3-Aminoindazole compounds and their applications

Abstract

The present invention provides a 3-aminoindazole compound of the structure shown in formula (Ⅰ) or a pharmaceutically acceptable salt thereof or a stereoisomer thereof or a prodrug molecule thereof, and its application. The compound and the pharmaceutically acceptable salt thereof related to the present invention can inhibit FGFR4 protease with high activity and high selectivity, can effectively inhibit the growth of various tumor cells, especially have a highly selective inhibitory effect on liver cancer cells and breast cancer cells, can be used to prepare anti-tumor drugs, and can be used to treat transitional proliferative diseases such as tumors in humans and other mammals.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical medicine, and in particular to a class of 3-aminoindazole compounds and applications thereof. Background Art

[0002] Fibroblast growth factor receptors (FGFRs) belong to the receptor tyrosine kinase family (RTKs), which contains four receptor subtypes: FGFR1, FGFR2, FGFR3 and FGFR4. The structure of FGFRs mainly consists of three parts: an extracellular region composed of three immunoglobulin-like domains, a single transmembrane region and an intracellular region containing a kinase domain. The activation mechanism of FGFR4 is the same as that of other receptor tyrosine kinases. The ligand binds tightly to the complex formed by the receptor and co-receptor, induces receptor dimerization, phosphorylates tyrosine residues in the intracellular region of the receptor, and activates receptor kinase activity. Activated FGFR4 can phosphorylate downstream target proteins to cause signal cascade reactions. Among them, the FGFR4 subtype is mainly distributed in the liver, lung, lymph and breast tissues. FGFR4 and its specific ligand fibroblast factor FGF19 play important physiological functions in the liver, lung and breast tissues. Studies have shown that FGF19 / FGFR4 is upregulated in 30-50% of liver cancers, and this upregulation is closely related to the poor prognosis of liver cancer, making it an effective new target for the treatment of liver cancer and other related tumors.

[0003] FGFR4 has become a hot target in anti-tumor research today, attracting the attention and R&D boom of many scientific research institutions and pharmaceutical companies. At present, several selective FGFR4 inhibitors have been reported. In 2015, Hagel et al. developed the first selective inhibitor BLU554 based on the difference between the key cysteine ​​at position 552 of FGFR4 and other members and entered clinical research (NCT02508467), showing good therapeutic effects in patients with liver cancer and cholangiocarcinoma. The FGFR4 selective inhibitor H3B-6527 developed by H3 Biomedicine has also entered clinical phase I (NCT03424577). Novartis has developed a class of reversible covalent inhibitors of selective FGFR4, among which the representative compound FGF401 has entered clinical phase I / II (NCT02325739) for the treatment of liver cancer with overexpression of FGFR4. Although many companies at home and abroad have also studied FGFR4 selective inhibitors, no FGFR4 selective inhibitors have been approved for marketing, and more effective FGFR4 selective inhibitors are urgently needed in clinical practice. In addition, due to the mutation of gatekeeping amino acids in the FGFR4 protein kinase region, existing clinical drugs have target-dependent drug resistance problems, and therefore there is an urgent need to study new FGFR4 inhibitors. Summary of the invention

[0004] In view of the above problems, the present invention provides a new class of 3-aminoindazole compounds, which can inhibit the activity of FGFR4 protein kinase with high selectivity and high activity, thereby inhibiting the proliferation of various tumor cells.

[0005] The specific technical solutions are as follows:

[0006] A 3-aminoindazole compound having a structure represented by formula (I) or a pharmaceutically acceptable salt thereof or a stereoisomer thereof or a prodrug molecule thereof:

[0007]

[0008] Wherein, X is selected from: CH, N;

[0009] Z is selected from: -O-, -N(H)-, -S-, -S(O)-, -S(O)2-;

[0010] R1, R2, R4 are independently selected from: H, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, halogen, halogen-substituted C1-C6 alkyl, nitro, cyano, hydroxyl, amino;

[0011] R3 is selected from: H, -NR 10 R 11 ;

[0012] R5 is selected from the group consisting of: H, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, halogen, halogen-substituted C1-C6 alkyl, nitro, cyano, hydroxyl, and amino;

[0013] R6, R7, R8, R9 are independently selected from: H, halogen;

[0014] R 10 , R 11 are independently selected from: H, C1-C 20 Alkyl, C3-C 20 Cycloalkyl, C1-C 20 Alkoxy substituted C1-C 20 Alkyl, C1-C 20 Alkylamino-substituted C1-C 20 Alkyl, or R 10 , R 11 Together with the nitrogen atom to which it is attached, it forms a substituted or unsubstituted heterocyclic group.

[0015] In some embodiments, the 3-aminoindazole compound has a structure shown in formula (II):

[0016]

[0017] In some embodiments, the 3-aminoindazole compound has a structure shown in formula (III):

[0018]

[0019] In some embodiments, R3 is selected from: H, -NR 10 R 11 ;

[0020] R 10 , R 11 are independently selected from: H, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkyl substituted with C1-C6 alkoxy, C1-C6 alkyl substituted with C1-C6 alkylamino, or, R 10 , R 11 Together with the nitrogen atom to which it is attached, it forms a substituted or unsubstituted 5-12 membered heterocyclic group.

[0021] In some embodiments, R3 is selected from: H, -NR 10 R 11 ;

[0022] R 10 , R 11 are independently selected from: H, C1-C3 alkyl, C1-C3 alkyl substituted with C1-C3 alkoxy, C1-C3 alkyl substituted with C1-C3 alkylamino, or, R 10 , R 11 Together with the nitrogen atom to which it is attached, it forms one or more R 12 a substituted or unsubstituted 5-6 membered heterocyclic group;

[0023] Each R 12 are independently selected from: H, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, halogen, halogen-substituted C1-C6 alkyl, nitro, cyano, hydroxyl, amino, C1-C6 alkylacyl, C1-C6 alkylsulfonyl, C1-C6 alkylamino, C3-C6 heterocyclyl, one or more R 13 Substituted C3-C6 heterocyclic group;

[0024] Each R 13 Each of them is independently selected from the group consisting of: H, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, halogen, halogen-substituted C1-C6 alkyl, nitro, cyano, hydroxyl, amino, C1-C6 alkylacyl, C1-C6 alkylsulfonyl, and C1-C6 alkylamino.

[0025] In some embodiments, R3 is selected from: H, -NR 10 R 11 ;

[0026] R 10 , R 11 are independently selected from: C1-C3 alkyl, methoxy-substituted C1-C3 alkyl, dimethylamino-substituted C1-C3 alkyl, or, R 10 , R 11 Together with the nitrogen atom to which it is attached, it forms one or more R 12 a substituted or unsubstituted 5-6 membered heterocyclic group;

[0027] Each R 12 Each of the following is independently selected from: H, C1-C3 alkyl, acetyl, methylsulfonyl, dimethylamino, oxetanyl, piperazinyl, C1-C3 alkyl-substituted piperazinyl, morpholinyl, piperidinyl, and tetrahydropyrrolyl.

[0028] In some embodiments, R3 is selected from: H, or any of the following groups:

[0029]

[0030] In some embodiments, R1, R2, and R4 are independently selected from: H, C1-C3 alkyl, C1-C3 alkoxy, cyclopropyl, and halogen.

[0031] In some embodiments, R1 is selected from: H, C1-C3 alkyl, C1-C3 alkoxy, cyclopropyl, halogen;

[0032] R2 is selected from: H, C1-C3 alkyl, C1-C3 alkoxy;

[0033] R4 is selected from: H.

[0034] In some embodiments, R1 is selected from: H, methyl, halogen; R2 is selected from: H; R4 is selected from: H.

[0035] In some embodiments, R5 is selected from: H, methyl, ethyl.

[0036] In some embodiments, the 3-aminoindazole compound has a structure shown in formula (IV):

[0037]

[0038] The present invention also provides the use of the above-mentioned 3-aminoindazole compound or its pharmaceutically acceptable salt or its stereoisomer or its prodrug molecule.

[0039] The specific technical solutions are as follows:

[0040] Use of the above-mentioned 3-aminoindazole compounds or pharmaceutically acceptable salts thereof or stereoisomers thereof or prodrug molecules thereof in the preparation of FGFR4 inhibitors.

[0041] Use of the above-mentioned 3-aminoindazole compounds or pharmaceutically acceptable salts thereof or stereoisomers thereof or prodrug molecules thereof in the preparation of drugs for preventing and / or treating transitional proliferative diseases caused by abnormal FGFR4 signaling pathway.

[0042] Use of the above-mentioned 3-aminoindazole compounds or pharmaceutically acceptable salts thereof or stereoisomers thereof or prodrug molecules thereof in the preparation of drugs for preventing and / or treating tumors.

[0043] In some embodiments, the tumor is: liver cancer, bile duct cancer, breast cancer, gastric cancer, prostate cancer.

[0044] The present invention also provides a pharmaceutical composition for preventing and / or treating tumors.

[0045] The specific technical solutions are as follows:

[0046] A pharmaceutical composition for preventing and / or treating tumors is prepared from active ingredients and pharmaceutically acceptable excipients, wherein the active ingredients include the above-mentioned 3-aminoindazole compounds or pharmaceutically acceptable salts or stereoisomers or prodrug molecules thereof.

[0047] The 3-aminoindazole compounds of the present invention or their pharmaceutically acceptable salts or stereoisomers or prodrug molecules can inhibit FGFR4 protease with high activity and high selectivity, can effectively inhibit the growth of various tumor cells, especially have a highly selective inhibitory effect on liver cancer cells and breast cancer cells, and have good pharmacokinetic properties, can be used to prepare anti-tumor drugs, and can be used to treat transitional proliferative diseases such as tumors in humans and other mammals. DETAILED DESCRIPTION

[0048] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0049] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0050] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0051] In the compounds described in the present invention, when any variable (such as R 12 If a (, etc.) compound occurs more than once in any component, its definition at each occurrence is independent of its definition at every other occurrence. Likewise, combinations of substituents and variables are permitted so long as such combinations result in stable compounds. A line drawn from a substituent into the ring system indicates that the indicated bond may be attached to any substitutable ring atom. If the ring system is polycyclic, this means that such bonds are attached only to any suitable carbon atom of an adjacent ring. It is to be understood that one of ordinary skill in the art can select substituents and substitution patterns for the compounds of the invention to provide compounds that are chemically stable and that can be readily synthesized from readily available raw materials by techniques in the art and the methods set forth below. If a substituent is itself substituted with more than one group, it is to be understood that these groups may be on the same carbon atom or on different carbon atoms so long as the structure is stable.

[0052] As used herein, the term "alkyl" is intended to include branched and straight chain saturated aliphatic hydrocarbon groups having a specific number of carbon atoms. For example, the definition of "C1-C6" in "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5 or 6 carbon atoms in a straight or branched arrangement. For example, "C1-C6 alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl.

[0053] The term "cycloalkyl" refers to a monocyclic saturated aliphatic hydrocarbon group having a specific number of carbon atoms. For example, "cycloalkyl" includes cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, etc.

[0054] The term "alkoxy" refers to a group having an -O-alkyl structure, such as -OCH3, -OCH2CH3, -OCH2CH2CH3, -O-CH2CH(CH3)2, -OCH2CH2CH2CH3, -O-CH(CH3)2, and the like.

[0055] The term "alkylamino" refers to a group having an -N-(alkyl)2 or -NH-alkyl structure, such as -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -NHCH2CH(CH3)2, -NHCH2CH2CH2CH3, -NHCH(CH3)2, -N(CH3)2, -N(CH2CH)2, -N(CH2CH)2, -N(CH2CH2CH3)2, etc.

[0056] The term "heterocycloalkyl" or "heterocyclyl" is a saturated or partially unsaturated monocyclic, fused, spiro or bridged cyclic substituent, wherein one or more ring atoms are selected from N, O or S(O)m (wherein m is an integer from 0 to 2) heteroatoms, and the remaining ring atoms are carbon, for example: morpholinyl, piperidinyl, tetrahydropyrrolyl, oxetanyl, piperazinyl, pyrrolidinyl, dihydroimidazolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothiophenyl, dihydrotriazolyl, dihydroazetidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, and the like, and N-oxides thereof. The attachment of the heterocyclic substituent may be achieved through carbon atoms or through heteroatoms.

[0057] As will be appreciated by those skilled in the art, "halo" or "halo" as used herein refers to chlorine, fluorine, bromine and iodine.

[0058] Unless otherwise defined, alkyl, cycloalkyl, aryl, heteroaryl and heterocycloalkyl substituents may be unsubstituted or substituted. For example, C1-C6 alkyl may be substituted with one, two or three substituents selected from OH, halogen, alkoxy, dialkylamino or heterocyclic radicals such as morpholinyl, piperidinyl, etc.

[0059] The present invention includes free forms of compounds of formula I-IV, as well as pharmaceutically acceptable salts and stereoisomers thereof. The term "free form" refers to amine compounds in non-salt form. The pharmaceutically acceptable salts included include not only the exemplary salts of the specific compounds described herein, but also typical pharmaceutically acceptable salts of the free forms of all compounds of formula I-IV. The free form of the specific salt of the compound can be isolated using techniques known in the art. For example, the free form can be regenerated by treating the salt with an appropriate dilute aqueous base solution, such as a dilute aqueous solution of NaOH, a dilute aqueous solution of potassium carbonate, a dilute aqueous ammonia, and a dilute aqueous solution of sodium bicarbonate. The free form is somewhat different from its respective salt form in certain physical properties, such as solubility in polar solvents, but for the purpose of the invention, such acid salts and base salts are equivalent to their respective free forms in other pharmaceutical aspects.

[0060] Pharmaceutically acceptable salts of the present invention can be synthesized from compounds of the present invention containing a basic or acidic moiety by conventional chemical methods. Typically, salts of basic compounds are prepared by ion exchange chromatography or by reacting a free base with a stoichiometric amount or excess of an inorganic or organic acid in the desired salt form in an appropriate solvent or combination of solvents. Similarly, salts of acidic compounds are formed by reacting with an appropriate inorganic or organic base.

[0061] Therefore, the pharmaceutically acceptable salts of the compounds of the present invention include conventional non-toxic salts of the compounds of the present invention formed by reacting the alkaline compounds of the present invention with inorganic or organic acids. For example, conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, nitric acid, etc., and also include salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, 2-acetoxy-benzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, trifluoroacetic acid, etc.

[0062] If the compound of the present invention is acidic, then suitable "pharmaceutically acceptable salts" refer to salts prepared by pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganic salts, manganous salts, potassium salts, sodium salts, zinc salts, etc. Ammonium salts, calcium salts, magnesium salts, potassium salts and sodium salts are particularly preferred. Salts derived from pharmaceutically acceptable organic non-toxic bases, including salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydroxocobalamin, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, guaiac, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.

[0063] Berg et al., "Pharmaceutical Salts," J. Pharm. Sci. '1977: 66: 1-19, describes in more detail the preparation of the above-mentioned pharmaceutically acceptable salts and other typical pharmaceutically acceptable salts.

[0064] Since deprotonated acidic moieties such as carboxyl groups in the compounds can be anionic under physiological conditions, and this charge can then be balanced by the internal protonated or alkylated basic moieties such as quaternary nitrogen atoms that carry cations, it should be noted that the compounds of the present invention are potential internal salts or zwitterions.

[0065] In some embodiments, the present invention provides a method for treating hyperproliferative diseases or symptoms such as tumors in humans or other mammals using compounds having formula I-IV and pharmaceutically acceptable salts thereof.

[0066] In some embodiments, the compounds of the present invention and pharmaceutically acceptable salts thereof can be used to treat or control hyperproliferative diseases such as liver cancer, bile duct cancer, breast cancer, gastric cancer, and prostate cancer.

[0067] Drug metabolites and prodrugs

[0068] The metabolites of the compounds involved in the present invention and their pharmaceutically acceptable salts, as well as prodrugs that can be converted into the structures of the compounds involved in the present invention and their pharmaceutically acceptable salts in vivo, are also included in the claims of the present invention.

[0069] Combination medication

[0070] The compounds of formula I-IV can be used in combination with other drugs known to treat or improve similar conditions. When administered in combination, the administration method and dosage of the original drug remain unchanged, and the compounds of formula I-IV are taken simultaneously or subsequently. When the compounds of formula I-IV are taken simultaneously with one or more other drugs, it is preferred to use a pharmaceutical composition containing one or more known drugs and the compounds of formula I-IV. Drug combination also includes taking the compounds of formula I-IV and one or more other known drugs in overlapping time periods. When the compounds of formula I-IV are used in combination with one or more other drugs, the dosage of the compounds of formula I-IV or the known drugs may be lower than when they are used alone.

[0071] Drugs or active ingredients that can be used in combination with the compounds of formula I-IV include but are not limited to:

[0072] Estrogen receptor modulators, androgen receptor modulators, retinal receptor modulators, cytotoxins / cytostatics, antiproliferative agents, protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protein kinase inhibitors, reverse transcriptase inhibitors, angiogenesis inhibitors, cell proliferation and survival signal inhibitors, drugs that interfere with cell cycle checkpoints and apoptosis inducers, cytotoxic drugs, tyrosine protein inhibitors, EGFR inhibitors, VEGFR inhibitors, serine / threonine protein inhibitors, Bcr-Abl inhibitors, c-Kit inhibitors, Met inhibitors, Raf inhibitors, MEK inhibitors, MMP inhibitors, topoisomerase inhibitors, histidine deacetylase inhibitors, proteasome inhibitors, CDK inhibitors, Bcl-2 family protein inhibitors, MDM2 family protein inhibitors, IAP family protein inhibitors, STAT family protein inhibitors, PI3K inhibitors, AKT inhibitors, integrin blockers, interferon-α, interleukin-12, COX-2 inhibitors, p53, p53 activators, VEGF antibodies, EGF antibodies, etc.

[0073] In some embodiments, the drugs or active ingredients that can be used in combination with the compounds of Formula I-IV include but are not limited to: aldesleukin, alendronic acid, interferon, atrenoin, allopurinol, allopurinol sodium, palonosetron hydrochloride, hexamethylmelamine, aminoglutethimide, amifostine, amrubicin, anacrine, anastrozole, dolasetron, aranesp, arglabin, arsenic trioxide, arnosin, 5-azacytidine, azathioprine, BCG or tice BCG, betadine, betamethasone acetate, betamethasone sodium phosphate preparation, bexarotene, bleomycin sulfate, bromourea, bortezomib, busulfan, Calcitonin, alezosumab injection, capecitabine, carboplatin, casodex, cefesone, simulleukin, daunorubicin, chlorambucil, cisplatin, cladribine, cladribine, clodronate, cyclophosphamide, cytarabine, dacarbazine, actinomycin D, daunorubicin liposome, dexamethasone, dexamethasone phosphate, estradiol valerate, denileukin 2, depomet, deslorelin, delazosen, diethylstilbestrol, diflucan, doxorubicin, doxorubicin, chin-166-chitosan complex, eligard, rasburicase, epirubicin hydrochloride, aprepitant, epirubicin, epoetin alfa, erythropoietin, epoetin, levamisole tablets, estradiol preparations, 17-β-estradiol, estramustine sodium phosphate, ethinyl estradiol, amifostine, hydroxyphosphonic acid, fenbifu, etoposide, fadrozole, tamoxifen preparations, filgrastim, finasteride, firapisti, floxuridine, fluconazole, fludarabine, 5-fluorodeoxyuridine nucleoside monophosphate, 5-fluorouracil, fluoxymesterone, flutamide, formestane, 1-β-D-arabinofuranosylcytosine-5'-stearoyl phosphate, fotemustine, fulvestrant, gamma globulin, gemcitabine, gemtuzumab, imatinib mesylate, carmustine rice paper capsules, goserelin, granisone hydrochloride, histrelin, and mexin, hydrocortisone, erythro-hydroxynonyl adenine, hydroxyurea, tentan isobetumomab, Idarubicin, ifosfamide, interferon α, interferon-α2, interferon α-2A, interferon α-2B, interferon α-n1, interferon α-n3, interferon β, interferon γ-la, interleukin-2, intron A, Iressa, irinotecan, Kateri, Lentinan sulfate, letrozole, leucovorin, leuprorelin, leuprorelin acetate, levomisole, levofolate calcium salt, levothyroxine sodium, levothyroxine sodium preparations, lomustine, lonidamine, nitrogen mustard, methylcobalamin, medroxyprogesterone acetate, megestrol acetate, melphalan, esterified estrogen, 6-mercaptopurine, mesna, methotrexate, methyl aminolevulinate, miltefosine, minocycline, mitomycin C, mitotane, mitoxantrone, trilostane, citric acidDoxorubicin liposome, nedaplatin, pegfilgrastim, oprelvekin, neupogen, nilutamide, tamoxifen, NSC-631570, recombinant human interleukin 1-β, octreotide, ondansetron hydrochloride, dehydrocortisone oral solution, oxaliplatin, paclitaxel, prednisone sodium phosphate preparation, pegaspargase, PEGASYS, pentostatin, lysin, pilocarpine hydrochloride, pirarubicin, plicamycin, porfimer sodium, prednimustine, prednisolone, prednisone, premarin, procarbazine, recombinant human erythropoietin, raltitrexed, ribavirin, etidronate rhenium-186, rituximab, Lidushen-A, romotide, pilocarpine hydrochloride tablets, octreotide, sarmustine, semustine, sizoran, sobuzosine, methylprednisolone sodium, pafosic acid, stem cell therapy, streptozocin, strontium-89 chloride, levothyroxine sodium, tamoxifen, tansulosin, tasonamin, tastolactone, taxotere, tesithionine, temozolomide, teniposide, testosterone propionate, methyltestosterone, thioguanine, thiotepa, pro- Thyroid hormone, tiludronate, topotecan, toremifene, tositumomab, trastuzumab, treosulfan, tretinoin, methotrexate tablets, trimethylmelamine, trimetrexate, triptorelin acetate, triptorelin pamoate, eufodine, uridine, valrubicin, vesellarinone, vinblastine, vincristine, vinblastine, vinorelbine, virulizine, dextrorotatory propionate, statins ester, zosin, paclitaxel protein-stabilized preparations, acolbifene, interferon r-lb, affinitak, aminopterin, arzoxifene, asoprisnil, atamestane, atrasentan, BAY 43-9006, Avastin, CCI-779, CDC-501, Celebrex, cetuximab, clenatol, cyproterone acetate, decitabine, DN-101, doxorubicin-MTC, dSLIM, dutasteride, edotecarin, eflornithine, exitecan, fenretinide, histamine dihydrochloride, histrelin hydrogel implant, holmium-166 DOTMP, ibandronic acid, interferon gamma, intron-PEG, ixabepilone, keyhole limpet hemocyanin, L-651582, lanreotide, lasofoxifene, libra, lonafamib, miproxifene, minocycline, MS-209, liposome MTP-PE, MX-6, nafarelin, nemorubicin, nevastatin, noratript, oblimersen, onco-TCS, osidem, paclitaxel polyglutamate, sodium bromoglutamate, PN-401, OS-21, quasi-amylase, R-154, raloxifene, ranpirnase, 13-cis retinoic acid, satraplatin, ciocalcitol, T-138067, tarceva, paclitaxel docosahexaenoic acid, thymosin αl, gazofurin, tipifamib, tirapazamine, TLK-286,Toremifene, trans-MID-lo7R, valspodar, vapreotide, vatalanib, verteporfin, vinflunine, Z-100 and zoledronic acid or a combination thereof.

[0074] The present invention is further described in the following examples, but the examples are not intended to limit the scope of protection of the present invention.

[0075] Example 1 Preparation of (R)-N-(2-((5-(1-(3,5-dichloropyridin-4-yl)ethoxy)1H-indazol-3-yl)amino)phenyl)acrylamide (Compound 1)

[0076]

[0077] Step 1 Preparation of Compound 1A

[0078]

[0079] Under ice bath, tert-butyldimethylsilyl chloride (42.2 g, 1.5 eq) and imidazole (19 g, 1.5 eq) were added to the N, N-dimethylformamide solution of 1H-indazole-5-ol (25 g, 1 eq), and then returned to room temperature for 6 hours. After the reaction was completed, water was added for dilution, ethyl acetate was extracted three times, the organic phases were combined and dried over anhydrous sodium sulfate, filtered and evaporated to obtain a crude product. The crude product was purified by flash chromatography on a silica gel column to obtain the title compound 1A (34.23 g, 74% yield).

[0080] 1 H NMR (400MHz, DMSO-d6) δ12.90(s,1H),7.92(s,1H),7.42(d,J=8.8Hz,1H),7.12(s,1H),6.92(d,J=8.8Hz,1H),0.97(s,9H),0.18(s,6H).MS(EI)m / z 248.8M + .

[0081] Step 2 Preparation of Compound 1B

[0082]

[0083] Under ice bath, N-iodosuccinimide (NIS, 34.1 g, 1.1 eq) was added to a dichloromethane solution of compound 1A (34.23 g, 1 eq), and then returned to room temperature for 4 hours. After the reaction was completed, saturated sodium thiosulfate aqueous solution was used for quenching, dichloromethane was extracted, and the combined organic phases were dried over anhydrous sodium sulfate, filtered and evaporated to obtain a crude product. The crude product was purified by flash chromatography on a silica gel column to obtain the title compound 1B (45.5 g, 88.2% yield).

[0084] 1 H NMR (400MHz, CDCl3) δ11.01(s,1H),7.43(d,J=8.9Hz,1H),7.06(dd,J=8.9,2.2Hz, 1H),6.88(d,J=2.0Hz,1H),1.04(s,9H),0.25(s,6H).MS(EI)m / z 374.9M + .

[0085] Step 3 Preparation of Compound 1C

[0086]

[0087] Under ice bath, p-toluenesulfonic acid (2.038 g, 0.2 eq) was added to a dichloromethane solution of compound 1B (45.5 g, 1 eq), 3,4-dihydropyran (16 ml, 3 eq) was added in batches, and the mixture was returned to room temperature for 7 hours. After the reaction, the reaction solution was washed with saturated sodium bicarbonate and extracted with dichloromethane to obtain a crude product. The crude product was purified by flash chromatography on a silica gel column to obtain the title compound 1C (40 g, 71.7% yield).

[0088] 1 H NMR (400MHz, CDCl3) δ7.44(d,J=8.9Hz,1H),7.04(d,J=8.9Hz,1H),6.84(s,1H), 5.68-5.62(d,J=9.3Hz,1H),4.10-4.00(m,1H),3.77-3.69(t,J=9.6Hz,1H),2.63–2.42(m,1H), 2.22-2.10(m,1H),2.10-2.02(m,1H),1.80-1.72(m,2H),1.67-1.60(m,1H),1.04(s,9H),0.24(s,6H).MS(EI)m / z 459.8[M+H] + .

[0089] Step 4 Preparation of Compound 1D

[0090]

[0091] At room temperature, tetrabutylammonium fluoride in tetrahydrofuran solution (87 ml, 1 M, 1 eq) was added to a tetrahydrofuran solution of compound 1C (40 g, 1 eq) and reacted for 6 h. After the reaction, the reaction solution was washed with water, extracted with ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate, filtered and evaporated to obtain the title compound 1D (25 g, 83% yield).

[0092] 1 H NMR (400MHz, CDCl3) δ7.47(d,J=8.9Hz,1H),7.06(d,J=8.8Hz,1H),6.82(s,1H), 5.66(d,J=9.3Hz,1H),5.19(s,1H),4.04(d,J=11.1Hz,1H),3.74(t,J=10.1Hz,1H),2.63– 2.42(m,1H),2.22-2.11(d,J=3.0Hz,1H),2.11-2.03(m,1H),1.80-1.72(m,2H),1.71-1.65(m, 1H).MS(EI)m / z 367.6[M+Na] + .

[0093] Step 5 Preparation of Compound 1E

[0094]

[0095] Under ice bath, triethylamine (11 mL, 1.5 eq) was added to a dichloromethane solution of (S)-1-(3,5-dichloropyridine-4-substituted)ethanol (10 g, 1 eq), and methanesulfonyl chloride (MsCl, 4.4 mL, 1.1 eq) was slowly added dropwise. After 5 minutes, the reaction solution was washed with saturated sodium bicarbonate solution, extracted with dichloromethane and dried to give the title compound 1E (14 g, 99.5% yield).

[0096] 1 H NMR (400MHz, CDCl3) δ8.51(s,2H),6.36(q,J=6.9Hz,1H),2.98(s,3H),1.81(d,J=6.9Hz,3H).MS(EI)m / z 293.8[M+Na] + .

[0097] Step 6 Preparation of Compound 1F

[0098]

[0099] Compound 1D (5.83 g, 1 eq) was dissolved in anhydrous acetonitrile, cesium carbonate (6.62 g, 1.2 eq) was added, the temperature was raised to 70°C, and an acetonitrile solution of compound 1E (5.49 g, 1.2 eq) was slowly added. After reacting for 5 hours, the reaction solution was spin-dried and the crude product was purified by flash chromatography on a silica gel column to obtain the title compound 1F (7 g, 79.7% yield).

[0100] 1H NMR (400MHz, CDCl3) δ8.44 (s, 2H), 7.46 (dd, J=9.1, 2.8Hz, 1H), 7.17 (dd, J=9.1, 2.4Hz,1H),6.72(d,J=2.3Hz,1H),6.08(q,J=6.7Hz,1H),5.65-5.59(m,1H),4.05-3.95(m, 1H),3.79-3.59(m,1H),2.57-2.41(m,1H),2.18-2.08(m,1H),2.05-1.98(m,1H),1.83(d,J=6.7 Hz,3H),1.75–1.65(m,3H).MS(EI)m / z 541.8[M+Na] + .

[0101] Step 7 Preparation of Compound 1G

[0102]

[0103] Compound 1F (1 g, 1 eq), o-nitroaniline (0.32 g, 1.2 eq), Pd2dba3 (0.088 g, 0.05 eq), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos, 0.112 g, 0.1 eq), and Cs2CO3 (1.26 g, 2 eq) were dissolved in anhydrous toluene and reacted at 100° C. for 10 hours under argon protection. The reaction solution was spin-dried and purified by flash chromatography on a silica gel column to obtain the title compound 1G (0.4157 g, 40.8% yield).

[0104] 1 H NMR (600MHz, CDCl3) δ10.12 (s, 1H), 8.39 (s, 2H), 8.29 (dd, J = 8.6, 1.5Hz, 1H), 8.00-7. 94(m,1H),7.47(d,J=9.1Hz,1H),7.44-7.38(m,1H),7.18(dd,J=9.1,2.3Hz,1H),6.91 -6.88(m,1H),6.80–6.76(m,1H),5.99(q,J=6.6Hz,1H),5.62–5.58(m,1H),4.08–4.02(m, 1H),3.78–3.69(m,1H),2.59–2.49(m,1H),2.21–2.11(m,1H),2.11–2.02(m,2H),1.80(d,J =6.6Hz,3H),1.78–1.72(m,2H).

[0105] Step 8 Preparation of Compound 1H

[0106]

[0107] Compound 1G (0.4157 g, 1 eq) was dissolved in ethanol (20 mL), and iron powder (0.2203 g, 5 eq) and ammonium chloride (0.3367, 8 eq) dissolved in water (4 mL) were added. After reacting at 65°C for 0.5 hour, saturated aqueous sodium bicarbonate solution was added for washing, and the mixture was extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, filtered and evaporated, and then purified by flash chromatography on a silica gel column to obtain the title compound 1H (0.0804 g, 21% yield).

[0108] 1 H NMR (400MHz, DMSO-d6) δ8.53 (s, 2H), 7.48 (d, J = 9.4Hz, 1H), 7.39 (s, 1H), 7.22 (d, J=7.8Hz,1H),7.09–7.04(m,2H),6.74-6.71(m,2H),6.47–6.41(m,1H),5.92(q,J=6.5Hz, 1H),5.60-5.55(m,1H),4.88(s,2H),3.88-3.80(m,1H),3.70–3.59(m,1H),2.37–2.24(m,1H), 2.04-1.95(m,1H),1.94-1.84(m,1H),1.71(d,J=6.6Hz,3H),1.69–1.63(m,1H),1.56–1.48(m, 2H).

[0109] Step 9: Preparation of (R)-N-(2-((5-(1-(3,5-dichloropyridin-4-yl)ethoxy)1H-indazol-3-yl)amino)phenyl)acrylamide

[0110]

[0111] Acryloyl chloride (13 μL) was added dropwise to a solution of compound 1H (0.0804 g, 1 eq) and triethylamine (0.11 mL, 5 eq) in anhydrous dichloromethane at low temperature, and the mixture was reacted for 30 minutes and then washed with a saturated sodium bicarbonate aqueous solution. The organic phase was dried under reduced pressure and redissolved in dichloromethane (3 mL), and trifluoroacetic acid (1 mL) was added. The reaction was stopped after stirring at room temperature for 5 hours. The mixture was washed with a saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, and the organic phase was dried under reduced pressure and then purified by flash chromatography on a silica gel column to obtain the title compound 1 (40 mg, 52.9% yield).

[0112] 1H NMR(400MHz,DMSO-d6)δ12.13(s,1H),9.87(s,1H),8.51(s,2H),7.67(s,1H),7.46 (dd,J=7.5,1.4Hz,1H),7.34(d,J=9.0Hz,1H),7.28(dd,J=7.9,1.5Hz,1H),7.05(d,J=9.0 Hz,1H),6.97–6.87(m,2H),6.73(d,J=2.0Hz,1H),6.58(dd,J=17.0,10.2Hz,1H),6.33(dd,J =17.0,1.9Hz,1H),5.89–5.80(m,2H),5.83(dd,J=10.0,1.8Hz),1.69(d,J=6.6Hz,3H). HRMS(ESI)m / z calcd for C 23 H 19 Cl2N5O2[M] + 468.0989,found468.0973.

[0113] Example 2 Preparation of (R)-N-(2-((5-(1-(3,5-dichloropyridin-4-yl)ethoxy)1H-indazol-3-yl)amino)-3-methylphenyl)acrylamide (Compound 2)

[0114]

[0115] The o-nitroaniline in step 7 of Example 1 was replaced by 2-methyl-6-nitroaniline, and the other raw materials, reagents and preparation methods were the same as those in Example 1 to prepare compound 2.

[0116] 1 H NMR(400MHz, CDCl3)δ9.08(s,1H),8.52(s,1H),8.41(s,2H),8.14(s,1H),7.25-7.19 (m,2H),7.13-7.07(m,1H),7.04-6.99(m,1H),6.56-6.55(m,1H),6.30(d,J=16.9Hz,1H),6.12 (dd,J=16.9,10.2Hz,1H),5.88(q,J=6.6Hz,1H),5.71(s,1H),5.67–5.61(d,J=10.2Hz,1H), 2.17(s,3H),1.76(d,J=6.7Hz,3H).HRMS(ESI)m / z calcd forC 24 H 21 Cl2N5O2[M] +482.1145, found 482.1120.

[0117] Example 3 Preparation of (R)-N-(2-((5-(1-(3,5-dichloropyridin-4-yl)ethoxy)1H-indazol-3-yl)amino)-3-ethylphenyl)acrylamide (Compound 3)

[0118]

[0119] The o-nitroaniline in step 7 of Example 1 was replaced by 2-ethyl-6-nitroaniline, and the other raw materials, reagents and preparation methods were the same as those in Example 1 to prepare compound 3.

[0120] 1 H NMR(400MHz, CDCl3)δ9.15(s,1H),8.52(s,1H),8.41(s,2H),8.17(s,1H),7.30-7.25 (m,1H),7.24-7.19(m,1H),7.14–7.03(m,2H),6.52(s,1H),6.27(d,J=16.8Hz,1H),6.09(dd,J =16.8,10.4Hz,1H),5.87(q,J=6.6Hz,1H),5.77(s,1H),5.63(d,J=10.2Hz,1H),2.56(q,J= 7.3Hz,2H),1.76(d,J=6.6Hz,3H),1.07(t,J=7.5Hz,3H).HRMS(ESI)m / zcalcd for C 25 H 23 Cl2N5O2[M] + 496.1302, found 496.1280.

[0121] Example 4 Preparation of (R)-N-(2-((5-(1-(3,5-dichloropyridin-4-yl)ethoxy)1H-indazol-3-yl)amino)-3-cyclopropylphenyl)acrylamide (Compound 4)

[0122]

[0123] The o-nitroaniline in step 7 of Example 1 was replaced by 2-cyclopropyl-6-nitroaniline, and the other raw materials, reagents and preparation methods were the same as those in Example 1 to prepare compound 4.

[0124] 1H NMR (400MHz, DMSO-d6) δ11.59(s,1H),9.53(s,1H),8.55(s,2H),7.70(d,J=7.7Hz, 1H),7.34(s,1H),7.20(d,J=8.8Hz,1H),7.12(t,J=8.0Hz,1H),6.99(d,J=8.9Hz,1H),6.76(s ,1H),6.69(d,J=7.9Hz,1H),6.40(dd,J=17.2,10.3Hz,1H),6.17(d,J=17.0Hz,1H),5.85(q,J =6.5Hz,1H),5.67(d,J=10.2Hz,1H),1.90-1.81(m,1H),1.69(d,J=6.5Hz,3H),0.57-0.51(m,2H),0.41-0.35(m,2H).HRMS(ESI)m / z calcd forC 26 H 23 Cl2N5O2[M] + 508.1302, found 508.1283.

[0125] Example 5 Preparation of (R)-N-(2-((5-(1-(3,5-dichloropyridin-4-yl)ethoxy)1H-indazol-3-yl)amino)-3-methoxyphenyl)acrylamide (Compound 5)

[0126]

[0127] The o-nitroaniline in step 7 of Example 1 was replaced by 2-methoxy-6-nitroaniline, and the other raw materials, reagents and preparation methods were the same as those in Example 1 to prepare compound 5.

[0128] 1 H NMR (400MHz, DMSO-d6) δ11.60(s,1H),9.49(s,1H),8.57(s,2H),7.55(d,J=8.0Hz, 1H),7.23–7.18(m,2H),7.17-7.11(m,1H),7.02-6.97(m,1H),6.92(s,1H),6.80(d,J=8.0Hz, 1H),6.37(dd,J=17.0,10.2Hz,1H),6.14(dd,J=17.0,1.7Hz,1H),5.89(q,J=6.6Hz,1H),5.65 (dd,J=10.2,1.6Hz,1H),3.59(s,3H),1.70(d,J=6.6Hz,3H).HRMS(ESI)m / z calcd for C24 H 21 Cl2N5O3[M] + 498.1094, found 498.1070.

[0129] Example 6 Preparation of (R)-N-(2-((5-(1-(3,5-dichloropyridin-4-yl)ethoxy)1H-indazol-3-yl)amino)-4-methoxyphenyl)acrylamide (Compound 6)

[0130]

[0131] The o-nitroaniline in step 7 of Example 1 was replaced by 3-methoxy-6-nitroaniline, and the other raw materials, reagents and preparation methods were the same as those in Example 1 to prepare compound 6.

[0132] 1 H NMR(400MHz,DMSO-d6)δ12.17(s,1H),9.75(s,1H),8.50(s,2H),7.71(s,1H),7.34 (d,J=8.9Hz,1H),7.27(d,J=8.7Hz,1H),7.10–7.00(m,2H),6.81(s,1H),6.55(dd,J=16.9, 10.2Hz,1H),6.49-6.44(m,1H),6.31(d,J=17.0Hz,1H),5.89(q,J=6.6Hz,1H),5.81(d,J= 10.1Hz,1H),3.57(s,3H),1.70(d,J=6.3Hz,3H).HRMS(ESI)m / z Calculate for C 24 H 21 Cl2N5O3[M] + 498.1094, found 498.1076.

[0133] Example 7 Preparation of (R)-N-(2-((5-(1-(3,5-dichloropyridin-4-yl)ethoxy)1H-indazol-3-yl)amino)-3-chlorophenyl)acrylamide (Compound 7)

[0134]

[0135] The o-nitroaniline in step 7 of Example 1 was replaced with 2-chloro-6-nitroaniline, and the other raw materials, reagents and preparation methods were the same as those in Example 1 to prepare compound 7.

[0136] 1H NMR(400MHz,DMSO-d6)δ11.70(s,1H),9.63(s,1H),8.56(s,2H),7.92-7.82(m,1H), 7.55(s,1H),7.27-7.15(m,3H),7.05-6.95(m,1H),6.85(s,1H),6.42(dd,J=16.9,10.3Hz,1H), 6.18(d,J=16.8Hz,1H),5.88(q,J=6.5Hz,1H),5.69(d,J=10.1Hz,1H),1.70(d,J=6.3Hz, 3H).HRMS(ESI)m / z calcd for C 23 H 18 Cl3N5O2[M] + 502.0599,found502.0583.

[0137] Example 8 Preparation of (R)-N-(2-((5-(1-(3,5-dichloropyridin-4-yl)ethoxy)1H-indazol-3-yl)amino)-3-fluorophenyl)acrylamide (Compound 8)

[0138]

[0139] The o-nitroaniline in step 7 of Example 1 was replaced with 2-fluoro-6-nitroaniline, and the other raw materials, reagents and preparation methods were the same as those in Example 1 to prepare compound 8.

[0140] 1 H NMR(400MHz, CDCl3)δ9.25(s,1H),9.01(s,1H),8.44(s,2H),8.02(s,1H),7.31–7.23 (m,1H),7.21–7.10(m,2H),6.97-6.87(m,1H),6.79(s,1H),6.31(d,J=16.9Hz,1H),6.14(dd,J =17.0,10.2Hz,1H),5.98(q,J=6.5Hz,1H),5.87(s,1H),5.67(d,J=10.2Hz,1H),1.81(d,J=6.6Hz,3H).HRMS(ESI)m / z calcd for C 23 H 18 Cl2FN5O2[M] + 486.0894, found 486.0891.

[0141] Example 9 Preparation of (R)-N-(2-((5-(1-(3,5-dichloropyridin-4-yl)ethoxy)1H-indazol-3-yl)amino)-5-(4-methylpiperazin-1-yl)phenyl)acrylamide (Compound 9)

[0142]

[0143] Compound 9 was prepared by replacing o-nitroaniline in step 7 of Example 1 with 4-(4-methylpiperazine-1-yl)-2-nitroaniline and using the same raw materials, reagents and preparation methods as in Example 1.

[0144] 1 H NMR(400MHz,DMSO-d6)δ11.90(s,1H),9.82(s,1H),8.51(s,2H),7.35(s,1H),7.28 (d,J=9.0Hz,1H),7.18(d,J=2.2Hz,1H),7.15(d,J=8.9Hz,1H),7.02(dd,J=9.0,2.3Hz,1H), 6.69(d,J=1.6Hz,1H),6.60(dd,J=8.9,2.6Hz,1H),6.54(J=17.0,10.2Hz,1H),6.31(d,J=16.9Hz 1H),5.84(q,J=6.7Hz,1H),5.80(d,J=10.1Hz,1H),3.10-3.04(m,4H),250-2.46(m, 4H),2.23(s,3H),1.68(d,J=6.6Hz,3H).HRMS(ESI)m / z calcd forC 28 H 29 Cl2N7O2[M] + 566.1833, found 566.1823.

[0145] Example 10 Preparation of (R)-N-(2-((5-(1-(3,5-dichloropyridin-4-yl)ethoxy)1H-indazol-3-yl)amino)-5-morpholinophenyl)acrylamide (Compound 10)

[0146]

[0147] The o-nitroaniline in step 7 of Example 1 was replaced with 4-(morpholin-1-yl)-2-nitroaniline, and the other raw materials, reagents and preparation methods were the same as those in Example 1 to prepare compound 10.

[0148] 1H NMR(400MHz,DMSO-d6)δ11.91(s,1H),9.81(s,1H),8.51(s,2H),7.38(s,1H),7.29 (d,J=9.0Hz,1H),7.19(d,J=8.9Hz,2H),7.02(dd,J=9.0,2.2Hz,1H),6.71(s,1H),6.62(dd,J =8.9,2.5Hz,1H),6.55(dd,J=16.9,10.2Hz,1H),6.31(d,J=17.0Hz,1H),5.85(q,J=6.6Hz, 1H),5.81(d,J=10.2Hz,1H),3.83–3.70(m,4H),3.10-3.00(m,4H),1.69(d,J=6.6Hz,3H). HRMS(ESI)m / z calcd for C27H26Cl2N6O3[M] + 553.1516, found 553.1507.

[0149] Example 11 Preparation of (R)-N-(2-((5-(1-(3,5-dichloropyridin-4-yl)ethoxy)1H-indazol-3-yl)amino)-5-(4-morpholinopiperidin-1-yl)phenyl)acrylamide (Compound 11)

[0150]

[0151] The o-nitroaniline in step 7 of Example 1 was replaced with 4-(4-morpholinopiperidin-1-yl)-2-nitroaniline, and the other raw materials, reagents and preparation methods were the same as those in Example 1 to prepare Compound 11.

[0152] 1H NMR(400MHz,DMSO-d6)δ11.90(s,1H),9.82(s,1H),8.51(s,2H),7.35(s,1H),7.28 (d,J=8.7Hz,1H),7.19(s,1H),7.15(d,J=8.9Hz,1H),7.02(d,J=8.2Hz,1H),6.69(s,1H), 6.61(d,J=7.8Hz,1H),6.52(dd,J=16.9,10.2Hz,1H),6.31(d,J=17.0Hz,1H),5.84(q,J=6.7 Hz,1H),5.80(d,J=9.9Hz,1H),3.65-3.55(m,6H),2.70-2.61(m,2H),2.58-2.52(m,4H), 2.42-2.25(m,1H),1.95-1.85(m,2H),1.68(d,J=5.6Hz,3H),1.60-1.50(m,2H).HRMS(ESI)m / z calcd for C 32 H 35 Cl2N7O3[M] + 636.2251, found 636.2251.

[0153] Example 12 Preparation of (R)-N-(2-((5-(1-(3,5-dichloropyridin-4-yl)ethoxy)1H-indazol-3-yl)amino)-3-fluoro-5-(4-morpholinopiperidin-1-yl)phenyl)acrylamide (Compound 12)

[0154]

[0155] The o-nitroaniline in step 7 of Example 1 was replaced with 6-fluoro-4-(4-morpholinopiperidin-1-yl)-2-nitroaniline, and the other raw materials, reagents and preparation methods were the same as those in Example 1 to prepare compound 12.

[0156] 1H NMR(400MHz,DMSO-d6)δ11.55(s,1H),9.52(s,1H),8.54(s,2H),7.49(s,1H),7.23– 7.15(m,2H),6.98(d,J=9.1Hz,1H),6.81(s,1H),6.61-6.55(m,1H),6.50(dd,J=16.9,10.4Hz, 1H),6.22(d,J=16.8Hz,1H),5.87(q,J=8.0Hz,1H),5.71(d,J=10.3Hz,1H),3.75-3.67(m, 2H),3.61-3.55(m,4H),2.78-2.69(m,2H),2.49–2.46(m,4H),2.36-2.26(m,1H), 1.92-1.84(m,2H),1.69(d,J=8.0Hz,3H),1.53–1.46(m,2H).HRMS(ESI)m / zcalcd for C 32 H 34 Cl2FN7O3[M] + 654.2157, found 654.2127.

[0157] Example 13 Preparation of (R)-N-(2-((5-(1-(3,5-dichloropyridin-4-yl)ethoxy)1H-indazol-3-yl)amino)-3-fluoro-5-(4-methylpiperazin-1-yl)phenyl)acrylamide (Compound 13)

[0158]

[0159] The o-nitroaniline in step 7 of Example 1 was replaced with 6-fluoro-4-(4-methylpiperazin-1-yl)-2-nitroaniline, and the other raw materials, reagents and preparation methods were the same as those in Example 1 to prepare compound 13.

[0160] 1H NMR (400MHz, DMSO-d6) δ11.56(s,1H),9.53(s,1H),8.54(s,2H),7.49(s,1H),7.24-7. 16(m,2H),6.99(d,J=8.9Hz,1H),6.80(s,1H),6.59(d,J=13.8Hz,1H),6.50(dd,J=16.8 ,10.2Hz,1H),6.22(d,J=17.0Hz,1H),5.86(q,J=6.7Hz,1H),5.71(d,J=10.3Hz,1H),3 .20-3.13(m,4H),2.49–2.43(m,4H),2.25(s,3H),1.69(d,J=6.4Hz,3H).HRMS(ESI)m / z calcd for C 28 H 28 Cl2FN7O2[M] + 584.1738, found 584.1712.

[0161] Example 14 Preparation of (R)-N-(2-((5-(1-(3,5-dichloropyridin-4-yl)ethoxy)1H-indazol-3-yl)amino)-3-fluoro-5-(4-formylpiperazin-1-yl)phenyl)acrylamide (Compound 14)

[0162]

[0163] The o-nitroaniline in step 7 of Example 1 was replaced with 6-fluoro-4-(4-formylpiperazin-1-yl)-2-nitroaniline, and the other raw materials, reagents and preparation methods were the same as those in Example 1 to prepare compound 14.

[0164] 1H NMR (400MHz, CDCl3) δ9.11(s,1H),8.83(s,1H),8.42(s,2H),7.93(s,1H),7.25(d,J= 9.0Hz,1H),7.13(dd,J=9.0,2.3Hz,1H),6.72(d,J=2.1Hz,1H),6.43(dd,J=12.6,2.7Hz,1H), 6.36(dd,J=16.9,1.1Hz,1H),6.17(dd,J=16.9,10.2Hz,1H),5.95(q,J=6.7Hz,1H),5.71(dd ,J=10.2,1.1Hz,1H),3.81–3.75(m,2H),3.67–3.58(m,2H),3.28–3.24(m,2H),3.23–3.19(m, 2H),2.16(s,3H),1.79(d,J=6.7Hz,3H).HRMS(ESI)m / z calcdfor C 29 H 28 Cl2FN7O3[M] + 612.1687, found 612.1682.

[0165] Example 15 Preparation of (R)-N-(2-((5-(1-(3,5-dichloropyridin-4-yl)ethoxy)1H-indazol-3-yl)amino)-3-fluoro-5-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)acrylamide (Compound 15)

[0166]

[0167] The o-nitroaniline in step 7 of Example 1 was replaced with 6-fluoro-4-(4-methylpiperazin-1-yl)piperidin-1-yl)-2-nitroaniline, and the other raw materials, reagents and preparation methods were the same as those in Example 1 to prepare Compound 15.

[0168] 1H NMR (400MHz, CDCl3) δ9.30 (s, 1H), 8.77 (s, 1H), 8.41 (s, 2H), 7.91 (s, 1H), 7.23 (d, J= 9.0Hz,1H),7.11(dd,J=9.0,2.1Hz,1H),6.68(d,J=1.9Hz,1H),6.45-6.39(m,1H),6.35(d,J= 16.8Hz,1H),6.16(dd,J=16.9,10.2Hz,1H),5.93(q,J=6.6Hz,1H),5.69(d,J=10.3Hz,1H), 5.52-5.48(m,1H),3.80(d,J=11.7Hz,2H),2.83-2.70(m,4H),2.63-2.48(m,4H),2.45-2.39(m, 1H),2.35(s,3H),1.95(d,J=11.9Hz,4H),1.77(d,J=6.7Hz,3H),1.70–1.62(m,2H).HRMS(ESI)m / z calcd for C 33 H 37 Cl2FN8O2[M] + 667.2473, found 667.2473.

[0169] Example 16 Preparation of (R)-N-(2-((5-(1-(3,5-dichloropyridin-4-yl)ethoxy)1H-indazol-3-yl)amino)-3-fluoro-5-(4-methylsulfonylpiperazin-1-yl)phenyl)acrylamide (Compound 16)

[0170]

[0171] The o-nitroaniline in step 7 of Example 1 was replaced with 6-fluoro-4-(4-methylsulfonylpiperazin-1-yl)-2-nitroaniline, and the other raw materials, reagents and preparation methods were the same as those in Example 1 to prepare compound 16.

[0172] 1H NMR (400MHz, CDCl3) δ9.01 (s, 1H), 8.83 (s, 1H), 8.43 (s, 2H), 7.95 (s, 1H), 7.25 (d, J= 9.0Hz,1H),7.14(dd,J=9.1,2.2Hz,1H),6.76-6.72(m,1H),6.48-6.42(m,1H),6.36(d,J=17.0 Hz,1H),6.17(dd,J=16.9,10.2Hz,1H),5.96(q,J=6.7Hz,1H),5.72(d,J=10.4Hz,1H),5.47 (s,1H),3.38(d,J=6.8Hz,8H),2.85(s,3H),1.80(d,J=6.7Hz,3H).HRMS(ESI)m / z calcd for C 28 H 28 Cl2FN7O4S[M] + 648.1357, found 648.1342.

[0173] Example 17 Preparation of (R)-N-(2-((5-(1-(3,5-dichloropyridin-4-yl)ethoxy)1H-indazol-3-yl)amino)-3-fluoro-5-morpholinophenyl)acrylamide (Compound 17)

[0174]

[0175] Compound 17 was prepared by replacing o-nitroaniline in step 7 of Example 1 with 6-fluoro-4-(morpholin-1-yl)-2-nitroaniline and using the same raw materials, reagents and preparation methods as in Example 1.

[0176] 1 H NMR (400MHz, CDCl3) δ9.09 (s, 1H), 8.78 (s, 1H), 8.42 (s, 2H), 7.93 (s, 1H), 7.24 (d, J= 9.0Hz,1H),7.12(dd,J=9.0,2.2Hz,1H),6.70(d,J=1.9Hz,1H),6.41(dd,J=12.8,2.6Hz,1H), 6.36(d,J=16.9Hz,1H),6.16(dd,J=16.9,10.2Hz,1H),5.94(q,J=6.6Hz,1H),5.70(d,J= 11.1Hz,1H),5.50(s,1H),3.90–3.83(m,4H),3.24–3.17(m,4H),1.78(d,J=6.7Hz,3H).HRMS(ESI)m / z calcd for C27 H 25 Cl2FN6O3[M] + 571.1422, found 571.1411.

[0177] Example 18 Preparation of (R)-N-(2-((5-(1-(3,5-dichloropyridin-4-yl)ethoxy)1H-indazol-3-yl)amino)-3-fluoro-5-(4-(dimethylamino)piperidin-1-yl)phenyl)acrylamide (Compound 18)

[0178]

[0179] The o-nitroaniline in step 7 of Example 1 was replaced with 6-fluoro-4-(4-(dimethylamino)piperidin-1-yl)-2-nitroaniline, and the other raw materials, reagents and preparation methods were the same as those in Example 1 to prepare compound 18.

[0180] 1 H NMR (400MHz, CDCl3) δ9.37(s,1H),8.79(s,1H),8.41(s,2H),7.90(s,1H),7.23(d,J= 9.0Hz,1H),7.10(dd,J=9.0,2.2Hz,1H),6.69(d,J=1.9Hz,1H),6.42(dd,J=13.0,2.4Hz,1H), 6.35(d,J=16.9Hz,1H),6.17(dd,J=16.9,10.2Hz,1H),5.93(q,J=6.6Hz,1H),5.69(d,J= 10.3Hz,1H),5.54(s,1H),3.79(d,J=12.4Hz,2H),2.82-2.72(m,2H),2.42-2.38(m,1H),2.36(s, 6H),1.96(d,J=12.9Hz,2H),1.77(d,J=6.7Hz,3H),1.70-1.62(m,2H).HRMS(ESI)m / z calcd for C 30 H 32 Cl2FN7O2[M] + 612.2051, found 612.2038.

[0181] Example 19 Preparation of (R)-N-(2-((5-(1-(3,5-dichloropyridin-4-yl)ethoxy)1H-indazol-3-yl)amino)-3-fluoro-5-((2-methoxyethyl)(methyl)amino)phenyl)acrylamide (Compound 19)

[0182]

[0183] The o-nitroaniline in step 7 of Example 1 was replaced with 6-fluoro-4-(2-methoxyethyl)(methyl)amino)-2-nitroaniline, and the other raw materials, reagents and preparation methods were the same as those in Example 1 to prepare Compound 19.

[0184] 1 H NMR (400MHz, CDCl3) δ9.03 (s, 1H), 8.65 (s, 1H), 8.41 (s, 2H), 7.76 (s, 1H), 7.22 (d, J= 9.0Hz,1H),7.10(dd,J=9.0,2.3Hz,1H),6.67(d,J=1.9Hz,1H),6.36(d,J=16.9Hz,1H), 6.25-6.20(m,1H),6.17(dd,J=16.9,10.2Hz,1H),5.92(q,J=6.7Hz,1H),5.69(d,J=10.3Hz ,1H),5.41(s,1H),3.67–3.51(m,4H),3.39(s,3H),3.05(s,3H),1.77(d,J=6.7Hz,3H).HRMS (ESI)m / z calcd for C 27 H 27 Cl2FN6O3[M] + 573.1578, found 573.1571.

[0185] Example 20 Preparation of (R)-N-(2-((5-(1-(3,5-dichloropyridin-4-yl)ethoxy)1H-indazol-3-yl)amino)-3-fluoro-5-(4-(oxetane-3-yl)piperazin-1-yl)phenyl)acrylamide (Compound 20)

[0186]

[0187] The o-nitroaniline in step 7 of Example 1 was replaced with 6-fluoro-4-(4-(oxetane-3-yl)piperazine-1-yl)-2-nitroaniline, and the other raw materials, reagents and preparation methods were the same as those in Example 1 to prepare compound 20.

[0188] 1H NMR (400MHz, CDCl3) δ9.25(s,1H),8.78(s,1H),8.41(s,2H),7.92(s,1H),7.23(d,J= 9.0Hz,1H),7.11(dd,J=9.0,1.9Hz,1H),6.68(s,1H),6.41(d,J=1.9Hz,1H),6.35(d,J=16.8 Hz,1H),6.16(dd,J=16.9,10.2Hz,1H),5.92(q,J=6.6Hz,1H),5.69(d,J=10.3Hz,1H),5.54 (s,1H),4.76-4.64(m,4H),3.61–3.53(m,1H),3.53-3.26(m,4H),2.55–2.43(m,4H),1.77(d,J =6.6Hz,3H).HRMS(ESI)m / z calcd for C 30 H 30 Cl2FN7O3[M] + 626.1844, found 626.1832.

[0189] Example 21 Preparation of N-(2-((5-((3,5-dichloropyridin-4-yl)methoxy)1H-indazol-3-yl)amino)phenyl)acrylamide (Compound 21)

[0190]

[0191] Step 13, Preparation of 5-dichloropyridine carboxaldehyde

[0192]

[0193] At -78 degrees Celsius, 25 ml of n-butyl lithium solution (1.2 eq, 1.6 M hexane solution) was added dropwise to 100 ml of dry tetrahydrofuran solution, and after stirring for 30 minutes, 20 ml of 1,3-dichloropyridine (5 g, 1 eq) tetrahydrofuran solution was added dropwise, and after continuing to stir for 30 minutes, 3 ml of dry N, N-dimethylformamide solution was added dropwise, and after continuing to stir for 60 minutes, saturated ammonium chloride was added to quench the reaction, extracted with ethyl acetate, and the organic phases were combined and dried over anhydrous sodium sulfate, filtered and evaporated to obtain a crude product. The crude product was purified by flash chromatography on a silica gel column to obtain 3,5-dichloropyridinecarboxaldehyde (3 g, 50% yield).

[0194] 1 H NMR (400MHz, DMSO-d6) δ10.31(s,1H),8.80(s,2H).

[0195] Step 2 Preparation of (3,5-dichloropyridine-4-substituted)methanol

[0196]

[0197] Under ice bath, sodium borohydride (0.97 g, 1.5 eq) was added to a methanol solution of 3,5-dichloropyridinecarboxaldehyde (3 g, 1 eq), and then the reaction was restored to room temperature. After the reaction was completed, the reaction solution was spin-dried and the crude product was purified by flash chromatography on a silica gel column to obtain (3,5-dichloropyridine-4-substituted)methanol (2.5 g, 83% yield).

[0198] 1 H NMR (400MHz, CDCl3) δ8.50 (s, 2H), 4.94 (s, 2H).

[0199] Step 3 Preparation of (3,5-dichloropyridine-4-substituted)methyl methanesulfonate

[0200]

[0201] Under ice bath, add 1.2 ml of triethylamine to a solution of (3,5-dichloropyridine-4-substituted)methanol (1 g, 1 eq) in dichloromethane, then add methylsulfonyl chloride (0.48 ml, 1.1 eq) dropwise. After reacting for 15 minutes, wash with saturated sodium bicarbonate aqueous solution, extract with dichloromethane, and remove the organic phase under reduced pressure to obtain the title compound.

[0202] Compound 21 was prepared by replacing compound 1E in step 6 of Example 1 with (3,5-dichloropyridine-4-substituted)methyl methanesulfonate and using the other raw materials, reagents and preparation methods the same as those of Example 1.

[0203] 1 H NMR(400MHz,DMSO)δ12.14(s,1H),9.83(s,1H),8.71(s,2H),7.85(s,1H),7.60(d,J =7.8Hz,1H),7.48(d,J=7.5Hz,1H),7.38(d,J=8.9Hz,1H),7.25(s,1H),7.14-7.06(m,2H), 6.93-6.86(m,1H),6.57(dd,J=16.7,10.1Hz,1H),6.30(d,J=16.8Hz,1H),5.78(d,J=10.0Hz, 1H),5.21(s,2H).HRMS(ESI)m / z calcd for C 22 H 17 Cl2N5O2[M] +454.0832, found 454.0826.

[0204] Example 22 Preparation of N-(2-((5-((3,5-dichloropyridin-4-yl)propoxy)1H-indazol-3-yl)amino)phenyl)acrylamide (Compound 22)

[0205]

[0206] The N,N-dimethylformamide in step 1 of Example 21 was replaced with propionaldehyde, and the remaining required raw materials, reagents and preparation methods were the same as those in Example 21 to prepare Compound 22.

[0207] 1 H NMR (400MHz, CDCl3) δ10.68(s,1H),9.52(s,1H),8.34(s,2H),8.03(s,1H),7.78(d,J =7.3Hz,1H),7.28-7.23(m,1H),7.16-7.09(m,2H),6.95(t,J=7.5Hz,1H),6.66-6.55(m,2H), 6.47(d,J=16.8Hz,1H),6.31(dd,J=16.7,10.1Hz,1H),5.78(d,J=10.2Hz,1H),5.62-5.52(m, 1H),2.36-2.22(m,1H),2.04-1.95(m,1H),1.06(t,J=7.4Hz,3H).HRMS(ESI)m / z calcd for C 24 H 21 Cl2N5O2[M] + 482.1145, found 482.1129.

[0208] Example 23 Preparation of N-(2-((5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)1H-indazol-3-yl)amino)phenyl)acrylamide (Compound 23)

[0209]

[0210] The (S)-1-(3,5-dichloropyridine-4-substituted)ethanol in step 5 of Example 1 was replaced with 1-(2,6-dichloro-3-fluorophenyl)ethanol, and the other raw materials, reagents and preparation methods were the same as those in Example 1 to prepare compound 23.

[0211] 1H NMR(400MHz,DMSO-d6)δ12.10(s,1H),9.88(s,1H),7.67(s,1H),7.49–7.28(m,5H), 7.07–7.01(m,1H),6.98(t,J=7.1Hz,1H),6.88(t,J=7.1Hz,1H),6.80(s,1H),6.58(dd,J =16.6,10.4Hz,1H),6.33(d,J=16.9Hz,1H),5.92(q,J=6.3Hz,1H),5.81(d,J=9.7Hz,1H), 1.70(d,J=6.1Hz,3H).HRMS(ESI)m / z calcd for C 24 H 19 Cl2FN4O2[M] + 485.0942, found 485.0919.

[0212] Example 24 Preparation of N-(2-((5-(1-(2,6-dichlorophenyl)ethoxy)1H-indazol-3-yl)amino)phenyl)acrylamide (Compound 24)

[0213]

[0214] The (S)-1-(3,5-dichloropyridine-4-substituted)ethanol in step 5 of Example 1 was replaced with 1-(2,6-dichlorophenyl)ethanol, and the remaining raw materials, reagents and preparation methods were the same as those in Example 1 to prepare compound 24.

[0215] 1 H NMR(400MHz,DMSO-d6)δ12.08(s,1H),9.89(s,1H),7.65(s,1H),7.46-7.42(m,1H), 7.39–7.23(m,5H),7.07–6.95(m,2H),6.89(t,J=7.4Hz,1H),6.83(s,1H),6.58(dd,J=17.0, 10.1Hz,1H),6.34(d,J=17.0Hz,1H),5.94(q,J=6.6Hz,1H),5.83(d,J=10.1Hz,1H),1.70(d,J=6.6Hz,3H).HRMS(ESI)m / z calcd for C 24 H 20 Cl2N4O2[M] + 467.1036, found 467.1027.

[0216] Experimental Example 1: Test of the inhibitory activity of 3-aminoindazole compounds on FGFRs kinase

[0217] FGFR1-4 kinase inhibition assay: The inhibitory activity of compounds against FGFR1-4 was determined using the FRET-based Z'-Lyte assay system according to the manufacturer's instructions (Life Technologies, Carlsbad, CA, USA). Reactions were performed in 384-well plates in 5 μL reaction volumes in 50 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EGTA, and 0.01% Brij-35 with appropriate amounts of kinase. Reactions were incubated at room temperature for 1.5 hours in the presence of 2 μM substrate and each kinase (corresponding to ATP concentrations of FGFR1: 25 μM, FGFR2: 5 μM, and FGFR3: 75 μM) and in the presence of various concentrations of compounds, followed by the addition of 2.5 μL of developer, followed by an additional incubation at room temperature for 1 hour, followed by the addition of 2.5 μL of stop solution. The fluorescence signal ratio of 445 nm (coumarin) / 520 nm (fluorescein) was detected using EnVision Multilabel Reader (Perkin Elmer, Inc.). The data were analyzed using Graphpad Prism 5 (Graphpad Software, Inc). The inhibition rate of the compound was calculated using the following formula:

[0218]

[0219] IC 50 The values ​​were calculated by four-parameter fitting of the inhibition curve. The kinase inhibitory activity results of the 3-aminoindazole compounds of the present invention are shown in Table 1. The compound numbers listed in Table 1 correspond to the compound numbers in Examples 1-24.

[0220] Table 1 Test results of the inhibitory activity of compounds on FGFRs kinases (IC 50 :nM)

[0221] Compound No. FGFR4 FGFR1 FGFR2 FGFR3 Compound 1 66 108 949 377 Compound 2 53 >10uM >10uM >10uM Compound 3 >10uM >10uM >10uM >10uM Compound 4 >10uM >10uM >10uM >10uM Compound 5 6432 >10uM >10uM >10uM Compound 6 >10uM >10uM >10uM >10uM Compound 7 19 >10uM >10uM >10uM Compound 8 21 >10uM >10uM >10uM Compound 9 9 >10uM >10uM >10uM Compound 10 12 >10uM >10uM >10uM Compound 11 33 >10uM >10uM >10uM Compound 12 0.5 >10uM >10uM >10uM Compound 13 1.4 >10uM >10uM >10uM Compound 14 72 >10uM >10uM >10uM Compound 15 11 >10uM >10uM >10uM Compound 16 16 >10uM >10uM >10uM Compound 17 26 >10uM >10uM >10uM Compound 18 18 >10uM >10uM >10uM Compound 19 30 >10uM >10uM >10uM Compound 20 8 >10uM >10uM >10uM Compound 21 352 762 >10uM 4032 Compound 22 273 811 >10uM 2792 Compound 23 1643 >10uM >10uM >10uM Compound 24 >10uM >10uM >10uM >10uM BLU9931 2.2 246 238 726

[0222] It can be seen from the data in Table 1 that the 3-aminoindazole compounds of the present invention have strong inhibitory activity against FGFR4 kinase, and weak inhibitory activity against the other three subtypes of the FGFR family, FGFR1-3, and have excellent selectivity.

[0223] Experimental Example 2: Cell proliferation inhibition activity test based on BaF3-FGFR4 wild type and Ba / F3-TEL-FGFR4 V550L, Ba / F3-TEL-FGFR4V550M drug-resistant mutant stable strains

[0224] The Ba / F3 cells (mouse pre-B cells) used in this experiment were purchased from ThermoFisher. The Ba / F3-TEL-FGFR4 wild-type and Ba / F3-TEL-FGFR4 V550L and Ba / F3-TEL-FGFR4 V550M drug-resistant mutant stable strains were all constructed by our laboratory and were completely correct through positive drug activity, protein expression, and gene sequencing experiments.

[0225] The brief steps for stable strain construction are as follows:

[0226] 1. The kinase domain gene of human FGFR4 (NM_002011.5) was fused to the N-terminus of ETV6 (NM_001987.4), and the fusion gene TEL-FGFR4 was cloned into the pMSCV vector. Positive clones were selected and verified by sequencing.

[0227] 2. Using the recombinant plasmid TEL-FGFR4-pMSCV as a template, construct the FGFR4 gatekeeper mutant plasmids TEL-FGFR4V550L-pMSCV and TEL-FGFR4 V550M-pMSCV.

[0228] 3. Transfect 293T cells with the three successfully constructed plasmids and the empty plasmid without the target gene, and collect and preserve the virus-containing supernatant culture medium.

[0229] 4. Use this supernatant medium to infect Ba / F3 cells for three days, change to normal medium containing puromycin for one day of recovery, and then select positive cell lines in the absence of interleukin-3 and with the addition of puromycin.

[0230] 5. The positive stable cell lines screened were verified by western-blot.

[0231] Cell proliferation inhibitory activity study:

[0232] 1. Add 2000 positive stable cell lines obtained by screening into a 96-well cell culture plate, and use the Ba / F3 cell line transfected with an empty plasmid without the target gene as a control.

[0233] 2. After 6-12 hours, add an equal volume of pre-diluted 3-aminoindazole compound (3-fold dilution).

[0234] 3. After 72 h, add Cell Counting Kit-8 (CCK-8) reagent to detect cell viability.

[0235] 4. Calculate IC of small molecule inhibition using GraphPad Prism software 50 .

[0236] The test results are shown in Table 2.

[0237] Table 2 Inhibitory activity test results of compounds on BaF3-FGFR4, BaF3-FGFR4 V550L, BaF3-FGFR4 V550M cell proliferation (IC 50 :nM)

[0238] Compound No. BaF3-FGFR4 BaF3-FGFR4 V550L BaF3-FGFR4 V550M Compound 12 3.5 4.7 8.1 Compound 13 2.3 2.5 3.7 BLU9931 2.4 >100 >100

[0239] It can be seen from the data in Table 2 that the 3-aminoindazole compounds of the present invention have strong inhibitory activity on the cell proliferation of BaF3-FGFR4, BaF3-FGFR4V550L and BaF3-FGFR4 V550M stable strains.

[0240] Experimental Example 3: Test of the inhibitory activity of compounds on tumor cell proliferation

[0241] In vitro cell proliferation inhibition was detected by CCK8 assay: Tumor cells (MDA-MB-453 (breast cancer cell line), Hep3B (liver cancer cell line) and H1299 (lung cancer cell line)) from the Maurice Wilkins Center of Excellence at the University of Auckland, New Zealand were stored in the corresponding growth medium and passaged at least two times before recovery. An appropriate number of cells in logarithmic growth phase were inoculated on a 6×7 96-well plate with a volume of 100 μL per well, and then cultured overnight in a 37°C incubator containing 5% CO2. In addition, the test compound was first dissolved in DMSO to make a 10 μM / L stock solution, and then diluted from 1×10 -5 M / L to1×10 -10 M / L dilution was performed in 10 gradients, and 2 μL of compound solutions of different concentrations were taken and mixed evenly with 998 μL of culture medium. Then, 100 μL of the culture medium mixed with the compound was taken and added to the corresponding 96-well plate. 2 μL of DMSO was used as a blank control. After 48 hours of co-incubation, 20 μL of Cell Counting Kit-8 (CCK-8) (5 mg / mL) reagent was added, and after shaking for 10 minutes, the number of stained cells was read using SynergyTMHT (Bio Tek) / OD570. Finally, the data was processed with GraphPad Prism, and the nonlinear regression curve was fitted using the S-type dose-response to obtain the IC 50 Values ​​(Table 3).

[0242] Table 3 Test results of the inhibitory activity of compounds on tumor cell proliferation (IC 50 :nM)

[0243] Compound No. MDA-MB-453 Hep3B H1299 Compound 12 5 2 3600 Compound 13 9 6 2500 BLU9931 40 190 800 BLU554 23 29 >10uM

[0244] As can be seen from the data in Table 3, the 3-aminoindazole compounds of the present invention have strong inhibitory activity on the proliferation of FGFR4-dependent MDA-MB-453 (breast cancer cell line) and Hep3B (liver cancer cell line) cells that highly express FGFR4, and have weaker inhibitory activity on the proliferation of FGFR4-independent H1299 (lung cancer cell line), showing good selectivity.

[0245] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the following embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0246] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A 3-aminoindazole compound having the structure represented by formula (I) or a pharmaceutically acceptable salt thereof or a stereoisomer thereof: in, X is selected from: CH, N; Z is selected from: -O-; R1, R2, R4 are independently selected from: H, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, halogen, halogen-substituted C1-C6 alkyl, nitro, cyano, hydroxyl, amino; R3 is selected from: H, -NR 10 R 11 ; R5 is selected from the group consisting of: H, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, halogen, halogen-substituted C1-C6 alkyl, nitro, cyano, hydroxyl, and amino; R6, R7, R8, R9 are independently selected from: H, halogen; R 10 , R 11 are independently selected from: H, C1-C 20 Alkyl, C3-C 20 Cycloalkyl, C1-C 20 Alkoxy substituted C1-C 20 Alkyl, C1-C 20 Alkylamino-substituted C1-C 20 Alkyl, or R 10 , R 11 Together with the nitrogen atom to which it is attached, it forms one or more R 12 a substituted or unsubstituted 5-6 membered heterocyclic group; Each R 12 are independently selected from: H, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, halogen, halogen-substituted C1-C6 alkyl, nitro, cyano, hydroxyl, amino, C1-C6 alkylacyl, C1-C6 alkylsulfonyl, C1-C6 alkylamino, C3-C6 heterocyclyl, one or more R 13 Substituted C3-C6 heterocyclic group; Each R 13 Each of them is independently selected from the group consisting of: H, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, halogen, halogen-substituted C1-C6 alkyl, nitro, cyano, hydroxyl, amino, C1-C6 alkylacyl, C1-C6 alkylsulfonyl, and C1-C6 alkylamino.

2. The 3-aminoindazole compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 1, characterized in that: It has the structure shown in formula (II):

3. The 3-aminoindazole compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 1, characterized in that: It has the structure shown in formula (III):

4. The 3-aminoindazole compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to any one of claims 1 to 3, characterized in that: R3 is selected from: H, -NR 10 R 11 ; R 10 , R 11 are independently selected from: H, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkyl substituted with C1-C6 alkoxy, C1-C6 alkyl substituted with C1-C6 alkylamino, or, R 10 , R 11 Together with the nitrogen atom to which it is attached, it forms one or more R 12 A substituted or unsubstituted 5-6 membered heterocyclic group.

5. The 3-aminoindazole compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 4, characterized in that: R3 is selected from: H, -NR 10 R 11 ; R 10 , R 11 are independently selected from: H, C1-C3 alkyl, C1-C3 alkyl substituted with C1-C3 alkoxy, C1-C3 alkyl substituted with C1-C3 alkylamino, or, R 10 , R 11 Together with the nitrogen atom to which it is attached, it forms one or more R 12 A substituted or unsubstituted 5-6 membered heterocyclic group.

6. The 3-aminoindazole compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 5, characterized in that: R3 is selected from: H, -NR 10 R 11 ; R 10 , R 11 are independently selected from: C1-C3 alkyl, methoxy-substituted C1-C3 alkyl, dimethylamino-substituted C1-C3 alkyl, or, R 10 , R 11 Together with the nitrogen atom to which it is attached, it forms one or more R 12 a substituted or unsubstituted 5-6 membered heterocyclic group; Each R 12 Each of the following is independently selected from: H, C1-C3 alkyl, acetyl, methylsulfonyl, dimethylamino, oxetanyl, piperazinyl, C1-C3 alkyl-substituted piperazinyl, morpholinyl, piperidinyl, and tetrahydropyrrolyl.

7. The 3-aminoindazole compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 6, characterized in that: R3 is selected from: H, or any of the following groups:

8. The 3-aminoindazole compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to any one of claims 1 to 3, characterized in that: R1, R2, and R4 are independently selected from the group consisting of: H, C1-C3 alkyl, C1-C3 alkoxy, cyclopropyl, and halogen.

9. The 3-aminoindazole compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 8, characterized in that: R1 is selected from: H, C1-C3 alkyl, C1-C3 alkoxy, cyclopropyl, halogen; R2 is selected from: H, C1-C3 alkyl, C1-C3 alkoxy; R4 is selected from: H.

10. The 3-aminoindazole compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 9, characterized in that: R1 is selected from: H, methyl, halogen; R2 is selected from: H; R4 is selected from: H.

11. The 3-aminoindazole compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to any one of claims 1 to 3, characterized in that: R5 is selected from: H, methyl, ethyl.

12. The 3-aminoindazole compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to any one of claims 1 to 3, characterized in that: It has the structure shown in formula (IV):

13. The 3-aminoindazole compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 1, characterized in that: The 3-aminoindazole compound is selected from the following compounds:

14. Use of the 3-aminoindazole compound according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof in the preparation of a FGFR4 inhibitor.

15. Use of the 3-aminoindazole compound according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof in the preparation of a medicament for preventing and / or treating a transient proliferative disease caused by abnormal FGFR4 signaling pathway.

16. The use according to claim 15, characterized in that The transitional proliferative disease caused by abnormal FGFR4 signaling pathway is a tumor.

17. The use according to claim 16, characterized in that The tumors are: liver cancer, bile duct cancer, breast cancer, stomach cancer, and prostate cancer.

18. A pharmaceutical composition for preventing and / or treating tumors, characterized in that: The invention is prepared from active ingredients and pharmaceutically acceptable excipients, wherein the active ingredients include the 3-aminoindazole compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof.

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