CN113527311B - FGFR4 inhibitor, composition and application thereof in preparation of medicines - Google Patents

Abstract

The invention provides an FGFR4 inhibitor which takes 3, 4-dihydropyrimidine [4,5-d ] pyrimidine-2 (1H) -ketone as a parent nucleus and has a covalent structure. 9 specific compounds are provided in the examples, and kinase inhibitory activity tests are carried out on the 9 compounds, wherein the half inhibitory concentration of LX08 on the kinase inhibitory activity of FGFR4 is only 7nM, and the half inhibitory concentration is lower than that of FIIN-2 of a specific activity control, so that the compound has potential application prospects. In addition, through MALDI-TOF mass spectrometry combination experiments on the synthesized compounds, the compounds such as LX01, LX05, LX06, LX07 and LX08 are found to be only capable of being covalently bonded with Cys552 in FGFR4 and not capable of being covalently bonded with Cys477 in FGFR4, and the compound such as LX09 is an FGFR4 inhibitor capable of being covalently bonded with two Cys552 and Cys477 of cysteine in LX FGFR 4.

Description

FGFR4 inhibitor, composition and application thereof in preparation of medicines

Technical Field

Described herein are compounds, methods of making the compounds, and methods of using the compounds and compositions to inhibit tyrosine kinase activity.

Background

Fibroblast Growth Factor Receptors (FGFRs) are a family of receptor tyrosine kinases, including FGFR1, FGFR2, FGFR3, FGFR4 and the high affinity receptors for 18 different FGF ligands. These ligand-receptor combinations modulate a variety of signaling and endocrine activities during human tissue development. Genetic alterations of FGFR, including mutations, fusions, and gene amplifications, result in aberrant signaling pathways that activate and drive the growth of cancer cells. Researchers have detected genetic alterations in FGFR in a variety of cancer types, including breast cancer, liver cancer, squamous non-small cell lung cancer, squamous head and neck cancer, cholangiocarcinoma, and the like. Clinical validation of FGFR as a therapeutic target has been demonstrated in bladder cancer, liver cancer, lung cancer, breast cancer, and gastric cancer.

In recent years, aberrant fibroblast growth factor receptor 4(FGFR4) signaling has been identified as a major driver of HCC tumorigenesis and progression. FGFR4 is the most highly expressed isoform in hepatocytes, with its ligand FGF19 exclusively binding to FGFR4 with the co-receptor β -Klotho to regulate hepatocyte proliferation, and FGF19 proteinexcess increasing the probability of proliferation and invasion of HCC cell lines. The liver cancer heterotypic transplanted mouse with high FGFR4 can effectively eliminate liver cancer in mouse model by inhibiting the generation of FGF19-FGFR4 or using FGFR4 antibody. Clinical studies have shown that half of all HCC patients have FGFR4 overexpressed, and most of HCC patients have both FGF19 and FGFR4 upregulated, whereas FGF19 levels were positively correlated with tumor size and post-hepatectomy recurrence, and HCC patients with FGF19 overexpressed survived five years shorter than HCC patients with low expression of FGF 19. Therefore, FGFR4 selective inhibitors can be developed to treat cancer patients driven by aberrant FGFR4 signaling.

The FGFR4 plays an important role in cancer cell metastasis and drug resistance, and the FGFR irreversible inhibitor with good FGFR4 inhibition effect has wide application prospect. Among the covalent inhibitors with high potency and selectivity for inhibition of FGFR4, BLU9931, BLU-554, and H3B-6527 all covalently bound to the Cys552 thiol group of the hinge region of the FGFR4 protein, whereas PRN1371, fin-2, and TAS-120 covalently bound to Cys477 in the p-loop of the FGFR4 protein, all covalently bound to only one cysteine residue therein, no inhibitor has been found that can covalently bind to both cysteine residues in the FGFR4 protein. And some of these irreversible inhibitors produced resistance mutations during clinical trials, such as Cys552 mutation in hepatoma cells found in phase I clinical trials of BLU-554. The FGFR4 double covalent inhibitor capable of being covalently bound with two cysteines in FGFR4 protein simultaneously is developed through strategies of drug combination, group replacement, carbon chain growth, structure simplification and the like.

Disclosure of Invention

The invention aims to provide a structurally optimized FGFR4 inhibitor which has excellent effect of inhibiting fibroblast growth factor receptor 4.

The invention also provides an inhibitor of FGFR4, which contains an FGFR4 double covalent inhibitor capable of being covalently combined with two cysteines (Cys477 and Cys552) in an FGFR4 protein at the same time.

The compounds of the present invention have the structure of formula I:

formula I

Wherein the content of the first and second substances,R₁refers to a moiety capable of forming a covalent bond with a nucleophile; r₂Is an aryl or heterocyclic group; l is- [ C (R5) (R6)]q-, wherein R5 and R6 are each independently H or C1-C6 alkyl, wherein q is 1-3; wherein A is phenyl, R₃Is hydrogen or methyl on the phenyl radical A.

In one embodiment, R₁Is an acryloyl group.

In one embodiment, L is independently C1-C3 alkyl.

In one embodiment, R₂Is phenyl.

In the synthesis examples of the present invention, the following compounds were synthesized:

n- (4- ((7- ((2-acrylamido-6-methylphenyl) amino) -3- (3, 5-dimethoxyphenyl) -2-)

Oxy-3, 4-dichloropyrimidine [4,5-d ] pyrimidin-1 (2H) -yl) methyl) phenyl) acrylamide;

2-cyano-N- (4- ((7- ((2- (2-cyano-3-methyl-2-butenamido) -6-methylphenyl) amino) -3- (3, 5-dimethoxyphenyl) -2-oxo-3, 4-dihydropyrimidin [4,5-d ] pyrimidin-1 (2H) -yl) methyl) phenyl) -3-methyl-2-butenamide;

n- (4- ((3- (3, 5-dimethoxyphenyl) -7- ((2-methyl-6- (ethenesulfonamido) phenyl) amino) -2-oxo-3, 4-dihydropyrimidine [4,5-d ] pyrimidin-1 (2H) -yl) methyl) phenyl) ethenesulfonamide;

2-chloro-N- (4- ((7- ((2- (2-chloroacetylamino) -6-methylphenyl) amino) -3- (3, 5-dimethylphenyl) -2-oxo-3, 4-dihydropyrimidine [4,5-d ] pyrimidin-1 (2H) -yl) methyl) phenyl) acetamide;

n- (3- ((7- ((2-acrylamido-6-methylphenyl) amino) -3- (3, 5-dimethoxyphenyl) -2-oxo-3, 4-dichloropyrimidine [4,5-d ] pyrimidin-1 (2H) -yl) methyl) phenyl) acrylamide;

n- (2- ((8(3- (4-acryloylpiperazin-1-yl) propyl) -6- (3, 5-dimethoxyphenyl) -7-oxo-5, 6,7, 8-tetrahydropyrimidinyl [4,5-d ] pyrimidin-2-yl) amino) -3-methylphenyl) acrylamide;

n- (2- ((8- (4-acrylamidobenzyl) -6- (3, 5-dimethoxyphenyl) -7-oxo-5, 6,7, 8-tetrahydropyrimidinyl [4,5-d ] pyrimidin-2-yl) amino) phenyl) acrylamide;

n- (2- ((8- (4-acrylamidophenethyl) -6- (3, 5-dimethoxyphenyl) -7-oxo-5, 6,7, 8-tetrahydropyrimidinyl [4,5-d ] pyrimidin-2-yl) amino) phenyl) acrylamide;

n- (2- ((8- (3- (4-acryloylpiperazin-1-yl) propyl) -6- (3, 5-dimethoxyphenyl) -7-oxo-5, 6,7, 8-tetrahydropyrimidinyl [4,5-d ] pyrimidin-2-yl) amino) phenyl) acrylamide;

the compounds according to the invention are highly potent covalent inhibitors of FGFR4 specificity.

Drawings

Fig. 1 is a comparison of mass spectra of compound LX01 before and after binding to the two cysteines (Cys477 and Cys552) of FGFR 4;

fig. 2 is a comparison of mass spectra of compound LX05 before and after binding to the two cysteines (Cys477 and Cys552) of FGFR 4;

fig. 3 is a comparison of mass spectra of compound LX06 before and after binding to the two cysteines (Cys477 and Cys552) of FGFR 4;

fig. 4 is a comparison of mass spectra of compound LX07 before and after binding to the two cysteines (Cys477 and Cys552) of FGFR 4;

fig. 5 is a comparison of mass spectra of compound LX08 before and after binding to the two cysteines (Cys477 and Cys552) of FGFR 4;

fig. 6 is a mass spectrum comparison of compound LX09 before and after binding to the two cysteines (Cys477 and Cys552) of FGFR 4.

Detailed Description

Unless otherwise specified, the term "cycloalkyl" as used herein alone or as part of another group includes saturated or partially unsaturated (containing 1 or more double bonds) cyclic hydrocarbon groups containing 1 to 2 rings, preferably 3 to 10 carbons, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and cyclodecyl. "substituted cycloalkyl" includes cycloalkyl optionally substituted with one or more substituents such as halogen, alkyl, alkoxy, hydroxy, aryl, aryloxy, arylalkyl, cycloalkyl, alkylamido, alkanoylamino, oxo, acyl, arylcarbonylamino, amino, nitro, cyano, thiol, and/or alkylthio and/or any of the substituents included in the definition of "substituted alkyl".

Unless otherwise specified, the terms "aryl" or "Ar" as used herein alone or as part of another group refer to monocyclic and polycyclic aromatic groups containing 6 to 10 carbons in the ring portion (e.g., phenyl or naphthyl, including 1-naphthyl and 2-naphthyl) and may optionally include one to three additional rings fused to carbocyclic or heterocyclic rings (e.g., aryl, cycloalkyl, heteroaryl, or cycloheteroalkyl rings).

As used herein, unless otherwise indicated, the term "heterocycle" or "heterocycle" means an unsubstituted or substituted stable 5-to 10-membered monocyclic ring system, which may be saturated or unsaturated, consisting of carbon atoms and 1 to 4 heteroatoms selected from N, O or S, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. Examples of such heterocyclic groups include piperidinyl, piperazinyl, oxopiperazinyl, pyrrolyl, pyrrolidinyl, furanyl, thienyl, pyrazolyl, pyrazolidinyl, imidazolyl.

It is also within the scope of the present invention that the compounds of formula I may exist as pharmaceutically acceptable salts. If the compounds of the formula I have, for example, at least one basic center, they can form acid addition salts. These are formed, for example, using strong inorganic acids, such as mineral acids, for example sulfuric acid, phosphoric acid or hydrohalic acids, strong organic carboxylic acids, such as unsubstituted or substituted (for example by halogen) alkane carboxylic acids of 1 to 4 carbon atoms, for example acetic acid, such as saturated or unsaturated dicarboxylic acids, for example oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid or terephthalic acid, such as hydroxycarboxylic acids, for example ascorbic acid, glycolic acid, lactic acid, malic acid, tartaric acid or citric acid, such as amino acids (for example aspartic acid or glutamic acid or lysine or arginine), or benzoic acid, or organic sulfonic acids, such as unsubstituted or substituted (for example by halogen) (C1-C4) alkyl or aryl sulfonic acids, for example methyl or p-toluene-sulfonic acid. If desired, it is also possible to additionally derivatize a basic center to form the corresponding acid addition salts.

The compounds of the invention may be used in the form of a pharmaceutical composition comprising a therapeutically effective amount of a compound of the invention as defined herein and a pharmaceutically acceptable carrier or diluent.

The medicaments of the invention are useful for the treatment of FGR4 mediated disorders, in particular cancer. These cancers include hepatocellular, bladder, breast, cervical, colorectal, endometrial, gastric, head and neck, kidney, liver, ovarian, prostate, esophageal, gall bladder, pancreatic, lung, mesothelioma, testicular, squamous cell carcinoma, thyroid, skin, leukemia, multiple myeloma, chronic lymphocytic lymphoma, adult T-cell leukemia, B-cell lymphoma, acute myelogenous leukemia, hodgkin's lymphoma, non-hodgkin's lymphoma, waldenstrom's macroglobulinemia, hairy cell lymphoma, burkitt's lymphoma, glioblastomas, melanoma, and rhabdomyosarcoma.

The form of the pharmaceutical composition according to the invention may be adapted to be administered to a patient in need of treatment, e.g. a mammal such as a human patient, by a variety of routes of administration, e.g. oral, intranasal, intraperitoneal, or parenteral, by intravenous, intramuscular, topical or subcutaneous routes, or by injection into a tissue. Such compositions and formulations should contain at least 0.01% of one or more of the compounds of the invention. The percentage of the compositions and formulations can, of course, vary and can be, for example, from about 0.05% to about 2% by weight of a given unit dosage form. The amount of the compound in such therapeutically useful compositions is such that an effective dosage level is obtained.

The compounds of the invention may be administered systemically, e.g., orally, in combination with a pharmaceutically acceptable carrier, e.g., an inert diluent or an assimilable edible carrier, or by inhalation or insufflation. They may be enclosed in hard or soft shell capsules, may be compressed into tablets, or may be mixed directly with food for consumption by the patient. For oral therapeutic administration, the compounds of the present invention may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The compounds may be combined with a fine inert powder carrier and inhaled by the patient or insufflated. Such compositions and formulations should contain at least 0.1% of one or more compounds of the invention.

Tablets, troches, pills, capsules and the like may also comprise: binders such as tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; disintegrating agents such as corn starch, potato starch, alginic acid, and the like; lubricants such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame, or an aromatic agent such as peppermint, oil of wintergreen or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a vegetable oil or polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For example, tablets, pills, or capsules can be coated with gelatin, wax, shellac, sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and a flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the compounds of the present invention may be incorporated into sustained release formulations and devices. For example, the compounds may be incorporated into time release capsules, time release tablets, and time release pills.

The compounds of the invention may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the compounds can be prepared in water, optionally mixed with a non-toxic surfactant. Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders. The liquid carrier can be a solvent or liquid medium including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), vegetable oils, non-toxic glycerides, and suitable mixtures thereof.

For topical administration, the compounds of the present invention may be used in pure form. However, it is generally desirable to administer them to the skin as a composition or formulation, together with a dermatologically acceptable carrier, which may be solid or liquid.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, bauxite, and the like. Other solid supports include non-toxic polymeric nanoparticles or microparticles. Useful liquid carriers include water, alcohols or glycols or water/alcohol/glycol blends in which the compounds of the present invention can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as perfumes and additional antimicrobial agents can be added to optimize performance for a given use. The resulting liquid composition can be applied from an absorbent pad, used to impregnate bandages and other dressings, or sprayed onto the affected area using a pump-type or aerosol sprayer.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be used with the liquid carrier to form spreadable pastes, gels, ointments, soaps, and the like, for direct application to the skin of a user.

The concentration of the compound in a liquid composition, such as a lotion, can be from about 0.1 to about 25% by weight, or from about 0.5 to about 10% by weight. The concentration in a semi-solid or solid composition such as a gel or powder may be from about 0.1 to about 5% by weight, or from about 0.5 to about 2.5% by weight.

The amount of a compound of the invention required for use in treatment will vary not only with the particular salt selected, but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will ultimately be at the discretion of the attendant physician or clinician.

The effective dosage and route of administration of the agents of the invention are conventional. The precise amount (effective dose) of an agent will vary from patient to patient, depending upon, for example, the type, age, weight, and general or clinical state of the patient, the severity or mechanism of any condition being treated, the particular agent or carrier employed, the method and extent of administration, and the like. Therapeutically effective dosages can be determined empirically by conventional procedures known to those skilled in the art.

Synthesis example 1 Synthesis of covalent Compound LX01

Step 1: synthesis of 5- (hydroxymethyl) pyrimidine-2, 4-diol

Uracil (20.0g, 178mmol), paraformaldehyde (6.50g, 72.1mmol), potassium hydroxide (6.50g, 116mmol) and 160mL of water were sequentially added to a 100mL single-neck flask, reacted at 60 ℃ for 72 hours, the solvent was removed at 65 ℃ under reduced pressure, 50mL of acetone was added to the residue, stirred for 2 hours, filtered and dried to obtain 26.3g of the objective compound as a white solid with a yield of 100%. mp 290 deg.C

Step 2: synthesis of 2, 4-dichloro-5- (chloromethyl) pyrimidine

5-hydroxymethylpyrimidine-2, 4-diol (5.00g, 35.2mmol) is added into a 100mL single-neck flask,

Phosphorus oxychloride (27.2g, 177mmol) and 10mL of toluene are slowly dropped into a single-mouth flask by a constant pressure dropper under ice bath, DIEA (14.5g, 112mmol) is stirred for 5min, then the temperature is raised to 115 ℃ for reaction for 1h, then the temperature is raised to 125 ℃ for reaction for 5h, TLC is used for monitoring until the reaction is complete (ethyl acetate: petroleum ether is 1:1), the reaction is cooled to room temperature, 50mL of ice water is slowly added for quenching reaction, toluene (50mL × 3) is extracted, organic phases are combined, anhydrous sodium sulfate is dried, the solvent is removed under reduced pressure, and the residue is purified by silica gel column chromatography to obtain 5.2 g of the target compound, colorless oily matter and the yield is 74.9%.¹H NMR(500MHz,DMSO-d₆):δ8.97 (s,1H),4.86(s,2H).

And step 3: synthesis of N- ((2, 4-dichloropyrimidine) -5-methyl) -3, 5-dimethoxyaniline (1)

2, 4-dichloro-5- (chloromethyl) pyrimidine (4.00g, 20.3 g) was added sequentially to a 50mL single-neck flask

mmol), potassium iodide (3.50g, 21.0mmol) and 25mL acetone, reacting at 25 ℃ for 15min, heating the reaction to 60 ℃ for reaction for 30min, filtering while the solution is hot to obtain a mother solution, cooling the mother solution to room temperature, adding 3, 5-dimethoxyaniline (3.70g, 24.2mmol) and potassium carbonate (4.80g, 34.5mmol), and stirring at 25 ℃ for 10 h. TLC monitoring reaction, decompression after reaction completely removing solvent, adding 25mL ethanol, stirring in ice bath for 30min,a white solid was precipitated, and the precipitate was filtered and dried to obtain 5.3g of Compound 1 as a white solid in a yield of 84.4%.¹H NMR(500MHz,CDCl₃):δ8.51(s,1H),5.92(s,1H),5.72 (s,2H),4.39(s,2H),4.27(s,1H),3.72(s,6H).

And 4, step 4: synthesis of 2-chloro-5- (((3, 5-dimethoxyphenyl) amino) methyl) -N- (4-nitrobenzyl) pyrimidin-4-amine (2)

Into a 50mL single-neck flask were added compound 1(2.51g, 8.00mmol), 4-nitrobenzylamine salt

Acid salt (2.00g, 10.6mmol), DIEA (3.28g, 25.4mmol) and 20mL dioxane. The reaction was carried out at 60 ℃ for 10h, monitored by TLC (petroleum ether: ethyl acetate: methanol: triethylamine: 20:8:1:1), the solvent was directly removed after the reaction was complete, the residue was purified by silica gel column chromatography to give 2.86g of crude product, which was then slurried with a small amount of ethyl acetate, filtered and dried to give 2.4g of compound 2 as a yellow solid with a yield of 70.0%.¹H NMR(500MHz,CDCl₃):δ8.12(d,J＝9.0Hz,1H),7.95 (s,1H),7.50(m,1H),7.46(d,J＝8.4Hz,1H),6.05(s,1H),5.98(s, 2H),4.78(d,J＝6.0Hz,2H),4.26(s,2H),3.71(s,6H).

And 5: synthesis of 7-chloro-3- (3, 5-dimethoxyphenyl) -1- (4-nitrobenzyl) -3, 4-dihydropyrimidine [4,5-d ] pyrimidin-2 (1H) -one (3)

Adding compound 2(2.00g, 4.65mmol), triphosgene (695mg, 2.31mmol) and 15mL of dry THF into a 25mL one-neck flask, slowly adding triethylamine (940mg, 9.32mmol) dropwise under ice bath, stirring for 1h, raising the temperature of the reaction to 70 ℃ for 10h, monitoring by TLC until the reaction of the raw materials is completed (petroleum ether: ethyl acetate ═ 2:1), adding 5mL of ice water to quench the reaction, removing the solvent THF under reduced pressure, extracting with ethyl acetate (20 mL. times.3), washing with saturated sodium bicarbonate and saturated NaCl in turn, combining the organic phases, drying with anhydrous sodium sulfate, and washing the residue with siliconPurification by gel column chromatography gave 1.76g of Compound 3 as a pale yellow solid with an yield of 82.9%.¹H NMR(500MHz,DMSO-d₆):δ8.41(s,1H),8.19(d,J＝8.5 Hz,2H),7.61(d,J＝8.5Hz,2H),6.61(d,J＝2.0Hz,2H),6.47(s, 1H),5.25(s,2H),4.92(s,2H),3.75(s,6H).¹³C NMR(125MHz,DMSO-d₆): δ160.51,158.06,157.60,154.72,151.33,146.56,145.32,143.54, 128.24,123.52,111.86,104.41,98.68,55.42,46.37,43.87.

And 6: synthesis of 3- (3, 5-dimethoxyphenyl) -7- ((2-methyl-6-nitrophenyl) amino) -1- (4-nitrobenzyl) -3, 4-dihydropyrimidine [4,5-d ] pyrimidin-2 (1H) -one (4)

Compound 3(1.14g, 2.5mmol), 2-methyl-6-nitroaniline (570mg, 3.75mmol), cesium carbonate (2.44g, 7.50mmol), XPhos (238mg, 0.50mmol) and Pd were weighed out in this order₂(dba)₃(²²⁹mg, 0.25mmol) was placed in a 25mL Schlenk tube and 4mL dry DMA was added and reacted at 110 ℃ for 3h under nitrogen. TLC monitored complete reaction of starting material (petroleum ether: ethyl acetate: methanol: 10:1), extracted with ethyl acetate (30mL × 3), washed with saturated sodium bicarbonate, combined organic phases, dried over anhydrous sodium sulfate, and the residue was purified by silica gel column chromatography to give 756mg of compound 4 as a pale yellow solid in 52.9% yield.¹H NMR(500MHz,DMSO-d₆):δ9.17(s,1H),8.07(s, 1H),8.01(s,2H),7.80(d,J＝7.5Hz,1H),7.62(d,J＝7.5Hz,1H), 7.40(t,J＝15.5Hz,1H),7.26(s,1H),6.60(s,2H),6.45(s,1H),5.04 (s,2H),4.74(s,2H),3.75(s,6H),2.18(s,3H).

And 7: synthesis of N- (4- ((7- ((2-acrylamido-6-methylphenyl) amino) -3- (3, 5-dimethoxyphenyl) -2-oxo-3, 4-dichloropyrimidine [4,5-d ] pyrimidin-1 (2H) -yl) methyl) phenyl) acrylamide (LX01)

Single-neck flask to 50mLTo this was added compound 4(500mg, 0.88mmol), 2mL Raney nickel/H₂And (3) carrying out hydrogen replacement on O and 20mL of methanol for three times, then carrying out reaction for 10h at 25 ℃, monitoring the reaction by TLC (thin layer chromatography) until the raw materials are completely reacted, filtering the reaction liquid by using kieselguhr to obtain a mother liquid, removing the solvent by decompression, and carrying out vacuum drying for 12h to obtain 430mg of compound 5 which is a light yellow solid with the yield of 96.1%. Compound 5(200mg, 0.39mmol) was taken in a 25mL two-necked flask, 10mL of dried DCM, triethylamine (87mg, 0.86mmol) and nitrogen were added, stirred for 10min under ice salt bath, a solution of acryloyl chloride (68mg, 0.76mmol) in dichloromethane (1mL) was slowly added dropwise, TLC monitored until the reaction was complete (petroleum ether: ethyl acetate: methanol 10:10:1), quenched with 2mL of ice water, extracted with ethyl acetate (20mL × 3), washed successively with saturated sodium bicarbonate and saturated NaCl, the organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to give 45mg of compound LX01 as a white solid with a yield of 18.6%.¹H NMR(500MHz,DMSO-d₆):δ10.05(s,1H),9.49(s,1H),8.28(s,1H), 8.01(s,1H),7.72(s,1H),7.45(s,2H),7.22(t,J＝7.6Hz,1H),7.12 (d,J＝4.3Hz,1H),6.54(d,J＝1.9Hz,2H),6.51(t,J＝6.3Hz,1H), 6.42(q,J＝4.3Hz,2H),6.23(m,J＝7.1Hz,2H),5.72(t,J＝11.8 Hz,2H),4.81(s,2H),4.66(s,2H),3.74(s,6H),2.09(s,3H).¹³C NMR (500MHz,MeOD-d₄):δ166.51,166.06,162.73,161.93,157.98,155.03, 145.36,139.08,138.47,136.22,135.37,132.49,132.24,131.77,130.37, 128.74,128.05,128.00,127.70,122.71,121.17,105.62,100.34,55.99, 44.67,18.68.HRMS[M+H]+m/z calculated for C₃₄H₃₃N₇O₅,620.2577；found, 620.2612.

Synthesis example 2 Synthesis of covalent Compound LX02

Step 1: synthesis of 2-cyano-3-methyl-2-butenoic acid (6)

A50 mL single-neck flask was charged with cyanoacetic acid (2.55g, 30.0mmol), acetone (3.48g, 60.0mmol) and 25mL of toluene, and reacted at 50 deg.CAfter 10h, the reaction was monitored by TLC until the starting material was completely reacted (ethyl acetate: petroleum ether ═ 2:1), extracted with toluene (20mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give 2.25g of compound 6 as white crystals in 60.0% yield.¹H NMR(500MHz, CDCl₃):δ10.03(s,1H),2.43(s,3H),2.37(s,3H).

Step 2: synthesis of 2-cyano-N- (4- ((7- ((2- (2-cyano-3-methyl-2-butenamido) -6-methylphenyl) amino) -3- (3, 5-dimethoxyphenyl) -2-oxo-3, 4-dihydropyrimidin [4,5-d ] pyrimidin-1 (2H) -yl) methyl) phenyl) -3-methyl-2-butenamide (LX02)

A25 mL one-neck flask was charged with 6(500mg, 4.0mmol) and 12mL of thionyl chloride, reacted at 85 ℃ for 4h,^TLCthe reaction was monitored until the starting material was completely reacted (petroleum ether: ethyl acetate: methanol: 10:1), and the solvent was removed under reduced pressure to give 550mg of compound 7. A25 mL two-necked flask was charged with 5(200mg, 0.39mmol), 12mL dry dichloromethane, triethylamine (87mg, 0.86mmol), nitrogen blanketed, stirred for 10min in an ice-salt bath, slowly added with a solution of compound 7(108mg, 0.76mmol) in dichloromethane (1mL), TLC monitored for completion of the reaction of the starting materials (petroleum ether: ethyl acetate: methanol 10:10:1), quenched with 2mL ice water, extracted with dichloromethane (20 mL. times.3), washed with saturated sodium bicarbonate, saturated NaCl in that order,^{the organic phases are combined}After drying over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to give 25mg of compound LX02 as a white solid in 8.8% yield.¹H NMR(500MHz,CDCl₃): δ8.75(s,1H),7.93(s,1H),7.89(d,J＝7.0Hz,2H),7.75(s,1H), 7.30(t,J＝7.9Hz,1H),7.19(d,J＝7.5Hz,2H),7.12(s,1H),6.88 (s,1H),6.47(d,J＝2.2Hz,2H),6.39(t,J＝2.1Hz,1H),5.05(s, 2H),4.64(s,2H),3.78(s,6H),2.40(d,J＝18.4Hz,6H),2.30(d,J ＝6.8Hz,6H),2.23(s,3H).¹³C NMR(500MHz,CDCl₃):δ171.23,170.98, 161.30,160.43,159.77,159.44,157.45,153.52,153.09,144.04,135.94, 134.94,130.11,127.65,120.51,117.03,116.80,106.80,106.53,104.37, 102.96,99.46,55.68,47.60,44.01,27.49,18.67.

Synthetic example 3 Synthesis of covalent Compound LX03

Step 1: synthesis of vinyl sulfonyl chloride (8)

A25 mL single-neck flask was charged with 2-chloroethanesulfonyl chloride (2.57g, 15.8mmol) and 10mL of diethyl ether, a solution of 2,4, 6-trimethylpyridine (2.30g, 19.0mmol) in diethyl ether (5mL) was slowly added dropwise at-60 ℃, the reaction was carried out for 10min, then the mixture was moved to 25 ℃ and reacted for 50min, 2mL of 1% sulfuric acid solution was added under ice bath to quench the reaction, extraction was carried out with ethyl acetate (2X 20mL), washing was carried out with saturated NaCl, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain 1.5g of Compound 8 as a colorless oil with a yield of 75.2%.

Step 2: synthesis of N- (4- ((3- (3, 5-dimethoxyphenyl) -7- ((2-methyl-6- (ethenesulfonamido) phenyl) amino) -2-oxo-3, 4-dihydropyrimidine [4,5-d ] pyrimidin-1 (2H) -yl) methyl) phenyl) ethenesulfonamide (LX03)

A25 mL two-necked flask was charged with 5(200mg, 0.39mmol), triethylamine (87mg, 0.86mmol) and 12mL dry dichloromethane, stirred for 10min under an ice salt bath under nitrogen blanket, a solution of compound 8(108mg, 0.76mmol) in dichloromethane (1mL) was slowly added for 4h reaction, TLC monitored until the reaction was complete (petroleum ether: ethyl acetate: methanol: 10:1), quenched with 5mL ice water, extracted with ethyl acetate (20 mL. times.3), washed with saturated sodium bicarbonate and saturated NaCl in that order, the organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to give 56mg compound LX03 as a white solid in 21.3% yield.¹H NMR(500MHz,DMSO-d₆):δ9.90 (s,1H),9.02(s,1H),8.42(s,1H),8.05(s,1H),7.22(t,J＝8.9Hz, 2H),7.13(d,J＝6.8Hz,1H),6.95(s,3H),6.73(q,J＝10Hz,2H),6.57 (d,J＝1.9Hz,2H),6.44(s,2H),6.09(d,J＝16.4Hz,1H),6.00(d, J＝9.9Hz,2H),5.66(s,1H),4.82(s,2H),4.69(s,2H),3.74(s,6H), 2.04(s,3H).HRMS[M+H]+m/z calculated for C₃₂H₃₃N₇O₇S₂,692.1916；found, 692.1954。

Synthesis example 4 Synthesis of covalent Compound LX04

Step 1: synthesis of 2-chloro-N- (4- ((7- ((2- (2-chloroacetylamino) -6-methylphenyl) amino) -3- (3, 5-dimethoxyphenyl) -2-oxo-3, 4-dihydropyrimidine [4,5-d ] pyrimidin-1 (2H) -yl) methyl) phenyl) acetamide (LX04)

To a 25mL two-necked flask were added 5(160mg, 0.31mmol), triethylamine (84mg, 0.78mmol) and 10mL dry dichloromethane, stirred under a nitrogen blanket in an ice salt bath for 10min, a solution of chloroacetyl chloride (70mg, 0.62mmol) in dichloromethane (1mL) was slowly added, TLC monitored until the reaction was complete (petroleum ether: ethyl acetate: methanol 10:10:1), quenched with 5mL ice water, extracted with ethyl acetate (20mL × 3), washed successively with saturated sodium bicarbonate, saturated NaCl, combined organic phases, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to give 36mg compound LX04 as a white solid in 17.3% yield.¹H NMR(500MHz,DMSO-d₆):δ10.23(s,1H), 9.43(s,1H),8.49(s,1H),8.04(s,1H),7.78(s,1H),7.35(q,J＝4.8 Hz,2H),7.23(t,J＝7.6Hz,2H),7.12(d,J＝6.9Hz,1H),6.81(s, 1H),6.54(d,J＝2.0Hz,2H),6.43(s,1H),4.67(s,2H),4.29(s,2H), 4.23(s,2H),3.74(s,6H),2.54(s,2H),2.10(s,3H).HRMS[M+H]+m/z calculated for C₃₂H₃₁Cl₂N₇O₅,664.1797；found,664.1838。

Synthesis example 5 Synthesis of covalent Compound LX05

Step 1: synthesis of 2-chloro-5- (((3, 5-dimethoxyphenyl) amino) methyl) -N- (3-nitrobenzyl) pyrimidin-4-amine (9)

To a 50mL single-neck flask were added compound 1(2.51g, 8.0mmol), 3-nitrobenzylamine hydrochloride (2.00g, 10.6mmol), DIEA (3.28g, 25.4mmol) and 20mL dioxane, reacted at 60 ℃ for 10h, TLC monitored until the reaction was complete (petroleum ether: ethyl acetate: methanol: triethylamine: 20:8:1:1), the solvent was removed, and the residue was purified by silica gel column chromatography to give 2.8g of compound 9 as a pale yellow oil in 51.0% yield.¹H NMR(500MHz,CDCl₃):δ8.11(s,1H),8.05(d, J＝8.5Hz,1H),7.88(s,1H),7.63(d,J＝7.6Hz,1H),7.46(t,J＝ 7.9Hz,1H),6.91(t,J＝5.9Hz,2H),5.93(s,1H),5.87(d,J＝1.8 Hz,2H),4.76(d,J＝5.9Hz,2H),4.11(d,J＝5.2Hz,2H),3.89(t, J＝5.2Hz,1H),3.70(s,6H).

Step 2: synthesis of 7-chloro-3- (3, 5-dimethoxyphenyl) -1- (3-nitrobenzyl) -3, 4-dihydropyrimidine [4,5-d ] pyrimidin-2 (1H) -one (10)

Compound 9(1.50g, 3.5mmol), triphosgene (517mg, 2.3mmol) and 15mL of dry THF were added to a 25mL single-neck flask, triethylamine (940mg, 7.0mmol) was slowly added dropwise in an ice bath, after stirring for 1h, the reaction was warmed to 70 ℃ for 10h, TLC monitored until the reaction was complete (petroleum ether: ethyl acetate ═ 2:1), 5mL of ice water was added to quench the reaction, ethyl acetate (20mL × 3) was extracted, washed with saturated sodium bicarbonate and saturated NaCl in that order, the organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to give 1.12g of compound 10 as a pale yellow solid in 70.2% yield.¹H NMR(500MHz,CDCl₃):δ8.40(s,1H),8.17(s,1H),8.13(d,J＝7.3 Hz,1H),7.88(d,J＝7.6Hz,1H),7.50(t,J＝7.9Hz,1H),6.46(d, J＝1.9Hz,2H),6.42(d,J＝1.9Hz,1H),5.36(s,2H),4.79(s,2H), 3.79(s,6H).

And step 3: synthesis of 3- (3, 5-dimethoxyphenyl) -7- ((2-methyl-6-nitrophenyl) amino) -1- (3-nitrobenzyl) -3, 4-dihydropyrimidine [4,5-d ] pyrimidin-2 (1H) -one (11)

Compound 10(1.00g, 2.5mmol), 2-methyl-6-nitroaniline (570mg, 3.8mmol), cesium carbonate (2.44g, 7.5mmol), XPhos (238mg, 0.50mmol) and Pd were weighed out in this order₂(dba)₃(229mg, 0.25mmol) was placed in a 25mL Schlenk tube, followed by addition of 4mL anhydrous DMA and reaction at 110 ℃ for 3h under nitrogen. TLC monitored complete reaction of starting material (petroleum ether: ethyl acetate: methanol 10:10:1), extraction with ethyl acetate (30mL × 3), washing with saturated sodium bicarbonate, combining the organic phases, drying over anhydrous sodium sulfate, solvent extraction under reduced pressure, and purification of the residue by silica gel column chromatography afforded 756mg of compound 11 as a pale yellow solid in 52.9% yield.¹H NMR(500MHz,CDCl₃):δ8.07(d,J＝7.2 Hz,2H),8.00(s,1H),7.88(d,J＝7.3Hz,1H),7.88(d,J＝7.9Hz, 1H),7.61(s,1H),7.54(d,J＝7.5Hz,1H),7.50(s,1H),7.37(t,J ＝8.0Hz,1H),7.32(t,J＝7.9Hz,1H),6.50(d,J＝2.1Hz,2H),6.43 (t,J＝2.1Hz,1H),5.18(s,2H),4.71(s,2H),3.82(s,6H),2.31(s, 3H).

And 4, step 4: synthesis of N- (3- ((7- ((2-acrylamido-6-methylphenyl) amino) -3- (3, 5-dimethoxyphenyl) -2-oxo-3, 4-dichloropyrimidine [4,5-d ] pyrimidin-1 (2H) -yl) methyl) phenyl) acrylamide (LX05)

To a 50mL single neck flask was added Compound 11(500mg, 0.87mmol), 2mL Raney nickel/H₂O and 20mL of methanol were reacted for 10 hours at 25 ℃ after three times of hydrogen substitution, and the reaction was monitored by TLC until the starting material was completely reacted (petroleum ether: ethyl acetate: methanol: 10:20: 1). The reaction solution was filtered through celite to obtain a mother liquor, the solvent was removed under reduced pressure and dried under vacuum for 12h to obtain 430mg of compound 12 as an off-white solid with a yield of 96.0%. Taking and combiningSubstance 12(400mg, 0.76mmol) was placed in a 25mL two-necked flask, triethylamine (87mg, 0.86mmol) and 10mL dry dichloromethane were added, nitrogen was used for protection, stirring was performed for 10min under ice salt bath, a dichloromethane (1mL) solution of acryloyl chloride (68mg, 0.76mmol) was slowly added dropwise, TLC monitored that the starting material reacted completely (petroleum ether: ethyl acetate: methanol 10:10:1), 2mL ice water was added to quench the reaction, extraction was performed with ethyl acetate (20mL × 3), washing was performed with saturated sodium bicarbonate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to purify the compound LX05 as a white solid in 18.6% yield.¹H NMR(500MHz,DMSO-d₆):δ8.27(s,1H),8.03 (s,1H),7.70(d,J＝6.4Hz,1H),7.55(d,J＝7.7Hz,2H),7.18(t, J＝7.8Hz,1H),7.06(d,J＝7.5Hz,2H),6.58(d,J＝2.2Hz,2H),6.48 (q,J＝10.2Hz,1H),6.42(q,J＝3.1Hz,2H),6.23(t,J＝1.7Hz,1H), 6.20(t,J＝1.8Hz,1H),5.71(m,J＝1.7Hz,2H),4.86(s,2H),4.70 (s,2H),3.74(s,6H),2.02(s,3H).HRMS[M+H]+m/z calculated for C₃₄H₃₃N₇O₅,620.2577；found,620.2618.

Synthesis example 6 Synthesis of covalent Compound LX06

Step 1: synthesis of tert-butyl 4- (3-aminopropyl) piperazine-1-carboxylate (14)

To a 50mL single-neck flask were added N- (3-bromopropyl) phenylenediamine (10.00g, 37.3mmol), 1-Boc-piperazine (7.00g, 37.6mmol), potassium iodide (12.40g, 74.6mmol), potassium carbonate (8.88g, 63.4mmol), and 50mL of N, N-dimethylacetamide, reacted at 30 ℃ for 18 hours, TLC monitored that the raw materials reacted completely (ethyl acetate: petroleum ether ═ 1:8), ethyl acetate (50mL × 3) was extracted, washed with saturated sodium bicarbonate and saturated NaCl in this order, the organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, 20mL of ethyl acetate was added, slurried, and filtered to obtain 12.2g of intermediate 13 as a white solid with an yield of 87.6%. Intermediate 13(7.40g, 19.8mmol) was taken in a 50mL single-neck flask, 30mL ethanol and 8mL hydrazine hydrate were added, reaction was carried out at 70 ℃ for 3h, and TLC monitored starting materialAnd (3) cooling the reaction liquid to normal temperature until the reaction is complete (ethyl acetate: petroleum ether ═ 1:5), carrying out vacuum filtration to obtain a mother liquid, carrying out vacuum filtration to remove the solvent, cooling, adding 20mL of diethyl ether and a small amount of anhydrous sodium sulfate, stirring at 0 ℃ for 10min, carrying out vacuum filtration to obtain the mother liquid, and carrying out vacuum filtration to obtain 3.9g of a compound 14 and a colorless oily substance, wherein the yield is 81.2%.¹H NMR(500MHz, CDCl₃):δ3.41(t,J＝4.8Hz,4H),2.74(t,J＝6.8Hz,4H),2.38(q, J＝7.2Hz,6H),1.62(m,J＝7.0Hz,2H),1.44(s,9H).

Step 2: synthesis of tert-butyl 4- (3- ((2-chloro-5- (((3,5 dimethoxyphenyl) amino) methyl) pyrimidin-4-yl) amino) propyl) piperazine-1-carboxylate (15)

To a 50mL single-neck flask were added compound 1(2.51g, 8.00mmol), compound 14(2.58 g, 10.6mmol), DIEA (3.28g, 25.4mmol) and 20mL dioxane, reacted at 60 ℃ for 10h, monitored by TLC until the starting materials reacted completely (petroleum ether: ethyl acetate: methanol: triethylamine: 16:8:1:1), the solvent was removed under reduced pressure, and the residue was purified by column chromatography to give 3.16g of compound 15 as a pale yellow oil in 76.0% yield.¹H NMR(500MHz,CDCl₃):δ7.89(d,J＝1.4Hz,1H),6.55 (s,1H),5.97(t,J＝2.0Hz,1H),5.86(d,J＝2.1Hz,2H),4.07(d, J＝3.3Hz,2H),3.74(s,6H),3.73(s,2H),3.55(q,J＝6.1Hz,2H), 3.37(s,4H),2.35(s,4H),1.45(s,2H),1.44(s,9H).

And step 3: synthesis of tert-butyl 4- (3- (7-chloro-3- (3, 5-dimethoxyphenyl) -2-oxo-3, 4-dihydropyrimidinyl [4,5-d ] pyrimidin-1 (2H) -yl) propyl) -1-carboxylate (16)

Into a 25mL one-neck flask were added compound 15(1.50g, 2.88mmol), triphosgene (427

mg, 1.44mmol) and 15mL of dry THF, triethylamine (58.2mg, 5.76mmol) was slowly added dropwise in an ice bath, the reaction was stirred for 1h, then the temperature was raised to 70 ℃ for 10h, the reaction was monitored by TLC (petroleum ether: ethyl acetate)Ethyl acetate (20mL × 3), washing with saturated sodium bicarbonate, combining the organic phases, drying with anhydrous sodium sulfate, removing the solvent under reduced pressure, purifying the residue by silica gel column chromatography to obtain 1.36g of a crude product, and recrystallizing with isopropanol/petroleum ether to obtain 920mg of compound 16 as a white solid with a yield of 58.4%.¹H NMR(500 MHz,CDCl₃):δ8.12(s,1H),6.45(d,J＝2.15Hz,2H),6.41(t,J＝ 2.15Hz,1H),4.74(s 2H),4.15(t,J＝7.25Hz,2H),3.79(s,6H),3.51 (s,4H),2.58(s,4H),2.52(s,2H),1.99(s,2H),1.45(s,9H).

And 4, step 4: synthesis of tert-butyl 4- (3- (3- (3, 5-dimethoxyphenyl) -7- ((2-methyl-6-nitrophenyl) amino) -2-oxo-3, 4-dihydropyrimidin [4,5-d ] pyrimidin-1 (2H) -yl) propyl) piperazine-1-carboxylate (17)

Compound 16(850mg, 1.55mmol), 2-methyl-6-nitroaniline (354mg, 2.33mmol), cesium carbonate (1.51g, 4.65mmol), XPhos (151mg, 0.31mmol) and Pd were weighed out in this order₂(dba)₃(146mg, 0.16mmol) was placed in a 25mL Schlenk tube, and 3mL of dried DMA was added and reacted at 110 ℃ for 3h under nitrogen. TLC monitored the completion of the reaction of the starting material (petroleum ether: ethyl acetate: methanol: 10:1), extracted with ethyl acetate (30mL × 3), washed with saturated sodium bicarbonate, washed with saturated NaCl in that order, the organic phases combined, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to give 620mg of compound 17 as a pale yellow solid in 60.4% yield.

And 5: synthesis of N- (2- ((8(3- (4-acryloylpiperazin-1-yl) propyl) -6- (3, 5-dimethoxyphenyl) -7-oxo-5, 6,7, 8-tetrahydropyrimidinyl [4,5-d ] pyrimidin-2-yl) amino) -3-methylphenyl) acrylamide (LX06)

Into a 50mL single neck flask was added compound 17(600mg, 0.91mmol), 1mL Raney nickel/H₂And (3) carrying out hydrogen replacement on O and 20mL of methanol for three times, then carrying out reaction at 25 ℃ for 10h, monitoring the reaction by TLC until the raw materials are completely reacted, filtering the reaction liquid by using kieselguhr to obtain a mother liquid, removing the solvent by decompression, and drying in vacuum for 12h to obtain 561mg of a compound 18 which is a white solid and has the yield of 97.1%. Compound 18(561mg, 0.89mmol) was taken in a 25mL single vial, 15mL dry dichloromethane and 1mL trifluoroacetic acid were added, stirred at 27 ℃ overnight, TLC monitored for completion of the reaction of the starting materials (petroleum ether: ethyl acetate: methanol: 10:1), the reaction solution was added to 30mL saturated sodium bicarbonate solution under ice bath, stirred for 10min, extracted with dichloromethane (20mL × 3), washed with saturated sodium bicarbonate, the combined organic phases were dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give 450mg compound 19. Taking compound 19(200mg, 0.38mmol) into a 25mL double-neck flask, adding 10mL of dried dichloromethane and triethylamine (94mg, 0.86mmol), protecting with nitrogen, stirring for 10min under an ice salt bath, slowly adding a dichloromethane (1mL) solution of acryloyl chloride (68mg, 0.76mmol) dropwise, TLC monitoring that the raw materials are completely reacted, adding 2mL of ice water to quench the reaction, extracting with ethyl acetate (20mL multiplied by 3), washing with saturated sodium bicarbonate and saturated NaCl in sequence, combining organic phases, drying with anhydrous sodium sulfate, removing the solvent under reduced pressure, and purifying the residue by silica gel column chromatography to obtain 32mg of compound LX06 as a white solid with the yield of 13.1%.¹H NMR(500MHz,CDCl₃):δ8.32(s,1H),7.93 (s,1H),7.91(s,1H),7.21(t,J＝7.8Hz,1H),7.07(d,J＝7.1Hz, 1H),6.88(s,1H),6.54(q,J＝10.6Hz,1H),6.44(d,J＝2.1Hz,2H), 6.37(m,J＝2.8Hz,2H),6.28(t,J＝1.7Hz,1H),6.20(q,J＝10.4 Hz,1H),5.69(m,J＝8.2Hz,2H),4.61(s,2H),3.86(s,2H),3.77(s, 6H),3.63(s,2H),3.51(s,2H),2.34(s,4H),2.25(s,3H),1.72(s, 3H).¹³C NMR(500MHz,CDCl₃):δ165.46,161.36,161.23,161.02,157.55, 153.14,152.85,143.88,136.23,131.48,128.00,127.73,127.55,127.38, 126.91,104.22,102.83,99.13,55.60,53.05,52.60,47.48,45.76,41.94, 40.12,24.77,18.74.HRMS[M+H]+m/z calculated for C₃₄H₄₀N₈O₅,641.3155； found,641.3198.

Synthesis example 7 covalent CompoundsSynthesis of LX07

Step 1: synthesis of 7- ((2-aminophenyl) amino) -3- (3, 5-dimethoxyphenyl) -1- (4-nitrobenzyl) -3, 4-dihydropyrimidinyl [4,5-d ] pyrimidin-2 (1H) -one (20)

Compound 3(500mg, 1.25mmol), o-phenylenediamine (570mg, 1.88mmol), trifluoroacetic acid (214mg, 1.88mmol) and 4mL of sec-butanol were sequentially weighed into a 25mL Schlenk tube and reacted at 100 ℃ for 10h under nitrogen. The reaction was monitored by TLC until the reaction was complete (ethyl acetate: petroleum ether: TEA ═ 10:10:1), extracted with ethyl acetate (30mL × 3), washed with saturated sodium bicarbonate, the organic phases combined, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to give 346mg of compound 20 as a yellow solid in 52.5% yield.¹H NMR(500MHz,DMSO-d₆):δ8.49(s, 1H),8.09(s,3H),7.46(d,J＝7.6Hz,2H),7.10(d,J＝7.4Hz,1H), 6.89(t,J＝6.9Hz,1H),6.75(d,J＝7.0Hz,1H),6.58(s,2H),6.47 (q,J＝7.0Hz,2H),5.14(s,2H),4.83(s,2H),4.73(s,2H),3.74(s, 6H),3.63(s,2H),3.51(s,2H),2.34(s,4H),2.25(s,3H),1.72(s, 3H).¹³C NMR(500MHz,DMSO-d₆):δ160.44,160.26,155.73,153.99,152.46, 146.35,146.15,144.16,142.54,128.89,125.69,125.23,124.66,123.25, 116.06,115.56,104.33,101.52,98.34,55.38,46.62,43.28。

Step 2: synthesis of N- (2- ((8- (4-acrylamidobenzyl) -6- (3, 5-dimethoxyphenyl) -7-oxo-5, 6,7, 8-tetrahydropyrimidinyl [4,5-d ] pyrimidin-2-yl) amino) phenyl) acrylamide (LX07)

Into a 50mL single-neck flask were added compound 20(346mg, 0.66mmol), 2mL Raney

nickel/H₂O and 20mL methanol, three times replaced with hydrogen and reacted at 25 ℃ for 10h TLC to monitor the reaction until the starting material was completely reacted (ethyl acetate:petroleum ether TEA-10: 5: 1). The reaction solution was filtered through celite to obtain a mother liquor, the solvent was removed under reduced pressure and dried under vacuum for 12h to obtain 312mg of compound 21 as a white solid with a yield of 97.6%. Compound 21(312mg, 0.63mmol) was taken in a 25mL two-necked flask, triethylamine (171mg, 1.57mmol) and 10mL dry dichloromethane were added, nitrogen was used for protection, stirring was performed for 10min under ice salt bath, then a solution of acryloyl chloride (115mg, 0.76mmol) in dichloromethane (1mL) was slowly added dropwise, the starting materials were monitored by TLC until the reaction was complete (petroleum ether: ethyl acetate: methanol: 10:1), 2mL ice water was added to quench the reaction, dichloromethane (30mL × 2) was extracted, washed with saturated sodium bicarbonate and saturated NaCl in order, the organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to give 56mg compound LX07 as a white solid with a yield of 14.6%.¹H NMR(500MHz,DMSO-d₆):δ10.11 (s,1H),9.88(s,1H),8.60(s,2H),8.11(s,1H),7.55(d,J＝7.6Hz, 4H),7.19(d,J＝7.7Hz,2H),7.11(s,2H),6.59(s,2H),6.52(q,J ＝10.3Hz,1H),6.42(q,J＝8.9Hz,2H),6.30(d,J＝17.0Hz,1H),6.24 (d,J＝16.9Hz,1H),5.78(d,J＝9.9Hz,1H),6.24(d,J＝9.9Hz,1H), 5.04(s,2H),4.74(s,2H),3.75(s,6H).¹³C NMR(500MHz,DMSO-d₆): δ163.78,163.03,160.45,159.28,155.95,153.78,152.50,144.25, 137.71,133.13,132.37,131.90,131.50,129.96,128.16,127.25,126.81, 125.17,124.57,124.44,123.67,119.19,104.32,102.81,98.38,55.40, 46.53,43.03.HRMS[M+H]+m/z calculated for C₃₃H₃₁N₇O₅,606.2420；found, 606.2615.

Synthesis example 8 Synthesis of covalent Compound LX08

Step 1: synthesis of 2-chloro-5- (((3, 5-dimethoxyphenyl) amino) methyl) -N- (4-nitrophenylethyl) pyrimidin-4-amine (22)

A50 mL one-neck flask was charged with Compound 1(2.00g, 6.41mmol), 4-nitrophenylethylamine hydrochloride (1.67g, 8.32mmol), DIEA (3.28g, 25.4mmol), and 20mL of dioxan,the reaction was carried out at 60 ℃ for 10h, TLC monitored until the starting material was completely reacted (petroleum ether: ethyl acetate: methanol: triethylamine: 20:8:1:1), the solvent was removed under reduced pressure, and the residue was purified by column chromatography to give 2.05g of compound 22 as a yellow oil in 72.3% yield.¹H NMR(500MHz,CDCl₃):δ8.01(s,1H), 7.99(s,1H),7.83(s,1H),7.28(s,1H),6.35(t,J＝5.3Hz,1H),5.99 (t,J＝2.0Hz,1H),5.77(d,J＝2.1Hz,2H),4.00(s,2H),3.78(q, J＝6.1Hz,2H),3.74(s,6H),3.00(t,J＝6.7Hz,2H).¹³C NMR(500MHz, CDCl₃):δ162.45,161.81,160.29,154.82,149.00,146.71,146.44, 129.65,123.79,112.72,93.288,91.42,55.256,43.78,41.41,35.00.

Step 2: synthesis of 7-chloro-3- (3, 5-dimethoxyphenyl) -1- (4-nitrophenylethyl) -3, 4-dihydropyrimidine [4,5-d ] pyrimidin-2 (1H) -one (23)

Compound 2(1.00g, 2.25mmol), triphosgene (335m g, 1.13mmol) and 15mL of dry THF were added to a 25mL single-neck flask, triethylamine (4.24g, 5.76mmol) was slowly added dropwise under ice bath, after stirring for 1h, the reaction was warmed to 70 ℃ to react for 10h, TLC monitored until the reaction was complete (petroleum ether: ethyl acetate ═ 2:1), 5mL of ice water was added to quench the reaction, solvent THF was removed under reduced pressure, ethyl acetate (20mL × 3) was extracted, washed with saturated sodium bicarbonate and saturated NaCl in order, the organic phases were combined, dried over anhydrous sodium sulfate, solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to give 812mg of compound 23 as a pale yellow solid in 76.9% yield.¹H NMR(500MHz,DMSO-d₆):δ8.35(s,1H), 8.16(d,J＝7.9Hz,2H),7.53(d,J＝7.9Hz,2H),6.50(s,2H),6.44 (s,1H),4.81(s,2H),4.20(t,J＝6.7Hz,2H),3.73(s,6H),3.06(t, J＝3.5Hz,2H).¹³C NMR(500MHz,DMSO-d₆):δ160.44,158.17,157.60, 154.48,151.17,147.27,146.16,143.58,130.24,123.45,111.71,104.16, 98.61,55.37,46.09,45.35,33.17.

And step 3: synthesis of 7- ((2-aminophenyl) amino) -3- (3, 5-dimethoxyphenyl) -1- (4-nitrophenylethyl) -3, 4-dihydropyrimidinyl [4,5-d ] pyrimidin-2 (1H) -one (24)

Compound 23(469mg, 1.00mmol), o-phenylenediamine (162mg, 1.50mmol), trifluoroacetic acid (171mg, 1.50mmol) and 3mL of sec-butanol were sequentially weighed out and reacted at 100 ℃ for 10h under nitrogen atmosphere in a 25mL Schlenk tube. The reaction was monitored by TLC until the starting material was reacted completely (ethyl acetate: petroleum ether: TEA ═ 10:10:1), extracted with ethyl acetate (30mL × 3), washed with saturated sodium bicarbonate, the organic phases combined, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to give 302mg of compound 24 as a yellow solid in 55.8% yield.¹H NMR(500MHz,CDCl₃):δ8.07(s,1H), 8.05(s,1H),7.98(s,1H),7.37(d,J＝7.8Hz,1H),7.19(t,J＝7.45 Hz,1H),7.14(d,J＝7.7Hz,2H),6.94(s,2H),6.88(d,J＝5.25Hz, 1H),6.84(t,J＝7.6Hz,1H),6.46(d,J＝1.95Hz,1H),6.41(s,1H), 4.64(s,2H),4.10(t,J＝8.25Hz,2H),3.79(s,6H),2.99(t,J＝8.0 Hz,2H).

And 4, step 4: synthesis of N- (2- ((8- (4-acrylamidophenethyl) -6- (3, 5-dimethoxyphenyl) -7-oxo-5, 6,7, 8-tetrahydropyrimidinyl [4,5-d ] pyrimidin-2-yl) amino) phenyl) acrylamide (LX08)

Into a 50mL single neck flask was added compound 24(200mg, 0.37mmol), 1mL Raney nickel/H₂O and 20mL of methanol, three times replaced with hydrogen, reacted at 25 ℃ for 10h, and monitored by TLC until the starting material was completely reacted (ethyl acetate: petroleum ether: TEA: 10: 1). The reaction solution was filtered through celite to obtain a mother liquor, the solvent was removed under reduced pressure and dried under vacuum for 12h to obtain 183mg of compound 25 as a white solid. Taking compound 25(180mg, 0.36mmol) and placing in a 25mL double-mouth bottle, adding 10mL dry dichloromethane, triethylamine (98mg, 0.89mmol) and nitrogen protection, stirring for 10min under ice salt bath, slowly dropping acryloyl chloride (65mg, 0.72mmol) in dichloromethane (1mL), reacting for 2h, TLCThe starting material was monitored until the reaction was complete (petroleum ether: ethyl acetate: methanol ═ 10:10:1), the reaction was quenched by the addition of 2mL of ice water, extracted with dichloromethane (30mL × 2), washed successively with saturated sodium bicarbonate and saturated NaCl, the organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to give 36mg of compound LX08 as a white solid in 16.1% yield.¹H NMR(500MHz,DMSO-d₆):δ10.10 (s,1H),9.94(s,1H),8.60(s,1H),8.11(s,1H),7.81(q,J＝6.5Hz, 1H),7.58(d,J＝8.4Hz,3H),7.18(m,J＝7.6Hz,2H),7.05(d,J＝ 8.3Hz,2H),6.54(m,J＝3.9Hz,3H),6.43(m,J＝2.2Hz,2H),6.31 (q,J＝17.0Hz,1H),6.25(q,J＝17.0Hz,1H),5.79(t,J＝10.9Hz, 1H),5.75(q,J＝10.0Hz,1H),4.68(s,2H),3.99(t,J＝7.7Hz,2H), 3.74(s,6H),2.78(t,J＝7.9Hz,2H).¹³C NMR(500MHz,DMSO-d₆):δ 164.29,163.45,160.86,159.99,156.57,154.14,152.63,144.68,137.74, 134.41,133.01,132.41,131.95,130.56,129.52,127.74,127.17,125.77, 125.11,124.17,119.80,104.62,103.37,98.83,55.84,46.90,42.87, 33.51.HRMS[M+H]+m/z calculated for C₃₄H₃₃N₇O₅,620.2577；found, 620.2616.

Synthesis example 9 Synthesis of covalent Compound LX09

Step 1: synthesis of tert-butyl 4- (3- (7- ((2-aminophenyl) amino) -3- (3, 5-dimethoxyphenyl) -2-oxo-3, 4-dihydropyrimidin [4,5-d ] pyrimidin-1 (2H) -yl) propyl) piperazine-1-carboxylate (26)

Compound 16(547mg, 1.00mmol), o-phenylenediamine (162mg, 1.50mmol), cesium carbonate (975mg, 3.00mmol), XPhos (98mg, 0.20mmol) and Pd were weighed in this order₂(dba)₃(92 mg, 0.10mmol) was placed in a 25mL Schlenk tube, followed by 3mL of anhydrous DMA and reacted at 110 ℃ for 3h under nitrogen. TLC monitored the starting material until the reaction was complete (ethyl acetate: petroleum ether: TEA ═ 10:10:1), extracted with ethyl acetate (30mL × 3), washed with saturated sodium bicarbonate, combined organic phases, dried over anhydrous sodium sulfate, and pumped off under reduced pressureThe solvent and residue were purified by silica gel column chromatography to give 328mg of compound 26 as a yellow oil in 53.1% yield.¹H NMR(500MHz,CDCl₃):δ7.93(d,J＝7.1Hz,1H), 7.03(d,J＝6.0Hz,2H),6.80(s,2H),6.45(s,2H),6.37(s,1H),4.59 (s,2H),3.97(s,2H),3.85(s,2H),3.77(s,6H),3.38(s,1H),2.32 (s,6H),1.84(s,2H),1.44(s,9H).¹³C NMR(500MHz,CDCl₃):δ161.07, 160.677,157.15,154.77,153.16,152.96,143.94,141.37,126.48,125.83, 125.68,119.14,116.98,104.05,102.27,99.01,79.60,55.99,55.50, 52.84,47.41,40.07,28.45,25.06.

Step 2: synthesis of N- (2- ((8- (3- (4-acryloylpiperazin-1-yl) propyl) -6- (3, 5-dimethoxyphenyl) -7-oxo-5, 6,7, 8-tetrahydropyrimidinyl [4,5-d ] pyrimidin-2-yl) amino) phenyl) acrylamide (LX09)

Compound 26(320mg, 0.52mmol) was taken in a 25mL single neck flask, 15mL of dried dichloromethane and 1mL of trifluoroacetic acid were added, stirred at 27 ℃ overnight, TLC monitored until the starting material reaction was complete (ethyl acetate: petroleum ether: TEA ═ 10:5:1), the reaction was added to 30mL of saturated sodium bicarbonate solution under ice bath, stirred for 10min, dichloromethane (20mL × 3) extracted, saturated sodium bicarbonate washed, the organic phases combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give 254mg of compound 19. Compound 19(200mg, 0.39mmol) was taken in a 25mL two-necked flask, triethylamine (94mg, 0.86mmol) and 10mL dry dichloromethane were added, stirred for 10min under an ice salt bath under nitrogen protection, a solution of acryloyl chloride (70mg, 0.78mmol) in dichloromethane (1mL) was slowly added dropwise, TLC monitored for completion of the reaction of the starting materials (petroleum ether: ethyl acetate: methanol ═ 10:10:1), 2mL ice water was added to quench the reaction, dichloromethane (20mL × 3) was extracted, washed successively with saturated sodium bicarbonate and saturated NaCl, the organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure,purification of the residue by silica gel column chromatography gave 36mg of compound LX09 as a white solid in 14.7% yield.¹H NMR(500MHz,CDCl₃):δ8.62(s,1H),7.92(s,1H),7.63 (q,J＝8.4Hz,3H),7.16(t,J＝8.2Hz,3H),6.53(q,J＝5.9Hz,1H), 6.45(s,2H),6.39(t,J＝12.2Hz,2H),6.25(t,J＝9.7Hz,2H),5.69 (q,J＝7.3Hz,2H),4.60(s,2H),3.97(s,2H),3.76(s,6H),3.58(s, 2H),3.47(s,2H),2.34(s,4H),2.30(s,2H),1.81(s,2H).HRMS[M+H]+ m/z calculated for C₃₄H₄₀N₈O₅,627.2999；found,627.3036.

Detection of inhibitory Activity of Compound on FGFR kinase

The inhibitory activity of the compounds on FGFR1-4 and FGFR4 mutants was determined using ADP-GloTMKinase Assay (promega, Part No # V9101). The specific experimental process is as follows:

1. the kinase reactions were performed in white, light-tight 384-well plates and incubated with the laboratory expressing purified kinase (0.1. mu.M), ATP (10. mu.M), substrate (50. mu.g/mL, Part No # ab204877, abcam) and equi-diluted compounds in kinase reaction buffer (40mM Tris-HCl pH 7.5,20mM MgCl2,20mM NaCl,0.1mg/mL BSA,1mM TCEP, and 4% DMSO) for 30min at room temperature;

2. adding reaction termination liquid ADP-Glo, incubating for 40 minutes at room temperature, and terminating the reaction;

3. adding a detection solution, incubating for 30-60 minutes at room temperature in a dark place, converting ADP generated by kinase reaction into ATP, and generating cold luminescence;

4. luminescence was measured in a microplate reader and IC50 for the compound was calculated using GraphPad Prism software, the results of which are shown in table 1 below.

TABLE 1 kinase Activity assay results (IC50, nM)

From the aspect of kinase activity, the compounds with the double covalent structures mainly have stronger inhibitory activity on FGFR4, but have weaker activity on FGFR 1-3. Comparing the kinase activity of LX01, LX02, LX03 and LX04 on FGFR4Acrylamide groups perform best among the electrophilic groups we have selected. Tail R₂The structures are six-membered rings which are about 3-6 fold less active than the benzene rings, e.g., LX01, LX05 compared to IC50 for LX06, LX08 compared to IC50 for LX 09. For the two cysteines (Cys477 and Cys552) in FGFR4, C552A had a greater effect on activity than C477A, compounds LX07, LX08 had at least about a 25-fold reduction in C552A activity over wild-type, LX09 had about a 12-fold reduction in C552A activity, and LX07, LX08, LX09 had only a 4-5-fold reduction in C477A activity over wild-type. Among the 9 compounds, compounds LX01, LX05, LX07, LX08 and LX09 have high inhibitory activity on FGFR4, and have potential medicinal prospects.

Proliferation assay of compounds on FGFR-highly expressed cells

The present invention assesses the inhibitory effect of compounds on the proliferation of cells dependent on the FGFR signaling pathway by survival assays as measured using CCK-8(Vazyme, Part No # A311). The Ba \ F3 cells with high FGFR expression constructed by the experiment are selected, and the specific experimental process is as follows:

1.50 μ L of Ba \ F3 cells were seeded in 96-well plates (about 2000/well) and cultured overnight in a 5% carbon dioxide incubator at 37 ℃;

2. adding 50 μ L of preheated compound diluted with culture medium the next day, mixing well, and culturing for 72 hr;

3. adding 10 mu L of CCK-8 detection reagent into each hole, uniformly mixing, and reacting in an incubator for 1-2 hours;

4. absorbance at 450nm was measured in a microplate reader and IC50 for the compounds was calculated using GraphPad Prism software, the results of which are shown in Table 2 below.

TABLE 2 results of cell Activity assays (IC50, nM)

From the aspect of cell activity, the inhibitory effect of the compounds on Ba \ F3 cells is consistent with the inhibitory effect of kinase, and the compounds have stronger proliferation inhibition effect on Ba \ F3 cells activated by FGFR4 signaling pathways. The results of kinase and cell activity are combined, and the series of compounds have very good selectivity on FGFR 4.

Covalent binding assay of compounds to FGFR4

In order to test the covalent binding of the compound having a significant inhibitory effect on FGFR4 and two cysteines (Cys477 and Cys552) in FGFR4, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) was used to detect the change in molecular weight of kinase mutants FGFR4(C477A) and FGFR4(C552A) before and after binding the compound. The specific experimental process is as follows:

1. protein and compound are mixed according to a molar ratio of 1:5 reacting at room temperature for 1 h;

2. by ddH₂Diluting O to 1mL, and concentrating to about 0.5-1mg/mL with 3kD ultrafilter tube;

3. selecting a primary mass spectrum method of linear positive ion mode for detecting large molecular weight, taking sinapic acid as a matrix (20mg/mL), mixing a sample and a saturated matrix solution according to a volume ratio of 1:1, and putting the mixture into a mass spectrometer (AB SCIEX, 5800MADI-TOF) for detection;

4. the data is processed in the data explorer and origin programs, and the results are shown in fig. 1-6.

From the mass spectrometry results, compounds LX01, LX05, LX06, LX07, LX08 all covalently bound to Cys552 in FGFR4 only and not to Cys477 in FGFR4, while compound LX09 can covalently bind to Cys552 and Cys477 of two cysteines in FGFR 4.

By integrating kinase activity, cell activity and mass spectrum results, the series of compounds have better selectivity on FGFR4, are covalently bound with single cysteine (Cys552) of FGFR4 or covalently bound with two cysteines (Cys477 and Cys552) of FGFR4 at the same time, are expected to be developed into a new generation of selective FGFR4 inhibitor, and meet the requirements of clinical application.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it will be appreciated that various changes or modifications may be made by those skilled in the art after reading the above teachings of the invention, and such equivalents will fall within the scope of the invention as defined in the appended claims.

Claims (18)

1. An FGFR4 inhibitor selected from the group consisting of:

2. a pharmaceutical composition comprising a therapeutically effective amount of the FGFR4 inhibitor of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

3. A pharmaceutical composition comprising a therapeutically effective amount of the FGFR4 inhibitor of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent.

4. A pharmaceutical composition comprising a therapeutically effective amount of the FGFR4 inhibitor of claim 1 or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable adjuvants.

5. Use of an inhibitor of FGFR4, or a pharmaceutically acceptable salt thereof, according to claim 1, in the manufacture of a medicament for the treatment of an FGFR 4-mediated disorder.

6. The use of claim 5, wherein the disorder is cancer.

7. The use of claim 6, wherein the cancer is selected from bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, ovarian cancer, prostate cancer, esophageal cancer, gall bladder cancer, pancreatic cancer, lung cancer, mesothelioma, testicular cancer, squamous cell carcinoma, thyroid cancer, skin cancer, leukemia, B-cell lymphoma, glioblastoma, melanoma, and rhabdomyosarcoma.

8. The use of claim 6, wherein the cancer is hepatocellular carcinoma.

9. The use of claim 6, wherein the cancer is adult T cell leukemia or acute myeloid leukemia.

10. The use of claim 6, wherein the cancer is Hodgkin's lymphoma, non-Hodgkin's lymphoma.

11. The use of claim 6, wherein the cancer is multiple myeloma.

12. The use of claim 6, wherein the cancer is chronic lymphocytic lymphoma.

13. The use of claim 6, wherein the cancer is waldenstrom's macroglobulinemia.

14. The use of claim 6, wherein the cancer is hairy cell lymphoma.

15. The use of claim 6, wherein the cancer is Burkitt's lymphoma.

16. Use of any one of the following compounds in the preparation of a medicament for inhibiting the activity of FGFR4 or a mutant thereof in a patient or a biological sample:

17. the use of claim 16, wherein the activity of FGFR4 or a mutant thereof is irreversibly inhibited.

18. The use of claim 17, wherein the activity of FGFR4 or a mutant thereof is irreversibly inhibited by covalently modifying Cys477 and Cys552 of FGFR 4.

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