CN113527311A - FGFR4 inhibitor, composition and application thereof in preparation of medicines - Google Patents

FGFR4 inhibitor, composition and application thereof in preparation of medicines Download PDF

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CN113527311A
CN113527311A CN202110965475.4A CN202110965475A CN113527311A CN 113527311 A CN113527311 A CN 113527311A CN 202110965475 A CN202110965475 A CN 202110965475A CN 113527311 A CN113527311 A CN 113527311A
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cancer
fgfr4
compound
pyrimidin
dimethoxyphenyl
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CN113527311B (en
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陈永恒
陈小娟
徐广宇
付莹
陈主初
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Xiangya Hospital of Central South University
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    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
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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. The 9 specific compounds are provided in the example 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 control, so that the compound has potential application prospect. 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 combined with Cys552 in FGFR4 and not capable of being covalently combined with Cys477 in FGFR4, and the compound such as LX09 is an FGFR4 inhibitor capable of being covalently combined with two Cys552 and Cys477 cysteines 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 the other 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, and its ligand FGF19 and co-receptor beta-Klotho bind exclusively to FGFR4 to regulate hepatocyte proliferation, and excessive FGF19 protein increases the probability of proliferation and invasion of HCC cell lines. The liver cancer heterotypic transplanted mouse with high FGFR4 expression can effectively eliminate liver cancer generation in a mouse model by inhibiting the generation of FGF19-FGFR4 or using an FGFR4 antibody. Clinical studies showed that half of all HCC patients had FGFR4 overexpressed, and most of the HCC patients had 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 are all covalently bound to Cys552 sulfhydryl group of the hinge region of FGFR4 protein, while PRN1371, FIIN-2 and TAS-120 are covalently bound to Cys477 in the p-loop of FGFR4 protein, all of which are covalently bound to only one cysteine residue, and no inhibitor has been found that can be covalently bound to both cysteine residues in 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.
The compounds of the present invention have the structure of formula I:
formula I
Figure BDA0003223782130000021
Wherein R is1Refers 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-)
Figure BDA0003223782130000022
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;
Figure BDA0003223782130000023
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;
Figure BDA0003223782130000031
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;
Figure BDA0003223782130000032
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;
Figure BDA0003223782130000033
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;
Figure BDA0003223782130000041
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;
Figure BDA0003223782130000042
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;
Figure BDA0003223782130000043
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;
Figure BDA0003223782130000051
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 may 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
Figure BDA0003223782130000071
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,
Figure BDA0003223782130000081
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
Figure BDA0003223782130000082
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 monitors the reaction, after the reaction is completed, the solvent is pumped out under reduced pressure, 25mL of ethanol is added, the mixture is stirred for 30min under ice bath, white solid is separated out, and 5.3g of compound 1 is obtained after filtration and drying, the white solid is obtained, and the yield is 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
Figure BDA0003223782130000083
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 completion of the reaction, 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)
Figure BDA0003223782130000091
Compound 2(2.00g, 4.65mmol), triphosgene (695mg, 2.31mmol) and 15mL of dry THF are added to a 25mL single neck flask, triethylamine (940mg, 9.32mmol) is slowly added dropwise in ice bath and stirred for 1h, the reaction is further heated to 70 ℃ to react for 10h, TLC monitors until the raw material reaction is complete (petroleum ether: ethyl acetate ═ 2:1), 5mL of quench reaction is added, solvent THF is removed under reduced pressure, ethyl acetate (20mL × 3) is extracted, the organic phases are washed successively with saturated sodium bicarbonate and saturated NaCl, combined, dried over anhydrous sodium sulfate, and the residue is purified by silica gel column chromatography to give 1.76g of compound 3, a pale yellow solid, yield 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.
Step 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)
Figure BDA0003223782130000092
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)
Figure BDA0003223782130000101
Into a 50mL single neck flask 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 at 25 ℃ for 10h, monitoring the reaction by TLC until the raw materials are completely reacted, filtering the reaction solution by using kieselguhr to obtain a mother solution, 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%. Taking compound 5(200mg, 0.39mmol) in a 25mL two-necked flask, adding 10mL dry DCM, triethylamine (87mg, 0.86mmol), nitrogen protection, stirring for 10min under ice salt bath, slowly adding acryloyl chloride (68mg, 0.76mmol) in dichloromethane (1mL) dropwise, TLC monitoring the raw materials until the reaction is complete (petroleum oil)Ether ethyl acetate methanol 10:10:1), 2mL of ice water was added to quench the reaction, ethyl acetate (20mL × 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, 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.
Synthetic example 2 Synthesis of covalent Compound LX02
Step 1: synthesis of 2-cyano-3-methyl-2-butenoic acid (6)
Figure BDA0003223782130000111
To a 50mL single-neck flask were added cyanoacetic acid (2.55g, 30.0mmol), acetone (3.48g, 60.0mmol) and 25mL of toluene, reacted at 50 ℃ for 10 hours, TLC monitored until the reaction was complete (ethyl acetate: petroleum ether ═ 2:1), toluene (20mL × 3) extracted, the organic phases 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)
Figure RE-GDA0003253399950000112
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 combinedAfter 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)
Figure BDA0003223782130000121
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)
Figure BDA0003223782130000122
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)
Figure BDA0003223782130000131
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)
Figure BDA0003223782130000132
A50 mL single-neck flask was charged with 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), solvent was removed, and the residue was chromatographed on silica gel columnPurification by chromatography gave 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)
Figure BDA0003223782130000141
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)
Figure BDA0003223782130000142
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, and 4mL of anhydrous DMA was added, and the mixture was reacted at 110 ℃ for 3 hours 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)
Figure BDA0003223782130000151
Into 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%. Compound 12(400mg, 0.76mmol) was taken in a 25mL two-necked flask, triethylamine (87mg, 0.86mmol) and 10mL dry dichloromethane were added, stirred for 10min under ice salt bath under nitrogen protection, a solution of acryloyl chloride (68mg, 0.76mmol) 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, extraction was then performed with ethyl acetate (20mL × 3), washing was performed with saturated sodium bicarbonate, the organic phases were combined, dried over anhydrous sodium sulfate, and purification by column chromatography under reduced pressure of the extraction solvent gave 45mg 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)
Figure BDA0003223782130000161
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%. Taking the intermediate 13(7.40g, 19.8mmol), putting the intermediate into a 50mL single-neck flask, adding 30mL ethanol and 8mL hydrazine hydrate, reacting for 3h at 70 ℃, monitoring raw materials by TLC until the reaction is complete (ethyl acetate: petroleum ether: 1:5), cooling the reaction liquid to normal temperature, carrying out vacuum filtration to obtain a mother liquid, carrying out vacuum filtration to remove the solvent, cooling, adding 20mL diethyl ether and a small amount of anhydrous sodium sulfate, stirring for 10min at 0 ℃, carrying out vacuum filtration to obtain the mother liquid, and carrying out vacuum filtration to obtain 3.9g of a compound 14, a colorless oily substance and a yield of 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)
Figure BDA0003223782130000171
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
Figure BDA0003223782130000172
mg, 1.44mmol) and 15mL of dry THF, slowly adding triethylamine (58.2mg, 5.76mmol) dropwise under ice bath, stirring for 1h, heating the reaction to 70 ℃ for 10h, monitoring by TLC (petroleum ether: ethyl acetate ═ 2:1), cooling to 0 ℃ after the reaction is complete, adding 5mL of ice water to quench the reaction, removing the solvent under reduced pressure, extracting with 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 crude product, 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, 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)
Figure BDA0003223782130000181
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 starting material reaction completed (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 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 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)
Figure BDA0003223782130000182
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, 1mL trifluoroacetic acid was added, stirred overnight at 27 ℃, TLC monitored for completion of the starting material reaction (petroleum ether: ethyl acetate: methanol: 10:1), and the reaction was added to 3 under ice-bath0mL of a saturated sodium bicarbonate solution was stirred for 10min, followed by extraction with dichloromethane (20 mL. times.3), washing with saturated sodium bicarbonate, combining the organic phases, drying over anhydrous sodium sulfate, and removing the solvent under reduced pressure to obtain 450mg of 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 Synthesis of covalent Compound 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)
Figure BDA0003223782130000191
Compound 3(500mg, 1.25mmol), o-phenylenediamine (570mg, 1.88mmol), trifluoroacetic acid (214mg, 1.88mmol) and 4mL of sec-butanol were weighed out in that orderThe reaction was carried out in a 25mL Schlenk tube under nitrogen at 100 ℃ for 10 h. 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
Figure BDA0003223782130000201
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%. Taking compound 21(312mg, 0.63mmol) in a 25mL two-port bottle, adding triethylamine (171mg, 1.57mmol) and 10mL dry dichloromethane, stirring for 10min under an ice salt bath under nitrogen protection, slowly adding a dichloromethane (1mL) solution of acryloyl chloride (115mg, 0.76mmol) dropwise, TLC monitoring the raw materials until the reaction is complete (petroleum ether: ethyl acetate: methanol ═ 10:10:1), adding 2mL ice water to quench the reaction, extracting dichloromethane (30mL multiplied by 2), and sequentially using saturated carbonic acidSodium hydrogen, saturated NaCl wash, combine the organic phases, dry over anhydrous sodium sulfate, pump off the solvent under reduced pressure, and purify the residue by silica gel column chromatography to give 56mg of compound LX07 as a white solid in 14.6% yield.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)
Figure BDA0003223782130000211
To a 50mL one-neck flask were added compound 1(2.00g, 6.41mmol), 4-nitrophenylethylamine hydrochloride (1.67g, 8.32mmol), DIEA (3.28g, 25.4mmol) and 20mL of dioxane, reacted at 60 ℃ for 10h, monitored by TLC 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)
Figure BDA0003223782130000212
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-nitrophenyl-ethyl) -3, 4-dihydropyrimidinyl [4,5-d ] pyrimidin-2 (1H) -one (24)
Figure BDA0003223782130000221
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. TLC monitored the reaction until the starting material was completely reacted (ethyl acetate: petroleum ether: TEA ═ 10:10:1), extracted with ethyl acetate (30mL × 3), and saturated carbonic acidWashed with sodium hydrogen, 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 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)
Figure BDA0003223782130000231
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. Compound 25(180mg, 0.36mmol) was taken in a 25mL two-necked flask, 10mL of dried dichloromethane, triethylamine (98mg, 0.89mmol) and nitrogen protection were added, stirred for 10min under ice salt bath, a solution of acryloyl chloride (65mg, 0.72mmol) in dichloromethane (1mL) was slowly added dropwise for 2h reaction, TLC monitored until the reaction was complete (petroleum ether: ethyl acetate: methanol ═ 10:10:1), 2mL of ice water was added to quench the reaction, dichloromethane (30mL × 2) was extracted, washed with saturated sodium bicarbonate and saturated NaCl in sequence, 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 with a yield of 16.1%.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)
Figure BDA0003223782130000241
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, 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 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)
Figure BDA0003223782130000242
Figure BDA0003223782130000251
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), quenched with 2mL ice water, extracted with dichloromethane (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 LX09 as a white solid with a yield of 14.7%.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.
Compound (I)Detection of inhibitory Activity 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 reaction was performed in white opaque 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 solution ADP-Glo, incubating at room temperature for 40 minutes, 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)
Figure BDA0003223782130000261
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, LX04 on FGFR4, acrylamide groups perform optimally in our chosen electrophilic groups. Tail R2The structure is about 3-6 times less active than the benzene ring for six-membered rings, e.g., LX01, LX05 compared to IC50 for LX06 and 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, while LX07, LX08, LX09 had only a 4-5-fold reduction in C477A activity over wild-type. Of these 9 compounds, compounds LX01, LX05, LX07, LX08 and LX09 have activity against FGFR4Has higher inhibitory activity and potential medicinal prospect.
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)
Figure BDA0003223782130000262
Figure BDA0003223782130000271
From the view 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 effect of inhibiting proliferation of Ba \ F3 cells activated by FGFR4 signal channels. 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
To test the covalent binding of the compounds having a significant inhibitory effect on FGFR4 according to the present invention to the two cysteines (Cys477 and Cys552) in FGFR4, changes in molecular weight of kinase mutants FGFR4(C477A) and FGFR4(C552A) before and after binding of the compounds were examined using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). 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, the compounds LX01, LX05, LX06, LX07, LX08 all covalently bind to Cys552 in FGFR4 only and not to Cys477 in FGFR4, while the 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 referred to herein are incorporated by reference into 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 (15)

1. An FGFR4 inhibitor having the structure of formula I:
Figure FDA0003223782120000011
or a pharmaceutically acceptable salt thereof,
wherein R is1Refers 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, q is a natural number from 1 to 3, A is phenyl, R is3Is hydrogen or methyl.
2. The FGFR4 inhibitor according to claim 1, wherein R1Is an acryloyl group.
3. The FGFR4 inhibitor according to claim 1, L is independently a C1-C3 alkyl group.
4. The FGFR4 inhibitor according to claim 1, R2Is phenyl.
5. The FGFR4 inhibitor of claim 1 selected from the group consisting 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,
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,
LX 08: 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,
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.
6. A pharmaceutical composition comprising a therapeutically effective amount of the FGFR4 inhibitor of any of claims 1 to 5, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
7. A pharmaceutical composition comprising a therapeutically effective amount of the FGFR4 inhibitor of any of claims 1 to 5, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent.
8. A pharmaceutical composition comprising a therapeutically effective amount of the FGFR4 inhibitor of any of claims 1 to 5, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.
9. 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 a FGR 4-mediated disorder.
10. The use according to claim 9, wherein the disorder is cancer.
11. The use of claim 10, wherein the cancer is selected from the group consisting of hepatocellular carcinoma, bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, gastric cancer, head and neck cancer, renal cancer, liver cancer, ovarian cancer, prostate cancer, esophageal cancer, gallbladder cancer, pancreatic cancer, lung cancer, mesothelioma, testicular cancer, squamous cell carcinoma, thyroid cancer, skin cancer, 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 lymphoma, glioblastoma, melanoma, and rhabdomyosarcoma.
12. The use of claim 11, wherein the cancer is hepatocellular carcinoma.
13. Use of a compound according to claim 1 in the manufacture of a medicament for inhibiting the activity of FGFR4 or a mutant thereof in a patient or a biological sample.
14. The use of claim 11, wherein the activity of FGFR4 or mutants thereof is irreversibly inhibited.
15. The use of claim 11, 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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