CN111410667A - Novel (1,2,4) triazolo (1,5-a) pyridylphosphine oxides and use thereof - Google Patents

Abstract

The present invention relates to novel (1,2,4) triazolo (1,5-a) pyridylphosphine oxides and their use. The compound is a compound shown as a formula I, or pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer or tautomer thereof, or prodrug thereof, wherein R is¹～R⁵As defined in the specification. The compound can be used for preparing a medicament for treating and/or preventing cancers.

Description

Novel (1,2,4) triazolo (1,5-a) pyridylphosphine oxides and use thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a novel (1,2,4) triazolo (1,5-a) pyridyl phosphorus oxide and application thereof.

Background

China's non-small cell lung cancer (NSC L C) patients account for about 85% of the total lung cancer cases, and about 30% -40% of the cases can generate Epidermal Growth Factor Receptor (EGFR) mutation.

Commonly used first and second generation EGFR-targeting drugs are gefitinib (iressa), erlotinib (tarceva), erlotinib (sodium chemeride), afatinib (getarari). However, most patients develop resistance after about one year using such targeted drugs. The drug resistance may be caused by the T790M mutation (about 60%) of EGFR gene, or c-MET amplification, HER2 mutation, activation of downstream KRAS or BRAF, etc., and also a part of patients transformed into small cell lung cancer. How to solve the problem of EGFR resistance? The answer is that symptomatic medication must be given. As against the T790M mutation, astrazen introduced the third generation EGFR TKI targeting drug, toretha (oxitinib, or AZD 9291). With the approval of Thoraisand on the market in China and the entrance of third-generation EGFR-TKI targeted drugs into conventional clinical treatment of lung cancer, more EGFR-mutated patients have the hope of obtaining treatment, but after the drug administration, a batch of lung cancer patients generate third-generation TKI resistance; genome sequencing found that drug resistance occurred mainly in the C797S mutation of EGFR. Therefore, the existing EGFR kinase inhibitor drugs still need to be improved, and the development of a new generation EGFR kinase inhibitor which is novel, safe and effective is urgently needed.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a novel (1,2,4) triazolo (1,5-a) pyridyl phosphorus oxide and application thereof, and the novel (1,2,4) triazolo (1,5-a) pyridyl phosphorus oxide provided by the invention can be used as an EGFR T790M/C797S mutant kinase inhibitor for treating and/or preventing tumors.

According to a first aspect of the present invention, there is provided a compound represented by formula I, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or a solvate thereof, or a metabolite thereof, or a stereoisomer thereof, or a tautomer thereof, or a prodrug thereof,

wherein the content of the first and second substances,

the A is selected from C, N, O or S;

the R1 is selected from H, C1-C8 alkyl, C1-C8 heteroalkyl, C3-C8 cycloalkyl, or C3-C8 heterocycloalkyl;

r2 is selected from H, C1-C8 alkyl, or C1-C8 heteroalkyl;

r3 is selected from H, halogen, cyano, C1-C8 alkyl, C1-C8 heteroalkyl, C3-C8 cycloalkyl, or C3-C8 heterocycloalkyl;

the R4 is selected from C1-C8 alkyl, C1-C8 heteroalkyl, C3-C8 cycloalkyl, or C3-C8 heterocycloalkyl;

r5 is selected from H, halogen, cyano, C1-C8 alkyl, C1-C8 heteroalkyl, C3-C8 cycloalkyl, or C3-C8 heterocycloalkyl.

Preferably, the "hetero" in "C1-C8 heteroalkyl", "C3-C8 heterocycloalkyl", "five-membered heteroaryl", or "six-membered heteroaryl" denotes a heteroatom or a group of heteroatoms. According to an embodiment of the invention, said heteroatom or heteroatom group is individually selected from-O-, -S-, -NH-, ═ O, -O-N ═ -C (═ O) O-, -C (═ O) -, -S (═ O) -, -O)₂-、-C(＝O)NH-、-S(＝O)₂NH-, or-NHC (═ O) NH-, in any of the above cases, the number of heteroatoms or groups of heteroatoms is independently selected from 1,2, or 3, respectively.

According to an embodiment of the present invention, the compound of formula I, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or a solvate thereof, or a metabolite thereof, or a stereoisomer thereof, or a tautomer thereof, or a prodrug thereof, is selected from any one of the following:

wherein, R is₁～R₅The definition of the A group is as described above.

Thus, throughout this specification, the skilled person will be able to refer to the R in the compounds of formula I₁～R₆The group a and its substituents are selected to provide a stable compound of formula I, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or a solvate thereof, or a metabolite thereof, or a stereoisomer thereof, or a tautomer thereof, or a prodrug thereof, as described in the examples of this invention.

According to an embodiment of the present invention, the compound of formula I, or a pharmaceutically acceptable salt, a hydrate, a solvate, a metabolite, a stereoisomer, a tautomer, or a prodrug thereof, is selected from any one of the following:

in the present invention, a person skilled in the art can select the groups and their substituents in the compound of formula I to provide stable compounds of formula I, or pharmaceutically acceptable salts thereof, or hydrates thereof, or solvates thereof, or metabolites thereof, or stereoisomers thereof, or tautomers thereof, or prodrugs thereof, including but not limited to I-1 to I-14 described in the examples of the present invention.

The reaction solvent used in each reaction step described in the present invention is not particularly limited, and any solvent that can dissolve the starting materials to some extent and does not inhibit the reaction is included in the present invention. Further, many equivalents, substitutions, or equivalents in the art to which this invention pertains, as well as different proportions of solvents, solvent combinations, and solvent combinations described herein, are deemed to be encompassed by the present invention.

According to a second aspect of the present invention, the present invention provides a pharmaceutical composition, which comprises an effective amount of a compound represented by formula I, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or a solvate thereof, or a metabolite thereof, or a stereoisomer thereof, or a tautomer thereof, or a prodrug thereof, and at least one pharmaceutical excipient.

The pharmaceutical excipients can be those widely used in the field of pharmaceutical production. The excipients are used primarily to provide a safe, stable and functional pharmaceutical composition and may also provide methods for dissolving the active ingredient at a desired rate or for promoting the effective absorption of the active ingredient after administration of the composition by a subject. The pharmaceutical excipients may be inert fillers or provide a function such as stabilizing the overall pH of the composition or preventing degradation of the active ingredients of the composition. The pharmaceutical excipients may include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrating agents, lubricants, antiadherents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, reinforcing agents, adsorbents, buffering agents, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

The pharmaceutical compositions of the present invention may be prepared according to the disclosure using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, levigating, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical compositions of the present invention may be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implant, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical compositions of the present invention may also be in a controlled release or delayed release dosage form (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum formulations. Examples of formulations for parenteral administration include, but are not limited to, solutions for injection, dry preparations which can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic formulations; aerosol: such as nasal sprays or inhalants; liquid dosage forms suitable for parenteral administration; suppositories and lozenges.

Oral administration of the compounds of the invention is preferred. Intravenous administration of the compounds of the invention is also preferred. Depending on the circumstances, other application routes may be applied or even preferred. For example, transdermal administration may be highly desirable for patients who are forgetful or whose oral medications are irritable. In particular cases, the compounds of the invention may also be administered by transdermal, intramuscular, intranasal or intrarectal routes. The route of administration may vary in any manner, limited by the physical nature of the drug, the convenience of the patient and caregiver, and other relevant circumstances.

According to a third aspect of the invention, the invention provides a compound shown in formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutical composition thereof, and the use of the compound or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof, or the tautomer thereof, in the preparation of a medicament for treating and/or preventing diseases caused by EGFR T790M/C797S abnormal mutation. The compound provided by the invention can be used for treating and/or preventing one or more diseases caused by EGFR T790M/C797S abnormal mutation, and has good clinical application and medical application.

The invention provides application of a compound shown as a formula I, or a pharmaceutically acceptable salt, a hydrate, a solvate, a metabolite, a stereoisomer, a tautomer, a prodrug or a pharmaceutical composition thereof in preparation of EGFR T790M/C797S inhibitor drugs. The compound provided by the invention has excellent EGFR T790M/C797S inhibitory activity, can be effectively used as an EGFR T790M/C797S inhibitor, and is used as a therapeutic drug of the EGFR T790M/C797S inhibitor.

According to a fifth aspect of the present invention, the present invention provides a compound represented by formula I, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or a solvate thereof, or a metabolite thereof, or a stereoisomer thereof, or a tautomer thereof, or a prodrug thereof, or a pharmaceutical composition thereof for use in the preparation of a medicament for the treatment and/or prevention of cancer. The compound provided by the invention can be used for preparing a medicament for treating and/or preventing cancers, wherein the cancers include but are not limited to lung cancer, glioma, renal cancer, prostate cancer, pancreatic cancer and breast cancer. As for various solid cancers, the compounds of the present patent can be used if there is an EGFR T790M/C797S mutation abnormality.

Terms and definitions

Unless stated to the contrary, the following terms used in the specification and claims have the following meanings.

"alkyl" refers to a saturated aliphatic hydrocarbon group including straight and branched chain groups of 1 to 20 carbon atoms, such as straight and branched chain groups of 1 to 18 carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In this context "alkyl" may be a monovalent, divalent or trivalent radical. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, and the various branched chain isomers thereof, and the like. Non-limiting examples also include methylene, methine, ethylene, ethylidene, propylidene, butylidene, and various branched chain isomers thereof. Alkyl groups may be optionally substituted or unsubstituted.

"cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising 3 to 12 ring atoms, which may be, for example, 3 to 12, 3 to 10, or 3 to 6 ring atoms, or may be a3, 4,5, 6 membered ring. Non-limiting examples of monocyclic radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl and the like. The cyclic group may be optionally substituted or unsubstituted.

"heterocycloalkyl" means a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent comprising 3 to 20 ring atoms, which may be, for example, 3 to 16, 3 to 12, 3 to 10, or 3 to 6 ring atoms, wherein one or more ring atoms are selected from nitrogen, oxygen, or a heteroatom of S (O) m (wherein m is 0, 1, or 2), but not including the ring moiety of-O-O-, -O-S-, or-S-S-, the remaining ring atoms being carbon. Preferably 3 to 12 ring atoms of which 1-4 are heteroatoms, more preferably a heterocycloalkyl ring comprising 3 to 10 ring atoms, most preferably a 5-or 6-membered ring of which 1-4 are heteroatoms, more preferably 1-3 are heteroatoms, most preferably 1-2 are heteroatoms. Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclic groups include spiro, fused, or bridged heterocyclic groups.

"halogen" means fluorine, chlorine, bromine and iodine, preferably fluorine, chlorine and bromine.

"optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. For example: "heterocyclic group optionally substituted with alkyl" means that alkyl may, but need not, be present, and this description includes the case where the heterocyclic group is substituted with alkyl and the heterocyclic group is not substituted with alkyl.

"substituted" means that one or more, preferably up to 5, more preferably 1 to 3, hydrogen atoms in a group are independently substituted with a corresponding number of substituents.

"pharmaceutically acceptable salts" refers to salts of the compounds of the present invention, prepared from the compounds of the present invention found to have particular substituents, with relatively nontoxic acids or bases. When compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonia or magnesium salts or similar salts. When compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, and the like; and salts of organic acids including acids such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, methanesulfonic, and the like; also included are Salts of amino acids (e.g., arginine, etc.), and Salts of organic acids such as glucuronic acid (see Berge et al, "Pharmaceutical Salts," Journal of Pharmaceutical Science 66:1-19 (1977)). Certain specific compounds of the invention contain both basic and acidic functionalities and can thus be converted to any base or acid addition salt. Preferably, the neutral form of the compound is regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. The parent form of the compound differs from the various salt forms by certain physical properties, such as solubility in polar solvents. According to an embodiment of the present invention, the pharmaceutically acceptable salt of the compound of formula I of the present invention is preferably hydrochloride, hydrobromide, phosphate, or sulfate, most preferably hydrochloride.

"pharmaceutical composition" means a mixture comprising one or more compounds of formula I, as described herein, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a tautomer thereof, in combination with other chemical components, as well as other components, such as pharmaceutically acceptable excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

The invention synthesizes a series of novel [1,2,4] triazolo [1,5-a ] pyridyl phosphorus oxides, and related enzyme and cell activity tests show that the compound has excellent cell activity, the IC50 value of the compound on cell proliferation in vitro reaches nM level, and the compound can be well applied to various tumors. According to the embodiment of the invention, the compound has a very good inhibition effect on human non-small cell lung cancer cell BaF3 abnormally expressed by EGFR T790M/C797S. And animal in vivo efficacy experiments further prove that the compound has excellent tumor inhibition effect. The compound has excellent EGFR T790M/C797S inhibitory activity, can be used for preparing a therapeutic drug of an EGFR T790M/C797S inhibitor, is used for treating and/or preventing one or more diseases caused by EGFR T790M/C797S abnormal mutation, and is used for preparing a drug for treating and/or preventing cancer.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Detailed Description

The preparation of the compound of formula I, or a pharmaceutically acceptable salt, a hydrate, a solvate, a metabolite, a stereoisomer, a tautomer, or a prodrug thereof according to the present invention can be accomplished by the following exemplary methods and relevant publications used by those skilled in the art, but the scope of the present invention is not limited by these examples. The present invention is described in detail below by way of examples, but is not meant to be limited to any of the disadvantages of the present invention.

The structure of the compounds of the invention is determined by Nuclear Magnetic Resonance (NMR) or Mass Spectrometry (MS). NMR was measured using a Bruker AVANCE-400 or Varian Oxford-300 nuclear magnetic instrument in deuterated dimethyl sulfoxide (DMSO-d)₆) Deuterated chloroform (CDC 1)₃) Deuterated methanol (CD)₃OD) internal standard as Tetramethylsilane (TMS) chemical shift is 10^-6(ppm) is given as a unit.

MS was measured using an Agilent SQD (ESI) mass spectrometer (manufacturer: Agilent, model 6110) or Shimadzu SQD (ESI) mass spectrometer (manufacturer: Shimadzu, model 2020).

HP L C was determined using an Agilent 1200DAD high pressure liquid chromatograph (Sunfirc C18, 150X4.6mm, 5wn, column) and Waters 2695-2996 high pressure liquid chromatograph (Gimini C18, 150X4.6mm, 5ym column).

The thin layer chromatography silica gel plate is Qingdao sea GF254 silica gel plate, the specification of the silica gel plate used by the thin layer chromatography (T L C) is 0.15mm-0.2mm, and the specification of the thin layer chromatography separation and purification product is 0.4mm-0.5 mm.

Column chromatography generally uses Qingdao ocean 200-mesh and 300-mesh silica gel as a carrier.

Known starting materials of the present invention can be synthesized using or following methods known in the art, companies such as Shaoyuan chemical technology (Accela ChemBio Inc), Beijing coupled chemicals, and the like.

In the examples, the reaction was carried out under an argon atmosphere or a nitrogen atmosphere unless otherwise specified. The hydrogenation reaction was usually evacuated and charged with hydrogen and repeated 3 times.

In the examples, the reaction temperature was room temperature and the temperature range was 20 ℃ to 30 ℃ unless otherwise specified.

The progress of the reaction in the examples was monitored by thin layer chromatography (T L C) using a developing solvent system of A dichloromethane and methanol and B petroleum ether and ethyl acetate, the volume ratio of the solvent being adjusted depending on the polarity of the compound.

The system of eluents for column chromatography and developing agents for thin layer chromatography used for purifying compounds include a: dichloromethane and methanol systems; b: the volume ratio of the solvent in the petroleum ether and ethyl acetate system is adjusted according to different polarities of the compounds, and a small amount of triethylamine, an acidic or basic reagent and the like can be added for adjustment.

Example 1: preparation of Compound represented by formula I-1

The synthetic route is as follows:

the first step is as follows: synthesis of Compound 1B

Dissolving the compound 1A (5g, 39.7mmol) in dichloromethane (100ml), cooling to 0-5 ℃, then slowly dropwise adding 70% nitric acid (5ml), keeping the internal temperature less than 5 ℃ in the dropwise adding process, raising the temperature to room temperature after the dropwise adding process, continuing to react for one hour, wherein T L C shows that the reaction is finished, washing the reaction solution with water (50m L× 2), drying the organic phase with anhydrous sodium sulfate, filtering, and concentrating under reduced pressure to obtain a crude compound 1B (4.48 g in a yellow oil state), wherein the yield is 66%, and the crude compound is directly used in the next step without further purification.

The second step is that: synthesis of Compound 1C

Compound 1B (2.83g,16.5mmol) obtained in the previous step was dissolved in DMF (25ml), potassium carbonate (3.4g,25mmol) and iodomethane (1.2m L, 20mmol) were added and stirred at room temperature overnight after the addition, T L C showed the reaction was complete, the reaction was poured into water and a large amount of solid was precipitated, filtered, and the filter cake was washed several times with water to obtain compound 1C (2.76g, yellow solid) in 90% yield.

The third step: synthesis of Compound 1D

Compound 1C (2.5g,13.5mmol) obtained in the previous step was dissolved in DMF (25ml) at room temperature, cesium carbonate (6.6g,20mmol)) and 1-ethyl-4- (4-piperidinyl) piperazine (2.47g,13.5mmol) were added, the reaction temperature was heated to 80 ℃ for 4 hours, T L C showed the reaction to be complete, then the reaction solution was poured into water, extracted with ethyl acetate (50m L× 2), the organic phases were combined, washed with saturated brine (50m L× 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was chromatographed on silica gel column (eluent petroleum ether: ethyl acetate 3:1-1:1) to obtain compound 1D (4.0g, yellow solid) with 85% yield.

MS m/z(ESI):349[M+1].

The fourth step: synthesis of Compound 1E

Compound 1D (4.0g,11.5mmol) obtained in the previous step was dissolved in methanol (50ml), 10% palladium on carbon (500mg) was added, hydrogen was replaced three times with a hydrogen balloon, then stirred overnight at room temperature while maintaining a hydrogen atmosphere, T L C indicated the reaction was complete, filtered over silica, washed with methanol, and the filtrate was concentrated to give crude compound 1E (3.42g) in 93.5% yield, which was used directly in the next step without further purification.

MS m/z(ESI):319[M+1].

The fifth step: synthesis of Compound 1G

Compound 1F (10G,51mmol) was dissolved in toluene (50ml) and benzophenone imine (10.9G,60 mmol) was added followed by cesium carbonate (32.6G,100mmol), Pd2dba3(2.3G,2.5mmol) and xanthphos (1.44G,2.5mmol) and after addition three times replacement with nitrogen, then warmed to 110 ℃ under nitrogen protection for 3 hours, T L C showed the reaction was complete, water was added, the organic layer was separated, the aqueous phase was extracted with ethyl acetate (50m L× 2), the organic phases were combined, the organic phase was dried over anhydrous sodium sulfate, filtered and the residue obtained by spin-dry concentration was chromatographed on a silica gel column (eluent petroleum ether: ethyl acetate ═ 3:1-1:1) to give compound 1G (12.7G, yellow solid) in 83% yield.

MS m/z(ESI):299[M+1]

And a sixth step: synthesis of Compound 1H

The intermediate 1G (12.5G,42mmol) obtained in the previous step was dissolved in THF (200ml), concentrated hydrochloric acid (20ml) was slowly added with stirring at room temperature, then stirring was continued for 2 hours at room temperature, T L C showed the reaction was complete, the reaction solution was slowly added to a saturated aqueous sodium bicarbonate solution, the internal temperature was protected to less than 10 ℃, and extraction was performed with ethyl acetate (50m L× 2), the organic phases were combined, and then dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was subjected to silica gel chromatography (eluent petroleum ether: ethyl acetate: 3:1-1:1) to obtain compound 1H (5.3G, yellow solid) in 93% yield.

MS m/z(ESI):135[M+1].

The seventh step: synthesis of Compound 1I

Compound 1H (5g,37mmol) obtained in the previous step was dissolved in DMF (50ml), sodium periodate (9.5g,44.4mmol) was added at room temperature, after stirring for 10 minutes, iodine (11.3g,44.4mmol) was added in portions, reaction was performed for 2 hours after completion of addition, T L C showed completion of reaction, then the reaction solution was added to a saturated sodium thiosulfate solution, sufficiently stirred until colorless, the resulting aqueous phase was extracted with ethyl acetate (50m L× 2), the organic phases were combined, and then dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was subjected to silica gel chromatography (eluent petroleum ether: ethyl acetate: 3:1-1:1) to obtain compound 1I (6.18g, yellow solid) in 64% yield.

MS m/z(ESI):261[M+1].

Eighth step: synthesis of Compound 1J

Compound 1I (6.1g,23.5mmol) obtained in the previous step was added to DMF (100ml), followed by the sequential addition of dimethylphosphine oxide (2.2g,28mmol), potassium phosphate (10g,47mmol), palladium acetate (515mg,2.3mmol) and Xanthpos (1.33g,2.3mmol), followed by three nitrogen replacements after the addition was completed, reaction at 150 ℃ for 3 hours under nitrogen protection, after T L C showed the completion of the reaction, cooling to room temperature, addition to water, extraction with ethyl acetate (50m L× 2), combination of organic phases, washing of the organic phases with saturated brine ((50m L× 2), drying of the organic phases with anhydrous sodium sulfate, filtration, spin-drying and concentration of the resulting residue on a silica gel column chromatography (eluent dichloromethane: methanol: 50:1-10:1) to give compound 1J (3.3g, yellow solid) in 67% yield.

MS m/z(ESI):211[M+1].

The ninth step: synthesis of Compound 1K

After compound 1J (3.3g,15.6mmol) obtained in the previous step was added to DMF (50ml) and stirred to dissolve, 5-bromo-2, 4-dichloropyrimidine (5.33g,23.4mmol) and potassium carbonate (4.3g,31.2mmol) were sequentially added, then heated to 60 ℃ for reaction for 4 hours, and after T L C showed that the reaction was completed, cooled to room temperature, the reaction solution was poured into water, extracted with ethyl acetate (50m L× 2), the organic phases were combined, the organic phases were washed with saturated brine (50m L× 2), the organic phases were dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-dry concentration was subjected to silica gel chromatography (eluent dichloromethane: methanol: 50:1 to 10:1) to obtain compound 1K (3.6g, yellow solid) with a yield of 57.8%.

MS m/z(ESI):401[M+1].

The tenth step: synthesis of Compound represented by the formula I-1

Compound 1K (200mg,0.5mmol) obtained in the previous step was dissolved in n-butanol (5ml), compound 1E (261mg,0.75mmol) and concentrated hydrochloric acid (0.5ml) were added in this order, the reaction solution was reacted at 90 ℃ for four days, T L C showed the reaction to be complete, spin-dried, added to an aqueous sodium bicarbonate solution (30ml), extracted with dichloromethane (10m L× 2), the organic phases were combined, washed with saturated brine (10m L× 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was passed through a silica gel chromatography column (eluent dichloromethane: methanol: 50:1 to 10:1) to obtain a crude product of the compound represented by formula I-1, the crude product obtained was purified over 30 × 100mm of a preparative column through reverse phase C18 (mobile phase was 10 to 100% acetonitrile (0.05% TFA)/water), flow rate was 20m L/min, 10 min to obtain the target product of the compound represented by formula I-1 (7.5mg, white solid).

MS m/z(ESI):697[M+1]

¹H NMR(400MHz,MeOD):8.72(d,J＝2.8Hz,1H,8.44(s,1H),8.14(s,1H),7.73(d,J＝9.6Hz,1H),7.50(s,1H),6.73(s,1H),3.83(s,3H),3.15(d,J＝11.6Hz,2H),2.77-2.35(m,13H),2.18(s,3H),2.15(s,3H),2.05(s,3H),2.04-2.01(m,2H),1.71-1.67(m,2H),1.13(t,J＝7.2Hz,3H).

Example 2: preparation of the Compound represented by the formula I-2

The synthetic route is as follows:

the first step is as follows: synthesis of Compound 2A

Compound 1C (2.5g,13.5mmol) was dissolved in DMF (25ml) at room temperature, cesium carbonate (6.6g,20mmol)) and tert-butyl 4- (piperidin-4-yl) piperazine-1-carboxylate (3.63g,13.5mmol) were then added, the reaction temperature was heated to 80 ℃ for 4 hours, T L C showed the reaction to be complete, the reaction solution was poured into water, extracted with ethyl acetate (50m L× 2), the organic phases were combined, washed with saturated brine (50m L× 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by rotary drying concentration was chromatographed on silica gel column (eluent petroleum ether: ethyl acetate 3:1-1:1) to give compound 2A (5.0g, yellow solid) in 85.5% yield.

MS m/z(ESI):435[M+1].

The second step is that: synthesis of Compound 2B

Compound 2A (5.0g,11.5mmol) from the previous step was dissolved in dichloromethane (50ml) and TFA (10ml) was added at room temperature, after which time stirring at room temperature was continued for 2h, with T L C indicating the end of the reaction and concentration by spin drying to give crude compound 2B (4.5g, brown oil) which was used directly in the next step without purification.

MS m/z(ESI):335[M+1].

The third step: synthesis of Compound 2C

Compound 2B (4.5g) was dissolved in Dichloromethane (DCM) (50ml), cooled to 0-5 ℃ in an ice-water bath, triethylamine (3.5g,34.5mmol) was added, a solution of methyl chloroformate (1.64g,17.3mmol) in DCM (5ml) was added dropwise while maintaining the internal temperature below 5 ℃, after completion of the dropwise addition, the reaction was allowed to warm to room temperature for 2 hours, T L C showed the reaction to be complete, the reaction was washed twice with water (15ml × 3), the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-dry concentration was chromatographed on silica gel (eluent petroleum ether: ethyl acetate 3:1-1:1) to give compound 2C (2.48g, yellow solid) in 55% yield over two steps.

MS m/z(ESI):393[M+1].

The fourth step: synthesis of Compound 2D

Compound 2C (2.4g,6.12mmol) obtained in the previous step was dissolved in methanol (25ml) and then 10% palladium on carbon (250mg) was added and hydrogen replaced three times with a hydrogen balloon and then stirred overnight at room temperature maintaining a hydrogen atmosphere, T L C indicated the reaction was complete, filtered over silica, washed with methanol and the filtrate was concentrated to give crude compound 2D (2.07g) in 93.5% yield which was used directly in the next step without further purification.

MS m/z(ESI):363[M+1].

The fifth step: synthesis of the Compound represented by the formula I-2

Compound 1K (200mg,0.5mmol) obtained in the previous step was dissolved in n-butanol (5ml), compound 2D (272mg,0.75mmol) and concentrated hydrochloric acid (0.5ml) were added in this order, the reaction solution was reacted at 90 ℃ for four days, T L C showed the reaction to be complete, spin-dried, added to an aqueous sodium bicarbonate solution (30ml), extracted with dichloromethane (10m L× 2), the organic phases were combined, washed with saturated brine (10m L× 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was passed through a silica gel chromatography column (eluent dichloromethane: methanol: 50:1 to 10:1) to obtain a crude product of the compound represented by formula I-2, the crude product obtained was purified over 30 × 100mm of a preparative column through reverse phase C18 (mobile phase using 10 to 100% acetonitrile (0.05% TFA)/water), flow rate was 20m L/min, and the target product of the compound represented by formula I-2 was obtained (5.5mg, white solid).

MS m/z(ESI):630[M+1].

¹H NMR(400MHz,MeOD):8.72(d,J＝2.4Hz,1H),8.44(s,1H),8.17(s,1H),7.73(d,J＝9.6Hz,1H),7.49(s,1H),6.73(s,1H),3.83(s,3H),3.70(s,3H),3.50(brs,4H),3.15(d,J＝11.6Hz,2H),2.71-2.63(m,6H),2.43-2.36(m,1H),2.18(s,3H),2.15(s,3H),2.05(s,3H),1.98(d,J＝12.4Hz,2H),1.73-1.69(m,2H).

Example 3: preparation of Compound represented by formula I-3

The synthetic route is as follows:

the first step is as follows: synthesis of Compound 3A

Dissolving crude compound 2B (1.00g,3mmol) in DCM (10ml), cooling to 0-5 ℃ in an ice-water bath, then adding triethylamine (910mg,9mmol), then dropwise adding a DCM (5ml) solution of acetyl chloride (351mg,4.5mmol), maintaining the internal temperature below 5 ℃ while dropwise adding, heating to room temperature after dropwise adding, reacting for 2 hours, T L C shows that the reaction is finished, washing the reaction solution twice with water (10ml × 2), separating out the organic phase, drying over anhydrous sodium sulfate, filtering, and concentrating by spin drying to obtain a residue, passing the residue through a silica gel chromatographic column (eluent petroleum ether: ethyl acetate ═ 3:1-1:1) to obtain compound 3A (959mg, yellow solid) with a yield of 85%.

MS m/z(ESI):377[M+1].

The second step is that: synthesis of Compound 3B

Compound 3A (950mg,2.53mmol) obtained in the previous step was dissolved in methanol (10ml), 10% palladium on carbon (100mg) was then added, hydrogen was replaced three times with a hydrogen balloon, then stirred overnight at room temperature under hydrogen atmosphere, T L C indicated the end of the reaction, filtered over silica, washed with methanol, and the filtrate was concentrated to give crude compound 3B (830mg) in 95% yield, which was used directly in the next step without further purification.

MS m/z(ESI):347[M+1].

The third step: synthesis of the Compound represented by the formula I-3

Compound 1K (200mg,0.5mmol) obtained in the previous step was dissolved in n-butanol (5ml), compound 3B (260,0.75mmol) and concentrated hydrochloric acid (0.5ml) were added in this order, the reaction solution was reacted at 90 ℃ for four days, T L C showed the reaction to be complete, spin-dried, added to an aqueous sodium bicarbonate solution (30ml), extracted with dichloromethane (10m L× 2), the organic phases were combined, washed with saturated brine (10m L× 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was passed through a silica gel chromatography column (eluent dichloromethane: methanol: 50:1 to 10:1) to obtain a crude product of the compound represented by formula I-3, the crude product obtained was purified by reverse phase C18 preparative column YMC ODSA 30 × 100mm (mobile phase was 10 to 100% acetonitrile (0.05% TFA)/water) at a flow rate of 20m L/min for 10 minutes to obtain the target product of the compound represented by formula I-3 (7.3mg, white solid).

MS m/z(ESI):711[M+1].

¹H NMR(400MHz,MeOD):8.73(dd,J＝2Hz,9.2Hz,1H),8.44(s,1H),8.17(s,1H),7.72(d,J＝9.6Hz,1H),7.50(s,1H),6.73(s,1H),3.83(s,3H),3.63-3.60(m,4H),3.17-3.11(m,2H),2.73-2.65(m,6H),2.50-2.48(m,1H),2.18(s,3H),2.15(s,3H),2.11(s,3H),2.02(s,3H),1.99-1.80(m,2H),1.78-1.68(m,2H).

Example 4: preparation of Compound represented by formula I-4

The synthetic route is as follows:

the first step is as follows: synthesis of Compound 4A

Crude compound 2B (1.00g,3mmol) was dissolved in DCM (10ml), cooled to 0-5 ℃ in an ice-water bath, and triethylamine (910mg,9mmol) and N, N' -Carbonyldiimidazole (CDI) (583mg,3.6mmol) were added, after completion of the addition, the temperature was raised to room temperature for 2 hours reaction, T L C showed completion of the reaction, and then triethylamine (303mg,3mmol) and methylamine hydrochloride (245mg,3.6mmol) were added, the reaction was continued overnight at room temperature, T L C showed completion of the reaction, 30ml of DCM was added, washed twice with water (15ml × 2), the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-dry concentration was chromatographed on a silica gel column (eluent petroleum ether: ethyl acetate: 10: 1-1:1) to give compound 4A (991mg, yellow solid) with a yield of 84.5%.

MS m/z(ESI):392[M+1].

The second step is that: synthesis of Compound 4B

The compound 4A (950mg,2.43mmol) obtained in the previous step was dissolved in methanol (10ml), 10% palladium on carbon (200mg) was added, hydrogen was replaced three times with a hydrogen balloon, and then the mixture was stirred overnight at room temperature under a hydrogen atmosphere, T L C indicated that the reaction was complete, filtered over silica, washed with methanol, and the filtrate was concentrated to give crude compound 4B (829mg) in 94.5% yield, which was used directly in the next step without further purification.

MS m/z(ESI):362[M+1].

The third step: synthesis of the Compound represented by the formula I-4

Compound 1K (200mg,0.5mmol) obtained in the previous step was dissolved in n-butanol (5ml), compound 4B (270mg,0.75mmol) and concentrated hydrochloric acid (0.5ml) were added in this order, the reaction solution was reacted at 90 ℃ for four days, T L C showed the reaction to be complete, spin-dried, added to an aqueous sodium bicarbonate solution (30ml), extracted with dichloromethane (10m L× 2), the organic phases were combined, washed with saturated brine (10m L× 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was passed through a silica gel chromatography column (eluent dichloromethane: methanol: 50:1 to 10:1) to obtain a crude product of the compound represented by formula I-4, the crude product obtained was purified over 30 × 100mm of a preparative column through reverse phase C18 (mobile phase was 10 to 100% acetonitrile (0.05% TFA)/water), flow rate was 20m L/min, and 10 min, the target product of the compound represented by formula I-4 was obtained (7.1mg, white solid).

MS m/z(ESI):726[M+1].

¹H NMR(400MHz,MeOD):8.73(d,J＝13.2Hz,1H),8.44(s,1H),8.17(s,1H),7.73(d,J＝9.6Hz,1H),7.49(s,1H),6.74(s,1H),3.83(s,3H),3.43-3.41(m,4H),3.16(d,J＝12Hz,2H),2.73(s,3H),2.72-2.63(m,6H),2.45-2.38(m,1H),2.18(s,3H),2.15(s,3H),2.06(s,3H),2.04-1.98(m,2H),1.73-1.70(m,2H).

Example 5: preparation of Compound represented by the formula I-5

The synthetic route is as follows:

the first step is as follows: synthesis of Compound 5A

Crude compound 2B (1.00g,3mmol) was dissolved in DCM (10ml), cooled to 0-5 ℃ in an ice-water bath, triethylamine (910mg,9mmol) and MsCl (410mg,3.6mmol) were added, after completion of the addition, the temperature was raised to room temperature for reaction for 2 hours, T L C showed the reaction to be complete, 30ml of DCM was added, washed twice with water (15ml × 2), the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-dry concentration was chromatographed on silica gel column (eluent petroleum ether: ethyl acetate: 10: 1-1:1) to give compound 5A (1.07g, yellow solid) in 86.3% yield.

MS m/z(ESI):413[M+1].

The second step is that: synthesis of Compound 5B

Compound 5A (1.0g,11.5mmol) obtained in the previous step was dissolved in methanol (20ml), 10% palladium on carbon (200mg) was then added, hydrogen was replaced three times with a hydrogen balloon, then stirred overnight at room temperature maintaining a hydrogen atmosphere, T L C indicated the reaction was complete, filtered over silica, washed with methanol, and the filtrate was concentrated to give crude compound 5B (0.89g) in 95.5% yield, which was used directly in the next step without further purification.

MS m/z(ESI):383[M+1].

The third step: synthesis of the Compound represented by the formula I-5

Compound 1K (200mg,0.5mmol) obtained in the previous step was dissolved in n-butanol (5ml), compound 5B (287mg,0.75mmol) and concentrated hydrochloric acid (0.5ml) were added in this order, the reaction solution was reacted at 90 ℃ for four days, T L C showed the reaction to be complete, spin-dried, added to an aqueous sodium bicarbonate solution (30ml), extracted with dichloromethane (10m L× 2), the organic phases were combined, washed with saturated brine (10m L× 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was passed through a silica gel chromatography column (eluent dichloromethane: methanol: 50:1 to 10:1) to obtain a crude product of the compound represented by formula I-5, the crude product obtained was purified over 30 × 100mm of a preparative column through reverse phase C18 (mobile phase was 10 to 100% acetonitrile (0.05% TFA)/water), flow rate was 20m L/min, and 10 min, to obtain the target product of the compound represented by formula I-5 (4.3mg, white solid).

MS m/z(ESI):747[M+1].

¹H NMR(400MHz,CDCl₃):11.33(s,1H),8.87(dd,J＝3.2Hz,10Hz,1H),8.30(s,1H),8.23(s,1H),7.88(s,1H),7.79(d,J＝10Hz,1H),7.30(s,1H),6.61(s,1H),3.85(s,3H),3.28(brs,4H),3.17(d,J＝10.8Hz,2H),2.80(s,6H),2.79-2.76(m,2H),2.68-2.62(m,1H),2.19(s,3H),2.15(d,J＝6Hz,6H),1.93-1.91(m,2H),1.74-1.62(m,2H).

Example 6: preparation of Compound represented by formula I-6

The synthetic route is as follows:

the first step is as follows: synthesis of Compound 6A

The crude compound 2B (1g,3mmol) was dissolved in DMF (15ml), then potassium carbonate (1.24g,9mmol) and bromoethanol (562mg,4.5mmol) were added in sequence, after completion of the addition, the temperature was raised to 60 ℃ to react for 4 hours, T L C showed the reaction to be completed, the reaction solution was poured into water (100ml), extracted with ethyl acetate (50ml × 3), the organic phases were combined and washed twice with water (50ml × 2), the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-dry concentration was subjected to silica gel chromatography (eluent petroleum ether: ethyl acetate ═ 3:1-1:1) to obtain compound 6A (737mg, yellow solid) with a yield of 65%.

MS m/z(ESI):379[M+1].

The second step is that: synthesis of Compound 6B

The compound 6A (730mg,1.9mmol) obtained in the previous step was dissolved in methanol (10ml), 10% palladium on carbon (200mg) was added, hydrogen was replaced three times with a hydrogen balloon, and then the mixture was stirred overnight at room temperature under a hydrogen atmosphere, T L C indicated that the reaction was complete, filtered over silica, washed with methanol, and the filtrate was concentrated to give crude compound 6B (625mg) in 94.5% yield, which was used directly in the next step without further purification.

MS m/z(ESI):349[M+1].

The third step: synthesis of the Compound represented by the formula I-6

Compound 1K (200mg,0.5mmol) obtained in the previous step was dissolved in n-butanol (5ml), compound 6B (261mg,0.75mmol) and concentrated hydrochloric acid (0.5ml) were added in this order, the reaction solution was reacted at 90 ℃ for four days, T L C showed the reaction to be complete, spin-dried, added to an aqueous sodium bicarbonate solution (30ml), extracted with dichloromethane (10M L× 2), the organic phases were combined, washed with saturated brine (10M L× 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was passed through a silica gel chromatography column (eluent dichloromethane: methanol: 50:1-10:1) to obtain a crude product of the compound represented by formula I-6, the crude product obtained was purified over a reverse phase C18 preparation column YMC sa 30 × 100mm (mobile phase was 10-100% acetonitrile (0.05% TFA)/water), flow rate was 20M L/min, 713 min to obtain the target product of the compound represented by formula I-6 (MS 7.5mg, esim + 1M) ((M): 1M): 713M).

1H NMR(400MHz,MeOD):8.72(d,J＝9.6Hz,1H),8.44(s,1H),8.17(s,1H),7.73(d,J＝8.8Hz,1H),7.49(s,1H),6.74(s,1H),3.83(s,3H),3.71-3.67(m,2H),3.17-3.12(m,3H),2.72-2.52(m,12H),2.18(s,3H),,2.15(s,3H),2.06(s,3H),2.02-1.80(m,2H),1.73-1.64(m,2H).

Example 7: preparation of Compound represented by formula I-7

The synthetic route is as follows:

the first step is as follows: synthesis of Compound 7A

Compound 1C (10g,54mmol) was dissolved in DMF (100ml) at room temperature, cesium carbonate (17.6g,20mmol) and 4-piperidone ethylene glycol (11.6g,81mmol) were then added, the reaction temperature was heated to 80 ℃ and reacted for 4 hours, T L C showed the reaction to be complete, then the reaction solution was poured into water (500ml), extracted with ethyl acetate (150m L× 2), the organic phases were combined, washed with saturated brine (150m L× 2), dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-dry concentration was passed through a silica gel column (eluent petroleum ether: ethyl acetate ═ 3:1-1:1) to give compound 7A (13.9g, yellow liquid) in 83.5% yield.

MS m/z(ESI):309[M+1].

The second step is that: synthesis of Compound 7B

Compound 7A (13.5g,43.8mmol) obtained in the previous step was added to water (500ml), p-toluenesulfonic acid hydrate (8.33g,43.8mmol) was then added, after completion of the addition, the temperature was raised to reflux, the reaction was carried out for 16 hours, T L C showed the completion of the reaction, the reaction was cooled to room temperature, the pH was adjusted to neutral with potassium carbonate, the mixture was extracted with ethyl acetate (150ml × 3), the organic phases were combined, the organic phases were dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-dry concentration was subjected to silica gel chromatography (eluent petroleum ether: ethyl acetate ═ 3:1-1:1) to obtain compound 7B (8.9g, yellow solid) with a yield of 77.2%.

MS m/z(ESI):265[M+1].

The third step: synthesis of Compound 7C

Compound 7B (1g,3.77mol) was added to methanol (20ml) at room temperature, followed by addition of acetic acid (0.2ml) and N-methyl homopiperazine (644mg,5.65mmol), stirring at room temperature for 2 hours, followed by further addition of sodium cyanoborohydride (356mg,5.65mmol), stirring at room temperature overnight, T L C showed completion of the reaction, followed by rotary drying to remove methanol, addition of 100ml of water, extraction with dichloromethane (25ml × 3), combining the organic phases, washing the organic phases with saturated brine (20m L× 2), drying the organic phases with anhydrous sodium sulfate, filtration, and rotary drying and concentration of the resulting residue on a silica gel chromatography column (eluent dichloromethane: methanol: 50:1 to 10:1) to give compound 7C (914mg, yellow solid) in 67% yield.

MS m/z(ESI):363[M+1].

The fourth step: synthesis of Compound 7D

The compound 7C (900mg,2.5mmol) obtained in the previous step was dissolved in methanol (10ml), 10% palladium on carbon (200mg) was then added, hydrogen was replaced three times with hydrogen balloon, then stirred overnight at room temperature maintaining a hydrogen atmosphere, T L C indicated the end of the reaction, filtered over silica, washed with methanol, and the filtrate was concentrated to give crude compound 7D (768mg) in 92.5% yield, which was used directly in the next step without further purification.

MS m/z(ESI):333[M+1].

The fifth step: synthesis of the Compound represented by the formula I-7

Compound 1K (200mg,0.5mmol) obtained in the previous step was dissolved in n-butanol (5ml), compound 7D (250mg,0.75mmol) and concentrated hydrochloric acid (0.5ml) were added in this order, the reaction solution was reacted at 90 ℃ for four days, T L C showed the reaction to be complete, spin-dried, added to an aqueous sodium bicarbonate solution (30ml), extracted with dichloromethane (10m L× 2), the organic phases were combined, washed with saturated brine (10m L× 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was passed through a silica gel chromatography column (eluent dichloromethane: methanol: 50:1 to 10:1) to obtain a crude product of the compound represented by formula I-7, the crude product obtained was purified over 30 × 100mm of a preparative column through reverse phase C18 (mobile phase using 10 to 100% acetonitrile (0.05% TFA)/water), flow rate was 20m L/min, 10 min to obtain the target product of the compound represented by formula I-7 (7.2mg, white solid).

MS m/z(ESI):697[M+1].

¹H NMR(400MHz,MeOD):8.73(d,J＝10Hz,1H),8.44(s,1H),8.17(s,1H),7.72(d,J＝10Hz,1H),7.49(s,1H),6.73(s,1H),3.82(s,3H),3.16-2.13(m,2H),2.94-2.91(m,4H),2.74-2.59(m,7H),2.38(s,3H),2.18(s,3H),2.15(s,3H),2.05(s,3H),1.93-1.86(m,4H),1.77-1.71(m,2H).

Example 8: preparation of Compound represented by formula I-8

The synthetic route is as follows:

the first step is as follows: synthesis of Compound 8A

Compound 7B (1g,3.77mol) was added to methanol (20ml) at room temperature followed by acetic acid (0.2ml) and thiomorpholine 1, 1-dioxide (763mg,5.65mmol), after stirring for 2 hours at room temperature, sodium cyanoborohydride (356mg,5.65mmol) was added in portions, the reaction was continued overnight with stirring at room temperature, T L C showed the reaction to be complete, then methanol was removed by spinning off, 100ml water was added, extraction was performed with dichloromethane (25ml × 3), the organic phases were combined, washed with saturated brine (20M L× 2), the organic phases were dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was passed through a silica gel column chromatography (eluent dichloromethane: methanol: 50:1-10:1) to give compound 8A (881mg, yellow solid) with a yield of 61%, MS M/z (384 [ M +1].

The second step is that: synthesis of Compound 8B

The compound 8A (800mg,2.1mmol) obtained in the previous step was dissolved in methanol (10ml), 10% palladium on carbon (500mg) was added, hydrogen was replaced three times with a hydrogen balloon, then the mixture was stirred overnight at room temperature under hydrogen atmosphere, T L C indicated completion of the reaction, filtered over silica, washed with methanol, and the filtrate was concentrated to give crude compound 8B (696mg) in 94.5% yield, which was used directly in the next step without further purification.

MS m/z(ESI):354[M+1].

The third step: synthesis of the Compound represented by the formula I-8

Compound 1K (200mg,0.5mmol) obtained in the previous step was dissolved in n-butanol (5ml), compound 8B (266mg,0.75mmol) and concentrated hydrochloric acid (0.5ml) were added in this order, the reaction solution was reacted at 90 ℃ for four days, T L C showed the reaction to be complete, spin-dried, added to an aqueous sodium bicarbonate solution (30ml), extracted with dichloromethane (10m L× 2), the organic phases were combined, washed with saturated brine (10m L× 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was passed through a silica gel chromatography column (eluent dichloromethane: methanol: 50:1-10:1) to obtain a crude product of the compound represented by formula I-8, the crude product obtained was purified over 30 × 100mm of a preparative column through reverse phase C18 (mobile phase using 10-100% acetonitrile (0.05% TFA)/water), flow rate was 20m L/min, 10 min to obtain the target product of the compound represented by formula I-8 (7.6mg, white solid).

MS m/z(ESI):718[M+1].

¹H NMR(400MHz,DMSO-d6):11.37(s,1H),8.67(d,J＝10Hz,1H),8.53(s,1H),8.23(d,J＝4Hz,1H),7.32(s,1H),6.74(s,1H),3.77(s,3H),3.08(brs,7H),3.03(brs,4H),2.69-2.64(m,3H),2.08-2.05(m,9H),1.92-1.79(m,2H),1.68-1.63(m,2H).

Example 9: preparation of Compound represented by formula I-9

The synthetic route is as follows:

the first step is as follows: synthesis of Compound 9A

Compound 7B (1g,3.77mol) was added to methanol (20ml) at room temperature followed by acetic acid (0.2ml) and morpholine (763mg,5.65mmol), stirred at room temperature for 2 hours then sodium cyanoborohydride (356mg,5.65mmol) was added in portions and the reaction continued overnight at room temperature with T L C showing the end of the reaction, then methanol was removed by spinning off, 100ml water was added, extracted with dichloromethane (25ml × 3), the organic phases were combined, washed with saturated brine (20m L× 2), dried over anhydrous sodium sulfate, filtered and the residue obtained by spin-dry concentration was chromatographed on silica gel (eluent dichloromethane: methanol: 50:1-10:1) to give compound 9A (881mg, yellow solid) in 61% yield.

MS m/z(ESI):336[M+1].

The second step is that: synthesis of Compound 9B

Compound 9A (850mg,2.5mmol) obtained in the previous step was dissolved in methanol (10ml), 10% palladium on carbon (200mg) was then added, hydrogen was replaced three times with a hydrogen balloon, then stirred overnight at room temperature maintaining a hydrogen atmosphere, T L C indicated the end of the reaction, filtered over silica, washed with methanol, and the filtrate was concentrated to give crude compound 9B (707mg) in 92.5% yield, which was used directly in the next step without further purification.

MS m/z(ESI):306[M+1].

The third step: synthesis of a Compound represented by the formula I-9

Compound 1K (200mg,0.5mmol) obtained in the previous step was dissolved in n-butanol (5ml), compound 9B (230mg,0.75mmol) and concentrated hydrochloric acid (0.5ml) were added in this order, the reaction solution was reacted at 90 ℃ for four days, T L C showed the reaction to be complete, spin-dried, added to an aqueous sodium bicarbonate solution (30ml), extracted with dichloromethane (10m L× 2), the organic phases were combined, washed with saturated brine (10m L× 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was passed through a silica gel chromatography column (eluent dichloromethane: methanol: 50:1 to 10:1) to obtain a crude product of the compound represented by formula I-9, the crude product obtained was purified over 30 × 100mm of a preparative column through reverse phase C18 (mobile phase using 10 to 100% acetonitrile (0.05% TFA)/water), flow rate was 20m L/min, and the target product represented by formula I-9 was obtained over 10 minutes (5.5mg, white solid).

MS m/z(ESI):670[M+1].

¹H NMR(400MHz,MeOD):8.73(d,J＝7.2Hz,1H),8.44(s,1H),8.17(s,1H),7.73(d,J＝10Hz,1H),7.50(s,1H),6.73(s,1H),3.82(s,3H),3.75-3.72(m,4H),3.15(d,J＝12Hz,2H),2.74-2.65(m,6H),2.35-2.30(m,1H),2.18(s,3H),2.15(s,3H),2.05(s,3H),2.01-1.92(m,2H),1.69-1.66(m,2H).

Example 10: preparation of Compound represented by formula I-10

The synthetic route is as follows:

the first step is as follows: synthesis of Compound 10B

Compound 10A (2.3g,13.5mmol) was dissolved in DMF (25ml) at room temperature, cesium carbonate (6.6g,20mmol)) and 1-methyl-4- (4-piperidinyl) piperazine (2.47g,13.5mmol) were added, the reaction temperature was heated to 80 ℃ and reacted for 4 hours, T L C showed the reaction to be completed, then the reaction solution was poured into water, extracted with ethyl acetate (50m L× 2), the organic phases were combined, washed with saturated brine (50m L× 2), dried over anhydrous sodium sulfate, filtered, and the resulting residue was concentrated by spin drying to give compound 10B (3.76g, yellow solid) in 83.5% yield.

MS m/z(ESI):335[M+1].

The second step is that: synthesis of Compound 10C

Compound 10B (2.0g,6mmol) obtained in the previous step was dissolved in methanol (20ml) and 10% palladium on carbon (400mg) was added, hydrogen replaced three times with hydrogen balloon, then kept under hydrogen atmosphere at room temperature and stirred overnight, T L C indicated the end of the reaction, filtered over silica, washed with methanol, and the filtrate was concentrated to give crude compound 10C (1.7g) in 95.6% yield, which was used directly in the next step without further purification.

MS m/z(ESI):305[M+1].

The third step: synthesis of Compound 10E

After compound 1J (2.1g,10mmol) obtained in the previous step was added to DMF (30ml) and stirred to dissolve, 2,4, 5-trichloropyrimidine (2.18g,12mmol) and potassium carbonate (2.76g,20mmol) were sequentially added, and then heated to 60 ℃ for reaction for 4 hours, after T L C showed the reaction was completed, the reaction solution was cooled to room temperature, and then poured into water (150ml), extracted with ethyl acetate (50m L× 2), the organic phases were combined, and washed with saturated brine (50m L× 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was subjected to silica gel chromatography (eluent dichloromethane: methanol ═ 50:1 to 10:1) to obtain compound 10E (2.09g, yellow solid) with a yield of 58.7%.

MS m/z(ESI):357[M+1].

The fourth step: synthesis of Compound represented by the formula I-10

Compound 10E (180mg,0.5mmol) obtained in the previous step was dissolved in n-butanol (5ml), compound 10C (228mg,0.75mmol) and concentrated hydrochloric acid (0.5ml) were added in this order, the reaction solution was reacted at 90 ℃ for four days, T L C showed the reaction to be completed, spin-dried, added to an aqueous sodium bicarbonate solution (30ml), extracted with dichloromethane (10m L× 2), the organic phases were combined, washed with saturated brine (10m L× 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was passed through a silica gel column chromatography (eluent dichloromethane: methanol: 50:1 to 10:1) to obtain a crude product of the compound represented by formula I-10, the crude product obtained was purified over 30 × 100mm of a preparative column through reverse phase C18 (mobile phase using 10 to 100% acetonitrile (0.05% TFA)/water), flow rate was 20m L/min, and 10 min, to obtain the target product of the compound represented by formula I-10 (7.2mg, white solid).

MS m/z(ESI):625[M+1].

¹H NMR(400MHz,MeOD):8.93(dd,J＝2Hz,5.6Hz,1H),8.42(s,1H),8.06(s,1H),7.75(d,J＝10Hz,1H),7.52(d,J＝8.8Hz,1H),6.69(d,J＝2.4Hz,1H),6.52(dd,J＝2.4Hz,8.8Hz,1H),3.84(s,3H),3.75(d,J＝12.4Hz,2H),2.76-2.37(m,11H),2.29(s,3H),2.18(s,3H),2.14(s,3H),2.04(d,J＝12Hz,2H),1.71-1.65(m,2H).

Example 11: preparation of Compound represented by formula I-11

The synthetic route is as follows:

the first step is as follows: synthesis of Compound 11A

Compound 1C (2.5g,13.5mmol) was dissolved in DMF (25ml) at room temperature, cesium carbonate (6.6g,20mmol) and NBoc piperazine (2.51g,13.5mmol) were then added, the reaction temperature was heated to 80 ℃ and reacted for 4 hours, T L C showed the reaction to be complete, then the reaction solution was poured into water (100ml), extracted with ethyl acetate (50m L× 2), the organic phases were combined, washed with saturated brine (50m L× 2), dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was chromatographed on silica gel column (eluent petroleum ether: ethyl acetate: 3:1-1:1) to give compound 11A (4.06g, yellow solid) in 85.6% yield.

MS m/z(ESI):352[M+1].

The second step is that: synthesis of Compound 11B

Compound 11A (4.0g,11.4mmol) from the previous step was dissolved in dichloromethane (50ml) and TFA (10ml) was added at room temperature, after which time stirring at room temperature was continued for 2h, T L C indicated the reaction was complete and was concentrated by spin-drying to give crude compound 11B (3.8g, brown oil) which was used in the next step without purification.

The third step: synthesis of Compound 11C

Crude compound 11B (1.9g) obtained in the previous step was added to methanol (20ml) at room temperature, followed by the addition of 3-oxetanone (792mg,11mmol), followed by stirring at room temperature for 2 hours, followed by the addition of sodium cyanoborohydride (704mg,11mmol) in portions, continued stirring at room temperature overnight, T L C indicating the end of the reaction, followed by spin-drying to remove methanol, addition of 100ml of water, extraction with dichloromethane (25ml × 3), combination of the organic phases, washing of the organic phases with saturated brine (20m L× 2), drying of the organic phases with anhydrous sodium sulfate, filtration, and spin-drying concentration of the resulting residue on a silica gel column (eluent dichloromethane: methanol ═ 50:1-10:1) to give compound 11C (717mg, yellow solid) in 41% yield over two steps.

MS m/z(ESI):308[M+1].

The fourth step: synthesis of Compound 11D

Compound 11C (700mg,2.28mmol) obtained in the previous step was dissolved in methanol (10ml), 10% palladium on carbon (150mg) was added, hydrogen was replaced three times with a hydrogen balloon, then stirred overnight at room temperature under hydrogen atmosphere, T L C indicated completion of the reaction, filtered over silica, washed with methanol, and the filtrate was concentrated to give crude compound 11D (597mg) in 94.2% yield, which was used directly in the next step without further purification.

MS m/z(ESI):278[M+1].

The fifth step: synthesis of a Compound represented by the formula I-11

Compound 1K (200mg,0.5mmol) obtained in the previous step was dissolved in n-butanol (5ml), compound 11D (208mg,0.75mmol) and concentrated hydrochloric acid (0.5ml) were added in this order, the reaction solution was reacted at 90 ℃ for four days, T L C showed the reaction to be complete, spin-dried, added to an aqueous sodium bicarbonate solution (30ml), extracted with dichloromethane (10m L× 2), the organic phases were combined, washed with saturated brine (10m L× 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was passed through a silica gel chromatography column (eluent dichloromethane: methanol: 50:1-10:1) to obtain a crude product of the compound represented by formula I-11, the crude product obtained was purified over 30 × 100mm of a preparative column through reverse phase C18 (mobile phase using 10-100% acetonitrile (0.05% TFA)/water), flow rate was 20m L/min, and the target product represented by formula I-11 was obtained over 10 minutes (6.1mg, white solid).

MS m/z(ESI):642[M+1].

¹H NMR(400MHz,MeOD):8.72(dd,J＝2.8Hz,10Hz,1H),8.44(s,1H),8.18(s,1H),7.73(d,J＝5.2Hz,1H),7.54(s,1H),6.77(s,1H),4.87-4.74(m,2H),4.67-4.64(m,2H),3.85(s,3H),3.62-3.58(m,1H),2.97-.295(m,4H),2.55(brs,4H),2.18(s,3H),2.14(s,3H),2.04(s,3H).

Example 12: preparation of Compound represented by formula I-12

The synthetic route is as follows:

the first step is as follows: synthesis of Compound 12B

Compound 12A (11.5g,53mmol) was dissolved in a mixed solvent of acetonitrile (150ml) and water (30ml), then aqueous formaldehyde (11m L, 160mmol) was added, after stirring at room temperature for 2 hours, sodium borohydride acetate (22.5g,110mmol) was added in portions, after completion of addition, reaction was carried out at room temperature overnight, T L C showed completion of reaction, then saturated sodium bicarbonate solution was added to the reaction solution to adjust pH to 8, dichloromethane (500ml) and methanol (50ml) were added to dilute the reaction solution, washing with saturated aqueous sodium carbonate solution (100m L× 2) was carried out, the organic phase was separated, dried over anhydrous sodium sulfate, and spin-dried to give product 12B (11.9g, white solid) which was used directly in the next step with no purification in 98% yield.

MS m/z(ESI):231[M+1].

The second step is that: synthesis of Compound 12C

Compound 2B (11g,47.8mmol) from the previous step was dissolved in dichloromethane (150ml) and TFA (30ml) was added at room temperature, after which stirring at room temperature was continued for 2h, T L C indicated the reaction was complete and was concentrated by spin-drying to give crude compound 12C (9.2g, brown oil) which was used in the next step without purification.

MS m/z(ESI):131[M+1].

The third step: synthesis of Compound 12D

Compound 1C (2.5g,13.5mmol) was dissolved in DMF (25ml) at room temperature, cesium carbonate (6.6g,20mmol) and compound 12C (1.75g,13.5mmol) were then added, the reaction temperature was heated to 80 ℃ and reacted for 4 hours, T L C showed the reaction to be complete, then the reaction solution was poured into water, extracted with ethyl acetate (50m L× 2), the organic phases were combined, washed with saturated brine (50m L× 2), dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-dry concentration was passed through a silica gel chromatography column (eluent petroleum ether: ethyl acetate ═ 3:1-1:1) to give compound 12D (3.4g, yellow solid) in 85.7% yield.

MS m/z(ESI):296[M+1].

The fourth step: synthesis of Compound 12E

Compound 12D (1.0g,3.39mmol) obtained in the previous step was dissolved in methanol (20ml), 10% palladium on carbon (200mg) was added, hydrogen was replaced three times with a hydrogen balloon, and then stirred overnight at room temperature while maintaining a hydrogen atmosphere, T L C indicated the reaction was complete, filtered over silica, washed with methanol, and the filtrate was concentrated to give crude compound 12E (858mg) in 95.2% yield, which was used directly in the next step without further purification.

MS m/z(ESI):266[M+1].

The fifth step: synthesis of a Compound represented by the formula I-12

Compound 1K (200mg,0.5mmol) was dissolved in n-butanol (5ml), compound 12E (200mg,0.75mmol) and concentrated hydrochloric acid (0.5ml) were added in this order, the reaction solution was reacted at 90 ℃ for four days, T L C showed the reaction to be complete, spin-dried, added to an aqueous sodium bicarbonate solution (30ml), extracted with dichloromethane (10m L× 2), the organic phases were combined, washed with saturated brine (10m L× 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was passed through a silica gel chromatography column (eluent dichloromethane: methanol ═ 50:1 to 10:1) to give compound crude compound I-12. the crude obtained was purified by reverse phase C18 preparative column YMC ODSA over 30 × 100mm (mobile phase was 10 to 100% acetonitrile (0.05% TFA)/water), flow rate 20m L/min, 10 min to give the target compound of formula I-12 (5.5mg, white solid).

MS m/z(ESI):630[M+1].

¹H NMR(400MHz,MeOD):8.71(dd,J＝2.8Hz,9.6Hz,1H),8.44(s,1H),8.18(s,1H),7.76(d,J＝9.6Hz,1H),7.54(s,1H),6.75(s,1H),3.84(s,3H),3.83-3.80(m,1H),3.63-3.58(m,1H),3.16(d,J＝11.6Hz,1H),2.99-2.85(m,3H),2.69-2.64(m,1H),2.58-2.56(m,1H),2.43(brs,4H),2.18(s,3H),2.15(s,3H),2.03(s,3H).

Example 13: preparation of Compound represented by formula I-13

The synthetic route is as follows:

the first step is as follows: synthesis of Compound 13A

Compound 7B (1g,3.77mol) was added to methanol (20ml) at room temperature followed by acetic acid (0.2ml) and compound 12C (734mg,5.65mmol), stirred at room temperature for 2 hours, then sodium cyanoborohydride (356mg,5.65mmol) was added in portions, the reaction was continued overnight at room temperature with continued stirring, T L C showed the reaction to be complete, then methanol was removed by spinning off, 100ml water was added, extraction was performed with dichloromethane (25ml × 3), the organic phases were combined, washed with saturated brine (20m L× 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-dry concentration was chromatographed over silica gel (eluent dichloromethane: methanol 50:1-10:1) to give compound 13A (892mg, yellow solid) in 62.6% yield.

MS m/z(ESI):379[M+1].

The second step is that: synthesis of Compound 13B

Compound 13A (800mg,2.12mmol) obtained in the previous step was dissolved in methanol (10ml) and 10% palladium on carbon (200mg) was added, hydrogen replaced three times with hydrogen balloon, then kept under hydrogen atmosphere at room temperature and stirred overnight, T L C showed the reaction was complete, filtered over silica, washed with methanol and the filtrate was concentrated to give crude compound 13B (683mg) in 92.7% yield, which was used directly in the next step without further purification.

MS m/z(ESI):349[M+1].

The third step: synthesis of the Compound represented by the formula I-13

Compound 1K (200mg,0.5mmol) obtained in the previous step was dissolved in n-butanol (5ml), compound 13B (261mg,0.75mmol) and concentrated hydrochloric acid (0.5ml) were added in this order, the reaction solution was reacted at 90 ℃ for four days, T L C showed the reaction to be complete, spin-dried, added to an aqueous sodium bicarbonate solution (30ml), extracted with dichloromethane (10m L× 2), the organic phases were combined, washed with saturated brine (10m L× 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was passed through a silica gel chromatography column (eluent dichloromethane: methanol: 50:1 to 10:1) to obtain a crude product of the compound represented by formula I-13, the crude product obtained was purified over 30 × 100mm of a preparative column through reverse phase C18 (mobile phase was 10 to 100% acetonitrile (0.05% TFA)/water), flow rate was 20m L/min, and 10 min, to obtain the target product of the compound represented by formula I-13 (5.4mg, white solid).

MS m/z(ESI):713[M+1].

¹H NMR(400MHz,MeOD):8.73(dd,J＝2.0Hz,8.8Hz,1H),8.44(s,1H),8.16(s,1H),7.73(d,J＝10.4Hz,1H),7.50(s,1H),6.74(s,1H),3.85(s,3H),3.76-3.72(m,1H),3.57-3.53(m,1H),3.17-3.14(m,3H),2.99-2.97(m,1H),2.88-2.86(m,1H),2.72-2.66(m,2H),2.42-2.37(m,2H),2.36(s,3H),2.23-2.20(m,3H),2.18(s,3H),2.14(s,3H),2.05(s,3H),2.04-2.01(m,2H),1.72-1.69(m,2H).

Example 14: preparation of the Compound represented by formula I-14

The synthetic route is as follows:

the first step is as follows: synthesis of Compound 14A

Compound 7B (1g,3.77mol) was added to methanol (20ml) at room temperature followed by acetic acid (0.2ml) and N-methylpiperazine (565mg,5.65mmol), stirred at room temperature for 2 hours, then sodium cyanoborohydride (356mg,5.65mmol) was added in portions, the reaction was continued overnight with stirring at room temperature, T L C showed completion of the reaction, then methanol was removed by spinning off, 100ml water was added, extraction was performed with dichloromethane (25ml × 3), the organic phases were combined, washed with saturated brine (20m L× 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-dry concentration was subjected to silica gel chromatography (eluent dichloromethane: methanol ═ 50:1-10:1) to give compound 14A (827mg, yellow solid) in 63% yield.

MS m/z(ESI):349[M+1].

The second step is that: synthesis of Compound 14B

Compound 14A (800mg,2.3mmol) obtained in the previous step was dissolved in methanol (10ml) and 10% palladium on carbon (200mg) was added, hydrogen replaced three times with hydrogen balloon, then kept under hydrogen atmosphere at room temperature and stirred overnight, T L C indicated the reaction was complete, filtered over silica, washed with methanol and the filtrate was concentrated to give crude compound 14B (669mg) in 91.5% yield, which was used directly in the next step without further purification.

MS m/z(ESI):319[M+1].

The third step: synthesis of a Compound represented by the formula I-14

Compound 1K (200mg,0.5mmol) obtained in the previous step was dissolved in n-butanol (5ml), compound 14B (238mg,0.75mmol) and concentrated hydrochloric acid (0.5ml) were added in this order, the reaction solution was reacted at 90 ℃ for four days, T L C showed the reaction to be complete, spin-dried, added to an aqueous sodium bicarbonate solution (30ml), extracted with dichloromethane (10m L× 2), the organic phases were combined, washed with saturated brine (10m L× 2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was passed through a silica gel chromatography column (eluent dichloromethane: methanol: 50:1 to 10:1) to obtain a crude product of the compound represented by formula I-14, the crude product obtained was purified over 30 × 100mm of a preparative column through reverse phase C18 (mobile phase using 10 to 100% acetonitrile (0.05% TFA)/water), flow rate was 20m L/min, 10 min to obtain the target product of the compound represented by formula I-14 (7.7mg, white solid).

MS m/z(ESI):683[M+1].

¹H NMR(400MHz,MeOD):8.72(d,J＝9.6Hz,1H),8.44(s,1H),8.16(s,1H),7.73(d,J＝10.4Hz,1H),7.50(s,1H),6.73(s,1H),3.83(s,3H),3.15(d.J＝12Hz,2H),2.75-2.66(m,10H),2.41-2.35(m,1H),2.32(s,3H),2.18(s,3H),2.14(s,3H),2.05(s,3H),2.04-2.01(m,2H),1.71-1.67(m,2H).

Effect example 1: cell antiproliferation assay

(1) Experimental materials:

RPMI1640, Glutamine, Interleukin 3, pancreatin were all purchased from L ife Technology.

Fetal bovine serum, dual antibody was purchased from Hyclone.

Blasticidin was purchased from Merck.

Phosphate buffer was purchased from Corning.

CellTiter-Glo kit was purchased from Promega.

H1975 cells were purchased from ATCC.

Ba/F3 cells were purchased from Riken.

Ba/F3 (EGFR. DELTA.19 del/T790M) cells and Ba/F3 (EGFR. DELTA.19 del/T790M/C797S) cells were constructed from Wuxi.

H1975 cell culture medium: 88% RPMI1640, 10% fetal bovine serum, 1% glutamine, 1% double antibody.

Ba/F3 cell culture medium including 88% RPMI1640, 10% fetal calf serum, 10ng/m L interleukin 3, 1% glutamine, and 1% double antibody.

Ba/F3 (EGFR. DELTA.19 del/T790M) and Ba/F3 (EGFR. DELTA.19 del/T790M/C797S) cell culture media: 87.9% RPMI1640, 10% fetal bovine serum, 0.1% blasticidin, 1% glutamine, 1% double antibody.

Experiment board: 781091(Greiner)

Plate reading instrument: envision (PerkinElmer)

(2) The experimental method comprises the following steps:

experimental method (H1975 cells) cells from T75 flask were digested, resuspended in medium, counted, and the cell concentration adjusted to 2.0 × 10 with medium⁴Cell/m L, adding 100 mu L phosphate buffer solution to the peripheral wells of 384-well plate, adding 50 mu L cell suspension to other wells, standing at room temperature for 15min, and standing at 37 ℃ with 5% CO₂Incubating for 24h in incubator, diluting test compound with DMSO on ECHO at 10 points and 3 times gradient, transferring 250n L compound to 384 well plate with final concentration of 5-0.00025 μ M, transferring 250n L DMSO as positive control and negative control, centrifuging at 1000rpm for 15s, and placing at 37 deg.C with 5% CO₂Incubate for 72h in incubator, add 25u L CellTiter-Glo to each well of 384-well plate, centrifuge for 15s at 1000rpm, shake for 15min at room temperature, and read with Envision.

Experimental methods (Ba/F3 cells, Ba/F3 (EGFR. DELTA.19 del/T790M) cells and Ba/F3 (EGFR. DELTA.19 del/T790M/C797S) cells) test compounds were diluted with DMSO in 10-point 3-fold gradient on ECHO, 250n L compounds were transferred to 384-well plates to a final concentration of 5-0.00025. mu.M, 250n L DMSO was transferred as positive and negative controls, cells in uniform T75 flasks were blown, counted, and the cell concentration was adjusted to 4 × 10 with medium⁴Cell/m L, adding 100 mu L phosphate buffer solution to the peripheral wells of 384-well plate, adding 50 mu L cell suspension to other wells, standing at room temperature for 15min, and standing at 37 ℃ with 5% CO₂Incubate in incubator for 72h, add 25. mu. L CellTiter-Glo to each well of 384-well plate, centrifuge at 1000rpm for 15s, chamberAfter 15min of warm shaking, the samples were read using Envision.

(3) And (3) data analysis:

the readings were converted to percent inhibition (%) (Sample value-HC)/(L C-HC) × 100 by the following equation parametric curve fitting (X L-fit software) to determine IC₅₀And (4) data.

(4) And (3) test results:

cell activity inhibition IC of EGFRba/F3 (delta 19del/T790M) of the compounds of the invention₅₀H1975 inhibition of cellular Activity IC₅₀EGFR Ba/F3(Δ 19del/T790M/C797S) cell activity inhibition IC₅₀Ba/F3 parent cell activity inhibition IC₅₀. The data are presented in table 1 below.

Wherein, IC₅₀Compounds between 1-100nm are identified by +++ IC₅₀Compounds between 100-1000nm are denoted by ++, IC₅₀Compounds greater than 1000nm are identified as + s.

Table 1: cell antiproliferative activity data (IC) of compounds of the examples of the invention₅₀)

From the experimental results in table 1 we can see that:

(1) most of the compounds have good inhibition effect on EGFR Ba/F3 (delta 19del/T790M/C797S) triple-mutation cells, and the cell antiproliferative activity of part of compounds is below 100 nm.

(2) EGFR Ba/F3 (delta 19del/T790M) is a cell model of EGFR double mutation, and is mainly used for the third generation of EGFR TKIs to generate resistance effect on the secondary mutation of T790M which is generated in an ATP receptor part, and tests show that most compounds have good cell anti-proliferation effect on the double mutation of EGFRBA/F3(A19del/T790M), which shows that the compounds have double inhibition effect on the triple mutation and the double mutation of EGFRBA/F3.

(3) The cell antiproliferative activity test of Ba/F3 parent and H1975(EGFRWT) is a test aiming at normal cells, mainly the selective effect of the compound on the normal cells is determined, and as can be seen from the table 1, the compound has weak inhibition effect on the normal cells when having very good inhibition effect in three mutations and two mutations, which indicates that the compound has very good cell selectivity, has weak toxicity effect and has very good antitumor application prospect.

Effect example 2: kinase inhibitory Activity assay

(1) Experimental materials:

EGFR T790M/C797S/L858R enzyme was purchased from Signalchem.

Both DTT and double distilled water were purchased from L ife Technology.

Magnesium chloride was purchased from Fluka.

ATP was purchased from Sigma.

HTRF KinEASE-TK Kit was purchased from Cisbio.

Reaction buffer: 1 Xenzymetic buffer, 5mM magnesium chloride, 1mM DTT, double distilled water.

Experiment board: 6007299(PerkinElmer)

Plate reading instrument: envision (PerkinElmer)

(2) The experimental method comprises the following steps:

the test compound was diluted with DMSO at 11 points in a 3-fold gradient, 100n L compound was transferred to 384 well plates to a final concentration of 0.5-0.0000085. mu.M, 100n L MSK-1403 was transferred as a positive control, 1. mu.M was transferred as a negative control, 100n L DMSO was transferred as a negative control, 1nM EGFR T790M/C797S/L858R enzyme and 2. mu.M TKSubstrate-biotin was prepared in reaction buffer, 5. mu. L was added to each well, 15s was centrifuged at 1000rpm and 15min was incubated at 23 ℃ for 15s, 20. mu.M ATP was prepared in reaction buffer, 5. mu. L was added to each well, 15s was centrifuged at 1000rpm and 60min was incubated at 23 ℃, 125nM Streptavidin-X L665 and 1/2X TKAntibot-Crytate mixture was added to each well, 10. mu.10 min was added to each well, L, 15min was incubated at 1000 ℃ for 15min, and the mixture was added to Detection buffer.

(3) And (3) data analysis:

the readings were converted to inhibition (%) (Sample value-HC)/(L C-HC) × 100% by the following equation IC was determined by parametric curve fitting (X L-fit software)₅₀And (4) data.

(4) And (3) test results:

IC (integrated circuit) for inhibiting activity of EGFR (T790M/C797S/L858R) and EGFR (d746-750/T790M/C797S) of the compound of the invention₅₀The data are presented in table 2 below.

Wherein, IC₅₀Compounds between 0.1 and 10nm are identified by +++ IC₅₀Compounds between 10 and 100nm are denoted by ++, IC₅₀Compounds greater than 100nm are identified as + s.

Table 2: kinase inhibitory data (IC) of Compounds of the examples of the invention₅₀)

As a result: as can be seen from Table 2, most of the compounds have good inhibitory effect on EGFR-C797S gene mutation kinase, the enzyme activity of most of the compounds is less than 10nM, and partial compounds are even less than 1nM, so that the compounds have good application prospect.

Effect example 3: drug metabolism test

1. Using the inventive Compound I-14 prepared in the above example, the oral drug was formulated as a 0.3mg/m L clear solution (2% DMSO + 30% PEG 300+ 2% Tween 80+ 66% H2O) and the intravenous drug was formulated as a 0.2mg/m L clear solution (2% DMSO + 30% PEG 300+ 2% Tween 80+ 66% H2)₂O)

2. Male CD-1 mice, 3 each per group, weighing 27-28g, were provided by Shanghai Si Laike laboratory animal responsibility Co., Ltd. The test mice are given an environmental adaptation period of 2-4 days before the experiment, are fasted for 8-12h before the administration, are fed with water after the administration for 2h, and are fed with food after 4 h.

3. After the mice are fasted but can drink water freely for 12 hours, blank plasma at 0 moment is adopted;

4. taking the mice in the step 1), and orally taking (PO) the compound to be detected for 3 mg/kg; intravenous (IV) administration of 1mg/kg of test compound;

5. continuously taking blood from fundus venous plexus 5min, 15min, 30min, 1h, 2h, 4h, 8h, 10h and 24h after oral administration, placing in an EP tube distributed with heparin, centrifuging at 8000rpm/min for 5min, taking upper layer plasma, freezing at-20 deg.C, and analyzing for L C-MS/MS;

6. and (3) calculating pharmacokinetic parameters by adopting WinNonlin software according to the blood concentration-time data obtained in the step (3), wherein the specific data are shown in a table 3.

TABLE 3 pharmacokinetic data for the compounds of the examples of the invention

The pharmacokinetic experimental data are shown in table 3, and the results show that the compounds shown in formulas I-9 and I-14 of the invention have very high exposure, very good half-life, area under the curve and bioavailability in animal plasma after being orally or intravenously administered to mice, and have good pharmaceutical property and good clinical application prospect.

Effect example 4: drug metabolism test

The experimental method comprises the following steps:

1. in vivo drug efficacy experiments were performed on xenograft (CDX) BA L B/C nude mice of Ba/F3(Δ 19del/T790M/C797S) origin subcutaneously.BA L B/C nude mice, females, 6-8 weeks, weighing about 18-22 grams, were housed in an SPF grade environment, each cage was vented separately (5 mice per cage). all cages, bedding and water were disinfected prior to use.all animals were free to obtain a standard certified commercial laboratory diet.48 total mice purchased from Tokyo Wilkinsonia were used for studies.each mouse was implanted with cells subcutaneously under the right flank for tumor growth.experiments were initiated when the average tumor volume reached about 150-200 cubic millimeters.test compounds were administered orally, with compound Birgnib (15 mg/kg), compound I-9(150 mg/10 mg) and compound I-14 kg continuously for 14 days.

2. Tumor volume was measured twice a week with a two-dimensional caliper, and the volume was measured in cubic millimeters and calculated by the formula V0.5 a × b²Wherein a and b are the major and minor diameters of the tumor, respectively. The anti-tumor efficacy was determined by dividing the mean tumor gain volume of animals treated with the compound by the mean tumor gain volume of untreated animals. Wherein TGI (the tumor volume inhibition value) is used for evaluating the inhibition effect of the test drug on the tumor growth in vivo, and the calculation company is as follows: TGI (100%) [ (1- (mean tumor volume at the end of the administration group-mean tumor volume at the start of the administration group)/(mean tumor volume at the end of the solvent control group-mean tumor volume at the start of the treatment in the solvent control group) ] -mean tumor volume at the start of the treatment in the solvent control group ]]100%. Wherein the TGI of group Birgatinib (15 mg/kg) is 8.6%, the TGI of group I-9(150 mg/kg) is 108%, and the TGI of group I-14(100 mg/kg) is 108%.

The experimental results are as follows: see table 4.

TABLE 4

In a mouse Ba/F3 (delta 19del/T790M/C797S) Derived Xenograft (CDX) subcutaneous transplanted tumor model, the compound has obvious inhibition effect on tumor growth, has the effect of shrinking tumor and shows good anti-tumor effect.

In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. The particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Variations, modifications, substitutions and alterations of the above-described embodiments may be made by those skilled in the art without departing from the principles and spirit of the invention.

Claims (10)

1. A compound comprising a compound of formula I, or a pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer, tautomer, or prodrug thereof;

wherein the content of the first and second substances,

the A is selected from C, N, O or S;

the R1 is selected from H, C1-C8 alkyl, C1-C8 heteroalkyl, C3-C8 cycloalkyl, or C3-C8 heterocycloalkyl;

r2 is selected from H, C1-C8 alkyl, or C1-C8 heteroalkyl;

r3 is selected from H, halogen, cyano, C1-C8 alkyl, C1-C8 heteroalkyl, C3-C8 cycloalkyl, or C3-C8 heterocycloalkyl;

the R4 is selected from C1-C8 alkyl, C1-C8 heteroalkyl, C3-C8 cycloalkyl, or C3-C8 heterocycloalkyl;

r5 is selected from H, halogen, cyano, C1-C8 alkyl, C1-C8 heteroalkyl, C3-C8 cycloalkyl, or C3-C8 heterocycloalkyl.

2. The compound of claim 1, wherein the compound of formula I is any one of the following:

wherein, R is₁～R₅The definition of the A group is defined in claim 2.

3. A compound according to claim 1, wherein the heteroatoms or groups of heteroatoms in C1-C8 heteroalkyl, C3-C8 heterocycloalkyl, five-membered heteroaryl or six-membered heteroaryl, are each independently selected from-O-, -S-, -NH-, ═ O, -O-N ═ C (═ O) O-, -C (═ O) -, -S (═ O) -, -O (═ O)₂-、-C(＝O)NH-、-S(＝O)₂NH-, or-NHC (═ O) NH-.

4. The compound of claim 1, wherein the number of heteroatoms or groups of heteroatoms is each independently selected from 1,2, or 3.

5. The compound of claim 1, wherein the compound of formula I is any one of the following:

6. a pharmaceutical composition comprising an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or a solvate thereof, or a metabolite thereof, or a stereoisomer thereof, or a tautomer thereof, or a prodrug thereof, as claimed in any one of claims 1-5, and at least one pharmaceutical excipient.

7. Use of a compound of formula I, or a pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer, tautomer, prodrug thereof, or a pharmaceutical composition according to any one of claims 1-5 for the manufacture of a medicament for the treatment of an EGFR T790M/C797S inhibitor.

8. Use of a compound of formula I, or a pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer, tautomer, prodrug or combination thereof, as claimed in any one of claims 1 to 5, in the manufacture of a medicament for the treatment and/or prevention of diseases caused by abnormal mutations in EGFR T790M/C797S.

9. Use of a compound of formula I according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer, tautomer, prodrug or pharmaceutical composition according to claim 6 for the manufacture of a medicament for the treatment and/or prophylaxis of cancer.

10. The use of claim 9, wherein the cancer comprises lung cancer, glioma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer.