CN111825658A

CN111825658A - Novel EGFR (epidermal growth factor receptor) triple-mutation inhibitor and application thereof

Info

Publication number: CN111825658A
Application number: CN201910315199.XA
Authority: CN
Inventors: 李洪林; 谢华; 赵振江; 丁健; 李圣青; 陈卓
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2019-04-18
Filing date: 2019-04-18
Publication date: 2020-10-27
Also published as: WO2020211853A1

Abstract

The invention relates to a compound shown in a general formula I or II.The compound is an EGFR inhibitory compound with a brand-new structure, can obviously inhibit three mutations of EGFR19del/T790M/C797S and L858R/T790M/C797S, and can be developed as a new generation of EGFR inhibitors and used for treating EGFR-mediated related diseases.

Description

Novel EGFR (epidermal growth factor receptor) triple-mutation inhibitor and application thereof

Technical Field

The present invention relates to the field of pharmaceutical chemistry; in particular to the application of novel indole and trisubstituted imidazole compounds as EGFR (epidermal growth factor receptor) triple mutation inhibitors and as EGFR inhibitors in preparation of drugs for treating tumor-related diseases.

Background

Epidermal Growth Factor Receptor (EGFR) is a transmembrane glycoprotein existing on the cell membrane of human tissues, has the molecular weight of 170-kDa, and belongs to the family of ErbB Receptor Tyrosine Kinases (RTK) with ErbB-2(HER2/Neu), ErbB-3(HER3) and ErbB-4(HER 4). These receptors are composed of an extracellular ligand-binding domain, which is then linked to an intracellular tyrosine kinase via a transmembrane domain. The tyrosine kinase domain in ErbB receptors is highly conserved, in contrast to the distinct differences in the extracellular domain, which also results in the receptor exhibiting different specificity when binding to a ligand. EGFR and ErbB-4 have intact extracellular ligand binding domains and activatable kinase domains, the extracellular domain of ErbB-2 may not have ligand binding capacity, ErbB-3, although it binds ATP, has no active tyrosine kinase receptor, and its homodimer is inactive. EGFR signaling is primarily through binding of specific ligands, causing dimerization, activation of its intracellular tyrosine kinase activity, autophosphorylation of some terminal tyrosine residues, and activation of downstream signaling pathways.

Research shows that the epidermal growth factor receptor plays an important role in cell proliferation, metabolism and the like. Because it has an overexpression phenomenon in various cancer cells, the EGFR is taken as a drug target, so that the EGFR has great research significance. The current tumor molecule targeted drugs aiming at EGFR are mainly divided into two main categories according to the properties: one class is monoclonal antibodies that act directly on the extracellular receptor region; another class are small molecule inhibitors that interfere with intracellular EGFR tyrosine kinase activity. The monoclonal antibody drug enables endogenous ligands such as EGF and the like to be incapable of being combined with EGFR through the action of the monoclonal antibody drug and an extramembranous ligand binding domain of the EGFR, so that signal transmission into cells is prevented; the small molecule drug is combined with the intracellular tyrosine kinase catalytic region to inhibit the catalytic activity of the small molecule drug, so that the cell proliferation signal is blocked.

Mutations in EGFR are mainly concentrated in the 18-21 exon, which are responsible for coding the tyrosine kinase domain of EGFR. In which the deletion of exon 19 occupies 44% of the EGFR tyrosine kinase sensitive mutation. The point mutation on the No. 21 exon-the L858R mutation accounted for 41% of the EGFR tyrosine kinase sensitive mutations. Residue 719 is mutated from glycine to serine, alanine or cysteine accounting for 10% of the total mutations, while the remaining 5% is occupied by the insertion or duplication mutation of exon 20. Among them, exon 19 deletion and L858R point mutation are the most common sensitive mutations. These mutations enhance the activity of EGFR kinase, thereby increasing downstream signaling pathways. It has also been reported that the T790M point mutation in exon 20 is found in 50% of patients with resistance to treatment with EGFR tyrosine kinase inhibitors. Such mutations are believed to be generated during treatment because they were not detected in untreated patients. A series of small molecule inhibitors were derived for these different mutations.

The first generation of EGFR small molecule inhibitors, such as Gefitinib and Erlotinib. These inhibitors are primarily directed against sensitive mutations, but with the discovery of T790M resistant mutations, patients develop resistance. Therefore, second-generation and third-generation EGFR inhibitors have been derived, which have been improved in inhibitory activity mainly by covalent binding of Michael acceptors on the molecule to cysteine 797 residues of proteins.

Although the use of third generation EGFR inhibitors to treat non-small cell lung cancer patients carrying the T790M mutation holds promise, resistance is also evolving. The research shows that the drug resistance is mainly caused by that Cys797 residues are mutated to form Ser797 residues, the binding force between small molecules and kinase is damaged, and the third-generation inhibitor basically loses the effect.

Therefore, there is an urgent need in the art to develop new generation inhibitors to overcome the three mutations of EGFR, L858R/T790M/C797S and EGFR19 del/T790M/C797S.

Disclosure of Invention

The invention aims to provide a novel EGFR (epidermal growth factor receptor) inhibitory compound which can inhibit the three mutations of EGFR19 del/T790M/C797S.

It is another object of the present invention to provide a pharmaceutical composition comprising the above compound.

The invention also aims to provide the application of the compound in preparing medicaments for treating EGFR related diseases or inhibiting EGFR.

In a first aspect, the present invention provides a compound represented by general formula I or a stereoisomer or an optical isomer thereof, or a pharmaceutically acceptable salt thereof:

in the formula (I), the compound is shown in the specification,

w is CH or N;

x is CH or N;

y is CH or N or C halogen;

z is CH or N;

R¹is an integer from 1 to 5, optionally in the 1', 2', 5 ', 6 ' or 7 ' position, and is independently selected from: H. halogen, C1-C6 substituted or unsubstituted alkyl,

Wherein n is an integer of 0-4;

p is 1-2C 1-C3 alkyl groups or absent;

q is-OH, -SH, -NH₂、-NHCH₃、-COOH、-CONH₂、-NHCONH₂、-NHCONHNH₂、-SO₃H、-SO₂NH₂；

Wherein when R is⁴Is composed of

When is at R⁴There may be one or more Q at each position;

x is O, S, NH;

R²selected from: hydrogen, C1-C5 substituted or unsubstituted alkylcarboxamide groups, C2-C5 substituted or unsubstituted alkenylcarboxamide groups;

R³selected from: hydrogen, halogen, NR⁷R⁸Substituted N-C1-C3 alkylpiperazino groups;

R⁷and R⁸Independently selected from: H. C1-C6 substituted or unsubstituted alkyl, NR⁹R¹⁰；

R⁹And R¹⁰Independently selected from: H. C1-C3 substituted or unsubstituted alkyl.

In a particular embodiment of the present invention,

w is CH;

x is CH or N;

y is CH or N;

z is CH;

R¹is in the 1 or 2, optionally in the 1 'or 5' position, and is independently selected from: H. C1-C6 substituted or unsubstituted alkylhydroxy or polyhydroxy, halogen;

R²selected from: hydrogen, C1-C3 substituted or unsubstituted alkylcarboxamide groups, C2-C3 substituted or unsubstituted alkenylcarboxamide groups;

R³selected from: halogen, NR⁷R⁸；

R⁷And R⁸Independently selected from: H. C1-C3 substituted or unsubstituted alkyl, NR⁹R¹⁰；

In a specific embodiment, the present invention provides the following compounds, or stereoisomers or optical isomers thereof, or pharmaceutically acceptable salts thereof:

in a second aspect, the present invention provides a compound represented by formula II or a stereoisomer or an optical isomer thereof, or a pharmaceutically acceptable salt thereof:

in the formula (I), the compound is shown in the specification,

R¹selected from:

wherein n is an integer of 0-4;

p is 1-2C 1-C3 alkyl groups or absent;

q is-OH, -SH, -NH₂、-NHCH₃,、-COOH、-CONH₂、-NHCONH₂、-NHCONHNH₂、-SO₃H、-SO₂NH₂；

Wherein when R is⁴Is composed of

When is at R⁴There may be one or more Q at each position;

x is O, S, NH;

R⁴selected from:

the number of A is any integer from 0 to 4 and is independently selected from: halogen, substituted or unsubstituted C1-C3 alkoxy;

v, S, T are each any integer from 0 to 4 and are independently selected from halogen, substituted or unsubstituted C1-C3 alkoxy;

R⁵selected from: H. substituted or unsubstituted

R⁶Selected from: H. substituted or unsubstituted C1-C3 alkyl.

In a particular embodiment of the present invention,

R⁴selected from:

v is an integer from 0 to 4 and is independently selected from halogen, substituted or unsubstituted C1-C3 alkoxy;

R⁵selected from: substituted or unsubstituted

R⁶Selected from: H.

in a specific embodiment, the present invention provides a compound selected from the group consisting of:

in a third aspect, the present invention provides a pharmaceutical composition comprising a compound of the first or second aspect, or a stereoisomer or an optical isomer thereof, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier or excipient.

In a preferred embodiment, the pharmaceutical composition is in a dosage form suitable for oral administration, including but not limited to tablets, solutions, suspensions, capsules, granules, powders.

In a fourth aspect, the present invention provides the use of a compound of the first or second aspects, or a stereoisomer or optical isomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the third aspect, in the manufacture of a medicament for the treatment or prevention of an EGFR-mediated disease or for the inhibition of EGFR.

In a specific embodiment, the EGFR-mediated disease is cancer.

In specific embodiments, the cancer is selected from the group consisting of: non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, breast cancer, prostate cancer, glioma, ovarian cancer, head and neck squamous carcinoma, cervical cancer, esophageal cancer, liver cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, leukemia, lymphoma, gastric cancer, multiple myeloma, and solid tumors.

In a fifth aspect, the present invention provides a method of treating or preventing an EGFR-mediated disease, comprising administering to a subject in need thereof a compound according to the first or second aspect or a pharmaceutical composition according to the third aspect.

In preferred embodiments, the EGFR-mediated disease is cancer; preferably, the cancer is selected from the group consisting of: non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, breast cancer, prostate cancer, glioma, ovarian cancer, head and neck squamous carcinoma, cervical cancer, esophageal cancer, liver cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, leukemia, lymphoma, gastric cancer, multiple myeloma, and solid tumors.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Detailed Description

The inventor unexpectedly discovers a series of EGFR inhibitory compounds with brand-new structures through extensive and intensive research, and the compounds can obviously inhibit three mutations of EGFR19del/T790M/C797S, so that a new generation of EGFR inhibitors can be developed, and a brand-new material basis is laid for developing the new generation of EGFR inhibitors. The present invention has been completed based on this finding.

Definition of terms

Some of the groups referred to herein are defined as follows:

as used herein, "alkyl" refers to a saturated, branched or straight-chain alkyl group having a carbon chain length of 1 to 10 carbon atoms, with preferred alkyl groups including those varying in length from 2 to 8, 1 to 6, 1 to 4, 3 to 8, 1 to 3 carbon atoms. Examples of alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, heptyl and the like. The alkyl group may be substituted with 1 or more substituents, for example, with halogen or haloalkyl. For example, the alkyl group may be an alkyl group substituted with 1 to 4 fluorine atoms, or the alkyl group may be an alkyl group substituted with a fluoroalkyl group.

Herein, "alkoxy" refers to an oxy group substituted with an alkyl group. Preferred alkoxy groups are alkoxy groups of 1 to 6 carbon atoms in length, more preferably 1 to 4 carbon atoms in length. Examples of alkoxy groups include, but are not limited to: methoxy, ethoxy, propoxy, and the like.

Herein, "halogen" refers to fluorine, chlorine, bromine and iodine.

As used herein, "amido" refers to a group of the formula "-R '-NH-C (O) -R", wherein R' may be selected from hydrogen or alkyl, and R may be selected from alkyl, alkenyl, alkynyl, or NR_cR_dSubstituted alkyl, by NR_cR_dSubstituted alkenyl and NR_cR_dSubstituted alkynyl, alkyl substituted by halogen, alkenyl substituted by cyano, wherein R_cAnd R_dCan be selected from alkyl and alkenyl.

Herein, "substituted or unsubstituted" or "optionally substituted" means that the substituent group it modifies may be optionally substituted with 1 to 5 (e.g., 1,2, 3, 4, or 5) substituents selected from the group consisting of: halogen, C_1-4Aldehyde group, C_1-6Straight or branched chain alkyl, cyano, nitro, amino, hydroxy, hydroxymethyl, halogen-substituted alkyl (e.g. trifluoromethyl), halogen-substituted alkoxy (e.g. trifluoromethoxy), carboxy, C_1-4Alkoxy, ethoxycarbonyl, N (CH)₃) And C_1-4An acyl group.

The term "substituted" as used herein means that one or more hydrogen atoms on a particular group are replaced with a particular substituent. The specific substituents may be those described above in relation to the corresponding substituents, or may be those specified in the examples. Therefore, in the present invention, the substituents in the general formula may each independently be the corresponding group in the specific compounds in the examples; that is, the present invention includes combinations of the respective substituents in the above general formulae, and also includes combinations of some of the substituents shown in the general formulae with other specific substituents appearing in the examples.

Compounds of the invention

The invention provides a series of EGFR (epidermal growth factor receptor) inhibitory compounds with brand-new structures, which can obviously inhibit three mutations of EGFR19 del/T790M/C797S. In a specific embodiment, the present invention provides a compound represented by formula I or II, or a stereoisomer or optical isomer thereof, or a pharmaceutically acceptable salt thereof:

wherein each substituent is as defined above.

On the basis of the compounds of the present invention, those skilled in the art can prepare pharmaceutically acceptable salts thereof. For example, the compounds of the present invention may be reacted with inorganic or organic acids to form conventional pharmaceutically acceptable salts. The inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, sulfamic acid, phosphoric acid and the like, and the organic acids include citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, isethionic acid and the like; or reacting a compound of the invention with an inorganic base to form a sodium, potassium, calcium, aluminum or ammonium salt; or with an organic base to form a methylamine salt, an ethylamine salt or an ethanolamine salt.

Unless otherwise specified, the structural formulae depicted herein are intended to include all isomeric forms (e.g., enantiomers, diastereomers and geometric isomers (or conformers): e.g., the R, S configuration containing an asymmetric center, (Z), (E) isomers of double bonds, etc. accordingly, a single stereochemical isomer of a compound of the present invention or a mixture of enantiomers, diastereomers or geometric isomers (or conformers) thereof is within the scope of the present invention.

Pharmaceutical compositions of the invention and methods of administration

The compounds of the invention can be used for the preparation of a medicament for the treatment of EGFR (especially EGFR19del/T790M/C797S triple mutation) mediated diseases, such as cancer, including but not limited to: non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, breast cancer, prostate cancer, glioma, ovarian cancer, head and neck squamous carcinoma, cervical cancer, esophageal cancer, liver cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, leukemia, lymphoma, gastric cancer, multiple myeloma, and solid tumors.

In view of this, the compounds of the present invention and their pharmaceutically acceptable salts are based on. The invention also provides pharmaceutical compositions comprising a compound of the invention, optionally comprising a pharmaceutically acceptable excipient.

In a specific embodiment, the pharmaceutical composition of the present invention comprises a compound of the present invention or a pharmaceutically acceptable salt thereof in a safe and effective amount range, and a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects.

"pharmaceutically acceptable excipient or carrier" means: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with and with the compounds of the present invention without significantly diminishing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties include cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame,Peanut oil, olive oil, etc.), polyol (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifier (such as

) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.

In addition to these inert diluents, the compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms for topical administration of the compounds of the present invention include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds. In the case of pharmaceutical compositions, a safe and effective amount of a compound of the invention is administered to a mammal (e.g., a human) in need of treatment at a dosage that is pharmaceutically acceptable for effective administration. The compounds and pharmaceutical compositions of the present invention may be administered by oral, nasal, dermal, pulmonary or gastrointestinal routes, and the like. Most preferably, it is administered orally, in a single dose or in divided doses. Regardless of the method of administration, the optimal dosage for an individual will depend on the particular treatment. Usually starting with a small dose and gradually increasing the dose until the most suitable dose is found. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The invention has the advantages that:

1. the compound of the invention is an EGFR inhibitory compound with a brand-new structure; and

2. the compound can obviously inhibit three mutations of EGFR19del/T790M/C797S, so that the compound can be developed as a new generation of EGFR inhibitors and lays a brand new material foundation for developing the new generation of EGFR inhibitors.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Examples

Material

The test materials and reagents used in the following examples are commercially available without specific reference.

Preparation method

The compounds of the present invention can be prepared by methods conventional in the art, or by synthetic routes according to the present invention.

Synthetic scheme of indole compounds

Wherein, the reaction reagents and conditions of each step are as follows:

(a)DMSO,KOH,25℃,1h,3-chloropropan-1-ol,8h,36％；

(b)DCE,AlCl3,0℃,2,4-dichloropyrimidine,55℃,1.5h,31％；

(c)TsOH,2-pentanol,4-fluoro-2-methoxy-5-nitroaniline,105℃,2.5h,76％；

(d)Fe,MeOH/DCM,AcOH,55℃,4h；

(e)acryloyl chloride,DCM,DIPEA,0℃,1.5h,35％；

(f)N,N,N-trimethylethane-1,2-diamine,DMF,K2CO3,110℃,3h,60％；

(g)Pd/C,H2,MeOH,25℃,5h.

example 1: synthesis of Compound 1

Synthesis of 3- (1H-indol-1-yl) propan-1-ol (2b) (step a)

Weighing potassium hydroxide (4.48g, 80.0mmol) in a 250mL single-neck bottle, adding 100mL of dimethyl sulfoxide, stirring at room temperature for 15min, adding 2a (4.46g, 40.0mmol), reacting at room temperature for 1h, dropwise adding 3-chloro-1-propanol (5.67g, 60.0mmol) into the solution, reacting at room temperature, monitoring by TLC, reacting for 8h, adding a large amount of water for quenching after the reaction is finished, extracting with ethyl acetate, collecting an organic phase, drying with anhydrous sodium sulfate, spin-drying a solvent, and separating a crude product by silica gel column chromatography with petroleum ether and ethyl acetate which are 20:1 to obtain 2.59g of 2c yellow liquid with the yield of 36%.

¹H NMR(400MHz,Chloroform-d):7.55(d,J＝7.9Hz,1H),7.29(d,J＝8.2Hz,1H),7.16–7.09(m,1H),7.06–6.97(m,2H),6.41(d,J＝3.1Hz,1H),4.17(t,J＝6.7Hz,2H),3.47(t,J＝6.0Hz,2H),1.94(p,J＝6.3Hz,2H),1.59(s,1H).LC-MS:m/z:176.2(M+H)⁺.

Synthesis of 3- (3- (2-chloropyrimidin-4-yl) -1H-indol-1-yl) propan-1-ol (2c) (step b)

Dissolving 2b (2.59g, 14.7mmol) in 50mL of 1, 2-dichloroethane, cooling to 0 ℃ in ice, adding anhydrous aluminum trichloride (2.81g, 21.0mmol), heating to room temperature after 10min, stirring for 15min, adding 2, 4-dichloropyrimidine (1.73g, 11.0mmol), heating to 55 ℃ and reacting for 1.5h, monitoring by TLC, finishing the reaction, standing and cooling to room temperature, adding methanol/water (20mL/10mL) in ice bath, returning to room temperature, stirring for 30min, filtering by suction, collecting the filtrate, extracting by dichloromethane, drying by anhydrous sodium sulfate, and separating a rotary-drying solvent by silica gel column chromatography with petroleum ether ethyl acetate ═ 20:1 to obtain 1.05g of 2c light yellow solid with the yield of 31%.

¹H NMR(400MHz,Chloroform-d):8.30(d,J＝5.4Hz,1H),8.26–8.19(m,1H),7.92(s,1H),7.45–7.31(m,2H),7.27–7.19(m,2H),4.27(t,J＝6.7Hz,2H),3.54(t,J＝5.7Hz,2H),2.02(p,J＝6.3Hz,2H).LC-MS:m/z:288.1(M+H)⁺.

Synthesis of 3- (3- (2- ((4-fluoro-2-methoxy-5-nitrophenyl) amino) pyrimidin-4-yl) -1H-indol-1-yl) propan-1-ol (2e) (step c)

4-Methylbenzenesulfonic acid hydrate (0.75g, 4.3mmol) was added in one portion to a solution of 2c (1.05g, 3.6mmol) and 4-fluoro-2-methoxy-5-nitroaniline (0.75g, 4.0mmol) in 25mL of 2-pentanol. The resulting mixture was stirred at 105 ℃ for 2.5 h. TLC, the reaction was stopped and the mixture was cooled to room temperature. Filtration and cake washing with ice 2-pentanol collected and dried in vacuo gave 1.225g of 2d yellow solid in 76% yield.

¹H NMR(400MHz,DMSO-d₆):10.21(s,1H),8.83(s,1H),8.73(d,J＝8.2Hz,1H),8.37(d,J＝6.6Hz,1H),8.16(s,1H),7.65(d,J＝8.3Hz,1H),7.58–7.48(m,2H),7.30(t,J＝7.4Hz,1H),7.11(q,J＝7.5,6.8Hz,1H),4.37(t,J＝7.0Hz,2H),4.00(s,3H),3.43(t,J＝6.0Hz,2H),2.52(p,J＝1.9Hz,1H),2.00(p,J＝6.5Hz,2H).LC-MS:m/z:438.2(M+H)⁺.

Synthesis of 3- (3- (2- ((5-amino-4-fluoro-2-methoxyphenyl) amino) pyrimidin-4-yl) -1H-indol-1-yl) propan-1-ol (2f) (step d)

2d (600.00mg, 1.4mmol) was dissolved in 40mL of a mixed solvent (dichloromethane: methanol ═ 3:1), and iron powder (2.92g, 52.1mmol) and 12mL of glacial acetic acid were added to the solution, followed by stirring and reaction at 55 ℃ for 4 hours. TLC, the reaction was complete, filtered, the filtrate was concentrated to dryness and the residue was adjusted to basic pH by addition of saturated sodium bicarbonate solution. Extraction was performed with ethyl acetate, and the organic phase was collected and dried over anhydrous sodium sulfate, and the solvent was dried by spinning and used directly for the next reaction.

Synthesis of N- (2-fluoro-5- ((4- (1- (3-hydroxypropyl) -1H-indol-3 yl) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide (1) (step e)

Acryloyl chloride (128.00mg, 1.4mmol) dissolved in 3.5mL of methylene chloride was added dropwise to a solution of 2e (500.00mg, 1.2mmol) and N, N-diisopropylethylamine (0.24mL, 1.4mmol) dissolved in 16mL of methylene chloride and cooled in an ice-water bath. The mixture was stirred for 1.5h, monitored by TLC, the reaction was complete, diluted with a small amount of dichloromethane and washed with saturated aqueous sodium bicarbonate. The organic phase was collected while the aqueous layer was re-extracted with dichloromethane, the organic phases were combined and dried over anhydrous sodium sulfate, and the rotary dried solvent was subjected to silica gel column chromatography and separated with dichloromethane: methanol ═ 200:1 to give compound 1 as a yellow solid 200mg, yield 35%.

¹H NMR(400MHz,DMSO-d₆):9.88(s,1H),8.61(d,J＝8.5Hz,1H),8.47(s,1H),8.32(dd,J＝8.7,6.5Hz,2H),8.09(s,1H),7.56(d,J＝8.2Hz,1H),7.31–7.18(m,2H),7.18–7.07(m,2H),6.59(dd,J＝17.0,10.2Hz,1H),6.26(dd,J＝17.1,2.0Hz,1H),5.76(dd,J＝10.2,2.0Hz,1H),4.69(s,1H),4.33(t,J＝7.0Hz,2H),3.87(s,3H),3.40(t,J＝6.2Hz,2H),1.96(p,J＝6.6Hz,2H).¹³C NMR(151MHz,DMSO):163.79,162.58,160.30,151.53,149.93,148.22,137.47,133.06,132.01,127.26,125.97,125.04,122.59,122.39,121.39,118.59,117.76,113.01,110.99,107.87,100.23,58.19,56.85,43.56,33.24.HRMS(EI)(m/z):calcd for C₂₅H₂₄FN₅O₃[M]⁺461.1863,found461.1862.

Example 2: synthesis of N- (2-fluoro-5- ((4- (1- (3-hydroxypropyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) -4-methoxyphenyl) propionamide (2) (step e)

Compound 2 was obtained according to the synthetic route for compound 1. Pale yellow solid, yield 23%.

¹H NMR(400MHz,DMSO-d₆):9.54(s,1H),8.49(d,J＝8.6Hz,1H),8.46(s,1H),8.35(d,J＝8.0Hz,1H),8.31(d,J＝5.4Hz,1H),8.00(s,1H),7.57(d,J＝8.2Hz,1H),7.27–7.21(m,2H),7.19–7.13(m,1H),7.08(d,J＝12.2Hz,1H),4.70(t,J＝4.9Hz,1H),4.34(t,J＝7.0Hz,2H),3.86(s,3H),3.42(q,J＝5.8Hz,2H),2.37(q,J＝7.5Hz,2H),1.97(p,J＝6.5Hz,2H),1.08(t,J＝7.6Hz,3H).¹³C NMR(151MHz,DMSO-d₆):172.49,162.45,160.50,157.76,151.61,150.02,147.90,137.45,132.90,126.00,125.07,122.56,121.31,118.90,118.12,113.02,110.97,107.84,100.13,58.20,56.81,43.53,33.25,29.28,10.19.HRMS(EI)(m/z):calcd for C₂₅H₂₄FN₅O₃[M]⁺463.2020,found 463.2021.

Synthesis of 3- (3- (2- ((4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxy-5-nitrophenyl) amino) pyrimidin-4-yl) -1H-indol-1-yl) propan-1-ol (2i)

N, N, N' -trimethylethylenediamine (35.00mg, 0.3mmol), N, N-diisopropylethylamine (46.00mg, 0.3mmol) and 2.5mL of N, N-dimethylacetamide were sequentially charged into a 10mL single-neck flask, reacted at 25 ℃ for 0.5h, followed by addition of 2d (108.00mg, 0.6mmol), warmed to 85 ℃ for 3h, monitored by TLC, stopped, allowed to stand to room temperature, extracted with saturated brine and ethyl acetate, the organic phase was collected and dried over anhydrous sodium sulfate, filtered, and the filtrate was subjected to silica gel column chromatography, separated with dichloromethane: methanol ═ 20:1 to give 84mg of 2i yellow-red solid, yield 65%.

¹H NMR(400MHz,Chloroform-d):9.50(s,1H),9.00(s,1H),8.30(d,J＝5.2Hz,1H),8.03–7.94(m,1H),7.67(s,1H),7.41–7.33(m,1H),7.19(dq,J＝8.0,5.3Hz,3H),6.71(s,1H),4.54(t,J＝5.8Hz,2H),3.73(s,3H),3.51(t,J＝5.2Hz,2H),2.95–2.88(m,2H),2.62(s,3H),2.43(q,J＝7.7Hz,2H),2.32(s,6H),2.00(p,J＝5.2Hz,2H).

Example 3: synthesis of N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -5- ((4- (1- (3 hydroxypropyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) -4-methoxyphenyl) propionamide (3)

Light orange solid, yield 26%.

¹H NMR(400MHz,Chloroform-d):9.84(s,1H),9.50(s,1H),9.00(s,1H),8.30(d,J＝5.2Hz,1H),8.03–7.94(m,1H),7.67(s,1H),7.41–7.33(m,1H),7.19(dq,J＝8.0,5.3Hz,3H),6.67(s,1H),4.44(t,J＝5.8Hz,2H),3.79(s,3H),3.48(t,J＝5.2Hz,2H),2.95–2.88(m,2H),2.59(s,3H),2.49(q,J＝7.7Hz,2H),2.38(s,2H),2.29(s,6H),2.00(p,J＝5.2Hz,2H),1.28(t,J＝7.6Hz,3H).¹³C NMR(151MHz,CDCl₃-d₆)173.20,161.94,159.52,158.07,144.35,137.12,134.86,134.36,129.30,127.77,126.26,121.84,121.03,120.37,113.58,110.55,109.76,107.95,104.66,57.49,57.23,56.11,55.79,45.29,44.32,42.54,32.54,31.26,10.90.HRMS(EI)(m/z):calcd forC₃₀H₃₉N₇O₃[M]⁺545.3114,found 545.3111.

Synthesis of N1- (3-methoxy-4-nitrophenyl) -N1, N2, N2-trimethylethane-1, 2-diamine (2g) (step f)

2f (425.00mg, 2.5mmol) was dissolved in 5mL of N, N-dimethylformamide, N, N, N' -trimethylethylenediamine (380.00mg, 3.72mmol) and potassium carbonate (750.00mg, 4.9mmol) were added sequentially thereto, the reaction was carried out at 110 ℃ for 3 hours, monitoring by TLC, the reaction was completed and cooled to room temperature, water was added and extraction was carried out with ethyl acetate, the organic phase was collected, dried over anhydrous sodium sulfate, suction filtration was carried out, and the rotary-dried filtrate was subjected to silica gel column chromatography and separated with dichloromethane: methanol at 100:1 to obtain 2g of a yellow-green liquid of 370mg in 60% yield.

¹H NMR(400MHz,Chloroform-d):8.00–7.86(m,1H),6.17(ddd,J＝9.5,6.8,2.4Hz,1H),6.11–6.00(m,1H),3.89(dd,J＝5.8,1.8Hz,3H),3.48(q,J＝6.8Hz,2H),3.09–2.98(m,3H),2.45(dt,J＝10.3,6.4Hz,2H),2.24(dd,J＝5.6,1.8Hz,6H)。LC-MS:m/z:254.3(M+H)⁺.

Synthesis of N1- (3-methoxy-4-aminophenyl) -N1, N2, N2-trimethylethane-1, 2-diamine (2h) (step g)

2g (250.00mg, 1.0mmol) was charged into a 50mL three-necked flask, 10mL methanol was added, palladium on carbon (50.00mg, 20%) was added after dissolution, hydrogen was substituted three times, reaction was carried out at 25 ℃ for 5h, celite was used to aid filtration, the filtrate was collected, spun dry and used directly in the next reaction.

Example 4: synthesis of 3- (3- (2- ((4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) pyrimidin-4-yl) -1H-indol-1-yl) propan-1-ol (4)

Green solid, yield 14%.

¹H NMR(400MHz,Chloroform-d):8.30–8.25(m,1H),8.23–8.17(m,2H),7.84(s,1H),7.38–7.31(m,1H),7.25(s,1H),7.22–7.13(m,3H),6.91(d,J＝5.3Hz,1H),6.38(dd,J＝8.8,2.7Hz,1H),6.32(d,J＝2.7Hz,1H),4.27(t,J＝6.7Hz,2H),3.80(s,3H),3.54(t,J＝5.8Hz,2H),3.47(t,J＝7.4Hz,2H),2.87(s,3H),2.63(t,J＝7.3Hz,2H),2.39(s,6H),2.01(p,J＝6.4Hz,2H).¹³C NMR(151MHz,DMSO-d₆):162.59,161.61,157.33,152.88,146.91,146.10,138.16,132.17,126.23,125.98,123.18,122.52,121.02,119.12,113.22,110.77,106.71,104.46,97.44,58.15,55.92,54.72,49.35,44.68,43.46,39.07,33.18,21.26.HRMS(EI)(m/z):calcd for C₂₇H₃₄N₆O₂[M]⁺474.2743,found 474.2745.

Synthesis of 7-vinyl-1H-indole (2k)

Methyl triphenyl iodide (1.30g, 3.2mmol) is filled into a 100mL three-neck flask, nitrogen is replaced three times, 10mL anhydrous tetrahydrofuran is added, a mixed solution of bis (trimethylsilyl) amino potassium (3.22mL, 3.2mmol) and 10mL anhydrous tetrahydrofuran is dropwise added into the three-neck flask through a constant pressure dropping funnel, the mixture reacts for 1h at 25 ℃, 2j (188.00mg, 1.3mmol) is added and reacts for 2h at 25 ℃, TLC monitoring is carried out, the reaction is stopped, a saturated ammonium chloride solution is quenched, water is added and ethyl acetate is extracted, anhydrous sodium sulfate is dried and suction filtration is carried out, and the dried filtrate is subjected to silica gel column chromatography and separated by petroleum ether ethyl acetate 15:1 to obtain an intermediate light green liquid 171mg with the yield of 90%. Under ice bath, filling the intermediate into a 100mL three-necked flask, adding 10mL anhydrous tetrahydrofuran, mixing, replacing with nitrogen for three times, dropwise adding borane tetrahydrofuran (5mL, 5.0mmol), returning to room temperature after dropwise addition, reacting for 1h, then dropwise and slowly adding sodium hydroxide solution (10mL, 1mol/L) and hydrogen peroxide (10mL, 30%), refluxing for 1h, monitoring by TLC, after the reaction is finished, cooling to room temperature, adding 15mL saturated sodium sulfite solution, extracting with ethyl acetate, collecting an organic phase, drying with anhydrous sodium sulfate, carrying out suction filtration, and carrying out silica gel column chromatography on a filtrate, and adding petroleum ether; ethyl acetate 20:1 separation gave 38mg of 2k light green liquid in 20% yield.

¹H NMR(400MHz,Chloroform-d):8.91(s,1H),7.46(d,J＝7.8Hz,1H),7.09(t,J＝2.8Hz,1H),6.98(t,J＝7.5Hz,1H),6.89(d,J＝7.0Hz,1H),6.47(dd,J＝3.2,2.0Hz,1H),3.87(t,J＝5.8Hz,2H),2.98(t,J＝5.7Hz,2H).LC-MS:m/z:162.2(M+H)⁺.

Example 5: synthesis of 2- (3- (2- ((4- ((2- (dimethylamino) ethyl) (methyl) amino) -2-methoxyphenyl) amino) pyrimidin-4-yl) -1H-indol-7-yl) ethanol (10)

Compound 10 was obtained according to the synthetic route for compound 4. Pale yellow solid, yield 23%.

¹H NMR(400MHz,DMSO-d₆):11.69(d,J＝3.0Hz,1H),8.24(d,J＝6.7Hz,1H),8.21(d,J＝3.0Hz,1H),8.18(d,J＝5.3Hz,1H),7.78(s,1H),7.63(d,J＝8.7Hz,1H),7.15(d,J＝5.3Hz,1H),6.99(q,J＝4.2Hz,2H),6.42(d,J＝2.6Hz,1H),6.32(dd,J＝8.8,2.6Hz,1H),4.71(t,J＝5.1Hz,1H),3.80(s,3H),3.71(td,J＝7.0,4.8Hz,2H),3.49(t,J＝7.2Hz,2H),3.03(t,J＝7.0Hz,2H),2.94(s,3H),2.57(s,2H),2.32(s,6H).¹³C NMR(151MHz,DMSO-d₆):167.54,164.71,156.53,153.88,147.54,144.32,139.21,134.32,127.63,126.32,122.78,122.62,120.32,119.54,113.73,110.81,106.82,103.86,97.74,60.25,55.52,54.42,49.43,44.78,43.46,33.32.HRMS(EI)(m/z):calcd for C₂₆H₃₂N₆O₂[M]⁺460.2587,found460.2581.

Example 6: synthesis of N- (2-fluoro-5- ((4- (6-fluoro-1- (3-hydroxypropyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) -4-methoxyphenyl) propionamide (5)

Compound 5 was obtained according to the synthetic route for compound 1. Yellow solid, yield 21%.

¹H NMR(400MHz,DMSO-d₆):9.54(s,1H),8.45(s,1H),8.33(dd,J＝21.5,6.9Hz,3H),8.17(s,1H),7.46(dd,J＝10.2,2.4Hz,1H),7.23(d,J＝5.5Hz,1H),7.09(d,J＝12.1Hz,1H),6.99(td,J＝9.2,2.3Hz,1H),4.30(t,J＝6.9Hz,2H),3.85(s,3H),3.41(t,J＝6.1Hz,2H),2.35(q,J＝7.5Hz,2H),1.96(p,J＝6.6Hz,2H),1.07(t,J＝7.6Hz,3H).¹³C NMR(151MHz,DMSO):172.50,162.42,160.42,158.85,152.08,150.45,148.58,137.74,133.58,124.70,123.93,122.68,119.81,118.12,113.25,109.64,107.61,100.35,97.58,58.09,56.79,43.67,33.09,29.24,10.13.HRMS(EI)(m/z):calcd for C₂₅H₂₅F₂N₅O₃[M]⁺481.1925,found 481.1927.

Synthesis of 2- (1H-indol-1-yl) ethanol (2l)

2-bromoethanol (230.00mg, 1.8mmol) was charged into a 25mL single-neck flask, dissolved by adding 4mL of dichloromethane, followed by sequentially adding tert-butyldimethylchlorosilane (300.00mg, 2.0mmol) and imidazole (140.00mg, 2.0mmol), reacted at 25 ℃ for 3h, suction filtered, washed with dichloromethane, collected the filtrate, and dried to give an intermediate, 2a (108.00mg, 0.9mmol) and sodium hydride (72.00mg, 1.8mmol) were weighed into a 50mL two-neck flask, replaced with nitrogen 3 times, anhydrous 7mL of N, N-dimethylformamide solution was slowly added under ice bath, after stirring for 15 minutes, the intermediate was added, warmed to 100 ℃ for reaction for 4h, monitored by TLC, cooled to room temperature after the reaction was completed, quenched with water and extracted with dichloromethane, the organic phase was collected, dried over anhydrous sodium sulfate, dried to give 230mg of 2l yellow liquid, 80% yield.

¹H NMR(400MHz,Chloroform-d):7.55(d,J＝7.9Hz,1H),7.29(d,J＝8.2Hz,1H),7.16–7.09(m,1H),7.06–6.97(m,2H),6.41(d,J＝3.1Hz,1H),4.09(t,J＝6.7Hz,2H),3.50(t,J＝6.0Hz,2H),.LC-MS:m/z:162.2(M+H)⁺.

Example 7: synthesis of N- (2-fluoro-5- ((4- (1- (2-hydroxyethyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) -4-methoxyphenyl) propanamide (6)

Compound 6 was obtained according to the synthetic route for compound 1. White solid, yield 23%.

¹H NMR(400MHz,DMSO-d₆):9.51(s,1H),8.48(d,J＝8.4Hz,1H),8.42(s,1H),8.32(dd,J＝13.6,6.7Hz,2H),7.97(s,1H),7.57(d,J＝8.1Hz,1H),7.22(t,J＝6.2Hz,2H),7.15(t,J＝7.5Hz,1H),7.07(d,J＝12.0Hz,1H),4.96(t,J＝5.1Hz,1H),4.32(t,J＝5.5Hz,2H),3.86(s,3H),3.79(q,J＝5.4Hz,2H),2.36(q,J＝7.5Hz,2H),1.07(t,J＝7.5Hz,3H).¹³C NMR(151MHz,DMSO):172.48,172.45,162.52,160.49,157.72,151.66,150.06,147.97,147.90,137.73,133.47,126.04,125.08,122.40,121.25,118.94,118.09,118.00,112.86,111.19,107.80,100.21,100.05,60.44,56.81,49.25,29.25,10.16.HRMS(EI)(m/z):calcdfor C₂₄H₂₄FN₅O₃[M]⁺449.1863,found449.1862.

Example 8: synthesis of N- (2-fluoro-5- ((4- (1- (4-hydroxybutyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) -4-methoxyphenyl) propanamide (7)

Compound 7 was obtained according to the synthetic route for compound 1. Yellowish solid in brown, yield 10%.

¹H NMR(400MHz,DMSO-d₆):9.54(s,1H),8.52(s,1H),8.48(d,J＝8.6Hz,1H),8.31(t,J＝7.3Hz,2H),8.13(s,1H),7.58(d,J＝8.2Hz,1H),7.28–7.21(m,2H),7.16(t,J＝7.5Hz,1H),7.08(d,J＝12.2Hz,1H),4.30(t,J＝7.0Hz,2H),3.86(s,3H),3.41(t,J＝6.4Hz,2H),2.37(q,J＝7.6Hz,2H),1.92–1.79(m,2H),1.51–1.35(m,2H),1.08(t,J＝7.6Hz,3H).¹³C NMR(151MHz,DMSO):172.52,137.54,126.01,122.79,122.52,121.61,112.83,111.21,107.61,100.44,100.27,60.71,56.84,46.54,30.14,29.29,26.88,10.18.HRMS(EI)(m/z):calcd for C₂₆H₂₈FN₅O₃[M]⁺477.2176,found 477.2179.

Synthesis of 1H-indole-2-carbaldehyde (2n)

Under ice bath, 2m (806.00mg, 5.0mmol) is put into a 100mL three-necked flask, 30mL of anhydrous tetrahydrofuran is added for dissolution, lithium aluminum hydride (380.00mg, 10.0mmol) is added in batches with stirring, the temperature is returned to room temperature after the addition, the reaction is carried out for 14h, the reaction is finished, a saturated ammonium chloride solution is quenched, the filtration is carried out by suction filtration, the filtrate is collected and extracted by ethyl acetate, dried by anhydrous sodium sulfate, and the solvent is dried by spinning to obtain an intermediate. The intermediate was dissolved in 10mL of dimethyl sulfoxide, 2-iodoxybenzoic acid (1.70g, 6.0mmol) was added, the reaction was carried out at 25 ℃ for 10h, monitored by TLC, a large amount of water was added to complete the reaction and extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the filtrate was subjected to silica gel column chromatography and separated with petroleum ether and ethyl acetate 20:1 to give 650mg of 2n white solid with a yield of 89%.

¹H NMR(400MHz,Chloroform-d):9.78(s,1H),9.30(s,1H),7.68(d,J＝8.0Hz,1H),7.43–7.37(m,1H),7.32(ddd,J＝8.2,6.8,1.1Hz,1H),7.23–7.16(m,1H),7.11(ddd,J＝8.0,6.9,1.0Hz,1H).LC-MS:m/z:146.1(M+H)⁺.

Synthesis of 3- (1H-indol-2-yl) propan-1-ol (2o)

Triethyl phosphonoacetate (6.67g, 29.7mmol) and sodium hydride (1.5g, 37.5mmol) were charged into a 100mL three-necked flask under ice bath, dissolved by slowly adding 20mL of anhydrous tetrahydrofuran, stirred for 1h, and then added dropwise to a 2n (3.60g, 24.8mmol) solution in 20mL of anhydrous tetrahydrofuran via a constant pressure dropping funnel, reacted at 25 ℃ for 10h, monitored by TLC, the reaction was quenched with saturated ammonium chloride solution, the solvent was dried by spinning, extracted with water and ethyl acetate, dried over anhydrous sodium sulfate, and the filtrate was dried by spinning to give an intermediate. Lithium aluminum hydride (2.00g, 52.6mmol) was slowly added to 30mL of anhydrous tetrahydrofuran solution under ice bath, followed by slowly adding intermediate solution dissolved in 30mL of anhydrous tetrahydrofuran thereto, reaction at 20 ℃ for 8h, TLC monitoring, reaction was terminated, quenching with water and extraction with ethyl acetate, drying over anhydrous sodium sulfate, and spin-drying the filtrate by silica gel column chromatography to obtain 2o white solid 2.58g with 60% yield by separating with petroleum ether ethyl acetate ═ 8: 1.

¹H NMR(400MHz,Chloroform-d):8.15(s,1H),7.44(dd,J＝7.2,1.6Hz,1H),7.20–7.13(m,1H),7.00(dtd,J＝16.2,7.2,1.3Hz,2H),6.16–6.09(m,1H),3.57(t,J＝6.1Hz,2H),2.70(t,J＝7.3Hz,2H),1.81(tt,J＝7.3,6.1Hz,2H).LC-MS:m/z:176.2(M+H)⁺.

Example 9: synthesis of N- (2-fluoro-5- ((4- (2- (3-hydroxypropyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) -4-methoxyphenyl) propanamide (8)

Compound 8 was obtained according to the synthetic route for compound 1. Tan solid, yield 23%.

¹H NMR(400MHz,DMSO-d₆):11.56(s,1H),9.45(s,1H),8.36(d,J＝5.3Hz,1H),8.29(d,J＝8.5Hz,1H),8.07–7.94(m,2H),7.36(d,J＝7.9Hz,1H),7.14–7.01(m,4H),4.60(d,J＝5.2Hz,1H),3.85(s,3H),3.44(q,J＝5.6Hz,2H),3.11(t,J＝7.8Hz,2H),2.31(q,J＝7.5Hz,2H),1.85(p,J＝6.7Hz,2H),1.05(t,J＝7.5Hz,3H).¹³CNMR(151MHz,DMSO):172.41,163.18,160.64,157.83,143.57,135.84,126.99,125.08,121.73,120.60,119.76,111.56,110.31,109.85,100.17,60.76,56.82,32.64,29.12,24.61,10.17.HRMS(EI)(m/z):calcdfor C₂₅H₂₆FN₅O₃[M]⁺463.2020,found463.2023.

Example 10: synthesis of N- (2-fluoro-5- ((4- (7- (2-hydroxyethyl) -1H-indol-3-yl) pyrimidin-2-yl) amino) -4-methoxyphenyl) propanamide (9)

Compound 9 was obtained according to the synthetic route for compound 1. Pale yellow solid, yield 22%.

¹H NMR(400MHz,DMSO-d₆):11.73(d,J＝3.0Hz,1H),9.50(s,1H),8.36(d,J＝8.6Hz,1H),8.30(d,J＝3.0Hz,1H),8.27(d,J＝5.4Hz,1H),8.25–8.19(m,1H),7.99(s,1H),7.29(d,J＝5.4Hz,1H),7.10–6.97(m,3H),4.71(t,J＝5.0Hz,1H),3.85(s,3H),3.72(td,J＝7.0,4.7Hz,2H),3.04(t,J＝7.0Hz,2H),2.33(q,J＝7.6Hz,2H),1.06(t,J＝7.5Hz,3H).¹³C NMR(151MHz,DMSO):172.48,172.45,162.52,160.49,157.72,151.66,150.06,147.97,148.91,137.73,133.47,126.04,125.08,122.40,121.25,118.95,118.06,118.00,112.86,112.19,107.80,100.21,99.05,60.44,56.81,49.25,29.25,10.16.HRMS(EI)(m/z):calcd for C₂₄H₂₄FN₅O₃[M]⁺449.1863,found449.1862.

Example 11: synthesis of N- (2-fluoro-5- ((6- (6-fluoro-1- (3-hydroxypropyl) -1H-indol-3-yl) pyrimidin-4-yl) amino) -4-methoxyphenyl) propionamide (16)

Compound 16 was obtained according to the synthetic route for compound 1. White solid, yield 30%.

¹H NMR(400MHz,DMSO-d₆):9.53(s,1H),8.72(s,1H),8.54(d,J＝1.0Hz,1H),8.31(dd,J＝8.9,5.6Hz,1H),8.26(d,J＝8.5Hz,1H),8.15(s,1H),7.47(dd,J＝10.2,2.4Hz,1H),7.29(s,1H),7.12–6.98(m,2H),4.67(t,J＝5.0Hz,1H),4.29(t,J＝6.8Hz,2H),3.87(s,3H),2.35(q,J＝7.5Hz,2H),1.92(q,J＝6.5Hz,2H),1.08(t,J＝7.6Hz,3H).¹³C NMR(151MHz,DMSO):172.59,161.36,158.75,158.30,137.65,131.87,124.23,122.90,122.51,120.49,117.99,113.68,109.42,109.27,100.36,97.58,58.04,56.80,43.43,33.03,29.15,10.18.HRMS(EI)(m/z):calcd for C₂₅H₂₅F₂N₅O₃[M]⁺481.1925,found481.1927.

Synthetic schemes for trisubstituted imidazoles

Wherein, the reaction reagents and conditions of each step are as follows:

(h)SeO2,1,4-dioxane,H2O,100℃,overnight；

(i)HCl,H2O,25℃,5h；

(j)NH4OAc,MeOH,25℃,overnight,6％；

(k)Pd/C,H2,MeOH,25℃,6h,69％；

(l)2-(1-oxoisoindolin-2-yl)-2-phenylacetic acid,HATU,DIPEA,DMF,25℃,6h,5％；

(m)NIS,MeCN,80℃,16h,30％；

(n)1,4-dioxane,K2CO3,H2O,Pd(dppf)Cl2·CH2Cl2,N-phenyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-amine,90℃,12h,32％.

synthesis of 3- (4- (4-nitrophenyl) -1H-imidazol-2-yl) propan-1-ol (2t) (step j)

Selenium dioxide (5.36g, 48.7mmol) is filled into a 250mL three-necked flask, 60mL of 1, 4-dioxane and 20mL of water are added to be stirred and dissolved at 55 ℃, after the selenium dioxide is completely dissolved, 2p (2.00g, 12.1mmol) is added, the temperature is raised to 100 ℃, the reaction is carried out for 10 hours, the reaction is cooled to room temperature, the filtration is carried out, the kieselguhr is used for assisting filtration, the filtrate is collected and extracted by ethyl acetate, anhydrous sodium sulfate is dried, and the filtrate is subjected to silica gel column chromatography to obtain 2q of intermediate. Under ice bath, weighing hydrochloric acid (14mL, 2mol/L) and filling into a 50mL three-necked bottle, dropwise adding 2r (1.40g, 20.0mmol), stirring for 0.5h, returning to room temperature, continuing to react for 5h, detecting by TLC, extracting with dichloromethane after the reaction is finished, collecting an organic phase, and spin-drying the solvent to obtain an intermediate 2 s. 2s and ammonium acetate (4.90g, 63.6mmol) are sequentially filled into a 250mL single-neck bottle, 30mL of methanol is added for dissolution, 2p is dissolved in 60mL of methanol, the single-neck bottle is dropwise added with the solution, the solution reacts at 25 ℃ overnight, TLC detection is carried out, the solvent is concentrated after the reaction is ended, saturated sodium bicarbonate is used for adjusting the pH value to 7, ethyl acetate is added for extraction, anhydrous sodium sulfate is used for drying, suction filtration is carried out, and the filtrate is subjected to silica gel column chromatography and is separated by dichloromethane and methanol which are 150:1, so that 260mg of 2t yellow liquid is obtained, and the yield is 6%.

¹H NMR(400MHz,DMSO-d₆):8.25–8.17(m,2H),8.02–7.94(m,2H),7.81(s,1H),3.48(t,J＝6.3Hz,2H),2.72(dd,J＝8.2,7.1Hz,2H),1.85(dq,J＝8.1,6.4Hz,2H)..LC-MS:m/z:248.1(M+H)⁺.

Synthesis of 3- (4- (4-aminophenyl) -1H-imidazol-2-yl) propan-1-ol (2u) (step k)

2t (184.00mg, 0.8mmol), palladium on carbon (40.00mg, 20%) and 12mL of methanol were sequentially charged into a 100mL single-neck flask, replaced with hydrogen three times, reacted for 6h at 25 ℃, monitored by TLC, the reaction was terminated, filtered through celite, and the filtrate was subjected to silica gel column chromatography, and separated with dichloromethane: methanol 40:1 to give 112mg of 2u of an orange-yellow liquid in 69% yield.

¹H NMR(400MHz,DMSO-d₆):7.39–7.31(m,2H),7.09(s,1H),6.60–6.51(m,2H),3.47(t,J＝6.3Hz,2H),2.67(t,J＝7.6Hz,2H),1.89–1.73(m,2H).LC-MS(m/z):218.1(M+H)⁺.

Synthesis of 2- (1-oxoisoindolin-2-yl) -2-phenylacetic acid (2w)

The method comprises the steps of filling phthalic dicarboxaldehyde (1.00g, 7.5mmol) into a 250mL single-neck bottle, dissolving the phthalic dicarboxaldehyde with 35mL acetonitrile, adding D, L-phenylglycine (1.13g, 7.5mmol) and 2mL acetic acid, carrying out nitrogen replacement for 3 times, carrying out reflux reaction for 3 hours, monitoring by TLC (thin layer chromatography), finishing the reaction, carrying out suction filtration, leaching a filter cake, and carrying out vacuum drying to obtain 1.3g of 2w black solid with the yield of 65%.¹H NMR(400MHz,DMSO-d₆):13.39(s,1H),7.75(d,J＝7.5Hz,1H),7.60(td,J＝7.4,1.2Hz,1H),7.57–7.49(m,2H),7.50–7.37(m,5H),6.00(s,1H),4.64(d,J＝17.4Hz,1H),3.92(d,J＝17.4Hz,1H).LC-MS:m/z:268.1(M+H)⁺.

Example 12: synthesis of N- (3- (2- (3-hydroxypropyl) -1H-imidazol-4-yl) phenyl) -2- (1-oxoisoindolin-2-yl) -2-phenylacetamide (11)

2u (176.00mg, 0.8mmol), 2w (108.00mg, 0.4mmol) and 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (308.00mg, 0.8mmol) were charged into a 25mL single-neck flask, 2.5mL of N, N-dimethylformamide was added to dissolve, N, N-diisopropylethylamine (212.00mg, 1.6mmol) was added under stirring, the reaction was carried out at 25 ℃ for 6 hours, TLC was monitored, the reaction was completed, water and ethyl acetate were used for extraction, the organic phase was collected, dried over anhydrous sodium sulfate and filtered, and the filtrate was subjected to silica gel column chromatography, and separated with dichloromethane: methanol: 200:1 to give 11 mg of a white solid with a yield of 5%.

¹H NMR(400MHz,DMSO-d₆):10.57(s,1H),8.01(d,J＝1.8Hz,1H),7.76(d,J＝7.5Hz,1H),7.65–7.35(m,12H),7.29(t,J＝7.9Hz,1H),6.24(s,1H),4.85(d,J＝17.7Hz,1H),3.97(d,J＝17.6Hz,1H),3.45(t,J＝6.3Hz,2H),2.70(t,J＝7.6Hz,2H),1.82(p,J＝6.8Hz,2H).¹³C NMR(151MHz,DMSO):168.63,168.18,149.08,142.88,139.32,135.93,132.16,132.00,129.49,129.08,128.93,128.43,124.12,123.41,120.28,117.77,115.72,60.62,59.02,48.90,31.58,24.94.HRMS(ESI)(m/z):calcd forC₃₉H₃₄N₆O₃[M+H]⁺467.2083,found 467.2084.

Example 13: synthesis of N- (4- (2- (3-hydroxypropyl) -1H-imidazol-4-yl) phenyl) -2- (1-oxoisoindolin-2-yl) -2-phenylacetamide (12)

Compound 12 was obtained according to the synthetic route for compound 11. Yellowish solid in brown, yield 8%.

¹H NMR(400MHz,DMSO-d₆):10.55(s,1H),7.76(d,J＝7.5Hz,1H),7.70–7.55(m,6H),7.54–7.37(m,7H),6.24(s,1H),4.85(d,J＝17.7Hz,1H),3.98(d,J＝17.7Hz,1H),3.46(t,J＝6.3Hz,2H),2.70(t,J＝7.6Hz,2H),1.83(p,J＝6.6Hz,2H).¹³C NMR(151MHz,DMSO)168.74,168.23,150.06,148.90,147.73,142.88,138.65,135.71,133.80,132.21,131.94,129.56,129.05,128.46,127.57,126.69,125.79,124.14,123.41,120.11,114.30,112.01,60.23,59.05,48.89,30.85,23.80.HRMS(ESI)(m/z):calcd for C₃₉H₃₄N₆O₃[M+H]⁺467.2083,found 467.2084.

Synthesis of 3-chloro-N-phenylpyridin-2-amine (2 ×)

Palladium acetate (13.5mg, 0.1mmol) and 2, 2 '-bis- (diphenylphosphino) -1, 1' -binaphthyl (37.5mg, 0.1mmol) were charged into a 50mL single-neck flask, replaced with nitrogen three times, dissolved in 10mL of toluene, stirred at 25 ℃ for 10min, and then the solution was transferred to a solution containing 2, 3-dichloropyridine (441.00mg, 3.0mmol), aniline (329.00uL, 3.6mmol), potassium carbonate (8.30g, 60.0mmol), and 17mL of toluene, replaced with nitrogen 3 times, refluxed for 4h, monitored by TLC, the reaction was cooled to room temperature, filtered with celite, the filtrate was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered with suction, the filtrate was collected, and the spin-dried filtrate was chromatographed on a silica gel column, separated with petroleum ether ethyl acetate 100:1 to give 267mg of 2X orange liquid with a yield of 43%.

¹H NMR(400MHz,Chloroform-d):8.03(dd,J＝4.8,1.7Hz,1H),7.57–7.51(m,2H),7.46(dd,J＝7.7,1.7Hz,1H),7.29–7.22(m,2H),7.00–6.93(m,1H),6.90(s,1H),6.60(dd,J＝7.7,4.8Hz,1H).LC-MS:m/z:205.1(M+H)⁺.

Synthesis of N-phenyl-3- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2-amine (2y)

Tris (dibenzylideneacetone) dipalladium (12.00mg, 0.013mmol), pinacol diboron (1.00g, 3.9mmol), 2-dicyclohexyl-p-2, 4, 6-triisopropylbiphenyl (13.00mg, 0.026mmol) and potassium acetate (385.00mg, 3.9mmol) were charged into a 50mL three-necked flask, replaced three times with nitrogen, 2x (267.00mg, 1.3mmol) was weighed and dissolved in 8mL of anhydrous 1, 4-dioxane and added to the three-necked flask, reacted at 110 ℃ for 16h, monitored by TLC, cooled to room temperature at the end of the reaction, filtered with celite as an aid, the filtrate was extracted with ethyl acetate, dried over anhydrous sodium sulfate, the dried filtrate was chromatographed on a silica gel column, separated with petroleum ether ethyl acetate ═ 20:1 to give 2y light liquid which was used directly in the next reaction.

Synthesis of N- (3- (2- (3-hydroxypropyl) -5-iodo-1H-imidazol-4-yl) phenyl) -2- (1-oxoisoindolin-2-yl) -2-phenylacetamide (2v) (step m)

11(104.00mg, 0.2mmol) was charged into a 25mL single neck flask, dissolved by addition of 6mL acetonitrile, added N-iodosuccinimide (61.00mg, 0.3mmol) portionwise with stirring, warmed to 80 ℃, reacted for 16h, monitored by TLC, stopped, cooled to room temperature, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the rotary filtrate was chromatographed on a silica gel column with dichloromethane: methanol 100:1 to give 40mg of 2v pale yellow solid in 30% yield.

¹H NMR(400MHz,DMSO-d₆):10.51(s,1H),8.11(d,J＝1.8Hz,1H),7.76(d,J＝7.5Hz,1H),7.65–7.35(m,11H),7.29(t,J＝7.9Hz,1H),6.25(s,1H),4.82(d,J＝17.7Hz,1H),3.82(d,J＝17.6Hz,1H),3.41(t,J＝6.3Hz,2H),2.81(t,J＝7.6Hz,2H),1.91(p,J＝6.8Hz,2H).LC-MS:m/z:593.1(M+H)⁺.

Example 14: synthesis of N- (3- (2- (3-hydroxypropyl) -5- (2- (phenylamino) pyridin-3-yl) -1H-imidazol-4-yl) phenyl) -2- (1-oxoisoindolin-2-yl) -2-phenylacetamide (13)

Potassium carbonate (24.00mg, 0.2mmol) and [1, 1' -bis (diphenylphosphino) ferrocene ] dichloropalladium dichloromethane complex (6.00mg, 0.007mmol) were sequentially charged into a 25mL three-necked flask, nitrogen was substituted three times, a 2v, 2y solution dissolved in 4mL of 1, 4-dioxane and 1mL of water was added, the reaction was carried out at 90 ℃ for 14 hours, TLC monitoring was carried out, the reaction was cooled to room temperature after completion, extraction was carried out with ethyl acetate, drying was carried out with anhydrous sodium sulfate, suction filtration was carried out, an organic phase was collected, and the spin-dried filtrate was subjected to silica gel column chromatography and separated with dichloromethane: methanol 100:1 to obtain 13 mg of a white solid with a yield of 32%.

¹H NMR(400MHz,Chloroform-d):9.95(s,1H),8.98(s,1H),8.03(d,J＝4.8Hz,1H),7.69(d,J＝7.6Hz,1H),7.61–7.32(m,11H),7.24–7.10(m,3H),6.97–6.73(m,3H),6.49(s,1H),6.44(q,J＝7.1,6.5Hz,1H),4.86(d,J＝17.5Hz,1H),4.00(d,J＝17.5Hz,1H),3.56(t,J＝5.5Hz,2H),2.83(d,J＝7.0Hz,2H),1.91(s,2H).¹³CNMR(151MHz,DMSO):168.54,168.31,154.02,149.33,144.23,143.29,141.73,139.49,135.23,132.71,132.11,131.84,129.37,129.17,129.07,129.01,128.85,128.31,128.10,126.36,124.35,124.11,120.72,120.33,118.34,116.37,114.64,60.24,59.34,48.69,31.70,23.87.HRMS(ESI)(m/z):calcd for C₃₉H₃₄N₆O₃[M+H]⁺635.2771,found 635.2772.

Example 15: synthesis of N- (4- (2- (3-hydroxypropyl) -5- (2- (phenylamino) pyridin-3-yl) -1H-imidazol-4-yl) phenyl) -2- (1-oxoisoindolin-2-yl) -2-phenylacetamide (14)

Compound 14 was obtained according to the synthetic route for compound 13. White solid, yield 25%.

¹H NMR(400MHz,DMSO-d₆):12.41(s,1H),10.72(s,1H),10.66(s,1H),8.05(dd,J＝4.8,1.9Hz,1H),7.76(d,J＝7.6Hz,1H),7.68(dd,J＝15.3,8.2Hz,3H),7.63–7.54(m,2H),7.54–7.40(m,5H),7.36(dd,J＝8.4,2.0Hz,3H),7.28(t,J＝7.7Hz,2H),6.89(t,J＝7.4Hz,1H),6.62(dd,J＝7.5,4.9Hz,1H),6.23(s,1H),4.83(d,J＝17.7Hz,1H),4.61(t,J＝5.1Hz,1H),3.98(d,J＝17.7Hz,1H),3.56(q,J＝6.0Hz,2H),2.82(t,J＝7.6Hz,2H),1.97(p,J＝6.9Hz,2H).¹³C NMR(151MHz,DMSO):168.74,168.21,153.08,148.23,145.73,142.89,141.89,138.39,135.73,132.20,132.01,131.94,129.57,129.17,129.08,129.02,128.95,128.46,128.00,126.66,124.16,123.41,120.88,120.00,118.60,115.27,114.34,60.60,59.04,48.89,31.40,24.77.HRMS(ESI)(m/z):calcd for C₃₉H₃₄N₆O₃[M+H]⁺635.2771,found 635.2772.

Synthesis of N-phenyl-3- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2-amine (2z)

Sequentially filling [1, 1' -bis (diphenylphosphino) ferrocene ] palladium dichloride dichloromethane complex (31.00mg, 0.04mmol), pinacol diboron (480.00mg, 2.0mmol), 4-bromo-7-azaindole (184.00mg, 1.0mmol) and potassium acetate (240.00mg, 3.0mmol) into a 25mL three-necked flask, performing nitrogen replacement three times, adding anhydrous 1, 4-dioxane, reacting at 90 ℃ for 18h, monitoring by TLC, stopping the reaction, cooling to room temperature, performing suction filtration, performing kieselguhr assisted filtration, extracting with ethyl acetate, drying with anhydrous sodium sulfate, performing suction filtration, and spin-drying the filtrate to obtain 2z which is directly used for the next reaction.

Example 16: synthesis of N- (3- (2- (3-hydroxypropyl) -5- (2- (phenylamino) pyridin-3-yl) -1H-imidazol-4-yl) phenyl) -2- (1-oxoisoindolin-2-yl) -2-phenylacetamide (15)

Potassium carbonate (59.00mg, 0.4mmol) and [1, 1' -bis (diphenylphosphino) ferrocene ] dichloropalladium dichloromethane complex (14.00mg, 0.017mmol) are sequentially put into a 25mL three-necked flask, nitrogen is replaced three times, 2v and 2z solutions dissolved in 5mL of 1, 4-dioxane and 1.25mL of water are added, the reaction is carried out at 90 ℃ for 12h, TLC monitoring is carried out, the reaction is finished and cooled to room temperature, ethyl acetate is used for extraction, anhydrous sodium sulfate is used for drying, suction filtration is carried out, and the spin-dried filtrate is subjected to silica gel column chromatography and is separated by dichloromethane and methanol at 100:1, so that 15 mg of white solid is obtained, and the yield is 10%.

¹H NMR(400MHz,Chloroform-d):9.95(s,1H),8.98(s,1H),8.03(d,J＝4.8Hz,1H),7.69(d,J＝7.6Hz,1H),7.61–7.32(m,11H),7.24–7.10(m,3H),6.97–6.73(m,3H),6.49(s,1H),6.44(q,J＝7.1,6.5Hz,1H),4.86(d,J＝17.5Hz,1H),4.00(d,J＝17.5Hz,1H),3.56(t,J＝5.5Hz,2H),2.83(d,J＝7.0Hz,2H),1.91(s,2H).¹³CNMR(151MHz,DMSO):168.68,168.17,149.57,142.86,139.22,136.37,135.79,132.18,131.93,130.07,129.53,128.97,128.44,126.64,124.37,124.13,123.40,118.83,117.59,115.26,100.73,60.53,58.87,48.84,29.48,22.56,14.43.HRMS(ESI)(m/z):calcd for C₃₉H₃₄N₆O₃[M+H]⁺635.2771,found635.2772.

The biological evaluation method comprises the following steps:

tyrosine kinase: EGFR (WT)

EGFRT790M/L858R(LR/TM)

EGFRT790M/L858R/C797S(LR/TM/CS)

ELISA kinase Activity detection

The inhibition of the kinase activity by the compound was calculated by measuring the ability of the kinase to phosphorylate substrates by Enzyme-Linked Immunosorbent Assay (ELISA). The kinase was EGFRL858R/T790M/C797S (from BPS Bioscience).

The ELISA main steps are as follows: an enzyme reaction substrate Poly (Glu, Tyr)4:1 is diluted into 2.5 mu g/hole by PBS without potassium ions, and reacts at 37 ℃ for 12-16h to coat an enzyme label plate for later use. Reaction buffer (50mM HEPES pH 7.4, 20mM MgCl) was added to each well₂，0.1mM MnCl₂，0.2mM Na₃VO₄1mM DTT) was added to a compound or solvent control, followed by addition of kinase to initiate the reaction, followed by shaking at 37 ℃ for 1 h. The plate was washed three times with T-PBS and 100. mu.L of antibody PY99 (T-PBS containing 5mg/mL BSA, 1:500 dilution) was added and subjected to shake reaction at 37 ℃ for 0.5 h. After washing the plate with T-PBS, 100. mu.L of goat anti-mouse IgG labeled with horseradish peroxidase (T-PBS containing 5mg/mL BSA, 1:2000 dilution) was added and subjected to shake reaction at 37 ℃ for 0.5 hour. After washing the plate again, 0.03% H was added₂O₂OPD (0.1mol/L, pH 5.4 citrate buffer solution) of 2mg/mL, 100 mu L/well of color development solution, and reaction at 25 ℃ for 1-10min in a dark place. Add 50. mu.L/well 2M H₂SO₄The reaction was stopped and read using a tunable wavelength microplate reader (SpectraMax Plus384, Molecular Devices) at a wavelength of 490 nm. IC50 values were obtained from the inhibition curves.

The results of the enzyme activity test are given in the following table:

^akinase activity assays were performed using ELISA-based EGFR-TK assays. Data are mean values of at least two independent determinations and are expressed as mean ± SD (standard deviation).^bDouble mutant (EGFR)^L858R/T790M).^cTriple mutant (EGFR)^L858R ^/T790M/C797S).

^aKinase activity assays were performed using ELISA-based EGFR-TK assays. Data are mean values of at least two independent determinations and are expressed as mean ± SD.

Cell proliferation inhibition assay

Tyrosine kinase: EGFR^{T790M/19DEL/C797S}

The test method comprises the following steps: the mouse original B cell strain BaF3 cell strain and EGFR are selected for experiments^{19del/T790M/C797S}-BaF 3. Cells in logarithmic growth phase are selected at the beginning of experiment, 3 groups of cells expressing 19del + T790M + C797S mutation are set in a 96-well plate, the number of each group of cells is 5000, the highest concentration of the drug is respectively 10 mu mol/L, the drug concentration is decreased progressively according to the ratio of 1:2, after 72 hours, CCK810ul is added into each well, the activity of the cells is measured at 450nm by using a thermomo enzyme-labeling instrument, and finally IC is obtained by fitting a curve₅₀The value is obtained.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A compound represented by the general formula I or a stereoisomer or an optical isomer or a pharmaceutically acceptable salt thereof:

in the formula (I), the compound is shown in the specification,

w is CH or N;

x is CH or N;

y is CH or N or C halogen;

z is CH or N;

Wherein n is an integer of 0-4;

p is 1-2C 1-C3 alkyl groups or absent;

Wherein when R is⁴Is composed of

When is at R⁴There may be one or more Q at each position;

x is O, S, NH;

2. The compound of claim 1, or a stereoisomer or optical isomer thereof, or a pharmaceutically acceptable salt thereof,

w is CH;

x is CH or N;

y is CH or N;

z is CH;

R³selected from: halogen, NR⁷R⁸；

3. A compound selected from the group consisting of stereoisomers or optical isomers thereof, or pharmaceutically acceptable salts thereof:

4. a compound represented by the general formula II:

in the formula (I), the compound is shown in the specification,

R¹selected from:

wherein n is an integer of 0-4;

p is 1-2C 1-C3 alkyl groups or absent;

Wherein when R is⁴Is composed of

When is at R⁴There may be one or more Q at each position;

x is O, S, NH;

R⁴selected from:

R⁵selected from: H. substituted or unsubstituted

R⁶Selected from: H. substituted or unsubstituted C1-C3 alkyl.

5. The compound of claim 4, or a stereoisomer or optical isomer thereof, or a pharmaceutically acceptable salt thereof,

R⁴selected from:

R⁵selected from: substituted or unsubstituted

R⁶Selected from: H.

6. a compound selected from the group consisting of stereoisomers or optical isomers thereof, or pharmaceutically acceptable salts thereof:

7. a pharmaceutical composition comprising a compound of any one of claims 1-6, or a stereoisomer or optical isomer thereof, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier or excipient.

8. Use of a compound of any one of claims 1-6, or a stereoisomer or an optical isomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 7, for the manufacture of a medicament for the treatment or prevention of an EGFR-mediated disease or for the inhibition of EGFR.

9. The use of claim 9, wherein the EGFR-mediated disease is cancer.

10. The use of claim 9, wherein the cancer is selected from the group consisting of: non-small cell lung cancer, lung adenocarcinoma, lung squamous carcinoma, breast cancer, prostate cancer, glioma, ovarian cancer, head and neck squamous carcinoma, cervical cancer, esophageal cancer, liver cancer, kidney cancer, pancreatic cancer, colon cancer, skin cancer, leukemia, lymphoma, gastric cancer, multiple myeloma, and solid tumors.