WO2023046114A1 - Pteridinone derivative and use thereof - Google Patents

Abstract

Disclosed in the present invention is a compound as represented by formula I or a pharmaceutically acceptable salt thereof. Such compounds exhibit an excellent effect of targeted inhibition of non-canonical mutations of EGFR and have a good anti-tumor activity. Further disclosed in the present invention are the use of the compound in the preparation of a drug for preventing or/and treating EGFR-mutation-mediated diseases, such as cancer, and a pharmaceutical composition containing the compound.

Description

Pteridinone derivatives and their applications technical field

The invention belongs to the field of medicinal chemistry; in particular, the invention relates to a compound targeting EGFR mutant protein, a preparation method thereof, and an application in treating EGFR-mediated diseases, such as tumors.

Background technique

Studies have found that EGFR plays an important role in the formation and development of various cancers, so the EGFR tyrosine kinase target has become the preferred target for cancer targeted therapy, especially for non-small cell lung cancer. In the course of treatment, small molecule inhibitors targeting EGFR have made good clinical progress, such as the first-generation reversible inhibitors gefitinib and erlotinib, the second-generation irreversible afatinib and Daco Tini and the third dioxitinib that has the activity of overcoming the acquired resistance mutation EGFR T790M mutation. But not all patients with EGFR mutations benefit. Among them, NSCLC with EGFR exon 20 insertion mutation (EGFR 20ins) has poor or ineffective response to most EGFR inhibitor targeted therapies, and these mutations are often classified as non-classic mutations. These non-canonical mutations mainly include insertion mutations and point mutations of exons 18-21, point mutations and insertion mutations of ERBB2.

Therefore, the research and development of drugs targeting non-canonical mutant EGFR inhibitors has great clinical significance and application prospects.

Contents of the invention

The object of the present invention is to provide a compound or a pharmaceutically acceptable salt thereof that targets and inhibits non-classical mutant EGFR. The compound should have excellent antitumor activity.

In a first aspect, the present invention provides a compound represented by formula I or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof:

In formula I

R ₁ is independently selected from hydrogen, substituted or unsubstituted C ₁ -C _{10 alkyl} , C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, optionally substituted C ₃ -C ₈ cycloalkyl, Optionally substituted or unsubstituted aryl, substituted or unsubstituted benzyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aromatic heterocyclic group;

R ₂ , R ₃ , R ₄ , R ₅ are independently selected from H, halogen, substituted (preferably halogen substituted, more preferably fluorine substituted) or unsubstituted C ₁ -C ₆ (preferably C ₁ -C ₃ ) alkoxy or Substituted (preferably halogen substituted, more preferably fluorine substituted) or unsubstituted C ₁ -C ₆ (preferably C ₁ -C ₃ ) deuterated alkoxy, substituted or unsubstituted C ₁ -C ₆ (preferably C ₁ -C ₃ ) Alkyl, _NRcRd ; wherein _Rc and _Rd are each independently selected from hydrogen _{, C1-3} alkyl;

G is a benzene ring, a five-membered or six-membered heterocyclic ring or a C ₃ -C ₈ cycloalkyl group or does not exist;

R ₆ is independently selected from hydrogen, unsubstituted or halogen-substituted C ₁ -C ₄ alkyl, nitro, amino, halogen, hydroxyl, C ₁ -C ₆ alkoxy, optionally substituted C ₁ -C ₆ acyl Oxygen, optionally substituted C ₁ -C ₆ amido, optionally substituted C ₁ -C ₆ acyl; wherein, when G is a benzene ring, R ₆ is meta-substituted;

m is an integer of 0-3;

R ₇ is independently selected from substituted or unsubstituted NH ₂ , substituted or unsubstituted heterocyclic group, substituted or unsubstituted aromatic heterocyclic group, substituted or unsubstituted C ₁ -C ₁₀ alkyl;

A is selected from the following group or is absent:

X is selected from the following group or does not exist: substituted or unsubstituted C _1-3 alkylene (preferably -CH ₂ -) or deuterated alkylene (preferably -CD ₂ -), -O-, -C(= O)-, -C(=O)NHN=-;

Y is selected from the following group or is absent: -NHC(=O)-, -C(=O)NH-, -=NNHC(=O)NH-, _-CH2- , -O-;

L is selected from the following group or absent: C ₁ -C ₁₀ alkylene, C ₁ -C ₁₀ heteroalkylene, -A'-(CH ₂ ) _m' -W-(CH ₂ ) _n' -, - (CH ₂ ) _m' -W-(CH ₂ ) _n' -O-(CH ₂ ) _V -and-(CH ₂ ) _m' -W-[(CH ₂ ) _n' -O] _u -(CH ₂ ) _v -;

A' is selected from the following group or does not exist: 5-membered arylene and 6-membered arylene;

W is selected from: phenylene, 5-membered heteroarylene, 6-membered heteroarylene, C ₁ -C ₁₀ heterocyclylene and C ₁ -C ₁₀ alkylene;

m' is 0, 1, 2, 3, 4, 5, 6, 7 or 8;

n' is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9;

each independent u is independently 2, 3 or 4;

v is 1, 2, 3 or 4

B is selected from the following group or absent:

R is selected from: hydrogen, methyl and fluorine;

Q ₁ is selected from: -C(R _2a )=and -N=;

Q ₂ is selected from: -C(R _2b )= and -N=;

Q ₃ is selected from: -C(R _2c )= and -N=;

R _2a , R _2b , and R _2c are each independently selected from: hydrogen, -C(=O)-;

Z is selected from: -CH ₂ -, -C(=O)-.

In a specific embodiment, the compound is a compound shown in the following formula II:

In formula II

R ⁷ is independently selected from: H, substituted (preferably halogen substituted, more preferably fluorine substituted) or unsubstituted C ₁ -C ₆ (preferably C ₁ -C ₃ ) alkyl, substituted (preferably halogen substituted, more preferably fluorine substituted) or unsubstituted C ₁ -C ₆ (preferably C ₁ -C ₃ ) deuterated alkyl;

R ⁸ is independently selected from: H, substituted (preferably halogen substituted, more preferably fluorine substituted) or unsubstituted C ₁ -C ₃ alkyl, substituted (preferably halogen substituted, more preferably fluorine substituted) or unsubstituted C ₁ -C ₃ deuterated alkyl.

In a specific embodiment, in formula II,

R ⁷ is independently selected from: methyl, deuteromethyl CD ₃ , trifluoromethyl, ethyl, deuteroethyl (eg, CD ₂ CH ₃ , CH ₂ CD ₃ ), CH ₂ CF ₃ ;

R ⁸ is independently selected from: hydrogen, methyl, deuteromethyl CD ₃ , ethyl, deuteroethyl (eg, CD ₂ CH ₃ , CH ₂ CD ₃ ).

In a specific embodiment, the compound is selected from:

In a second aspect, the present invention provides a pharmaceutical composition comprising the compound described in the first aspect, or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof and optionally pharmaceutically acceptable excipients.

In the third aspect, the present invention provides the compound described in the first aspect, or its pharmaceutically acceptable salt, solvate, stereoisomer or prodrug in the preparation of prevention or treatment of abnormal expression of EGFR protein activity, ERBB2 overexpression Use in medicine for diseases related to point mutations thereof.

In a specific embodiment, said EGFR is mutant EGFR.

In a specific embodiment, the mutant EGFR includes at least one of the following mutations: EGFR sensitivity mutation L858R and 19del, EGFR T790M mutation, EGFR 18-21 exon point mutation and insertion mutation, ERBB2 overexpression and point mutation Mutations and insertional mutations.

In a specific embodiment, the EGFR 18-21 exon point mutation and insertion mutation contain:

Point mutations of exon 18 G719X, E709X, K716A, K728A and deletion mutation at codon 709;

Exon 19 insertion mutation I744-K745insKIPVAI, K745-E746insIPVAIK, K745-E746insVPVAIK, K745-E746insTPVAIK and point mutation D761Y;

Insertion mutations and point mutations in exon 20 include: A763-Y764insFQEA, A763-Y764insFHEA, V769-D770insASV, V769-D770insDNP, D770-N771insNPG, D770-N771insNPH, D770-N771insSVD, D770-N771insP7insASVDN, D771SVG-N7 、N771-H773dupNPH、P772-H773insPNP、P772-H773insPR、H773-V774insH、A763-Y764insFQEA、H773-V774insPH、H773-V774insNPH、N771-P772insH、H771-P772insN、H773-V774insAH、D770delinsGY、V774-C775insHV等以及20号外Exon point mutation S768I;

Exon 21 point mutation L861Q;

ERBB2 point mutations V777L, D769Y, R896C, P1170A and insertion mutations V777-G778insCG, P780-Y781insGSP, etc.

In a specific embodiment, the disease is cancer and is selected from one or more of the following: non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, pancreatic cancer, prostate cancer, ovarian cancer Carcinoma, glioma, head and neck squamous cell carcinoma, cervical cancer, esophageal cancer, liver cancer, kidney cancer, colon cancer, skin cancer, leukemia, lymphoma, stomach cancer or multiple myeloma.

In the fourth aspect, the present invention provides a method for preventing or treating diseases related to abnormal expression of EGFR protein activity, ERBB2 overexpression and point mutations thereof, the method comprising treating a therapeutically effective amount of the compound described in the first aspect, or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof, or the pharmaceutical composition described in the second aspect to a subject in need thereof.

In a preferred embodiment, said subject is a mammal, preferably a human.

In a preferred embodiment, the EGFR is a mutant EGFR.

In a preferred embodiment, the mutant EGFR includes at least one of the following mutations: EGFR sensitivity mutation L858R and 19del, EGFR T790M mutation, EGFR 18-21 exon point mutation and insertion mutation, ERBB2 overexpression and point mutation Mutations and insertional mutations.

In a preferred embodiment, the EGFR 18-21 exon point mutation and insertion mutation contain:

Point mutations of exon 18 G719X, E709X, K716A, K728A and deletion mutation at codon 709;

Exon 19 insertion mutation I744-K745insKIPVAI, K745-E746insIPVAIK, K745-E746insVPVAIK, K745-E746insTPVAIK and point mutation D761Y;

Insertion mutations and point mutations in exon 20 include: A763-Y764insFQEA, A763-Y764insFHEA, V769-D770insASV, V769-D770insDNP, D770-N771insNPG, D770-N771insNPH, D770-N771insSVD, D770-N771insP7insASVDN, D771SVG-N7 、N771-H773dupNPH、P772-H773insPNP、P772-H773insPR、H773-V774insH、A763-Y764insFQEA、H773-V774insPH、H773-V774insNPH、N771-P772insH、H771-P772insN、 H773-V774insAH、D770delinsGY、V774-C775insHV等以及20号外Exon point mutation S768I;

Exon 21 point mutation L861Q;

ERBB2 point mutations V777L, D769Y, R896C, P1170A and insertion mutations V777-G778insCG, P780-Y781insGSP, etc.

In a preferred embodiment, the disease is cancer and is selected from one or more of the following: non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, pancreatic cancer, prostate cancer, ovarian Carcinoma, glioma, head and neck squamous cell carcinoma, cervical cancer, esophageal cancer, liver cancer, kidney cancer, colon cancer, skin cancer, leukemia, lymphoma, stomach cancer or multiple myeloma.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1 shows the in vivo drug efficacy (EGFR ^L858R/T790M xenograft tumor) activity of compound 2 and compound 3;

Figure 2 shows the in vivo drug effect (EGFR ^L858R/T790M xenograft tumor) activity of compound 5;

Figure 3 shows the drug-time curves of compounds 0, 1, 2, 3, 4, 5 in SD rat plasma.

Detailed ways

After extensive and in-depth research, the inventors have discovered a batch of small molecular compounds with brand-new structures that can target and inhibit non-classical mutant EGFR. These compounds have the activity of inhibiting EGFR protein and good antitumor activity, so these compounds can be used to prevent or/and treat various cancers, and have great application prospects in the field of medicine. The present invention has been accomplished on this basis.

Definition of Terms:

Some groups involved in this paper are defined as follows:

Herein, "alkyl" refers to a saturated branched or linear alkyl group with a carbon chain length of 1-10 carbon atoms, and preferred alkyl groups include 2-8 carbon atoms, 1-6, 1-4 carbon atoms, alkyl groups ranging from 1 to 3 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isoisoyl, heptyl, and the like. Alkyl groups may be substituted by one or more substituents, such as halogen or haloalkyl. For example, the alkyl group may be an alkyl group substituted with 1-4 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 those with a length of 1 to 6 carbon atoms, more preferably those with a length of 1 to 3 carbon atoms. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, and the like. Alkoxy may be substituted by one or more substituents, such as halogen or haloalkyl. For example, an alkoxy group can be an alkyl group substituted with 1-4 fluorine atoms, or an alkyl group can be an alkyl group substituted with a fluoroalkyl group.

Herein, "alkenyl" generally means a monovalent hydrocarbon group having at least one double bond, usually containing 2 to 8 carbon atoms, preferably containing 2 to 6 carbon atoms, and may be straight or branched. Examples of alkenyl groups include, but are not limited to, vinyl, propenyl, isopropenyl, butenyl, isobutenyl, hexenyl, and the like.

Herein, "acylamino" refers to a group with the structural formula "-R'-NH-C(O)-R", wherein R' can be selected from hydrogen or alkyl, and R can be selected from alkyl, alkenyl , alkynyl, alkyl substituted by NRcRd, alkenyl substituted by NRcRd and alkynyl substituted by NRcRd, alkyl substituted by halogen, alkenyl substituted by cyano, wherein Rc and Rd can be selected from alkyl group and alkenyl group.

Herein, "aryl" refers to a monocyclic, bicyclic or tricyclic aromatic group containing 6 to 14 carbon atoms, including phenyl, naphthyl, phenanthrenyl, anthracenyl, indenyl, fenyl, tetralin base, indanyl, etc. Aryl may be optionally substituted with 1-5 (eg, 1, 2, 3, 4 or 5) substituents selected from the group consisting of halogen, C1-4 aldehyde, C1-6 alkyl, cyano, Nitro, amino, amido, hydroxy, hydroxymethyl, halogen substituted alkyl (eg trifluoromethyl), halogen substituted alkoxy (eg trifluoromethoxy), carboxyl, C1-4 alkoxy , ethoxy formyl, N(CH3) and C1-4 acyl, etc., heterocyclyl or heteroaryl, etc.

Herein, "heterocyclic group" includes, but is not limited to, 5-membered or 6-membered heterocyclic groups containing 1-3 heteroatoms selected from O, S or N, including but not limited to furyl, thienyl, pyrrolyl , pyrrolidinyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, pyranyl, pyridyl, pyrimidinyl, pyrazinyl, piperidinyl, morpholinyl, etc.

Herein, "aromatic heterocyclic group" refers to a group containing 5-14 ring atoms, and 6, 10 or 14 electrons are shared on the ring system. And the contained ring atoms are carbon atoms and optional 1-3 heteroatoms selected from oxygen, nitrogen and sulfur. Useful aromatic heterocyclic groups include piperazinyl, morpholinyl, piperidinyl, pyrrolidinyl, thienyl, furyl, pyryl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, including but not limited to Yu pyrimidinyl and so on. The aromatic heterocyclic group can be optionally substituted by 1-5 (for example, 1, 2, 3, 4 or 5) substituents selected from the group consisting of halogen, C1-4 aldehyde, C1-6 straight chain or branched Alkanyl, cyano, nitro, amino, hydroxy, hydroxymethyl, halogen substituted alkyl (eg trifluoromethyl), halogen substituted alkoxy (eg trifluoromethoxy), carboxyl, C1- 4 alkoxy, ethoxy formyl, N(CH3) and C1-4 acyl.

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

Herein, "optionally substituted" means that the substituent it modifies may be optionally substituted with 1-5 (eg, 1, 2, 3, 4 or 5) substituents selected from the group consisting of halogen, C1 -4 aldehyde, C1-6 straight chain or branched alkyl, cyano, nitro, amino, hydroxyl, hydroxymethyl, halogen substituted alkyl (such as trifluoromethyl), halogen substituted alkoxy ( Examples include trifluoromethoxy), carboxy, C1-4 alkoxy, ethoxyformyl, N(CH3) and C1-4 acyl.

Compounds of the invention

Herein, the compound of the present invention refers to a compound represented by the following general formula I, or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof:

The substituents in the formula are as defined above.

Further, the present invention provides a compound represented by formula II, or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof:

The substituents in the formula are as defined above.

In a specific embodiment, the present invention provides the following compounds capable of inhibiting EGFR activity, and these compounds have significant inhibitory activity on mutant EGFR (L858R/T790M, 20ins):

Based on the teaching of the present invention and common knowledge in the field, those skilled in the art will understand that each group in the compound of the present invention can be further substituted, so as to obtain derivatives that can have the same or similar activity as the compound disclosed in the present invention. things. Each group in the compound of the present invention can be substituted by various conventional substituents in the art, as long as the substitution does not violate the rules of chemical synthesis or valence.

The term "substituted" as used herein refers to the replacement of one or more hydrogen atoms on a specified group with a specified substituent. The specific substituents may be correspondingly described substituents above, specific substituents appearing in each embodiment or conventional substituents in the art. Therefore, in the present invention, the substituents in the general formula can also be independently the corresponding groups in the specific compounds in the examples; Combinations of some of the substituents shown in and other specific substituents appearing in the examples. It is not difficult for those skilled in the art to prepare a compound having such a combination of substituents and to detect whether the obtained compound is active based on conventional technical means in this field. In other words, based on the teachings of the present invention, those skilled in the art can synthesize various compounds falling within the protection scope of the present invention, and these compounds are not limited to the specific compounds disclosed in the Examples section of the specification; The disclosed specific compounds also include various compounds composed of specific substituents at a certain substituting position in these specific compounds and substituents at other substituting positions in the general formula. Due to space limitations, they are not listed here.

The term "pharmaceutically acceptable salt" as used herein refers to a salt formed of a compound of the present invention with an acid or a base which is suitable for use as a medicine. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts are the salts of the compounds of the invention with acids. Acids suitable for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, Maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenemethanesulfonic acid, benzenesulfonic acid and other organic acids; and acidic amino acids such as aspartic acid and glutamic acid.

Unless otherwise specified, the structural formulas described in the present invention are intended to include all isomeric forms (such as enantiomers, diastereoisomers and geometric isomers (or conformational isomers)): for example, containing asymmetric The R, S configuration of the center, the (Z), (E) isomer of the double bond, etc. Accordingly, individual stereochemical isomers of the compounds of the present invention or mixtures thereof as enantiomers, diastereomers or geometric isomers (or conformers) are within the scope of the present invention.

As the term is used herein, "tautomer" means that structural isomers with different energies can cross a low energy barrier and thereby interconvert. For example, proton tautomers (ie, prototropism) include interconversions via migration of a proton, such as 1H-indazole and 2H-indazole. Valence tautomers include interconversion by recombination of some of the bonding electrons.

The term "solvate" as used herein refers to a complex in which the compound of the present invention coordinates with solvent molecules to form a specific ratio.

The term "hydrate" as used herein refers to a complex formed by coordination between the compound of the present invention and water.

Furthermore, as a compound with pharmaceutical activity, the compound of the present invention can obviously be used as a medicine. Therefore, in addition to the various properties that have been examined in the Examples, the compounds of the present invention can also possess various activities inherent as drugs. For example, in vivo activity, bioavailability, druggability, toxicity, differential inhibitory activity, etc. Based on the teachings of the present invention and conventional technical means in the art, those skilled in the art know how to obtain various compounds within the scope of the present invention and detect various activities of various compounds within the scope of the present invention; in other words, based on the teachings of the present invention With conventional technical means in the field, those skilled in the art know how to repeat, verify and implement the present invention.

Pharmaceutical compositions and methods of administration

Since the compounds of the present invention have targeted inhibition of non-classical mutant EGFR, especially for mutant EGFR

(L858R/T790M, 20ins) has significant inhibitory activity, the compound of the present invention and its various crystal forms, pharmaceutically acceptable inorganic or organic salts, prodrugs or solvates or hydrates, and compounds containing the present invention as the main The pharmaceutical composition of active ingredients can be used to prevent and/or treat diseases related to abnormal expression of EGFR protein activity, ERBB2 overexpression and point mutations thereof.

In a specific embodiment, said EGFR is mutant EGFR. For example, the mutant EGFR includes at least one of the following mutations: EGFR sensitivity mutations L858R and 19del, EGFR T790M mutation, EGFR exon 18-21 point mutation and insertion mutation, ERBB2 point mutation and insertion mutation.

In a preferred example, the EGFR exon 18-21 point mutation and insertion mutation include: exon 18 G719X, E709X, K716A, K728A point mutation and codon 709 deletion mutation; exon 19 insertion mutation I744 -K745insKIPVAI, K745-E746insIPVAIK, K745-E746insVPVAIK, K745-E746insTPVAIK and point mutation D761Y; exon 20 insertion mutation and point mutation include: A763-Y764insFQEA, A763-Y764insFHEA, V769-D770insASV, V710insNPD-ND777 D770-N771insNPH、D770-N771insSVD、D770-N771insASVDN、D770-N771insG、N771-P772insSVDNP、N771-H773dupNPH、P772-H773insPNP、P772-H773insPR、H773-V774insH、A763-Y764insFQEA、H773-V774insPH、H773-V774insNPH、N771- P772insH, H771-P772insN, H773-V774insAH, D770delinsGY, V774-C775insHV, etc. and exon 20 point mutation S768I; exon 21 point mutation L861Q; ERBB2 point mutation V777L, D769Y, R896C, P1170A and insertion mutation V777-G778insC , P780-Y781insGSP, etc.

In a preferred embodiment, the disease is cancer, for example, one or more selected from the following: non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, pancreatic cancer, prostate cancer, Ovarian cancer, glioma, head and neck squamous cell carcinoma, cervical cancer, esophageal cancer, liver cancer, kidney cancer, colon cancer, skin cancer, leukemia, lymphoma, stomach cancer, or multiple myeloma.

The pharmaceutical composition of the present invention comprises the compound of the present invention and pharmaceutically acceptable excipients or carriers in a safe and effective amount range. Wherein, "safe and effective dose" refers to: the amount of the compound is sufficient to obviously improve the condition without causing severe side effects. Usually, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably 10-200 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.

"Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use, and must have sufficient purity and low toxicity. "Compatibility" herein means that the components of the composition can be blended with the compound of the present invention and with each other without significantly reducing the efficacy of the compound. Examples of pharmaceutically acceptable carrier parts include cellulose and derivatives thereof (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid , magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween

), wetting agent (such as sodium lauryl sulfate), coloring agent, flavoring agent, stabilizer, antioxidant, preservative, pyrogen-free water, etc.

The administration method of the compound or pharmaceutical composition of the present invention is not particularly limited, and representative administration methods include (but not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous).

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

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shell materials, such as enteric coatings and others well known in the art. They may contain opacifying agents and, in such compositions, the release of the active compound or compounds may be in a certain part of the alimentary canal in a delayed manner. Examples of usable embedding components are polymeric substances and waxy substances. The active compounds can also be in microencapsulated form, if desired, 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 compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, etc.

Besides such inert diluents, the compositions can 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, 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 non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols, and suitable mixtures thereof.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

When administered in combination, the pharmaceutical composition also includes one or more (2, 3, 4, or more) other pharmaceutically acceptable compounds. One or more of the other pharmaceutically acceptable compounds may be administered simultaneously, separately or sequentially with the compound of the present invention.

When using a pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage when administered, for a person with a body weight of 60kg, the daily The dosage is usually 1-2000 mg, preferably 20-500 mg. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

Advantages of the present invention:

1. The compound of the present invention has the activity of inhibiting EGFR protein, especially mutant EGFR (L858R/T790M, 20ins). Therefore, the compound of the present invention can be used for the treatment of diseases related to abnormal expression of EGFR protein, such as various cancers.

2. The compounds of the present invention have excellent pharmacokinetic properties;

3. The compound of the present invention has excellent antitumor activity and antitumor activity, so it has great application prospects in the field of medicine.

The technical scheme of the present invention is further described below in conjunction with specific implementation examples, but the following examples do not constitute a limitation to the present invention, and all various application methods adopted according to the principles and technical means of the present invention all belong to the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, the conventional conditions or the conditions suggested by the manufacturer are usually followed. Percentages and parts are by weight unless otherwise indicated.

Example 1: N-(3-(2-((2-(methoxy-d ₃ )-4-(4-methylpiperazin-1-yl)phenyl)amino)-7-oxo- 6-phenylpterin-8(7H)-yl)phenyl)acrylamide synthetic route (compound 1):

Synthesis of 4-fluoro-2-(methoxy-d ₃ )-1-nitrobenzene

NaH (0.20g, 8.28mmol) and anhydrous tetrahydrofuran (3mL) were mixed in a 50mL two-necked flask, protected by N ₂ , and stirred in an ice bath. 5-Fluoro-2-nitrophenol (1.00g, 6.37mmol) was dissolved in anhydrous tetrahydrofuran (4mL), slowly added to the reaction solution, stirred at 0°C for 10min, and CD ₃ I (2.78g, 19.11mmol) was added rapidly. The temperature was raised to reflux, and the reaction was continued for 19h. After cooling to room temperature, the mixture was quenched with water, extracted with ethyl acetate, washed with saturated aqueous sodium chloride three times, the organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo, and subjected to column chromatography to obtain the product (0.84 g, 72.00%).

Synthesis of 1-(3-(methoxy-d ₃ )-4-nitrophenyl)-4-methylpiperazine

4-fluoro-2-(methoxy-d ₃ )-1-nitrobenzene (11.52g, 66.22mmol), N-methylpiperazine (6.62g, 62.34mmol), N,N-diisopropyl Ethylamine (12.81g, 99.33mmol) and acetonitrile (20.00mL) were mixed in a 100mL one-necked bottle, and stirred at 90°C for 12h. The reaction mixture was spin-dried, extracted with ethyl acetate, washed three times with saturated aqueous sodium chloride, the organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo, and recrystallized from petroleum ether to obtain a yellow solid (16.30 g, 87.00%).

Synthesis of 2-(methoxy-d ₃ )-4-(4-methylpiperazin-1-yl)aniline

Mix 4-fluoro-2-(methoxy-d ₃ )-1-nitrobenzene (1.67g, 6.56mmol), 10% palladium on carbon (0.65g, 0.65mmol) and ethanol (8.00mL) in a 50mL In the two-neck flask, protected by hydrogen, stirred at 100°C for 24 hours. The reaction mixture was suction filtered, then extracted with dichloromethane, washed 3 times with saturated aqueous sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo, and subjected to column chromatography (PE-DCM:MeOH30:1) to obtain a brown oil .

N-(3-((2-((2-(methoxy-d ₃ )-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-nitropyrimidine-4- Synthesis of base) amino) phenyl) acrylamide

Weigh 2-(methoxy-d ₃ )-4-(4-methylpiperazin-1-yl)aniline (1.22 g, 5.45 mmol) into a 50 mL single-necked bottle, add THF and stir well. Weigh N-(3-((2-chloro-5-nitropyrimidin-4-yl)amino)phenyl)acrylamide (1.67g, 5.45mmol) into another 50mL single-necked bottle, add THF, DIPEA (1.05g, 8.17mmol) was slowly added dropwise and stirred at room temperature for 1h. The reaction mixture was spin-dried, extracted with dichloromethane, washed three times with saturated aqueous sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo. Methanol was recrystallized to obtain a reddish-brown solid.

N-(3-((5-amino-2-((2-(methoxy-d ₃ )-4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl ) amino) phenyl) acrylamide synthesis

N-(3-((2-((2-(methoxy-d ₃ )-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-nitropyrimidine-4 -yl)amino)phenyl)acrylamide (1.18g, 2.33mmol), iron powder (0.50g, 9.32mmol), ammonium chloride (0.62g, 11.65mmol) and ethanol:water (8mL, 4:1) mixed In a 100mL single-necked bottle, stir at 80°C for 24h. The reaction mixture was suction-filtered, the filtrate was adjusted to alkali, extracted with ethyl acetate, washed three times with saturated aqueous sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, and concentrated in vacuo to obtain a purple-black solid (0.45 g, 36.00%).

N-(3-(2-((2-(methoxy-d ₃ )-4-(4-methylpiperazin-1-yl)phenyl)amino)-7-oxo-6-phenyl Synthesis of pterin-8(7H)-yl)phenyl)acrylamide

N-(3-((5-amino-2-((2-(methoxy-d ₃ )-4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidine-4- Base)amino)phenyl)acrylamide (0.36g, 0.75mmol), ethyl benzoylformate (0.26g, 1.12mmol) and ethanol (10mL, 4:1) were mixed in a microwave reaction flask, and a few drops Acetic acid was reacted under microwave conditions (200PSI, 50W, 120°C) for 2h. The reaction mixture was adjusted to base, extracted with ethyl acetate, washed three times with saturated aqueous sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo, and subjected to column chromatography (DCM:MeOH=25:1) to obtain a reddish-brown solid (0.15 g, 30.00%).

¹ H NMR (400MHz, DMSO-d ₆ ) δ10.42(s, 1H), 8.87(s, 1H), 8.42(s, 1H), 8.20(dd, J=6.8, 3.1Hz, 2H), 7.89( d,J=7.0Hz,1H),7.75(s,1H),7.54(t,J=8.0Hz,1H),7.51-7.47(m,3H),7.35(d,J=8.3Hz,1H), 7.15(d, J=8.0Hz, 1H), 6.54(d, J=2.5Hz, 1H), 6.46(dd, J=16.9, 10.1Hz, 1H), 6.27(dd, J=16.9, 2.1Hz, 1H ),6.04(s,1H),5.78(dd,J=10.0,2.1Hz,1H),3.05(s,4H),2.45(s,4H),2.23(s,3H).HRMS(ESI):[ M+H] ⁺ calcd for C ₃₃ H ₂₆ D ₆ N ₈ O ₃ , 592.2864; found 592.2866. HPLC purity: 97.54%, retention time＝6.844min.

Embodiment 2: N-(3-(2-((2-methoxy-4-(piperazin-1-yl) phenyl) amino)-7-oxo-6-phenylpteridine-8( Synthesis of 7H)-yl)phenyl)acrylamide (intermediate)

Preparation of N-(3-nitrophenyl)acrylamide

Weigh 3-nitroaniline (10.00g, 72.4mmol) into a 1000mL three-necked flask, add 250mL of dichloromethane, stir at 0°C, and then add triethylamine (14g, 108.6mmol) dropwise. Another acrylamide (7.8g, 86.87mmol) was dissolved in 100mL of dichloromethane, and added dropwise to the above reaction solution. After the dropwise addition was completed, stirring was continued at room temperature for 1 h, and TLC traced that the raw material was almost completely converted. Rotary evaporation under reduced pressure, adding 100ml of 20% potassium hydroxide solution to the residue, a large amount of white solids were separated out, suction filtered after ultrasonication, and then the filter cake was washed with 50mL of dichloromethane and dried in vacuo to obtain 9.4g of crude product with a yield of about 68 %. Dichloromethane was recovered under reduced pressure.

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.73(t, J=2.1Hz, 1H), 7.98(dd, J=8.1, 1.2Hz, 1H), 7.91(dd, J=8.2, 1.6Hz ,1H),7.62(t,J=8.2Hz,1H),6.47(dd,J=17.0,10.1Hz,1H),6.31(dd,J=17.0,1.9Hz,1H),5.81(dd,J= 10.1,1.9Hz,1H).

Preparation of N-(3-aminophenyl)acrylamide

Weigh N-(3-nitrophenyl)acrylamide (5g, 26.1mmol) in a 250mL round bottom flask, add solvent 150mL ethanol to dissolve, 20ml water, then add iron powder (5.8g, 104.4mmol), chlorination Ammonium (6.7g, 130.5mmol) was reacted with stirring at 80°C, followed by TLC, and the reaction was completed after 3h. The reaction solution was filtered with diatomaceous earth, the filtrate was rotary evaporated, and the residue was extracted with EA and 20ml 20% NaOH water, repeated three times. Several layers were combined and evaporated under reduced pressure to obtain 3.5 g of a light green solid with a yield of about 83%. The product was directly used in the next step without purification.

¹ H NMR (400MHz, DMSO-d ₆ ): ¹ H NMR (400MHz, DMSO): δ8.73 (t, J = 2.1Hz, 1H), 7.98 (dd, J = 8.1, 1.2Hz, 1H), 7.91 (dd, J=8.2,1.6Hz,1H),7.62(t,J=8.2Hz,1H),6.47(dd,J=17.0,10.1Hz,1H),6.31(dd,J=17.0,1.9Hz, 1H), 5.81(dd, J=10.1, 1.9Hz, 1H), 5.62(s, J=13.2, 1.9Hz, 1H).

Preparation of N-(3-((2-chloro-5-nitropyrimidin-4-yl)amino)phenyl)acrylamide

Weigh 2,4-dichloro-5-nitropyrimidine (5g, 25.77mmol) and DIPEA (4.99g, 38.66mmol) into a 100mL round-bottomed three-necked flask, add 40mL DMF to dissolve and place at room temperature to stir for reaction. N-(3-aminophenyl)acrylamide (4.1g, 25.77mmol) was weighed and dissolved in 20ml DMF, and the mixture was slowly added dropwise into a round bottom flask, and the reaction was detected by TLC. After 5h, the reaction was completed. Acetonitrile was removed by rotary evaporation under reduced pressure, and the residue was extracted with DCM for three times. The organic layers were combined and rotary evaporated under reduced pressure. The residue was crystallized with EA:PE=1:4, filtered with suction, the filter cake was washed with 20ml of EA:PE=1:4 mixture, and the filter residue was vacuum-dried to obtain 6.1g of orange-red solid with a yield of about 84%.

¹ H NMR (400MHz, DMSO-d6) δ10.45(s,1H),10.33(s,1H),9.15(s,1H),7.96–7.85(m,1H),7.55(dd,J=7.9, 2.1Hz, 1H), 7.40(t, J=8.1Hz, 1H), 7.26(dd, J=7.8, 2.1Hz, 1H), 6.48(dd, J=17.0, 10.1Hz, 1H), 6.28(dd, J=17.0,2.1Hz,1H),5.78(dd,J=10.0,2.1Hz,1H).

tert-butyl 4-(4-nitro-3-methoxyphenyl)piperazine-1-carboxylate

Weigh 2-nitro-5-fluoroanisole (10g, 58.47mmol) and DIEA (11.31g, 87.7mmol) into a 50mL round-bottomed three-neck flask, add 100mL DMF to dissolve, and then add piperazine-1- Tert-butyl carboxylate (16.31 g, 87.7 mmol) was stirred at 90° C. for 6 h, followed by TLC until complete conversion of the starting material. Under stirring at room temperature, 50 mL of deionized water was added to the reaction solution, a large amount of yellow solids precipitated out, filtered with suction, the filter cake was washed with 50 mL of deionized water, and dried in vacuo to obtain pure 1-(3-methoxy-4 -Nitrophenyl)-4-methylpiperazine yellow solid 18g, yield about 95%.

¹ H NMR (400MHz, DMSO-d6) δ7.91(d, J=9.3Hz, 1H), 6.59(dd, J=9.4, 2.5Hz, 1H), 6.53(d, J=2.5Hz, 1H), 3.91(s,3H),1.43(s,8H).

tert-butyl 4-(4-amino-3-methoxyphenyl)piperazine-1-carboxylate

Weigh 1-(3-methoxy-4-nitrophenyl)-4-methylpiperazine (2.5g, 10mmol) in a 250mL round bottom flask, add solvent 150mL ethanol to dissolve, then add 530mg Pd/C (containing 10% Pd), hydrogen gas was introduced with a hydrogen balloon, and after stirring at room temperature for about 3 h, TLC tracked until the raw material was completely converted. The reaction solution was suction-filtered with celite, washed with 50 mL of ethanol, and then the solvent was removed by rotary evaporation to obtain 2-methoxy-4-(4-methyl-1-piperazine)aniline as a solid with a purity of about 99%. The product was directly used in the next step without purification.

¹ H NMR (400MHz, DMSO-d6) δ6.52(d, J=4.5Hz, 1H), 6.51(d, J=1.3Hz, 1H), 6.31(dd, J=8.3, 2.4Hz, 1H), 4.26(s, 2H), 3.74(s, 3H), 3.43(d, J=10.1Hz, 4H), 2.87(t, J=5.1Hz, 4H), 1.41(s, 9H).

N-(3-((2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-nitropyrimidin-4-yl)amino)benzene Preparation of amides

Weigh N-(3-((2-chloro-5-nitropyrimidin-4-yl)amino)phenyl)acrylamide (2g, 7.38mmol) into a 100mL round bottom flask, dissolve with 50mL tetrahydrofuran, and then Add tert-butyl 4-(4-amino-3-methoxyphenyl)piperazine-1-carboxylate (2.18g, 7.38mmol) and N,N-diisopropylethylamine (1.06g, 8.3mmol ), under argon protection, stirred at room temperature for about 8 h, followed by TLC until the complete conversion of the raw material. Under stirring at room temperature, 50 mL of deionized water was added to the reaction solution, a large amount of reddish-brown solids precipitated, filtered with suction, the filter cake was washed with 50 mL of deionized water, and vacuum-dried to obtain 3.4 g of reddish-brown solids, with a yield of about 86%. .

¹ H NMR(400MHz,DMSO-d6)δ10.30(s,1H),10.19(s,1H),9.22(s,1H),9.04(s,1H),7.70(s,1H),7.48(d ,J=8.2Hz,1H),7.40(d,J=8.7Hz,1H),7.33(d,J=8.2Hz,1H),7.19(t,J=8.1Hz,1H),6.63(s,1H ),6.44(dd,J=16.9,10.1Hz,1H),6.28(t,J=13.4Hz,2H),5.77(dd,J=10.0,2.1Hz,1H),3.76(s,3H),3.46 (t,J=5.0Hz,4H),3.08(s,4H),1.44(s,9H).

4-(4-((((4-((3-acrylamidophenyl)amino)-5-aminopyrimidin-2-yl)amino)-3-methoxyphenyl)piperazine-1-carboxylic acid tert Preparation of butyl ester

Weigh N-(3-((2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-nitropyrimidin-4-yl)amino ) phenylamide (1.5g, 2.61mmol) was placed in a 100mL round-bottomed flask, dissolved in 50mL of ethanol as a solvent, 10ml of water, then iron powder (0.58g, 10.44mmol), ammonium chloride (0.67g, 13.05mmol) , stirring reaction at 80°C, TLC tracking reaction, and the reaction ended after 3h. The reaction solution was filtered with diatomaceous earth, the filtrate was rotary evaporated, and the residue was extracted with EA and 10ml 20% NaOH water, repeated three times. There were several layers combined, and the vacuum rotary After steaming, 0.92 g of a light green solid was obtained, with a yield of about 61%. The product was directly used in the next step without purification.

¹ H NMR (400MHz, DMSO-d6) δ10.60(s,1H),8.68(s,1H),8.02–7.95(m,1H),7.90–7.76(m,3H),7.57(t,J= 8.1Hz, 1H), 7.29(d, J=8.2Hz, 1H), 6.63(d, J=2.5Hz, 1H), 6.52(dd, J=16.9, 10.1Hz, 1H), 6.36(dd, J= 8.8,2.5Hz,1H),6.30(dd,J=17.0,1.9Hz,1H),5.81(dd,J=10.0,2.0Hz,1H),3.79(s,3H),3.45(t,J=5.1 Hz,4H),3.01(t,J=5.1Hz,4H),2.44(s,3H),1.42(s,9H).

4-(4-(((8-(3-acrylamidophenyl)-7-oxo-6-phenyl-7,8-dihydropterin-2-yl)amino)-3-methoxy Preparation of tert-butyl phenyl)piperazine-1-carboxylate

Weigh 4-(4-(((4-((3-acrylamidinyl)amino)-5-aminopyrimidin-2-yl)amino)-3-methoxyphenyl)piperazine-1- Put tert-butyl formate (1.5g, 2.7mmol) in a 50mL round bottom flask, add 1mL glacial acetic acid, 15mL absolute ethanol, then add ethyl benzoylformate (515mg, 3mmol), heat to reflux and stir for about 8h, The solvent was removed by rotary evaporation under reduced pressure, and the residue was extracted with DCM and 10ml of 20% NaOH water for three times. The organic layers were combined and subjected to column chromatography to obtain 0.71 g of an orange-red solid with a yield of 40%.

¹ H NMR (400MHz, DMSO-d6) δ10.41(s, 1H), 8.87(s, 1H), 8.44(s, 1H), 8.20(dd, J=6.7, 3.1Hz, 2H), 7.89(d ,J=8.0Hz,1H),7.75(d,J=2.1Hz,1H),7.55(t,J=8.1Hz,1H),7.51–7.45(m,3H),7.39–7.32(m,1H) ,7.16(dt,J=7.9,1.3Hz,1H),6.58(d,J=2.6Hz,1H),6.47(dd,J=16.9,10.1Hz,1H),6.27(dd,J=16.9,2.1 Hz,1H),6.04(d,J=5.4Hz,1H),5.77(dd,J=10.1,2.1Hz,1H),3.78(s,3H),3.45(t,J=5.1Hz,4H), 3.00(s,4H),1.43(s,9H).

N-(3-(2-((2-methoxy-4-(piperazin-1-yl)phenyl)amino)-7-oxo-6-phenylpteridin-8(7H)-yl ) Preparation of phenyl) acrylamide

Weigh 4-(4-(((8-(3-acrylamidophenyl)-7-oxo-6-phenyl-7,8-dihydropterin-2-yl)amino)-3- Methoxyphenyl)piperazine-1-carboxylate tert-butyl (50mg, 0.07mmol) was placed in a 25mL round-bottomed flask, 20mL of DCM was added to dissolve, then trifluoroacetic acid (24mg, 0.21mmol) was added, stirred at room temperature, After TLC tracking the reaction for 2 hours, the reaction was completed. Rotary evaporation under reduced pressure, the residue was extracted with DCM and saturated aqueous sodium bicarbonate solution, the organic layers were combined, and rotary evaporation gave a reddish-brown solid, 16 mg, yield 42%, which was immediately used for the next step.

¹ H NMR (400MHz, DMSO-d6) δ10.43(s,1H),8.87(s,1H),8.41(s,1H),8.20(dd,J＝6.5,3.2Hz,2H),7.79–7.70 (m,2H),7.49(h,J=4.1Hz,4H),7.35(dd,J=22.3,15.9,7.2Hz,3H),7.15(d,J=8.2Hz,1H),6.51(d, J＝7.8Hz, 1H), 6.49–6.41(m, 1H), 6.31–6.22(m, 1H), 6.04(d, J＝5.4Hz, 1H), 5.78(dd, J＝10.1, 2.1Hz, 1H ).

Example 3: N-(3-(2-((2-methoxy-4-(4-(methyl-d ₃ )piperazin-1-yl)phenyl)amino)-7-oxo- Synthesis of 6-phenylpterin-8(7H)-yl)phenyl)acrylamide (compound 2):

NaH (0.20g, 8.28mmol) and anhydrous tetrahydrofuran (3mL) were mixed in a 50mL two-necked flask, protected by N ₂ , and stirred in an ice bath. N-(3-(2-((2-methoxy-4-(piperazin-1-yl)phenyl)amino)-7-oxo-6-phenylpterin-8(7H)- yl)phenyl)acrylamide (3.66g, 6.37mmol) was dissolved in anhydrous tetrahydrofuran (4mL), slowly added to the reaction solution, stirred at 0°C for 10min, and CD ₃ I (2.78g, 19.11mmol) was added rapidly. The temperature was raised to reflux, and the reaction was continued for 19h. After cooling to room temperature, the mixture was quenched with water, extracted with ethyl acetate, washed with saturated aqueous sodium chloride three times, the organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo, and subjected to column chromatography to obtain the product (1.21 g, 32.00%).

¹ H NMR (400MHz, DMSO-d ₆ )δ10.41(s,1H),8.86(s,1H),8.42(s,1H),8.19(s,2H),7.88(d,J＝7.7Hz, 1H), 7.75(s, 1H), 7.54(t, J=8.1Hz, 1H), 7.51-7.46(m, 3H), 7.35(d, J=8.3Hz, 1H), 7.15(d, J=8.2 Hz,1H),6.54(s,1H),6.46(dd,J=16.9,10.1Hz,1H),6.27(d,J=18.1Hz,1H),6.06(s,1H),5.78(d,J =10.4Hz,1H),3.77(s,3H),3.05(s,4H),2.45(s,4H).HRMS(ESI):[M+H] ⁺ calcd for C ₃₃ H ₂₆ D ₆ N ₈ O ₃ , 592.2864; found 592.2862.HPLC purity: 98.77%, retention time＝6.839min.

Synthesis of the mesylate salt of compound 2:

Weigh compound Compound 2 (100mg, 17mmol) in a 100mL round bottom flask, add 10mL of dichloromethane to form orange-red turbidity, stir at 30°C for 10min, add methanesulfonic acid (16mg, 17mmol), the mixture gradually turns orange The yellow clear solution slowly became turbid after 3 minutes, continued to stir at room temperature for 1 hour, then stopped, and stood overnight at room temperature, a small amount of dichloromethane volatilized, and a yellow solid precipitated on the wall. After ultrasonication, it was suction filtered and washed with a small amount of dichloromethane to obtain 112 mg of yellow Solid, 96.5% yield.

Salts of other compounds were also obtained in a similar manner.

Example 4: N-(3-(2-((2-(methoxy-d ₃ )-4-(4-(methyl-d ₃ )piperazin-1-yl)phenyl)amino)- 7-oxo-6-phenylpterin-8(7H))-yl)phenyl)acrylamide synthetic route (compound 3):

Synthesis of tert-butyl 4-(3-(methoxy-d ₃ )-4-nitrophenyl)piperazine-1-carboxylate

4-fluoro-2-(methoxy-d ₃ )-1-nitrobenzene (14.90g, 66.22mmol), tert-butoxycarbonylpiperazine (11.60g, 62.34mmol), N,N-diisopropyl Ethylamine (12.81g, 99.33mmol) and acetonitrile (20.00mL) were mixed in a 100mL one-necked bottle, and stirred at 90°C for 12h. The reaction mixture was spin-dried, extracted with ethyl acetate, washed three times with saturated aqueous sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo, and recrystallized from petroleum ether to obtain a yellow solid (19.60 g, 87.00%).

Synthesis of tert-butyl 4-(4-amino-3-(methoxy-d ₃ )phenyl)piperazine-1-carboxylate

tert-butyl 4-(3-(methoxy-d ₃ )-4-nitrophenyl)piperazine-1-carboxylate (2.21g, 6.56mmol), 10% palladium on carbon (0.65g, 0.65mmol ) and ethanol (8.00mL) were mixed in a 50mL two-neck flask, protected by hydrogen, and stirred at 100°C for 24h. The reaction mixture was suction filtered, then extracted with dichloromethane, washed 3 times with saturated aqueous sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo, and subjected to column chromatography (PE-DCM:MeOH30:1) to obtain a brown oil .

4-(4-((4-((3-acrylamidephenyl)amino)-5-nitropyrimidin-2-yl)amino)-3-(methoxy-d ₃ )phenyl)piperazine- Synthesis of tert-butyl 1-carboxylate

(1) Weigh tert-butyl 4-(4-amino-3-(methoxy-d ₃ )phenyl)piperazine-1-carboxylate (1.74g, 5.45mmol) into a 50mL single-necked bottle, Add THF and stir well, then drop into the reaction system (2).

(2) Weigh N-(3-((2-chloro-5-nitropyrimidin-4-yl)amino)phenyl)acrylamide (1.67g, 5.45mmol) into a 50mL single-necked bottle, add THF , DIPEA (1.05g, 8.17mmol), slowly drop the reaction system (1), and stir at room temperature for 1h. The reaction mixture was spin-dried, extracted with dichloromethane, washed three times with saturated aqueous sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo. Methanol was recrystallized to obtain a reddish-brown solid.

4-(4-((4-((3-acrylamidephenyl)amino)-5-aminopyrimidin-2-yl)amino)-3-(methoxy-d ₃ )phenyl)piperazine-1 -Synthesis of tert-butyl carboxylate

4-(4-((4-((3-acrylamidophenyl)amino)-5-nitropyrimidin-2-yl)amino)-3-(methoxy-d ₃ )phenyl)piperazine - tert-butyl 1-carboxylate (1.38g, 2.33mmol), iron powder (0.50g, 9.32mmol), ammonium chloride (0.62g, 11.65mmol) and ethanol: water (8mL, 4:1) were mixed in 100mL In a one-necked bottle, stir at 80°C for 24h. The reaction mixture was suction-filtered, the filtrate was adjusted to alkali, extracted with ethyl acetate, washed three times with saturated aqueous sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, and concentrated in vacuo to obtain a purple-black solid (0.47 g, 36.00%).

tert-Butyl 4-(4-((8-(3-acrylamidophenyl)-7-oxo-6-phenyl-7,8-dihydropterin-2-yl)amino)-3-( Synthesis of methoxy-d ₃ )phenyl)piperazine-1-carboxylate

4-(4-((4-((3-acrylamide phenyl)amino)-5-aminopyrimidin-2-yl)amino)-3-(methoxy-d ₃ )phenyl)piperazine- 1-tert-butylcarboxylate (0.42g, 0.75mmol), ethyl benzoylformate (0.26g, 1.12mmol) and ethanol (10mL, 4:1) were mixed in a microwave reaction flask, and a few drops of acetic acid were added dropwise, React under microwave conditions (200PSI, 50W, 120°C) for 2h. The reaction mixture was adjusted to alkali, extracted with ethyl acetate, washed 3 times with saturated aqueous sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo, and subjected to column chromatography (DCM:MeOH=25:1) to obtain a reddish-brown solid (0.15 g, 30.00%).

N-(3-(2-((2-(methoxy-d ₃ )-4-(piperazin-1-yl)phenyl)amino)-7-oxo-6-phenylpterin-8 Synthesis of (7H)-yl)phenyl)acrylamide

tert-Butyl 4-(4-((8-(3-acrylamidophenyl)-7-oxo-6-phenyl-7,8-dihydropterin-2-yl)amino)-3- (Methoxy-d ₃ )phenyl)piperazine-1-carboxylate (0.42g, 0.75mmol) and dichloromethane (3mL) were mixed in a 25mL reaction flask, and a few drops of trifluoroacetic acid were added dropwise under ice cooling , reaction at room temperature. The reaction mixture was adjusted to base, extracted with ethyl acetate, washed three times with saturated aqueous sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo, and subjected to column chromatography (DCM:MeOH=25:1) to obtain a reddish-brown solid (0.30 g, 70.00%).

N-(3-(2-((2-(methoxy-d ₃ )-4-(4-(methyl-d ₃ )piperazin-1-yl)phenyl)amino)-7-oxo Synthesis of -6-phenylpterin-8(7H))-yl)phenyl)acrylamide (compound 3)

NaH (0.20g, 8.28mmol) and anhydrous tetrahydrofuran (3mL) were mixed in a 50mL two-necked flask, protected by N ₂ , and stirred in an ice bath. N-(3-(2-((2-(methoxy-d ₃ )-4-(piperazin-1-yl)phenyl)amino)-7-oxo-6-phenylpterin- 8(7H)-yl)phenyl)acrylamide (3.66g, 6.37mmol) was dissolved in anhydrous tetrahydrofuran (4mL), slowly added to the reaction solution, stirred at 0°C for 10min, and CD ₃ I (2.78g, 19.11mmol) was quickly added . The temperature was raised to reflux, and the reaction was continued for 19h. After cooling to room temperature, the mixture was quenched with water, extracted with ethyl acetate, washed with saturated aqueous sodium chloride three times, the organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo, and subjected to column chromatography to obtain the product (1.21 g, 32.00%).

¹ H NMR (600MHz,DMSO-d ₆ )δ10.42(s,1H),8.87(s,1H),8.41(s,1H),8.19(s,2H),7.89(s,1H),7.75( s,1H),7.54(t,J=8.1Hz,1H),7.51-7.47(m,3H),7.35(s,1H),7.15(d,J=7.8Hz,1H),6.53(d,J =2.6Hz,1H),6.46(dd,J=16.9,10.2Hz,1H),6.27(dd,J=16.9,2.0Hz,1H),6.02(s,1H),5.78(dd,J=10.1, 2.0Hz,1H),3.05(s,4H),2.46(s,4H).HRMS(ESI):[M+H] ⁺ calcd for C ₃₃ H ₂₆ D ₆ N ₈ O ₃ ,595.3052; found 595.3057.HPLC Purity: 98.82%, retention time = 6.825min.

Example 5: N-(3-(7-oxo-6-phenyl-2-((4-(piperazin-1-yl)-2-(2,2,2-trifluoroethoxy) Synthesis of phenyl)amino)pteridin-8(7H))-yl)phenyl)acrylamide (intermediate 2)

Synthesis of tert-butyl 4-(4-nitro-3-(2,2,2-trifluoroethoxy)phenyl)piperazine-1-carboxylate

4-Fluoro-1-nitro-2-(2,2,2-trifluoroethoxy)benzene (1.00g, 4.184mmol), piperazine-1-carboxylate tert-butyl ester (1.17g, 6.276mmol ), N,N-diisopropylethylamine (0.648g, 5.021mmol) and DMF (15.00mL) were mixed in a 100mL one-necked bottle, and stirred at 90°C for 12h. The reaction mixture was spin-dried, extracted with dichloromethane, washed with saturated aqueous sodium chloride, and the organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo to obtain a yellow pure product (2.2 g, 90.2%).

Synthesis of tert-butyl 4-(4-amino-3-(2,2,2-trifluoroethoxy)phenyl)piperazine-1-carboxylate

tert-butyl 4-(4-nitro-3-(2,2,2-trifluoroethoxy)phenyl)piperazine-1-carboxylate (1.52g, 3.76mmol), 10% palladium on carbon ( 0.39g, 0.38mmol) and ethanol (8.00mL) were mixed in a 100mL three-neck flask, protected by hydrogen, and stirred at 80°C for 24h. The reaction mixture was suction-filtered, then extracted with dichloromethane, washed 3 times with saturated aqueous sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo, and subjected to column chromatography (DCM:MeOH=50:1) to obtain the product (1.02 g, 78%).

4-(4-((4-((3-acrylamidephenyl)amino)-5-nitropyrimidin-2-yl)amino)-3-(2,2,2-trifluoroethoxy)benzene Synthesis of tert-butyl piperazine-1-carboxylate

Weigh tert-butyl 4-(4-amino-3-(2,2,2-trifluoroethoxy)phenyl)piperazine-1-carboxylate (0.90g, 2.42mmol) into a 50mL single-necked bottle , add THF and stir well, then drop into the reaction system. Weigh N-(3-((2-chloro-5-nitropyrimidin-4-yl)amino)phenyl)acrylamide (0.78g, 2.42mmol) and place it in a 100mL single-necked bottle, add THF, DIPEA ( 0.47g, 3.63mmol), the reaction system was slowly added dropwise, and stirred at room temperature for 1h. The reaction mixture was spin-dried, extracted with dichloromethane, washed three times with saturated aqueous sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo. An aqueous methanol solution was recrystallized to obtain a reddish-brown solid (0.70 g, 50.72%).

4-(4-((4-((3-acrylamidephenyl)amino)-5-aminopyrimidin-2-yl)amino)-3-(2,2,2-trifluoroethoxy)phenyl ) Synthesis of tert-butyl piperazine-1-carboxylate

N-(3-((2-((4-(4-methylpiperazin-1-yl)-2-(2,2,2 trifluoroethoxy)phenyl)amino)-5-nitro Pyrimidin-4-yl) amino) phenyl) acrylamide (0.80g, 1.22mmol), iron powder (0.27g, 4.90mmol), ammonium chloride (0.33g, 6.12mmol) and ethanol: water (25mL, 4 : 1) Mix in a 100mL single-necked bottle, and stir for 24h at 80°C. The reaction mixture was suction-filtered, the filtrate was adjusted to alkali, extracted with ethyl acetate, washed 3 times with saturated aqueous sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, and concentrated in vacuo to obtain a dark brown solid (0.56 g, 75.75%).

N-(3-(7-oxo-6-phenyl-2-((4-(piperazin-1-yl)-2-(2,2,2-trifluoroethoxy)phenyl)amino Synthesis of )pteridin-8(7H))-yl)phenyl)acrylamide

N-(3-((2-((4-(4-methylpiperazin-1-yl)-2-(2,2,2 trifluoroethoxy)phenyl)amino)-5-amine Pyrimidin-4-yl)amino)phenyl)acrylamide (0.33g, 0.52mmol), ethyl benzoylformate (0.19g, 1.05mmol) and ethanol were mixed in a microwave reaction flask, and a few drops of acetic acid were added dropwise, Microwave conditions (200PSI, 50W, 100°C) reacted for 2h. The reaction mixture was adjusted to alkali, extracted with dichloromethane, washed three times with saturated aqueous sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo, and subjected to column chromatography (DCM:MeOH=25:1) to obtain a reddish-brown solid (0.3 g, 78.78%).

¹ H NMR (400MHz,DMSO-d ₆ )δ10.43(s,1H),8.90–8.80(m,1H),8.40(s,1H),8.18(dd,J＝6.1,2.6Hz,2H), 7.84(d, J=8.1Hz, 1H), 7.74(s, 1H), 7.54(d, J=8.0Hz, 1H), 7.51–7.47(m, 3H), 7.33(s, 1H), 7.13(d ,J=7.6Hz,1H),6.65(s,1H),6.45(dd,J=17.0,10.1Hz,1H),6.30–6.23(m,1H),6.17(s,1H),5.78(d, J=10.0Hz, 1H), 4.71(q, J=8.9Hz, 2H), 3.06(s, 4H), 2.44(s, 4H).

Example 6: N-(3-(2-((4-(4-(methyl-d3)piperazin-1-yl)-2-(2,2,2-trifluoroethoxy)phenyl Synthesis of )amino)-7-oxo-6-phenylpteridin)-8(7H)-yl)phenyl)acrylamide (Compound 4)

NaH (0.20g, 8.28mmol) and anhydrous tetrahydrofuran (3mL) were mixed in a 50mL two-necked flask, protected by N ₂ , and stirred in an ice bath. N-(3-(7-oxo-6-phenyl-2-((4-(piperazin-1-yl)-2-(2,2,2 trifluoroethoxy)phenyl)amino )pteridin-8(7H)-yl))phenyl)acrylamide (4.10g, 7.12mmol) was dissolved in anhydrous tetrahydrofuran (4mL), slowly added to the reaction solution, stirred at 0°C for 10min, and quickly added CD ₃ I (2.78 g, 19.11 mmol). The temperature was raised to reflux, and the reaction was continued for 19h. After cooling to room temperature, the mixture was quenched with water, extracted with ethyl acetate, washed with saturated aqueous sodium chloride three times, the organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo, and subjected to column chromatography to obtain the product (1.21 g, 32.00%).

LC-MS: m/z=660[M+H] ⁺ .1H NMR (600MHz, DMSO-d6) δ10.43(s, 1H), 8.88(s, 1H), 8.37(s, 1H), 8.21( dd,J=6.7,3.1Hz,2H),7.87(d,J=8.2Hz,1H),7.76(d,J=2.2Hz,1H),7.54(t,J=8.1Hz,1H),7.52– 7.45(m, 3H), 7.43–7.27(m, 1H), 7.15(dd, J＝7.8, 1.9Hz, 1H), 6.69(s, 1H), 6.48(dd, J＝16.9, 10.2Hz, 1H) ,6.28(dd,J=16.9,2.0Hz,1H),6.23–6.09(m,1H),5.79(dd,J=10.1,2.0Hz,1H),4.75(q,J=8.9Hz,2H), 3.09(s,4H),2.49(s,4H).

Example 7: N-(3-(2-((4-(4-methylpiperazin-1-yl)-2-(2,2,2-trifluoroethoxy)phenyl)amino)- Synthesis of 7-oxo-6-phenylpteridin-8(7H))-yl)phenyl)acrylamide (Compound 5)

Synthesis of tert-butyl 4-(4-nitro-3-(2,2,2-trifluoroethoxy)phenyl)piperazine-1-carboxylate

4-fluoro-1-nitro-2-(2,2,2-trifluoroethoxy)benzene (1.00g, 4.184mmol), N-methylpiperazine (0.63g, 6.276mmol), N, N-Diisopropylethylamine (0.648g, 5.021mmol) and DMF (15.00mL) were mixed in a 100mL one-necked bottle, and stirred at 90°C for 12h. The reaction mixture was spin-dried, extracted with dichloromethane, washed three times with saturated aqueous sodium chloride, and the organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo to obtain a yellow pure product (1.20 g, 90.2%).

LC-MS: m/z=320[M+H] ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ7.91(d, J=9.4Hz, 1H), 6.69(dd, J=9.4,2.4 Hz,1H),6.63(d,J=2.3Hz,1H),4.92(q,J=8.8Hz,2H),3.47–3.42(m,4H),2.44–2.40(m,4H),2.22(s ,3H).

Synthesis of 4-(4-methylpiperazin-1-yl)-2-(2,2,2-trifluoroethoxy)aniline

1-methyl-4-(4-nitro-3-(2,2,2-trifluoroethoxy)phenyl)piperazine (1.20g, 3.76mmol), 10% palladium carbon (0.39g, 0.38mmol) and ethanol (8.00mL) were mixed in a 100mL three-neck flask, protected by hydrogen, and stirred at 80°C for 24h. The reaction mixture was suction-filtered, then extracted with dichloromethane, washed 3 times with saturated aqueous sodium chloride, the organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo, column chromatography (DCM:MeOH=50:1), and the product (0.85 g, 78%).

N-(3-((2-((4-(4-methylpiperazin-1-yl)-2-(2,2,2-trifluoroethoxy)phenyl)amino)-5-nitro Synthesis of ylpyrimidin-4-yl)amino)phenyl)acrylamide

Weigh 4-(4-methylpiperazin-1-yl)-2-(2,2,2-trifluoroethoxy)aniline (0.70g, 2.42mmol) into a 50mL single-necked bottle, add THF Stir well. Weigh N-(3-((2-chloro-5-nitropyrimidin-4-yl)amino)phenyl)acrylamide (0.78g, 2.42mmol) and place it in a 100mL single-necked bottle, add THF, DIPEA ( 0.47g, 3.63mmol), the reaction solution was mixed, stirred at room temperature for 1h. The reaction mixture was spin-dried, extracted with dichloromethane, washed three times with saturated aqueous sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo. Recrystallized from methanol: water to obtain a reddish-brown solid (0.70 g, 50.72%).

N-(3-((2-((4-(4-methylpiperazin-1-yl)-2-(2,2,2 trifluoroethoxy)phenyl)amino)-5-amino pyrimidin-4-yl)amino)phenyl)acrylamide

N-(3-((2-((4-(4-methylpiperazin-1-yl)-2-(2,2,2 trifluoroethoxy)phenyl)amino)-5-nitro Pyrimidin-4-yl) amino) phenyl) acrylamide (0.70g, 1.22mmol), iron powder (0.27g, 4.90mmol), ammonium chloride (0.33g, 6.12mmol) and ethanol: water (25mL, 4 : 1) Mix in a 100mL single-necked bottle, and stir for 24h at 80°C. The reaction mixture was filtered with suction, the filtrate was adjusted to alkali, extracted with ethyl acetate, washed with saturated aqueous sodium chloride three times, the organic phase was dried over anhydrous sodium sulfate, and concentrated in vacuo to obtain a dark brown solid (0.50 g, 75.75%).

N-(3-(2-((4-(4-methylpiperazin-1-yl)-2-(2,2,2-trifluoroethoxy)phenyl)amino)-7-oxo -6-phenylpteridin-8(7H))-yl)phenyl)acrylamide

N-(3-((2-((4-(4-methylpiperazin-1-yl)-2-(2,2,2 trifluoroethoxy)phenyl)amino)-5-amine Pyrimidin-4-yl)amino)phenyl)acrylamide (0.28g, 0.52mmol), ethyl benzoylformate (0.19g, 1.05mmol) and ethanol were mixed in a microwave reaction flask, and a few drops of acetic acid were added dropwise, Microwave conditions (200PSI, 50W, 100°C) reacted for 2h. The reaction mixture was adjusted to base, extracted with dichloromethane, washed three times with saturated aqueous sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, concentrated in vacuo, and subjected to column chromatography (DCM:MeOH=25:1) to obtain a reddish-brown solid (0.26 g, 78.78%).

LC-MS: m/z=657[M+H] ^{+ .1} H NMR (400MHz, DMSO-d ₆ )δ10.43(s,1H),8.86(s,1H),8.41(s,1H), 8.18(dd, J=6.8,3.0Hz,2H),7.84(d,J=8.3Hz,1H),7.74(t,J=2.0Hz,1H),7.54(d,J=8.0Hz,1H), 7.51–7.46(m,3H),7.33(s,1H),7.13(d,J=8.2Hz,1H),6.65(s,1H),6.45(dd,J=17.0,10.2Hz,1H),6.26 (dd,J=16.9,2.0Hz,1H),6.15(s,1H),5.78(dd,J=10.0,2.1Hz,1H),4.71(q,J=8.9Hz,2H),3.06(s, 4H), 2.44(t, J=5.0Hz, 4H), 2.22(s, 3H), 1.22(d, J=2.6Hz, 3H).

Example 8. Kinase and Cell Viability Assays

Test Methods

a) Kinase activity test method

EGFR-TK can catalyze the transfer of a phosphate group of adenosine triphosphate (ATP) to the polypeptide substrate poly(Glu,Tyr) _4:1 , and the polypeptide substrate is labeled with two fluorescent groups coumarin ( coumarin) and fluorescein. Based on the fluorescence resonance energy transfer (FRET) method, EGFR-TK catalyzes the reaction of ATP to cause two fluorescent groups to approach, the donor (coumarin) is excited at 400nM, part of the energy is released, the emission wavelength is 445nM, and the other part of the energy is transferred To fluorescein, the emission wavelength is 520nM. Different compounds have different degrees of inhibition of EGFR-TK, resulting in different degrees of substrate phosphorylation, so the inhibition rate of different compounds is calculated by measuring the ratio of the enzyme-catalyzed substrate phosphorylation percentage.

Add 2.5 μL Test Compounds, 5 μL Kinase/Peptide Substrate Mixture, 2.5 μL ATP Solution to the 384-well plate, shake the 10 μL reaction system for 30 seconds, and incubate at room temperature for 1 hour; add 5 μL Development Solution, shake the reaction system for 30 seconds, and mix at room temperature Incubate for 1 h; add 5 μL Stop Reagent, shake the reaction system for 30 s with a total volume of 20 μL, and use a microplate reader to detect the fluorescence signal. The excitation wavelength is 400 nm, and the emission wavelength is 445 nm and 520 nm, respectively. The inhibitory rate of the compound under 7 concentration gradients was determined, and the IC ₅₀ value of each compound was calculated by the Origin8.0 fitting curve. During the experiment, a positive control was carried out to confirm the feasibility of the reaction system, and each experiment was performed in triplicate.

In vitro enzyme activity analysis: wild-type and mutant (L858/T790M, Del19) EGFR were purchased from Invitrogen. Ten concentration gradients from 5.1×10 ^-11 mol/L to 1.0×10 ^-6 mol/L were set up for all the compounds to be tested.

Concentrations of different kinases were determined by optimization experiments and compounds were diluted three-fold from 5.1x10 ^-9 M to 1x l0 ^-4 M in DMSO. 4 μL of compound was dissolved in 96 μL of water to obtain a 4x compound solution. 40 μM ATP is dissolved in 1.33x Kinase Buffer, the Kinase/Peptide Mix contains 2x Kinase, 4 μM Acetine 4-Peptide is ready for use. A 10 μL kinase reaction consists of 2.5 μL compound solution, 5 μL kinase/peptide mix, 2.5 μL ATP solution. 5 μL of phosphorylated peptide solution was used instead of the kinase/peptide mixture as a 100% phosphorylated control. 2.5 μL of 1.33x kinase buffer instead of ATP solution was used as 100% inhibition control, and 2.5 μL of 4% DMSO instead of compound solution was used as 0% inhibition control. The solution in the plate was mixed well and incubated at room temperature for 1.5 hours. Add 5 μL of Development Solution to each well and continue to incubate at room temperature for 1 hour, during which time the non-phosphorylated peptide is cleaved. Finally, 5 μL of Stop Reagent was added to end the reaction. Well plates were measured with an EnVision Multilabel Reader (Perkin Elmer). Experimental data were calculated using GraphPad Prismversion 4.0. Each experiment was repeated more than 3 times.

b) Cell Proliferation Inhibitory Activity Test Method

Cell proliferation and growth inhibition analysis: H1975 (EGFR ^L858R/T790M ) cells were obtained from ATCC. Cell proliferation activity was assessed by MTS assay. The cells were exposed to the treatment conditions for 72 hours, and the number of cells used in each experiment of each cell line was adjusted according to the absorbance value (absorbance value at 490 nm was 1.3-2.2). Six concentration gradients (0.1 nM-10 μM) were set up for the compounds to be tested, and at least six groups of parallel controls were used for each concentration value.

H1975 cells were cultured in the corresponding medium, and the cells were passaged at least twice after recovery, and then used in experiments. Cells in log phase are trypsinized and resuspended in medium. H1975 (1000 cells per well) were seeded in a 96-well plate with a volume of 100 μL; 6 parallel groups and 7 columns were set up. The orifice plate was placed in an incubator with 5% carbon dioxide at 37°C overnight. The compounds were dissolved in DMSO to prepare a concentration of 10 μM per liter, and then the compound concentrations were gradually diluted to obtain compound concentrations of 10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM, and 0.0001 μM per liter, respectively. 2 μL of compound solution was added to 998 μL of culture medium, and the mixture was mixed well. 100 μL of the mixture was added to a 96-well plate. 2 μL of DMSO was used instead of the compound solution as a 0% inhibition control. After culturing for 68 hours, 20 μL of MTT (5 mg/mL) was added. After 4 hours, discard the supernatant and add 150 μL DMSO. After shaking for 10 minutes, the plate was read with Synergy HT (Bio TeK) (OD490). Data were calculated using GraphPad Prism version 4.0, and _IC50 values were adjusted by non-linear regression models using dose-response curves.

The results are shown in Table 1:

Table 1. Inhibitory activity of compounds on EGFR(L858R/T790M) kinase and H1975 cells

From the data in the table, it can be seen that the IC ₅₀ (nM) of the deuterated compounds of the present invention for inhibiting EGFR L858R/T790M at the kinase level is between 1.0-1.5, which is similar to that of non-deuterated compounds. It shows that the deuterated derivatives do not affect the interaction between the inhibitor and the kinase. However, compound 4 and compound 5 substituted by trifluoroethoxy group will reduce the kinase inhibitory activity by about 1 times, and it is understandable that the introduction of a larger group will lead to a decrease in the interaction between the two.

The inhibitory activity of the compounds of the present invention on H1975 cells is not regular, which is related to the changes in the physical and chemical properties and metabolic properties of the derivatives, and there are many factors. The cellular activity of deuterated compounds was similar to that of non-deuterated compounds.

Example 9. EGFR 20ins and rare mutation kinase activity test results

EGFR 20ins and rare mutation kinase activity test (entrust foreign testing company: Reaction Biology testing, domestic agent company: Creus Bio)

Test Methods:

RBC--EGFR Rare Mutation Kinase Detection Protocol (Protocol)

a) Prepare substrate in freshly prepared reaction buffer

b) Delivery of any desired cofactors into the above substrate solution

c) Delivery of the kinase into the substrate solution and mixing gently

d) Delivery of compounds in 100% DMSO into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range), incubation at room temperature for 20 min

e) Delivery of 33P-ATP into the reaction mixture to initiate the reaction

f) Incubate at room temperature for 2 hours

g) Detection of kinase activity by P81 filter binding method

From the data in the above table, it can be seen that the compounds of the present invention have excellent inhibitory activity on kinases with rare EGFR mutations and 20ins insertion mutations, which is better than the 20ins insertion mutation-positive drug TAK788 that will be launched in 2021. Among them, compound 2 performed best and had good application prospects.

Example 10. Animal in vivo drug efficacy (EGFR L858R/T790M transplanted tumor) activity test results

Animal in vivo activity evaluation method (in vivo drug effect evaluation of compound on H1975 cell xenograft tumor):

Purpose of the experiment: Orally (PO) administered the test substance to H1975 transplanted tumor BALB/c nude mice for 14 consecutive days, recorded and observed the changes in the body weight and tumor size of the nude mice over time, and observed the changes in the physiological activities of the nude mice after administration, to investigate Antitumor efficacy of the test substance.

H1975 cell preparation

H1975 cells were transferred from Shanghai Institute of Materia Medica, Chinese Academy of Sciences. The condition of the cell culture medium is 1640+10% FBS (Gibco), the condition of the incubator is a constant temperature carbon dioxide incubator at 37°C, the concentration of carbon dioxide is 5%, the cells are changed and passaged once every 2-3 days, and the culture is continuously expanded. After transfer to the required amount of cells for cell transplantation, the cells were digested with trypsin for 4 min, and then the digestion was terminated with medium. Collect the obtained cells, centrifuge at 1000r/min for 3min, remove the supernatant, wash twice with serum-free 1640, the cell viability should be maintained above 95% before transplantation, and use serum-free 1640 as a solvent to suspend the cells, the concentration of the suspended cells is 20 million/mL, put the prepared cell suspension on ice for use, in order to maintain cell viability, the cell suspension should be inoculated within 0.5-1h.

H1975 xenograft modeling

Seventy-two male BALB/c nude mice, 4-5 weeks old, weighing 17-23g, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Cell inoculation was carried out after 2-3 days of adaptation to the animal room environment. Select the rich capillaries on the upper side of the forelimb of nude mice to inject the cell suspension subcutaneously, and the volume of the cell suspension injected per mouse is 0.1 mL.

Experimental conditions in the animal room: laboratory temperature: 23±3°C;

Laboratory humidity: 30-60%;

Each 12-hour light on/off setting;

The transplanted tumors can be seen in about 1-2 weeks. When the average size of the tumors reaches 100-200mm ³ , they are randomly divided into groups, with 8 mice in each group, so that the tumor volume in each group is basically the same. Then it can be administered, and the administration is carried out according to the administration compound, administration dose and administration method of each group. After that, it was administered every day, and the tumor volume and mouse body weight were recorded once every 2 days. The same amount of solvent (0.1 mL) was given to the solvent control. The tumor volume and weight of mice need to be recorded before administration. Data were plotted using GraphPad Prism version 4.0. During the whole experiment, the diameter of the transplanted tumor was measured every other day, and the body weight of the mice was weighed at the same time. The formula for calculating tumor volume (TV) is: TV=1/2×L×W2, where L and W represent length and width, respectively, in mm. Wherein, V0 is the tumor volume measured at the time of cage administration (ie, d0), and Vt is the tumor volume at each measurement. The evaluation index of anti-tumor activity is tumor volume growth inhibition rate TGI, and the calculation formula is as follows: %TGI={[1-(TVt/TV0)/(CVt/CV0)]/(1-CV0/CVt)}×100, TVt = mean tumor volume of the treatment group at t, TV0 = mean tumor volume of the treatment group before drug addition, CVt = mean tumor volume of the solvent control group at time t, CV0 = mean tumor volume of the solvent control group before drug addition .

The results are shown in Figures 1 and 2. As can be seen from Figure 1, compound 2 and compound 3 have excellent drug efficacy (EGFR L858R/T790M transplanted tumor) activity in animals, and compound 3 (10mg/Kg) active TGI has just reached 96.2%, which is close to 100%; compound 2 (10mg/Kg) Kg) active TGI reached 106.2%. The active TGI of compound 2 (5mg/Kg) also reached nearly 80%, the effect is obvious. It can be seen from Figure 2 that compound 5 has a general pharmacological effect (EGFR L858R/T790M xenograft tumor) activity in animals.

Example 11. Inhibitory activity of compounds on cells with high expression of Her2: breast cancer cell SKBR3, lung cancer cell H2170, gastric cancer cell N87

Cell Proliferation Inhibitory Activity Test Method

Analysis of cell proliferation and growth inhibition: Breast cancer cells SKBR3, lung cancer cells H2170, and gastric cancer cells N87 were obtained from the Cell Bank of the Chinese Academy of Sciences. Cell proliferation activity was assessed using a CCK8 assay. Cells were exposed to the treatment conditions for 72 hours, and the number of cells used in each experiment for each cell line was adjusted according to the absorbance value (absorbance value at 450 nm was 1-1.2). Eight concentration gradients (0.1 nM-10 μM) were set up for the compounds to be tested, and at least six groups of parallel controls were used for each concentration value.

The cells were cultured in the corresponding medium, and the cells were subcultured at least twice after thawing, and the cell growth was in the logarithmic growth phase, and then used for the experiment. 3,000 SKBR3 cells/well, 6,000/well H2170 cells, and 9,000/well N87 cells were spread in a 96-well plate with a volume of 90 μL; 6 parallel groups and 7 columns were set up. The orifice plate was placed in an incubator with 5% carbon dioxide at 37°C overnight.

Dissolve the compound in DMSO. For the SKBR3 cell line, prepare the stock solution at a concentration of 10 mM per liter, then dilute the compound 100 times with the medium to obtain a maximum concentration of 100 μM, and then serially dilute the compound concentration by 2 times to gradually dilute 8 concentration gradients; for H2170 For cell lines, the concentration of the mother solution is prepared to be 100 μM per liter, and then the compound is diluted 100 times with the medium to obtain a maximum concentration of 1 μM, and then the compound concentration is serially diluted by 3 times to gradually dilute 8 concentration gradients; for the N87 cell line, the concentration of the prepared mother solution is 1 μM per liter. Then the compound was diluted 100 times with medium to obtain a maximum concentration of 1 μM, and then the compound concentration was serially diluted 5 times and gradually diluted to 8 concentration gradients; 10 μL of the compound solution was added to the laid 96-well plate. Control supplemented with 10ul medium containing 1% DMSO instead of the compound solution was used as 0% inhibition control. After culturing for 72 hours, 10 μl of CCK8 was added. After 1.5 hours, the plate was read with a microplate reader. Data were calculated using GraphPad Prism version 4.0, and _IC50 values were adjusted by non-linear regression models using dose-response curves.

compound	SKBR3 (μM)	H2170(nM)	N87(nM)
Poziotinib	11.16±3.27	0.74±0.086	0.069±0.035
Compound 0	2.68±0.91	17.51±3.09	3.39±1.26
Compound 1	2.37±0.79	24.67±4.16	2.72±0.70
Compound 2	2.14±0.60	20.29±3.64	2.95±1.36
Compound 3	1.92±0.41	21.77±6.91	2.92±1.65
Compound 4	2.79±0.76	25.68±4.45	8.02±2.43
Compound 5	3.32±0.91	35.73±2.34	8.29±3.22

It can be seen from the data in the above table that compound 1, compound 2 and compound 3 have the best cell activity on lung cancer H2170 and gastric cancer N87 cells. The cellular activity of the deuterated compound was slightly lower compared to Poziotinib. On breast cancer SKBR3 cells, the inhibitory activity IC ₅₀ of the deuterated compound is between 2-3 μM, which is better than the IC ₅₀ (11 μM) activity of Poziotinib.

Embodiment 12. Pharmacokinetic property test of compound

The pharmacokinetic properties test of the compound of the present invention is entrusted to Hangzhou Leading Pharmaceutical Technology Co., Ltd. and Medicipia Pharmaceutical Technology (Shanghai) Co., Ltd. to conduct.

As mentioned above, the pharmacokinetic properties of Compound 1-Compound 6 were all significantly improved. Taking compound 2, which performed well in the double mutation EGFR L858R/T790M and performed best in the main insertion mutation of EGFR 20ins, as an example, its bioavailability reached 29.4%; Cmax was 223.96ng/mL; AUC _(0-∞) was 2643.35 ng/mL*h. As the salt form of compound 2, the pharmacokinetic properties of its mesylate salt will be better. These properties have great advantages for this type of inhibitor in the later clinical application, and the advantages are more obvious in reducing the effective dose and improving safety.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. Compound shown in formula I or its pharmaceutically acceptable salt, solvate, stereoisomer or prodrug:

   

   In formula I

   R ₁ is independently selected from hydrogen, substituted or unsubstituted C ₁ -C ₁₀ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, optionally substituted C ₃ -C ₈ cycloalkyl, Optionally substituted or unsubstituted aryl, substituted or unsubstituted benzyl, substituted or unsubstituted heterocyclic group, substituted or unsubstituted aromatic heterocyclic group;

   R ₂ , R ₃ , R ₄ , R ₅ are independently selected from H, halogen, substituted (preferably halogen substituted, more preferably fluorine substituted) or unsubstituted C ₁ -C ₆ (preferably C ₁ -C ₃ ) alkoxy or Substituted (preferably halogen substituted, more preferably fluorine substituted) or unsubstituted C ₁ -C ₆ (preferably C ₁ -C ₃ ) deuterated alkoxy, substituted or unsubstituted C ₁ -C ₆ (preferably C ₁ -C ₃ ) Alkyl, _NRcRd ; wherein _Rc and _Rd are each independently selected from hydrogen _{, C1-3} alkyl;

   G is a benzene ring, a five-membered or six-membered heterocyclic ring or a C ₃ -C ₈ cycloalkyl group or does not exist;

   R ₆ is independently selected from hydrogen, unsubstituted or halogen-substituted C ₁ -C ₄ alkyl, nitro, amino, halogen, hydroxyl, C ₁ -C ₆ alkoxy, optionally substituted C ₁ -C ₆ acyl Oxygen, optionally substituted C ₁ -C ₆ amido, optionally substituted C ₁ -C ₆ acyl; wherein, when G is a benzene ring, R ₆ is meta-substituted;

   m is an integer of 0-3;

   R ₇ is independently selected from substituted or unsubstituted NH ₂ , substituted or unsubstituted heterocyclic group, substituted or unsubstituted aromatic heterocyclic group, substituted or unsubstituted C ₁ -C ₁₀ alkyl;

   A is selected from the following group or is absent:

   

   X is selected from the following group or does not exist: substituted or unsubstituted C _1-3 alkylene (preferably -CH ₂ -) or deuterated alkylene (preferably -CD ₂ -), -O-, -C(= O)-, -C(=O)NHN=-;

   Y is selected from the following group or is absent: -NHC(=O)-, -C(=O)NH-, -=NNHC(=O)NH-, _-CH2- , -O-;

   L is selected from the following group or absent: C ₁ -C ₁₀ alkylene, C ₁ -C ₁₀ heteroalkylene, -A'-(CH ₂ ) _m' -W-(CH ₂ ) _n' -, - (CH ₂ ) _m' -W-(CH ₂ ) _n' -O-(CH ₂ ) _V -and-(CH ₂ ) _m' -W-[(CH ₂ ) _n' -O] _u -(CH ₂ ) _v -;

   A' is selected from the following group or does not exist: 5-membered arylene and 6-membered arylene;

   W is selected from: phenylene, 5-membered heteroarylene, 6-membered heteroarylene, C ₁ -C ₁₀ heterocyclylene and C ₁ -C ₁₀ alkylene;

   m' is 0, 1, 2, 3, 4, 5, 6, 7 or 8;

   n' is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9;

   each independent u is independently 2, 3 or 4;

   v is 1, 2, 3 or 4

   B is selected from the following group or absent:

   

   R is selected from: hydrogen, methyl and fluorine;

   Q ₁ is selected from: -C(R _2a )=and -N=;

   Q ₂ is selected from: -C(R _2b )= and -N=;

   Q ₃ is selected from: -C(R _2c )= and -N=;

   R _2a , R _2b , and R _2c are each independently selected from: hydrogen, -C(=O)-;

   Z is selected from: -CH ₂ -, -C(=O)-.
2. The compound or its pharmaceutically acceptable salt, solvate, stereoisomer or prodrug as claimed in claim 1, is characterized in that, described compound is the compound shown in following formula II:

   

   In formula II

   R ⁷ is independently selected from: H, substituted (preferably halogen substituted, more preferably fluorine substituted) or unsubstituted C ₁ -C ₆ (preferably C ₁ -C ₃ ) alkyl, substituted (preferably halogen substituted, more preferably fluorine substituted) or unsubstituted C ₁ -C ₆ (preferably C ₁ -C ₃ ) deuterated alkyl;

   R ⁸ is independently selected from: H, substituted (preferably halogen substituted, more preferably fluorine substituted) or unsubstituted C ₁ -C ₃ alkyl, substituted (preferably halogen substituted, more preferably fluorine substituted) or unsubstituted C ₁ -C ₃ deuterated alkyl.
3. The compound or its pharmaceutically acceptable salt, solvate, stereoisomer or prodrug as claimed in claim 2, is characterized in that, in formula II,

   R ⁷ is independently selected from: methyl, deuteromethyl CD ₃ , trifluoromethyl, ethyl, deuteroethyl (eg, CD ₂ CH ₃ , CH ₂ CD ₃ ), CH ₂ CF ₃ ;

   R ⁸ is independently selected from: hydrogen, methyl, deuteromethyl CD ₃ , ethyl, deuteroethyl (eg, CD ₂ CH ₃ , CH ₂ CD ₃ ).
4. The compound shown in the following group, or its pharmaceutically acceptable salt, solvate, stereoisomer or prodrug, is characterized in that, the compound is selected from:

   

   
5. A pharmaceutical composition comprising the compound according to any one of claims 1-4, or a pharmaceutically acceptable salt, solvate, stereoisomer or prodrug thereof and optional pharmaceutical acceptable excipients.
6. Compounds as claimed in any one of claims 1-4, or pharmaceutically acceptable salts, solvates, stereoisomers or prodrugs thereof in the preparation of prevention or treatment of abnormal expression of EGFR protein activity, ERBB2 overexpression and Use in medicine for diseases associated with point mutations.
7. The use according to claim 6, wherein the EGFR is a mutant EGFR.
8. The use according to claim 7, wherein the mutant EGFR comprises at least one of the following mutations: EGFR sensitivity mutation L858R and 19del, EGFR T790M mutation, EGFR 18-21 exon point mutation and insertion mutation, ERBB2 overexpression and its point and insertion mutations.
9. purposes as claimed in claim 8, is characterized in that, described EGFR 18-21 exon point mutation and insertion mutation contain:

   Point mutations of exon 18 G719X, E709X, K716A, K728A and deletion mutation at codon 709;

   Exon 19 insertion mutation I744-K745insKIPVAI, K745-E746insIPVAIK, K745-E746insVPVAIK, K745-E746insTPVAIK and point mutation D761Y;

   Insertion mutations and point mutations in exon 20 include: A763-Y764insFQEA, A763-Y764insFHEA, V769-D770insASV, V769-D770insDNP, D770-N771insNPG, D770-N771insNPH, D770-N771insSVD, D770-N771insP7insASVDN, D771SVG-N7 、N771-H773dupNPH、P772-H773insPNP、P772-H773insPR、H773-V774insH、A763-Y764insFQEA、H773-V774insPH、H773-V774insNPH、N771-P772insH、H771-P772insN、H773-V774insAH、D770delinsGY、V774-C775insHV等以及20号外Exon point mutation S768I;

   Exon 21 point mutation L861Q;

   ERBB2 point mutations V777L, D769Y, R896C, P1170A and insertion mutations V777-G778insCG, P780-Y781insGSP, etc.
10. The use according to any one of claims 6-9, wherein the disease is cancer and is selected from one or more of the following: non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, squamous lung cancer cancer, breast cancer, pancreatic cancer, prostate cancer, ovarian cancer, glioma, head and neck squamous cell carcinoma, cervical cancer, esophageal cancer, liver cancer, kidney cancer, colon cancer, skin cancer, leukemia, lymphoma, gastric cancer or Multiple myeloma.

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