CN114539269A - Nitrogen-containing macrocyclic compound, preparation method and application thereof - Google Patents

Abstract

The invention discloses a nitrogen-containing macrocyclic compound, a preparation method and application thereof. The invention provides a nitrogen-containing macrocyclic compound shown as a formula I or a pharmaceutically acceptable salt thereof, wherein the compound has high inhibitory activity on EGFR-T790M mutation and has certain inhibitory activity on wild type EGFR.

Description

Nitrogen-containing macrocyclic compound, preparation method and application thereof

Technical Field

The invention relates to a nitrogen-containing macrocyclic compound, a preparation method and application thereof.

Background

The morbidity and mortality of lung cancer is the first of all malignancies, and there is a constant need for new compounds with better activity/selectivity in the field of lung cancer drugs. Non-small cell lung cancer is a common form of lung cancer, and the most common type of gene mutation in patients with non-small cell lung cancer is an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) gene mutation, accounting for approximately 50%. Mutations or overexpression of EGFR are important in connection with tumor development.

The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) on the market at present are mainly divided into three generations: first generation EGFR-targeting drugs: gefitinib was approved by the FDA for marketing in 2003. Erlotinib was approved by the FDA in 2004 for marketing, and is used as a first-line therapeutic for non-small cell lung cancer patients. However, gefitinib and erlotinib develop acquired resistance after a treatment period of about 12 months, and patients with near 2/3 develop a secondary acquired mutation of EGFR-T790M. Third generation EGFR-targeting drugs: the drug is a targeted drug developed specially aiming at the problem of drug resistance of non-small cell lung cancer patients to gefitinib and erlotinib, and is used for treating EGFR-T790M mutant non-small cell lung cancer patients. Unfortunately, oxitinib causes EGFR-C797S mutation, thereby losing its therapeutic potency. The feature of almost all targeted drugs, or the so-called drawback, is the problem of resistance, which once developed, presents the patient with pain.

The important position of EGFR targeting drugs in the treatment of non-small cell lung cancer, the development of compounds with EGFR-T790M mutation, high inhibitory activity and low inhibitory activity to wild type EGFR, is still a difficult point to be solved in the pharmaceutical field

Disclosure of Invention

The technical problem to be solved by the invention is the defect that the existing EGFR targeted drug has a single structure. Therefore, the invention provides a nitrogen-containing macrocyclic compound, a preparation method and application thereof. The compounds have high inhibitory activity on EGFR-T790M mutation and low inhibitory activity on wild type EGFR.

The invention provides a nitrogen-containing macrocyclic compound shown as a formula I or pharmaceutically acceptable salt thereof,

wherein ,R¹Is hydrogen, C₁～C₃Alkoxy, halogen or C substituted by one or more halogens₁～C₃An alkoxy group;

R²is hydrogen or C substituted by one or more halogens₁～C₃An alkoxy group;

R³is hydrogen or halogen;

R⁴is hydrogen or halogen;

r is hydrogen or

n is 0, 1, 2, 3, 4, 5 or 6.

In one embodiment, in the nitrogen-containing macrocyclic compound of formula I or a pharmaceutically acceptable salt thereof, certain groups may be defined as follows, and other groups may be defined as described in any of the above embodiments (hereinafter "in one embodiment"): r¹Is hydrogen, C₁～C₃Alkoxy, halogen or C substituted by one or more fluorine₁～C₃An alkoxy group.

In a certain embodiment, R²Is hydrogen or C substituted by one or more fluorine₁～C₃An alkoxy group.

In a certain embodiment, R³Is hydrogen or chlorine.

In a certain embodiment, R⁴Is hydrogen or chlorine.

In one embodiment, n is 2, 3 or 4.

In a certain embodiment, R¹Is hydrogen, C₁～C₃Alkoxy, halogen or C substituted by one or more fluorine₁～C₃An alkoxy group;

R²is hydrogen or is substituted byC substituted by one or more fluorine₁～C₃An alkoxy group;

R³is hydrogen or chlorine;

R⁴is hydrogen or chlorine;

r is hydrogen or

n is 2, 3 or 4.

In a certain aspect, wherein R¹Is hydrogen, C₁～C₃Alkoxy, halogen or C substituted by one or more halogens₁～C₃An alkoxy group;

R²is hydrogen or C substituted by one or more halogens₁～C₃An alkoxy group;

R³is hydrogen or halogen;

R⁴is hydrogen or halogen;

r is

n is 0, 1, 2, 3, 4, 5 or 6.

In a certain embodiment, R¹Is hydrogen, fluorine or C substituted by one or more fluorine₁～C₃An alkoxy group;

R²is hydrogen or C "substituted by one or more fluorine₁～C₃Alkoxy groups ";

R³is hydrogen;

R⁴is hydrogen or chlorine;

r is

n is 2 or 3.

In a certain embodiment, R¹Is hydrogen, fluorine or C substituted by one or more fluorine₁～C₃An alkoxy group;

R²is hydrogen;

R³is hydrogen orChlorine;

R⁴is hydrogen or chlorine;

r is

n is 2 or 3.

In one embodiment, R is

In one aspect, when R is said¹Is C₁～C₃When alkoxy, said C₁～C₃The alkoxy group may be methoxy, ethoxy, n-propoxy or isopropoxy, and may also be methoxy.

In one aspect, when R is said¹When halogen is used, the halogen may be fluorine, chlorine, bromine or iodine, and may also be fluorine.

In one aspect, when R is said¹Is C substituted by one or more halogens₁～C₃At alkoxy, said C₁～C₃The alkoxy group may be methoxy, ethoxy, n-propoxy or isopropoxy, and may also be methoxy.

In one aspect, when R is said¹Is C substituted by one or more halogens₁～C₃In the case of alkoxy, the halogen may be fluorine, chlorine, bromine or iodine, or may be fluorine.

In one aspect, when R is said¹Is C substituted by one or more halogens₁～C₃In the case of an alkoxy group, the number of the alkoxy groups is 3.

In one aspect, when R is said¹Is C substituted by more than one halogen₁～C₃When alkoxy, said C substituted by more than one halogen₁～C₃Alkoxy is trifluoromethoxy.

In a certain embodiment, R¹Is hydrogen, fluorine or trifluoromethoxy.

In one aspect, when R is said²Is "substituted by plural halogensSubstituted C₁～C₃Alkoxy "said C₁～C₃The alkoxy group may be methoxy, ethoxy, n-propoxy or isopropoxy, and may be n-propoxy.

In one aspect, when R is said²Is "C substituted by one or more halogens₁～C₃Alkoxy "said halogen may be fluorine, chlorine, bromine or iodine, and may also be fluorine.

In one aspect, when R is said²Is "C substituted by one halogen₁～C₃Alkoxy, said "C substituted by one halogen₁～C₃The alkoxy radical "may be a monofluoro-n-propoxy radical or may be

In a certain embodiment, R²Is composed of

Or hydrogen.

In one aspect, when R is said³When halogen is used, the halogen may be fluorine, chlorine, bromine or iodine, and may also be chlorine.

In one aspect, when R is said⁴When halogen is used, the halogen may be fluorine, chlorine, bromine or iodine, and may also be chlorine.

In one embodiment, the nitrogen-containing macrocyclic compound of formula I can be any of the following compounds:

the invention provides a nitrogen-containing macrocyclic compound shown as a formula II or a salt thereof,

wherein ,

in cis configuration, trans configuration or mixtures thereof;

R¹、R²、R³、R⁴r and n are as defined above.

In one embodiment, the nitrogen-containing macrocyclic compound of formula II can be any of the following compounds:

or its cis isomer,

Or its cis isomer,

Or its cis-isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or the cis form thereofIsomers of,

Or its cis isomer,

Or its cis-isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis-isomer,

Or its cis isomer,

Or cis-iso thereofA structure body,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis-isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or cis isomer thereof、

Or its cis isomer,

Or its cis isomer,

Or its cis isomer or

Or a cis isomer thereof.

The invention also provides a preparation method of the nitrogen-containing macrocyclic compound shown in the formula I, which is a scheme I or a scheme ii;

scheme i, which includes the steps of subjecting a compound of formula II to a hydrogenation reduction as shown below;

r is hydrogen;

in cis configuration, trans configuration or mixtures thereof;

R¹、R²、R³、R⁴and n is as defined above.

Scheme ii, the method comprises the following steps of carrying out acylation reaction on a compound shown as a formula A and acryloyl chloride shown as a formula;

r is

R¹、R²、R³、R⁴And n is as defined above.

The invention also provides a pharmaceutical composition comprising substance X and a pharmaceutical excipient; the substance X is a nitrogen-containing macrocyclic compound shown as a formula I or pharmaceutically acceptable salt thereof.

In the pharmaceutical composition, the substance X can be a therapeutically effective amount of substance X.

The pharmaceutical composition can be prepared by combining the compound of the application with a suitable pharmaceutical adjuvant, and can be prepared into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powder, granules, paste, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres, aerosols and the like.

Typical routes of administration of a compound of the present application or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration.

The invention also provides an application of the substance X in preparing the EGFR inhibitor; the EGFR inhibitor is used in vitro; the substance X is a nitrogen-containing macrocyclic compound shown as a formula I, a pharmaceutically acceptable salt thereof or the pharmaceutical composition.

Further, the EGFR is a wild-type EGFR or an EGFR mutant.

Further, the EGFR mutant may have a T mutation at position 790 to M and/or a L mutation at position 858 to T.

The invention also provides the application of the substance X in preparing the medicine for treating and/or preventing the diseases related to the EGFR; the substance X is a nitrogen-containing heterocyclic compound shown as a formula I or pharmaceutically acceptable salt thereof.

Further, the EGFR is a wild-type EGFR or an EGFR mutant.

Further, the EGFR mutant may have a T mutation at position 790 to M and/or a L mutation at position 858 to T.

Preferably, in the application, the disease related to the EGFR inhibitor and the mutant thereof can be lung cancer; more preferably, the lung cancer is non-small cell lung cancer.

The invention also provides the application of the substance X in preparing the medicine for treating and/or preventing cancer-related diseases; the substance X is a nitrogen-containing heterocyclic compound shown as a formula I or pharmaceutically acceptable salt thereof.

Preferably, in said use, said cancer-related disease may be lung cancer; and can also be non-small cell lung cancer induced by EGFR mutation.

In the invention, the mutant is obtained by mutating the amino acid residue at a certain position of the wild EGFR into other amino acid residues.

The above preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention without departing from the common general knowledge in the art.

The reagents and starting materials used in the present invention are commercially available.

The term "treating" means administering a compound or formulation described herein to prevent, ameliorate or eliminate a disease or one or more symptoms associated with the disease, and includes:

(i) preventing the occurrence of a disease or condition in a mammal, particularly when such mammal is susceptible to the disease condition, but has not yet been diagnosed as having the disease condition;

(ii) inhibiting the disease or disease state, i.e., arresting its development;

(iii) alleviating the disease or condition, i.e., causing regression of the disease or condition.

The term "plurality" means 2, 3, 4 or 5.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "therapeutically effective amount" means an amount of a compound of the present application that (i) treats or prevents a particular disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of a compound of the present application that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by those skilled in the art with their own knowledge and this disclosure.

The term "pharmaceutically acceptable" is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. As the pharmaceutically acceptable salt, for example, a metal salt, an ammonium salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid, and the like can be mentioned.

The organic acid may be any of various organic acids capable of forming a salt, which are conventional in the art, and is preferably one or more of methanesulfonic acid, trifluoromethanesulfonic acid, phenylmethanesulfonic acid, p-toluenesulfonic acid, maleic acid, fumaric acid, succinic acid, citric acid, tartaric acid, malic acid, lactic acid, formic acid, acetic acid, propionic acid, trifluoroacetic acid, oxalic acid, succinic acid, benzoic acid, phenylacetic acid, isethionic acid, 1-naphthalenesulfonic acid, 2-naphthalenesulfonic acid, mandelic acid, and salicylic acid. The inorganic acid may be any of various inorganic acids capable of forming a salt, which are conventional in the art, and preferably one or more of hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid. The organic base can be various organic bases which are conventional in the field and can form salts, and one or more of pyridine, imidazole, pyrazine, indole, purine, tertiary amine and aniline is/are preferable. The tertiary amine organic base is preferably triethylamine and/or N, N-diisopropylethylamine. The aniline organic base is preferably N, N-dimethylaniline. The pyridine organic base is preferably one or more of pyridine, picoline, 4-dimethylamino pyridine and 2-methyl-5-ethyl pyridine. The inorganic base may be any of various inorganic bases capable of forming a salt, which are conventional in the art, and preferably one or more of alkali metal hydride, alkali metal hydroxide, alkali metal alkoxide, potassium carbonate, sodium carbonate, lithium carbonate, cesium carbonate, potassium hydrogen carbonate and sodium hydrogen carbonate. The alkali metal hydride is preferably sodium hydride and/or potassium hydride. The alkali metal hydroxide is preferably one or more of sodium hydroxide, potassium hydroxide and lithium hydroxide. The alkoxide of the alkali metal is preferably one or more of sodium methoxide, sodium ethoxide, potassium tert-butoxide and sodium tert-butoxide.

The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present application or salts thereof and pharmaceutically acceptable excipients. The purpose of the pharmaceutical composition is to facilitate administration of the compounds of the present application to an organism.

The term "pharmaceutically acceptable adjuvants" refers to those adjuvants which do not have a significant irritating effect on the organism and do not impair the biological activity and properties of the active compound. Suitable adjuvants are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like.

The words "comprise" or "comprise" and variations thereof such as "comprises" or "comprising," are to be understood in an open, non-exclusive sense, i.e., "including but not limited to.

The compounds and intermediates of the present application may also exist in different tautomeric forms, and all such forms are included within the scope of the present application. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can interconvert via a low energy barrier. For example, proton tautomers (also referred to as proton transfer tautomers) include interconversion via proton migration, such as keto-enol and imine-enamine isomerizations. A specific example of a proton tautomer is an imidazole moiety, wherein the proton can migrate between two ring nitrogens. Valence tautomers include interconversion by recombination of some of the bonding electrons.

The pharmaceutical compositions of the present application can be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, lyophilizing, and the like.

In some embodiments, the pharmaceutical composition is in an oral form. For oral administration, the pharmaceutical compositions may be formulated by mixing the active compounds with pharmaceutically acceptable excipients well known in the art. These adjuvants enable the compounds of the present application to be formulated as tablets, pills, lozenges, dragees, capsules, liquids, gels, slurries, suspensions and the like, for oral administration to a patient.

Solid oral compositions may be prepared by conventional mixing, filling or tableting methods. For example, it can be obtained by the following method: the active compounds are mixed with solid adjuvants, optionally the mixture obtained is milled, if desired with further suitable adjuvants, and the mixture is then processed to granules, to give tablets or dragee cores. Suitable excipients include, but are not limited to: binders, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents, and the like.

The pharmaceutical compositions may also be adapted for parenteral administration, as sterile solutions, suspensions or lyophilized products in suitable unit dosage forms.

Therapeutic dosages of the compounds of the present application may be determined, for example, by: the particular use of the treatment, the mode of administration of the compound, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of the compound of the present application in the pharmaceutical composition may not be fixed, depending on a variety of factors including dosage, chemical properties (e.g., hydrophobicity), and the route of administration. For example, the compounds of the present application can be provided for parenteral administration by a physiological buffered aqueous solution containing about 0.1-10% w/v of the compound. Some typical dosage ranges are from about 1. mu.g/kg to about 1g/kg body weight/day. In certain embodiments, the dosage range is from about 0.01mg/kg to about 100mg/kg body weight/day. The dosage will likely depend on such variables as the type and extent of progression of the disease or disorder, the general health status of the particular patient, the relative biological efficacy of the selected compound, the excipient formulation and its route of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

The compounds of the present application may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other chemical synthetic methods, and equivalents thereof known to those skilled in the art, with preferred embodiments including, but not limited to, the examples of the present application.

The positive progress effects of the invention are as follows:

in order to solve the problem of drug resistance caused by EGFR-T790M mutation, the applicant designs and synthesizes a series of macrocyclic derivatives through a conformation restriction research strategy, wherein the macrocyclic derivatives have high inhibitory activity to EGFR-T790M mutation and certain inhibitory activity to wild-type EGFR. In addition, the compounds have better pharmacological effect.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Some experimental material sources in the examples of the present invention are shown in Table 1, and the raw materials are not described as being commercially available.

TABLE 1

EXAMPLE 1 Synthesis of Compound A1

Synthetic route

1) Indole (5.00g,42.7mmol) was dissolved in DMF (90mL) at 0 deg.C, 60% NaH (2.05g,51.2mmol) was added and stirred for 30 minutes to form a clear solution, 6-bromo-1-hexene (7.65g,46.9mmol) was added dropwise, followed by stirring at room temperature for 5 hours, quenching with water (50mL), extraction with methyl tert-butyl ether (90mL), collection of the organic phase, drying of the organic phase over anhydrous sodium sulfate, filtration, spin drying to give the crude product. The crude product was purified by column chromatography (PE/EA ═ 20/1) to give intermediate a1-1 (light yellow oil; 8.37 g; yield: 98%; MS (ES +): 200.1).

2) Ethylene glycol dimethyl ether (250mL) and 2, 4-dichloropyrimidine (7.48g,50.2mmol) were sequentially added to a reaction flask, the temperature was raised to 60 ℃, ferric trichloride (8.14g,50.2mmol) was added in portions, the mixture was reacted at 60 ℃ for 3.5 hours, A1-1(10.0g,50.2mmol) was added, and the reaction was continued for 6 hours. After the completion of the TLC monitoring reaction, the reaction mixture was naturally cooled to room temperature, methanol and water (methanol/water: 1/2.7, 250mL) were added, and the mixture was stirred for 3 hours to precipitate a solid, which was then filtered and dried to obtain intermediate A1-2 (pale yellow solid; 6.10 g; yield: 38%; MS (ES +): 312.1).

3) Anhydrous acetonitrile (325mL), KF (6.60g,113.1mmol) and 3-nitroaniline (15.0g,108.6mmol) were added sequentially to the reaction flask, the temperature was raised to 80 ℃ and then 3-bromopropylene (10.9g,90.5mmol) was added dropwise, and the reaction was refluxed for 3 hours under nitrogen protection. After the reaction was monitored by TLC, it was cooled to room temperature and filtered to remove insoluble solids, washed with dichloromethane (150mL), and the filtrate was collected and spin dried to give the crude product. The crude product was purified by column chromatography (PE/EA ═ 10/1) to give intermediate a1-3 (orange solid; 8.51 g; yield: 52%; MS (ES +): 179.1).

4) A1-3(0.50g,2.80mmol) was added to a mixed DCM/methanol (1/1, 30mL), cooled to 0 deg.C and SnCl was added₂.2H₂O (6.35g,28.0mmol), and the reaction solution was gradually raised to room temperature and stirred at room temperature for 7 hours. After TLC monitoring of the reaction, the solvent of the reaction solution was removed under reduced pressure, and the residue was taken up in a mixed solution of dichloromethane and saturated sodium carbonate (1/1, 60mL), filtered, the organic phase was separated, the aqueous layer was extracted with DCM (100mL), the organic phases were collected and combined, dried over anhydrous sodium sulfate, filtered, and dried by spin-drying to give intermediate A1-4 (dark brown liquid; 0.40 g; yield: 96%; MS (ES +): 149.1).

5) A1-2(1.09g,3.50mmol), A1-4(0.40g,2.70mmol), p-toluenesulfonic acid monohydrate (0.49g,2.60mmol) and a1, 4-dioxane solution (15mL) were added to a reaction flask in this order under nitrogen protection, and the mixture was heated to 85 ℃ for 8 hours. After TLC monitoring reaction, the reaction solution was cooled to room temperature, mixed solution of ammonia water/ice water (1/4, 15mL) was added, stirred for crystallization for 4 hours, filtered, the filter cake was washed three times with dioxane/water (3/1, 20mL), and the filter cake was dried to give intermediate A1-5 (yellow solid; 0.55 g; yield: 36%; MS (ES +): 424.2).

6) Anhydrous dichloromethane (500mL), A1-5(0.50g,1.30mmol) and Grubbs 2 were combined in that order^ndcatalyst (0.12g,0.13mmol) was added to the reaction flask and the pH was adjusted to around 2 using HCl-dioxane. The reaction temperature was raised to 45 ℃ and reacted overnight. After the completion of the TLC monitoring reaction, the solvent of the reaction solution was removed under reduced pressure to give a crude product, which was purified by column chromatography (PE/EA ═ 4/1) to give intermediate a1-6 (pale green solid; 0.27 g; yield: 52%; MS (ES +): 396.2).

7) A1-6(0.10g,0.25mmol) was dissolved in methanol (10mL), wet 10% Pd/C (0.01g) was added, hydrogen was added for reaction for 10 hours, and after the reaction was monitored by HPLC, the crude product was obtained by filtration and spin-drying. The crude product was purified by column chromatography (PE/EA ═ 4/1) to give the title compound a1 (white solid; 0.07 g; yield: 69%).¹H-NMR(600MHz,CDCl₃,δppm):8.38(d,J＝5.2Hz,1H),8.07(s,1H),8.02(dd,J＝6.0,3.0Hz,1H),7.91(t,J＝2.4Hz,1H),7.43(dd,J＝6.6,2.9Hz,1H),7.32–7.27(m,2H),7.20(d,J＝5.4Hz,1H),7.16(s,1H),7.08(t,J＝7.8Hz,1H),6.34(dd,J＝8.0,2.0Hz,1H),6.30(dd,J＝7.8,1.2Hz,1H),4.26–4.18(m,2H),3.15(t,J＝6.6Hz,2H),2.01–1.94(m,2H),1.67(dd,J＝12.6,6.6Hz,2H),1.55–1.46(m,4H),1.38–1.32(m,2H).ES-API(m/z):[M+H]⁺:(m/z):[M+H]⁺:398.2.

Example 2 Synthesis of Compound A2

Synthetic route

1) Indole (11.7g,0.10mol) was dissolved in DMF (200mL) at 0 deg.C, 60% NaH (4.80g,0.12mol) was added and stirred for 30 minutes to form a clear solution, 5-bromo-1-pentene (16.4g,0.11mol) was added dropwise, followed by stirring at room temperature for 5 hours, quenching the reaction with water (100mL), extraction with methyl tert-butyl ether (200mL), the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and spun dried to give the crude product. The crude product was purified by column chromatography (PE/EA ═ 20/1) to give intermediate a2-1 (light yellow oil; 18.0 g; yield: 97%; MS (ES +): 186.1).

2) Ethylene glycol dimethyl ether (500mL) and 2, 4-dichloropyrimidine (16.1g,0.108mol) are sequentially put into a reaction bottle, heated to 60 ℃, ferric trichloride (17.5g,0.108mol) is added in batches, reaction is continued for 3.5 hours at 60 ℃, A2-1(20.0g.0.108mol) is added, and reaction is continued for 6 hours. After completion of the TLC monitoring reaction, the reaction mixture was allowed to cool to room temperature, methanol and water (methanol/water 1/2.7, 500mL) were added, and the mixture was stirred for 3 hours to precipitate a solid, which was then filtered and dried to obtain intermediate A2-2 (pale yellow solid; 17.3 g; yield: 53%; MS (ES +): 298.1).

3) Under the protection of nitrogen, adding A2-2(2.30g,7.80mmol), A1-4(0.97g,6.50mmol) (refer to step 3-4 of example 1), p-toluenesulfonic acid monohydrate (1.20g.6.50mmol) and 1, 4-dioxane (20mL) into a reaction bottle in sequence, heating to 85 ℃ for reaction for 8 hours, naturally cooling the reaction liquid to room temperature after TLC monitoring of the reaction is finished, adding a mixed solution of ammonia water/ice water (1/4, 20mL), stirring for crystallization for 4 hours, filtering, washing a filter cake with dioxane/water (3/1, 30mL) for three times, and drying the filter cake to obtain an intermediate A2-5 (light yellow solid; 1.40 g; yield: 53 percent; MS (ES +): 410.2).

4) Anhydrous dichloromethane (500mL), A2-5(0.50g,1.20mmol) and Grubbs 2 were combined in that order^ndThe catalst (0.11g,0.12mmol) was added to the reaction flask and the pH of the reaction was adjusted to about 2 using hydrochloric acid-dioxane. The reaction temperature was raised to 45 ℃ and reacted overnight. After TLC monitoring the reaction was completed, the solvent of the reaction solution was removed under reduced pressure to give crude product, which was purified by column chromatography (PE/EA ═ 4/1) to give intermediate a2-6 (light crude product)A green solid; 0.27 g; yield: 59 percent of water; MS (ES +): 382.2).

5) A2-6(0.11g,0.30mmol) was dissolved in methanol (10mL), wet 10% Pd/C (0.01g) was added, and the reaction was allowed to proceed with hydrogen gas for 10 hours. After the reaction is monitored by HPLC, the crude product is obtained by filtration and spin-drying. The crude product was purified by column chromatography (PE/EA ═ 4/1) to give the title compound a2 (white solid; 0.07 g; yield: 69;).¹H-NMR(400MHz,CDCl₃,δppm):8.37–8.33(m,1H),8.31(d,J＝2.4Hz,1H),8.11(d,J＝2.4Hz,1H),8.02–7.97(m,1H),7.44(dd,J＝6.8,2.4Hz,2H),7.33–7.27(m,2H),7.21(d,J＝5.2Hz,1H),7.06(t,J＝8.0Hz,1H),6.32(t,J＝8.0Hz,2H),4.31–4.23(m,2H),3.35–3.23(m,2H),1.99–1.92(m,2H),1.79–1.72(m,2H),1.67(dd,J＝10.0,4.4Hz,2H),1.43–1.38(m,2H).ES-API(m/z):[M+H]⁺:384.2.

EXAMPLE 3 Synthesis of Compound A3

Synthetic route

1) Indole (11.7g,0.10mol) was dissolved in DMF (200mL) at 0 deg.C, 60% NaH (4.80g,0.12mol) was added and stirred for 30 minutes to form a clear solution, 7-bromo-1-heptene (19.5g,0.11mol) was added dropwise, followed by stirring at room temperature for 5 hours, the reaction was quenched with water (100mL), extracted with methyl tert-butyl ether (200mL), the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and spun dry to give the crude product. The crude product was purified by column chromatography (PE/EA ═ 20/1) to give intermediate A3-1 (light yellow oil; 21.3 g; yield: 99%; MS (ES +): 214.1).

2) Ethylene glycol dimethyl ether (500mL) and 2, 4-dichloropyrimidine (14.9g,0.10mol) are sequentially put into a reaction bottle, heated to 60 ℃, ferric trichloride (16.3g,0.10mol) is added in batches to react for 3.5 hours at 60 ℃, A3-1(21.3g,0.10mol) is added to continue the reaction for 6 hours. After completion of the TLC monitoring reaction, the reaction mixture was naturally cooled to room temperature, methanol and water (methanol/water: 1/2.7, 500mL) were added, and the mixture was stirred for 3 hours to precipitate a solid, which was filtered and dried to obtain intermediate A3-2 (pale yellow solid; 15.0 g; yield: 46%; MS (ES +): 326.1).

3) A3-2(2.30g,7.30mmol), A1-4(0.90g,6.10mmol) (see example 1, steps 3-4), p-toluenesulfonic acid monohydrate (1.20g,6.10mmol) and 1, 4-dioxane (20mL) were added sequentially to a reaction flask under nitrogen protection and heated to 85 ℃ for 8 hours. After TLC monitoring of the reaction, the reaction mixture was allowed to cool to room temperature, a mixed solution of ammonia water/ice water (1/4, 20mL) was added, crystallization was performed for four hours with stirring, filtration was performed, the filter cake was washed three times with dioxane/water (3/1, 30mL), and the filter cake was dried to give intermediate A3-5 (pale yellow solid; 1.51 g; yield: 57%; MS (ES +): 438.2).

4) Anhydrous dichloromethane (500mL), A3-5(0.48g,1.10mmol) and Grubbs 2 were combined in this order^ndcatalyst (0.11g,0.11mmol) was added to the reaction flask and the pH was adjusted to around 2 using HCl-dioxane. The reaction temperature was raised to 45 ℃ and reacted overnight. After the completion of the TLC monitoring reaction, the solvent of the reaction solution was removed under reduced pressure to give a crude product, which was purified by column chromatography (PE/EA ═ 4/1) to give intermediate A3-6 (pale green solid; 0.37 g; yield: 79%; MS (ES +): 410.2).

5) A3-6(0.16g,0.40mmol) was dissolved in methanol (10mL), wet 10% Pd/C (0.02g) was added, and the reaction was allowed to proceed with hydrogen gas for 10 hours. After the reaction is monitored by HPLC, the crude product is obtained by filtration and spin-drying. The crude product was purified by column chromatography (PE/EA ═ 4/1) to give the title compound A3 (white solid; 0.11 g; yield: 67%).¹H-NMR(400MHz,CDCl₃,δppm):8.37(d,J＝5.2Hz,1H),8.05(s,1H),8.04–7.99(m,1H),7.82(t,J＝2.0Hz,1H),7.42(dd,J＝6.8,2.0Hz,1H),7.34–7.27(m,2H),7.20(d,J＝5.2Hz,1H),7.10(d,J＝8.0Hz,1H),7.07(d,J＝2.8Hz,1H),6.31(td,J＝8.0,2.0Hz,2H),4.19(t,J＝6.8Hz,2H),3.20(t,J＝6.8Hz,2H),1.95(t,J＝6.8Hz,2H),1.74–1.67(m,2H),1.60(s,4H),1.50(dd,J＝12.8,6.4Hz,2H),1.41(s,2H).ES-API(m/z):[M+H]⁺:412.2.

Example 4 Synthesis of Compound A4

Synthetic route

1) Indole (11.7g,0.10mol) was dissolved in DMF (200mL) at 0 deg.C, 60% NaH (4.80g,0.12mol) was added and stirred for 30 minutes to form a clear solution, 8-bromo-1-octene (21.0g,0.11mol) was added dropwise, followed by stirring at room temperature for 5 hours, quenching the reaction with water (100mL), extraction with methyl tert-butyl ether (200mL), the organic phase was collected, dried over anhydrous sodium sulfate, filtered and spun dried to give the crude product. The crude product was purified by column chromatography (PE/EA ═ 20/1) to give intermediate a4-1 (light yellow oil; 22.1 g; yield: 97%; MS (ES +): 228.1).

2) Ethylene glycol dimethyl ether (500mL) and 2, 4-dichloropyrimidine (14.9g,0.10mol) are sequentially put into a reaction bottle, heated to 60 ℃, ferric trichloride (16.3g,0.10mol) is added in batches to react for 3.5 hours at 60 ℃, A4-1(22.7g,0.10mol) is added to continue the reaction for 6 hours. After completion of the TLC monitoring reaction, the reaction mixture was naturally cooled to room temperature, methanol and water (methanol/water: 1/2.7, 500mL) were added, and the mixture was stirred for 3 hours to precipitate a solid, which was filtered and dried to obtain intermediate A4-2 (pale yellow solid; 15.4 g; yield: 45%; MS (ES +): 340.1).

3) A4-2(1.40g,4.10mmol), A1-4(0.50g,3.40mmol) (see example 1, step 3-4), p-toluenesulfonic acid monohydrate (0.65g,3.40mmol) and 1, 4-dioxane (20mL) were added sequentially to a reaction flask under nitrogen protection and heated to 85 ℃ for 8 hours. After TLC monitoring reaction, the reaction solution was cooled to room temperature, mixed solution of ammonia water/ice water (1/4, 20mL) was added, crystallization was performed for four hours with stirring, filtration was performed, the filter cake was washed three times with dioxane/water (3/1, 30mL), and the filter cake was dried to give intermediate A4-5 (pale yellow solid; 1.50 g; yield: 97%; MS (ES +): 452.2).

4) Anhydrous dichloromethane (500mL), A4-5(0.60g,1.30mmol) andGrubbs 2^ndcatalyst (0.12g,0.13mmol) was added to the reaction flask and the pH was adjusted to around 2 using HCl-dioxane. The reaction temperature was raised to 45 ℃ and reacted overnight. After the completion of the TLC monitoring reaction, the solvent of the reaction solution was removed under reduced pressure to give a crude product, which was purified by column chromatography (PE/EA ═ 4/1) to give intermediate a4-6 (pale green solid; 0.25 g; yield: 45%; MS (ES +): 424.2).

5) A4-6(0.13g,0.30mmol) was dissolved in methanol (10mL), wet 10% Pd/C (0.01g) was added, and the reaction was allowed to proceed with hydrogen gas for 10 hours. After the HPLC monitoring reaction is finished, filtering and spin-drying to obtain a crude product. The crude product was purified by column chromatography (PE/EA ═ 4/1) to give the title compound a4 (white solid; 0.10 g; yield: 76%).¹H-NMR(400MHz,CDCl₃,δppm):8.35(d,J＝5.2Hz,1H),8.27(d,J＝7.6Hz,1H),7.94(t,J＝2.0Hz,1H),7.84(s,1H),7.42(d,J＝7.6Hz,1H),7.35–7.27(m,2H),7.25–7.20(m,1H),7.08(dd,J＝9.2,6.8Hz,2H),6.29(dd,J＝7.6,2.0Hz,2H),4.21(t,J＝6.8Hz,2H),2.92(t,J＝7.2Hz,2H),1.92(dd,J＝13.2,6.8Hz,2H),1.49(p,J＝6.8Hz,2H),1.31–1.25(m,2H),1.21(dd,J＝11.2,3.6Hz,4H),1.09(dd,J＝14.8,7.2Hz,2H),0.92(dd,J＝14.4,7.2Hz,2H)ES-API(m/z):[M+H]⁺:426.2.

EXAMPLE 5 Synthesis of Compound A5

Synthetic route

1) Anhydrous acetonitrile (400mL), KF (8.65g,0.149mol) and 3-nitro-4-methoxyaniline (20.0g,0.119mol) were added to the reaction flask in sequence, warmed to 82 deg.C, then 3-bromopropene (14.3g,0.119mol) was added dropwise and reacted under reflux under nitrogen for 3 hours. After TLC monitoring reaction, the reaction liquid is cooled to room temperature and filtered to remove insoluble solid, and then the insoluble solid is washed by dichloromethane, and the filtrate is collected and spin-dried to obtain a crude product. The crude product was purified by column chromatography (PE/EA ═ 10/1) to give intermediate a5-3 (dark red liquid; 11.0 g; yield: 44%; MS (ES +): 209.1).

2) A5-3(2.08g,0.10mol) was added to a DCM/MeOH (1/1, 90mL) mixed solution, cooled to 0 deg.C and SnCl was added₂.2H₂O (22.6g,0.10mol), the reaction mixture was gradually warmed to room temperature and stirred for 7 hours. After completion of the TLC monitoring reaction, the solvent in the reaction mixture was removed under reduced pressure, and the residue was taken up in a mixed solution of methylene chloride and saturated sodium carbonate (1/1, 90mL), filtered, the organic phase was separated, the aqueous layer was extracted with DCM (50mL), the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and dried by spin-drying to give intermediate A5-4 (dark brown liquid; 1.43 g; yield: 80%; MS (ES +): 179.1).

3) A1-2(3.00g,9.60mmol) (see step 1-2 of example 1), A5-4(1.43g,8.0mmol), p-toluenesulfonic acid monohydrate (1.50g,8.0mmol) and 1, 4-dioxane (40mL) were added in this order to a reaction flask under nitrogen protection and reacted at 85 ℃ for 8 hours. After TLC monitoring reaction, the reaction solution was cooled to room temperature naturally, mixed solution of ammonia water/ice water (1/4, 40mL) was added, crystallization was performed for four hours with stirring, filtration was performed, the filter cake was washed three times with dioxane/water (3/1, 60mL), and the filter cake was dried to obtain intermediate A5-5 (pale yellow solid; 1.73 g; yield: 48%; MS (ES +): 454.2).

4) Anhydrous dichloromethane (500mL), A5-5(0.60g,1.30mmol) and Grubbs 2 were combined in that order^ndcatalyst (0.11g,0.13mmol) was added to the reaction flask and the pH was adjusted to around 2 using HCl-dioxane. The reaction temperature was raised to 45 ℃ and left to react overnight. After the completion of the TLC monitoring reaction, the solvent of the reaction solution was removed under reduced pressure to give a crude product, which was purified by column chromatography (PE/EA ═ 4/1) to give intermediate a5-6 (pale green solid; 0.18 g; yield: 32%; MS (ES +): 426.2).

5) A5-6(0.13g,0.30mmol) was dissolved in methanol (10mL), wet 10% Pd/C (0.02g) was added, and the reaction was allowed to proceed with hydrogen gas for 10 hours. After the reaction is monitored by HPLC, the crude product is obtained by filtration and spin-drying. The crude product was purified by column chromatography (PE/EA ═ 4/1) to give the title compound a5 (white solid; 0.09 g; yield: 68%).¹H-NMR(400MHz,CDCl₃,δppm):8.39(d,J＝5.2Hz,1H),8.20(d,J＝2.8Hz,1H),8.08(s,1H),8.02(dd,J＝5.6,3.2Hz,1H),7.66(s,1H),7.44(dd,J＝6.0,3.2Hz,1H),7.32–7.27(m,2H),7.20(d,J＝5.2Hz,1H),6.78(d,J＝8.6Hz,1H),6.28(dd,J＝8.6,2.8Hz,1H),4.30–4.15(m,2H),3.85(s,3H),3.14(t,J＝6.8Hz,2H),1.98(dd,J＝12.4,6.2Hz,2H),1.68(d,J＝5.8Hz,2H),1.51(dd,J＝9.6,6.8Hz,4H),1.42–1.36(m,2H).ES-API(m/z):[M+H]⁺:428.2.

EXAMPLE 6 Synthesis of Compound A6

Synthetic route

1) A3-3(2.60g,8.10mmol) (see step 1-2 of example 3), A5-4(1.20g,6.70mmol) (see step 1-2 of example 5), p-toluenesulfonic acid monohydrate (1.30g,6.70mmol) and 1, 4-dioxane (40mL) were added sequentially to a reaction flask under nitrogen atmosphere and reacted at 85 ℃ for 8 hours. After TLC monitoring of the reaction, the reaction mixture was allowed to cool to room temperature, a mixed solution of ammonia/ice water (1/4, 40mL) was added, the mixture was stirred for four hours for crystallization, filtered, washed three times with dioxane/water (3/1, 60mL), and the filter cake was dried to give intermediate A6-5 (white solid; 1.23 g; yield: 39%; MS (ES +): 468.2).

2) Anhydrous dichloromethane (500mL), A6-5(0.60g,1.30mmol) and Grubbs 2 were combined in this order^ndcatalyst (0.11g,0.13mmol) was added to the reaction flask and the pH was adjusted to around 2 using HCl-dioxane. The reaction temperature was raised to 45 ℃ and reacted overnight. After the completion of the TLC monitoring reaction, the solvent of the reaction solution was removed under reduced pressure to give a crude product, which was purified by column chromatography (PE/EA ═ 4/1) to give intermediate a6-6 (pale green solid; 0.30 g; yield: 52%; MS (ES +): 440.2).

3) A6-6(0.13g,0.30mmol) was dissolved in methanol (10mL), wet 10% Pd/C (0.02g) was added, and the reaction was allowed to proceed with hydrogen gas for 10 hours. After the reaction is monitored by HPLC, the crude product is obtained by filtration and spin-drying. CoarseThe product was purified by column chromatography (PE/EA: 4/1) to give the objective compound A6 (white solid; 0.12 g; yield: 90%).¹H-NMR(400MHz,CDCl₃,δppm):8.39(d,J＝5.2Hz,1H),8.10(d,J＝2.8Hz,1H),8.08(s,1H),8.03(d,J＝7.2Hz,1H),7.71(s,1H),7.43(d,J＝7.6Hz,1H),7.31–7.27(m,2H),7.21(d,J＝5.2Hz,1H),6.79–6.76(m,1H),6.25(dd,J＝8.4,2.8Hz,1H),4.20(t,J＝7.2Hz,2H),3.84(s,3H),3.17(t,J＝6.4Hz,2H),1.98–1.94(m,2H),1.73–1.68(m,2H),1.54–1.50(m,2H),1.46(d,J＝6.4Hz,4H),1.36(d,J＝22.8Hz,2H).ES-API(m/z):[M+H]+:442.2.

EXAMPLE 7 Synthesis of Compound A10

Synthetic route

1) Anhydrous acetonitrile (500mL), KF (9.70g,0.167mol) and 3-nitro-4-fluoroaniline (25.0g,0.16mol) were added to the reaction flask in this order, warmed to 82 ℃ and then 3-bromopropene (16.0g,0.133mol) was added dropwise and reacted under reflux for 3 hours under nitrogen. After TLC monitoring reaction, the reaction liquid is cooled to room temperature and filtered to remove insoluble solid, and then the insoluble solid is washed by dichloromethane, and the filtrate is collected and spin-dried to obtain a crude product. The crude product was purified by column chromatography (PE/EA ═ 10/1) to give intermediate a10-3 (dark red liquid; 10.2 g; yield: 39%; MS (ES +): 197.1).

2) A10-3(1.70g,8.67mmol) was added to a mixed DCM/methanol (1/1, 90mL), cooled to 0 deg.C and SnCl was added₂.2H₂O (22.6g,0.10mol), the reaction mixture was gradually warmed to room temperature and stirred for 7 hours. After TLC monitoring of the reaction, the solvent was removed from the reaction mixture under reduced pressure, the residue was taken up in a mixed solution of dichloromethane and saturated sodium carbonate (1/1, 90mL), filtered, the organic phase was separated, the aqueous layer was extracted with DCM (50mL), the organic phase was collected, dried over anhydrous sodium sulfate, filtered and spun-dried to give intermediate A10-4 (dark brown liquid; 1.38 g; yield: 96%; MS (ES +): 167.1).

3) A1-2(3.50g,11.4mmol) (see step 1-2 of example 1), A10-4(1.38g,8.30mmol), p-toluenesulfonic acid monohydrate (1.80g,9.50mmol) and 1, 4-dioxane (40mL) were added in this order to a reaction flask under nitrogen protection and reacted at 85 ℃ for 8 hours. After TLC monitoring of the reaction, the reaction mixture was allowed to cool to room temperature, a mixed solution of ammonia water/ice water (1/4, 40mL) was added, crystallization was performed for four hours with stirring, filtration was performed, the filter cake was washed three times with dioxane/water (3/1, 60mL), and the filter cake was dried to give intermediate A10-5 (pale yellow solid; 1.56 g; yield: 43%; MS (ES +): 442.2).

4) Anhydrous dichloromethane (500mL), A10-5(0.53g,1.20mmol) and Grubbs 2 were combined in that order^ndcatalyst (0.11g,0.12mmol) was added to the reaction flask and the pH was adjusted to around 2 using HCl-dioxane. The reaction temperature was raised to 45 ℃ and reacted overnight. After the completion of the TLC monitoring reaction, the solvent of the reaction solution was removed under reduced pressure to give a crude product, which was purified by column chromatography (PE/EA ═ 4/1) to give intermediate a10-6 (pale green solid; 0.23 g; yield: 46%; MS (ES +): 414.2).

5) A10-6(0.20g,0.48mmol) was dissolved in methanol (12mL), wet 10% Pd/C (0.03g) was added, and the reaction was allowed to proceed with hydrogen gas for 10 hours. After the reaction is monitored by HPLC, the crude product is obtained by filtration and spin-drying. The crude product was purified by column chromatography (PE/EA ═ 4/1) to give the title compound a10 (white solid; 0.18 g; yield: 90%).¹H-NMR(400MHz,CDCl₃,δppm):8.40(d,J＝5.2Hz,1H),8.20(d,J＝2.8Hz,1H),8.08(s,1H),8.04(dd,J＝5.6,3.2Hz,1H),7.64(s,1H),7.44(dd,J＝6.0,3.2Hz,1H),7.32–7.28(m,2H),7.20(d,J＝5.2Hz,1H),6.78(d,J＝8.6Hz,1H),6.28(dd,J＝8.6,2.8Hz,1H),4.30–4.15(m,2H),3.14(t,J＝6.8Hz,2H),1.98(dd,J＝12.4,6.2Hz,2H),1.68(d,J＝5.8Hz,2H),1.51(dd,J＝9.6,6.8Hz,4H),1.42–1.36(m,2H).ES-API(m/z):[M+H]⁺:416.2.

EXAMPLE 8 Synthesis of Compound A13

Synthetic route

1) Anhydrous acetonitrile (350mL), KF (3.90g,0.067mol) and 3-nitro-4-trifluoromethoxy aniline (14.4g,0.065mol) were added to a reaction flask in this order, warmed to 82 ℃, and then 3-bromopropylene (6.53g, 0.054mol) was added dropwise, and the reaction was refluxed for 3 hours under nitrogen protection. After the reaction was monitored by TLC, the reaction solution was cooled to room temperature, filtered to remove insoluble solids, washed with dichloromethane, and the filtrate was collected and spin-dried to give a crude product. The crude product was purified by column chromatography (PE/EA ═ 10/1) to give intermediate a13-3 (dark red liquid; 8.0 g; yield: 56%; MS (ES +): 263.1).

2) A13-3(2.50g,9.50mmol) was added to a mixed DCM/methanol (1/1, 90mL), cooled to 0 deg.C and SnCl was added₂.2H₂O (22.6g,0.10mol), the reaction mixture was gradually warmed to room temperature and stirred for 7 hours. After completion of the TLC monitoring reaction, the solvent in the reaction mixture was removed under reduced pressure, and the residue was taken up in a mixed solution of methylene chloride and saturated sodium carbonate (1/1, 90mL), filtered, the organic phase was separated, the aqueous layer was extracted with DCM (50mL), the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and dried by spin-drying to give intermediate A13-4 (dark brown liquid; 1.80 g; yield: 81%; MS (ES +): 233.1).

3) A1-2(3.40g,10.9mmol) (see step 1-2 of example 1), A13-4(2.20g,9.50mmol), p-toluenesulfonic acid monohydrate (1.80g,9.50mmol) and 1, 4-dioxane (30mL) were added in this order to a reaction flask under nitrogen protection and reacted at 85 ℃ for 8 hours. After TLC monitoring reaction, the reaction solution was cooled to room temperature naturally, mixed solution of ammonia water/ice water (1/4, 40mL) was added, crystallization was performed for four hours with stirring, filtration was performed, the filter cake was washed three times with dioxane/water (3/1, 60mL), and the filter cake was dried to obtain intermediate A13-5 (pale yellow solid; 1.73 g; yield: 36%; MS (ES +): 508.2).

4) Anhydrous dichloromethane (500mL), A13-5(0.61g,1.20mmol) and Grubbs 2 were combined in that order^ndcatalyst (0.11g,0.12mmol) was added to the reaction flask,the pH was adjusted to around 2 using hydrochloric acid-dioxane. The reaction temperature was raised to 45 ℃ and left to react overnight. After the completion of the TLC monitoring reaction, the solvent of the reaction solution was removed under reduced pressure to give a crude product, which was purified by column chromatography (PE/EA: 4/1) to give intermediate A13-6 (pale green solid; 0.18 g; yield: 31%; MS (ES +): 480.2).

5) A13-6(0.14g,0.30mmol) was dissolved in methanol (10mL), wet 10% Pd/C (0.02g) was added, and the reaction was allowed to proceed with hydrogen gas for 10 hours. After the reaction is monitored by HPLC, the crude product is obtained by filtration and spin-drying. The crude product was purified by column chromatography (PE/EA ═ 4/1) to give the title compound a13 (white solid; 0.09 g; yield: 64%).¹H-NMR(400MHz,CDCl₃,δppm):8.43(d,J＝5.2Hz,1H),8.23(d,J＝2.8Hz,1H),8.04(s,1H),7.84(dd,J＝5.6,3.2Hz,1H),7.64(s,1H),7.43(dd,J＝6.0,3.2Hz,1H),7.30–7.25(m,2H),7.20(d,J＝5.2Hz,1H),6.64(d,J＝8.6Hz,1H),6.28(dd,J＝8.6,2.8Hz,1H),4.30–4.15(m,2H),3.85(s,3H),3.18(t,J＝6.8Hz,2H),1.98(dd,J＝12.4,6.2Hz,2H),1.68(d,J＝5.8Hz,2H),1.59(dd,J＝9.6,6.8Hz,4H),1.48–1.36(m,2H).ES-API(m/z):[M+H]⁺:482.2.

Example 9 Synthesis of Compound A15

Synthetic route

1) Anhydrous acetonitrile (150mL), cesium carbonate (41.7g,0.128mol) and 2-fluoro-5-nitroaniline (10.0g,0.064mol), 3-fluoropropanol (5.0g,0.064mol) were added to a reaction flask in this order, the temperature was raised to 82 ℃ and the reaction was carried out under nitrogen protection for 5 hours, TLC monitored for the completion of the reaction, the reaction solution was cooled to room temperature, then filtered through celite to remove insoluble solids, washed with water (50mL), extracted with EA (150mL), the organic phases were combined and purified by column chromatography (PE/EA ═ 10/1) to give intermediate a15-2 (brown solid; 3.60 g; yield: 22 percent; MS (ES +): 215.1).

2) Anhydrous acetonitrile (450mL), KF (5.0g,0.087mol) and A15-2(18.0g,0.084mol) were added to the reaction flask in this order, warmed to 82 deg.C, then 3-bromopropene (8.50g,0.07mol) was added dropwise and the reaction refluxed for 3 hours under nitrogen. After TLC monitoring reaction, the reaction liquid is cooled to room temperature and filtered to remove insoluble solid, and then the insoluble solid is washed by dichloromethane, and the filtrate is collected and spin-dried to obtain a crude product. The crude product was purified by column chromatography (PE/EA ═ 10/1) to give intermediate a15-3 (black liquid; 7.0 g; yield: 39%; MS (ES +): 255.1).

3) A15-3(2.0g,7.80mmol) was added to a mixed DCM/methanol (1/1, 90mL), cooled to 0 deg.C and SnCl was added₂.2H₂O (22.6g,0.10mol), the reaction mixture was gradually warmed to room temperature and stirred for 7 hours. After completion of the TLC monitoring reaction, the solvent in the reaction solution was removed under reduced pressure, and the residue was taken up in a mixed solution of methylene chloride and saturated sodium carbonate (1/1, 90mL), filtered, the organic phase was separated, the aqueous layer was extracted with DCM (50mL), the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and dried by spin-drying to give intermediate A15-4 (dark brown liquid; 1.11 g; yield: 63%; MS (ES +): 225.1).

4) A1-2(1.87g,6.0mmol) (see step 1-2 of example 1), A15-4(1.12g,5.0mmol), p-toluenesulfonic acid monohydrate (0.95g,5.0mmol) and 1, 4-dioxane (20mL) were added in this order to a reaction flask under nitrogen protection and reacted at 85 ℃ for 8 hours. After TLC monitoring of the reaction, the reaction mixture was allowed to cool to room temperature, a mixed solution of ammonia/ice water (1/4, 40mL) was added, crystallization was performed for four hours with stirring, filtration was performed, the filter cake was washed three times with dioxane/water (3/1, 60mL), and the filter cake was dried to give intermediate A15-5 (yellow solid; 0.8 g; yield: 32%; MS (ES +): 500.3).

5) Anhydrous dichloromethane (500mL), A15-5(0.50g,1.0mmol) and Grubbs 2 were combined in that order^ndcatalyst (0.11g,0.10mmol) was added to the reaction flask and the pH was adjusted to around 2 using HCl-dioxane. The reaction temperature was raised to 45 ℃ and left to react overnight. After the completion of the TLC monitoring reaction, the solvent of the reaction solution was removed under reduced pressure to give a crude product, which was purified by column chromatography (PE/EA ═ 4/1) to give intermediate a15-6 (pale green solid; 0.21 g; yield: 45%; MS (ES +): 472.3).

6) A15-6(0.21g,0.44mmol) was dissolved in methanol (10mL), wet 10% Pd/C (0.02g) was added, and the reaction was allowed to proceed with hydrogen gas for 10 hours. After the reaction is monitored by HPLC, the crude product is obtained by filtration and spin-drying. The crude product was purified by column chromatography (PE/EA: 4/1) to give the objective compound A15 (white solid; 0.10 g; yield: 48%).¹H NMR(400MHz,CDCl₃,δppm)：8.32(dd,J＝5.4,1.8Hz,1H),8.06–7.98(m,2H),7.71(d,J＝2.4Hz,1H),7.44–7.41(m,1H),7.34(s,1H),7.31–7.27(m,2H),7.19(dd,J＝5.4,2.8Hz,1H),6.73(dd,J＝8.4,3.6Hz,1H),6.28(dd,J＝8.4,2.4Hz,1H),4.77–4.59(m,2H),4.22(dd,J＝6.8,4.8Hz,2H),4.17–4.13(m,2H),3.13(t,J＝6.8Hz,2H),2.35–2.08(m,4H),1.94(dd,J＝11.8,6.0Hz,2H),1.69(dd,J＝12.8,6.4Hz,2H),1.56–1.43(m,4H).ES-API(m/z):[M+H]⁺:474.3.

EXAMPLE 10 Synthesis of Compound A20

Synthetic route

1) After 6-chloroindole (15.2g,0.10mol) was dissolved in DMF (200mL) at 0 deg.C, 60% NaH (4.80g,0.12mol) was added and stirred for 30 minutes to form a clear solution, 5-bromo-1-pentene (16.4g,0.11mol) was added dropwise and stirred at room temperature for 5 hours, the reaction was quenched with water (100mL), extracted with methyl tert-butyl ether (200mL), the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and spun dried to give the crude product. The crude product was purified by column chromatography (PE/EA ═ 20/1) to give intermediate a20-1 (light yellow oil; 23.0 g; yield: 97%; MS (ES +): 220.1).

2) Ethylene glycol dimethyl ether (500mL) and 2, 4-dichloropyrimidine (16.1g,0.108mol) are sequentially put into a reaction bottle, heated to 60 ℃, ferric trichloride (17.5g,0.108mol) is added in batches, the reaction is continued for 3.5 hours at 60 ℃, A20-1(23.6g,0.108mol) is added, and the reaction is continued for 6 hours. After completion of the TLC monitoring reaction, the reaction mixture was cooled to room temperature, methanol and water (methanol/water 1/2.7, 500mL) were added, and the mixture was stirred for 3 hours to precipitate a solid, which was then filtered and dried to obtain intermediate A20-2 (pale yellow solid; 15.4 g; yield: 42%; MS (ES +): 332.1).

3) Under the protection of nitrogen, adding A20-2(2.59g,7.80mmol), A10-4(1.08g,6.50mmol) (refer to step 3-4 of example 1), p-toluenesulfonic acid monohydrate (1.23g,6.50mmol) and 1, 4-dioxane (20mL) into a reaction bottle in sequence, heating to 85 ℃ for reaction for 8 hours, naturally cooling the reaction liquid to room temperature after TLC monitoring of the reaction is finished, adding a mixed solution of ammonia water/ice water (1/4, 20mL), stirring for crystallization for 4 hours, filtering, washing a filter cake with dioxane/water (3/1, 30mL) for three times, and drying the filter cake to obtain an intermediate A20-5 (light yellow solid; 0.70 g; yield: 20 percent; MS (ES +): 444.3).

4) Anhydrous dichloromethane (500mL), A20-5(0.54g,1.20mmol) and Grubbs 2 were combined in that order^ndThe catalst (0.11g,0.12mmol) was added to the reaction flask and the pH of the reaction was adjusted to about 2 using hydrochloric acid-dioxane. The reaction temperature was raised to 45 ℃ and reacted overnight. After the completion of the TLC monitoring reaction, the solvent of the reaction solution was removed under reduced pressure to give a crude product, which was purified by column chromatography (PE/EA ═ 4/1) to give intermediate a20-6 (pale green solid; 0.27 g; yield: 54%; MS (ES +): 416.2).

5) A20-6(0.10g,0.24mmol) was dissolved in methanol (10mL), wet 10% Pd/C (0.01g) was added, and the reaction was allowed to proceed with hydrogen gas for 10 hours. After the reaction is monitored by HPLC, the crude product is obtained by filtration and spin-drying. The crude product was purified by column chromatography (PE/EA ═ 4/1) to give the title compound a20 (white solid; 0.07 g; yield: 69%).¹H-NMR(400MHz,CDCl₃,δppm):8.30(d,J＝2.4Hz,1H),8.08(d,J＝2.4Hz,1H),8.02–7.97(m,1H),7.44(dd,J＝6.8,2.4Hz,2H),7.33–7.27(m,2H),7.21(d,J＝5.2Hz,1H),7.06(t,J＝8.0Hz,1H),6.32(t,J＝8.0Hz,2H),4.31–4.23(m,2H),3.35–3.23(m,2H),1.99–1.92(m,2H),1.79–1.72(m,2H),1.67(dd,J＝10.0,4.4Hz,2H),1.43–1.38(m,2H).ES-API(m/z):[M+H]⁺:418.2.

In the invention, the 6 compounds (such as A1-6, A2-6 and A3-6) in A1 to A20 are prepared to be a mixture of cis and trans.

EXAMPLE 11 Synthesis of Compound B1

Synthetic route

Under the protection of nitrogen, A1(120mg,0.30mmol) is dissolved in acetone (12mL), potassium carbonate (30mg,0.20mmol) is added, the mixture is cooled to 0 ℃, acryloyl chloride (30mg,0.30mmol) is added dropwise, after reaction for half an hour, water (15mL) and dichloromethane (15mL) are added into reaction liquid, organic phases are extracted and collected, and the organic phases are dried through anhydrous sodium sulfate, filtered and dried in a rotary mode to obtain crude products. The crude product was purified by column chromatography (PE/EA ═ 4/1) to give the target compound B1 (white solid; 50 mg; yield: 36%).

¹H-NMR(400MHz,CDCl₃,δppm):8.40–8.37(m,2H),7.98(dd,J＝6.4,2.4Hz,1H),7.89(s,1H),7.62(s,1H),7.42(dd,J＝6.8,2.4Hz,1H),7.35(d,J＝8.0Hz,1H),7.31–7.27(m,2H),6.92(dd,J＝8.0,1.2Hz,1H),6.87(dd,J＝8.0,1.2Hz,1H),6.31(dd,J＝16.8,2.0Hz,1H),6.06(dd,J＝16.8,10.4Hz,1H),5.46(dd,J＝10.4,2.0Hz,1H),4.27–4.23(m,2H),3.75(t,J＝6.8Hz,2H),1.88(dd,J＝10.0,4.4Hz,2H),1.67–1.61(m,2H),1.50(dd,J＝14.8,7.2Hz,2H),1.42–1.36(m,2H),1.25–1.19(m,2H).ES-API(m/z):[M+H]⁺:452.2.

EXAMPLE 12 Synthesis of Compound B2

Synthetic route

A2(115mg,0.30mmol) and acryloyl chloride (30mg,0.30mmol) were purified under nitrogen (see synthetic procedure in example 11) to give the title compound B2 (white solid; 40 mg; yield: 31%).

¹H-NMR(400MHz,CDCl₃,δppm):8.68(s,1H),8.40(d,J＝5.2Hz,1H),8.03(s,1H),7.99(dd,J＝6.4,2.4Hz,1H),7.67(s,1H),7.42(dd,J＝6.4,2.4Hz,1H),7.35(d,J＝7.6Hz,1H),7.28(d,J＝5.2Hz,2H),6.93(dd,J＝8.0,1.3Hz,1H),6.82(d,J＝7.6Hz,1H),6.40(dd,J＝16.8,2.0Hz,1H),6.13(dd,J＝16.8,10.0Hz,1H),5.53(dd,J＝10.0,1.6Hz,1H),4.35(d,J＝5.2Hz,2H),1.92(s,6H),1.60(d,J＝7.6Hz,4H).ES-API(m/z):[M+H]⁺:438.2.

EXAMPLE 13 Synthesis of Compound B3

Synthetic route

A3(123mg,0.30mmol) and acryloyl chloride (30mg,0.30mmol) (see the synthetic procedure of example 11) were reacted under nitrogen atmosphere to give compound B3 (white solid; 70 mg; yield: 50%).

¹H-NMR(400MHz,CDCl₃,δppm):8.70(s,1H),8.38(d,J＝5.2Hz,1H),8.06(d,J＝8.0Hz,1H),7.91(s,1H),7.59(s,1H),7.44(d,J＝8.0Hz,1H),7.31(d,J＝8.0Hz,2H),7.23(d,J＝5.6Hz,1H),6.87(dd,J＝26.8,8.0Hz,2H),6.42(d,J＝14.8Hz,1H),6.30(d,J＝10.0Hz,1H),5.55(d,J＝10.0Hz,1H),4.23(t,J＝5.6Hz,2H),1.99–1.68(m,8H),1.44(d,J＝8.8Hz,2H),1.25(d,J＝7.2Hz,4H).ES-API(m/z):[M+H]+:466.3.

EXAMPLE 14 Synthesis of Compound B4

Synthetic route

A4(127mg,0.30mmol) and acryloyl chloride (30mg,0.30mmol) (see the synthetic procedure of example 11) were reacted under nitrogen atmosphere to give compound B4 (white solid; 50 mg; yield: 35%).

¹H-NMR(400MHz,CDCl₃,δppm):8.59(s,1H),8.37(d,J＝5.2Hz,1H),8.08(d,J＝8.0Hz,1H),7.83(s,1H),7.43(d,J＝8.0Hz,1H),7.36–7.30(m,2H),7.22(d,J＝7.6Hz,1H),7.18(d,J＝5.2Hz,1H),6.91(d,J＝8.0Hz,1H),6.84(d,J＝7.6Hz,1H),6.40(dd,J＝16.8,2.0Hz,1H),6.27(dd,J＝16.8,10.0Hz,1H),5.52(dd,J＝10.0,2.0Hz,1H),4.25(t,J＝6.0Hz,2H),3.92–3.70(m,2H),1.96–1.87(m,2H),1.24(s,6H),1.01(d,J＝7.2Hz,6H).ES-API(m/z):[M+H]+:480.3.

EXAMPLE 15 Synthesis of Compound B5

Synthetic route

A5(128mg,0.30mmol) and acryloyl chloride (0.03mg,0.30mmol) (see synthetic procedure in example 11) B5 (white solid; 40 mg; yield: 28%) were reacted under nitrogen.

¹H-NMR(400MHz,CDCl₃,δppm):8.57(d,J＝2.4Hz,1H),8.39(d,J＝5.2Hz,1H),7.98(dd,J＝6.8,2.4Hz,1H),7.91(s,1H),7.81(s,1H),7.42(dd,J＝6.8,2.4Hz,1H),7.28(t,J＝2.4Hz,1H),7.24(d,J＝5.2Hz,1H),6.91(d,J＝8.4Hz,1H),6.78(dd,J＝8.4,2.4Hz,1H),6.30(dd,J＝16.8,2.0Hz,1H),6.07(dd,J＝16.8,10.0Hz,1H),5.44(dd,J＝10.0,2.0Hz,1H),4.28–4.24(m,2H),3.96(s,3H),1.92–1.87(m,2H),1.67–1.62(m,2H),1.54–1.47(m,2H),1.42–1.36(m,2H),1.26(dd,J＝12.8,5.2Hz,4H).ES-API(m/z):[M+H]+:482.3.

EXAMPLE 16 Synthesis of Compound B6

Synthetic route

A6(132mg,0.30mmol) and acryloyl chloride (30mg,0.30mmol) (see the synthetic procedure of example 11) were reacted under nitrogen atmosphere to give compound B6 (white solid; 80 mg; yield: 53%).

¹H-NMR(400MHz,CDCl₃,δppm):8.79(d,J＝2.4Hz,1H),8.39(d,J＝5.2Hz,1H),8.06(d,J＝8.0Hz,1H),7.97(d,J＝17.6Hz,1H),7.91(s,1H),7.44(d,J＝8.0Hz,1H),7.31–7.27(m,1H),7.24(s,1H),6.90(d,J＝8.4Hz,1H),6.76(dd,J＝8.4,2.4Hz,1H),6.41(dd,J＝16.8,2.4Hz,1H),6.29(dd,J＝16.8,10.0Hz,1H),5.53(dd,J＝10.0,2.4Hz,1H),4.23(s,2H),3.96(s,3H),2.30–2.10(m,2H),1.91(s,2H),1.44(dd,J＝8.4,4.4Hz,2H),1.26(dd,J＝12.8,4.8Hz,8H).ES-API(m/z):[M+H]+:496.3.

Compound B7-16, prepared by the method of preparation of example 11, has a nuclear magnetism as shown in Table 2:

TABLE 2

Effect experimental example 1 in vitro kinase inhibitory Activity

1.1EGFR (WT) inhibitory Activity Screen

With kinase buffer (50mM HEPES, 10mM MgCl)₂2mM DTT, 1mM EGTA, 0.01% Tween 20) 50 ng/. mu.L of EGFR (WT) stock solution was diluted, 6. mu.l of 1.67X 0.00835 ng/. mu.l working solution (final concentration of 0.005 ng/. mu.l) was added to each well, DMSO-dissolved different compounds were added to the wells using a nanoliter loading apparatus such that the compounds to be tested had a final concentration of 1000nM to 0.24nM, a positive of 100nM to 0.024nM, a 4-fold gradient, a total of 7 concentrations, blank control empty (no enzyme) and negative control wells (enzyme-containing, vehicle DMSO), and 2 replicate wells were set. After the enzyme reacts with the compound or the solvent for 30min, 5 × 25 μ M ATP (final concentration of 5 μ M) prepared with a kinase buffer and 5 × 0.5 μ M substrate (final concentration of 0.1 μ M, U Light-poly GT) were mixed in a ratio of 1:1 and added to the wells at 4 μ L per well; after sealing the plate and membrane, after reacting for 2h at room temperature, 5. mu.L of 4X 40mM EDTA (final concentration 10mM) was added to each well for 5min at room temperature, and 5. mu.L of 4X 8nM detection reagent (final concentration 2nM, E. mu. -anti-phospho-tyrosine antibody) was added to each well and incubated for 1 h at room temperature; the PE instrument reads the plate (excitation 320or 340nm, emission 665nm) and calculates IC50 using a four parameter fit.

1.2EGFR (T790M L858R) inhibition Activity screening

With kinase buffer (50mM HEPES, 10mM MgCl)₂2mM DTT, 1mM EGTA, 0.01% Tween 20) 50ng/uL of EGFR (T790M L858R) stock solution was diluted, 6. mu.l of 1.67 X0.167 ng/uL working solution (final concentration of 0.1 ng/. mu.L) was added to each well, DMSO-dissolved different compounds were added to the wells using a nanoliter loader to give final concentrations of 1000nM-0.24nM, 100nM-0.024nM positivity, 4-fold gradient, 7 concentrations in total, while blank control wells (no enzyme) and negative control wells (enzyme-containing, vehicle DMSO) were set. EnzymeAfter 30min reaction with compound or vehicle, 5 × 25 μ M ATP (final concentration of 5 μ M) prepared in kinase buffer and 5 × 0.5 μ M substrate (final concentration of 0.1 μ M, U Light-poly GT) were mixed at a ratio of 1:1 and added to the wells at 4 μ L per well; after sealing the plate and membrane, after reacting for 2h at room temperature, 5. mu.L of 4X 40mM EDTA (final concentration 10mM) was added to each well for 5min at room temperature, and 5. mu.L of 4X 8nM detection reagent (final concentration 2nM, E. mu. -anti-phospho-tyrosine antibody) was added to each well and incubated for 1 h at room temperature; the PE instrument reads the plate (excitation 320or 340nm, emission 665nm) and calculates IC50 using a four parameter fit.

The results are shown in Table 3.

Table 3: results of in vitro kinase inhibitory Activity

EGFR (WT) kinase inhibition IC of the compounds of the invention₅₀(nm) is less than 40nm, wherein some compounds are less than 10nm, and some compounds are less than 1 nm. EGFR (T790M/L858R) kinase inhibition IC of the compounds of the invention₅₀(nm) is less than 40nm, wherein some compounds are less than 10nm, and some compounds are less than 1 nm.

Effect experiment example 2 in vitro cell proliferation inhibitory Activity

2.1 inhibitory Effect on proliferation of LOVO cells

A bottle of LOVO cells in exponential growth phase is taken, 5mL of PBS is added for washing once, and 2mL of pancreatin is added. The cells were digested in a cell incubator, occasionally removed and observed under a microscope, and when the cells just shed, 2mL of complete medium was added to stop the digestion. Low speed desk centrifuge, 1500 rpm, centrifuge for 3 min. The cell digest was decanted and the cells resuspended by adding 2mL of complete medium using a pipette. The cells were counted using a cytometer, diluted in complete medium and adjusted to a cell density of 8 x 104 cells/mL. The cells were inoculated in a 96-well plate using a line gun at 100. mu.L/well and incubated in a constant temperature CO2 incubator for 24 hours. And (3) carrying out compound loading by using a nano-liter loading instrument, adding CCK-8 at 10 mu L/hole after 72 hours, detecting the light absorption value at 450nm of an Envision microplate reader after 3 hours, calculating the inhibition rate, and calculating IC50 by adopting four-parameter fitting.

2.2 proliferation inhibition of NCI-H1975 cells

A vial of NCI-H1975 cells in the exponential growth phase was washed with 5mL of PBS and 2mL of pancreatin. The cells were digested in a cell incubator, occasionally removed and observed under a microscope, and when the cells just shed, 2mL of complete medium was added to stop the digestion. Low speed desk centrifuge, 1500 rpm, centrifuge for 3 min. The cell digest was decanted and the cells resuspended by adding 2mL of complete medium using a pipette. The cells were counted using a cytometer, diluted in complete medium and adjusted to a cell density of 5 x 104 cells/mL. The cells were inoculated in a 96-well plate using a line gun at 100. mu.L/well and incubated in a constant temperature CO2 incubator for 24 hours. And (3) carrying out compound loading by using a nano loading instrument, adding CCK-8 at a concentration of 10 mu L/hole after 72 hours, detecting the light absorption value at 450nm of an Envision microplate reader after 3 hours, calculating the inhibition rate, and calculating IC50 by adopting four-parameter fitting. The results are shown in Table 4.

Table 4: inhibition of cell proliferation

Inhibitory IC of compounds of the invention on LOVO cell proliferation₅₀(nm) are all less than 10nm, and some of the partial compounds are less than 1 nm. Inhibition of NCI-H1975 cell proliferation by the compounds of the invention IC₅₀(nm) are all less than 10nm, and some of the partial compounds are less than 1 nm.

Claims (13)

1. A nitrogen-containing macrocyclic compound as shown in formula I or a pharmaceutically acceptable salt thereof,

wherein ,R¹Is hydrogen, C₁～C₃Alkoxy, halogen or C substituted by one or more halogens₁～C₃An alkoxy group;

R²is hydrogen or C substituted by one or more halogens₁～C₃An alkoxy group;

R³is hydrogen or halogen;

R⁴is hydrogen or halogen;

r is hydrogen or

n is 0, 1, 2, 3, 4, 5 or 6.

2. The nitrogen-containing macrocyclic compound of formula I of claim 1, or a pharmaceutically acceptable salt thereof, wherein R is¹Is hydrogen, C₁～C₃Alkoxy, halogen or C substituted by one or more fluorine₁～C₃An alkoxy group;

and/or, R²Is hydrogen or C substituted by one or more fluorine₁～C₃An alkoxy group;

and/or, R³Is hydrogen or chlorine;

and/or, R⁴Is hydrogen or chlorine;

and/or n is 2, 3 or 4;

and/or R is

3. A nitrogen-containing macrocyclic compound of formula I as claimed in claim 1, wherein the nitrogen-containing macrocyclic compound is as defined in any one of the following schemes:

scheme 1:

R¹is hydrogen, C₁～C₃Alkoxy, halogen or C substituted by one or more fluorine₁～C₃An alkoxy group;

R²is hydrogen or C substituted by one or more fluorine₁～C₃An alkoxy group;

R³is hydrogen or chlorine;

R⁴is hydrogen or chlorine;

r is hydrogen or

n is 2, 3 or 4;

scheme 2:

wherein ,R¹Is hydrogen, C₁～C₃Alkoxy, halogen or C substituted by one or more halogens₁～C₃An alkoxy group;

R²is hydrogen or C substituted by one or more halogens₁～C₃An alkoxy group;

R³is hydrogen or halogen;

R⁴is hydrogen or halogen;

r is

n is 0, 1, 2, 3, 4, 5 or 6;

scheme 3:

wherein ,R¹Is hydrogen, fluorine or C substituted by one or more fluorine₁～C₃An alkoxy group;

R²is hydrogen or C "substituted by one or more fluorine₁～C₃Alkoxy groups ";

R³is hydrogen;

R⁴is hydrogen or chlorine;

r is

n is 2 or 3;

scheme 4:

R¹is hydrogen, fluorine or C substituted by one or more fluorine₁～C₃An alkoxy group;

R²is hydrogen;

R³is hydrogen or chlorine;

R⁴is hydrogen or chlorine;

r is

n is 2 or 3.

4. The nitrogen-containing macrocyclic compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3,

when said R is¹Is C₁～C₃At alkoxy, said C₁～C₃The alkoxy is methoxy, ethoxy, n-propoxy or isopropoxy, and can also be methoxy;

and/or, when said R is¹When the halogen is fluorine, chlorine, bromine or iodine, the halogen can be fluorine;

and/or, when said R is¹Is C substituted by one or more halogens₁～C₃At alkoxy, said C₁～C₃The alkoxy is methoxy, ethoxy, n-propoxy or isopropoxy, and can also be methoxy;

and/or, when said R is¹Is C substituted by one or more halogens₁～C₃When alkoxy, the halogen is fluorine, chlorine, bromine or iodine, and can also be fluorine;

and/or, when said R is¹Is C substituted by one or more halogens₁～C₃When alkoxy, said plurality is 3;

and/or, when said R is²Is "C substituted by one or more halogens₁～C₃Alkoxy "said C₁～C₃The alkoxy is methoxy, ethoxy, n-propoxy or isopropoxy, and can also be n-propoxy;

and/or, when said R is²Is "quilt oneC substituted by one or more halogens₁～C₃When the alkoxy is adopted, the halogen is fluorine, chlorine, bromine or iodine, and can also be fluorine;

and/or, when said R is³When the halogen is fluorine, chlorine, bromine or iodine, and also can be chlorine;

and/or, when said R is⁴When halogen is used, the halogen is fluorine, chlorine, bromine or iodine, and can also be chlorine.

5. The nitrogen-containing macrocyclic compound of formula I or a pharmaceutically acceptable salt thereof of claim 4, wherein R is²Is "C substituted by one halogen₁～C₃Alkoxy, said "C substituted by one halogen₁～C₃The alkoxy radical "may be a monofluoro-n-propoxy radical or may be

And/or, when said R is¹Is C substituted by more than one halogen₁～C₃When alkoxy, said C is substituted by more than one halogen₁～C₃Alkoxy is trifluoromethoxy.

6. The nitrogen-containing macrocyclic compound of formula I or a pharmaceutically acceptable salt thereof according to claim 4,

R¹is hydrogen, fluorine or trifluoromethoxy;

and/or, R²Is composed of

Or hydrogen.

7. The nitrogen-containing macrocyclic compound of formula I, or a pharmaceutically acceptable salt thereof, of claim 1, wherein the nitrogen-containing macrocyclic compound of formula I is any one of:

8. a nitrogen-containing macrocyclic compound shown as a formula II or a salt thereof, which is characterized in that,

wherein ,

in cis configuration, trans configuration or mixtures thereof;

R¹、R²、R³、R⁴r and n are as defined in any one of claims 1 to 7.

9. The nitrogen-containing macrocyclic compound of formula II, or a salt thereof, as claimed in claim 8, wherein the nitrogen-containing macrocyclic compound of formula II is any one of the following:

or its cis isomer,

Or its cis-isomer,

Or its cis isomer,

Or its cis-isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis-isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis-isomer,

Or its cis isomer,

Or its cis-isomer,

Or its cis isomer,

Or its cisThe isomers of the formula,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis isomer,

Or its cis-isomer,

Or its cis isomer or

Or a cis isomer thereof.

10. A process for the preparation of a nitrogen containing macrocyclic compound of the formula I according to any of claims 1 to 7, which is scheme I or scheme ii;

the scheme i comprises the following steps: carrying out hydrogenation reduction on the compound shown in the formula II;

r is hydrogen;

in cis configuration, trans configuration or mixtures thereof;

the scheme ii comprises the following steps that the compound shown as the formula A and acryloyl chloride are subjected to acylation reaction shown as the following formula;

r is

11. A pharmaceutical composition comprising a substance X and a pharmaceutical excipient, wherein the substance X is a nitrogen-containing macrocyclic compound of formula I as defined in any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof.

12. Use of a substance X for the preparation of an EGFR inhibitor, wherein said EGFR inhibitor is for use in vitro; substance X is a nitrogen containing macrocyclic compound of formula I as described in any of claims 1-7, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in claim 11;

further, the EGFR is a wild type EGFR or an EGFR mutant;

furthermore, the EGFR mutant has T mutation at the 790 th site of M and/or L mutation at the 858 th site of T.

13. Use of substance X in the manufacture of a medicament for the treatment and/or prevention of a disease or cancer associated with EGFR, wherein substance X is a nitrogen containing macrocyclic compound of formula I as defined in any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof; preferably, in said use, the disease or cancer associated with an EGFR inhibitor is lung cancer; more preferably, the lung cancer is non-small cell lung cancer;

further, the EGFR is a wild type EGFR or an EGFR mutant;

furthermore, the EGFR mutant has T mutation at the 790 th site of M and/or L mutation at the 858 th site of T.