present application can be used alone, in combination with other compounds of the present application, or in combination with one or more other therapeutic agents (e.g., an anti-cancer agent or other pharmaceutically active substance).

[0126] Regardless of the route of delivery selected, the compounds of the present application, which can be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present application, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.

[0127] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present application can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0128] The selected dosage level will depend on a variety of factors including the activity of the particular compound of the present application employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and pre-existing medical conditions of the patient being treated, and like factors well known in the medical arts.

[0129] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the application employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a compound of the application will be in the range from about 0.01 to about 50 mg / kg body weight per day. Generally, oral, intravenous, intracerebroventricular, and subcutaneous doses of the compounds of the application for patients will range from about 0.01 to about 50 mg / kg body weight per day. If desired, the effective daily dose can be divided into two, three, four, five, six, or more sub-doses that are administered separately at appropriate intervals during the day, optionally, in unit dosage forms. In certain aspects of the application, dosing is once daily.

[0130] While it is possible for a compound of the present application to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).

[0131] Kits / Products Packets

[0132] Kits / Products Packets are also described herein for use in the treatment of the indications described above. These kits can consist of a carrier, a packet, or a container box, which can be divided into compartments, for containing one or more containers, such as vials, test tubes, and the like, each containing one of the ingredients in the method. Suitable containers include bottles, vials, syringes, and test tubes. The containers are made of a material that is acceptable to the intended recipient of the pharmaceutical composition. For example, the containers can be formed from plastic, glass, or other material.

[0133] For example, a container can contain one or more of the compounds described herein, which can be present as a pharmaceutical composition, in combination with one or more of the other ingredients described herein. The container can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Such kits can further include one or more compounds, and instructions, a label, or packaging insert describing the use of the compounds in the methods described herein.

[0134] A typical kit can include one or more containers into which one or more of the materials described herein can be placed, for example, for commercial sale and use. Such kits can include, for example, one or more containers as noted above, containing one or more of the compounds described herein, optionally in a composition form, and / or one or more other ingredients described herein. Such kits can further include, for example, one or more containers containing one or more of the other ingredients described herein, and / or one or more containers containing compositions / formulations including the compounds described herein. Such kits can further include, for example, one or more containers containing one or more of the other ingredients described herein, and / or one or more containers containing compositions / formulations including the compounds described herein. Such kits can further include, for example, instructions for using the compounds described herein in the methods described herein.

[0135] Labels suitable for use in containers and boxes can be, for example, printed labels, embossed labels, or engraved labels. Labels can be present on or associated with the container. The label is indicative of a proposed therapeutic use or uses of the contents; for example, the label can indicate that the contents are useful in treating a particular disease, such as a disease described herein. The label can also indicate directions for use, such as in the methods described herein.

[0136] All of the features described in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process described herein, can be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0137] All of the features described in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process described herein, can be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0138] The application will be further described with reference to the following examples. It should be appreciated that these examples are for illustrative purposes only and are not meant to limit the scope of the application. Unless otherwise indicated, the methods of preparing the examples below generally follow conventional procedures or those described in the Examples section. Unless otherwise indicated, all percentages, ratios, proportions, or parts are by weight.

[0139] The units in the weight / volume percentages in the present application are well known to those skilled in the art, for example, refer to the weight of the solute in 100 ml of solution. Unless otherwise defined, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application belongs. In addition, all methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. The preferred methods and materials described herein are illustrative only and not intended to be limiting.

[0140] Examples

[0141] General procedures

[0142] When not included in the preparation route, the starting materials and reagents used in the present application are known products, which can be synthesized according to the methods known in the art, or can be obtained by purchasing commercially available products. The commercially available reagents used do not need further purification.

[0143] Room temperature refers to 20-30°C.

[0144] Unless otherwise specified in the reaction examples, the reactions are carried out under a nitrogen atmosphere. The nitrogen atmosphere refers to the reaction bottle connected to a nitrogen balloon of about 1 L.

[0145] The hydrogenation reaction is usually vacuumed and filled with hydrogen, and the operation is repeated 3 times. The hydrogen atmosphere refers to the reaction bottle connected to a hydrogen balloon of about 1 L.

[0146] Microwave reaction uses Initiator + microwave reactor.

[0147] The structure of the compounds of the present application is determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS). The NMR shift (δ) is given in units of 10 -6 (ppm). The NMR is measured by a (Bruker Ascend TM 500 type) nuclear magnetic instrument, and the measuring solvent is deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD), and the internal standard is tetramethylsilane (TMS). The following abbreviations are used for the multiplicity of NMR signals: s = singlet, br s = broad singlet, d = doublet, t = triplet, m = multiplet. The coupling constant is listed as J value, measured in Hz.

[0148] The measurement of LC-MS uses Thermo liquid chromatograph-mass spectrometer (UltiMate 3000+MSQ PLUS). The measurement of HPLC uses Thermo high pressure liquid chromatograph (UltiMate 3000). The reverse phase preparative chromatography uses Thermo (UltiMate 3000) reverse phase preparative chromatograph. The flash column chromatography uses Ajinomoto (FS-9200T) automatic column machine, and the silica gel pre-packed column uses San Tai (S-1000) silica gel pre-packed column. Pre-packed column. Thin layer chromatography silica gel plate with Yantai Yellow Sea HSGF254 or Qingdao GF254 silica gel plate, the specification used for thin layer chromatography separation and purification of the product is 0.4mm-0.5mm.

[0149] The synthesis method of some intermediates in the application is as follows:

[0150] Intermediate 1

[0151]

[0152] Intermediate 1 is prepared by the following steps:

[0153]

[0154] Step 1: 1-methyl-3,5-dinitropyridin-2-one Int-1a (1.0 g, 5.02 mmol) was dissolved in methanol (50 mL), and then ammonium methanol solution (7 mol / L, 8.61 mL, 60.27 mmol) and 1-methylpiperidin-4-one Int-1b (625 mg, 5.52 mmol) were added in sequence. The reaction mixture was heated to 50°C and stirred for 5 hours. After cooling to room temperature, it was left to stand for 48 hours, and the reaction solution was concentrated under reduced pressure. The residue was filtered after adding ethyl acetate (50 mL). The filtrate was concentrated under reduced pressure to obtain red solid Int-1c (1.0 g), which was directly used in the next step reaction. ESI-MS (m / z): 194.4 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 9.14 (d, J = 2.5 Hz, 1H), 8.36 (d, J = 2.5 Hz, 1H), 3.64 (s, 2H), 3.02 (t, J = 6.0 Hz, 2H), 2.74 (t, J = 6.0 Hz, 2H), 2.39 (s, 3H).

[0155] Step 2: The compound Int-1c (1.0 g) obtained in the previous step was dissolved in methanol (30 mL), and 10% Pd-C (400 mg) was added. The reaction was carried out under hydrogen atmosphere at room temperature for 6 hours. The palladium carbon was removed by filtration, and the filtrate was concentrated to obtain yellow solid Int-1d (800 mg, yield 94.70%). ESI-MS (m / z): 164.2 [M+H] + .

[0156] Step 3: Compound Int-1d (100 mg, 0.61 mmol) was dissolved in acetic acid (3 mL), N-bromosuccinimide (109 mg, 0.61 mmol) was added, and the reaction mixture was stirred at room temperature for 1 hour. The reaction was quenched with saturated aqueous sodium bicarbonate until no gas bubbles were generated, and the aqueous phase was extracted with methanol / dichloromethane (1 / 20, 50 mL x 2), the combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give compound Int-1e (38 mg, yield 25%). ESI-MS (m / z): 242.3 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 6.77 (s, 1H), 5.25 (s, 2H), 3.37 (s, 2H), 2.69 (t, J = 6.0 Hz, 2H), 2.60 (t, J = 6.0 Hz, 2H), 2.32 (s, 3H).

[0157] Step 4: Compound Int-1e (37 mg, 0.15 mmol) was dissolved in methanol (1 mL), and cuprous iodide (3 mg, 0.015 mmol), 1,10-phenanthroline (3 mg, 0.03 mmol), and cesium carbonate (99 mg, 0.30 mmol) were added. The reaction mixture was stirred at 100 °C for 2 hours after being purged with nitrogen and heated with a microwave. The reaction was cooled to room temperature, the reaction was concentrated, and the residue was purified by preparative thin layer chromatography (methanol / dichloromethane / triethylamine = 1 / 10 / 0.1) to give Int-1 as a yellow solid (20 mg, yield 67%). ESI-MS (m / z): 194.5 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 6.54 (s, 1H), 4.68 (s, 2H), 3.80 (s, 3H), 3.30 (s, 2H), 2.64 (t, J = 5.6 Hz, 2H), 2.59 (t, J = 5.7 Hz, 2H), 2.31 (s, 3H).

[0158] Intermediate 2

[0159]

[0160] Intermediate 2 was prepared from the following steps:

[0161]

[0162] Step 1: Compound Int-1e (230 mg, 0.94 mmol) was dissolved in ethanol (2 mL), and cuprous iodide (18 mg, 0.095 mmol), 1,10-phenanthroline (34 mg, 0.18 mmol), and cesium carbonate (619 mg, 1.90 mmol) were added. The reaction mixture was purged with nitrogen and then microwaved to 100 °C with stirring for 5 hours. The reaction mixture was cooled to room temperature, filtered, the filtrate was concentrated, and the residue was purified by silica gel column chromatography (methanol / dichloromethane / triethylamine = 1 / 50 / 0.1) to give a yellow solid Int-2 (113 mg, yield 57%). ESI-MS (m / z): 208.5 [M+H] + .

[0163] Intermediate 3

[0164]

[0165] Intermediate 3 is prepared by the following steps:

[0166]

[0167] Step 1: Compound Int-1e (100 mg, 0.41 mmol) and trimethylcycloborane (148 mg, 1.19 mmol) were dissolved in dioxane (1.5 mL) and water (0.15 mL). Potassium carbonate (171 mg, 1.24 mmol) and Pd(dppf)Cl2 (30 mg, 0.041 mmol) were added. The reaction system was purged with nitrogen and then heated to 140 °C in a microwave oven with stirring for 1 hour. The reaction was cooled to room temperature, and the reaction mixture was filtered through diatomaceous earth. The filtrate was concentrated. The residue was purified by silica gel column chromatography (methanol / dichloromethane = 1 / 20) to give a yellow solid Int-3 (50 mg, yield 68%). ESI-MS (m / z): 178.6 [M+H] + .

[0168] Intermediate 4

[0169]

[0170] Intermediate 4 is prepared by the following steps:

[0171]

[0172] First Step: Compound Int-1e (350 mg, 1.45 mmol) and vinylpotassium trifluoroborate (387 mg, 2.89 mmol) were dissolved in 1,4-dioxane (1.5 mL) and water (0.15 mL), potassium carbonate (399 mg, 2.89 mmol) and Pd(dppf)Cl2(105 mg, 0.14 mmol) were added. The reaction was heated to 120 °C in a microwave reactor for 1 hour after purging with nitrogen. After the reaction was cooled to room temperature, it was filtered with celite, the filtrate was concentrated, and the residue was separated by column chromatography (methanol / dichloromethane = 1 / 20) to give Int-4a (136 mg, yield 49%) as a yellow solid. ESI-MS (m / z): 190.7 [M+H] + ; 1 H NMR (500 MHz, CDC13) δ 6.85 (dd, J = 17.2, 11.0 Hz, 1H), 6.66 (s, 1H), 6.16 (dd, J = 17.3, 1.9 Hz, 1H), 5.49 (dd, J = 11.0, 1.9 Hz, 1H), 3.67-3.63 (m, 2H), 3.55 (s, 2H), 3.00 (t, J = 6.1 Hz, 2H), 2.80 (t, J = 6.1 Hz, 2H), 2.49 (s, 3H).

[0173] Second Step: Compound Int-4a (60 mg, 0.31 mmol) was dissolved in methanol (5 mL), 10% palladium on carbon (20 mg) was added, and the mixture was stirred at room temperature under a hydrogen atmosphere for 1 hour. The reaction was filtered with celite, and the filtrate was concentrated to give intermediate 4 (37 mg, yield 61%). ESI-MS (m / z): 192.7 [M+H] + .

[0174] Intermediate 5

[0175]

[0176] Intermediate 5 was prepared by the following steps:

[0177]

[0178] First Step: Dissolve compound Int-1e (100 mg, 0.41 mmol) in a mixture solvent of toluene (3 mL) and water (0.3 mL), add cyclopropylboronic acid (42 mg, 0.49 mmol), potassium phosphate (306 mg, 1.45 mmol), tricyclohexylphosphine (23 mg, 0.082 mmol) and palladium acetate (9 mg, 0.041 mmol). Replace the reaction system with nitrogen and heat to 100 °C for 18 hours with stirring. After the reaction is cooled to room temperature, filter the reaction solution with diatomite, concentrate the filtrate, and separate the residue by column chromatography (methanol / dichloromethane = 1 / 20) to obtain yellow solid Int-5 (61 mg, yield 72%). ESI-MS (m / z): 204.2 [M+H] + .

[0179] Intermediate 6

[0180]

[0181] Intermediate 6 is prepared by the following steps:

[0182]

[0183] First Step: Dissolve N-tert-butoxycarbonyl-4-piperidinone Int-6a (4.4 g, 22.1 mmol) and 1-methyl-3,5-dinitro-2-pyridinone Int-1a (4.0 g, 20.1 mmol) in methanol (150 mL), and add an ammonia methanol solution (7 N, 34.4 mL, 240.8 mmol). Stir at 60 °C for 6 hours under nitrogen protection. After the reaction solution is cooled to room temperature, continue stirring for 2 days. Monitor the end of the reaction by LCMS, concentrate the reaction solution, add ethyl acetate (150 mL), stir for half an hour, then filter, and concentrate the filtrate to obtain yellow solid Int-6b (5.1 g, yield 91%). ESI-MS (m / z): 280.1 [M+H] + .

[0184] Second Step: Dissolve compound Int-6b (5.0 g, 17.9 mmol) in methanol (50 mL), and add 10% palladium on carbon (500 mg). Stir the mixture at room temperature for 16 hours under a hydrogen atmosphere (hydrogen balloon). After the reaction is completed, filter the reaction solution, and concentrate the filtrate to obtain light yellow solid Int-6c (3.7 g, yield 84%). ESI-MS (m / z): 250.2 [M+H] + .

[0185] Third Step: Compound Int-6c (3.7 g, 14.8 mmol) was dissolved in DMF (20 mL), N-bromosuccinimide (2.78 g, 15.6 mmol) and acetic acid (370 mg) were added. The reaction mixture was stirred at room temperature for 2 hours, and the reaction was monitored by LCMS. Water (100 mL) was added, and the aqueous phase was extracted with ethyl acetate (150 mL*3). The organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was separated by silica gel column chromatography to obtain yellow solid Int-6d (3.6 g, yield 74%). ESI-MS (m / z): 328.2 [M+H] + .

[0186] Fourth Step: Compound Int-6d (500 mg, 1.53 mmol) was dissolved in methanol (5 mL), and sodium methoxide methanol solution (5 N, 0.33 mL, 1.65 mmol) was added. The reaction mixture was heated to 100 °C with microwave for 3 hours. The reaction was cooled to room temperature, and the reaction solution was concentrated. The residue was separated by silica gel column chromatography to obtain yellow solid Int-6 (330 mg, yield 77%). ESI-MS (m / z): 280.2 [M+H] + .

[0187] Example 1

[0188] 3-((5-chloro-2-((2-methoxy-6-methyl-5,6,7,8-tetrahydro-l,6-naphthyridin-3-yl)amino)pyrimidin-4-yl)amino)-N,2,2-trimethylpropanamide

[0189]

[0190] Compound 1 was prepared by the following steps:

[0191]

[0192] First Step: Boc-3-amino-2,2-dimethyl-propanoic acid la (50 mg, 0.23 mmol) and methylamine hydrochloride (77 mg, 1.15 mmol) were dissolved in N,N-dimethylformamide (5 mL), and HATU (105 mg, 0.27 mmol) and N,N-diisopropylethylamine (297 mg, 2.30 mmol) were added in turn. The reaction mixture was stirred at room temperature for 14 hours. TLC detection showed that the starting material la was completely converted. The reaction mixture was diluted with water (5 mL) and extracted with ethyl acetate (15 mL*3). The organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product lb, which was directly used in the next step.

[0193] Second Step: The crude compound 1b from the previous step was dissolved in hydrochloric acid in dioxane (4 mol / L, 5 mL), and the reaction was stirred at room temperature for 2 hours. TLC showed that the reaction of compound 1b was complete. The reaction was concentrated under reduced pressure to obtain the crude compound 1c, which was directly used in the next step.

[0194] Third Step: The crude compound 1c from the previous step was dissolved in isopropanol (5 mL), and 2,4,5-trichloropyrimidine 1d (40 mg, 0.21 mmol) and N,N-diisopropylethylamine (84 mg, 0.65 mmol) were added. The reaction was heated to 90°C and stirred overnight. LCMS detection showed that the reaction of compound 1d was complete. The reaction was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2 / 1) to obtain compound 1e (52 mg, 82% yield for three steps). ESI-MS (m / z): 278.4 [M+H] + .

[0195] Fourth Step: Compound 1e (52 mg, 0.18 mmol) and intermediate Int-1 (36 mg, 0.18 mmol) were dissolved in 1,4-dioxane (5 mL), and BrettPhos G3Pd (17 mg, 0.018 mmol), BrettPhos (10 mg, 0.018 mmol), and cesium carbonate (122 mg, 0.37 mmol) were added in turn. After nitrogen replacement, the reaction mixture was stirred at 100°C overnight. LCMS detection showed that the reaction of compound 1e was complete. The reaction was filtered through diatomite, and the filtrate was concentrated. The residue was purified by preparative thin layer chromatography (petroleum ether / ethyl acetate = 1 / 1) to obtain the crude compound 1, which was further purified by reverse phase preparative HPLC to obtain compound 1 (6 mg, 7% yield). ESI-MS (m / z): 434.2 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 8.18 (s, 1H), 7.97 (s, 1H), 7.69 (d, J = 4.5 Hz, 1H), 7.65 (s, 1H), 6.93 (t, J = 5.9 Hz, 1H), 3.89 (s, 3H), 3.53-3.48 (m, 4H), 2.80-2.69 (m, 4H), 2.59 (s, 3H), 2.40 (s, 3H), 1.11 (s, 6H).

[0196] Example 2

[0197] 3-((5-chloro-2-((2-methoxy-6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)amino)pyrimidin-4-yl)amino)-N,2-dimethylpropanamide

[0198]

[0199] Compound 2 was obtained by replacing Boc-3-amino-2,2-dimethyl-propionic acid la in the first step of Example 1 with Boc-DL-3-aminoisobutyric acid, using similar methods and reaction procedures. ESI-MS (m / z): 420.2 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.94 (s, 1H), 7.78 (d, J = 4.0 Hz, 1H), 7.60 (s, 1H), 7.20 (t, J = 5.8 Hz, 1H), 3.89 (s, 3H), 3.55 - 3.48 (m, 1H), 3.41 (d, J = 7.2 Hz, 2H), 3.37 (s, 1H), 2.74 (t, J = 5.6 Hz, 2H), 2.71 - 2.64 (m, 3H), 2.57 (d, J = 4.5 Hz, 3H), 2.34 (s, 3H), 1.02 (d, J = 7.0 Hz, 3H).

[0200] Example 3

[0201] 3-((5-chloro-2-((2-methoxy-6-methyl-5,6,7,8-tetrahydro-l,6-naphthyridin-3- yl)amino)pyrimidin-4-yl)amino)-N,N,2,2-tetramethylpropanamide

[0202]

[0203] Compound 3 was obtained by replacing methylamine hydrochloride in the first step of Example 1 with dimethylamine hydrochloride, using similar methods and reaction procedures. ESI-MS (m / z): 448.2 [M+H] + ; 1 H NMR (500 MHz, CDCl3) δ 8.36 (s, 1H), 7.87 (s, 1H), 7.27 (s, 1H), 6.49 (s, 1H), 3.98 (s, 3H), 3.70 (d, J = 6.2 Hz, 2H), 3.58 (s, 2H), 3.05 (s, 6H), 2.89 (d, J = 5.6 Hz, 2H), 2.80 (t, J = 5.4 Hz, 2H), 2.50 (s, 3H), 1.35 (s, 6H).

[0204] Example 4

[0205] 3-((2-((2-methoxy-6-methyl-5,6,7,8-tetrahydro-l,6-naphthyridin-3-yl)amino)-5- methylpyrimidin-4-yl)amino)-N,2,2-trimethylpropanamide

[0206]

[0207] Compound 4 was prepared by the following steps:

[0208]

[0209] First step: 2,4-dichloro-5-methyl-pyrimidine (652 mg, 4 mmol) and 3-amino-2,2- dimethylpropionic acid methyl ester hydrochloride (670 mg, 4 mmol) were dissolved in isopropanol (15 mL), and DIEA (1.55 g, 12.00 mmol) was added to the reaction mixture. The reaction mixture was stirred at 85 °C overnight. LCMS showed the starting material was completely converted. The reaction mixture was concentrated to get the crude product, which was purified by column chromatography (petroleum ether / ethyl acetate = 1 / 1) to give the target compound 4c (835 mg, yield 81%) as colorless oil. ESI-MS (m / z): 258.3 [M+H] + .

[0210] Second step: Compound 4c (540 mg, 2.10 mmol) was dissolved in a mixture of tetrahydrofuran (10 mL) and water (5 mL), and lithium hydroxide (100 mg, 4.19 mmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature overnight. LCMS showed the starting material was completely converted. The reaction mixture was concentrated to remove tetrahydrofuran, and then the pH value of the solution was adjusted to 3 with 1 N aqueous hydrochloric acid solution. The resulting white solid was filtered and dried to give compound 4d (340 mg, yield 66%). ESI-MS (m / z): 244.3 [M+H] + .

[0211] Third step: Compound 4d (121 mg, 0.5 mmol), methylamine hydrochloride (33 mg, 0.5 mmol), diisopropylethylamine (129 mg, 1 mmol) and HATU (285 mg, 0.75 mmol) were dissolved in dichloromethane (8 mL), and the reaction mixture was stirred at room temperature overnight. LCMS showed the starting material was completely converted. The reaction mixture was concentrated to get the crude product, which was purified by column chromatography (dichloromethane / ethyl acetate = 1 / 1) to give compound 4e (50 mg, yield 38%). ESI-MS (m / z): 257.3 [M+H] + .

[0212] Fourth Step: Compound 4e (50 mg, 0.19 mmol), Int-1 (37 mg, 0.19 mmol), BrettPhos G3 Pd (17 mg, 0.019 mmol), Brettphos (20 mg, 0.038 mmol), cesium carbonate (126 mg, 0.39 mmol) were dissolved in dioxane (10 mL), the reaction was stirred at 110 °C under nitrogen atmosphere overnight. LCMS showed the starting material was consumed completely. The reaction was filtered and concentrated, the crude was purified by reverse phase preparative HPLC to give compound 4 (8 mg, 10% yield). ESI-MS (m / z): 414.4 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 8.36 (s, 1H), 7.68 (s, 1H), 7.60 (q, J = 4.6 Hz, 1H), 7.20 (s, 1H), 6.42 (t, J = 6.0 Hz, 1H), 3.90 (s, 3H), 3.54 (d, J = 6.0 Hz, 2H), 3.43 (s, 2H), 2.73 (d, J = 6.0 Hz, 2H), 2.64 (t, J = 6.0 Hz, 2H), 2.58 (d, J = 4.4 Hz, 3H), 2.34 (s, 3H), 1.92 (s, 3H), 1.11 (s, 6H).

[0213] Example 5

[0214] 5-Chloro-N2-(2-methoxy-6-methyl-5,6,7,8-tetrahydro-l,6-naphthyridin-3-yl)-N4-((l- (methylsulfonyl)cyclobutyl)methyl)pyrimidine-2,4-diamine

[0215]

[0216] Compound 5 was prepared by the following steps:

[0217]

[0218] First Step: 2,4,5-trichloropyrimidine (46 mg, 0.25 mmol) was dissolved in isopropanol (2 mL), then 1-(methylsulfonyl)cyclobutyl)methanamine hydrochloride 5a (51 mg, 0.25 mmol) and N,N-diisopropylethylamine (97 mg, 0.75 mmol) were added, the reaction was stirred at 25 °C for 16 h. After the reaction was completed, the reaction was concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2 / 1) to give white solid 5b (48 mg, 58% yield). ESI-MS (m / z): 310.1 [M+H] + .

[0219] Step 2: Compound 5b (48 mg, 0.16 mmol) and Int-1 (30 mg, 0.16 mmol) were dissolved in 1,4-dioxane (5 mL), BrettPhos Pd G3 (14 mg, 0.016 mol), BrettPhos (17 mg, 0.032 mol) and cesium carbonate (101 mg, 0.32 mol) were added. The reaction system was replaced by nitrogen and heated to 110 °C for 18 hours with stirring. After the reaction solution was cooled to room temperature, the reaction solution was filtered with diatomite, and the filtrate was concentrated. The residue was purified by reverse phase preparative HPLC to obtain compound 5 (6 mg, yield 8%). ESI-MS (m / z): 467.2 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 8.02 (s, 1H), 7.99 (s, 1H), 7.84 (s, 1H), 7.13-7.07 (m, 1H), 3.98 (d, J = 6.0 Hz, 3H), 3.87 (s, 3H), 3.43 (s, 2H), 2.93 (s, 3H), 2.77-2.72 (m, 2H), 2.67-2.61 (m, 2H), 2.48-2.42 (m, 2H), 2.34 (s, 3H), 2.18-2.10 (m, 2H), 1.90-1.80 (m, 2H).

[0220] Example 6

[0221] 1-(((5-Chloro-2-((2-methoxy-6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)amino)pyrimidin-4-yl)amino)methyl)-N-methylcyclobutane-1-carboxamide

[0222]

[0223] Compound 6 can be obtained by replacing 2,2-dimethyl-3-aminopropanoic acid methyl ester 4a and 2,4-dichloro-5-methylpyrimidine 4b in the first step of Example 4 with 1-(aminomethyl)cyclobutane carboxylic acid methyl ester and 2,4,5-trichloropyrimidine, respectively, by using similar methods and reaction procedures. ESI-MS (m / z): 446.2 [M+H] + ; 1H NMR (500 MHz, DMSO-d6) δ 8.30 (s, 1H), 7.98 (s, 1H), 7.77 (s, 1H), 7.74 (d, J = 4.5 Hz, 1H), 7.08 (t, J = 5.3 Hz, 1H), 3.93 (s, 3H), 3.70 (d, J = 5.8 Hz, 2H), 2.89 (s, 3H), 2.55 (d, J = 4.5 Hz, 3H), 2.22 (dd, J = 20.1, 9.2 Hz, 2H), 2.02 - 1.96 (m, 2H), 1.90 - 1.80 (m, 1H), 1.75 - 1.67 (m, 1H).

[0224] Example 7

[0225] 3-((2-((2-methoxy-6-methyl-5,6,7,8-tetrahydro-l,6-naphthyridin-3-yl)amino)-5- methylpyrimidin-4-yl)amino)-2,2-dimethylpropanoic acid

[0226]

[0227] Example 8

[0228] 3-((2-((2-methoxy-6-methyl-5,6,7,8-tetrahydro-l,6-naphthyridin-3-yl)amino)-5- methylpyrimidin-4-yl)amino)-2,2-dimethylpropanamide

[0229]

[0230] Compounds 7 and 8 were prepared from the following steps:

[0231]

[0232] First Step: Compound 4c (410 mg, 1.59 mmol), Int-1 (338 mg, 1.75 mmol), cesium carbonate (1.04 g, 3.18 mmol), Brettphos Pd G3 (144 mg, 0.159 mmol), Brettphos (170 mg, 0.318 mmol) were dissolved in dioxane (20 mL), the reaction was stirred at 110 degree Celsius under nitrogen atmosphere for 16 hours. LCMS detected the starting material was completely converted. The reaction was filtered, the filtrate was concentrated to get the crude product and purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1) to get compound 7a (190 mg, yield 28%). ESI-MS (m / z): 415.3 [M+H] + .

[0233] Second Step: Dissolve compound 7a (15 mg, 0.036 mmol) in tetrahydrofuran (3 mL) and water (3 mL), then add lithium hydroxide (3 mg, 0.072 mmol) to the above solution, stir the reaction mixture at room temperature for 3 hours. LCMS shows the starting material is completely converted. Concentrate the reaction mixture to remove tetrahydrofuran, then adjust the pH of the solution to 3 with 1 N aqueous hydrochloric acid solution, concentrate the aqueous solution to obtain compound 7 (15 mg). ESI-MS (m / z): 401.3 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 8.38 (s, 1H), 7.69 (s, 1H), 7.20 (s, 1H), 6.40 (d, J = 6.3 Hz, 1H), 3.90 (s, 3H), 3.61 (d, J = 6.0 Hz, 3H), 2.74-2.67 (m, 6H), 2.37 (s, 3H), 1.93 (s, 3H), 1.12 (s, 6H).

[0234] Third Step: Dissolve compound 7 (75 mg, 187 umol), ammonium chloride (142 mg, 0.37 mmol), HATU (142 mg, 0.37 mmol), DIPEA (72 mg, 0.56 mmol) in DMF (8 mL), stir the reaction mixture at room temperature overnight. LCMS shows the starting material is completely converted. Concentrate the reaction mixture to obtain crude product and purify it by reverse phase preparative HPLC to obtain compound 8 (10 mg, yield 14%). ESI-MS (m / z): 400.3 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 8.37 (s, 1H), 7.69 (s, 1H), 7.20 (s, 1H), 6.97 (s, 1H), 6.30 (t, J = 6.1 Hz, 1H), 3.90 (s, 3H), 3.53 (d, J = 6.0 Hz, 2H), 3.44 (s, 2H), 2.73 (t, J = 6.0 Hz, 2H), 2.64 (t, J = 6.0 Hz, 2H), 2.34 (s, 3H), 1.92 (s, 3H), 1.12 (s, 6H).

[0235] Example 9

[0236] 1-(((5-chloro-2-((2-methoxy-6-methyl-5,6,7,8-tetrahydro-l,6-naphthyridin-3- yl)amino)pyrimidin-4-yl)amino)methyl)-N-ethylcyclobutane-l-carboxamide

[0237]

[0238] Compound 9 was obtained by a similar method and procedures to Example 4, using 1- (aminomethyl)cyclobutane carboxylic acid methyl ester and 2,4,5-trichloropyrimidine to replace methyl 2,2-dimethyl-3-aminopropanoate 4a and 2,4-dichloro-5-methylpyrimidine 4b in the first step, respectively, and then using ethylamine hydrochloride to replace methylamine hydrochloride in the third step. ESI-MS (m / z): 460.2 [M+H] + ; 1 HNMR (500 MHz, DMSO-d6) δ 8.17 (s, 1H), 7.96 (s, 1H), 7.72 (t, J = 5.5 Hz, 1H), 7.63 (s, 1H), 6.81 (t, J = 5.4 Hz, 1H), 3.88 (s, 3H), 3.71 (d, J = 5.6 Hz, 2H), 3.44 (s, 2H), 3.11 - 2.99 (m, 2H), 2.75 (t, J = 5.9 Hz, 2H), 2.64 (t, J = 5.8 Hz, 2H), 2.34 (s, 3H), 2.23 (dd, J = 18.7, 9.9 Hz, 2H), 1.98 - 1.90 (m, 2H), 1.88 - 1.81 (m, 1H), 1.76 - 1.68 (m, 1H), 0.94 (t, J = 7.2 Hz, 3H).

[0239] Example 10

[0240] 4-((5-chloro-2-((2-methoxy-6-methyl-5,6,7,8-tetrahydro-l,6-naphthyridin-3-yl)amino)pyrimidin-4-

[0241] yl)amino)-3,3-dimethylbutanoic acid

[0242]

[0243] Compound 10 was prepared by the following steps:

[0244]

[0245] First Step: Compound 10a (200 mg, 1.02 mmol) and 2,4,5-trichloropyrimidine (43 mg, 1.33 mmol) 1d were dissolved in isopropanol (8 mL), and DIPEA (264 mg, 2.04 mmol) was added to the above reaction solution, which was stirred at room temperature overnight. LCMS detection showed that the starting material was completely converted. The reaction solution was concentrated to obtain a crude product, which was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 3 / 1) to obtain compound 10b (312 mg, yield 100%) as colorless oil. ESI-MS (m / z): 306.3 [M+H] + .

[0246] Second Step: Dissolve compound 10b (360 mg, 1.18 mmol) and Int-1 (227 mg, 1.18 mmol) in dioxane (10 mL), add cesium carbonate (766 mg, 2.35 mmol), Brettphos Pd G3 (213 mg, 0.23 mmol) and Brettphos (126 mg, 0.23 mmol) successively, stir the reaction mixture at 110 °C under nitrogen atmosphere overnight. LCMS detection shows the starting material is completely converted. Concentrate the reaction mixture to get the crude product and purify it by silica gel column chromatography (dichloromethane / methanol = 10 / 1) to get brown oil 10c (280 mg, yield 51%). ESI-MS (m / z): 463.5 [M+H] + .

[0247] Third Step: Dissolve compound 10c (260 mg, 0.56 mmol) in a mixture of tetrahydrofuran (5 mL) and water (5 mL), add lithium hydroxide (23 mg, 0.56 mmol) to the reaction mixture, stir the reaction mixture at room temperature for four hours. Acidify the reaction mixture to pH = 3 with 1 N aqueous hydrochloric acid solution, then extract it with ethyl acetate. Concentrate the aqueous phase after extraction to get the crude product. Purify the crude product by reverse phase preparative HPLC to get compound 10 (3.59 mg, yield 1.47%). ESI-MS (m / z): 435.2 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) d 8.13 (s, 1H), 7.95 (s, 1H), 7.62 (s, 1H), 7.25 (s, 1H), 3.88 (s, 2H), 3.45 (s, 3H), 3.38 (s, 2H), 2.75 (d, J = 6.0 Hz, 2H), 2.67 (d, J = 6.0 Hz, 2H), 2.35 (s, 3H), 2.15 (s, 2H), 0.96 (s, 6H).

[0248] Example 11

[0249] 1-(((5-chloro-2-((2-methoxy-6-methyl-5,6,7,8-tetrahydro-l,6-naphthyridin-3- yl)amino)pyrimidin-4-yl)amino)methyl)-N-methylcyclopropane-l-carboxamide

[0250]

[0251] Compound 11 can be obtained by using 1-(aminomethyl)cyclopropyl acetate and 2,4,5-trichloropyrimidine to replace 2,2-dimethyl-3-aminopropionic acid methyl ester 4a and 2,4-dichloro-5-methylpyrimidine 4b in the first step of Example 4, respectively, in a similar manner and by using similar reaction procedures. ESI-MS (m / z): 432.2 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 8.03 (s, 1H), 7.96 (s, 1H), 7.81 (s, 1H), 7.69 (d, J = 4.2 Hz, 1H), 7.15 (t, J = 6.0 Hz, 1H), 3.88 (s, 3H), 3.67 (d, J = 5.9 Hz, 2H), 3.38 (s, 2H), 2.75 (t, J = 5.6 Hz, 2H), 2.64 (t, J = 5.7 Hz, 2H), 2.55 (d, J = 4.3 Hz, 3H), 2.34 (s, 3H), 0.93 (dd, J = 6.2, 3.9 Hz, 2H), 0.78 (dd, J = 6.5, 4.0 Hz, 2H).

[0252] Example 12

[0253] 1-(((5-chloro-2-((2-methoxy-6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)amino)pyrimidin-4-yl)amino)methyl)-N-methylcyclopentane-1-carboxamide

[0254] -4-yl)amino)methyl)-N-methylcyclopentane-1-carboxamide

[0255]

[0256] Compound 12 can be obtained by using Boc-1-aminomethylcyclopentane carboxylic acid to replace Boc-3-amino-2,2-dimethyl-propionic acid 1a in the first step of Example 1, in a similar manner and by using similar reaction procedures. ESI-MS (m / z): 460.2 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 8.09 (s, 1H), 7.96 (s, 1H), 7.68 (s, 1H), 7.66 (d, J = 4.7 Hz, 1H), 6.83 (t, J = 5.6 Hz, 1H), 3.88 (s, 3H), 3.52 (d, J = 5.8 Hz, 2H), 3.42 (s, 2H), 2.75 (t, J = 5.9 Hz, 2H), 2.64 (t, J = 5.7 Hz, 2H), 2.57 (d, J = 4.4 Hz, 3H), 2.34 (s, 3H), 1.86 (dt, J = 7.7, 5.5 Hz, 2H), 1.61 - 1.55 (m, 6H).

[0257] Example 13

[0258] 1-(((5-chloro-2-((2-methoxy-6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)amino)pyrimidin-4-yl)amino)methyl)cyclobutane-1-carboxylic acid

[0259] -4-yl)amino)methyl)cyclobutane-1-carboxylic acid

[0260]

[0261] Compound 13 was obtained by using 4-(aminomethyl)cyclobutane carboxylic acid methyl ester hydrochloride instead of ethyl 3,3-dimethyl-4-aminobutanoate hydrochloride 10a in the first step of Example 10, in a similar manner and reaction procedure. ESI-MS (m / z): 433.3 [M+H] + ; 1 H NMR (500 MHz, DMSO-d6) δ 8.15 (s, 1H), 7.97 (s, 1H), 7.63 (s, 1H), 7.01 (t, J = 6.1 Hz, 1H), 3.88 (s, 3H), 3.75 (d, J = 6.0 Hz, 2H), 3.44 (s, 2H), 2.75 (t, J = 5.8 Hz, 2H), 2.66 (t, J = 5.8 Hz, 2H), 2.35 (s, 3H), 2.28-2.21 (m, 2H), 2.06-1.99 (m, 2H), 1.84-1.76 (m, 2H).

[0262] The following compounds of the examples were prepared according to the synthetic routes and methods of synthesis of intermediates described above.

[0263]

[0264]

[0265]

[0266] Biological screening of HPK1 inhibitors and results

[0267] Test Example 1: Detection of the ability of compounds to inhibit HPK1 kinase activity (Method 1)

[0268] The reagents used are as follows

[0269]

[0270] Experimental procedure

[0271] The specific operation is as follows: the enzyme reaction system buffer (10 mM MOPS, pH 7.2, 5 mM β-glycerol-phosphate, 10 mM MgCl2, 0.8 mM EDTA, 2 mM EGTA, 0.1 mM DTT) is configured; the tested compound (1 mM compound stock solution configured in DMSO) is diluted with the buffer to a maximum concentration of 60 uM (containing 6% DMSO), and the compound is diluted 5 times with the buffer containing 6% DMSO to obtain 8 gradient concentrations; then the HPK1 kinase is diluted to 30 nM using the buffer. 2 ul of the HPK1 kinase diluent is added to each well of a Greiner 384-well microplate (item number: 784075), and 2 ul of the buffer is added to the control wells; after centrifugation, 1 ul of the diluted compound is added to the reaction wells, and 1 ul of the buffer containing 6% DMSO is added to the control wells; after centrifugation, incubate in a 25℃ constant temperature incubator (Shanghai Yiheng Scientific Instrument Co., Ltd., item number: LRH-150) for 20 min. 3 ul of the reaction substrate (10 uM MBP and 20 uM ATP dissolved in distilled water) is added to each well, and after centrifugation, incubate in a 25℃ constant temperature incubator for 60 min. The ADP-Glo Kinase Assay Kit is used to detect the enzyme activity, and the ADP-Glo Kinase Assay Kit detection is performed according to the operation instruction of the kit. The data is described by the half inhibitory concentration IC50 of the compound.

[0272]

[0273] The above results show that the compound of the present application has excellent inhibitory ability on HPK1 kinase activity.

[0274] Test Example 2: Detection of the agonistic ability of the compound on the secretion of the cytokine interleukin-2 (IL-2) of Jurkat cells and the influence of the compound on the activity of Jurkat cells (Method 2)

[0275] The required reagents and cells are as follows:

[0276] Experimental reagents:

[0277]

[0278] Experimental cells:

[0279] Cells Cell type Brand Jurkat E6-1 Human T lymphocytic leukemia cells Institute of Cell Biology, Chinese Academy of Sciences

[0280] Experimental steps

[0281] The specific operation is as follows: compound powder is dissolved in DMSO to 10 mM, 2 μl of compound is added to 998 μl of RPMI 1640 medium (10% FBS is contained in this test), and after vortex mixing, the highest concentration point is obtained. The compound solution is gradually diluted with 0.2% DMSO medium by 3 times, and a total of 8 concentration points are obtained. The RPMI 1640 medium solution containing 0.1% DMSO is treated as a control. 1×105 Jurkat E6-1 cells are added to each well of a Corning 96-well cell culture plate (item number: 3599), and then an equal volume of compound diluent is added, and the control group is added with 0.2% DMSO in RPMI 1640 medium, and then placed in a 37°C cell incubator (Thermo Fisher Scientific, model number: 3111) for 1 h. Then 1 μg / ml Anti-human CD3 Antibody and 1 μg / ml Anti-human CD28 Antibody are added to a final concentration, and then placed in a 37°C cell incubator for 24 h. The culture supernatant is collected, and the IL-2 content in the cell supernatant is detected by Human IL-2 DuoSet ELISA KIT. The Human IL-2 DuoSet ELISA detection is carried out according to the operating instructions of the kit. The IL-2 secretion data is described by the highest multiple ratio of the stimulation signal of the compound to the signal of 0.1% DMSO; the cells are collected, and the cell viability is detected by using the Luminescent Cell Viability Assay kit. The cell viability data is described by the half-inhibitory concentration IC50 of the compound. Luminescent Cell Viability Assay kit, and the cell viability data is described by the half-inhibitory concentration IC50 of the compound.

[0282]

[0283] NA: indicates that the release of IL-2 is not detected.

[0284] The above results show that, compared with the control, the compound of the present application significantly increases the level of the cytokine interleukin-2 secreted by Jurkat cells, and has no adverse effect on the cell viability of Jurkat.

[0285] Test Example 3: Detection of the agonistic ability of the compound on the secretion of the cytokine interleukin-2 (IL-2) by human PBMC cells

[0286] The reagents required for use are as follows

[0287]

[0288] Information on the source of experimental cells:

[0289]

[0290] Experimental steps

[0291] The specific procedures are as follows: Human PBMCs were removed from liquid nitrogen according to standard procedures and thawed in a 37°C water bath. The cells were resuspended in RPMI 1640 medium (containing 10% FBS in this experiment), and washed twice by centrifugation. The human PBMCs were then resuspended in RPMI 1640 medium for later use. The compound powder was dissolved in DMSO to a concentration of 10 mM. 2 μl of the compound was added to 998 μl of RPMI 1640 medium and vortexed to obtain the highest concentration. The compound solution was gradually diluted 3-fold with 0.2% DMSO medium, resulting in 8 concentration points. A RPMI 1640 medium solution containing 0.1% DMSO was used as a control. One × 10⁵ human PBMC cells were added to each well of a Corning 96-well cell culture plate (catalog number: 3599), followed by an equal volume of compound dilution buffer. The control group was added to RPMI 1640 medium containing 0.2% DMSO. The plates were incubated at 37°C in a Thermo Fisher Scientific (model: 3111) for 1 hour. Antibody at a final concentration of 0.01 μg / ml and antibody at 1 μg / ml for 24 hours were then added, and the plates were incubated at 37°C. The IL-2 content in the cell supernatant was detected using the Human IL-2 DuoSet ELISA kit, performed according to the kit's instructions. Data are described as the fold-over ratio of the compound's stimulus signal to the signal from 0.1% DMSO. Cells were collected and used... The Luminescent CellViability Assay kit was used to detect cell viability, and cell viability data were described using the half-maximal inhibitory concentration (IC50) of the compound.

[0292]

[0293]

[0294] NA: Indicates that no enhanced IL-2 release was detected.

[0295] The above results indicate that, compared with the control, the compound of the present invention significantly increased the level of the cytokine interleukin-2 secreted by PBMC cells, without adversely affecting the cell viability of PBMCs.

Claims

1. Compounds having the structure of Formula I or pharmaceutically acceptable salts: in R1 represents hydrogen, (C1-C6) alkyl, or (C3-C8) cycloalkyl; R2 indicates (C1-C6) alkyl or halogenated (C1-C6) haloalkyl; R3 represents OCH3; A represents B represents -(C0-C6)alkylene-, -O-(C1-C6)alkyl-, -S-(C1-C6)alkyl-, or -S(O)-(C1-C6)alkyl-; R4 and R4' each independently represent hydrogen; R5 represents CH3; R6 and R 6’ Each represents hydrogen independently; X1 represents N; R M and R N Each can independently represent hydrogen, (C1-C6) alkyl, or R M and R N Together with the attached carbon atom ring, they form 3-6 membered rings, and R M and R N They are not both hydrogen; in, R a Indicates hydrogen or (C1-C6) alkyl; m represents 1 and n represents 2.

2. A compound having the following structure:

3. A pharmaceutical composition comprising the compound of claim 1 or 2 or a pharmaceutically acceptable salt and a pharmaceutically usable carrier.

4. Use of the compound or pharmaceutically acceptable salt of claim 1 or 2, or the pharmaceutical composition of claim 3, in the preparation of a medicament for the prevention and / or treatment of leukemia.

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