CN114853735A

CN114853735A - Compound for target ubiquitination degradation of TRK and preparation method, composition and application thereof

Info

Publication number: CN114853735A
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: 2022-08-05
Anticipated expiration: 2042-07-06
Also published as: CN114853735B

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 ₁ Represents carbon or nitrogen; 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

(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. 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 proteasome. 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 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, not takeSubstituted 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 Heterocyclyl 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, five-membered heteroaryl, six-membered 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 base under the action of a second catalyst to obtain an intermediate V;

s3, when the 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;

(ii) S4, the intermediate having the structure of formula vii participating in a reaction to give a compound having the structure of formula i-a or a pharmaceutically acceptable salt thereof, or the intermediate having the structure of formula ix participating in a reaction to give a compound having the structure of 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 group as described in 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 and the like 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 represented by the prefix designating the minimum and maximum number of carbon atoms for that moiety, i.e., 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 from 3 to 8 ring atoms, of which 1-3 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 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 Heterocyclyl 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 radical, C _3-7 Heterocyclic group,

、

、

、

、

、

、

Or

(ii) a D represents

、

、

Or

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 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 In the case of alkylamino, the substituent is one or more of hydroxyl, methyl, amino, methoxy, dimethylamino, halogen and ethyl; when said R is ₃ Represents substituted C _1-4 When the alkyl is selected, the substituent is one or more of hydroxyl, methyl, amino, methoxy, dimethylamino, halogen and ethyl; when said R is ₅ Represents substituted C _1-4 Alkyl, substituted C _3-7 Cycloalkyl, substituted C _3-7 When the heterocyclic group, the substituted phenyl group, the substituted five-membered heteroaryl group or the substituted six-membered heteroaryl group is substituted, the substituent is methyl, ethyl, hydroxyl, halogen, methoxy, n-hydroxy, n-or n-hydroxy, n-or n-hydroxy, n,

、

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 one or more of methyl, ethyl, hydroxyl, halogen, methoxy, dimethylamino and diethylamino; when Y 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 A is ₁ 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 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 of methyl, ethyl, hydroxy, halogen, methoxy, dimethylamino, and diethylaminoOr a plurality of them.

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、

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 palladium tetratriphenylphosphine.

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 palladium tetratriphenylphosphine.

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, so that the solubility of the second base can be increased, and the reaction yield can be improved.

Further, in step S2, the reaction temperature is 50 ℃ to 120 ℃, for example, the reaction temperature may 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, embodiments of the present invention provide a pharmaceutical composition 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.

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 or pharmaceutical compositions thereof include, but are not limited to, oral, rectal, transmucosal, enteral, 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 preparation for targeted ubiquitination degradation of TRK, which comprises 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 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 taken in conjunction with specific examples.

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

to a three-necked flask was added N, N-dimethylformamide (DMF, 1.75g,24.0 mmol), cooling to 0 ℃ in an ice water bath, and then slowly adding phosphorus oxychloride (3.68 g, 24.0 mmol) dropwise, wherein an exothermic phenomenon and bubbles are generated. After the dropwise addition, the reaction solution was reacted at room temperature for 30min, then 1, 2-dichloroethane (20 mL) was added, the temperature was reduced to 0 ℃ and the compound a5-1 (2.8 g, 20.1 mmol) was dissolved in 1, 2-dichloroethane (5 mL), then slowly dripping the mixture into the reaction solution, heating to reflux after finishing dripping, reacting for 30min, monitoring the reaction by TLC, after the reaction is finished, the reaction was quenched by adding dropwise water under ice-water bath conditions, purified water (100 mL) was added, the mixture was extracted three times with dichloromethane, 50mL of dichloromethane was used for each extraction, the organic phases were combined, washed with 50mL of saturated sodium bicarbonate solution and 50mL of saturated brine, and then dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography to give 1.8g of intermediate a5, intermediate a5 was a pale yellow solid, and ESI (+) M/z was 168.1[ M + H ].] ⁺ 。

EXAMPLE 1 preparation of Compound 1

Synthesis of S1, intermediate a 2:

compound a1 (5.0 g, 23.58 mmol), pinacol diboron (7.2 g, 28.3 mmol) and potassium acetate (4.6 g, 47.00 mmol) were dissolved in 1, 4-dioxane (50 mL) and dispersed with stirring, Pd (dppf) Cl was added ₂ ([ 1,1' -bis (diphenylphosphino) ferrocene)]1.7g of palladium dichloride and 2.36 mmol), 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 to dryness to obtain concentrated residue, and adding the concentrated residue into the concentrated residueThe residue was added with ethyl acetate to dissolve all of it, washed twice with water, once with saturated aqueous sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure, to give 6.6g of intermediate a 2.

Synthesis of S2, intermediate a 4:

intermediate a2 (6.6 g), a3 (5.86 g, 28.30 mmol), potassium phosphate (10.0 g, 47.16 mmol) and Pd (Pph) ₃ ) ₄ (Tetratriphenylphosphine palladium, 2.7g, 2.36 mmol) is dissolved in a mixed solution of 1, 4-dioxane (150 mL) and water (37.5 mL), nitrogen is replaced three times, the temperature is raised to 100 ℃, the reaction is carried out for 4 hours, TLC monitoring reaction is carried out, after the reaction is finished, the temperature is reduced to room temperature, liquid separation is carried out, water phase is discarded, organic phase is subjected to pressure concentration to dryness, concentrated residue is obtained, a small amount of methanol is added into the concentrated residue until the methanol is completely dissolved, dichloromethane is dripped at the room temperature, a large amount of solid is separated out, stirring is carried out for 2 hours, filtering is carried out, filter cake is collected, the filter cake is dried to obtain 2.37g of solid product 1, filtrate is collected, the filtrate is subjected to pressure reduction concentration to obtain solid product 2, the solid product 1 and the solid product 2 are combined, column chromatography separation is carried out to obtain 4.5g of intermediate a4, and the total yield of step S1 and step S2 is 73.6%.

Synthesis of S3, intermediate a 6:

intermediate a4 (2.1 g, 8.10 mmol) and compound a5 (1.6 g, 9.57 mmol) were dissolved in absolute ethanol (100 mL), piperidine (63 drops) was added at room temperature, the temperature was raised to 85 ℃, the reaction was carried out for 4 hours, TLC monitored, the reaction was cooled to room temperature after completion of the reaction, filtered, the filter cake was washed with 30mL n-heptane, pumped dry and dried in vacuo to give 2.92g intermediate a6, intermediate a6 as a yellow solid in 88.3% yield.

Synthesis of S4, intermediate a 7:

dissolving intermediate a6 (2.6 g, 6.37 mmol) 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 not 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 intermediate a7, wherein the intermediate a7 is yellow solids, and the yield is 86.6%.

Synthesis of S5, intermediate a 8:

intermediate a7 (1.0 g, 2.63 mmol) 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, 2.36 mmol) were added at room temperature and reacted for 2h at room temperature with TLC monitoring. After the reaction, ethyl acetate was added to the reaction mixture, which was washed with water three times, the organic phase was dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and subjected to column chromatography to obtain 1.0g of intermediate a8, intermediate a8 was a yellow solid, yield was 85%, ESI (+) M/z =499[ M + H ] (yield: 85%)] ⁺ 。

Synthesis of S6, intermediate a 11:

dissolving compound a9 (500 mg, 1.83 mmol) and compound a10 (616 mg, 2.2 mmol) in DMF (5.0 mL), adding potassium carbonate (530 mg, 3.83 mmol) at room temperature, reacting at room temperature for 16 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 735mg of intermediate a11, intermediate a11 being a yellow solid with a yield of 85.0%, ESI (+) M/z =473[ M + H ], (ESI (+) M/z =473] ⁺ 。

Synthesis of S7, intermediate a 12:

intermediate a11 (735 mg, 1.55 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 16h, the reaction was monitored by TLC, and after completion of the reaction, concentration to dryness under reduced pressure gave 576.6mg of intermediate a 12.

S8, synthesis of compound 1:

intermediate a8 (50 mg, 0.10 mmol) and intermediate a12 (60 mg, 0.16 mmol) were dissolved in DMF (2.0 mL) and DBU (1, 8-diazabicyclo [5.4.0 ] was added at room temperature]Undec-7-ene, 45mg, 0.30 mmol), 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 and 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 45mg of compound 1, wherein the compound 1 is yellow solid, the yield is 61.2%, ESI (+) M/z =735.7[ M + H ] (E + M) (+) M/z)] ⁺ 。

EXAMPLE 2 preparation of Compound 2

Synthesis of S1, intermediate b 6:

intermediate a4 (2.1 g, 8.10 mmol) and compound b5 (1.6 g, 9.72 mmol) were dissolved in absolute ethanol (100 mL), piperidine (63 drops) was added at room temperature, the temperature was raised to 85 ℃ for 4 hours, the reaction was monitored by TLC, after completion of the reaction, the temperature was lowered to room temperature, filtered, the filter cake was washed with 30mL n-heptane, pumped dry and dried in vacuo to give 2.6g of intermediate b6, intermediate b6 as a yellow solid in 78.6% yield.

Synthesis of S2, intermediate b 7:

dissolving intermediate b6 (2.6 g, 6.37 mmol) 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 not 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 intermediate b7, wherein the intermediate b7 is yellow solids, and the yield is 86.6%.

Synthesis of S3, intermediate b 8:

intermediate b7 (1.0 g, 2.63 mmol) was dissolved in DMF (10 mL) and DIEA (1.0 mL) and HATU (900 mg, 2.36 mmol) were added at room temperature and reacted for 2h at room temperature. The reaction was monitored by TLC, after completion of the reaction, ethyl acetate was added to the reaction solution, washed with water three times, the organic phase was dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and separated by column chromatography to give 0.75g of intermediate b8, intermediate b8 was a yellow solid with a yield of 63.81%, ESI (+) M/z =499[ M + H%] ⁺ 。

S4, synthesis of compound 2:

intermediate b8 (50 mg, 0.10 mmol) and intermediate a12 (60 mg, 0.16 mmol) were dissolved in DMF (2.0 mL), DBU (45 mg, 0.30 mmol) was added at room temperature, the reaction was carried out at room temperature for 2 hours, TLC was used to monitor the reaction, and acetic acid was added to the reaction solution after completion of the reactionEthyl ester, water was added and washed three times, the organic phase was dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and separated by column chromatography to give 32mg of compound 2, compound 2 as a yellow solid in 43.5% yield, ESI (+) M/z =735.7[ M + H ] (M + H) (%)] ⁺ 。

EXAMPLE 3 preparation of Compound 3

Synthesis of S1, intermediate c 3:

intermediate a9 (500 mg, 1.83 mmol) and compound c2 (477 mg, 2.19 mmol) were dissolved in pyridine (5.0 mL), cooled to-10 deg.C, phosphorus oxychloride (1.4 g, 9.15 mmol) was added dropwise to the reaction mixture, and the reaction was incubated at-10 deg.C for 2 h. Monitoring the reaction by TLC, concentrating the reaction solution to dryness under reduced pressure 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 anhydrous sodium sulfate, concentrating to dryness under reduced pressure, and separating by column chromatography to obtain 674mg of an intermediate c3, wherein the yield is 78.0%, and ESI (+) M/z =473.5[ M + H ] (E) (+) M/z)] ⁺ 。

Synthesis of S2, intermediate c 4:

intermediate c3 (674 mg, 1.43 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 16h, the reaction was monitored by TLC, and after completion of the reaction, concentration to dryness under reduced pressure gave 531mg of intermediate c 4.

S3, synthesis of compound 3:

dissolving intermediate a8 (50 mg, 0.10 mmol) and intermediate c4 (56 mg, 0.15 mmol) in DMF (2.0 mL), adding DBU (45 mg, 0.30 mmol) 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 and washing three times, drying the organic phase with anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and separating by column chromatography to obtain 40mg of compound 3, wherein the compound 3 is yellow solid, the yield is 54.4%, and ESI (+) M/z =735.7[ M + H ] M/z] ⁺ 。

EXAMPLE 4 preparation of Compound 4

S1, synthesis of compound 4:

intermediate b8 (50 mg, 0.10 mmol) and intermediate c4 (56 mg, 0.15 mmol) were dissolved in DMF (2.0 mL), DBU (45 mg, 0.30 mmol) was added at room temperature, the reaction was carried out at room temperature for 2 hours, TLC monitored, ethyl acetate was added to the reaction solution after completion of the reaction, water was added and washed three times, the organic phase was dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and column chromatography was carried out to obtain 43mg of compound 4, compound 4 was a yellow solid with a yield of 58.5%, ESI (+) M/z =735.7[ M + H ], (M + H) (+)] ⁺ 。

EXAMPLE 5 preparation of Compound 5

Synthesis of S1, intermediate d 3:

compound d1 (2.0 g, 8.8 mmol) and compound d2 (1.24 g, 9.69 mmol) were dissolved in acetic acid (10.0 mL), potassium acetate (2.7 g, 27.31 mmol) was added at room temperature, and the reaction was allowed to warm to 90 ℃ 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%.

Synthesis of S2, intermediate d 5:

intermediate d3 (500 mg, 1.48 mmol), cuprous iodide (30.0 mg, 0.15 mmol), compound d4 (322 mg, 1.63 mmol) and Pd (Pph) ₃ ) ₂ Cl ₂ (1.60 g, 2.28 mmol) was dissolved in DMF (5.0 mL), triethylamine (1.50 g, 14.83 mmol) was added at room temperature, the mixture was replaced with nitrogen three times, and the mixture was heated to 80 ℃ for reaction 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 using anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and carrying out column chromatographyIsolation gave 346mg of intermediate d5, intermediate d5 as a yellow solid in 51.6% yield ESI (+) M/z =454.5[ M + H [] ⁺ 。

Synthesis of S3, intermediate d 6:

intermediate d5 (346 mg, 0.76 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 16h, the reaction was monitored by TLC, and after completion of the reaction, concentration to dryness under reduced pressure gave 268mg of intermediate d 6.

S4, synthesis of compound 5:

intermediate a8 (60 mg, 0.12 mmol) and intermediate d6 (49.5 mg, 0.14 mmol) were dissolved in DMF (3.0 mL), DBU (45 mg, 0.30 mmol) was added at room temperature, the reaction was carried out at room temperature for 2 hours, the reaction was monitored by TLC, ethyl acetate was added to the reaction solution after completion of the reaction, water was added and washed three times, the organic phase was dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and column chromatography was carried out to give 50mg of compound 5, compound 5 was a yellow solid with a yield of 58.2%, ESI (+) M/z =716.7[ M + H ], (ESI M/z) (+)] ⁺ 。

EXAMPLE 6 preparation of Compound 6

S1, synthesis of compound 6:

intermediate b8 (60 mg, 0.12 mmol) and intermediate d6 (49.5 mg, 0.14 mmol) were dissolved in DMF (3.0 mL), DBU (45 mg, 0.30 mmol) was added at room temperature, the reaction was carried out at room temperature for 2 hours, the reaction was monitored by TLC, ethyl acetate was added to the reaction solution after completion of the reaction, water was added and washed three times, the organic phase was dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and column chromatography was carried out to give 44.5mg of compound 6, compound 6 was yellow solid at 51.8% yield ESI (+) M/z =716.7[ M + H ], (ESI (+) M/z) (+)] ⁺ 。

EXAMPLE 7 preparation of Compound 7

Synthesis of S1, intermediate e 3:

dissolving compound e1 (500 mg, 1.93 mmol) and compound a10 (650 mg, 2.31 mmol) in DMF (5.0 mL), adding potassium carbonate (530 mg, 3.83 mmol) at room temperature, reacting at room temperature for 16 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, and separating by column chromatography to obtain 742mg of intermediate e3 with a yield of 84%, ESI (+) M/z =459.5[ M + H ] (M + H + K)] ⁺ 。

Synthesis of S2, intermediate e 4:

intermediate e3 (742 mg, 1.62 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 to dryness under reduced pressure gave 579.96mg of intermediate e 4.

S3, synthesis of compound 7:

intermediate a8 (50 mg, 0.10 mmol) and intermediate e4 (43 mg, 0.12 mmol) were dissolved in DMF (3.0 mL), DBU (45 mg, 0.30 mmol) was added at room temperature, the reaction was carried out at room temperature for 2 hours, TLC monitored, ethyl acetate was added to the reaction solution after completion of the reaction, water was added and washed three times, the organic phase was dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and column chromatography was carried out to give 36mg of compound 7, compound 7 was a yellow solid with a yield of 49.9%, ESI (+) M/z =721.7[ M + H ], (M + H) (+)] ⁺ 。

EXAMPLE 8 preparation of Compound 8

S1, synthesis of compound 8:

intermediate b8 (50 mg, 0.10 mmol) and intermediate e4 (43 mg, 0.12 mmol) were dissolved in DMF (3.0 mL), DBU (45 mg, 0.30 mmol) was added at room temperature, the reaction was carried out at room temperature for 2 hours, TLC monitored, ethyl acetate was added to the reaction solution after completion of the reaction, water was added and washed three times, the organic phase was dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and column chromatography was carried out to obtain 39.5mg of compound 8, compound 8 was a yellow solid with a yield of 54.8%, ESI (+) M/z =721.7[ M + H ], (ESI M + z) (+)] ⁺ 。

EXAMPLE 9 preparation of Compound 9

S1, synthesis of intermediate f 2:

dissolving compound f1 (474 mg, 1.83 mmol) and compound c2 (477 mg, 2.19 mmol) in pyridine (5.0 mL), cooling to-10 deg.C, adding phosphorus oxychloride (1.4 g, 9.15 mmol) dropwise into the reaction solution, keeping the temperature at-10 deg.C for 2H, monitoring the reaction by TLC, concentrating the reaction solution 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 anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and performing column chromatography to obtain 600mg of intermediate f2 with the yield of 71.5%, ESI (+) M/z = 71.5 [ M + H459.5 ]] ⁺ 。

S2, synthesis of intermediate f 3:

intermediate f2 (600 mg, 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 to dryness under reduced pressure gave 466mg of intermediate f 3.

S3, synthesis of compound 9:

intermediate a8 (50 mg, 0.10 mmol) and intermediate f3 (43 mg, 0.12 mmol) were dissolved in DMF (2.0 mL), DBU (45 mg, 0.30 mmol) was added at room temperature, the reaction was carried out at room temperature for 2 hours, TLC monitored, ethyl acetate was added to the reaction solution after completion of the reaction, water was added and washed three times, the organic phase was dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and column chromatography was carried out to give 40mg of compound 9, compound 9 was a yellow solid with a yield of 55.5%, ESI (+) M/z =721.7[ M + H ], (M + H) (+)] ⁺ 。

EXAMPLE 10 preparation of Compound 10

S1, synthesis of compound 10:

intermediate b8 (50 mg, 0.10 mmol) and intermediate f3 (43 mg, 0.12 mmol) were dissolved in DMF (3.0 mL), DBU (45 mg, 0.30 mmol) was added at room temperature, the reaction was carried out at room temperature for 2 hours, TLC detection was carried out, ethyl acetate was added to the reaction solution after completion of the reaction, water was added and washing was carried out three times, the organic phase was dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, column chromatography was carried out to obtain 39.5mg of compound 10, compound 10 was a yellow solid, yield was 54.8%, ESI (+) M/z =721.7[ M + H ], (ESI M + z) (+)] ⁺ 。

EXAMPLE 11 preparation of Compound 11

Synthesis of S1, intermediate g 3:

compound g1 (5.00 g, 16.2 mmol) and compound g2 (2.67 g, 16.2 mmol) were dissolved in acetonitrile (50.0 mL), DIEA (2.72 g, 21.1 mmol) was added at room temperature, and the reaction was allowed to warm to 70 ℃ for 7 hours. The reaction was monitored by TLC, after the reaction was complete, cooled to 0 ℃, filtered, the filter cake was rinsed with acetonitrile, drained and dried in vacuo to give 3.20g of intermediate g3 with a yield of 61.1%.

Synthesis of S2, intermediate g 5:

intermediate g3 (300 mg, 0.93 mmol), compound d4 (200 mg, 1.02 mmol), cuprous iodide (20 mg, 0.10 mmol), Pd (Pph) ₃ ) ₂ Cl ₂ (22 mg, 0.03 mmol) was dissolved in DMF (5.0 mL), triethylamine (940 mg, 9.29 mmol) was added at room temperature, and the reaction was allowed to warm to 80 ℃ for 6 hours. TLC monitoring reaction, after the reaction is finished, adding ethyl acetate into the reaction solution, adding water and 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 290mg of intermediate g5, wherein the intermediate g5 is yellow solid, the yield is 71%, and ESI (+) M/z =440.5[ M + H ], (ESI (+) M/z)] ⁺ 。

Synthesis of S3, intermediate g 6:

intermediate g5 (290 mg, 0.66 mmol) 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 to dryness under reduced pressure gave 223.7mg intermediate g 6.

S4, synthesis of compound 11:

intermediate a8 (50 mg, 0.10 mmol) and intermediate g6 (40.7 mg, 0.12 mmol) were dissolved in DMF (3.0 mL), DBU (45 mg, 0.30 mmol) was added at room temperature, the reaction was carried out at room temperature for 2 hours, the reaction was monitored by TLC, ethyl acetate was added to the reaction solution after completion of the reaction, water was added and washed three times, the organic phase was dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and column chromatography was carried out to give 38mg of compound 11, compound 11 was a yellow solid with a yield of 54.1%, ESI (+) M/z =702.7[ M + H ], (ESI M + z) (+)] ⁺ 。

EXAMPLE 12 preparation of Compound 12

S1, synthesis of compound 12:

dissolving intermediate b8 (50 mg, 0.10 mmol) and intermediate g6 (40.7 mg, 0.12 mmol) in DMF (3.0 mL), adding DBU (45 mg, 0.30 mmol) 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, washing with water three times, drying the organic phase with 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 with the yield of 57.0%, and ESI (+) M/z =702.7[ M + H ] (M + H) ()] ⁺ 。

EXAMPLE 13 preparation of Compound 13

S1 and synthesis of an intermediate h 3:

compound h1 (5.00 g, 16.2 mmol) and compound g2 (2.67 g, 16.2 mmol) were dissolved in acetonitrile (50.0 mL), DIEA (2.72 g, 21.1 mmol) was added at room temperature, the temperature was raised to 70 ℃ for 7 hours, the reaction was monitored by TLC, after completion of the reaction, the temperature was lowered to 0 ℃, filtered, the filter cake was rinsed with acetonitrile, drained and dried in vacuo to give 3.20g of intermediate h3 in 61.1% yield.

S2 and synthesis of an intermediate h 5:

intermediate h3 (300 mg, 0.93 mmol), compound d4 (200 mg, 1.02 mmol), cuprous iodide (20 mg, 0.09 mmol) and Pd (pph) ₃ ) ₂ Cl ₂ (22 mg, 0.03 mmol) was dissolved in DMF (5.0 mL), triethylamine (940 mg, 9.29 mmol) was added at room temperature, and the reaction was allowed to warm to 80 ℃ 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 an organic phase by using anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and carrying out column chromatography separation 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 ]/z] ⁺ 。

S3 and synthesis of an intermediate h 6:

intermediate h5 (290 mg, 0.66 mmol) 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 under reduced pressure was carried out to dryness to give 223.7mg of intermediate h 6.

S4, synthesis of compound 13:

intermediate a8 (50 mg, 0.10 mmol) and intermediate H6 (40.7 mg, 0.12 mmol) were dissolved in DMF (3.0 mL), DBU (45 mg, 0.30 mmol) was added at room temperature, the reaction was carried out at room temperature for 2 hours, the reaction was monitored by TLC, ethyl acetate was added to the reaction solution after completion of the reaction, water was added and washed three times, the organic phase was dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and column chromatography was carried out to give 38mg of compound 13, compound 13 was a yellow solid with a yield of 54.1%, ESI (+) M/z =702.7[ M + H ], (ESI M + z) (+)] ⁺ 。

EXAMPLE 14 preparation of Compound 14

S1, synthesis of compound 14:

intermediate b8 (50 m)g, 0.10 mmol) and intermediate H6 (40.7 mg, 0.12 mmol) were dissolved in DMF (3.0 mL), DBU (45 mg, 0.30 mmol) was added at room temperature, the reaction was carried out at room temperature for 2 hours, the reaction was monitored by TLC, ethyl acetate was added to the reaction solution after completion of the reaction, water was added and washed three times, the organic phase was dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, column chromatography was carried out to give 40mg of compound 14, compound 14 was a yellow solid with a yield of 57.0%, ESI (+) M/z =702.7[ M + H ]/] ⁺ 。

EXAMPLE 15 preparation of Compound 15

S1, synthesis of intermediate i 1:

dissolving a compound sm (5.00 g, 16.2 mmol) and a compound g2 (2.67 g, 16.2 mmol) in acetonitrile (50.0 mL), adding DIEA (2.72 g, 21.1 mmol) at room temperature, raising the temperature to 70 ℃ for reaction for 7 hours, 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 3.12g of an intermediate i1 with the yield of 59.6%.

S2, synthesis of intermediate i 2:

intermediate i1 (300 mg, 0.93 mmol) and compound d4 (200 mg, 1.02 mmol), cuprous iodide (20 mg, 0.09 mmol), Pd (pph) ₃ ) ₂ Cl ₂ (22 mg, 0.03 mmol) was dissolved in DMF (5.0 mL), and triethylamine (940 mg, 9.29 mmol) was added at room temperature, and the reaction was allowed to warm to 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 using anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and carrying out column chromatography separation to obtain 300mg of an intermediate i2, wherein the intermediate i2 is a yellow solid product, the yield is 73.4%, and ESI (+) M/z =440.5[ M + H ]/z] ⁺ 。

S3, synthesis of intermediate i 3:

intermediate i2 (300 mg, 0.68 mmol) 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, concentration was carried out under reduced pressure to dryness to obtain 230.8mg of intermediate i 3.

S4, synthesis of compound 15:

dissolving intermediate a8 (50 mg, 0.10 mmol) and intermediate i3 (40.7 mg, 0.12 mmol) in DMF (3.0 mL), adding DBU (45 mg, 0.30 mmol) 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 three times, drying the organic phase with anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and separating by column chromatography to obtain 36mg of compound 15, wherein the compound 15 is yellow solid, the yield is 51.3%, ESI (+) M/z =702.7[ M + H ], (yield)] ⁺ 。

EXAMPLE 16 preparation of Compound 16

S1, synthesis of compound 16:

dissolving intermediate b8 (50 mg, 0.10 mmol) and intermediate i3 (40.7 mg, 0.12 mmol) in DMF (3.0 mL), adding DBU (45 mg, 0.30 mmol) at room temperature, reacting at room temperature for 2 hours, detecting by TLC, adding ethyl acetate to 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 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 =702.7[ M + H ], (yield)] ⁺ 。

EXAMPLE 17 preparation of Compound 17

S1, synthesis of intermediate j 2:

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

S2, synthesis of intermediate j 3:

dissolving intermediate j2 (2.5 g, 5.73 mmol) in methanol (81 mL), adding 2mol/L aqueous sodium hydroxide (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 not 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 yellow solid, and the yield is 85.5%.

S3, synthesis of intermediate j 4:

intermediate j3 (1.0 g, 2.45 mmol) was dissolved in DMF (10 mL) and DIEA (1.0 mL) and HATU (1.4 g, 3.67 mmol) were added at room temperature and reacted for 2h at room temperature. The reaction was monitored by TLC, ethyl acetate was added to the reaction solution after the reaction was complete, water was added and washed three times, the organic phase was dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure and separated by column chromatography to give 1.0g of intermediate j4, intermediate j4 was a yellow solid with a yield of 77.5%, ESI (+) M/z =527.5[ M + H ], (ii)] ⁺ 。

S4, synthesis of compound 17:

intermediate j4 (50 mg, 0.1 mmol) and intermediate g6 (38.7 mg, 0.11 mmol) were dissolved in DMF (2.0 mL), DBU (45 mg, 0.30 mmol) was added at room temperature, the reaction was carried out at room temperature for 2 hours, the reaction was monitored by TLC, after completion of the reaction, ethyl acetate was added to the reaction solution, water was added and washed three times, the organic phase was dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and column chromatography was carried out to give 45mg of compound 17, compound 17 was a yellow solid with a yield of 61.6%, ESI (+) M/z =730.7[ M + H ], (yield)] ⁺ 。

EXAMPLE 18 preparation of Compound 18

S1, synthesis of compound 18:

intermediate j4 (50 mg, 0.1 mmol) and h6 (38.7 mg, 0.11 mmol) were dissolved in DMF (2.0 mL), DBU (45 mg, 0.30 mmol) was added at room temperature, the reaction was allowed to proceed for 2 hours at room temperature, the reaction was monitored by TLC, and after completion of the reaction, acetic acid was added to the reaction mixtureEthyl ester, water was added and washed three times, the organic phase was dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and subjected to column chromatography to give 41.9mg of compound 18 as a yellow solid, compound 18 in 57.4% yield, ESI (+) M/z =730.7[ M + H = 730.7%] ⁺ 。

EXAMPLE 19 preparation of Compound 19

S1, synthesis of intermediate k 2:

intermediate a8 (150 mg, 0.3 mmol) and compound k1 (72 mg, 0.36 mmol) were dissolved in DMF (5.0 mL) and DBU (138 mg, 0.9 mmol) was added at room temperature and reacted at room temperature for 3 h. 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 using anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and carrying out column chromatography separation to obtain 137mg of an intermediate k2, wherein the intermediate k2 is a yellow solid, the yield is 81.2%, and ESI (+) M/z =563.6[ M + H ] (ESI (+) M/z = 563.6)] ⁺ 。

S2, synthesis of intermediate k 3:

intermediate k2 (137 mg, 0.24 mmol) 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 to dryness under reduced pressure gave 111mg of intermediate k 3.

S3, synthesis of intermediate k 5:

intermediate h3 (500 mg, 1.55 mmol), compound k4 (167 mg, 1.70 mmol), cuprous iodide (30 mg, 0.15 mmol), Pd (pph) ₃ ) ₂ Cl ₂ (35 mg, 0.05 mmol) was dissolved in DMF (10.0 mL), triethylamine (1.57 g, 15.52 mmol) 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 an organic phase by using anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and carrying out column chromatography separation 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 ] (ESI (+) M/z = 341)] ⁺ 。

S4, synthesis of intermediate k 6:

intermediate k5 (160 mg, 0.48 mmol) was dissolved in a mixed solution of dichloromethane (3 mL) and tetrahydrofuran (3 mL), sodium bicarbonate (20 mg, 0.24 mmol) was added thereto at room temperature, dessimidine oxidant (0.3 g, 0.70 mmol) was added thereto after cooling to 0 ℃ and substitution with nitrogen was carried out three times, and the mixture was heated to 40 ℃ 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:

intermediate k3 (100 mg, 0.2 mmol) and intermediate k6 (74 mg, 0.22 mmol) were dissolved in a mixed solvent of DMF and methanol (volume ratio 1:4, 5.0 mL), glacial acetic acid (13 mg, 0.22 mmol) was added at room temperature, cooled to 0 ℃ for reaction for 40min, and sodium cyanoborohydride (30 mg, 0.47 mmol) was added at room temperature for reaction at 0 ℃ for 4 h. 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 with anhydrous sodium sulfate, concentrating under reduced pressure to dryness, and separating by column chromatography to obtain 42mg of compound 19, wherein the compound 19 is yellow solid, the yield is 26.7%, and ESI (+) M/z =785.8[ M + H ], (ESI (+) M/z = 785.8)] ⁺ 。

EXAMPLE 20 preparation of Compound 20

S1, synthesis of intermediate m 1:

intermediate b8 (150 mg, 0.3 mmol) and compound k1 (72 mg, 0.36 mmol) were dissolved in DMF (5.0 mL) and DBU (138 mg, 0.9 mmol) was added at room temperature and reacted at room temperature for 3 h. 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 a yellow solid, the yield is 81.2%, and ESI (+) M/z =563.6[ M + H ] (ESI (+) M/z = 563.6)] ⁺ 。

S2, synthesis of intermediate m 2:

intermediate m1 (137 mg, 0.24 mmol) 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 m 2.

S3, synthesis of compound 20:

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

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

The first generation TRK kinase inhibitor Entrectinib and compounds 1-20 were diluted in 100% DMSO in 4-fold gradient starting at 10000nM each (total of 9 concentrations). Entrectinib and a compound 1-20 were diluted 50-fold into 1 Xkinase reaction buffer, and shaken on an oscillator for 20 minutes. A mixture of 2X TrkA, TrkB, TrkC kinase (purchased from Carna Biosciences 08-186, 08-187, 08-197) and 4X Substrate (ATP + TK Substrate-biotin polypeptide Substrate) was prepared 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 substrate mixture of 4x prepared as described above was added to the 384 reaction plate, the plate was 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, and incubate at 25 ℃ for 40 min. Transfer 8. mu.L of assay solution to 384 reaction plates, 1000rpm/min, centrifuge for 1min, and incubate at 25 ℃ for 40 min. RLU (relative luminescence unit) signals are read by a Biotek multifunctional plate reader, and the signal strength is used for a watchCharacterizing 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 interval 50nM-500nM 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, each of the compounds 1 to 20 binds to TRKA, TRKB and TRKC to 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 detecting the effect of 1-20 of the compounds prepared in examples 1-20 on the in vitro cell proliferation activity in 1 tumor cell line (KM12-Luc), wherein Entrectinib is an internal control compound.

The specific method comprises the following steps: (1) harvesting cells in logarithmic growth phase and counting the cells with platelet counter, adjusting cell concentration to 3-6 × 10 ⁴ cell/mL, 100. mu.L of cell suspension per well in the plate, medium without cells (containing 0.1% DMSO) in Min control well, and plate at 37 ℃ with 5% CO ₂ And culturing for 24h 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 hours of culture, removing 50 muL of supernatant by suction, adding 45 muL of fresh culture medium, taking the solution in a5 muL 96-hole transparent medicine plate, and adding the solution into the culture plateAdding 5 mu LDMSO cell culture solution mixed solution into Max control in plate culture, and placing the cells in the dosed culture plate 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), an equal volume of CTG solution was added to each well, and the wells were 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 the data using non-linear sigmoidal regression to derive a dose-effect curve, and IC was calculated therefrom ₅₀ The results are shown in Table 2.

TABLE 2

As can be seen from Table 2, the compounds 1-20 all have certain inhibitory effects 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 Entrectinib prepared in examples 1 to 20 on the TRKA content in the tumor cell line (KM12-Luc) was evaluated, and the content of internal reference GAPDH was analyzed.

The specific method is (1) collecting each tumor cell after compound intervention into 1.5ml of LEP tube, removing the culture medium, adding a certain volume (about 80 uL/2E 6 cells according to the cell amount) of lysine Buffer (RIPA: protease/phosphatase inhibitor =100: 1). (2) After addition of Lysis 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 total. (3) Crack (crack)After the time for decomposition, the EP tube was placed in a centrifuge precooled to 4 ℃ and centrifuged at 12000rpm for 10 min. (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 Lysis 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 30 min. (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 electrotransfer: soaking the PVDF membrane in methanol for 30s to wake up the membrane, 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 1h30 min. (11) Blocking and incubating primary antibody: after the electrotransfer is finished, putting the PVDF membrane into 5% skimmed milk sealing solution, slowly shaking on a decoloring shaking table, and sealing at room temperature for 1h(ii) a Slowly washing the sealed PVDF membrane in TBST for 3 times, 10min each time; after washing, 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, 10min each time; putting the washed PVDF membrane into a secondary antibody (1: 3000) diluent (containing 5% skimmed milk) of a corresponding species, and incubating for 1h at room temperature by a mild shaking table; after the secondary antibody incubation is finished, taking out the PVDF membrane, and slowly washing the PVDF membrane 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 value of each band was analyzed using Image J software to calculate the inhibitor concentration DC at 50% protein degradation 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 interval 50nM-500nM 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 verify the protein degradation capability of the compounds 1 to 20 provided by the present application, and the degraded PROTACs can cyclically and continuously degrade other protein kinases, and have the advantages of low clinical drug dosage requirement and low 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, 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 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 is 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 radical, C _3-7 A heterocyclic group,

、

、

、

、

、

、

Or

；

D represents

、

、

Or

2. A compound or pharmaceutically acceptable salt thereof according to claim 1, wherein when 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 In the case of alkylamino, the substituent is one or more of hydroxyl, methyl, amino, methoxy, dimethylamino, halogen and ethyl; when said R is ₃ Represents substituted C _1-4 When the alkyl is selected, the substituent is one or more of hydroxyl, methyl, amino, methoxy, dimethylamino, halogen and ethyl; when said R is ₅ Represents substituted C _1-4 Alkyl, substituted C _3-7 Cycloalkyl, substituted C _3-7 When the heterocyclic group, the substituted phenyl group, the substituted five-membered heteroaryl group or the substituted six-membered heteroaryl group is substituted, the substituent is methyl, ethyl, hydroxyl, halogen, methoxy, n-hydroxy, n-or n-hydroxy, n-or n-hydroxy, n,

、

Dimethylamino group, diethylamino group,

、

、

、

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 group is substituted, 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 pentabasic heteroaryl or the substituted hexabasic heteroaryl is adopted, the substituent is methyl, ethyl, hydroxyl, halogen, methoxy, dimethylamino, diethylaminoOne or more of the groups; when Y 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 A is ₁ 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 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.

3. A compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R is ₅ Selected from one of the following structures:

。

4. a compound or pharmaceutically acceptable salt thereof according to claim 1, wherein R is ₇ Selected from one of the following structures:

；

the R is ₈ Selected from one of the following structures:

；

the R is ₆ Selected from one of the following structures:

。

5. the compound or pharmaceutically acceptable salt thereof according to claim 1, wherein L is selected from one of the following structures:

；

d is selected from one of the following structures:

。

6. the compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound having the structure of formula i-a or formula i-b is selected from the group consisting of:

。

7. a process for the preparation of a compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, comprising the steps of:

S1、

S2、

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

in the formula, R ₁ 、R ₂ 、M、Q ₁ 、Q ₂ 、Q ₃ 、W ₁ 、R ₃ And R ₄ Is as defined in any one of claims 1 to 6 for R ₁ 、R ₂ 、M、Q ₁ 、Q ₂ 、Q ₃ 、W ₁ 、R ₃ And R ₄ The definition of (1).

8. A pharmaceutical composition comprising a therapeutically effective amount of a compound selected from the group consisting of compounds according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

9. A formulation for targeted ubiquitination of TRK, comprising a therapeutically effective amount of a compound selected from the group consisting of any of claims 1-6, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

10. Use of a compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention and/or treatment of a tumour.