CN113582994A - Compound with TRK kinase inhibitory activity, preparation method, composition and application thereof - Google Patents

Abstract

The invention belongs to the field of medicines, and particularly relates to a compound with TRK kinase inhibitory activity, a preparation method, a composition and application thereof, wherein the compound is a compound with a structure shown in a formula I:

in which ring A represents C_3‑7Cycloalkyl radical, C_3‑7A heterocyclic group,Phenyl, a five-membered heteroaryl or a six-membered heteroaryl; r₁And R₂Identical or different and each independently represents hydrogen, fluorine, chlorine, bromine, iodine, unsubstituted C_1‑4Alkyl, substituted C_1‑4Alkyl, unsubstituted C_1‑4Alkoxy, substituted C_1‑4Alkoxy, unsubstituted C_1‑4Alkylthio, substituted C_1‑4Alkylthio, unsubstituted C_1‑4Alkylamino or substituted C_1‑4An alkylamino group. The compound provided by the invention can be used as a main component of a pharmaceutical composition, can effectively treat, relieve and/or prevent tumors by inhibiting the activity of TRK kinase, and has important significance for researching the development of tumor drugs.

Description

Compound with TRK kinase inhibitory activity, preparation method, composition and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a compound with TRK kinase inhibitory activity, 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 nerve growth factor receptors, are present in a variety of tissues, and play an important role in the processes of proliferation, differentiation, and survival of cells. TRK kinase belongs to receptor tyrosine kinase family, mainly comprises TRKA, TRKB and TRKC, and is respectively encoded by NTRK1, NTRK2 and NTRK3 genes.

Each TRK kinase has a neurotrophic factor ligand associated with it. The ligand of TRKA is NGF (nerve growth factor), the ligand of TRKB comprises BDGF (woven-derived growth factor) and NT-4/5 (neurotropin-4/5), and the ligand of TRKC is NT-3. These neurotrophic factor ligands specifically bind to TRKs, trigger receptor dimerization and phosphorylation of specific tyrosine residues of kinases, thereby activating downstream signal pathways including Ras/MAPK, PLC gamma/PKC and PI3K/AKT, and further regulating and controlling a series of physiological processes of cell proliferation, differentiation, metabolism, apoptosis and the like.

The TRK kinase signaling pathway is normally precisely regulated and, when abnormally activated by gene fusion, overexpression of proteins, or single nucleotide mutation, etc., causes various tumors to occur independently of the tissue origin and type of the tumor. The rapid development of genomics allows more and more NTRK fusion genes to be discovered, such as ETV6-NTRK3, MPRIP-NTRK1, CD74-NTRK1, and the like. The development of the TRK kinase inhibitor can treat various tumors of NTRK fusion protein, such as lung cancer, malignant hematological diseases, prostatic cancer, breast cancer, ovarian cancer, brain glioma, pancreatic cancer, hepatobiliary duct type liver cancer, papillary thyroid cancer, colon cancer, head and neck squamous cell carcinoma, melanoma and the like, and has huge potential and wide market prospect.

Therefore, there is an urgent need to identify a compound having TRK inhibitory activity, and the preparation of the compound is of great significance in the research of the development of tumor drugs.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a compound with TRK kinase inhibitory activity, 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:

；

in the formula, ring A represents C_3-7Cycloalkyl radical, C_3-7A heterocyclic group, a phenyl group, a five-membered heteroaryl group or a six-membered heteroaryl group;

R₁and R₂Identical or different and each independently represents hydrogen, fluorine, chlorine, bromine, iodine, unsubstituted C_1-4Alkyl, substituted C_1-4Alkyl, unsubstituted C_1-4Alkoxy, substituted C_1-4Alkoxy, unsubstituted C_1-4Alkylthio, substituted C_1-4Alkylthio, unsubstituted C_1-4Alkylamino or substituted C_1-4An alkylamino group.

The compound provided by the invention can be used as a main component of a pharmaceutical composition, can effectively treat, relieve and/or prevent tumors by inhibiting the activity of TRK kinase, and has important significance for researching the development of 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:

in the formula, ring A, R₁And R₂Is defined as the first aspect facing ring A, R₁And R₂The definition of (1);

s1, synthesis of intermediate III:

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, synthesis of intermediate V:

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, synthesis of intermediate VI:

reacting the intermediate V, ammonium salt and hydrogen in a third reaction solvent in the presence of a third alkali under the action of a third catalyst to obtain an intermediate VI;

s4, synthesis of an intermediate VIII:

reacting the intermediate VI with a compound VII in a fourth reaction solvent in the presence of a fourth base to obtain an intermediate VIII;

s5, synthesis of intermediate IX:

reacting the intermediate VIII in a fifth reaction solvent in the presence of a fifth base and water to obtain a first product, and reacting the first product with an acid to obtain an intermediate IX;

synthesis of S6, intermediate x:

reacting the intermediate IX in a sixth reaction solvent in the presence of a sixth base under the action of a condensing agent to obtain an intermediate X;

s7, Synthesis of Compound I:

the intermediate X is subjected to manual resolution to obtain the compound I.

The preparation method provided by 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.

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 TRK kinase inhibitor 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 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 5 to 6 carbon atoms. Non-limiting examples of cycloalkyl groups include cyclopentenyl, cyclohexenyl, 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 nitrogen, oxygen or sulfur, the remaining ring atoms being carbon. Preferably 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; most preferably 3 to 8 ring atoms, of which 1 to 3 are heteroatoms; most preferably 5 to 6 ring atoms, of which 1 is a heteroatom. Non-limiting examples of monocyclic heterocyclic groups include dihydropyrrolyl, tetrahydropyridinyl and the like.

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 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; preferred are, for example, furyl, pyrrolyl, thienyl, pyridyl, imidazolyl, thiazolyl, pyrazolyl, oxazolyl, pyrimidinyl, thiadiazole, pyrazinyl and the like, and more preferred is furyl, pyrrolyl, thienyl or pyridyl.

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. More preferred are lower alkyl groups containing 1 to 4 carbon atoms, non-limiting examples include methyl, ethyl, n-propyl, isopropyl, and the like. The alkyl group may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, independently optionally substituted with one or more substituents selected from the group consisting of halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, alkylamino, hydroxy, cyano, amino.

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, substituents may be substituted at any available point of attachment, independently optionally substituted with one or more substituents selected from halogen, alkyl, haloalkyl, alkoxy, haloalkoxy, alkylamino, hydroxy, cyano, amino.

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)₂A N- "group wherein alkyl is as defined above. Non-limiting examples of alkylamino groups include: dimethylamino, methylamino, and the like.

Various hydrocarbon containing moietiesIs represented by the prefix labeled minimum and maximum number of carbon atoms for that portion, i.e., prefix C_i~jThe number of carbon atoms representing the moiety is from the integer "i" to the integer "j" (inclusive). Thus, for example, C_1~4Alkyl refers to alkyl groups of 1 to 4 carbon atoms (including 1 and 4).

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:

；

in the formula, ring A represents C_3-7Cycloalkyl radical, C_3-7A heterocyclic group, a phenyl group, a five-membered heteroaryl group or a six-membered heteroaryl group;

R₁and R₂Identical or different and each independently represents hydrogen, fluorine, chlorine, bromine, iodine, unsubstituted C_1-4Alkyl, substituted C_1-4Alkyl, unsubstituted C_1-4Alkoxy, substituted C_1-4Alkoxy, unsubstituted C_1-4Alkylthio, substituted C_1-4Alkylthio, unsubstituted C_1-4Alkylamino or substituted C_1-4An alkylamino group.

The compound provided by the embodiment of the invention has a novel structure, can be used as a main component of a pharmaceutical composition, can effectively treat, relieve and/or prevent tumors by inhibiting the activity of TRK kinase, and has important significance for researching the development of tumor drugs.

The first generation TRK kinase inhibitors Larotretinib and Entretinib are approved by FDA in 2018 and 2019 respectively, can simultaneously inhibit TRKA, TRKB and TRKC 3 subtypes, can treat various tumors, and are known as 'broad-spectrum anticancer drugs'. However, clinically, the first generation of TRK kinase inhibitors have presented significant adverse reactions and drug resistance problems and have been shown to be caused by NTRK mutations, such as G595R or G667C mutations of NTRK1, which greatly limit later development and use. Once resistance occurs, the drug needs to be replaced immediately, and many times, a batch of drug cannot be used, and if iterative drug use is good, if not, the condition of the patient will rapidly deteriorate and cannot be controlled. Therefore, the study of a new generation of TRK kinase inhibitors is expected to solve these problems.

The compound provided by the embodiment of the invention has a novel structure and small molecular weight, can inhibit the activities of TRKA, TRKB and TRKC simultaneously, can be used as a novel TRK kinase inhibitor, can improve the selectivity of tumor treatment medicines, is expected to solve the problem of drug resistance of the existing anticancer medicines, and has great social value and economic benefit.

In the process of researching a novel TRK kinase inhibitor, the inventor of the invention unexpectedly finds that the compound provided by the embodiment of the invention can compete with an ATP pocket in TRK kinase for binding due to the macrocyclic structure, so that the kinase activity is inhibited, the space loss caused by mutation of a TRK kinase structural domain is avoided, and the defect that the parent nucleus of a first generation TRK kinase inhibitor is easy to generate drug resistance due to the non-cyclic structure can be overcome.

The structure of the first generation TRK kinase inhibitor Larotrectinib is as follows:

。

further, when said R is₁Represents substituted C_1-4Alkyl, substituted C_1-4Alkoxy, substituted C_1-4Alkylthio or substituted C_1-4In the case of alkylamino, the substituent is hydroxyl, methyl, amino, methoxy, dimethylamino, halogen or ethyl; when said R is₂Represents substituted C_1-4Alkyl, substituted C_1-4Alkoxy, substituted C_1-4Alkylthio or substituted C_1-4In the case of alkylamino, the substituent is hydroxy, methyl, amino, methoxy, dimethylamino, halogen or ethyl.

Further, the ring a is selected from one of the following structures:

。

further, said R₁And R₂Are identical or different and each independently represents hydrogen, fluorine, chlorine, methyl, methoxy or

。

Further, the compound may be selected from, 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:

in the formula, ring A, R₁And R₂Is defined as the first aspect facing ring A, R₁And R₂The definition of (1);

s1, synthesis of intermediate III:

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, synthesis of intermediate V:

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, synthesis of intermediate VI:

reacting the intermediate V, ammonium salt and hydrogen in a third reaction solvent in the presence of a third alkali under the action of a third catalyst to obtain an intermediate VI;

s4, synthesis of an intermediate VIII:

reacting the intermediate VI with a compound VII in a fourth reaction solvent in the presence of a fourth base to obtain an intermediate VIII;

s5, synthesis of intermediate IX:

reacting the intermediate VIII in a fifth reaction solvent in the presence of a fifth base and water to obtain a first product, and reacting the first product with an acid to obtain an intermediate IX;

synthesis of S6, intermediate x:

reacting the intermediate IX in a sixth reaction solvent in the presence of a sixth base under the action of a condensing agent to obtain an intermediate X;

s7, Synthesis of Compound I:

the intermediate X is subjected to manual resolution to obtain the compound I.

The preparation method provided by the embodiment of the invention is simple, mild in condition, convenient to operate, low in equipment condition requirement, 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 is at least one of palladium acetate, [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium, and tetratriphenylphosphine palladium.

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

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

Further, in step S1, the reaction temperature is 50 ℃ to 100 ℃, for example, the reaction temperature may be 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃ or 100 ℃.

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

Further, in step S2, the second base is at least one of sodium carbonate, potassium carbonate, cesium carbonate and potassium phosphate, and the second base cannot be sodium acetate and potassium acetate, and the sodium acetate and potassium acetate are too weak to react the intermediate iii with the compound iv.

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 20 ℃ to 100 ℃, for example, the reaction temperature may be 20 ℃, 40 ℃, 60 ℃, 80 ℃ or 100 ℃.

Further, in step S3, the ammonium salt is at least one of ammonium formate, ammonium acetate, ammonium chloride, ammonium bromide, ammonium iodide, ammonium hydroxide, and hydroxylamine hydrochloride.

Further, in step S3, the third catalyst is palladium on carbon.

Further, in step S3, the third base is at least one of sodium acetate and potassium acetate, the third base is weak, if the third base is too strong, more impurities are generated, and the yield of intermediate VI is low, for example, if sodium acetate or potassium acetate is replaced by any one of sodium carbonate, potassium phosphate and cesium carbonate, the yield is reduced.

Further, in step S3, the third reaction solvent is at least one of methanol, ethanol, isopropanol, and n-butanol.

Further, in step S3, the reaction temperature is 20 ℃ to 80 ℃, for example, the reaction temperature may be 20 ℃, 40 ℃, 60 ℃ or 80 ℃.

Further, in step S4, the fourth base is at least one of potassium carbonate, cesium carbonate, sodium tert-butoxide, potassium phosphate, triethylamine, N-diisopropylethylamine.

Further, in step S4, the fourth reaction solvent is at least one of isopropyl alcohol and n-butyl alcohol, and if the fourth reaction solvent is methanol or ethanol, side reactions are more likely to occur, and the reaction yield is lowered.

Further, in step S4, the reaction temperature is 40 ℃ to 120 ℃, for example, the reaction temperature may be 40 ℃, 60 ℃, 80 ℃, 100 ℃ or 120 ℃.

Further, in step S5, the fifth base is at least one of lithium hydroxide monohydrate, sodium hydroxide, and potassium hydroxide.

Further, in step S5, the fifth reaction solvent is at least one of methanol, ethanol, and tetrahydrofuran.

Further, in step S5, the first product has the structure of formula B:

B

in the formula, R₃Represents lithium, sodium or potassium.

Further, in step S5, the acid is hydrochloric acid.

Further, in step S5, the reaction temperature is 20 ℃ to 80 ℃, for example, the reaction temperature may be 20 ℃, 40 ℃, 60 ℃ or 80 ℃.

Further, in step S6, the condensing agent is at least one of 2- (7-azabenzotriazole) -N, N ' -tetramethylurea hexafluorophosphate, O-benzotriazol-N, N ' -tetramethylurea hexafluorophosphate, and N, N ' -carbonyldiimidazole.

Further, in step S6, the sixth base is at least one of triethylamine and N, N-diisopropylethylamine.

Further, in step S6, the sixth reaction solvent is at least one of dichloromethane, tetrahydrofuran, N-dimethylformamide, and N, N-dimethylacetamide.

Further, in step S7, the chiral resolution is performed using a high performance liquid chromatography chiral column.

Further, in step S7, the high performance liquid chromatography chiral column is a Kromasil AmyCoat chiral column.

Further, in step S7, the mobile phase used is methanol and water in a volume ratio of 4: 1.

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, embodiments of the present invention provide a TRK kinase inhibitor 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 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 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, renal parenchymal cancer, cervical 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, small cell lung cancer, non-small cell lung 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.

EXAMPLE 1 preparation of (R,63E,64E) -15-fluoro-8-methyl-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidine-1 (2,3) -pyridine 3-3(1,2) -benzocyclooctadecan-5-one

S1, synthesis of intermediate 1 b:

dissolving compound 1a (3.47 g, 20.0 mmol), pinacol diboron (5.6 g, 22.0 mmol), [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (1.47 g, 2.0 mmol) and potassium acetate (3.93 g, 40.0 mmol) in 1, 4-dioxane (50 mL), heating to 100 ℃ under nitrogen protection, refluxing for 5 hours, TLC monitoring the reaction, after the reaction is completed, concentrating under reduced pressure to remove the solvent, adding 200mL of purified water, extracting three times with ethyl acetate, wherein the volume of ethyl acetate used for each extraction is 50mL, combining the organic phases, washing the organic phase with 50mL of purified water, washing the organic phase with 50mL of saturated saline water, drying with anhydrous sodium sulfate, concentrating under reduced pressure, and separating to obtain 4.56g of intermediate 1b, wherein the yield is 86.0%, and the intermediate 1b is a light yellow solid by column chromatography.

S2, synthesis of intermediate 1 d:

dissolving the intermediate 1b (4.56 g, 17.2 mmol) in 1, 4-dioxane (50 mL), adding the compound 1c (4.46 g, 20.6 mmol), palladium tetratriphenylphosphine (2.00 g, 1.72 mmol), potassium phosphate (7.30 g, 34.4 mmol), and 12.5mL of purified water at room temperature, heating to 100 ℃ under nitrogen protection, refluxing and stirring for 4 hours, monitoring the reaction by TLC, concentrating under reduced pressure to remove the solvent after the reaction is finished, adding 200mL of purified water, extracting with ethyl acetate three times, wherein the volume of ethyl acetate used in each extraction is 50mL, combining the organic phases, washing the organic phases with 50mL of purified water, washing the organic phases with 50mL of saturated saline water, drying with anhydrous sodium sulfate column chromatography, concentrating under reduced pressure, and isolating to obtain 3.87g of the intermediate 1d, wherein the yield is 82.0%, and the intermediate 1d is yellow solid.

S3, synthesis of intermediate 1 e:

dissolving intermediate 1d (3.87 g, 14.1 mmol), ammonium hydroxide (1 g, 28.2 mmol) and sodium acetate (4.62 g, 56.4 mmol) in ethanol (50 mL), heating to 60 ℃ for 6 hours, TLC monitoring the reaction, concentrating under reduced pressure after the reaction is finished to remove the solvent, adding 200mL of purified water, extracting three times with ethyl acetate, wherein the volume of ethyl acetate used for each extraction is 50mL, combining the organic phases, washing the organic phase with 50mL of purified water, washing the organic phase with 50mL of saturated saline, drying over anhydrous sodium sulfate, concentrating under reduced pressure to obtain a yellow solid intermediate, adding methanol (50 mL) and 10% palladium-carbon (500 mg), introducing hydrogen, stirring at room temperature for 3 hours, TLC monitoring the reaction, filtering after the reaction is finished, pouring the filtrate into 200mL of purified water, extracting three times with ethyl acetate, wherein the volume of ethyl acetate used for each extraction is 50mL, combining the organic phases, then, the organic phase was washed with 50mL of purified water, and then with 50mL of saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography to obtain 2.77g of intermediate 1e, with a yield of 80.0%, and intermediate 1e as a yellow solid.

S4, synthesis of intermediate 1 g:

dissolving intermediate 1e (2.77 g, 11.28 mmol), compound 1f (2.55 g, 11.28 mmol) and N, N-diisopropylethylamine (DIEA, 2.18g, 16.92 mmol) in N-butanol (30 mL), heating to 80 ℃ for 10 hours, monitoring the reaction by TLC, concentrating under reduced pressure after the reaction is finished to remove the solvent, adding 200mL of purified water, extracting three times with dichloromethane, wherein the volume of dichloromethane used in each extraction is 50mL, combining the organic phases, washing the organic phases with 50mL of purified water, washing the organic phases with 50mL of saturated saline water, drying with anhydrous sodium sulfate, concentrating under reduced pressure, and performing column chromatography to obtain 4.59g of intermediate 1g, wherein the yield is 93.6% and the intermediate 1g is yellow solid.

S5, synthesis of intermediate 1 h:

dissolving 1g (4.59 g, 10.56 mmol) of the compound in methanol (25 mL) and purified water (25 mL), adding lithium hydroxide monohydrate (0.89 g, 21.12 mmol), heating to 50 ℃ for reaction for 3 hours, monitoring the reaction by TLC, removing the solvent by concentration under reduced pressure after the reaction is finished, adding 100mL of purified water, extracting three times by using dichloromethane, wherein the volume of dichloromethane used for each extraction is 50mL, combining the water phases, dropwise adding 22mL of 1M hydrochloric acid, precipitating yellow solid, filtering, drying a filter cake to obtain 3.58g of an intermediate for 1h, the yield is 83.5%, and the intermediate for 1h is yellow solid.

S6, synthesis of intermediate 1 j:

intermediate 1h (3.58 g, 8.82 mmol), N-diisopropylethylamine (DIEA, 2.28g, 17.64 mmol) was dissolved in N, N-dimethylformamide (DMF, 40 mL), 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU, 6.71g, 17.64 mmol) was added at room temperature to react at room temperature for 6 hours, the reaction was monitored by TLC, 200mL of purified water was added after the reaction was completed, the mixture was extracted three times with ethyl acetate, 50mL of ethyl acetate was used for each extraction, the organic phases were combined, then the organic phase was washed with 50mL of purified water, then with 50mL of saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography to give 1.75g of intermediate 1j, yield 51.2%, intermediate 1j was a pale yellow solid, and ESI (+) m/z = 388.4.

S7, synthesis of compound 1:

intermediate 1j (1.75 g, 4.52 mmol) was resolved on preparative high performance liquid chromatography chiral column (Kromasil AmyCoat chiral column) using methanol and water as mobile phases (flow rate 20mL/min, volume ratio 4: 1) to give 600mg of compound 1 (ee > 98%), yield 68.6%, compound 1 as a pale yellow solid, ESI (+) m/z = 388.4.

EXAMPLE 2 preparation of (R,63E,64E) -15, 36-difluoro-8-methyl-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3) -pyridin-3 (1,2) -benzocyclooctadecan-5-one

Compound 2 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 1.68g of intermediate 2j was obtained in 49.8% yield, intermediate 2j was a light yellow solid, ESI (+) m/z = 406.4.

In step S7, referring to step S7 of example 1, 1.68g of intermediate 2j was resolved to give 569mg of compound 2 (ee > 98%), yield 67.8%, compound 2 was a pale yellow solid, ESI (+) m/z = 406.4.

EXAMPLE 3 preparation of (R,63E,64E) -15-fluoro-36, 8-dimethyl-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3) -pyridin-3 (1,2) -benzocyclooctadecan-5-one

Compound 3 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 1.26g of intermediate 3j was obtained in 47.2% yield, intermediate 3j was a light yellow solid, ESI (+) m/z = 402.4.

In step S7, referring to step S7 of example 1, 1.26g of intermediate 3j was resolved to give 454mg of compound 3 (ee > 98%) with a yield of 72.1%, compound 3 was a pale yellow solid, ESI (+) m/z = 402.4.

EXAMPLE 4 preparation of (R,63E,64E) -15, 34-difluoro-36, 8-dimethyl-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3) -pyridin-3 (1,2) -benzocyclooctadecan-5-one

Compound 4 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 1.48g of intermediate 4j was obtained in 48.3% yield, intermediate 4j was a light yellow solid, ESI (+) m/z = 420.4.

In step S7, referring to step S7 of example 1, 1.48g of intermediate 4j was resolved to afford 559mg of compound 4 (ee > 98%) with a yield of 75.6%, compound 4 was a pale yellow solid, ESI (+) m/z = 420.4.

EXAMPLE 5 preparation of (R,63E,64E) -15-fluoro-34-methoxy-36, 8-dimethyl-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3) -pyridinyl-3 (1,2) -benzocyclooctadecan-5-one

Compound 5 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 1.57g of intermediate 5j was obtained in 62.4% yield, intermediate 5j was a light yellow solid, ESI (+) m/z = 432.5.

In step S7, referring to step S7 of example 1, 1.57g of intermediate 5j was resolved to give 490mg of compound 5 (ee > 98%) with a yield of 62.4%, compound 5 was a pale yellow solid, ESI (+) m/z = 432.5.

EXAMPLE 6 preparation of (R,63E,64E) -15-fluoro-36, 8-dimethyl-34- (methylamino) -4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3) -pyridinyl-3 (1,2) -benzocyclooctadecan-5-one

Compound 6 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 1.65g of intermediate 6j was obtained in 63.7% yield, intermediate 6j was a light yellow solid, ESI (+) m/z = 431.5.

In step S7, referring to step S7 of example 1, 1.65g of intermediate 6j was resolved to afford 447mg of compound 6 (ee > 98%) with a yield of 54.2%, compound 6 was a pale yellow solid, ESI (+) m/z = 431.5.

EXAMPLE 7 preparation of (R,63E,64E) -15-fluoro-8-methyl-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3),3(4,3) -bipyridylcyclooctadecan-5-one

Compound 7 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 1.44g of intermediate 7j was obtained in 42% yield, intermediate 7j was a light yellow solid, ESI (+) m/z = 389.4.

In step S7, referring to step S7 of example 1, 1.44g of intermediate 7j was resolved to yield 373mg of compound 7 (ee > 98%) with a yield of 51.8%, compound 7 was a pale yellow solid, ESI (+) m/z = 389.4.

EXAMPLE 8 preparation of (R,63E,64E) -15, 35-difluoro-8-methyl-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3),3(4,3) -bipyridylcyclooctadecan-5-one

Compound 8 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 1.46g of intermediate 8j was obtained in 40.5% yield, intermediate 8j was a light yellow solid, ESI (+) m/z = 407.4.

In step S7, referring to step S7 of example 1, 1.46g of intermediate 8j was resolved to give 395mg of compound 8 (ee > 98%), yield 54.1%, compound 8 was a pale yellow solid, ESI (+) m/z = 407.4.

Example 9 preparation of (R,63E,64E) -15-fluoro-35, 8-dimethyl-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3),3(4,3) -bipyridylcyclooctadecan-5-one

Compound 9 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 1.89g of intermediate 9j was obtained in 53.2% yield, intermediate 9j was a light yellow solid, ESI (+) m/z = 403.4.

In step S7, referring to step S7 of example 1, 1.89g of intermediate 9j was resolved to give 581mg of compound 9 (ee > 98%) with a yield of 61.5%, compound 9 was a pale yellow solid, ESI (+) m/z = 403.4.

EXAMPLE 10 preparation of (R,63E,64E) -35-chloro-15-fluoro-8-methyl-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3),3(4,3) -bipyridino cyclooctadecane-5-one

Compound 10 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 1.79g of intermediate 10j was obtained in 47.8% yield, intermediate 10j was a light yellow solid, ESI (+) m/z = 423.8.

In step S7, referring to step S7 of example 1, 1.79g of intermediate 10j was resolved to give 575mg of compound 10 (ee > 98%), yield 64.2%, compound 10 was a pale yellow solid, ESI (+) m/z = 423.8.

EXAMPLE 11 preparation of (R,63E,64E) -15-fluoro-35-methoxy-8-methyl-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3),3(4,3) -bipyridino cyclooctadecane-5-one

Compound 11 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 1.78g of intermediate 11j was obtained in 48.2% yield, intermediate 11j was a light yellow solid, ESI (+) m/z = 419.4.

In step S7, referring to step S7 of example 1, 1.78g of intermediate 11j was resolved to give 509mg of compound 11 (ee > 98%), yield 57.2%, compound 11 was a pale yellow solid, ESI (+) m/z = 419.4.

Example 12 preparation of (R,63E,64E) -15, 36-difluoro-35, 8-dimethyl-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3),3(4,3) -bipyridino cyclooctadecane-5-one

Compound 12 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 1.74g of intermediate 12j was obtained in 46.9% yield, intermediate 12j was a light yellow solid, ESI (+) m/z = 421.4.

In step S7, referring to step S7 of example 1, 1.74g of intermediate 12j was resolved to give 572mg of compound 12 (ee > 98%), yield 65.8%, compound 12 was a pale yellow solid, ESI (+) m/z = 421.4.

EXAMPLE 13 preparation of (R,63E,64E) -15-fluoro-8-methyl-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3) -pyridine 3-3(2,3) -furocyclooctadecane-5-one

Compound 13 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 2.28g of intermediate 13j was obtained in 68.2% yield, intermediate 13j was a light yellow solid, ESI (+) m/z = 378.4.

In step S7, referring to step S7 of example 1, 2.28g of intermediate 13j was resolved to give 822mg of compound 13 (ee > 98%) with a yield of 72.1%, compound 13 was a pale yellow solid, ESI (+) m/z = 378.4.

Example 14 preparation of (R,63E,64E) -15-fluoro-35, 8-dimethyl-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3) -pyridin-3 (2,3) -furocyclooctadecane-5-one

Compound 14 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 2.50g of intermediate 14j was obtained in 72.1% yield, intermediate 14j was a light yellow solid, ESI (+) m/z = 392.4.

In step S7, referring to step S7 of example 1, 2.50g of intermediate 14j was resolved to give 806mg of compound 14 (ee > 98%), yield 64.5%, compound 14 was a pale yellow solid, ESI (+) m/z = 392.4.

EXAMPLE 15 preparation of (R,63E,64E) -15-fluoro-8-methyl-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3) -pyridine 3-3(3,2) -furocyclooctadecane-5-one

Compound 15 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 2.35g of intermediate 15j was obtained in 70.3% yield, intermediate 15j was a light yellow solid, ESI (+) m/z = 378.4.

In step S7, referring to step S7 of example 1, 2.35g of intermediate 15j was resolved to give 820mg of compound 15 (ee > 98%) with a yield of 69.8%, compound 15 was a pale yellow solid, ESI (+) m/z = 378.4.

EXAMPLE 16 preparation of (R,63E,64E) -15-fluoro-35, 8-dimethyl-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3) -pyridin-3 (3,2) -furocyclooctadecane-5-one

Compound 16 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 2.52g of intermediate 16j was obtained in 72.8% yield, intermediate 16j was a light yellow solid, ESI (+) m/z = 392.4.

In step S7, referring to step S7 of example 1, 2.52g of intermediate 16j was resolved to give 816mg of compound 16 (ee > 98%), yield 64.8%, compound 16 was a pale yellow solid, ESI (+) m/z = 392.4.

EXAMPLE 17 preparation of (R,63E,64E) -15-fluoro-8-methyl-31H-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3) -pyridin-3 (2,3) -pyrrolocyclooctadecane-5-one

Compound 17 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 1.64g of intermediate 17j was obtained in 49.2% yield, intermediate 17j was a light yellow solid, ESI (+) m/z = 377.4.

In step S7, referring to step S7 of example 1, 1.64g of intermediate 17j was resolved to give 515mg of compound 17 (ee > 98%), yield 62.8%, compound 17 was a pale yellow solid, ESI (+) m/z = 377.4.

EXAMPLE 18 preparation of (R,63E,64E) -15-fluoro-31, 8-dimethyl-31H-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3) -pyridin-3 (2,3) -pyrrolocyclooctadecane-5-one

Compound 18 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 1.85g of intermediate 18j was obtained in 53.6% yield, intermediate 18j was a light yellow solid, ESI (+) m/z = 391.4.

In step S7, referring to step S7 of example 1, 1.85g of intermediate 18j is resolved to give 536mg of compound 18 (ee > 98%) with a yield of 57.9%, compound 18 is a pale yellow solid, ESI (+) m/z = 391.4.

EXAMPLE 19 preparation of (R,63E,64E) -15-fluoro-8-methyl-31H-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3) -pyridin-3 (3,2) -pyrrolocyclooctadecane-5-one

Compound 19 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 1.73g of intermediate 19j was obtained in 52.1% yield, intermediate 19j was a light yellow solid, ESI (+) m/z = 377.4.

In step S7, referring to step S7 of example 1, 1.73g of intermediate 19j was resolved to give 554mg of compound 19 (ee > 98%) with a yield of 64.1%, compound 19 was a pale yellow solid, ESI (+) m/z = 377.4.

EXAMPLE 20 preparation of (R,63E,64E) -15-fluoro-31, 8-dimethyl-31H-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3) -pyridin-3 (3,2) -pyrrolocyclooctadecane-5-one

Compound 20 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 1.95g of intermediate 20j was obtained in 56.4% yield, intermediate 20j was a light yellow solid, ESI (+) m/z = 391.4.

In step S7, referring to step S7 of example 1, 1.95g of intermediate 20j was resolved to give 613mg of compound 20 (ee > 98%) with a yield of 62.9%, compound 20 was a pale yellow solid, ESI (+) m/z = 391.4.

EXAMPLE 21 preparation of (R,63E,64E) -15-fluoro-8-methyl-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3) -pyridine 3-3(2,3) -thienocyclooctadecane-5-one

Compound 21 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 2.15g of intermediate 21j was obtained in 61.7% yield, intermediate 21j was a light yellow solid, ESI (+) m/z = 394.4.

In step S7, referring to step S7 of example 1, 2.15g of intermediate 21j was resolved to give 698mg of compound 21 (ee > 98%), yield 64.9%, compound 21 was a pale yellow solid, ESI (+) m/z = 394.4.

EXAMPLE 22 preparation of (R,63E,64E) -15-fluoro-8-methyl-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3) -pyridine 3(3,2) -thienocyclooctadecane-5-one

Compound 22 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 2.01g of intermediate 22j was obtained in 57.9% yield, intermediate 22j was a light yellow solid, ESI (+) m/z = 394.4.

In step S7, referring to step S7 of example 1, 2.01g of intermediate 22j is resolved to give 621mg of compound 22 (ee > 98%) with a yield of 61.8%, compound 22 is a pale yellow solid, ESI (+) m/z = 394.4.

EXAMPLE 23 preparation of (R,63E,64E) -15-fluoro-8-methyl-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidine-1 (2,3) -pyridine 3-3(1,2) -cyclopentenocyclooctadecan-5-one

Compound 23 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 1.39g of intermediate 23j was obtained in 41.5% yield, intermediate 23j was a light yellow solid, ESI (+) m/z = 378.4.

In step S7, referring to step S7 of example 1, 1.39g of intermediate 23j was resolved to give 395mg of compound 23 (ee > 98%) with a yield of 56.8%, compound 23 was a pale yellow solid, ESI (+) m/z = 378.4.

EXAMPLE 24 preparation of (63E,64E,8R) -15-fluoro-35, 8-dimethyl-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidine-1 (2,3) -pyridine-3 (1,2) -cyclopentenocyclooctadecan-31-5-one

Compound 24 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 1.53g of intermediate 24j was obtained in 44.2% yield, intermediate 24j was a light yellow solid, ESI (+) m/z = 392.4.

In step S7, referring to step S7 of example 1, 1.53g of intermediate 24j was resolved to give 405mg of compound 24 (ee > 98%) with a yield of 52.9%, compound 24 was a pale yellow solid, ESI (+) m/z = 392.4.

EXAMPLE 25 preparation of (R,63E,64E) -15-fluoro-8-methyl-32, 33-dihydro-31H-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3) -pyridin-3 (5,4) -pyrrolocyclooctadecane-5-one

Compound 25 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 1.29g of intermediate 25j was obtained in 38.6% yield, intermediate 25j was a light yellow solid, ESI (+) m/z = 379.4.

In step S7, referring to step S7 of example 1, 1.29g of intermediate 25j is resolved to give 402mg of compound 25 (ee > 98%) with a yield of 62.3%, compound 25 is a pale yellow solid, ESI (+) m/z = 379.4.

Example 26 preparation of (R,63E,64E) -15-fluoro-31, 8-dimethyl-32, 33-dihydro-31H-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3) -pyridin-3 (5,4) -pyrrolocyclooctadecane-5-one

Compound 26 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 1.45g of intermediate 26j was obtained in 41.8% yield, intermediate 26j was a light yellow solid, ESI (+) m/z = 393.4.

In step S7, referring to step S7 of example 1, 1.45g of intermediate 26j was resolved to give 433mg of compound 26 (ee > 98%), yield 59.7%, compound 26 was a pale yellow solid, ESI (+) m/z = 393.4.

EXAMPLE 27 preparation of (R,63E,64E) -15-fluoro-8-methyl-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidine-1 (2,3) -pyridine 3-3(1,2) -cyclohexenocyclooctadecane-5-one

Compound 27 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 1.81g of intermediate 27j was obtained in 52.3% yield, intermediate 27j was a light yellow solid, ESI (+) m/z = 392.4.

In step S7, referring to step S7 of example 1, 1.81g of intermediate 27j was resolved to give 556mg of compound 27 (ee > 98%) with a yield of 61.4%, compound 27 was a pale yellow solid, ESI (+) m/z = 392.4.

EXAMPLE 28 preparation of (63E,64E,8R) -15-fluoro-36, 8-dimethyl-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3) -pyridin-3 (1,2) -cyclohexenocyclooctadecane-5-one

Compound 28 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 2.03g of intermediate 28j was obtained in 56.7% yield, intermediate 28j was a light yellow solid, ESI (+) m/z = 406.5.

In step S7, referring to step S7 of example 1, 2.03g of intermediate 28j was resolved to give 591mg of compound 28 (ee > 98%), yield 58.2%, compound 28 was a pale yellow solid, ESI (+) m/z = 406.5.

Example 29 preparation of (R,63E,64E) -15-fluoro-8-methyl-31, 32,33, 34-tetrahydro-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3), 3(6,5) -bipyridylcyclooctadecan-5-one

Compound 29 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 1.65g of intermediate 29j was obtained in 47.6% yield, intermediate 29j was a light yellow solid, ESI (+) m/z = 393.4.

In step S7, referring to step S7 of example 1, 1.65g of intermediate 29j was resolved to give 535mg of compound 29 (ee > 98%) with a yield of 64.8%, compound 29 was a pale yellow solid, ESI (+) m/z = 393.4.

Example 30 preparation of (R,63E,64E) -15-fluoro-31, 8-dimethyl-31, 32,33, 34-tetrahydro-4, 7-diaza-6 (3,5) -pyrazolo [1,5-a ] pyrimidin-1 (2,3), 3(6,5) -bipyridylcyclooctadecan-5-one

Compound 30 was prepared according to the synthetic route provided in this example, with reference to the procedure for preparation of compound 1 in example 1.

In step S6, 1.93g of intermediate 30j was obtained in 53.7% yield, intermediate 30j was a light yellow solid, ESI (+) m/z = 407.5.

In step S7, referring to step S7 of example 1, 1.93g of intermediate 30j was resolved to give 576mg of compound 30 (ee > 98%) with a yield of 59.7%, compound 30 was a pale yellow solid, ESI (+) m/z = 407.5.

Example 31TRKA, TRKB, TRKC kinase in vitro Activity assay

First generation TRK kinase inhibitor Larotrectinib and compounds 1-30 were diluted in 100% DMSO in 4-fold gradient starting at 10000nM (9 concentrations total). Larotrectinib and a compound 1-30 times diluted into 1 Xkinase reaction buffer respectively, 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 enzyme 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.L ADP-Glo to 384 reaction plates, 1000rpm/min, centrifuge for 1min, incubate for 40min at 25 ℃. 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 were read using a Biotek multifunctional plate reader, and the signal intensity was used to characterize the degree of kinase activity. Data were obtained using GraFit6.0 software (Erithacu)s Software) calculation of IC for this Compound₅₀Value, IC₅₀Specific results of the values are shown in table 1.

Example 32 mutant TRKA (G595R) and TRKA (G667C) kinase in vitro Activity assay

Recombinant human TRKA (G595R) and TRKA (G667C) kinases were purchased from SignalChem, and their in vitro activity assay methods RLU (relative luminescence unit) signals were read using a Biotek multifunctional plate reader, and the signal intensities were used to characterize the activity levels of the kinases, in reference to the in vitro activity assay method in example 31. Data IC of the compound was calculated using grafit6.0 Software (Erithacus Software)₅₀Value, IC₅₀Specific results of the values are shown in table 1.

A＜50nM，50nM≤B≤300nM，300nM＜C＜500nM，500nM≤D

TABLE 1

As shown in Table 1, the compounds 1-30 have strong TRK kinase inhibitory activity, and particularly the compounds 18, 24, 25 and 28 have the strongest inhibitory activity on TRK kinase and are obviously superior to Larotrectinib. Calculated by IC50, the inhibition activity intensity of compound 24 on TRKA is 4 times that of Larotrectinib, the inhibition activity intensity on TRKB is 2 times that of Larotrectinib, and the inhibition activity intensity on TRKC is 2 times that of Larotrectinib.

The inhibitory activity of the compounds 1-30 on mutated TRKA (G595R) and TRKA (G667C) kinase is far stronger than that of Larotretinib, the inhibitory activity of the compound 24 is strongest calculated by IC50, the inhibitory activity of the compound 28 is inferior, the inhibitory activity of the compound 24 on TRKA (G595R) is 5 times that of the compound 28, and the inhibitory activity of the compound 24 on TRKA (G667C) is 4 times that of the compound 28.

It has been found that the first generation TRK kinase inhibitor Larotrectinib has certain drug resistance phenomenon after being treated for a period of time (mainly relating to NTRK1/NTRK 3). Further research shows that the domain mutation is similar to the mutation of ALK and ROS1 kinase, and mainly relates to amino acid substitution of 3 main regions, namely, a solvent front locus, a bayonet residue and xFG-motif. The solvent front site mutation mainly comprises TRKA-G595R, TRKB-G639R and TRKC-G623R; the bayonet residue mutations comprise TRKA-F589L, TRKB-F633L and TRKC-F617L; the xFG-motif mutations include TRKA-G667C, TRKB-G709C, and TRKC-G696A.

The occurrence of mutation can reduce the inhibitory activity of the drug on TRK kinase, thereby causing the problem of drug resistance.

The inhibitory activity of the compounds 1-30 on mutated TRKA (G595R) and TRKA (G667C) kinases is far stronger than that of Larotrectinib, and the drug resistance defect in the process of using a first-generation TRK kinase inhibitor Larotrectinib is overcome.

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 (10)

1. A compound having the structure of formula I:

；

in the formula, ring A represents C_3-7Cycloalkyl radical, C_3-7A heterocyclic group, a phenyl group, a five-membered heteroaryl group or a six-membered heteroaryl group;

R₁and R₂Identical or different and each independently represents hydrogen, fluorine, chlorine, bromine, iodine, unsubstituted C_1-4Alkyl, substituted C_1-4Alkyl, unsubstituted C_1-4Alkoxy, substituted C_1-4Alkoxy, unsubstituted C_1-4Alkylthio, substituted C_1-4Alkylthio, unsubstituted C_1-4Alkylamino or substituted C_1-4An alkylamino group.

2. A compound or pharmaceutically acceptable salt thereof according to claim 1, wherein when R is₁Represents substituted C_1-4Alkyl, substituted C_1-4Alkoxy, substituted C_1-4Alkylthio or substituted C_1-4In the case of alkylamino, the substituent is hydroxyl, methyl, amino, methoxy, dimethylamino, halogen or ethyl;

when said R is₂Represents substituted C_1-4Alkyl, substituted C_1-4Alkoxy, substituted C_1-4Alkylthio or substituted C_1-4In the case of alkylamino, the substituent is hydroxy, methyl, amino, methoxy, dimethylamino, halogen or ethyl.

3. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein ring a is selected from one of the following structures:

；

the R is₁And R₂Are identical or different and each independently represents hydrogen, fluorine, chlorine, methyl, methoxy or

。

4. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is selected from the group consisting of:

。

5. a process for the preparation of a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, comprising the steps of:

in the formula, ring A, R₁And R₂Is as defined in any one of claims 1 to 4 for ring A, R₁And R₂The definition of (1);

s1, synthesis of intermediate III:

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, synthesis of intermediate V:

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, synthesis of intermediate VI:

reacting the intermediate V, ammonium salt and hydrogen in a third reaction solvent in the presence of a third alkali under the action of a third catalyst to obtain an intermediate VI;

s4, synthesis of an intermediate VIII:

reacting the intermediate VI with a compound VII in a fourth reaction solvent in the presence of a fourth base to obtain an intermediate VIII;

s5, synthesis of intermediate IX:

reacting the intermediate VIII in a fifth reaction solvent in the presence of a fifth base and water to obtain a first product, and reacting the first product with an acid to obtain an intermediate IX;

synthesis of S6, intermediate x:

reacting the intermediate IX in a sixth reaction solvent in the presence of a sixth base under the action of a condensing agent to obtain an intermediate X;

s7, Synthesis of Compound I:

the intermediate X is subjected to manual resolution to obtain the compound I.

6. The production method according to claim 5, wherein in step S1:

the first catalyst is a palladium catalyst, and the palladium catalyst is at least one of palladium acetate, [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride and tetratriphenylphosphine palladium;

the first alkali is at least one of sodium acetate, potassium acetate, sodium carbonate, potassium phosphate and cesium carbonate;

the first reaction solvent is at least one of 1, 4-dioxane, toluene and tetrahydrofuran;

the reaction temperature is 50-100 ℃.

7. The production method according to claim 5, wherein in step S2:

the second catalyst is a palladium catalyst, and the palladium catalyst is at least one of palladium acetate, [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride and tetratriphenylphosphine palladium;

the second base is at least one of sodium carbonate, potassium carbonate, cesium carbonate and potassium phosphate;

the second reaction solvent is tetrahydrofuran and water or 1, 4-dioxane and water;

the reaction temperature is 20-100 ℃.

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 4, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

9. A TRK kinase inhibitor comprising a therapeutically effective amount of a compound selected from the group consisting of the compounds of any one of claims 1-4, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

10. Use of a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention and/or treatment of tumours.