CN114853735B

CN114853735B - Compound for target ubiquitination degradation of TRK and preparation method, composition and application thereof

Info

Publication number: CN114853735B
Application number: CN202210785777.8A
Authority: CN
Inventors: 蒋兴凯; 王岩; 范伏田; 汪小涧; 潘显道; 毕续海; 马明阳; 苏小庭; 戴信敏
Original assignee: Beijing Xinkaiyuan Pharmaceuticals Co Ltd
Current assignee: Beijing Xinkaiyuan Pharmaceuticals Co Ltd
Priority date: 2022-07-06
Filing date: 2022-07-06
Publication date: 2022-10-21
Anticipated expiration: 2042-07-06
Also published as: CN114853735A

Abstract

The invention belongs to the field of medicines, and particularly relates to a compound for target ubiquitination and TRK degradation, and a preparation method, a composition and an application thereof, wherein the compound has a structure shown in a formula I-a or a formula I-b:

，R ₁ and R ₂ Represents hydrogen, fluorine, chlorine, bromine, iodine, unsubstituted C _1‑4 Alkyl, substituted C _1‑4 Alkyl, unsubstituted C _1‑4 Alkoxy, substituted C _1‑4 Alkoxy, unsubstituted C _1‑4 Alkylthio, substituted C _1‑4 Alkylthio, unsubstituted C _1‑4 Alkylamino or substituted C _1‑4 An alkylamino group; m represents carbon, nitrogen, oxygen or sulfur; q ₁ 、Q ₂ And Q ₃ Represents carbon or nitrogen; w is a group of ₁ Represents carbon or nitrogen; r is ₃ Represents hydrogen, fluorine, chlorine, bromine, iodine, aldehyde group, carboxyl, hydroxyl, amino, sulfydryl or unsubstituted C _1‑4 Alkyl, substituted C _1‑4 Alkyl, aryl, heteroaryl, and heteroaryl,

、

、

Or

(ii) a L represents

(ii) a D represents

、

、

Or

Description

Compound for target ubiquitination degradation of TRK and preparation method, composition and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a compound for targeted ubiquitination and TRK degradation, and a preparation method, a composition and application thereof.

Background

Tropomyosin-related kinases (TRKs, which may also be referred to as "tropomyosin-receptor kinases") are a class of neurotrophic factor receptors belonging to the receptor tyrosine kinase family, including the TRKA, TRKB and TRKC 3 subtypes, encoded by the NTRK1 (neurolytic receptor tyrosine kinase 1), NTRK2 and NTRK3 genes, respectively. When the NTRK gene is fused with other genes, dimerization and phosphorylation of a receptor can be induced, high expression of TRK or continuous increase of TRK activity is caused, and downstream PI3K/Akt/mTOR, PLC gamma and Ras/Raf/MEK/ERK signal cascade pathways are activated, so that growth, proliferation, invasion, migration, angiogenesis and drug resistance of tumor cells are regulated.

The first generation of TRK inhibitors Larotrectinib and Entrectinib are approved by FDA to be marketed in 2018 and 2019 respectively, can simultaneously inhibit 3 subtypes TRKA, TRKB and TRKC, and can treat various types of tumors, wherein 4 histological forms (breast analogue secretory cancer, secretory breast cancer, pediatric fibroma sarcoma and congenital mesodermal nephroma) which are abundant due to NTRK gene fusion are included, and other malignant tumors comprise lung cancer, gastrointestinal cancer, breast cancer, thyroid cancer, melanoma and soft tissue sarcoma. However, the two TRK inhibitors also have the problem of drug resistance at present, once the drug resistance occurs, the drugs need to be replaced immediately to solve the problems of drug resistance and off-target, in order to achieve better tumor treatment effect and better meet the requirements of clinic and market, and the development of safer and more efficient novel TRK inhibitor drugs has great social value and economic benefit.

Aiming at the drug resistance defect, a technology of targeted protein degradation chimeric molecules (PROTACs) is developed to construct PROTACs compounds, which comprise three functional structures: a target protein ligand, an E3 ubiquitin ligase ligand and a linker chain. They can recognize the target protein and E3 ubiquitin ligase by the target protein ligand and E3 ubiquitin ligase ligand, respectively, polyubiquitinate the target protein, so that the target protein is recognized and degraded by proteasomes. The novel PROTACs can be expected to overcome the drug resistance of the traditional targeted drugs, are also an advantage of the combination therapy in the future, and have promising prospects. Therefore, a compound for target ubiquitination degradation of TRK is determined, so that the compound has important significance for induced degradation of TRK.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a compound for target ubiquitination degradation of TRK, a preparation method, a composition and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a compound having the structure of formula I-a or formula I-b:

；

in the formula, R ₁ And R ₂ Identical or different and each independently represents hydrogen, fluorine, chlorine, bromine, iodine, unsubstituted C _1-4 Alkyl, substituted C _1-4 Alkyl, unsubstituted C _1-4 Alkoxy, substituted C _1-4 Alkoxy, unsubstituted C _1-4 Alkylthio, substituted C _1-4 Alkylthio, unsubstituted C _1-4 Alkylamino or substituted C _1-4 An alkylamino group; m represents carbon, nitrogen, oxygen or sulfur; q ₁ 、Q ₂ And Q ₃ Are the same or different and each independently represents carbon or nitrogen; w is a group of ₁ Represents carbon or nitrogen; when W is ₁ When represents nitrogen, R ₄ Is absent when W ₁ When represents carbon, R ₄ Represents hydrogen, unsubstituted C _1-4 Alkyl, fluoro, chloro, bromo or iodo; r ₃ Represents hydrogen, fluorine, chlorine, bromine, iodine, aldehyde group, carboxyl, hydroxyl, amino, sulfydryl or unsubstituted C _1-4 Alkyl, substituted C _1-4 Alkyl, aryl, heteroaryl, and heteroaryl,

、

、

Or

Wherein R is ₅ Represents unsubstituted C _1-4 Alkyl, substituted C _1-4 Alkyl, substituted C _3-7 Cycloalkyl, unsubstituted C _3-7 Cycloalkyl, substituted C _3-7 Heterocyclic radical, unsubstituted C _3-7 Heterocyclyl, substituted phenyl, unsubstituted phenyl, substituted five-membered heteroaryl, unsubstituted five-membered heteroaryl, substituted six-membered heteroaryl or unsubstituted six-membered heteroaryl, R ₇ Represents unsubstituted C _1-4 Alkyl, substituted C _1-4 Alkyl, substituted C _3-7 Cycloalkyl, unsubstituted C _3-7 Cycloalkyl, substituted C _3-7 Heterocyclyl or unsubstituted C _3-7 Heterocyclyl radical, R ₈ Represents unsubstituted C _1-4 Alkyl, substituted C _1-4 Alkyl, substituted C _3-7 Cycloalkyl, unsubstituted C _3-7 Cycloalkyl, substituted C _3-7 Heterocyclyl or unsubstituted C _3-7 A heterocyclic group; r ₆ Represents unsubstituted C _1-4 Alkyl, substituted C _1-4 Alkyl, substituted C _3-7 Cycloalkyl, unsubstituted C _3-7 Cycloalkyl, substituted C _3-7 Heterocyclyl radical, unsubstituted C _3-7 A heterocyclic group, a substituted phenyl group, an unsubstituted phenyl group, a substituted five-membered heteroaryl group, an unsubstituted five-membered heteroaryl group, a substituted six-membered heteroaryl group, or an unsubstituted six-membered heteroaryl group; l represents

In the formula, Y represents CH ₂ 、SiH ₂ NH, PH, O, S, substituted CH ₂ Substituted SiH ₂ Substituted NH or substituted PH, A ₁ And A ₂ Each independently represents unsubstituted C _1-5 Alkyl, substituted C _1-5 Alkyl, unsubstituted C _1-5 Alkoxy, substituted C _1-5 Alkoxy, unsubstituted C _1-5 Alkylthio, substituted C _1-5 Alkylthio, unsubstituted C _1-5 Alkylamino radical, substituted C _1-5 Alkylamino, phenyl, pentabasic heteroaryl, hexabasic heteroaryl, C _3-7 Cycloalkyl radical, C _3-7 A heterocyclic group,

、

、

、

、

、

、

Or

(ii) a D represents

、

、

Or

In the formula, Z represents hydrogen or CH ₂ 、SiH ₂ 、NH、PH、O、S、

Substituted CH ₂ Substituted SiH ₂ Substituted NH or substituted PH, R ₉ 、R ₁₀ 、R ₁₁ And R ₁₂ Independently represent hydrogen and unsubstituted C _1-4 Alkyl, substituted C _1-4 Alkyl, unsubstituted C _1-4 Alkoxy, substituted C _1-4 Alkoxy, unsubstituted C _1-4 Alkylthio, substituted C _1-4 Alkylthio, unsubstituted C _1-4 Alkylamino or substituted C _1-4 An alkylamino group.

The compound provided by the invention can enable TRK to be identified by proteasomes, realizes the induced degradation of TRK, and has important significance for researching tumor drugs.

In a second aspect, the present invention provides a process for the preparation of a compound according to the first aspect, or a pharmaceutically acceptable salt thereof, comprising the steps of:

S1、

reacting the compound II with pinacol diboron in a first reaction solvent in the presence of a first base under the action of a first catalyst to obtain an intermediate III;

S2、

reacting the intermediate III with a compound IV in a second reaction solvent in the presence of a second alkali under the action of a second catalyst to obtain an intermediate V;

s3, when said compound or pharmaceutically acceptable salt thereof has the structure of formula I-a:

reacting the intermediate V and the compound with the structure of the formula VI in a third reaction solvent in the presence of a third base to obtain an intermediate with the structure of the formula VII;

when the compound or pharmaceutically acceptable salt thereof has the structure of formula i-b:

reacting the intermediate V and the compound with the structure of the formula VIII in a fourth reaction solvent in the presence of a fourth base to obtain an intermediate with the structure of the formula IX;

s4, the intermediate with the structure of the formula VII participates in a reaction to obtain a compound with the structure of the formula I-a or a pharmaceutically acceptable salt thereof, or the intermediate with the structure of the formula IX participates in a reaction to obtain a compound with the structure of the formula I-b or a pharmaceutically acceptable salt thereof;

in the formula, R ₁ 、R ₂ 、M、Q ₁ 、Q ₂ 、Q ₃ 、W ₁ 、R ₃ And R ₄ Is as defined for the first aspect R ₁ 、R ₂ 、M、Q ₁ 、Q ₂ 、Q ₃ 、W ₁ 、R ₃ And R ₄ The definition of (1).

In a third aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound selected from the compounds according to the first aspect, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In a fourth aspect, the present invention provides a formulation for targeted ubiquitination degradation of TRK, comprising a therapeutically effective amount of a compound selected from the group consisting of the compounds according to the first aspect, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In a fifth aspect, the present invention provides a compound according to the first aspect or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for the prevention and/or treatment of a tumour.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the raw materials, instruments, equipment, etc. used in the following examples are either commercially available or available by existing methods; the dosage of the experimental reagent is the dosage of the reagent in the conventional experimental operation if no special description exists; the experimental methods are conventional methods unless otherwise specified.

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

The term "pharmaceutically acceptable salts" refers to those salts that retain the biological effectiveness and properties of the parent compound. The salt comprises: acid addition salts obtained by reaction of the free base of the parent compound with an inorganic acid or with an organic acid; such as hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, phosphoric acid, sulfuric acid, perchloric acid, and the like; such as acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, p-toluenesulfonic acid, salicylic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, fumaric acid, succinic acid, tartaric acid, malonic acid, or the like; preferably hydrochloric acid or (L) -malic acid; or when the acid proton present in the parent compound is replaced by a metal ion, such as an alkali metal ion, an alkaline earth metal ion, or an aluminum ion, or coordinated with an organic base, a salt is formed; such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like.

The carbon atom content of various hydrocarbon-containing moieties is indicated by the prefix designating the minimum and maximum number of carbon atoms for that moiety, i.e., the prefix C _i~j The number of carbon atoms representing the moiety is from the integer "i" to the integer "j" (inclusive). Thus, for example, C _1~4 Alkyl refers to alkyl groups of 1 to 4 carbon atoms (including 1 and 4).

The term "alkyl" refers to a saturated aliphatic hydrocarbon group which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 4 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, and various branched chain isomers thereof, and the like. Alkyl groups may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available point of attachment.

The term "substituted" means that any available point of attachment on the structure may be substituted with a substituent.

The term "alkoxy" refers to-O- (alkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy. Alkoxy groups may be optionally substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment.

The term "alkylthio" refers to (alkyl) -S-, non-limiting examples of alkylthio include: methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, and the like.

The term "alkylamino" refers to an amino group having one or two alkyl substituents, such as "alkyl-NH-" or "(alkyl) ₂ A N- "group wherein alkyl is as defined above. Non-limiting examples of alkylamino groups include: dimethylamino, methylamino, and the like.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic cyclic hydrocarbon group, the cycloalkyl ring containing from 3 to 20 carbon atoms, preferably from 3 to 12 carbon atoms, more preferably from 3 to 7 carbon atoms. Non-limiting examples of cycloalkyl groups include cyclohexyl, cyclopentyl, and the like.

The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic cyclic hydrocarbon group comprising 3 to 20 ring atoms, wherein one or more ring atoms is a heteroatom selected from silicon, phosphorus, nitrogen, oxygen or sulfur, the remaining ring atoms being carbon. Preferably 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; most preferably 3 to 8 ring atoms, of which 1 to 3 are heteroatoms.

The term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, with a completely conjugated pi-electron system, wherein the heteroatoms are selected from silicon, phosphorus, oxygen, sulfur and nitrogen. Heteroaryl is preferably 5 to 10 membered, containing 1 to 3 heteroatoms; more preferably 5 or 6 membered, containing 1 to 2 heteroatoms.

The term "pharmaceutical composition" refers to a mixture of one or more of the compounds described herein or a physiologically acceptable salt thereof with other chemical ingredients such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compound to the organism.

The term "pharmaceutically acceptable carrier" refers to a pharmaceutical carrier that is conventional in the pharmaceutical art, a carrier that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound, such as: diluents such as water and the like; fillers, such as starch, sucrose, and the like; binders such as cellulose derivatives, alginates, gelatin, polyvinylpyrrolidone; humectants, such as glycerol; disintegrating agents such as agar, calcium carbonate and sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and bentonite clay; lubricants, such as talc, calcium stearate and magnesium stearate, and polyethylene glycol, and the like. In addition, other adjuvants such as flavoring agent and sweetener can also be added into the above medicinal composition.

The term "therapeutically effective amount" refers to an amount of a compound of the present invention sufficient to effect the intended use. The therapeutically effective amount may vary depending on the following factors: the intended application (in vitro or in vivo), or the subject and disease condition being treated, such as the weight and age of the subject, the severity of the disease condition and the mode of administration, etc., can be readily determined by one of ordinary skill in the art. The specific dosage will vary depending on the following factors: the particular compound selected, the dosing regimen according to, whether to administer in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system carried.

The term "room temperature" as used herein has the meaning well known in the art and generally means 24-28 ℃.

In a first aspect, embodiments of the present invention provide a compound having the structure of formula I-a or formula I-b:

；

in the formula, R ₁ And R ₂ Identical or different and each independently represents hydrogen, fluorine, chlorine, bromine, iodine, unsubstituted C _1-4 Alkyl, substituted C _1-4 Alkyl, unsubstituted C _1-4 Alkoxy, substituted C _1-4 Alkoxy, unsubstituted C _1-4 Alkylthio, substituted C _1-4 Alkylthio, unsubstituted C _1-4 Alkylamino or substituted C _1-4 An alkylamino group; m represents carbon, nitrogen, oxygen or sulfur; q ₁ 、Q ₂ And Q ₃ Are the same or different and each independently represents carbon or nitrogen; w ₁ Represents carbon or nitrogen; when W is ₁ When represents nitrogen, R ₄ Is absent when W ₁ When represents carbon, R ₄ Represents hydrogen, unsubstituted C _1-4 Alkyl, fluoro, chloro, bromo or iodo; r is ₃ Represents hydrogen, fluorine, chlorine, bromine, iodine, aldehyde group, carboxyl, hydroxyl, amino, sulfydryl or unsubstituted C _1-4 Alkyl, substituted C _1-4 Alkyl, aryl, heteroaryl, and heteroaryl,

、

、

Or

Wherein R is ₅ Represents unsubstituted C _1-4 Alkyl, substituted C _1-4 Alkyl, substituted C _3-7 A cycloalkyl group, a,Unsubstituted C _3-7 Cycloalkyl, substituted C _3-7 Heterocyclic radical, unsubstituted C _3-7 Heterocyclyl, substituted phenyl, unsubstituted phenyl, substituted five-membered heteroaryl, unsubstituted five-membered heteroaryl, substituted six-membered heteroaryl or unsubstituted six-membered heteroaryl, R ₇ Represents unsubstituted C _1-4 Alkyl, substituted C _1-4 Alkyl, substituted C _3-7 Cycloalkyl, unsubstituted C _3-7 Cycloalkyl, substituted C _3-7 Heterocyclyl or unsubstituted C _3-7 Heterocyclic radical, R ₈ Represents unsubstituted C _1-4 Alkyl, substituted C _1-4 Alkyl, substituted C _3-7 Cycloalkyl, unsubstituted C _3-7 Cycloalkyl, substituted C _3-7 Heterocyclyl or unsubstituted C _3-7 A heterocyclic group; r ₆ Represents unsubstituted C _1-4 Alkyl, substituted C _1-4 Alkyl, substituted C _3-7 Cycloalkyl, unsubstituted C _3-7 Cycloalkyl, substituted C _3-7 Heterocyclyl radical, unsubstituted C _3-7 A heterocyclic group, a substituted phenyl group, an unsubstituted phenyl group, a substituted five-membered heteroaryl group, an unsubstituted five-membered heteroaryl group, a substituted six-membered heteroaryl group, or an unsubstituted six-membered heteroaryl group; l represents

In the formula, Y represents CH ₂ 、SiH ₂ NH, PH, O, S, substituted CH ₂ Substituted SiH ₂ Substituted NH or substituted PH, A ₁ And A ₂ Each independently represents unsubstituted C _1-5 Alkyl, substituted C _1-5 Alkyl, unsubstituted C _1-5 Alkoxy, substituted C _1-5 Alkoxy, unsubstituted C _1-5 Alkylthio, substituted C _1-5 Alkylthio, unsubstituted C _1-5 Alkylamino radical, substituted C _1-5 Alkylamino, phenyl, five-membered heteroaryl, six-membered heteroaryl, C _3-7 Cycloalkyl, C _3-7 Heterocyclic group,

、

、

、

、

、

、

Or

(ii) a D represents

、

、

Or

Substituted CH ₂ Substituted SiH ₂ Substituted NH or substituted PH, R ₉ 、R ₁₀ 、R ₁₁ And R ₁₂ Each independently represents hydrogen, unsubstituted C _1-4 Alkyl, substituted C _1-4 Alkyl, unsubstituted C _1-4 Alkoxy, substituted C _1-4 Alkoxy, unsubstituted C _1-4 Alkylthio, substitutedC _1-4 Alkylthio, unsubstituted C _1-4 Alkylamino or substituted C _1-4 An alkylamino group.

The compound provided by the invention is a novel targeted protein degradation chimeric molecule (PROTACs), can enable TRK to be recognized by proteasomes, realizes induced degradation of TRK, reduces TRK activity by directly inducing the proteasomes to degrade TRK, and has important significance for researching development of tumor drugs.

The D fragment in the compound with the structure shown in the formula I-a or the formula I-b can interact with ubiquitin ligase E3, the L fragment is a connecting arm between the D fragment and the fragment capable of being combined with TRK, and the compound with the structure shown in the formula I-a or the formula I-b can be combined with TRK and E3 simultaneously, so that TRK which cannot be combined with E3 originally is ubiquitinated and then is recognized and degraded by proteasome. As long as the compound has certain binding capacity with TRK, the over-expressed TRK can be degraded, the influence of the mutation of the kinase is small, after the TRK connected with the compound is degraded, the compound can also circularly and continuously degrade other TRKs, the requirement on the dosage of the medicine is small, and the toxic and side effects are small.

If the fragment capable of being combined with TRK in the compound with the structure of the formula I-a or the formula I-b is replaced by other structures, the stability, the solubility and other properties of the compound are influenced, and the activity of inducing the TRK to be degraded is reduced or even disappears.

Further, when said R is ₁ Represents substituted C _1-4 Alkyl, substituted C _1-4 Alkoxy, substituted C _1-4 Alkylthio or substituted C _1-4 In the case of alkylamino, the substituent is one or more of hydroxyl, methyl, amino, methoxy, dimethylamino, halogen and ethyl; when said R is ₂ Represents said substituted C _1-4 Alkyl, substituted C _1-4 Alkoxy, substituted C _1-4 Alkylthio or substituted C _1-4 When the alkylamino radical is adopted, the substituent is one or more of hydroxyl, methyl, amino, methoxyl, dimethylamino, halogen and ethyl; when said R is ₃ Represents substituted C _1-4 When the alkyl group is substituted, the substituent is a hydroxyl group, a methyl group, an amino group, a methoxy group, a dimethylamino group, a halogen,One or more of ethyl groups; when said R is ₅ Represents substituted C _1-4 Alkyl, substituted C _3-7 Cycloalkyl, substituted C _3-7 Heterocyclic group, substituted phenyl group, substituted five-membered heteroaryl group or substituted six-membered heteroaryl group, the substituent is methyl, ethyl, hydroxyl, halogen, methoxy,

、

Dimethylamino group, diethylamino group,

、

One or more of; when said R is ₇ Represents substituted C _1-4 Alkyl, substituted C _3-7 Cycloalkyl or substituted C _3-7 In the case of a heterocyclic group, the substituent is methyl, ethyl, hydroxy, halogen, methoxy, dimethylamino, diethylamino, etc,

、

、

One or more of; when said R is ₈ Represents substituted C _1-4 Alkyl, substituted C _3-7 Cycloalkyl or substituted C _3-7 When the heterocyclic radical is adopted, the substituent is one or more of methyl, ethyl, hydroxyl, halogen, methoxy, dimethylamino and diethylamino; when said R is ₆ Represents substituted C _1-4 Alkyl, substituted C _3-7 Cycloalkyl, substituted C _3-7 When the heterocyclic radical, the substituted phenyl, the substituted five-membered heteroaryl or the substituted six-membered heteroaryl are adopted, the substituent is methyl, ethyl,One or more of hydroxy, halogen, methoxy, dimethylamino, diethylamino; when Y represents substituted CH ₂ Substituted SiH ₂ Substituted NH or substituted PH, the substituent group is one or more of methyl, ethyl, hydroxyl, halogen, methoxyl, dimethylamino and diethylamino; when A is ₁ Represents substituted C _1-5 Alkyl, substituted C _1-5 Alkoxy, substituted C _1-5 Alkylthio or substituted C _1-5 When the alkylamino group is selected, the substituent is one or more of methyl, ethyl, hydroxyl, halogen, methoxy, dimethylamino and diethylamino; when A is ₂ Represents substituted C _1-5 Alkyl, substituted C _1-5 Alkoxy, substituted C _1-5 Alkylthio or substituted C _1-5 In the case of alkylamino, the substituent is one or more of methyl, ethyl, hydroxyl, halogen, methoxy, dimethylamino and diethylamino; when Z represents substituted CH ₂ Substituted SiH ₂ Substituted NH, or substituted PH, the substituent is one or more of methyl, ethyl, hydroxy, halogen, methoxy, dimethylamino, diethylamino; when R is ₉ 、R ₁₀ 、R ₁₁ And R ₁₂ Each independently represents substituted C _1-4 Alkyl, substituted C _1-4 Alkoxy, substituted C _1-4 Alkylthio or substituted C _1-4 In the case of alkylamino, the substituent is one or more of methyl, ethyl, hydroxy, halogen, methoxy, dimethylamino, and diethylamino.

Further, said R ₅ Selected from one of the following structures:

。

further, said R ₇ Selected from one of the following structures:

。

further, said R ₈ Selected from one of the following structures:

。

further, said R ₆ Selected from one of the following structures:

。

further, L is selected from one of the following structures:

；

d is selected from one of the following structures:

。

further, the compound having the structure of formula i-a may be, but is not limited to, the following compounds:

。

further, the compound having the structure of formula i-b may be, but is not limited to, the following compounds:

。

in a second aspect, the embodiments of the present invention provide a method for preparing a compound according to the first aspect, or a pharmaceutically acceptable salt thereof, comprising the steps of:

S1、

S2、

reacting the intermediate III with a compound IV in a second reaction solvent in the presence of a second base under the action of a second catalyst to obtain an intermediate V;

s4, the intermediate with the structure of the formula VII participates in the reaction to obtain a compound with the structure of the formula I-a or a pharmaceutically acceptable salt thereof, or the intermediate with the structure of the formula IX participates in the reaction to obtain a compound with the structure of the formula I-b or a pharmaceutically acceptable salt thereof;

in the formula, R ₁ 、R ₂ 、M、Q ₁ 、Q ₂ 、Q ₃ 、W ₁ 、R ₃ And R ₄ Is as defined for the first aspect R ₁ 、R ₂ 、M、Q ₁ 、Q ₂ 、Q ₃ 、W ₁ 、R ₃ And R ₄ The definition of (2).

The sequence number of each step in the embodiment of the invention does not limit the sequence of the steps in the embodiment of the invention, and the preparation method provided by the embodiment of the invention is simple, mild in condition, convenient to operate, low in requirement on equipment condition, easy to realize, simple in post-treatment, high in yield and suitable for industrial large-scale production.

Further, in step S1, the first catalyst is a palladium catalyst, and the palladium catalyst includes at least one of [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride, palladium acetate, and tetratriphenylphosphine palladium.

Further, in step S1, the first base includes at least one of sodium acetate, potassium acetate, sodium carbonate, potassium phosphate, and cesium carbonate.

Further, in step S1, the first reaction solvent includes at least one of 1,4-dioxane, toluene, and tetrahydrofuran.

Further, in step S2, the second catalyst is a palladium catalyst, and the palladium catalyst includes at least one of [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride, palladium acetate, and tetratriphenylphosphine palladium.

Further, in step S2, the second base includes at least one of sodium carbonate, potassium phosphate, and cesium carbonate.

Further, in step S2, the second reaction solvent is tetrahydrofuran and water or 1,4-dioxane and water, and the second reaction solvent includes water, which can increase the solubility of the second base and improve the reaction yield.

Further, in step S2, the reaction temperature is 50 ℃ to 120 ℃, for example, the reaction temperature can be 50 ℃, 60 ℃, 70 ℃, 80 ℃,90 ℃,100 ℃, 110 ℃ or 120 ℃.

Further, in step S3, the third base is piperidine.

Further, in step S3, the third reaction solvent includes at least one of ethanol, methanol, and propanol.

Further, in step S3, the fourth base is piperidine.

Further, in step S3, the fourth reaction solvent includes at least one of ethanol, methanol, and propanol.

In a third aspect, the embodiments provide a pharmaceutical composition comprising a therapeutically effective amount of a compound selected from the compounds described in the first aspect, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

The pharmaceutical composition of the embodiment of the present invention can be prepared by combining the compound of the present invention or a salt thereof with a suitable pharmaceutically acceptable carrier, and can be formulated into, for example, solid, semi-solid, liquid or gaseous formulations such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, solutions, suppositories, injections, inhalants, gels, microspheres, aerosols, and the like.

Typical routes of administration of the compounds of the embodiments of the invention or pharmaceutically acceptable salts thereof or pharmaceutical compositions thereof include, but are not limited to, oral, rectal, transmucosal, intestinal administration, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration.

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

For oral administration, the pharmaceutical compositions may be formulated by mixing the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, slurries, suspensions and the like, for oral administration to a patient.

In a fourth aspect, the embodiments of the present invention provide a formulation for targeted ubiquitination degradation of TRK, which comprises a therapeutically effective amount of a compound selected from the compounds described in the first aspect, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In a fifth aspect, the embodiments of the present invention provide a compound according to the first aspect or a pharmaceutically acceptable salt thereof for use in the preparation of a medicament for the prevention and/or treatment of a tumor.

Further, the present embodiment provides a use of the compound according to the first aspect or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and/or treating tumors associated with abnormal expression of TRK activity.

Further, the tumor is selected from skin cancer, bladder cancer, ovarian cancer, breast cancer, stomach cancer, prostate cancer, colon cancer, lung cancer, bone cancer, brain cancer, rectal cancer, esophageal cancer, tongue cancer, kidney cancer, cervical cancer, uterine corpus cancer, endometrial cancer, testicular cancer, urinary cancer, melanoma, astrocytic cancer, meningioma, hodgkin's lymphoma, non-hodgkin's lymphoma, acute lymphatic leukemia, chronic lymphatic leukemia, acute myeloid leukemia, chronic myeloid leukemia, adult T-cell leukemia lymphoma, hepatocellular carcinoma, bronchial cancer, multiple myeloma, basal cell tumor, seminoma, rhabdomyosarcoma, chondrosarcoma, myosarcoma, fibrosarcoma.

The invention is tested for a plurality of times in sequence, and the invention is carried out by taking part of test results as reference

In one detailed description, reference is made to the following detailed description in connection with specific embodiments.

The preparation of compound a5 in the following examples is as follows:

n, N-dimethylformamide (DMF, 1.75g,24.0 mmol) was added to a three-necked flask, and then cooled to 0 ℃ in an ice-water bath, and then phosphorus oxychloride (3.68g, 24.0 mmol) was slowly added dropwise, resulting in heat release and bubble generation. After the dropwise addition, the reaction solution reacts at room temperature for 30min, then 1,2-dichloroethane (20 mL) is added, the temperature is reduced to 0 ℃, a compound a5-1 (2.8g, 20.1mmol) is dissolved in 1,2-dichloroethane (5 mL), then the compound a5-1 is slowly dripped into the reaction solution, after the dropwise addition is finished, the heating is carried out till the reflux, the reaction is 30min, the TLC monitoring reaction is carried out, after the reaction is finished, water is dripped under the condition of ice water bath for quenching reaction, and purified water (10 mL) is added0 mL) and extracted with dichloromethane three times, the volume of dichloromethane used in each extraction is 50mL, the organic phases are combined, 50mL of saturated sodium bicarbonate solution and 50mL of saturated saline are used for washing the organic phases, then anhydrous sodium sulfate is used for drying, reduced pressure concentration and column chromatography separation are carried out to obtain 1.8g of an intermediate a5, the intermediate a5 is light yellow solid, ESI (+) m/z =168.1[ M ] +H] ⁺ 。

EXAMPLE 1 preparation of Compound 1

S1, synthesis of an intermediate a 2:

compound a1 (5.0g, 23.58mmol), pinacol diboron ester (7.2g, 28.3mmol) and potassium acetate (4.6g, 47.00mmol) were dissolved in 1,4-dioxane (50 mL), dispersed with stirring, and Pd (dppf) Cl was added ₂ ([ 1,1' -bis (diphenylphosphino) ferrocene)]Palladium dichloride, 1.7g, 2.36mmol), replacing with nitrogen for three times, heating to 100 ℃, reacting for 4 hours, monitoring the reaction by TLC, cooling to room temperature after the reaction is finished, filtering, discarding a filter cake, collecting filtrate, concentrating the filtrate under reduced pressure till dryness to obtain concentrated residue, adding ethyl acetate into the concentrated residue till all the ethyl acetate is dissolved, washing twice by water, washing once by saturated sodium chloride aqueous solution, drying an organic phase by anhydrous sodium sulfate, concentrating under reduced pressure till dryness to obtain 6.6g of an intermediate a2.

S2, synthesis of an intermediate a 4:

intermediate a2 (6.6 g), a3 (5.86g, 28.30mmol), potassium phosphate (10.0g, 47.16mmol) and Pd (Pph) ₃ ) ₄ (Tetratriphenylphosphine Palladium, 2.7g, 2.36mmol) was dissolved in 1,4-dioxane (150 mL) and water (R: (R))37.5 mL) of the reaction solution, replacing the reaction solution with nitrogen for three times, heating the reaction solution to 100 ℃, reacting for 4 hours, monitoring the reaction by TLC, cooling the reaction solution to room temperature after the reaction is finished, separating the solution, discarding the aqueous phase, concentrating the organic phase under reduced pressure to dryness to obtain a concentrated residue, adding a small amount of methanol into the concentrated residue until the methanol is completely dissolved, dripping dichloromethane at room temperature to precipitate a large amount of solid, stirring for 2 hours, filtering, collecting the filter cake, drying the filter cake to obtain 2.37g of a solid product 1, collecting the filtrate, concentrating the filtrate under reduced pressure to obtain a solid product 2, combining the solid product 1 and the solid product 2, and then carrying out column chromatography to obtain 4.5g of an intermediate a4, wherein the total yield of the step S1 and the step S2 is 73.6%.

S3, synthesis of an intermediate a 6:

dissolving the intermediate a4 (2.1g, 8.10mmol) and the compound a5 (1.6g, 9.57mmol) in absolute ethyl alcohol (100 mL), adding piperidine (63 drops) at room temperature, raising the temperature to 85 ℃, reacting for 4 hours, monitoring the reaction by TLC, cooling to room temperature after the reaction is finished, filtering, soaking and washing a filter cake by using 30mL of n-heptane, pumping, and drying in vacuum to obtain 2.92g of the intermediate a6, wherein the intermediate a6 is yellow solid and the yield is 88.3%.

S4, synthesis of an intermediate a 7:

dissolving the intermediate a6 (2.6 g, 6.37mmol) in methanol (81 mL), adding 2mol/L sodium hydroxide aqueous solution (27 mL), heating to 65 ℃ for reaction for 7 hours, monitoring the reaction by TLC, cooling to room temperature after the reaction is finished, concentrating under reduced pressure until methanol is almost remained, supplementing 80mL water to the reaction solution, dropwise adding 1mol/L hydrochloric acid, adjusting the pH value to 6, stirring for 30min, precipitating solids, filtering, washing a filter cake with water, and drying to obtain 2.1g of the intermediate a7, wherein the intermediate a7 is yellow solid, and the yield is 86.6%.

S5, synthesis of an intermediate a 8:

intermediate a7 (1.0g, 2.63mmol) was dissolved in DMF (N, N-dimethylformamide, 10 mL), DIEA (N, N-diisopropylethylamine, 1.0 mL) and HATU (2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, 900mg, 2.36mmol) were added at room temperature, the reaction was reacted for 2h, and the reaction was monitored by TLC. After the reaction is finished, adding ethyl acetate into the reaction solution, adding water to wash for three times, and using organic phaseDrying with sodium sulfate, concentrating under reduced pressure to dryness, and separating by column chromatography to obtain 1.0g of intermediate a8, wherein the intermediate a8 is yellow solid with yield of 85%, ESI (+) m/z =499[ M ] +H] ⁺ 。

S6, synthesis of an intermediate a 11:

dissolving compound a9 (500mg, 1.83mmol) and compound a10 (616mg, 2.2mmol) in DMF (5.0 mL), adding potassium carbonate (530mg, 3.83mmol) at room temperature, reacting at room temperature for 16 hours, monitoring the reaction by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, adding water, washing three times, drying the organic phase with anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and carrying out column chromatography separation to obtain 735mg of intermediate a11, wherein the intermediate a11 is yellow solid, the yield is 85.0%, and ESI (+) m/z = 473M + H] ⁺ 。

S7, synthesis of an intermediate a 12:

intermediate a11 (735mg, 1.55mmol) was dissolved in dichloromethane (2.0 mL), trifluoroacetic acid (0.4 mL) was added at room temperature, the reaction was carried out at room temperature for 16 hours, the reaction was monitored by TLC, and after completion of the reaction, concentration was carried out under reduced pressure to dryness to give 576.6mg of intermediate a12.

S8, synthesis of a compound 1:

intermediate a8 (50mg, 0.10mmol) and intermediate a12 (60mg, 0.16mmol) were dissolved in DMF (2.0 mL) and DBU (1,8-diazabicyclo [5.4.0 ] was added at room temperature]Undec-7-ene, 45mg, 0.30mmol), reacting at room temperature for 2 hours, monitoring the reaction by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, adding water to wash for three times, drying the organic phase by anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and separating by column chromatography to obtain 45mg of compound 1, wherein the compound 1 is yellow solid, the yield is 61.2%, ESI (+) m/z =735.7[ M + H ]] ⁺ 。

EXAMPLE 2 preparation of Compound 2

S1, synthesis of an intermediate b 6:

dissolving the intermediate a4 (2.1g, 8.10mmol) and the compound b5 (1.6g, 9.72mmol) in absolute ethyl alcohol (100 mL), adding piperidine (63 drops) at room temperature, heating to 85 ℃ for reaction for 4 hours, monitoring the reaction by TLC, cooling to room temperature after the reaction is finished, filtering, soaking and washing a filter cake by using 30mL of n-heptane, pumping, and drying in vacuum to obtain 2.6g of the intermediate b6, wherein the intermediate b6 is yellow solid and the yield is 78.6%.

S2, synthesis of an intermediate b 7:

dissolving the intermediate b6 (2.6 g, 6.37mmol) in methanol (81 mL), adding 2mol/L sodium hydroxide aqueous solution (27 mL), heating to 65 ℃ for reaction for 7 hours, monitoring the reaction by TLC, cooling to room temperature after the reaction is finished, concentrating under reduced pressure until methanol is almost remained, supplementing 80mL water into the reaction solution, dropwise adding 1mol/L hydrochloric acid, adjusting the pH value to 6, stirring for 30min, precipitating solids, filtering, washing a filter cake with water, and drying to obtain 2.1g of the intermediate b7, wherein the intermediate b7 is yellow solid, and the yield is 86.6%.

S3, synthesis of an intermediate b 8:

intermediate b7 (1.0 g, 2.63mmol) was dissolved in DMF (10 mL), DIEA (1.0 mL) and HATU (900mg, 2.36mmol) were added at room temperature, and the mixture was reacted at room temperature for 2h. Monitoring the reaction by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, adding water to wash for three times, drying the organic phase by anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and separating by column chromatography to obtain 0.75g of intermediate b8, wherein the intermediate b8 is yellow solid, the yield is 63.81%, ESI (+) m/z =499[ M + H ])] ⁺ 。

S4, synthesis of a compound 2:

dissolving intermediate b8 (50mg, 0.10mmol) and intermediate a12 (60mg, 0.10mmol) in DMF (2.0 mL), adding DBU (45mg, 0.30mmol) at room temperature, reacting at room temperature for 2 hours, monitoring the reaction by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, adding water for washing for three times, drying an organic phase by anhydrous sodium sulfate, concentrating under reduced pressure to be dry, and carrying out column chromatography separation to obtain 32mg of compound 2, wherein the compound 2 is yellow solid, the yield is 43.5%, ESI m/z =735.7 (+) M + H + and the yield is 43.5%, and the ESI m/z =735.7 (+) is obtained] ⁺ 。

EXAMPLE 3 preparation of Compound 3

S1, synthesis of an intermediate c 3:

the intermediate a9 (500mg, 1.83mmol) and the compound c2 (477mg, 2.19mmol) are dissolved in pyridine (5.0 mL), the temperature is reduced to-10 ℃, phosphorus oxychloride (1.4g, 9.15mmol) is added into the reaction liquid dropwise, and the temperature is kept at-10 ℃ for 2h after the dropwise addition. Monitoring the reaction by TLC, after the reaction is finished, concentrating the reaction solution under reduced pressure to be dry to obtain a concentrated residue, adding ethyl acetate into the concentrated residue until the ethyl acetate is completely dissolved, washing the organic phase with 1mol/L hydrochloric acid aqueous solution for three times, then washing the organic phase with purified water for one time, drying the organic phase with anhydrous sodium sulfate, concentrating under reduced pressure to be dry, and separating by column chromatography to obtain 674mg of an intermediate c3, wherein the yield is 78.0%, and ESI (+) m/z = 473.5M + H] ⁺ 。

S2, synthesis of an intermediate c 4:

intermediate c3 (674mg, 1.43mmol) was dissolved in dichloromethane (2.0 mL), trifluoroacetic acid (0.4 mL) was added at room temperature, the reaction was carried out at room temperature for 16 hours, the reaction was monitored by TLC, and after completion of the reaction, concentration was carried out under reduced pressure to dryness, yielding 531mg of intermediate c4.

S3, synthesis of a compound 3:

dissolving intermediate a8 (50mg, 0.10mmol) and intermediate c4 (56mg, 0.15mmol) in DMF (2.0 mL), adding DBU (45mg, 0.30mmol) at room temperature, reacting at room temperature for 2 hours, monitoring the reaction by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, adding water for washing for three times, drying an organic phase by anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and carrying out column chromatography separation to obtain 40mg of compound 3, wherein the compound 3 is yellow solid, the yield is 54.4%, ESI m/z =735.7 (+) M + H +, (M + H) +] ⁺ 。

EXAMPLE 4 preparation of Compound 4

S1, synthesis of a compound 4:

intermediate b8 (50mg, 0.10mmol) and intermediate c4 (56mg, 0.15mmol) were dissolved in DMF (2.0 mL), and DBU (45mg, 0.30m) was added at room temperaturemol), reacting at room temperature for 2 hours, monitoring the reaction by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, adding water to wash for three times, drying the organic phase by anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and separating by column chromatography to obtain 43mg of a compound 4, wherein the compound 4 is a yellow solid, the yield is 58.5 percent, and ESI (+) m/z =735.7[ M + H ] +] ⁺ 。

EXAMPLE 5 preparation of Compound 5

S1, synthesis of an intermediate d 3:

compound d1 (2.0g, 8.8mmol) and compound d2 (1.24g, 9.69mmol) were dissolved in acetic acid (10.0 mL), and potassium acetate (2.7g, 27.31mmol) was added thereto at room temperature, and the mixture was heated to 90 ℃ to react for 9 hours. The reaction was monitored by TLC and after completion the reaction was allowed to cool to room temperature. Adding water into the reaction solution for dilution, controlling the temperature of an ice water bath to be 0 ℃, stirring for 30min, filtering, washing a filter cake by using ice water, pumping, and drying the filter cake in vacuum to obtain 2.86g of an intermediate d3, wherein the intermediate d3 is a purple solid, and the yield is 96.3%.

S2, synthesis of an intermediate d 5:

intermediate d3 (500mg, 1.48mmol), cuprous iodide (30.0mg, 0.15mmol), compound d4 (322mg, 1.63mmol) and Pd (Pph) ₃ ) ₂ Cl ₂ (1.60g, 2.28mmol) was dissolved in DMF (5.0 mL), triethylamine (1.50g, 14.83mmol) was added thereto at room temperature, the mixture was replaced with nitrogen three times, and the mixture was heated to 80 ℃ to react for 3 hours. Monitoring the reaction by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, adding water to wash for three times, drying the organic phase by anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and separating by column chromatography to obtain 346mg of an intermediate d5, wherein the intermediate d5 is a yellow solid, the yield is 51.6%, and ESI (+) m/z = 454.5M + H +] ⁺ 。

S3, synthesis of an intermediate d 6:

intermediate d5 (346mg, 0.76mmol) was dissolved in dichloromethane (2.0 mL), trifluoroacetic acid (0.4 mL) was added at room temperature, the reaction was monitored by TLC for 16h, and after completion of the reaction, concentration to dryness under reduced pressure gave 268mg of intermediate d6.

S4, synthesis of a compound 5:

dissolving intermediate a8 (60mg, 0.12mmol) and intermediate d6 (49.5mg, 0.14mmol) in DMF (3.0 mL), adding DBU (45mg, 0.30mmol) at room temperature, reacting at room temperature for 2 hours, monitoring the reaction by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, washing with water for three times, drying the organic phase with anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and separating by column chromatography to obtain 50mg of compound 5, wherein the compound 5 is yellow solid, the yield is 58.2%, ESI (+) m/z = 716.7M + H] ⁺ 。

EXAMPLE 6 preparation of Compound 6

S1, synthesis of compound 6:

dissolving intermediate b8 (60mg, 0.12mmol) and intermediate d6 (49.5mg, 0.14mmol) in DMF (3.0 mL), adding DBU (45mg, 0.30mmol) at room temperature, reacting at room temperature for 2 hours, monitoring the reaction by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, adding water, washing for three times, drying the organic phase by anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and separating by column chromatography to obtain 44.5mg of compound 6, wherein the compound 6 is yellow solid, the yield is 51.8%, and ESI (+) m/z = 716.7M + H] ⁺ 。

EXAMPLE 7 preparation of Compound 7

S1, synthesis of an intermediate e 3:

dissolving compound e1 (500mg, 1.93mmol) and compound a10 (650mg, 2.31mmol) in DMF (5.0 mL), adding potassium carbonate (530mg, 3.83mmol) at room temperature, reacting at room temperature for 16h, monitoring the reaction by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, adding water, washing for three times, drying the organic phase with anhydrous sodium sulfate, concentrating under reduced pressure to dryness, separating by column chromatography,742mg of intermediate e3 is obtained in 84% yield, ESI (+) m/z =459.5[ 2 ] +H] ⁺ 。

S2, synthesis of an intermediate e 4:

intermediate e3 (742mg, 1.62mmol) was dissolved in dichloromethane (2.0 mL), trifluoroacetic acid (0.4 mL) was added at room temperature, the reaction was carried out at room temperature for 16 hours, the reaction was monitored by TLC, and after completion of the reaction, concentration was carried out under reduced pressure to dryness to give 579.96mg of intermediate e4.

S3, synthesis of a compound 7:

dissolving intermediate a8 (50mg, 0.10mmol) and intermediate e4 (43mg, 0.12mmol) in DMF (3.0 mL), adding DBU (45mg, 0.30mmol) at room temperature, reacting at room temperature for 2 hours, monitoring the reaction by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, adding water, washing for three times, drying the organic phase with anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and carrying out column chromatography separation to obtain 36mg of compound 7, wherein the compound 7 is yellow solid, the yield is 49.9%, and ESI (+) m/z = 721.7M + H] ⁺ 。

EXAMPLE 8 preparation of Compound 8

S1, synthesis of compound 8:

dissolving intermediate b8 (50mg, 0.10mmol) and intermediate e4 (43mg, 0.12mmol) in DMF (3.0 mL), adding DBU (45mg, 0.30mmol) at room temperature, reacting at room temperature for 2 hours, monitoring the reaction by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, adding water, washing for three times, drying the organic phase by anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and carrying out column chromatography separation to obtain 39.5mg of compound 8, wherein the compound 8 is yellow solid, the yield is 54.8%, ESI (+) m/z = 721.7M + H] ⁺ 。

EXAMPLE 9 preparation of Compound 9

S1, synthesis of an intermediate f 2:

dissolving a compound f1 (474mg, 1.83mmol) and a compound c2 (477mg, 2.19mmol) in pyridine (5.0 mL), cooling to-10 ℃, dropwise adding phosphorus oxychloride (1.4g, 9.15mmol) into the reaction liquid, keeping the temperature at-10 ℃ for reaction for 2h after dropwise adding, monitoring the reaction by TLC, concentrating the reaction liquid under reduced pressure to dryness after the reaction is finished to obtain a concentrated residue, adding ethyl acetate into the concentrated residue until the ethyl acetate is completely dissolved, washing the organic phase with 1mol/L hydrochloric acid aqueous solution for three times, washing the organic phase with purified water once, drying the organic phase with sodium sulfate, concentrating under reduced pressure to dryness, and carrying out column chromatography to obtain 600mg of an intermediate f2, wherein the yield is 71.5%, and ESI (+) m/z = 459.5M + H] ⁺ 。

S2, synthesis of an intermediate f 3:

intermediate f2 (600mg, 1.3 mmol) was dissolved in dichloromethane (2.0 mL), trifluoroacetic acid (0.4 mL) was added at room temperature, the reaction was carried out at room temperature for 16 hours, the reaction was monitored by TLC, and after completion of the reaction, concentration was carried out under reduced pressure to dryness to give 466mg of intermediate f3.

S3, synthesis of a compound 9:

dissolving intermediate a8 (50mg, 0.10mmol) and intermediate f3 (43mg, 0.12mmol) in DMF (2.0 mL), adding DBU (45mg, 0.30mmol) at room temperature, reacting at room temperature for 2 hours, monitoring the reaction by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, adding water, washing for three times, drying the organic phase with anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and carrying out column chromatography separation to obtain 40mg of compound 9, wherein the compound 9 is yellow solid, the yield is 55.5%, and ESI (+) m/z = 721.7M + H] ⁺ 。

EXAMPLE 10 preparation of Compound 10

S1, synthesis of compound 10:

intermediate b8 (50mg, 0.10mmol) and intermediate f3 (43mg, 0.12mmol) is dissolved in DMF (3.0 mL), DBU (45mg, 0.30mmol) is added at room temperature, the reaction is carried out for 2 hours at room temperature, TLC detection reaction is carried out, ethyl acetate is added into reaction liquid after the reaction is finished, water is added for washing for three times, an organic phase is dried by anhydrous sodium sulfate, reduced pressure concentration is carried out until dryness, column chromatography separation is carried out, 39.5mg of compound 10 is obtained, the compound 10 is yellow solid, the yield is 54.8%, ESI (+) m/z =721.7[ M ] +H ], and] ⁺ 。

EXAMPLE 11 preparation of Compound 11

S1, synthesis of an intermediate g 3:

compound g1 (5.00g, 16.2 mmol) and compound g2 (2.67g, 16.2 mmol) were dissolved in acetonitrile (50.0 mL), DIEA (2.72g, 21.1 mmol) was added at room temperature, and the mixture was heated to 70 ℃ to react for 7 hours. And monitoring the reaction by TLC, reducing the temperature to 0 ℃ after the reaction is finished, filtering, leaching a filter cake with acetonitrile, draining, and drying in vacuum to obtain 3.20g of an intermediate g3 with the yield of 61.1%.

S2, synthesis of an intermediate g 5:

intermediate g3 (300mg, 0.93mmol), compound d4 (200mg, 1.02mmol), cuprous iodide (20mg, 0.10mmol), pd (Pph ₃ ) ₂ Cl ₂ (22mg, 0.03mmol) was dissolved in DMF (5.0 mL), and triethylamine (940mg, 9.29mmol) was added thereto at room temperature, and the mixture was heated to 80 ℃ to react for 6 hours. Monitoring the reaction by TLC, after the reaction is finished, adding ethyl acetate into the reaction solution, adding water to wash for three times, drying the organic phase by anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and carrying out column chromatography separation to obtain 290mg of an intermediate g5, wherein the intermediate g5 is a yellow solid, the yield is 71%, and ESI (+) m/z =440.5[ M ] +H ] +] ⁺ 。

S3, synthesis of an intermediate g 6:

intermediate g5 (290mg, 0.66mmol) was dissolved in dichloromethane (5.0 mL), trifluoroacetic acid (0.1 mL) was added at room temperature, the reaction was carried out at room temperature for 16 hours, the reaction was monitored by TLC, and after completion of the reaction, concentration was carried out under reduced pressure to dryness to give 223.7mg intermediate g 6.

S4, synthesis of a compound 11:

dissolving intermediate a8 (50mg, 0.10mmol) and intermediate g6 (40.7mg, 0.12mmol) in DMF (3.0 mL), adding DBU (45mg, 0.30mmol) at room temperature, reacting at room temperature for 2 hours, monitoring the reaction by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, adding water, washing for three times, drying the organic phase by anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and carrying out column chromatography separation to obtain 38mg of compound 11, wherein the compound 11 is yellow solid, the yield is 54.1%, and ESI (+) m/z = 702.7M + H] ⁺ 。

EXAMPLE 12 preparation of Compound 12

S1, synthesis of a compound 12:

dissolving intermediate b8 (50mg, 0.10mmol) and intermediate g6 (40.7mg, 0.12mmol) in DMF (3.0 mL), adding DBU (45mg, 0.30mmol) at room temperature, reacting at room temperature for 2 hours, monitoring the reaction by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, adding water for washing for three times, drying the organic phase by anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and separating by column chromatography to obtain 40mg of compound 12, wherein the compound 12 is yellow solid, the yield is 57.0%, and ESI (+) m/z = 702.7M + H] ⁺ 。

EXAMPLE 13 preparation of Compound 13

S1, synthesis of an intermediate h 3:

dissolving the compound h1 (5.00g, 16.2mmol) and the compound g2 (2.67g, 16.2mmol) in acetonitrile (50.0 mL), adding DIEA (2.72g, 21.1 mmol) at room temperature, heating to 70 ℃ for 7 hours, monitoring the reaction by TLC, reducing the temperature to 0 ℃ after the reaction is finished, filtering, leaching the filter cake with acetonitrile, draining, and drying in vacuum to obtain 3.20g of an intermediate h3 with the yield of 61.1%.

S2, synthesis of an intermediate h 5:

intermediate h3 (300mg, 0.93mmol), compound d4 (200mg, 1.02mmol), cuprous iodide (20mg, 0.09mmol) and Pd (pph) ₃ ) ₂ Cl ₂ (22mg, 0.03mmol) was dissolved in DMF (5.0 mL), and triethylamine (940mg, 9.29mmol) was added thereto at room temperature, and the mixture was heated to 80 ℃ to react for 6 hours. Monitoring the reaction by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, adding water to wash for three times, drying the organic phase by anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and separating by column chromatography to obtain 290mg of an intermediate h5, wherein the intermediate h5 is a yellow solid, the yield is 71%, and ESI (+) m/z =440.5[ M ] +H ],] ⁺ 。

s3, synthesis of an intermediate h 6:

intermediate h5 (290mg, 0.66mmol) was dissolved in dichloromethane (5.0 mL), trifluoroacetic acid (0.1 mL) was added at room temperature, the reaction was carried out at room temperature for 16h, the reaction was monitored by TLC, and after completion of the reaction, concentration was carried out under reduced pressure to dryness to give 223.7mg intermediate h6.

S4, synthesis of a compound 13:

dissolving intermediate a8 (50mg, 0.10mmol) and intermediate h6 (40.7mg, 0.12mmol) in DMF (3.0 mL), adding DBU (45mg, 0.30mmol) at room temperature, reacting at room temperature for 2 hours, monitoring the reaction by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, adding water, washing for three times, drying the organic phase by anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and carrying out column chromatography separation to obtain 38mg of compound 13, wherein the compound 13 is yellow solid, the yield is 54.1%, and ESI (+) m/z = 702.7M + H] ⁺ 。

EXAMPLE 14 preparation of Compound 14

S1, synthesis of compound 14:

dissolving intermediate b8 (50mg, 0.10mmol) and intermediate h6 (40.7mg, 0.12mmol) in DMF (3.0 mL), adding DBU (45mg, 0.30mmol) at room temperature, reacting at room temperature for 2 hours, monitoring the reaction by TLC, adding ethyl acetate to the reaction solution after the reaction is finished, adding water, washing for three times,the organic phase is dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and separated by column chromatography to obtain 40mg of compound 14, which is a yellow solid with yield of 57.0% and ESI (+) m/z =702.7[ M +H ]] ⁺ 。

EXAMPLE 15 preparation of Compound 15

S1, synthesis of an intermediate i 1:

dissolving a compound sm (5.00g, 16.2mmol) and a compound g2 (2.67g, 16.2mmol) in acetonitrile (50.0 mL), adding DIEA (2.72g, 21.1mmol) at room temperature, heating to 70 ℃, reacting for 7 hours, monitoring the reaction by TLC, reducing the temperature to 0 ℃ after the reaction is finished, filtering, leaching a filter cake by acetonitrile, draining, and drying in vacuum to obtain 3.12g of an intermediate i1 with the yield of 59.6%.

S2, synthesis of an intermediate i 2:

intermediate i1 (300mg, 0.93mmol) and compound d4 (200mg, 1.02mmol), cuprous iodide (20mg, 0.09mmol), pd (pph) ₃ ) ₂ Cl ₂ (22mg, 0.03mmol) was dissolved in DMF (5.0 mL), and triethylamine (940mg, 9.29mmol) was added thereto at room temperature, and the reaction was allowed to proceed at 80 ℃ for 6 hours. TLC detection reaction, adding ethyl acetate into the reaction solution after the reaction is finished, adding water to wash for three times, drying the organic phase by anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and separating by column chromatography to obtain 300mg of intermediate i2, wherein the intermediate i2 is a yellow solid product, the yield is 73.4%, ESI (+) m/z =440.5[ M + H ] +] ⁺ 。

S3, synthesis of an intermediate i 3:

the intermediate i2 (300mg, 0.68mmol) was dissolved in dichloromethane (5.0 mL), trifluoroacetic acid (0.1 mL) was added at room temperature, the reaction was carried out at room temperature for 16 hours, the reaction was checked by TLC, and after completion of the reaction, the reaction was concentrated to dryness under reduced pressure to obtain 230.8mg of intermediate i3.

S4, synthesis of compound 15:

intermediate a8 (50mg, 0.10mmol) and intermediate i3 (40.7mg, 0.12mmol) were dissolved in DMF (3.0 mL), and DBU (45 m, 0.12mmol) was added at room temperatureg,0.30 mmol) at room temperature for 2 hours, TLC detection reaction, adding ethyl acetate into the reaction solution after the reaction is finished, adding water to wash for three times, drying the organic phase by anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and separating by column chromatography to obtain 36mg of a compound 15, wherein the compound 15 is a yellow solid, the yield is 51.3%, and ESI (+) m/z = 702.7M + H +] ⁺ 。

EXAMPLE 16 preparation of Compound 16

S1, synthesis of compound 16:

dissolving intermediate b8 (50mg, 0.10mmol) and intermediate i3 (40.7mg, 0.12mmol) in DMF (3.0 mL), adding DBU (45mg, 0.30mmol) at room temperature, reacting at room temperature for 2 hours, detecting by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, adding water, washing for three times, drying the organic phase with anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and separating by column chromatography to obtain 46.5mg of compound 16, wherein the compound 16 is yellow solid, the yield is 66.3%, and ESI (+) m/z = 702.7M & lt, & gt H & lt & gtH & lt & gtH & gt] ⁺ 。

EXAMPLE 17 preparation of Compound 17

S1, synthesis of an intermediate j 2:

intermediate a4 (2.1g, 8.10mmol) and compound j1 (1.87g, 9.57mmol) were dissolved in absolute ethanol (100 mL), piperidine (63 drops) was added at room temperature, the temperature was raised to 85 ℃ for reaction for 4 hours, the reaction was monitored by TLC, after the reaction was completed, the temperature was lowered to room temperature, filtration was performed, the filter cake was bubble-washed with 30mL of n-heptane, and then the mixture was vacuum-dried to obtain 3.0g of intermediate j2, intermediate j2 was yellow solid, and the yield was 84.8%.

S2, synthesis of an intermediate j 3:

dissolving the intermediate j2 (2.5g, 5.73mmol) in methanol (81 mL), adding 2mol/L aqueous sodium hydroxide solution (27 mL), heating to 65 ℃ for reaction for 7 hours, monitoring the reaction by TLC, cooling to room temperature after the reaction is finished, concentrating under reduced pressure until methanol is almost remained, supplementing 80mL water to the reaction solution, adjusting the pH value to 6, stirring for 30min, precipitating a solid, filtering, washing a filter cake with water, and drying to obtain 2.0g of intermediate j3, wherein the intermediate j3 is a yellow solid, and the yield is 85.5%.

S3, synthesis of an intermediate j 4:

intermediate j3 (1.0g, 2.45mmol) was dissolved in DMF (10 mL), and DIEA (1.0 mL) and HATU (1.4g, 3.67mmol) were added at room temperature, and reacted at room temperature for 2 hours. Monitoring the reaction by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, adding water to wash for three times, drying an organic phase by anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and carrying out column chromatography separation to obtain 1.0g of intermediate j4, wherein the intermediate j4 is a yellow solid, the yield is 77.5%, and ESI (+) m/z =527.5[ M + H ] +] ⁺ 。

S4, synthesis of compound 17:

dissolving intermediate j4 (50mg, 0.1mmol) and intermediate g6 (38.7mg, 0.11mmol) in DMF (2.0 mL), adding DBU (45mg, 0.30mmol) at room temperature, reacting at room temperature for 2 hours, monitoring the reaction by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, washing with water for three times, drying the organic phase with anhydrous sodium sulfate, concentrating under reduced pressure to dryness, separating by column chromatography to obtain 45mg of compound 17, wherein the compound 17 is yellow solid, the yield is 61.6%, ESI (+) m/z = 730.7M + H ], (M + H)] ⁺ 。

EXAMPLE 18 preparation of Compound 18

S1, synthesis of Compound 18:

dissolving intermediate j4 (50mg, 0.1mmol) and intermediate h6 (38.7mg, 0.11mmol) in DMF (2.0 mL), adding DBU (45mg, 0.30mmol) at room temperature, reacting at room temperature for 2 hours, monitoring the reaction by TLC, adding ethyl acetate to the reaction solution after the reaction is completed, washing with water three times, drying the organic phase with anhydrous sodium sulfate, concentrating under reduced pressure to dryness, separating by column chromatography to obtain 41.9mg of compound 18 as a yellow solid,the yield is 57.4%, ESI (+) m/z =730.7[ M ] +H] ⁺ 。

EXAMPLE 19 preparation of Compound 19

S1, synthesis of an intermediate k 2:

intermediate a8 (150mg, 0.3mmol) and compound k1 (72mg, 0.36mmol) were dissolved in DMF (5.0 mL), DBU (138mg, 0.9mmol) was added at room temperature and reacted at room temperature for 3h. Monitoring the reaction by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, adding water to wash for three times, drying the organic phase by anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and separating by column chromatography to obtain 137mg of intermediate k2, wherein the intermediate k2 is yellow solid, the yield is 81.2%, ESI (+) m/z =563.6[ M + H ], "] ⁺ 。

S2, synthesis of an intermediate k 3:

intermediate k2 (137mg, 0.24mmol) was dissolved in dichloromethane (5.0 mL), trifluoroacetic acid (0.1 mL) was added at room temperature, the reaction was monitored by TLC for 16 hours at room temperature, and after completion of the reaction, it was concentrated to dryness under reduced pressure to give 111mg of intermediate k3.

S3, synthesis of an intermediate k 5:

intermediate h3 (500mg, 1.55mmol), compound k4 (167mg, 1.70mmol), cuprous iodide (30mg, 0.15mmol), pd (pph) ₃ ) ₂ Cl ₂ (35mg, 0.05mmol) was dissolved in DMF (10.0 mL), triethylamine (1.57g, 15.52mmol) was added at room temperature, and the reaction was allowed to warm to 80 ℃ for 4 hours. Monitoring the reaction by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, adding water to wash for three times, drying the organic phase by anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and separating by column chromatography to obtain 376mg of an intermediate k5, wherein the intermediate k5 is a white solid, the yield is 71.3%, and ESI (+) m/z =341[ M + ] H +] ⁺ 。

S4, synthesis of an intermediate k 6:

dissolving the intermediate k5 (160mg, 0.48mmol) in a mixed solution of dichloromethane (3 mL) and tetrahydrofuran (3 mL), supplementing sodium bicarbonate (20mg, 0.24mmol) at room temperature, cooling to 0 ℃, adding dessimidine oxidant (0.3g, 0.70mmol), replacing with nitrogen for three times, and heating to 40 ℃ for reacting for 4 hours. And monitoring the reaction by TLC, adding a sodium bicarbonate aqueous solution into the reaction solution after the reaction is finished, washing for 5min, discarding a water phase, adding a sodium thiosulfate aqueous solution into an organic phase, washing for 5min, discarding a water phase, drying the organic phase by using anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and performing column chromatography separation to obtain 104mg of an intermediate k6, wherein the intermediate k6 is a white solid, and the yield is 64%.

S5, synthesis of compound 19:

dissolving intermediate k3 (100mg, 0.2mmol) and intermediate k6 (74mg, 0.22mmol) in a mixed solvent of DMF and methanol (volume ratio 1,4, 5.0 mL), adding glacial acetic acid (13mg, 0.22mmol) at room temperature, cooling to 0 ℃ for reaction for 40min, adding sodium cyanoborohydride (30mg, 0.47mmol) at room temperature, and reacting at 0 ℃ for 4h. Monitoring the reaction by TLC, adding sodium bicarbonate water solution into the reaction solution after the reaction is finished, washing for 5min, discarding the water phase, drying the organic phase by anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and separating by column chromatography to obtain 42mg of a compound 19, wherein the compound 19 is a yellow solid, the yield is 26.7%, ESI (+) m/z =785.8[ M + H ], ]] ⁺ 。

EXAMPLE 20 preparation of Compound 20

S1, synthesis of an intermediate m 1:

intermediate b8 (150mg, 0.3mmol) and compound k1 (72mg, 0.36mmol) were dissolved in DMF (5.0 mL), DBU (138mg, 0.9mmol) was added at room temperature, and 3h was reacted at room temperature. Monitoring the reaction by TLC, adding ethyl acetate into the reaction solution after the reaction is finished, adding water to wash for three times, drying the organic phase by anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and separating by column chromatography to obtain 137mg of intermediate m1, wherein the intermediate m1 is yellow solid, the yield is 81.2%, ESI (+) m/z =563.6[ M + H ], "] ⁺ 。

S2, synthesis of an intermediate m 2:

intermediate m1 (137mg, 0.24mmol) was dissolved in dichloromethane (5.0 mL), trifluoroacetic acid (0.1 mL) was added at room temperature, the reaction was carried out at room temperature for 16 hours, the reaction was monitored by TLC, and after completion of the reaction, concentration was carried out under reduced pressure to dryness to give 120mg of intermediate m2.

S3, synthesis of compound 20:

intermediate m2 (100mg, 0.2mmol) and intermediate k6 (74mg, 0.22mmol) were dissolved in a mixed solvent of DMF and methanol (volume ratio 1,4, 5.0 ml), glacial acetic acid (13mg, 0.22mmol) was added at room temperature, and the mixture was cooled to 0 ℃ for reaction for 40min, and sodium cyanoborohydride (30mg, 0.47mmol) was added at room temperature for reaction at 0 ℃ for 4h. Monitoring the reaction by TLC, adding sodium bicarbonate water solution into the reaction solution after the reaction is finished, washing for 5min, discarding the water phase, drying the organic phase by anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and separating by column chromatography to obtain 42mg of a compound 20, wherein the compound 20 is a yellow solid, the yield is 26.7%, ESI (+) m/z =785.8[ M + H ], ]] ⁺ 。

Example 21 TRKA, TRKB, TRKC kinase in vitro Activity assay

Starting from 10000nM for the first generation of the TRK kinase inhibitor Entrectiniband compounds 1 to 20, respectively, a 4-fold gradient dilution (9 concentrations in total) was performed with 100% DMSO. Entrectinib and compounds 1-20 were diluted 50-fold into 1 Xkinase reaction buffer, and shaken on an oscillator for 20 minutes. 2X trka, trkB, trkC kinase (purchased from carba Biosciences 08-186, 08-187, 08-197) and 4X Substrate mixture (ATP + TK Substrate-biotin polypeptide Substrate) were formulated with 1X kinase reaction buffer. mu.L of the prepared TRK kinase and 1. Mu.L of the diluted compound in the buffer were added to each well of the 384 reaction plate, the plate was sealed with a sealing plate, centrifuged at 1000g for 30 seconds, and left at room temperature for 10 minutes. mu.L of the 4 Xsubstrate mixture prepared above was added to the 384 reaction plates, the plates were sealed with a sealing plate, centrifuged at 1000g for 30 seconds, and reacted at room temperature for 60 minutes. Transfer 4. Mu.LADP-Glo to 384 reaction plates, 1000rpm/min, centrifuge for 1min, incubate for 40min at 25 ℃. Transfer 8. Mu.L of detection solution to 384 reaction plates, 1000rpm/min, centrifuge for 1min, incubate for 40min at 25 ℃. RLU (Relative luminescence unit) signals were read using a Biotek multifunctional plate reader, and signal intensity was used to characterize the degree of kinase activity. Data IC of the compound was calculated using grafit6.0 Software (Erithacus Software) ₅₀ Value, IC ₅₀ Specific results of the values are shown in table 1.

IC ₅₀ Values in the interval 0.1-5nM are indicated by the letter A, IC ₅₀ Values in the 5nM-50nM interval are indicated by the letter B, IC ₅₀ Values in the 50nM-500nM interval are indicated by the letter C, IC ₅₀ Values greater than 500nM are indicated by the letter D.

TABLE 1

As shown in Table 1, compounds 1 to 20 all bind to TRKA, TRKB, and TRKC, and inhibit the activity of TRKA, TRKB, and TRKC.

Example 22 tumor cell proliferation inhibitory Activity assay

In this example, the effect of the compounds on inhibiting cell proliferation was investigated by examining the influence of the compounds 1 to 20 prepared in examples 1 to 20 on the in vitro cell proliferation activity in 1 tumor cell line (KM 12-Luc), and Entrectinib is an internal control compound.

The specific method comprises the following steps: (1) Harvesting cells in logarithmic growth phase, counting cells with platelet counter, and adjusting cell concentration to 3-6 × 10 ⁴ cell/mL, 100. Mu.L of cell suspension per well in the plate, cell-free (0.1% DMSO) in Min control wells, plate at 37 ℃ 5% CO ₂ And culturing for 24 hours under the condition of 95% humidity. (2) Diluting an internal control compound Entrectinib to 1mM by using a culture medium containing 1% DMSO, preparing a compound to be detected to 10mM, and sequentially diluting by 3 times to obtain 9-concentration drug solutions; adding 98 muL of cell culture solution into a 96-well flat-bottom transparent drug plate, adding 2 muL of liquid medicine of a compound to be detected and an internal control compound into the cell culture solution of the 96-well transparent drug plate by sucking, adding 2 muL of LDMSO into a solvent control, and blowing and uniformly mixing the mixture with a discharging gun after adding the compound or DMSO; taking out the culture plate after 24-hour culture, removing 50 muL of supernatant, adding 45 muL of fresh culture medium, adding the solution in a5 muL 96-hole transparent drug plate into the culture plate, adding 5 muL of LDMSO cell culture solution mixed solution in Max control, and adding the dosed culture plateThe cells in (A) are incubated at 37 ℃ and 5% CO ₂ And further cultured under 95% humidity conditions for 72 hours, followed by CTG analysis (CellTiter-Glo luminescence cell activity assay). (3) Detection was performed according to the instructions of Promega CellTiter-Glo luminescence cell activity detection kit (Promega-G7573), and an equal volume of CTG solution was added to each well, and the well was left at room temperature for 20 minutes to stabilize the luminescence signal, and the luminescence value was read. (4) Data were analyzed using GraphPad prism7.0 software, fitted to data using nonlinear S-curve regression to derive dose-response curves, and IC was calculated therefrom ₅₀ The results are shown in Table 2.

IC ₅₀ Values in the interval 0.1-5nM are indicated by the letter A, IC ₅₀ Values in the 5nM-50nM interval are indicated by the letter B, IC ₅₀ Values in the interval 50nM-500nM are indicated by the letter C, IC ₅₀ Values greater than 500nM are indicated by the letter D.

TABLE 2

As can be seen from Table 2, compounds 1 to 20 all had a certain inhibitory effect on the proliferation of KM12-Luc tumor cells.

Example 23 Activity assay for Targeted degradation of TRKA

In this example, the influence of the compounds 1 to 20 and Entrecinib prepared in examples 1 to 20 on the TRKA content in the tumor cell line (KM 12-Luc) was evaluated, and the content of internal control GAPDH was analyzed.

The specific method is (1) each tumor cell that had been intervened by the compound is collected into a 1.5mLEP tube, the medium is removed, and a volume of Lysis Buffer (RIPA: protease/phosphatase inhibitor = 100). (2) After addition of lysine Buffer, the EP tube was quickly placed on ice and vortexed using a vortexer to lyse the cells thoroughly. Note that the vortex time was not too long, and the sample tubes were quickly placed on ice for lysis, shaking once every 10min, three times in total. (3) After the lysis time was complete, the EP tube was placed in a precooled to 4 ℃ centrifuge and centrifuged at 12000rpm for 10min.(4) After centrifugation, the supernatant was transferred to a new 1.5ml lep tube and labeled. (5) Protein quantification was performed on tissue lysis supernatants using the BCA protein concentration assay kit according to kit instructions. (6) After completion of the protein concentration determination, the lowest concentration of Lysis supernatant was used as a reference, and the remaining Lysis supernatants were diluted to this concentration using lysine Buffer after calculation according to the BCA standard curve. (7) To the diluted lysis supernatant was added a corresponding volume of 5Xloading Buffer, heated in a warm metal bath at 95 ℃ for 10min, then placed on ice to cool, and the samples were stored in a refrigerator at-20 ℃ for a short period. (8) preparing glue: cleaning glass plates for glue making, aligning the bottoms of the two glass plates, placing the two glass plates into a bracket, and fixing the two glass plates by a clamp; preparing separation gel according to 12% or 15% separation gel formula, mixing well, adding into glass plate, adding about 3/4 glass plate, and pressing with 75% ethanol; after 30min, the separation gel is solidified, 75% ethanol is poured out, the residual ethanol solution on the glass plate is sucked by filter paper, 5% concentrated gel is prepared, the mixture is uniformly mixed and added into the glass plate, a sample adding comb is inserted, and the gel is prepared after 30min. (9) protein loading and electrophoresis: fixing the rubber plate in an electrophoresis tank, filling the electrophoresis tank with 1x electrophoresis solution, wherein the outer tank exceeds the metal wire; pulling out the sample adding comb, and adding a 5uL protein sample and a protein ladder; and (3) switching on a power supply, performing electrophoresis for 30min by using a constant voltage of 80V, continuing electrophoresis for about 1h by using a constant voltage of 120V until the bromophenol blue indicator band reaches the bottom, and stopping electrophoresis. (10) protein electrotransformation: soaking the PVDF membrane in methanol for 30s for membrane standing, and then putting the PVDF membrane, 4 pieces of filter paper and 2 pieces of sponge into a membrane rotating box; pouring pre-prepared 1x electric conversion liquid into the film conversion box; taking out the gel, cutting the gel into required size, sequentially placing the sponge, the filter paper, the gel, the PVDF membrane, the filter paper and the sponge in a membrane rotating clamp in sequence, removing bubbles, placing the membrane on an anode surface, placing the gel on a cathode surface (black gel and white membrane), inserting the gel into an electrophoresis tank, pouring a membrane rotating buffer solution, placing the gel into an ice bag, and placing the ice bag around the electrophoresis tank for cooling; constant voltage 100V electrophoresis transfer for 1h30min. (11) blocking and incubating primary antibody: after the electrotransformation is finished, putting the PVDF membrane into 5% skimmed milk sealing solution, slowly shaking on a decoloring shaking table, and sealing for 1h at room temperature; slowly washing the sealed PVDF membrane in TBST for 3 times, 10min each time; washing knotAfter completion, the PVDF membrane was placed in an antibody cassette for primary antibodies, which were expressed according to a 1:1000, diluted with primary antibody diluent, and the antibody cassette incubated at 4 ℃ in a gentle shaker (10-16 h). (12) incubation of secondary antibody: taking the antibody box of the PVDF membrane incubated overnight out of the refrigerator, and sucking away the primary antibody incubation liquid; slowly washing the PVDF membrane in TBST for 3 times, and 10min each time; putting the washed PVDF membrane into a diluent (containing 5% skimmed milk) of a secondary antibody (1; after the secondary antibody incubation is finished, taking out the PVDF membrane, and slowly washing in TBST for 3 times, 10min each time; mixing the equal volumes of the solution A and the solution B in the ECL kit, uniformly adding the mixture on the surface of the membrane, placing the membrane into a Tanon5200 luminescence imager (precooling is started for 5min in advance), exposing and developing, and taking a picture to store the picture. The grey values of each band were analyzed using Image J software and the inhibitor concentration DC at 50% protein degradation was calculated ₅₀ The results obtained are shown in table 3 below.

DC ₅₀ Values in the interval 0.1-5nM are indicated by the letter A, DC ₅₀ Values in the 5nM-50nM interval are indicated by the letter B, DC ₅₀ Values in the 50nM-500nM range are indicated by the letter C, DC ₅₀ Values greater than 500nM are indicated by the letter D.

TABLE 3

As can be seen from Table 3, the compounds 1 to 20 prepared in examples 1 to 20 can effectively degrade over-expressed TRKA in cells, and the protein degradation capability of the compounds 1 to 20 provided by the application is verified, so that degraded PROTACs can circularly and continuously degrade other protein kinases, and the compounds have the advantages of small clinical drug dosage requirement and small toxic and side effects. Wherein, in the compounds in all intervals A, the degradation capacities of the compound 17 and the compound 18 are equivalent and strongest, the degradation capacity of the compound 17 is 12 times that of the compound 13 after 13 times; among the compounds in all intervals B, compound 1 is the least degradable; among the compounds in all intervals C, compound 4 had the strongest degradation ability; compound 1 had 4 times the ability to degrade compared to compound 4.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A compound having the structure of formula I-a or formula I-b:

；

in the formula:

when R is ₁ And R ₂ Each independently represents fluorine, M, Q ₁ 、Q ₂ 、Q ₃ And W ₁ Each independently represents carbon, R ₃ And R ₄ Each independently represents hydrogen, L is selected from one of the following structures:

(ii) a The D is selected from one of the following structures:

；

when R is ₁ And R ₂ Each independently represent fluorine, M, Q ₁ 、Q ₂ 、Q ₃ And W ₁ Each independently represents carbon, R ₃ And R ₄ Each independently represents unsubstituted C ₁ Alkyl, L represents

When D represents

Or

；

The compound having the structure of formula I-a or formula I-b is selected from the following compounds:

。

2. a process for preparing a compound according to claim 1, or a pharmaceutically acceptable salt thereof, comprising the steps of:

S1、

S2、

reacting the intermediate V with a compound with a structure shown in the formula VI in a third reaction solvent in the presence of a third base to obtain an intermediate with a structure shown in the formula VII;

reacting the intermediate V with a compound with a structure of a formula VIII in a fourth reaction solvent in the presence of a fourth base to obtain an intermediate with a structure of a formula IX;

in the formula, R ₁ 、R ₂ 、M、Q ₁ 、Q ₂ 、Q ₃ 、W ₁ 、R ₃ And R ₄ Are as defined in claim 1 for R ₁ 、R ₂ 、M、Q ₁ 、Q ₂ 、Q ₃ 、W ₁ 、R ₃ And R ₄ The definition of (1).

3. A pharmaceutical composition comprising a therapeutically effective amount of a compound selected from the group of compounds of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

4. A formulation for targeted ubiquitination of TRK, comprising a therapeutically effective amount of a compound selected from the group consisting of the compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

5. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention and/or treatment of a tumour.