CN111410667B

CN111410667B - Novel (1,2,4)triazolo(1,5-a)pyridylphosphorus oxide and its use

Info

Publication number: CN111410667B
Application number: CN201911199979.9A
Authority: CN
Inventors: 方华祥; 李芳芳; 许勇; 黄璐
Original assignee: Wuhan Yukeyuan Medical Biotechnology Co ltd
Current assignee: Wuhan Yukeyuan Medical Biotechnology Co ltd
Priority date: 2019-01-08
Filing date: 2019-11-29
Publication date: 2024-11-22
Anticipated expiration: 2039-11-29
Also published as: CN111410667A

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 in a formula I, or pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer, tautomer or prodrug thereof, wherein R ¹～R⁵ is defined as in the specification. The compound provided by the invention can be used for preparing medicines for treating and/or preventing cancers.

Description

Novel (1, 2, 4) triazolo (1, 5-a) pyridylphosphine oxides and uses 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) pyridylphosphine oxide and application thereof.

Background

Non-small cell lung cancer (NSCLC) patients in China account for about 85% of the total lung cancer cases, with about 30% -40% of them suffering from Epidermal Growth Factor Receptor (EGFR) mutations. EGFR-TKI is a standard treatment for first line treatment of EGFR mutant NSCLC patients. Therefore, the development of EGFR inhibitors is a great research hotspot in the field of lung cancer treatment.

Common first and second generation EGFR-targeted drugs are gefitinib (iressa), erlotinib (tarceva), icotinib (sodium kemel), afatinib (Ji Tairui). However, most patients develop drug resistance after about one year of use of such targeted drugs. The cause of drug resistance may be T790M mutation (about 60%) in EGFR gene, or c-MET amplification, HER2 mutation, activation of downstream KRAS or BRAF, etc., and some patients are transformed into small cell lung cancer. How to solve the problem of EGFR resistance? The answer is that the patient must be placed in symptomatic medication. As for the T790M mutation, assirinotecan has evolved the third generation EGFR TKI targeting drug, tedaphne (austinib, or AZD 9291). With the approval of the Tadapsa on the market in China and the introduction of the third-generation EGFR-TKI targeting drugs into the conventional clinical treatment of lung cancer, more EGFR mutant patients have hope of getting treatment, but a batch of lung cancer patients generate the third-generation TKI resistance after the administration; genomic sequencing found that resistance occurred mainly at the C797S mutation of EGFR. Accordingly, there is a need for new and improved EGFR kinase inhibitors, and there is an urgent need to develop new and effective EGFR kinase inhibitors.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a novel (1, 2, 4) triazolo (1, 5-a) pyridylphosphine oxide and application thereof, and the novel (1, 2, 4) triazolo (1, 5-a) pyridylphosphine oxide provided by the invention can be used as 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 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,

Wherein,

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

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

The R2 is selected from H, C-C8 alkyl, or C1-C8 heteroalkyl;

The 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;

The 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" represents a heteroatom or a heteroatom group. According to an embodiment of the present invention, the heteroatoms or heteroatom groups are independently selected from-O-, -S-, -NH-, =o, -O-n=, -C (=o) O-, -C (=o) -, -S (=o) ₂-、-C(＝O)NH-、-S(＝O)₂ NH-, or-NHC (=o) NH-, respectively, in any of the above cases, the number of heteroatoms or heteroatom groups is independently selected from 1, 2 or 3.

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, according to the present invention is any one selected from the group consisting of:

wherein the R ₁～R₅ and A groups are defined as above.

Thus, throughout this specification, the R ₁～R₆, A groups, and substituents thereof, in the compounds of formula I may be selected by those skilled in the art to provide stable compounds of formula I, or pharmaceutically acceptable salts, or hydrates, or solvates, or metabolites, or stereoisomers, or tautomers, or prodrugs thereof, as described in the examples of the invention.

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:

In the present invention, the groups and substituents thereof in the compounds of formula I may be selected by those skilled in the art 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 as described in the examples of the present invention.

The reaction solvent used in each of the reaction steps described in the present invention is not particularly limited, and any solvent which dissolves the starting materials to some extent and does not inhibit the reaction is included in the present invention. In addition, many similar modifications, equivalent substitutions, or equivalent solvents, combinations of solvents, and different proportions of solvent combinations described herein are considered to be encompassed by the present invention.

According to a second aspect of the present invention, there is provided 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, and at least one pharmaceutically acceptable adjuvant.

The pharmaceutical excipients can be those which are widely used in the field of pharmaceutical production. Adjuvants are used primarily to provide a safe, stable and functional pharmaceutical composition, and may also provide means for allowing the subject to dissolve at a desired rate after administration, or for promoting effective absorption of the active ingredient after administration of the composition. 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 can comprise one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, sizing agents, disintegrants, lubricants, anti-adherents, glidants, wetting agents, gelling agents, absorption retarders, dissolution inhibitors, enhancing agents, adsorbents, buffering agents, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

The pharmaceutical compositions of the present invention may be prepared in accordance with the disclosure using any method known to those of skill 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, implantation, subcutaneous, intravenous, intra-arterial, intramuscular). The pharmaceutical compositions of the invention may also be in controlled or delayed release dosage forms (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 formulations which may 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; a liquid dosage form suitable for parenteral administration; suppositories and lozenges.

The compounds of the present invention are preferably administered orally. Intravenous administration of the compounds of the present invention is also preferred. Other routes of administration may be applied or even preferred, depending on the circumstances. For example, transdermal administration may be highly desirable for patients who are forgetful or are irritated with oral medications. In particular cases, the compounds of the invention may also be administered by the transdermal, intramuscular, intranasal or intrarectal route. The route of administration may vary in any manner, limited by the physical nature of the drug, the convenience of the patient and caretaker, and other relevant circumstances.

According to a third aspect of the present invention, there is provided the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a tautomer thereof, or a pharmaceutical composition thereof, in the manufacture of a medicament for the treatment and/or prophylaxis of a disease caused by an abnormal mutation in EGFR T790M/C797S. 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 an application of a compound shown in a 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 in preparing EGFR T790M/C797S inhibitor drugs. The compound provided by the invention has excellent EGFR T790M/C797S inhibition activity, can be effectively used as EGFR T790M/C797S inhibitor and as a therapeutic drug of EGFR T790M/C797S inhibitor.

According to a fifth aspect of the present invention, there is provided the use 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, or a pharmaceutical composition thereof, in the manufacture of a medicament for the treatment and/or prophylaxis of cancer. The compound provided by the invention can be used for preparing medicines for treating and/or preventing cancers, wherein the cancers comprise, but are not limited to, lung cancer, glioma, renal cancer, prostatic cancer, pancreatic cancer and breast cancer. Because of the variety of solid cancers, the compounds of this patent can be used as long as there is an EGFR T790M/C797S aberrant mutation.

Terminology and definitions

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

"Alkyl" refers to saturated aliphatic hydrocarbon groups, including straight and branched chain groups of 1 to 20 carbon atoms, which may be, for example, 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. "alkyl" herein 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 various branched isomers thereof, and the like. Non-limiting examples also include methylene, ethylene, propylene, butylene, and various branched isomers thereof. The alkyl group 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 a 3,4,5, 6 membered ring. Non-limiting examples of monocyclic cycloalkenyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like. The cyclic group may be optionally substituted or unsubstituted.

"Heterocyclyl" refers to 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 heteroatoms of S (O) m (where m is 0, 1, or 2), but excluding the ring portion of-O-, -O-S-, or-S-S-, and the remaining ring atoms are carbon. Preferably 3 to 12 ring atoms, of which 1-4 are heteroatoms, more preferably the heterocycloalkyl ring contains 3 to 10 ring atoms, most preferably 5-or 6-membered rings, 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 heterocyclyl 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: by "optionally alkyl-substituted heterocyclic group" is meant that an alkyl group may be, but is not required to be, present, and this description includes both cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

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

By "pharmaceutically acceptable salts" is meant salts of the compounds of the invention prepared from the compounds of the invention which have been found to have a particular substituent with a relatively non-toxic acid or base. When the compounds of the present invention contain relatively acidic functional groups, base addition salts may be obtained by contacting neutral forms of such compounds with a sufficient amount of a base in pure 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 the compounds of the present invention contain relatively basic functional groups, the acid addition salts may be obtained by contacting the neutral form of such compounds with a sufficient amount of an acid in pure 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 organic acid salts including acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; also included are salts of amino acids such as arginine and the like, 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 basic and acidic functionalities that can be converted to either base or acid addition salts. Preferably, the salt is contacted with a base or acid in a conventional manner to isolate the parent compound, thereby regenerating the neutral form of the compound. The parent form of a compound differs from its various salt forms in certain physical properties, such as solubility in polar solvents. According to an embodiment of the present invention, preferably the pharmaceutically acceptable salt of the compound of formula I of the present invention is a hydrochloride, hydrobromide, phosphate, or sulfate salt, most preferably a hydrochloride salt.

"Pharmaceutical composition" means a composition comprising one or more compounds of formula I as described herein, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a mixture of its tautomer and other chemical components, as well as other components, such as pharmaceutically acceptable excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.

A series of novel [1,2,4] triazolo [1,5-a ] pyridylphosphine oxides are synthesized, and the activity test of related enzymes and cells shows that the compounds have excellent cell activity, the IC50 value of the compounds for cell proliferation in vitro reaches nM level, and the compounds can be well applied to various tumors. According to the embodiment of the invention, the compound has very good inhibition effect on the EGFR T790M/C797S abnormally expressed human non-small cell lung cancer cell BaF 3. And the in vivo efficacy experiment of the animal further proves the excellent tumor inhibition effect of the compound. The compound provided by the invention has excellent EGFR T790M/C797S inhibition activity, can be used as a therapeutic drug for preparing EGFR T790M/C797S inhibitors, 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 cancers.

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 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, according to the present invention, may be accomplished by the exemplary methods described in the following examples and related published literature procedures used by those skilled in the art, but these examples are not limiting the scope of the invention. The present invention is described in detail below by way of examples, but is not meant to be limiting in any way.

The structure of the compounds of the present invention is determined by Nuclear Magnetic Resonance (NMR) or Mass Spectrometry (MS). NMR measurements were performed using Bruker AVANCE-400 or Varian Oxford-300 nuclear magnetic instruments with deuterated dimethyl sulfoxide (DMSO-d ₆) as the measurement solvent and deuterated chloroform (CDC 1 ₃) deuterated methanol (CD ₃ OD) as the internal standard Tetramethylsilane (TMS) chemical shifts given in units of 10 ^-6 (ppm).

MS was measured using AGILENT SQD (ESI) mass spectrometer (manufacturer: agilent, model: 6110) or Shimadzu SQD (ESI) mass spectrometer (manufacturer: shimadzu, model: 2020).

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

The thin layer chromatography silica gel plate uses Qingdao ocean GF254 silica gel plate, the specification of the silica gel plate used by Thin Layer Chromatography (TLC) is 0.15mm-0.2mm, and the specification of the thin layer chromatography separation and purification product is 0.4mm-0.5mm silica gel plate.

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

The known starting materials of the present invention can be synthesized using or according to methods known in the art, from Shaoguan chemical technology (Accela ChemBio Inc), beijing coupling chemicals, and the like.

In the examples, unless otherwise specified, the reaction was carried out under an argon atmosphere or a nitrogen atmosphere. The hydrogenation reaction is usually vacuumized, filled with hydrogen and repeatedly operated for 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 (TLC) using a system of developing agents of a: methylene chloride and methanol systems; b: petroleum ether and ethyl acetate systems, and the volume ratio of the solvent is adjusted according to the polarity of the compounds.

The system of eluent for column chromatography and the system of developing agent for thin layer chromatography used for purifying the compound include a: methylene chloride and methanol systems; b: petroleum ether and ethyl acetate system, the volume ratio of the solvent is regulated according to the polarity of the compound, and small amount of triethylamine, acidic or alkaline reagent and the like can be added for regulation.

Example 1: preparation of Compounds of formula I-1

The synthetic route is as follows:

the first step: synthesis of Compound 1B

Compound 1A (5 g,39.7 mmol) was dissolved in dichloromethane (100 mL), cooled to 0 to 5℃and then 70% nitric acid (5 mL) was slowly added dropwise, keeping the internal temperature less than 5℃during the addition, and after completion of the addition, the reaction was continued at room temperature for one hour, TLC showed the end of the reaction, the reaction solution was washed with water (50 mL. Times.2), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give crude compound 1B (yellow oil state, 4.48 g), 66% yield, which was used directly in the next step without further purification.

And a second step of: synthesis of Compound 1C

Compound 1B (2.83 g,16.5 mmol) obtained in the previous step was dissolved in DMF (25 ml), then potassium carbonate (3.4 g,25 mmol) and methyl iodide (1.2 mL,20 mmol) were added, stirring overnight at room temperature after the addition was completed, TLC showed the end of the reaction, then the reaction solution was poured into water, a large amount of solids was eluted, filtration and washing of the filter cake with water several times gave compound 1C (2.76 g, yellow solid) in 90% yield.

And a third step of: synthesis of Compound 1D

Compound 1C (2.5 g,13.5 mmol) obtained in the previous step was dissolved in DMF (25 mL), cesium carbonate (6.6 g,20 mmol)) and 1-ethyl-4- (4-piperidinyl) piperazine (2.47 g,13.5 mmol) were added at room temperature, the reaction temperature was heated to 80℃for 4 hours, TLC showed the end of the reaction, then the reaction solution was poured into water, extracted with ethyl acetate (50 mL. Times.2), the organic phases were combined, washed with saturated brine (50 mL. Times.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 (eluent petroleum ether: ethyl acetate=3:1 to 1:1) to give compound 1D (4.0 g, yellow solid) in 85% yield.

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

Fourth step: synthesis of Compound 1E

Compound 1D (4.0 g,11.5 mmol) obtained in the previous step was dissolved in methanol (50 ml), then 10% palladium on carbon (500 mg) was added, hydrogen balloon was replaced three times, then the hydrogen atmosphere was kept stirring overnight at room temperature, TLC showed the end of the reaction, silica-filled filtration, methanol washing, filtrate concentration gave crude compound 1E (3.42 g), yield 93.5%, which was used directly in the next step without further purification.

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

Fifth step: synthesis of Compound 1G

Compound 1F (10G, 51 mmol) was dissolved in toluene (50 mL), then benzophenone imine (10.9G, 60 mml) was added, then cesium carbonate (32.6G, 100 mmol), pd2dba3 (2.3G, 2.5 mmol) and xantphos (1.44G, 2.5 mmol) were added, nitrogen was replaced three times after the addition was completed, then the reaction was warmed to 110℃under nitrogen protection for 3 hours, TLC showed the end of the reaction, water was added, the organic layer was separated, the aqueous phase was extracted with ethyl acetate (50 mL. Times.2), the organic phases were combined, and then dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was passed through a silica gel column (eluent petroleum ether: ethyl acetate=3:1-1:1) to obtain compound 1G (12.7G, yellow solid) in 83% yield.

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

Sixth step: synthesis of Compound 1H

Intermediate 1G (12.5G, 42 mmol) obtained in the previous step was dissolved in THF (200 mL), concentrated hydrochloric acid (20 mL) was slowly added with stirring at room temperature, then stirring was continued at room temperature for 2 hours, TLC showed the reaction was ended, the reaction was slowly added to saturated aqueous sodium bicarbonate, the internal temperature was protected to less than 10 ℃ and extracted with ethyl acetate (50 ml×2), the organic phases were combined, 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 (eluent petroleum ether: ethyl acetate=3:1 to 1:1) to give compound 1H (5.3G, yellow solid) in 93% yield.

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

Seventh step: synthesis of Compound 1I

Compound 1H (5 g,37 mmol) obtained in the previous step was dissolved in DMF (50 mL), sodium periodate (9.5 g,44.4 mmol) was added at room temperature, after stirring for 10 min, iodine (11.3 g,44.4 mmol) was added in portions, after completion of the reaction for 2 hours, TLC showed the end of the reaction, then the reaction solution was added to saturated sodium thiosulfate solution, stirring was complete until colorless, the resulting aqueous phase was extracted with ethyl acetate (50 mL. Times.2), the organic phases were combined, 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 (eluent petroleum ether: ethyl acetate=3:1 to 1:1) to give compound 1I (6.18 g, yellow solid) in 64% yield.

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

Eighth step: synthesis of Compound 1J

Compound 1I (6.1 g,23.5 mmol) obtained in the previous step was added to DMF (100 mL), followed by addition of dimethylphosphine oxide (2.2 g,28 mmol), potassium phosphate (10 g,47 mmol), palladium acetate (515 mg,2.3 mmol) and Xanthpos (1.33 g,2.3 mmol) in this order, the addition was completed and replaced three times with nitrogen, the reaction was carried out at 150℃for 3 hours under nitrogen protection, TLC showed that the reaction was completed, cooled to room temperature, added to water, extracted with ethyl acetate (50 mL. Times.2), the organic phases were combined, and then washed ((50 mL. Times.2) with saturated brine, 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 (eluent dichloromethane: methanol=50:1 to 10:1) to give Compound 1J (3.3 g, yellow solid) in 67% yield.

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

Ninth step: synthesis of Compound 1K

Compound 1J (3.3 g,15.6 mmol) obtained in the previous step was added to DMF (50 mL), after stirring and dissolution, 5-bromo-2, 4-dichloropyrimidine (5.33 g,23.4 mmol) and potassium carbonate (4.3 g,31.2 mmol) were added sequentially, then heated to 60℃for 4 hours, after TLC showed that the reaction was completed, cooled to room temperature, the reaction solution was poured into water, extracted with ethyl acetate (50 mL. Times.2), the organic phases were combined, the organic phases were washed with saturated brine (50 mL. Times.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 (eluent dichloromethane: methanol=50:1 to 10:1) to give compound 1K (3.6 g, yellow solid) in 57.8% yield.

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

Tenth step: synthesis of Compound of formula I-1

Compound 1K (200 mg,0.5 mmol) obtained in the previous step is dissolved in n-butanol (5 mL), compound 1E (261 mg,0.75 mmol) and concentrated hydrochloric acid (0.5 mL) are sequentially added, the reaction solution is reacted at 90 ℃ for four days, TLC shows the end of the reaction, spin-drying is carried out, sodium bicarbonate aqueous solution (30 mL) is added, extraction is carried out with dichloromethane (10 ml×2), the organic phases are combined, the organic phases are washed with saturated saline (10 ml×2), the organic phases are dried with anhydrous sodium sulfate, filtration is carried out, and the residue obtained by spin-drying concentration is subjected to a silica gel chromatographic column (eluent dichloromethane: methanol=50:1-10:1) to obtain a crude product of the compound shown in the formula I-1. The crude product obtained was purified by column YMC ODSA 30X 100mm prepared by reverse phase C18 (mobile phase with 10-100% acetonitrile (0.05% TFA)/water, flow rate 20mL/min, over 10 min to give the desired product of the compound of formula I-1 (7.5 mg, 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 Compounds of formula I-2

The synthetic route is as follows:

The first step: synthesis of Compound 2A

Compound 1C (2.5 g,13.5 mmol) was dissolved in DMF (25 mL), cesium carbonate (6.6 g,20 mmol) and tert-butyl 4- (piperidin-4-yl) piperazine-1-carboxylate (3.63 g,13.5 mmol) were then added at room temperature, the reaction temperature was heated to 80℃for 4 hours, TLC showed the end of the reaction, then the reaction solution was poured into water, extracted with ethyl acetate (50 mL. Times.2), the organic phases were combined, washed with saturated brine (50 mL. Times.2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the concentrated residue was spin-dried over silica gel column (eluent petroleum ether: ethyl acetate=3:1-1:1) to give compound 2A (5.0 g, yellow solid) in 85.5% yield.

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

And a second step of: synthesis of Compound 2B

Compound 2A (5.0 g,11.5 mmol) obtained in the previous step was dissolved in dichloromethane (50 ml) and TFA (10 ml) was added at room temperature, stirring was continued at room temperature for 2 hours after the addition was completed, TLC showed the end of the reaction, and concentration by spin-drying gave crude compound 2B (4.5 g, brown oil) which was used directly in the next step without purification.

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

And a third step of: synthesis of Compound 2C

Compound 2B (4.5 g) was dissolved in Dichloromethane (DCM) (50 ml), cooled to 0-5℃in an ice-water bath, triethylamine (3.5 g,34.5 mmol) was then added, followed by dropwise addition of a solution of methyl chloroformate (1.64 g,17.3 mmol) in DCM (5 ml), the internal temperature was kept at less than 5℃when the addition was completed, the reaction was allowed to warm to room temperature for 2 hours after completion of the addition, TLC indicated the end of the reaction, the reaction solution was washed twice with water (15 ml. Times.3), the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was passed through a silica gel column (eluent petroleum ether: ethyl acetate=3:1-1:1) to give Compound 2C (2.48 g, yellow solid) in two steps yield 55%.

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

Fourth step: synthesis of Compound 2D

Compound 2C (2.4 g,6.12 mmol) obtained in the previous step was dissolved in methanol (25 ml), then 10% palladium on carbon (250 mg) was added, hydrogen balloon was replaced three times, then the hydrogen atmosphere was kept stirring overnight at room temperature, TLC showed the end of the reaction, silica-filled filtration, methanol washing, filtrate concentration gave crude compound 2D (2.07 g), yield 93.5%, which was used directly in the next step without further purification.

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

Fifth step: synthesis of Compound of formula I-2

Compound 1K (200 mg,0.5 mmol) obtained in the previous step is dissolved in n-butanol (5 mL), compound 2D (272 mg,0.75 mmol) and concentrated hydrochloric acid (0.5 mL) are sequentially added, the reaction solution is reacted for four days at 90 ℃, TLC shows the end of the reaction, spin-drying is carried out, sodium bicarbonate aqueous solution (30 mL) is added, extraction is carried out with dichloromethane (10 ml×2), the organic phases are combined, the organic phases are washed with saturated saline (10 ml×2), the organic phases are dried with anhydrous sodium sulfate, filtration is carried out, and the residue obtained by spin-drying concentration is subjected to a silica gel chromatographic column (eluent dichloromethane: methanol=50:1-10:1) to obtain a crude product of the compound shown in the formula I-2. The crude product obtained was purified by column YMC ODSA 30X 100mm prepared by reverse phase C18 (mobile phase with 10-100% acetonitrile (0.05% TFA)/water, flow rate 20mL/min, over 10 min to give the desired product of the compound of formula I-2 (5.5 mg, 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 Compounds of formula I-3

The synthetic route is as follows:

The first step: synthesis of Compound 3A

The crude compound 2B (1.00 g,3 mmol) was dissolved in DCM (10 ml), cooled to 0-5 ℃ in an ice-water bath, triethylamine (910 mg,9 mmol) was then added, and a solution of acetyl chloride (351 mg,4.5 mmol) in DCM (5 ml) was added dropwise, keeping the internal temperature at less than 5 ℃ when the dropwise addition was completed, and after the completion of the dropwise addition, the reaction was allowed to proceed to room temperature for 2 hours, TLC showed the end of the reaction, the reaction solution was washed twice with water (10 ml×2), the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was passed through a silica gel column (eluent petroleum ether: ethyl acetate=3:1 to 1:1) to give compound 3A (959 mg, yellow solid) in 85% yield.

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

And a second step of: synthesis of Compound 3B

Compound 3A (950 mg,2.53 mmol) obtained in the previous step was dissolved in methanol (10 ml), then 10% palladium on carbon (100 mg) was added, hydrogen balloon was replaced three times, then the hydrogen atmosphere was kept stirring overnight at room temperature, TLC showed the end of the reaction, silica-filled filtration, methanol washing, and filtrate concentration gave crude compound 3B (830 mg), yield 95% which was used directly in the next step without further purification.

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

And a third step of: synthesis of Compound of formula I-3

Compound 1K (200 mg,0.5 mmol) obtained in the previous step is dissolved in n-butanol (5 mL), compound 3B (260, 0.75 mmol) and concentrated hydrochloric acid (0.5 mL) are sequentially added, the reaction solution is reacted at 90 ℃ for four days, TLC shows the end of the reaction, spin-drying is carried out, sodium bicarbonate aqueous solution (30 mL) is added, extraction is carried out with dichloromethane (10 ml×2), the organic phases are combined, the organic phases are washed with saturated saline (10 ml×2), the organic phases are dried with anhydrous sodium sulfate, filtration is carried out, and the residue obtained by spin-drying concentration is subjected to a silica gel chromatographic column (eluent dichloromethane: methanol=50:1-10:1) to obtain a crude product of the compound shown in the formula I-3. The crude product obtained was purified by column YMC ODSA 30X 100mm prepared by reverse phase C18 (mobile phase with 10-100% acetonitrile (0.05% TFA)/water, flow rate 20mL/min, over 10 min to give the desired product of the compound of formula I-3 (7.3 mg, 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 Compounds of formula I-4

The synthetic route is as follows:

The first step: synthesis of Compound 4A

The crude compound 2B (1.00 g,3 mmol) was dissolved in DCM (10 ml), cooled to 0-5 ℃ in an ice water bath, then triethylamine (910 mg,9 mmol) and N, N' -Carbonyldiimidazole (CDI) (583 mg,3.6 mmol) were added, the reaction was completed by heating to room temperature after completion, TLC showed the end of the reaction, then triethylamine (303 mg,3 mmol) and methylamine hydrochloride (245 mg,3.6 mmol) were added, the reaction was continued at room temperature overnight, TLC showed the end of the reaction, 30ml of DCM was further added, washed twice with water (15 ml×2), the organic phase was separated, dried over anhydrous sodium sulfate, filtered and the residue obtained by spin-drying concentration was purified by silica gel chromatography column (eluent petroleum ether: ethyl acetate=10:1 to 1:1) to give compound 4A (991 mg, yellow solid) in 84.5% yield.

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

And a second step of: synthesis of Compound 4B

Compound 4A (950 mg,2.43 mmol) obtained in the previous step was dissolved in methanol (10 ml), then 10% palladium on carbon (200 mg) was added, hydrogen balloon was replaced three times, then the mixture was stirred overnight at room temperature maintaining the hydrogen atmosphere, TLC showed the end of the reaction, silica-filled filtration, methanol washing, and filtrate concentration gave crude compound 4B (829 mg), yield 94.5%, which was used directly in the next step without further purification.

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

And a third step of: synthesis of Compound of formula I-4

Compound 1K (200 mg,0.5 mmol) obtained in the previous step is dissolved in n-butanol (5 mL), compound 4B (270 mg,0.75 mmol) and concentrated hydrochloric acid (0.5 mL) are sequentially added, the reaction solution is reacted for four days at 90 ℃, TLC shows the end of the reaction, spin-drying is carried out, sodium bicarbonate aqueous solution (30 mL) is added, extraction is carried out with dichloromethane (10 ml×2), the organic phases are combined, the organic phases are washed with saturated saline (10 ml×2), the organic phases are dried with anhydrous sodium sulfate, filtration is carried out, and the residue obtained by spin-drying concentration is subjected to a silica gel chromatographic column (eluent dichloromethane: methanol=50:1-10:1) to obtain a crude product of the compound shown in the formula I-4. The crude product obtained was purified by column YMC ODSA 30X 100mm prepared by reverse phase C18 (mobile phase with 10-100% acetonitrile (0.05% TFA)/water, flow rate 20mL/min, over 10 min to give the desired product of the compound of formula I-4 (7.1 mg, 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 Compounds of formula I-5

The synthetic route is as follows:

The first step: synthesis of Compound 5A

The crude compound 2B (1.00 g,3 mmol) was dissolved in DCM (10 ml), cooled to 0-5 ℃ in an ice-water bath, triethylamine (910 mg,9 mmol) and MsCl (410 mg,3.6 mmol) were then added, the reaction was warmed to room temperature after completion for 2 hours, TLC showed the end of the reaction, 30ml of DCM was further added, washed twice with water (15 ml×2), the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was passed through a silica gel column (eluent petroleum ether: ethyl acetate=10:1 to 1:1) to give compound 5A (1.07 g, yellow solid) in 86.3% yield.

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

And a second step of: synthesis of Compound 5B

Compound 5A (1.0 g,11.5 mmol) obtained in the previous step was dissolved in methanol (20 ml), then 10% palladium on carbon (200 mg) was added, hydrogen balloon was replaced three times, then the hydrogen atmosphere was kept stirring overnight at room temperature, TLC showed the end of the reaction, silica-filled filtration, methanol washing, filtrate concentration gave crude compound 5B (0.89 g), yield 95.5% and was used directly in the next step without further purification.

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

And a third step of: synthesis of Compound of formula I-5

Compound 1K (200 mg,0.5 mmol) obtained in the previous step is dissolved in n-butanol (5 mL), compound 5B (287 mg,0.75 mmol) and concentrated hydrochloric acid (0.5 mL) are sequentially added, the reaction solution is reacted at 90 ℃ for four days, TLC shows the end of the reaction, spin-drying is carried out, sodium bicarbonate aqueous solution (30 mL) is added, extraction is carried out with dichloromethane (10 ml×2), the organic phases are combined, the organic phases are washed with saturated saline (10 ml×2), the organic phases are dried with anhydrous sodium sulfate, filtration is carried out, and the residue obtained by spin-drying concentration is subjected to a silica gel chromatographic column (eluent dichloromethane: methanol=50:1-10:1) to obtain a crude product of the compound shown in the formula I-5. The crude product obtained was purified by column YMC ODSA 30X 100mm prepared by reverse phase C18 (mobile phase with 10-100% acetonitrile (0.05% TFA)/water, flow rate 20mL/min, over 10 min to give the desired product of the compound of formula I-5 (4.3 mg, 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 Compounds of formula I-6

The synthetic route is as follows:

The first step: synthesis of Compound 6A

The crude compound 2B (1 g,3 mmol) was dissolved in DMF (15 ml), then potassium carbonate (1.24 g,9 mmol) and bromoethanol (560 mg,4.5 mmol) were added sequentially, the reaction was warmed to 60 ℃ after completion for 4 hours, TLC showed the end of the reaction, the reaction solution was poured into water (100 ml), extracted with ethyl acetate (50 ml×3), the organic phases were combined and washed twice with water (50 ml×2), the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was passed through a silica gel column (eluent petroleum ether: ethyl acetate=3:1 to 1:1) to give compound 6A (737 mg, yellow solid) in 65% yield.

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

And a second step of: synthesis of Compound 6B

Compound 6A (730 mg,1.9 mmol) obtained in the previous step was dissolved in methanol (10 ml), then 10% palladium on carbon (200 mg) was added, hydrogen balloon was replaced three times, then the hydrogen atmosphere was kept stirring overnight at room temperature, TLC showed the end of the reaction, silica-filled filtration, methanol washing, filtrate concentration gave crude compound 6B (625 mg), yield 94.5% which was used directly in the next step without further purification.

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

And a third step of: synthesis of Compound of formula I-6

Compound 1K (200 mg,0.5 mmol) obtained in the previous step was dissolved in n-butanol (5 mL), compound 6B (261 mg,0.75 mmol) and concentrated hydrochloric acid (0.5 mL) were sequentially added, the reaction solution was reacted at 90℃for four days, TLC showed the end of the reaction, dried by spinning, added to an aqueous sodium bicarbonate solution (30 mL), extracted with methylene chloride (10 mL. Times.2), the organic phases were combined, the organic phase was further washed with saturated brine (10 mL. Times.2), the organic phase was dried with anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was subjected to a silica gel column (eluent methylene chloride: methanol=50:1-10:1) to obtain a crude product of the compound represented by formula I-6. The crude product obtained was purified by column YMC ODSA 30X 100mm prepared by reverse phase C18 (mobile phase with 10-100% acetonitrile (0.05% TFA)/water), flow rate 20mL/min, over 10 min, to give the desired product of the compound of formula I-6 (7.5 mg, white solid), MS m/z (ESI): 713[ M+1].

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 Compounds of formula I-7

The synthetic route is as follows:

The first step: synthesis of Compound 7A

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

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

And a second step of: synthesis of Compound 7B

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

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

And a third step of: synthesis of Compound 7C

Compound 7B (1 g,3.77 mol) was added to methanol (20 mL) at room temperature, then acetic acid (0.2 mL) and N-methyl homopiperazine (640 mg,5.65 mmol) were added, stirring was performed at room temperature for 2 hours, then sodium cyanoborohydride (356 mg,5.65 mmol) was added in portions, stirring was continued at room temperature overnight, TLC showed the end of the reaction, then methanol was removed by spin-drying, 100mL of water was added, extraction with dichloromethane (25 mL. Times.3), the organic phases were combined, the organic phase was washed with saturated brine (20 mL. Times.2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was purified by a silica gel column (eluent dichloromethane: methanol=50:1 to 10:1) to give compound 7C (914 mg, yellow solid) in 67% yield.

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

Fourth step: synthesis of Compound 7D

Compound 7C (900 mg,2.5 mmol) obtained in the previous step was dissolved in methanol (10 ml), then 10% palladium on carbon (200 mg) was added, hydrogen balloon was replaced with hydrogen three times, then stirring overnight at room temperature was maintained under hydrogen atmosphere, TLC showed the end of the reaction, silica-filled filtration, methanol washing, filtrate concentration gave crude compound 7D (768 mg), yield 92.5%, which was used directly in the next step without further purification.

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

Fifth step: synthesis of Compound of formula I-7

Compound 1K (200 mg,0.5 mmol) obtained in the previous step is dissolved in n-butanol (5 mL), compound 7D (250 mg,0.75 mmol) and concentrated hydrochloric acid (0.5 mL) are sequentially added, the reaction solution is reacted at 90 ℃ for four days, TLC shows the end of the reaction, spin-drying is carried out, sodium bicarbonate aqueous solution (30 mL) is added, extraction is carried out with dichloromethane (10 ml×2), the organic phases are combined, the organic phases are washed with saturated saline (10 ml×2), the organic phases are dried with anhydrous sodium sulfate, filtration is carried out, and the residue obtained by spin-drying concentration is subjected to a silica gel chromatographic column (eluent dichloromethane: methanol=50:1-10:1) to obtain a crude product of the compound shown in the formula I-7. The crude product obtained was purified by column YMC ODSA 30X 100mm prepared by reverse phase C18 (mobile phase with 10-100% acetonitrile (0.05% TFA)/water, flow rate 20mL/min, over 10 min to give the desired product of the compound of formula I-7 (7.2 mg, 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 Compounds of formula I-8

The synthetic route is as follows:

The first step: synthesis of Compound 8A

Compound 7B (1 g,3.77 mol) was added to methanol (20 mL) at room temperature, then acetic acid (0.2 mL) and thiomorpholine 1, 1-dioxide (763 mg,5.65 mmol) were added, stirring was carried out at room temperature for 2 hours, then sodium cyanoborohydride (356 mg,5.65 mmol) was added in portions, stirring was continued at room temperature overnight, TLC showed the end of the reaction, then methanol was removed by spin-drying, 100mL of water was added, extraction with dichloromethane (25 mL. Times.3), the organic phases were combined, washed with saturated brine (20 mL. Times.2), the organic phases were dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was purified by silica gel chromatography (eluent dichloromethane: methanol=50:1-10:1) to give compound 8A (881 mg, yellow solid), yield 61%, MS m/z (ESI): 384[ M+1].

And a second step of: synthesis of Compound 8B

Compound 8A (800 mg,2.1 mmol) obtained in the previous step was dissolved in methanol (10 ml), then 10% palladium on carbon (500 mg) was added, hydrogen balloon was replaced three times, then the mixture was stirred overnight at room temperature, TLC showed the end of the reaction, silica-filled filtration, methanol washing, and concentration of the filtrate gave crude compound 8B (696 mg), yield 94.5%, which was used directly in the next step without further purification.

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

And a third step of: synthesis of Compound of formula I-8

Compound 1K (200 mg,0.5 mmol) obtained in the previous step is dissolved in n-butanol (5 mL), compound 8B (266 mg,0.75 mmol) and concentrated hydrochloric acid (0.5 mL) are sequentially added, the reaction solution is reacted at 90 ℃ for four days, TLC shows the end of the reaction, spin-drying is carried out, sodium bicarbonate aqueous solution (30 mL) is added, extraction is carried out with dichloromethane (10 ml×2), the organic phases are combined, the organic phases are washed with saturated saline (10 ml×2), the organic phases are dried with anhydrous sodium sulfate, filtration is carried out, and the residue obtained by spin-drying concentration is subjected to a silica gel chromatographic column (eluent dichloromethane: methanol=50:1-10:1) to obtain a crude product of the compound shown in the formula I-8. The crude product obtained was purified by column YMC ODSA 30X 100mm prepared by reverse phase C18 (mobile phase with 10-100% acetonitrile (0.05% TFA)/water, flow rate 20mL/min, over 10 min to give the desired product of the compound of formula I-8 (7.6 mg, 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 Compounds of formula I-9

The synthetic route is as follows:

The first step: synthesis of Compound 9A

Compound 7B (1 g,3.77 mol) was added to methanol (20 mL) at room temperature, then acetic acid (0.2 mL) and morpholine (763 mg,5.65 mmol) were added, stirring was carried out at room temperature for 2 hours, then sodium cyanoborohydride (356 mg,5.65 mmol) was added in portions, stirring was continued at room temperature overnight, TLC showed the end of the reaction, then methanol was removed by spin-drying, 100mL of water was added, dichloromethane was used for extraction (25 mL. Times.3), the organic phases were combined, washed with saturated brine (20 mL. Times.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 (eluent dichloromethane: methanol=50:1-10:1) to give compound 9A (881 mg, yellow solid) in 61% yield.

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

And a second step of: synthesis of Compound 9B

Compound 9A (850 mg,2.5 mmol) obtained in the previous step was dissolved in methanol (10 ml), then 10% palladium on carbon (200 mg) was added, hydrogen balloon was replaced three times, then the mixture was stirred overnight at room temperature maintaining the hydrogen atmosphere, TLC showed the end of the reaction, silica-filled filtration, methanol washing, and concentration of the filtrate afforded crude compound 9B (707 mg), yield 92.5%, which was used directly in the next step without further purification.

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

And a third step of: synthesis of Compound of formula I-9

Compound 1K (200 mg,0.5 mmol) obtained in the previous step is dissolved in n-butanol (5 mL), compound 9B (230 mg,0.75 mmol) and concentrated hydrochloric acid (0.5 mL) are sequentially added, the reaction solution is reacted at 90 ℃ for four days, TLC shows the end of the reaction, spin-drying is carried out, sodium bicarbonate aqueous solution (30 mL) is added, extraction is carried out with dichloromethane (10 ml×2), the organic phases are combined, the organic phases are washed with saturated saline (10 ml×2), the organic phases are dried with anhydrous sodium sulfate, filtration is carried out, and the residue obtained by spin-drying concentration is subjected to a silica gel chromatographic column (eluent dichloromethane: methanol=50:1-10:1) to obtain a crude product of the compound shown in the formula I-9. The crude product obtained was purified by column YMC ODSA 30X 100mm prepared by reverse phase C18 (mobile phase with 10-100% acetonitrile (0.05% TFA)/water, flow rate 20mL/min, over 10 min to give the desired product of the compound of formula I-9 (5.5 mg, 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 Compounds of formula I-10

The synthetic route is as follows:

the first step: synthesis of Compound 10B

Compound 10A (2.3 g,13.5 mmol) was dissolved in DMF (25 mL), cesium carbonate (6.6 g,20 mmol) and 1-methyl-4- (4-piperidinyl) piperazine (2.47 g,13.5 mmol) were then added at room temperature, the reaction temperature was heated to 80℃and the reaction was completed for 4 hours, TLC showed the completion of the reaction, then the reaction solution was poured into water, extracted with ethyl acetate (50 mL. Times.2), the organic phases were combined, washed with saturated brine (50 mL. Times.2), and the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was purified over a silica gel column (eluent petroleum ether: ethyl acetate=3:1 to 1:1) to give compound 10B (3.76 g, yellow solid) in 83.5% yield.

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

And a second step of: synthesis of Compound 10C

Compound 10B (2.0 g,6 mmol) obtained in the previous step was dissolved in methanol (20 ml), then 10% palladium on carbon (400 mg) was added, hydrogen balloon was replaced three times, then the reaction was stirred overnight at room temperature maintaining the hydrogen atmosphere, TLC showed the end of the reaction, silica-filled filtration, methanol washing, filtrate concentration gave crude compound 10C (1.7 g), yield 95.6% and was used directly in the next step without further purification.

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

And a third step of: synthesis of Compound 10E

The compound 1J (2.1 g,10 mmol) obtained in the previous step was added to DMF (30 mL), and after stirring and dissolution, 2,4, 5-trichloropyrimidine (2.18 g,12 mmol) and potassium carbonate (2.76 g,20 mmol) were added sequentially, then heated to 60℃and reacted for 4 hours, after which TLC showed the end of the reaction, cooled to room temperature, the reaction solution was poured into water (150 mL), extracted with ethyl acetate (50 mL. Times.2), the organic phases were combined, and then the organic phases were washed with saturated brine (50 mL. Times.2), the organic phases were dried with anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was purified by a silica gel column (eluent dichloromethane: methanol=50:1 to 10:1) to give compound 10E (2.09 g, yellow solid) in 58.7% yield.

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

Fourth step: synthesis of Compound of formula I-10

Compound 10E (180 mg,0.5 mmol) obtained in the previous step was dissolved in n-butanol (5 mL), compound 10C (228 mg,0.75 mmol) and concentrated hydrochloric acid (0.5 mL) were sequentially added, the reaction solution was reacted at 90℃for four days, TLC showed the end of the reaction, dried by spinning, added to an aqueous sodium bicarbonate solution (30 mL), extracted with methylene chloride (10 mL. Times.2), the organic phases were combined, the organic phase was further washed with saturated brine (10 mL. Times.2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was subjected to a silica gel column (eluent methylene chloride: 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 by column YMC ODSA 30X 100mm prepared by reverse phase C18 (mobile phase with 10-100% acetonitrile (0.05% TFA)/water, flow rate 20mL/min, over 10min to give the desired product of the compound of formula I-10 (7.2 mg, 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 Compounds of formula I-11

The synthetic route is as follows:

the first step: synthesis of Compound 11A

Compound 1C (2.5 g,13.5 mmol) was dissolved in DMF (25 mL), cesium carbonate (6.6 g,20 mmol) and NBoc piperazine (2.51 g,13.5 mmol) were then added at room temperature, the reaction temperature was heated to 80℃and the reaction was completed for 4 hours, TLC showed the reaction completion, then the reaction solution was poured into water (100 mL), extracted with ethyl acetate (50 mL. Times.2), the organic phases were combined, the organic phase was washed with saturated brine (50 mL. Times.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 (eluent petroleum ether: ethyl acetate=3:1 to 1:1) to give compound 11A (4.06 g, yellow solid) in 85.6% yield.

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

And a second step of: synthesis of Compound 11B

Compound 11A (4.0 g,11.4 mmol) obtained in the previous step was dissolved in dichloromethane (50 ml), TFA (10 ml) was then added at room temperature, stirring was continued at room temperature for 2 hours after the addition was completed, TLC showed the end of the reaction, and concentration by spin-drying afforded crude compound 11B (3.8 g, brown oil) which was used directly in the next step without purification.

And a third step of: synthesis of Compound 11C

Crude compound 11B (1.9 g) obtained in the previous step was added to methanol (20 mL) at room temperature, then 3-oxetanone (792 mg,11 mmol) was added, after stirring at room temperature for 2 hours, sodium cyanoborohydride (704 mg,11 mmol) was added in portions, stirring at room temperature was continued overnight, TLC showed the reaction to end, then methanol was removed by spin-drying, 100mL of water was added, extracted with dichloromethane (25 ml×3), the organic phases were combined, washed with saturated brine (20 ml×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 (eluent dichloromethane: methanol=50:1-10:1) to give compound 11C (717 mg, yellow solid) in two steps of 41%.

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

Fourth step: synthesis of Compound 11D

Compound 11C (700 mg,2.28 mmol) obtained in the previous step was dissolved in methanol (10 ml), then 10% palladium on carbon (150 mg) was added, hydrogen balloon was replaced three times, then stirring overnight at room temperature was maintained under hydrogen atmosphere, TLC showed the end of the reaction, silica-filled filtration, methanol washing, filtrate concentration gave crude compound 11D (597 mg), yield 94.2%, which was used directly in the next step without further purification.

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

Fifth step: synthesis of Compound of formula I-11

Compound 1K (200 mg,0.5 mmol) obtained in the previous step is dissolved in n-butanol (5 mL), compound 11D (208 mg,0.75 mmol) and concentrated hydrochloric acid (0.5 mL) are sequentially added, the reaction solution is reacted at 90 ℃ for four days, TLC shows the end of the reaction, spin-drying is carried out, sodium bicarbonate aqueous solution (30 mL) is added, extraction is carried out with dichloromethane (10 ml×2), the organic phases are combined, the organic phases are washed with saturated saline (10 ml×2), the organic phases are dried with anhydrous sodium sulfate, filtration is carried out, and the residue obtained by spin-drying concentration is subjected to a silica gel chromatographic column (eluent dichloromethane: methanol=50:1-10:1) to obtain a crude product of the compound shown in the formula I-11. The crude product obtained was purified by column YMC ODSA 30X 100mm prepared by reverse phase C18 (mobile phase with 10-100% acetonitrile (0.05% TFA)/water, flow rate 20mL/min, over 10min to give the desired product of the compound of formula I-11 (6.1 mg, 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 Compounds of formula I-12

The synthetic route is as follows:

the first step: synthesis of Compound 12B

Compound 12A (11.5 g,53 mmol) was dissolved in a mixed solvent of acetonitrile (150 mL) and water (30 mL), then aqueous formaldehyde (11 mL,160 mmol) was added, after stirring at room temperature for 2 hours, sodium borohydride acetate (22.5 g,110 mmol) was added in portions, the reaction was allowed to proceed overnight at room temperature after the addition was completed, TLC showed the end of the reaction, then saturated sodium bicarbonate solution was added to the reaction solution to adjust the pH to pH=8, dichloromethane (500 mL) and methanol (50 mL) were further added to dilute the reaction solution, the reaction solution was washed with saturated aqueous sodium carbonate solution (100 mL. Times.2), the organic phase was separated, dried over anhydrous sodium sulfate and dried by spin-drying to give product 12B (11.9 g, white solid) which was directly used in the next step without purification in 98% yield.

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

And a second step of: synthesis of Compound 12C

Compound 2B (11 g,47.8 mmol) obtained in the previous step was dissolved in dichloromethane (150 ml) and TFA (30 ml) was added at room temperature, stirring was continued at room temperature for 2 hours after the addition was completed, TLC showed the end of the reaction and concentration by spin-drying gave crude compound 12C (9.2 g, brown oil) which was used directly in the next step without purification.

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

And a third step of: synthesis of Compound 12D

Compound 1C (2.5 g,13.5 mmol) was dissolved in DMF (25 mL), then cesium carbonate (6.6 g,20 mmol) and compound 12C (1.75 g,13.5 mmol) were added at room temperature, the reaction temperature was heated to 80 ℃, the reaction was completed for 4 hours, TLC showed the reaction completion, then the reaction solution was poured into water, extracted with ethyl acetate (50 ml×2), the organic phases were combined, the organic phases were washed with saturated brine (50 ml×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 (eluent petroleum ether: ethyl acetate=3:1 to 1:1) to give compound 12D (3.4 g, yellow solid) in 85.7% yield.

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

Fourth step: synthesis of Compound 12E

Compound 12D (1.0 g,3.39 mmol) obtained in the previous step was dissolved in methanol (20 ml), then 10% palladium on carbon (200 mg) was added, hydrogen balloon was replaced three times, then the hydrogen atmosphere was kept stirring overnight at room temperature, TLC showed the end of the reaction, silica-filled filtration, methanol washing, and filtrate concentration gave crude compound 12E (858 mg), yield 95.2%, which was used directly in the next step without further purification.

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

Fifth step: synthesis of Compound of formula I-12

Compound 1K (200 mg,0.5 mmol) was dissolved in n-butanol (5 mL), compound 12E (200 mg,0.75 mmol) and concentrated hydrochloric acid (0.5 mL) were sequentially added, the reaction solution was reacted at 90℃for four days, TLC showed the end of the reaction, dried by spinning, added to an aqueous sodium hydrogen carbonate solution (30 mL), extracted with methylene chloride (10 mL. Times.2), the organic phases were combined, the organic phase was further washed with saturated brine (10 mL. Times.2), the organic phase was dried with anhydrous sodium sulfate, filtered, and the residue obtained by concentration by spinning was subjected to a silica gel column (eluent methylene chloride: methanol=50:1 to 10:1) to obtain crude compound I-12. The crude product obtained was purified by column YMC ODSA 30X 100mm prepared by reverse phase C18 (mobile phase with 10-100% acetonitrile (0.05% TFA)/water, flow rate 20mL/min, over 10 min to give the desired product of the compound of formula I-12 (5.5 mg, 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 Compounds of formula I-13

The synthetic route is as follows:

The first step: synthesis of Compound 13A

Compound 7B (1 g,3.77 mol) was added to methanol (20 mL) at room temperature, then acetic acid (0.2 mL) and compound 12C (284 mg,5.65 mmol) were added, stirring was performed at room temperature for 2 hours, then sodium cyanoborohydride (356 mg,5.65 mmol) was added in portions, stirring was continued at room temperature overnight, TLC showed the end of the reaction, then methanol was removed by spin-drying, 100mL of water was added, extraction with dichloromethane (25 mL. Times.3), the organic phases were combined, the organic phase was washed with saturated brine (20 mL. Times.2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was purified by a silica gel column (eluent dichloromethane: methanol=50:1-10:1) to give compound 13A (892 mg, yellow solid) in 62.6% yield.

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

And a second step of: synthesis of Compound 13B

Compound 13A (800 mg,2.12 mmol) obtained in the previous step was dissolved in methanol (10 ml), then 10% palladium on carbon (200 mg) was added, hydrogen balloon was replaced three times, then the reaction was stirred overnight at room temperature while maintaining the hydrogen atmosphere, TLC showed the end of the reaction, silica-filled filtration, methanol washing, and concentration of the filtrate gave crude compound 13B (683 mg), yield 92.7%, which was used directly in the next step without further purification.

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

And a third step of: synthesis of Compound of formula I-13

Compound 1K (200 mg,0.5 mmol) obtained in the previous step is dissolved in n-butanol (5 mL), compound 13B (261 mg,0.75 mmol) and concentrated hydrochloric acid (0.5 mL) are sequentially added, the reaction solution is reacted at 90 ℃ for four days, TLC shows the end of the reaction, spin-drying is performed, sodium bicarbonate aqueous solution (30 mL) is added, extraction is performed with dichloromethane (10 ml×2), the organic phases are combined, the organic phases are washed with saturated saline (10 ml×2), the organic phases are dried with anhydrous sodium sulfate, filtration is performed, and the residue obtained by spin-drying concentration is subjected to a silica gel chromatographic column (eluent dichloromethane: methanol=50:1-10:1) to obtain a crude product of the compound shown in the formula I-13. The crude product obtained was purified by column YMC ODSA 30X 100mm prepared by reverse phase C18 (mobile phase with 10-100% acetonitrile (0.05% TFA)/water, flow rate 20mL/min, over 10min to give the desired product of the compound of formula I-13 (5.4 mg, 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 Compounds of formula I-14

The synthetic route is as follows:

The first step: synthesis of Compound 14A

Compound 7B (1 g,3.77 mol) was added to methanol (20 mL) at room temperature, then acetic acid (0.2 mL) and N-methylpiperazine (560 mg,5.65 mmol) were added, stirring was performed at room temperature for 2 hours, then sodium cyanoborohydride (356 mg,5.65 mmol) was added in portions, stirring was continued at room temperature overnight, TLC showed the end of the reaction, then methanol was removed by spin-drying, 100mL of water was added, extraction with dichloromethane (25 mL. Times.3), the organic phases were combined, the organic phase was washed with saturated brine (20 mL. Times.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 (eluent dichloromethane: methanol=50:1-10:1) to give compound 14A (827 mg, yellow solid) in 63% yield.

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

And a second step of: synthesis of Compound 14B

Compound 14A (800 mg,2.3 mmol) obtained in the previous step was dissolved in methanol (10 ml), then 10% palladium on carbon (200 mg) was added, hydrogen balloon was replaced three times, then the reaction was stirred overnight at room temperature while maintaining the hydrogen atmosphere, TLC showed the end of the reaction, silica-filled filtration, methanol washing, and filtrate concentration gave crude compound 14B (669 mg), yield 91.5%, which was used directly in the next step without further purification.

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

And a third step of: synthesis of Compound of formula I-14

Compound 1K (200 mg,0.5 mmol) obtained in the previous step was dissolved in n-butanol (5 mL), compound 14B (238 mg,0.75 mmol) and concentrated hydrochloric acid (0.5 mL) were sequentially added, the reaction solution was reacted at 90℃for four days, TLC showed the end of the reaction, dried by spinning, added to an aqueous sodium bicarbonate solution (30 mL), extracted with methylene chloride (10 mL. Times.2), the organic phases were combined, the organic phase was further washed with saturated brine (10 mL. Times.2), the organic phase was dried with anhydrous sodium sulfate, filtered, and the residue obtained by spin-drying concentration was subjected to a silica gel column (eluent methylene chloride: 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 by column YMC ODSA 30X 100mm prepared by reverse phase C18 (mobile phase with 10-100% acetonitrile (0.05% TFA)/water, flow rate 20mL/min, over 10min to give the desired product of the compound of formula I-14 (7.7 mg, 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 antiproliferative assay

(1) Experimental materials:

RPMI1640, glutamine, interleukin 3, pancreatin was purchased from Life Technology.

Fetal bovine serum, diabody, 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 (EGFRΔ19del/T790M) cells and Ba/F3 (EGFRΔ19del/T790M/C797S) cells were constructed from WuXi.

H1975 cell culture medium: 88% RPMI 1640, 10% fetal bovine serum, 1% glutamine, 1% diabody.

Ba/F3 cell culture Medium: 88% RPMI 1640, 10% fetal bovine serum, 10ng/mL interleukin 3,1% glutamine, 1% diabody.

Ba/F3 (EGFRΔ19del/T790M) cells and Ba/F3 (EGFRΔ19del/T790M/C797S) cell culture medium: 87.9% RPMI 1640, 10% fetal bovine serum, 0.1% blasticidin, 1% glutamine, 1% diabody.

Experiment plate: 781091 (Greiner)

Plate reading instrument: envision (PerkinElmer) A

(2) The experimental method comprises the following steps:

Experimental method (H1975 cells): digesting and separating cells in a T75 culture flask, re-suspending the cells by using a culture medium, counting, regulating the concentration of the cells to 2.0X10 ⁴ cells/mL by using the culture medium, adding 100 mu L of phosphate buffer solution into peripheral holes of a 384-well plate, adding 50 mu L of cell suspension into other holes, standing for 15min at room temperature, and then placing the cells in a 5% CO ₂ incubator at 37 ℃ for incubation for 24h; the test compounds were subjected to 10-point 3-fold gradient dilutions on ECHO with DMSO and transferred to 384 well plates at a final concentration of 5-0.00025. Mu.M, positive and negative control transfers 250nL DMSO,1000rpm centrifuged for 15s, and incubated in a 5% CO ₂ incubator at 37℃for 72h; 25uL CellTiter-Glo was added to each well of 384 well plates, centrifuged at 1000rpm for 15s, and after shaking at room temperature for 15min, read with Envision.

Experimental methods (Ba/F3 cells, ba/F3 (EGFRΔ19del/T790M) cells and Ba/F3 (EGFRΔ19del/T790M/C797S) cells): the test compounds were 3-fold gradient diluted with DMSO at 10 points on ECHO, transferring 250nL of the compound to 384 well plates, respectively, at a final concentration of 5-0.00025 μm, positive and negative controls transferring 250nL of DMSO; blowing cells in a uniform T75 culture bottle, counting, regulating the cell concentration to 4X 10 ⁴ cells/mL by using a culture medium, adding 100 mu L of phosphate buffer solution into peripheral holes of a 384-well plate, adding 50 mu L of cell suspension into other holes, standing at room temperature for 15min, and then placing in a 5% CO ₂ incubator at 37 ℃ for incubation for 72h; 25 mu L CELLTITER-Glo was added to each well of 384 well plates, centrifuged at 1000rpm for 15s, and after shaking at room temperature for 15min, read with Envision.

(3) Data analysis:

The readings were converted to inhibition (%) (Sample value-HC)/(LC-HC) 100. Parametric curve fitting (XL-fit software) to measure IC ₅₀ data by the following formula.

(4) Test results:

EGFRBa/F3 (Delta19 del/T790M) cell activity inhibition IC ₅₀, H1975 cell activity inhibition IC ₅₀, EGFR Ba/F3 (Delta19 del/T790M/C797S) cell activity inhibition IC ₅₀, ba/F3 parent cell activity inhibition IC ₅₀ of the compounds of the invention. The data are shown in table 1 below.

Wherein compounds with IC ₅₀ between 1-100nm are identified with++, compounds with IC ₅₀ between 100-1000nm are identified with++, and compounds with IC ₅₀ greater than 1000nm are identified with+.

Table 1: cell antiproliferative activity data (IC ₅₀) of the compounds of the present examples

From the experimental results in table 1 we can see:

(1) Most of the compounds of the invention have good inhibition effect on EGFR Ba/F3 (delta 19 del/T790M/C797S) three-mutant cells, and the antiproliferative activity of cells of some compounds is below 100 nm.

(2) EGFR Ba/F3 (delta 19 del/T790M) is a cell model of EGFR double mutation, and is a main object of third generation EGFRTKIs and mainly aims at generating a tolerance effect on secondary mutation of T790M generated by an ATP receptor part, and through tests, most of the compounds have better cell proliferation resistance on double mutation of EGFRBa/F3 (A19 del/T790M), so that the compounds have double inhibition on the triple mutation and double mutation of EGFRBa/F3.

(3) The cell antiproliferative activity test of Ba/F3 parent and H1975 (EGFRWT) is one 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 double mutations, which indicates that the compound has very good cell selectivity, weak toxic effect and very good antitumor application prospect.

Effect example 2: kinase inhibition Activity assay

(1) Experimental materials:

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

DTT and double distilled water were both purchased from Life Technology.

Magnesium chloride was purchased from Fluka.

ATP was purchased from Sigma.

HTRF KINEASE-TK Kit was purchased from Cisbio.

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

Experiment plate: 6007299 (Perkinelmer)

Plate reading instrument: envision (PerkinElmer) A

(2) The experimental method comprises the following steps:

The test compounds were 3-fold gradient diluted with DMSO at 11 spots on ECHO, 100nL of the compounds were transferred to 384 well plates at a final concentration of 0.5-0.0000085. Mu.M, positive control transferred to 100nL of MSK-1403, and negative control transferred to 100nL of DMSO; a mixture of 1nM EGFR T790M/C797S/L858R enzyme and 2. Mu.M TK Substrate-biotin was prepared with reaction buffer and 5. Mu.L was added to each well, shaking at room temperature for 15S, centrifuging at 1000rpm for 15S, incubating at 23℃for 15min; preparing 20 mu M ATP with reaction buffer, adding 5 mu L of ATP into each well, shaking for 15s at room temperature, centrifuging for 15s at 1000rpm, and incubating at 23 ℃ for 60min; a mixture of 125nM strepitavidin-XL 665 and 1/2 XTK Antibody-Cryptate was formulated with Detection buffer and 10. Mu.L of each well was added, shaken 15s at room temperature, centrifuged 15s at 1000rpm, and incubated at 23℃for 60min; read with Envision.

(3) Data analysis:

IC ₅₀ data were measured by converting the readings to inhibition (%) (Sample value-HC)/(LC-HC). Times.100% by the following formula parametric curve fitting (XL-fit software).

(4) Test results:

The EGFR (T790M/C797S/L858R) and EGFR (d 746-750/T790M/C797S) activity inhibiting activity IC ₅₀ data for the compounds of the invention are shown in Table 2 below.

Wherein compounds with IC ₅₀ between 0.1-10nm are identified with++, compounds with IC ₅₀ between 10-100nm are identified with++, and compounds with IC ₅₀ greater than 100nm are identified with+.

Table 2: kinase inhibitory data (IC ₅₀) for the compounds of the examples

Results: as shown in Table 2, most of the compounds of the invention have good inhibition effect on EGFR-C797S gene mutant 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: experiment of drug metabolism

1. Using the compound I-14 of the present invention prepared in the above example, an oral drug was formulated as a clear solution (2%DMSO+30%PEG 300+2%Tween 80+66%H2O) of 0.3mg/mL, and a intravenous drug was formulated as a clear solution (2%DMSO+30%PEG 300+2%Tween 80+66%H ₂ O) of 0.2mg/mL

2. Male CD-1 mice, 3 each, weighing 27-28g, supplied by Shanghai Laike laboratory animal liability Co. The mice to be tested were given an environmental adaptation period of 2-4 days prior to the experiment, fasted for 8-12 hours prior to administration, fed water 2 hours after administration, and fed after 4 hours.

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

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

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

6. based on the blood concentration-time data obtained in step 3, the pharmacokinetic parameters were calculated using WinNonlin software, and the specific data are shown in table 3.

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

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

Effect example 4: experiment of drug metabolism

The experimental method comprises the following steps:

1. In vivo efficacy experiments were performed on xenograft (CDX) BALB/C nude mice subcutaneously implanted with Ba/F3 (. DELTA.19 del/T790M/C797S) sources. BALB/c nude mice, females, weighing about 18-22 g, were housed in SPF-class environment with separate ventilation per cage (5 mice per cage). All cages, bedding and water were sterilized prior to use. All animals were free to obtain a standard certified commercial laboratory diet. A total of 48 mice purchased from beijing veteran rituximab were used for the study. Each mouse was subcutaneously implanted with cells in the right flank for tumor growth. Experiments were started when the average tumor volume reached about 150-200 cubic millimeters. The test compounds were orally administered daily, wherein compound Birgatinib (15 mg/kg), compound I-9 (150 mg 10/kg) and compound I-14 (100 mg/kg) were administered for 14 consecutive days.

2. Tumor volumes were measured twice a week with two-dimensional calipers, the volumes were measured in cubic millimeters, and calculated by the following formula: v=0.5a×b ², where a and b are the long and short diameters of the tumor, respectively. Antitumor efficacy was determined by dividing the average tumor-increasing volume of animals treated with the compound by the average tumor-increasing volume of untreated animals. Wherein TGI (the tumor inhibition value, tumor volume inhibition) is used to evaluate the inhibition of tumor growth by the test drug in vivo, calculated as: TGI (100%) = [ (1- (mean tumor volume at the end of dosing group-mean tumor volume at the start of dosing group)/(mean tumor volume at the end of solvent control group-mean tumor volume at the start of treatment of solvent control group) ] × 100%. Wherein TGI was 8.6% for compound Birgatinib (15 mg/kg), 108% for compound I-9 (150 mg 10/kg) and 108% for compound I-14 (100 mg/kg).

Experimental results: see table 4.

TABLE 4 Table 4

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

In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. 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 are now within the scope of this disclosure for those of ordinary skill in the art without departing from the principles and spirit of the invention.

Claims

1. A compound comprising a compound of formula I, or a pharmaceutically acceptable salt thereof; the compound shown in the formula I is any one of the following compounds:

。

2. A pharmaceutical composition comprising an effective amount of a compound of formula I according to claim 1, and at least one pharmaceutically acceptable adjuvant.

3. Use of a compound of formula I as defined in claim 1, or a pharmaceutically acceptable salt thereof, or a hydrate thereof, or a pharmaceutical composition as defined in claim 2, for the preparation of an EGFR T790M/C797S inhibitor medicament.

4. Use of a compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 2, for the manufacture of a medicament for the treatment and/or prophylaxis of diseases caused by abnormal mutations in EGFR T790M/C797S.

5. Use of a compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 2, for the manufacture of a medicament for the treatment and/or prophylaxis of cancer.

6. The use of claim 5, wherein the cancer comprises lung cancer, glioma, renal cancer, prostate cancer, pancreatic cancer, breast cancer.