CN111253314B - Vinyl sulfonamide substituted pyrazolyl benzamides - Google Patents

Abstract

The invention provides a vinyl sulfonamide substituted pyrazolyl benzamide compound. Specifically, the invention provides a compound shown in formula I or a pharmaceutically acceptable salt thereof, and a preparation method and application thereof. The compound of formula I has excellent FGFR inhibitory activity and can be used for preventing or treating diseases related to the activity or expression level of FGFR kinase, such as cancers.

Description

Vinyl sulfonamide substituted pyrazolyl benzamides

Technical Field

The invention belongs to the field of medicines, and particularly relates to a novel pyrazole compound, a preparation method of the compound, a pharmaceutical composition containing the compound, and application of the compound as an irreversible inhibitor of a Fibroblast Growth Factor Receptor (FGFR) in preventing and treating diseases such as cancer.

Technical Field

Fibroblast Growth Factors (FGFs) are a class of structurally related polypeptides consisting of 150-200 amino acids, of which there are approximately twenty-several members, involved in developmental regulation, and expressed in a variety of tissues. Fibroblast growth factors stimulate cell proliferation, migration and differentiation and play an important role in bone and limb development, wound healing, tissue repair, hematopoiesis, angiogenesis, and tumorigenesis.

The biological actions of fibroblast growth factors are mediated through the Fibroblast Growth Factor Receptor (FGFR), a receptor type protein tyrosine kinase (RPTK). The protein kinases all include an extracellular ligand binding domain, a single transmembrane domain and an intracellular tyrosine kinase domain which phosphorylate and activate downstream signaling pathways upon binding to fibroblast growth factor. Currently known FGFRs mainly comprise 4 subtypes: FGFR1, FGFR2, FGFR3 and FGFR4. Different subtypes of receptors have different affinities for different fibroblast growth factors.

The abnormal expression and/or activation of FGFR of tumor tissues are accompanied in the occurrence of various cancers through clinical findings, so that the FGFR is widely regarded as an important drug target for resisting tumors. Therefore, many medicinal chemists have attracted research on the FGFR in terms of medicinal chemistry, and a plurality of clinical compound molecules are born. FGFR inhibitor Erdafitinib of hadenberg is a reversible inhibitor, which has been approved for urothelial cancer, but the resulting drug resistance problem remains to be solved. It has recently been reported that the irreversible inhibitor TS120 can be used to treat drug resistance developed by reversible inhibitors. Therefore, the development of the FGFR irreversible inhibitor can meet the urgent clinical requirement.

Disclosure of Invention

The object of the present invention is to further develop new compounds with better FGFR inhibitory activity by structural improvement on the basis of the previous work. To achieve the object, in a first aspect of the present invention, there is provided a compound of formula (I):

in the formula R ^a 、R ^b 、R ^c 、R ^d Each independently of the others hydrogen, halogen, hydroxy, -C1-C6 alkoxy, -NR ¹ R ² Or C1-C6 alkyl; wherein said C1-C6 alkoxy and C1-C6 alkyl may be optionally substituted with one or more groups selected from: halogen, hydroxy, cyano, C1-C3 alkoxy, C1-C3 alkylthio, -NR ³ R ⁴ 、-CONR ⁵ R ⁶ ；

R ¹ 、R ² 、R ³ 、R ⁴ 、R ⁵ 、R ⁶ Each independently hydrogen, C1-C3 alkyl, C1-C3 alkoxy or C1-C3 alkylthio.

In another preferred embodiment, R ^a 、R ^b 、R ^c 、R ^d Each independently of the other is hydrogen, halogen or C1-C6An alkoxy group; wherein said C1-C6 alkoxy group may be optionally substituted with one or more groups selected from: halogen, hydroxy, cyano, C1-C3 alkoxy, C1-C3 alkylthio, -NR ³ R ⁴ 、-CONR ⁵ R ⁶ ；R ³ 、R ⁴ 、R ⁵ 、R ⁶ Each independently hydrogen or C1-C3 alkyl.

In another preferred embodiment, R ^a 、R ^b 、R ^c 、R ^d Each independently hydrogen, fluorine, chlorine or unsubstituted C1-C3 alkoxy.

In another preferred embodiment, R ^a 、R ^b 、R ^c 、R ^d Each independently hydrogen, fluorine, methoxy, ethoxy or isopropoxy.

In another preferred embodiment, R ^a 、R ^b 、R ^c 、R ^d Each independently hydrogen, fluorine or methoxy.

In another preferred embodiment, R ^a 、R ^b 、R ^c 、R ^d Each independently is hydrogen, fluoro or methoxy; and R is ^a 、R ^b 、R ^c 、R ^d At least two of which are hydrogen.

In another preferred embodiment, R ^a 、R ^b 、R ^c 、R ^d Each independently is hydrogen, fluoro or methoxy; and R is ^a 、R ^b 、R ^c 、R ^d Three of which are hydrogen.

In another preferred embodiment, the compound is selected from the group consisting of:

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in a second aspect of the present invention, there is provided a pharmaceutical composition comprising (a) the above-mentioned compound or a pharmaceutically acceptable salt thereof as an active ingredient, and (b) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition further comprises (c) a second active ingredient.

In a third aspect of the invention, the invention provides the use of the compound or the pharmaceutically acceptable salt thereof and a pharmaceutical composition in the preparation of a pharmaceutical composition for treating or preventing diseases related to the activity or expression amount of FGFR kinase and/or in the preparation of an FGFR kinase inhibitor.

In another preferred embodiment, the FGFR kinase is selected from one or more of the group consisting of: FGFR1, FGFR2, FGFR3 and FGFR4.

In another preferred embodiment, the disease associated with FGFR kinase activity or expression level is selected from the group consisting of: bladder cancer, liver cancer, brain cancer, breast cancer, colon cancer, kidney cancer, lung cancer, ovary cancer, pancreas cancer, prostate cancer, stomach cancer, cervical cancer, colon cancer, thyroid cancer, skin cancer, bile duct cancer, acute lymphocytic leukemia, B-cell lymphoma, burketts lymphoma, acute myelogenous leukemia, chronic myelogenous leukemia, promyelocytic leukemia, fibrosarcoma, rhabdomyoma, melanoma, seminoma, teratoma, neuroblastoma, glioma.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Detailed Description

The present inventors have made intensive studies and have unexpectedly found that a vinylsulfonamide group-substituted pyrazolyl benzamide compound has more excellent FGFR kinase inhibitory activity, particularly, FGFR1 and FGFR4 inhibitory activity, and thus is useful for treating or preventing diseases related to FGFR kinase, and have completed the present invention based on this finding.

Compounds of formula (I) and uses thereof

The present invention is a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof:

wherein the compounds of formula (I) may be converted into pharmaceutically acceptable salts, such as acid addition salts: such as hydrochloride, hydrobromide, phosphate, acetate, fumarate, maleate, tartrate, citrate, oxalate, methanesulfonate, p-toluenesulfonate; or an alkali metal salt: such as sodium or potassium salts.

The compounds of formula (I) may also exist as stereoisomers, etc., such as tautomers, geometric isomers, meso forms, racemates, enantiomers, diastereomers, or mixtures thereof, at ambient temperature.

The hydrogen atom in the compounds of formula (I) according to the invention may be present in the form of deuterium as its isotope, for example-CH ₃ Can be represented by-CD ₃ Form exists of-CH ₂ Can be in the form of a CD ₂ -in the form.

We have found that the compounds defined in the present invention, or pharmaceutically acceptable salts thereof, are potent anticancer agents, the properties of which are believed to result from modulation or inhibition of FGFR activity. The compounds of the invention are therefore expected to be useful in the treatment of diseases or medical conditions induced in whole or in part by FGFR, i.e. the compounds may be used to produce an FGFR inhibitory effect in a warm-blooded animal in need of such treatment. Preferably, said FGFR comprises: FGFR1, FGFR2, FGFR3 and FGFR4.

The compounds of the invention are expected to have a broad spectrum of anti-cancer properties, since uncontrolled expression or abnormal activation of FGFR has been observed in a number of human cancers, including but not limited to bladder, liver, stomach, breast, prostate and multiple myeloma. It is therefore expected that the compounds of the present invention will have anti-cancer activity against these cancers. Furthermore, it is expected that the compounds of the invention will have activity against leukemias, lymphoid malignancies and solid tumors, for example in tissue: such as carcinomas and sarcomas in the liver, kidney, bladder, prostate, breast and pancreas. In one embodiment, the compounds of the present invention are expected to advantageously delay the growth of primary and recurrent solid tumors, such as skin, colon, thyroid, lung and ovary. More specifically, the compounds of the present invention, or pharmaceutically acceptable salts thereof, are expected to inhibit the growth of tumors associated with FGFR, particularly those tumors whose growth and spread is significantly dependent on FGFR, including, for example, certain bladder, liver, stomach, breast and prostate tumors and multiple myeloma.

In a further aspect of the invention there is provided the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof, as defined herein, in the manufacture of a medicament for the treatment of: melanoma, papillary thyroid carcinoma, cholangiocarcinoma, colon cancer, ovarian cancer, lung cancer, leukemia, lymphoid malignancies, multiple myeloma; carcinomas and sarcomas in the liver, kidney, bladder, prostate, breast and pancreas; and primary and recurrent solid tumors of the skin, colon, thyroid, lung, and ovary.

Pharmaceutical composition

The present invention also provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, as defined herein, in association with a pharmaceutically acceptable adjuvant, diluent or carrier. Said pharmaceutical composition is for use in the production of an FGFR inhibitory effect or an anti-cancer effect in a warm-blooded animal such as man.

The present invention provides a pharmaceutical composition for use in the treatment of a warm-blooded animal such as man suffering from: melanoma, papillary thyroid carcinoma, cholangiocarcinoma, colon cancer, ovarian cancer, lung cancer, leukemia, lymphoid malignancies, multiple myeloma; carcinomas and sarcomas in the liver, kidney, bladder, prostate, breast and political glands; and primary and recurrent solid tumors of the skin, colon, thyroid, lung. The composition comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof as defined herein and a pharmaceutically acceptable diluent or carrier.

The compounds of formula (I) and their pharmaceutically acceptable salts may be used alone but are generally administered in the form of a pharmaceutical composition in which the compound of formula (I) or its pharmaceutically acceptable salt (active ingredient) is in association with a pharmaceutically acceptable adjuvant, diluent or carrier. The pharmaceutical composition may comprise 0.01-99wt% (weight percent), 0.05-80wt%, 0.10-70wt%, and/or even 0.10-50wt% of the active ingredient by weight of the total composition, depending on the mode of administration.

The present invention further provides a process for the preparation of a pharmaceutical composition according to the invention which comprises mixing a compound of formula (I) or a pharmaceutically acceptable salt thereof as defined herein with a pharmaceutically acceptable adjuvant, diluent or carrier.

The pharmaceutical compositions may be administered topically (e.g., to the skin or lung and/or airways), for example, in the form of creams, solutions, suspensions, hexafluoroalkane aerosols and dry powder formulations; or systemic administration, for example oral administration in the form of tablets, capsules, syrups, powders or granules; or gastrointestinal administration in the form of a solution or suspension; or subcutaneously; or rectal administration in the form of suppositories; or administered transdermally.

The compositions of the present invention may be obtained by conventional methods using conventional pharmaceutical excipients well known in the art. Thus, compositions intended for oral use may contain, for example, one or more coloring, sweetening, flavoring and/or preservative agents.

Suitable pharmaceutically acceptable excipients for the preparation of tablets include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate; granulating and disintegrating agents such as corn starch or alginic acid; binders such as starch; lubricants such as magnesium stearate, stearic acid or talc; preservatives such as ethyl or propyl p-hydroxybenzoate, and antioxidants such as ascorbic acid. The tablets may be uncoated or coated using conventional coatings and techniques well known in the art to improve disintegration and subsequent absorption of the active ingredient in the gastrointestinal tract, or to improve stability and/or appearance.

Compositions for oral use may be in the form of hard gelatin capsules wherein the active ingredient is mixed with an inert diluent, for example calcium carbonate, calcium phosphate or kaolin; or may be in the form of a soft gelatin capsule wherein the active ingredient may be mixed with water, or an oil: such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions typically contain the active ingredient in finely divided powder form and one or more suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as lecithin or condensation products of 1, 2-alkylene oxides with fatty acids (e.g. polyoxyethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols (e.g. heptadecaethyleneoxycetanol), or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and liver of hexitols such as polyoxyethylene sorbitan monooleate. The aqueous suspension may also contain one or more preservatives (e.g., ethyl or propyl parabens, antioxidants (e.g., ascorbic acid), coloring, flavoring and/or sweetening agents (e.g., sucrose, saccharin or aspartame)).

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may also contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water generally comprise the active ingredient and a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents have been exemplified by those mentioned above. Other excipients, such as sweetening, flavoring and coloring agents, may also be present.

The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin, or a mixture of any of these. Suitable emulsifying agents may be, for example, naturally-occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides (for example sorbitan monooleate) and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavoring and preservative agents.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, aspartame or sucrose, and may also contain analgesics, preservatives, flavouring and/or colouring agents.

The pharmaceutical compositions may also be in the form of a sterile injectable aqueous or oleaginous suspension, which may be formulated according to known methods using one or more of the suitable dispersing or wetting agents and suspending agents already mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a toxic, parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol.

Suppositories can be prepared by mixing the active ingredient with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Suitable excipients include, for example, cocoa butter and polyethylene glycols.

Topical formulations such as creams, ointments, gels, or aqueous or oily solutions or suspensions may generally be formulated using conventional methods well known in the art using conventional excipients or diluents for the active ingredient.

Compositions for insufflation may be administered, for example, as a finely divided powder having a mean particle size of 30 μm or less, either alone or diluted with one or more physiologically acceptable carriers such as lactose. The powder for insufflation is then placed in capsules containing, for example, 1-50mg of active ingredient, for use with a turbo-inhaler device (turbo-inhaler device), for example for insufflation of the known drug sodium cromoglycate.

Compositions for administration by inhalation may be in the form of conventional pressurized aerosols, which formulate the active ingredient as an aerosol comprising finely divided solids or liquid droplets. Conventional aerosol propellants such as volatile fluorinated hydrocarbons or hydrocarbons may be used and the aerosol device is conveniently capable of determining the amount of active ingredient.

The size of the dose of the compounds of the invention employed for therapeutic purposes will naturally vary according to the nature and severity of the condition, the age and sex of the animal or patient and the route of administration, according to well-known principles of medicine.

In general, the compounds of the invention are administered so as to obtain a daily dose of active ingredient per kg body weight, for example in the range from 0.1mg to 1000mg, and if desired in divided doses. However, the daily dosage will necessarily vary depending upon the host treated, the particular route of administration, and the severity of the condition being treated. Thus, the optimal dosage may be determined by the physician treating any particular patient. Generally, lower doses are administered when the parenteral route is used. Thus, for example, for intravenous administration, dosages in the range of, for example, 0.1mg to 30mg of active ingredient per kg of body weight will generally be used. Similarly, for administration by inhalation, dosages in the range of, for example, 0.1mg to 25mg of active ingredient per kg of body weight will generally be used. But oral administration is preferred. For example, formulations intended for oral administration in humans will generally contain from 0.1mg to 2g of active ingredient.

For further information on the formulation, route of administration and dosage regimen, reference may be made to the disclosure of Comprehensive Medicinal Chemistry (Corwin Hansch; chairman of Editorial Board).

Combination therapy

The anti-cancer treatments as defined hereinbefore may be used as monotherapy or as combination therapy, i.e. in addition to treatment with the compounds of the invention, together with conventional surgery or radiotherapy or chemotherapy. Such chemotherapy may include one or more of the following classes of antineoplastic agents (either included together as a second active ingredient in the pharmaceutical composition or packaged separately as a second active ingredient with the other ingredients):

(1) Other antiproliferative/antineoplastic agents and combinations thereof for oncology: for example alkylating agents (such as cisplatin, oxaliplatin, carboplatin, cyclophosphamide, mechlorethamine, melphalan, chlorambucil, busulfan, temozolomide and nitrourea), antimetabolites (such as gemcitabine and antifolates such as fluoropyrimidines such as 5-fluorouracil and tegafur, raltitrexed, methotrexate, cytarabine and hydroxyurea), antitumor antibiotics (such as anthracyclines such as doxorubicin, bleomycin, doxorubicin, daunorubicin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin), antimitotics (vinca alkaloids such as vincristine, vinblastine, vindesine and vinorelbine, taxanes such as paclitaxel and docetaxel and a polokinase inhibitor), and topoisomerase inhibitors (podophyllotoxins such as etoposide and teniposide, ambridine, topotecan and camptothecin);

(2) A cell growth inhibitor: antiestrogens (such as tamoxifen, fulvestrant, toremifene, raloxifene, droloxifene and iodoxyfene), antiandrogens (such as bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH antagonists or LHRH agonists (such as goserelin, leuprorelin and buserelin), progestogens (such as megestrol acetate), aromatase inhibitors (such as anastrozole, letrozole, fluorochlorazole and exemestane) and 5 x-reductase inhibitors such as finasteride;

(3) Drugs that inhibit cancer cell invasion: for example inhibitors of the c-Src kinase family such as 4- (6-chloro-2,3-methylenedioxyanilino) -7- [2- (4-methylpiperazin-1-yl) ethoxy ] -5-tetrahydropyran-4-yloxyquinazoline (AZD 0530; international patent application WO 01194341) and N- (2-chloro-6-methylphenyl) -2- {6- [4- (2-hydroxyethyl) piperidin-1-yl ] -2-methylimidazol-4-ylamino } thiazole-5-carboxamide (dasatinib, BMS-354825, J.Med.Chem.,2004,47,6658-6661), and metalloproteinase inhibitors such as marimastat, and inhibitors of the urokinase plasminogen activator receptor function or antibodies to Heparanase;

(4) Growth factor function inhibitors: including growth factor antibodies and growth factor receptor antibodies (e.g., anti-erbB 2 antibody trastuzumab [ Herceptin ] ^TM ]anti-EGFR antibody panitumumab, anti-erbB 1 antibody cetuximab [ Erbitux, C225]) And any growth factor or growth factor receptor antibody disclosed by Stem et al in clinical reviews in oncology/hematology,2005, vol 54, p 11-29), which also include tyrosine kinase inhibitors such as epidermal growth factor family inhibitors (e.g., EGFR family tyrosine kinase inhibitors such as N- (3-chloro-4-fluorophenyl) -7-methoxy-6- (3-morpholinopropoxy) quinazolin-4-amine (gefitinib, AZD 1839), N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxy-5- ((4- (1-methylamino) ethyl) (methyl) amino)-1H-indol-3-yl) pyrimidin-2-yl) amino) phenyl) acrylamide (AZD 9291), N- (3-ethylblockylphenyl) -6, 7-bis (2-methoxyethoxy) quinazolin-4-amine (erlotinib, OSI-774) and 6-acylamido-N- (3-chloro-4-fluorophenyl) -7- (3-morpholinopropoxy) quinazolin-4-amine (CI 1033)), erbB2 tyrosine kinase inhibitors such as lapatinib, platelet derived growth factor family inhibitors such as imatinib, serine/threonine kinase inhibitors (e.g. Ras/Raf signaling inhibitors such as farnesyl transferase inhibitors, e.g. sorafenib (BAY 43-9006)), cell signaling inhibitors by MEK and/or AKT kinases, hepatocyte growth factor family inhibitors, c-kit inhibitors, abl kinase inhibitors, insulin like growth factor kinase inhibitors, aurora kinase inhibitors (e.g. AZD1152, VX 7358, VX-2-yl) amino) phenyl) acrylamide inhibitors (AZD 9291), N- (R) inhibitors such as CDK/x kinase inhibitors, VX-kinase inhibitors, IGF-4-kinase inhibitors such as IGF/CDK/R764 and/CDK inhibitors, IGF/R766;

(5) Anti-angiogenic agents: for example, anti-angiogenic agents which inhibit the action of vascular endothelial growth factor (e.g., anti-VEGF antibody Bevacizumab [ Avastin ] ^TM ]And VEGF receptor tyrosine kinase inhibitors such as 4- (4-bromo-2-fluoroanilino) -6-methoxy-7- (1-methylpiperidin-4-ylmethoxy) quinazoline (AZD 6474; example 2 in WO 01132651), 4- (4-fluoro-2-methylindol-5-yloxy) -6-methoxy-7- (3-pyrrolidin-1-ylpropoxy) quinazoline (AZD 2171; example 240 in WO 00/47212), vatalanib (PTK 787; WO 98/35985) and SU 1123548 (sunitinib; WO 11201160814), such as the compounds disclosed in International patent applications WO 97/22596, WO 97/30035, WO 97/32856 and WO 98/856 54 and compounds which act by other mechanisms (such as linoglossil, integrin α vb3 functional inhibitors and angiostatin);

(6) A vascular damaging agent: for example combretastatin A4 and the compounds disclosed in International patent applications WO99/02166, WO 00/40529, WO 00/41669, WO 01192224, WO 02/04434 and WO 02/08213;

(7) Antisense therapy: such as therapies directed against the above targets, such as ISIS 2503 (anti-ras antisense);

(8) Gene therapy: including, for example, methods of replacing aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2, GDEPT (gene-directed enzyme prodrug therapy) methods such as methods using cytosine deaminase, thymidine kinase, or bacterial nitroreductase, and methods of increasing a patient's resistance to chemotherapy or radiation therapy such as multidrug resistance gene therapy; and

(9) And (3) immunotherapy: including, for example, immune checkpoint blockade methods such as PD-1 antibodies such as Opdivo and Keytruda and PD-L1 antibodies such as Tecntriq, chimeric antigen receptor T-cell immunotherapy (Chimeric antigen receptor T-cell immunotherapy), ex vivo and in vivo therapies to increase the immunogenicity of patient tumor cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor, methods to reduce T-cell anergy, methods to transfect dendritic cells with transfected immune cells such as cytokines, methods to transfect tumor cell lines with cytokines, and methods to transfect tumor cell lines with anti-idiotypic antibodies.

The main advantages of the invention include:

a compound of formula I or a pharmaceutically acceptable salt thereof is provided having a novel structure. The compound has more excellent FGFR kinase inhibition activity and hepatoma cell Hep3B inhibition activity, and can be used as an FGFR kinase inhibitor. The inhibitor can inhibit the activity of various FGFR kinases, especially FGFR1 and FGFR4 kinases, at extremely low concentration.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, the starting materials are all commercially available.

Wherein, unless otherwise specified:

(1) The operation is carried out at room or ambient temperature, i.e. at a temperature in the range of 18-25 ℃;

(2) After drying the organic solution over anhydrous sodium sulfate, evaporation of the solvent was carried out at a bath temperature of up to 60 ℃ under reduced pressure (600-4000Pascals, 4.5-30 mmHg) using a rotary evaporator;

(3) The column chromatography is to carry out chromatography on a silica gel column; thin Layer Chromatography (TLC) is chromatography on silica gel plates;

(4) Typically, the course of the reaction is monitored by TLC, and the reaction times are given for illustration only;

(5) The final product has good proton Nuclear Magnetic Resonance (NMR) spectrum and/or mass spectrum data;

(6) The yields given are for illustration only and are not necessarily yields obtained by continuous process modifications; the preparation can be repeated if more raw materials are needed;

(7) Unless otherwise indicated, NMR data are given in the form of delta values for the primary labeled protons, measured in parts per million (ppm) relative to the internal standard Tetramethylsilane (TMS), in the corresponding indicated deuterated solvents at 400 MHz;

(8) Chemical symbols have their usual meanings; SI units and symbols are used;

(9) The solvent ratios given are volume: volume ratio (v/v); and

(10) The following abbreviations are used:

THF tetrahydrofuran

DMF N, N-dimethylformamide

EtOAc ethyl acetate

DCM dichloromethane

DMSO dimethyl sulfoxide

DIPEA N, N-diisopropylethylamine

PBS phosphate buffer

ADP adenosine diphosphate (ADP-ADP)

ATP adenosine triphosphate

BSA bovine serum albumin

(11) Compounds were named using the appropriate naming software chembioffice 2014, using IUPAC naming rules.

An intermediate A:3- (3, 5-Dimethoxyphenethyl) -1H-pyrazolyl-5-amine

The method comprises the following steps: in a circlePlacing 2000mg (9.5 mmol) of 3- (3, 5-dimethoxyphenyl) propionic acid into a bottom burning bottle, adding 15ml of ethanol for dissolving, then dropwise adding 3ml of concentrated sulfuric acid, placing the mixture into an oil bath at 90 ℃ for reflux reaction for 6 hours, then spin-drying the reaction solution under reduced pressure, adding ethyl acetate for dissolving, then using saturated NaHCO for dissolving ₃ Solution washing, saturated NaCl solution washing, anhydrous Na ₂ SO ₄ And (5) drying. Column chromatography on silica gel (gradient elution, petroleum ether: ethyl acetate =30:1 to 5) gave ethyl 3- (3, 5-dimethoxyphenyl) propionate (1621mg, 73%) as a yellow transparent liquid.

Step two: a round bottom flask was charged with a stirring bar, N ₂ 8.6ml of THF was added under protection, the round-bottomed flask was placed at-78 ℃ and stirred for 10min, then 5.8ml of n-BuLi (2.5M, 15.4 mmol) was added, 690mg (16.8 mmol) of acetonitrile was dissolved in 16ml of THF and added dropwise to the reaction solution, and after stirring for 1h, 1621mg of the compound ethyl 3- (3, 5-dimethoxyphenyl) propionate (7 mmol) was dissolved in 2ml of THF and then added dropwise to the reaction solution. Stirring at-78 deg.C for 3 hr, slowly heating to room temperature, adding acetic acid to quench reaction, extracting with ethyl acetate, washing the obtained solution with saturated NaCl, and adding anhydrous Na ₂ SO ₄ And (5) drying. Column chromatography on silica gel (gradient elution, petroleum ether: ethyl acetate =30:1 to 5) gave 5- (3, 5-dimethoxyphenyl) -3-oxopentanenitrile (910mg, 53%) as a yellow powder.

Step three: in a round-bottom flask was placed 820mg (3.5 mmol) of 5- (3, 5-dimethoxyphenyl) -3-oxovaleronitrile, N ₂ Adding 35ml ethanol for dissolution under protection, then adding 1095mg (17.5 mmol) hydrazine hydrate dropwise, and heating to 80 ℃ for reaction. After 24H, the reaction mixture was spin-dried under reduced pressure, extracted with ethyl acetate and the resulting solution was taken up in H ₂ O washing, saturated NaCl solution washing, anhydrous Na ₂ SO ₄ And (5) drying. Column chromatography on silica gel (gradient elution, dichloromethane: methanol =200:1 to 50) gave intermediate a (760mg, 88%) as a yellow powder.

Intermediate B1: synthesis of ethyl 2- (vinylsulfonamido) benzoate

Adding a stirrer N into a round-bottom flask ₂ Adding 2g (12.1 mmol) of ethyl anthranilate into a reaction bottle under protection, then adding 12ml of anhydrous dichloromethane for dissolution, placing the reaction solution into an ice-water bath for stirring for 10min, then adding 1.8g (18.15 mmol) of triethylamine, then adding 1.98g (15.7 mmol) of ethylene sulfonyl chloride, continuing stirring for 5H in the ice-water bath, then raising the temperature to room temperature, adding H, and stirring ₂ O quench the reaction, neutralize with 10% HCl, extract with ethyl acetate, and the resulting organic layer was saturated NaHCO ₃ Solution washing, saturated NaCl solution washing, anhydrous Na ₂ SO ₄ And (5) drying. Column chromatography on silica gel (gradient elution, petroleum ether: ethyl acetate =50:1 to 8) gave the title intermediate B1 (1.94 g, yield 63%) as a white solid. LCMS m/z =256.1[ m + H ]] ⁺ . ¹ H NMR(400MHz,DMSO-d6)δ11.35(br.s,1H),7.36(m,1H),7.01(m,1H),6.94(m,1H),6.58(m,1H),6.44(m,1H),6.18(m,1H),5.61(m,1H),4.35(q,J＝6.4,2H),1.29(t,J＝6.4,3H).

The intermediates B2 to B10 were obtained as described above using ethyl anthranilates having different substituents as starting materials.

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Example 1: synthesis of N- (5- (3, 5-dimethoxyphenethyl) -1H-pyrazol-3-yl) -2- (vinylsulfonamido) benzamide

Into a round-bottomed flask was placed 152mg (0.62 mmol) of intermediate A, N ₂ Adding 4ml of dry toluene for dissolving under protection, stirring for 10min in ice bath, adding 0.58ml of trimethylaluminum solution (1.6M, 0.92mmol) dropwise, reacting for 1h, adding 235mg (0.92 mmol) of intermediate B1, stirring for 20min, removing ice bath, and 65 minAfter reacting at the temperature of 24 hours, cooling and dropwise adding H ₂ Quenching the reaction, extracting with ethyl acetate, washing the obtained solution with saturated NaCl solution, and removing anhydrous Na ₂ SO ₄ And (5) drying. Column chromatography on silica gel (gradient elution, dichloromethane: methanol =250:1 to 100) gave N- (5- (3, 5-dimethoxyphenethyl) -1H-pyrazol-3-yl) -2- (vinylsulfonamido) benzamide (compound 1,116mg, 41%) as a light yellow powdery solid. LCMS m/z =457.2[ m ] +H] ⁺ ； ¹ HNMR(400MHz,DMSO-d6)δ11.35(br.s,2H),10.56(s,1H),7.36(m,1H),7.01(m,1H),6.94(m,1H),6.75(m,1H),6.58(m,1H),6.43(m,1H),6.25(m,2H),6.18(m,1H),6.08(m,1H),5.62(m,1H),3.68(s,6H),2.97(m,2H),2.84(m,2H)。

Following the above procedure, intermediates B2-B10 react with intermediate a to give compounds 2-10:

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the reference substances D1-D4 can be prepared according to the synthetic method in CN110317173

Enzymatic activity assay for FGFR inhibitors

The inhibition of the FGFR enzyme activity by the compound is detected by a Caliper technology. The reagents used in the model were as follows: FGFR4 (qiagen, cat.no. PR4380A, lot.no. 1856505a); FGFR1 (Carna, cat.No. 08-133, lot.No. 12CBS-0123K); p22 polypeptide (GL Biochem, cat. No.112393, lot. No.

P170622-SL 112393); ATP (Sigma, cat. No. A7699-1G, CAS No. 987-65-5); 96-well plates (corning, cat. No.3365, lot. No. 22008026); 384-well plates (kangning, cat. No.3573, lot. No. 12608008).

The specific operation is as follows:

1. 1 Xkinase buffer (20mM HEPES, pH 7.5,0.0015% Brij-35) and reaction termination buffer (100mM HEPES, pH 7.5,0.015% Brij-35,0.2% Coating Reagent (Coating Reagent) #3,50mM EDTA) were prepared

2. Compounds were first serially diluted in 5% DMSO solution and 5 μ L compound solution was added to 384-well plates with a maximum final concentration of 1 μ M of compound, three-fold diluted, 10 concentrations.

3. mu.L of kinase solution was added to the 384 well plate and incubated at room temperature for 10 minutes.

4. mu.L of P22 polypeptide and ATP solution were added to the 384-well plate, and after a specific reaction time at 28 ℃, 25. Mu.L of reaction termination buffer was added.

5. Data were collected using Caliper readings and inhibition was calculated:

inhibition rate = (max-conversion)/(max-min) × 100.

Where max is the DMSO reference, conversion is the compound treatment reading, and min is the maximum inhibition reference.

Compound IC was calculated using XLFit excel add-in version 5.4.0.8 software ₅₀ The value is obtained.

The results of in vitro inhibition assays of FGFR1 and FGFR4 with the example compounds and the control compounds are shown in table 1:

TABLE 1 inhibitory Activity on FGFR1 and FGFR4

Compound numbering	FGFR1(nM)	FGFR4(nM)
			1	1.4	1.9
2	1.1	1.6
			3	0.63	0.74
4	1.5	1.8
			5	0.81	0.93
6	5.3	6.1
			7	0.84	0.92
8	4.5	3.8
			9	3.6	4.1
10	3.0	4.2
			D1	52	＞1000
D2	3.4	3.5
			D3	19	11
D4	2.2	2.3

From the results in table 1, it can be seen that when the phenyl group linked to the amide is substituted with a vinylsulfonamide group, the inhibitory activity of the compound on FGFR1 and FGFR4 is significantly improved, and the introduction of the vinylsulfonamide group improves the inhibitory activity of the compound on FGFR1 and FGFR4 as a whole compared to the acrylamide substituent. The research also finds that the type and the substitution position of the substituent have obvious influence on the activity of the compound, and particularly when the substituent is in the meta position of the vinyl sulfonamide group, the activity is obviously improved. Furthermore, the activity is significantly increased when the substituent is fluorine compared to methoxy.

Human hepatoma cell Hep3B survival assay

The human liver cancer Hep3B cell strain is derived from ATCC. The cells were cultured in DMEM liquid medium, and fetal bovine serum (10% FBS) and penicillin-streptomycin (100,000U/L) were additionally added. The cells were maintained in culture at 37 ℃, 95% humidity and 5% carbon dioxide. For the experiment, hep3B cells were plated in 96-well plates at a density of 3000 cells per well, with a cell suspension volume of 100 μ L per well, and the cells were cultured overnight to allow the cells to attach. The following day, each compound was diluted in DMSO in a three-fold gradient, and 1 μ Ι _ compound DMSO solution was added to the cell culture medium, while 1 μ Ι _ DMSO was used as a control, with three parallel side wells for each concentration of compound. Cells were then placed in a 37 ℃ incubator and after 72 consecutive hours of compound treatment, 50 μ L of CellTiter-Glo (Promega, madison WI) was added to the cell culture medium and the Relative Luminescence Units (RLU) of each well were determined and cell viability and compound activity (IC 50) were calculated. The results of the inhibitory activity of the example compounds on Hep3B cells are shown in table 2 below:

TABLE 2 inhibitory Activity on Hep3B cells

Compound number	IC50(nM)
		1	2.1
2	3.0
		3	0.8
4	1.5
		5	0.5
6	2.0
		7	0.6
8	4.2
		9	1.3
10	8.0
		D3	21.3

From the results in table 2, it can be seen that the vinylsulfonamide group substituted compound also has strong inhibitory activity on Hep3B cells, and the inhibitory activity is also obviously improved compared with the acrylamide substituted compound.

All documents cited in the present application are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.

Claims (5)

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof:

the compound is selected from the group consisting of:

2. a pharmaceutical composition, comprising: (a) A compound according to claim 1, or a pharmaceutically acceptable salt thereof, as an active ingredient, and (b) a pharmaceutically acceptable carrier.

3. The pharmaceutical composition of claim 2, wherein said pharmaceutical composition further comprises (c) a second active ingredient.

4. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 2 for: (1) Preparing a pharmaceutical composition for treating or preventing diseases related to the activity or expression amount of FGFR kinase; (2) preparing FGFR kinase inhibitor.

5. The use of claim 4, wherein the disease associated with FGFR kinase activity or expression is selected from the group consisting of: bladder cancer, liver cancer, brain cancer, breast cancer, colon cancer, kidney cancer, lung cancer, ovary cancer, pancreas cancer, prostate cancer, stomach cancer, cervical cancer, thyroid cancer, skin cancer, bile duct cancer, acute lymphocytic leukemia, B-cell lymphoma, burketts lymphoma, acute myelogenous leukemia, chronic myelogenous leukemia, promyelocytic leukemia, fibrosarcoma, rhabdomyoma, melanoma, seminoma, teratoma, neuroblastoma, glioma.