WO2018157737A1 - Multi-target kinase inhibitor - Google Patents

Abstract

The present invention provides a multi-target protein kinase inhibitor, a pharmaceutical composition thereof, and a use of the multi-target kinase inhibitor in the preparation of a medicament for treating diseases caused by abnormal protein kinase activity. The multi-target kinase inhibitor of the present invention has the general structural formula represented by formula (I). The multi-target kinase inhibitor has an ideal inhibitory effect and specific selectivity, and has a great prospect in the treatment of complex heterogeneous diseases and overcoming of drug resistance.

Description

Multi-target kinase inhibitor Technical field

The invention belongs to the field of medicine and relates to a novel multi-target protein kinase inhibitor.

Background technique

Protein kinases are a class of enzymes that catalyze the phosphorylation of proteins. The most important life activities in cells are related to the phosphorylation of proteins. By mediating cell signal transduction, phosphorylation of proteins regulates cell fate, for example. Cell proliferation, differentiation and apoptosis. Some human fatal diseases, such as tumors, are significantly associated with abnormal protein kinase activity. Therefore, protein kinases have become a hot drug target, and kinase inhibitor drugs have become the most important component of tumor-targeted therapy.

Most tumors have significant heterogeneity, probably because most tumors are not caused by a single molecular abnormality, but rather by the synergistic effects of multiple abnormal molecules. On the other hand, kinase inhibitors have been widely used in tumor targeting therapy, inflammation therapy, etc. However, with the widespread use of kinase inhibitors, drug resistance has become one of the key issues facing the current clinical research. The data show that activation of the bypass compensatory signaling pathway is one of the important reasons for kinase inhibitor resistance. The development of multi-target kinase inhibitors that can act on multiple signaling pathways simultaneously can not only effectively cope with the biological characteristics of tumor multi-molecular abnormalities, but also alleviate drug resistance problems to some extent.

At present, it has been found that the human genome encodes 518 protein kinases. According to the structure and function, the protein kinases of eukaryotic cells can be divided into five categories, namely serine/threonine protein kinase, tyrosine protein kinase, group/ Lai/arginine protein kinase, cysteine protein kinase and aspartate/glutamate protein kinase. Most protein kinases have a more conserved catalytic binding domain, and these catalytic domains are sequence-dependent, thus structurally ensuring the feasibility of multi-target kinase inhibitor design.

For some heterogeneous diseases, multi-targeted drugs may be the only effective drug treatment. For example, liver cancer is a type of cancer with great heterogeneity. It is not sensitive to most chemotherapy drugs. A number of targeted drugs, including Brivanib, Suntinib, and Linifanib, have failed in clinical trials of liver cancer, and only a drug of sorafenib has been approved by the FDA. Sorafenib is a multi-target kinase inhibitor, the main targets include c-Raf, VEGFR2, c-kit, p38α, etc. The multi-target synergistic mechanism of this drug may be an important reason for its effectiveness in advanced liver cancer. Recently, a number of ongoing Alzheimer's disease drugs have experienced major setbacks in clinical phase III, suggesting that the single target assumed in the early stage may not be an effective therapeutic target.

On the other hand, in the field of treatment of diseases that may rapidly develop drug resistance, such as the field of AIDS treatment, "cocktail"-type mixed drug therapy has proven to be the most effective treatment for AIDS. However, the development of drug combinations not only faces complex "drug-drug interaction" problems, but also may have patent disputes, and the development of a single multi-target drug has a significant comparative advantage.

VEGFR, the epidermal growth factor receptor, is a class of receptor tyrosine kinases that play a key role in tumor angiogenesis and is an effective therapeutic target for some vascular-dependent tumors. c-Met, a hepatocyte growth factor receptor, is an important regulator of normal hepatocyte growth and differentiation. Raf kinase is involved in the Ras-Raf-Mek-ERK signal transduction cascade, which regulates cell cycle, differentiation, proliferation and apoptosis. c-Kit is a stem cell growth factor receptor that plays an important role in hematopoietic cell function. Abnormally high expression of these protein kinases has been found in a variety of tumors, such as acute myeloid leukemia, liver cancer, renal cell carcinoma, melanoma, gastrointestinal stromal tumors, etc., multi-target kinases targeting the above kinase combinations Inhibitors have good development potential.

Chinese patent application CN105541798A discloses an N ¹ -(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)-N ² -substituted heterocyclic ring-N having antitumor activity ³ -Phenylmalonamide quinoline multi-target kinase inhibitor and preparation method thereof. The compound has strong inhibitory activity against tumor cell line human thyroid carcinoma SW579, human liver cancer HepG2, human lung adenocarcinoma A549, human intestinal cancer HCT116 and human gastric cancer MKN45. Most of the target compounds have been confirmed by in vitro cell experiments for two kinases. KDR and MET showed strong inhibitory activity.

Chinese patent application CN103214489A discloses a N-(6-substituted-3,4,6,7-tetrahydro-2H-pyrimido[1,6-c]quinazolin-2-alkenyl) substituted aniline An antitumor compound that has broad-spectrum inhibitory activity against a variety of tumor-associated kinases, but some of which have significant cytotoxic effects.

The development of multi-targeted kinase inhibitors with moderate target selectivity is challenging, with sorafenib as an example, its effectiveness in clinical use is still not high, in part because it inhibits some targets that "inhibit the development of liver cancer" Point, for example p38α. P38α has been reported to be a liver cancer suppressor protein. Therefore, inhibition of its activity is not conducive to the treatment of liver cancer, and it is likely to bring about unexpected side effects.

In view of the above discussion and existing problems, the present inventors have devised the development of a multi-target protein kinase inhibitor with moderate selectivity, which has great prospects in the treatment of complex heterogeneous diseases and overcoming drug resistance.

Summary of the invention

A first object of the invention is to provide a novel multi-target protein kinase inhibitor;

A second object of the present invention is to provide a pharmaceutical composition of a multi-target protein kinase inhibitor;

A third object of the present invention is to provide a novel multi-target protein kinase inhibitor for use in the preparation of a medicament for treating a disease caused by abnormal protein kinase activity.

According to one aspect of the invention, a multi-target protein kinase inhibitor having moderate selectivity (ie, capable of acting on a plurality of target protein kinases while maintaining certain specificity) is designed to treat heterogeneous diseases and alleviate Drug resistance.

The multi-target protein kinase inhibitor designed and synthesized for this purpose is a compound of formula (I):

Wherein, R ¹ is H, or is in the ortho, meta or para-halogen atom, non-cyclic alkyl, C3-C6 substituted or non-substituted heterocyclic, C3-C7 cycloalkyl;

R ² is H, or a halogen atom or an acyclic alkyl group in the ortho or meta or para position of the linker;

M ¹ is O or NH in the linker alignment or meta position;

M ² is one selected from the group consisting of formula (II), (III) and (IV):

Wherein, in the formula (II), R ³ is a spiro ring, or an unsubstituted or substituted C3-C6 heterocyclic ring; X ¹ is CH or N; Y ¹ is CH or N; Z ¹ is CH, S, NH, or O; Z ² is N, CH or NH;

In the formula (III), X ² is CH or N; Z ³ is N or S; Z ⁴ is N or S; R ⁴ is an unsubstituted or substituted C3-C6 heterocyclic ring, or a group of the formula (V)

in,

X ⁴ is NH, S or O; M ³ is S or O; R ⁶ is a C3-C7 substituted or unsubstituted cycloalkane, or a substituted or unsubstituted C3-C6 heterocyclic ring;

In the formula (IV), X ³ is CH or N; Y ² is CH or N; and R ⁵ is an unsubstituted or substituted C3-C6 heterocyclic ring;

Linker is selected from one of formula (VI) and formula (VII):

In the formula I, R ¹ is H, or a halogen atom at a different substitution position, an alkyl group, a C3-C6 unsubstituted or substituted heterocyclic ring, a C3-C7 cycloalkane; R ² is H, or is at a different substitution position. Halogen atom, alkyl group; M ¹ is O or NH; M ² is

or

or

Linker is

or

Further, R ^{3 is} selected from one of the following structures:

Or one selected from the following structures,

Among them, the general formula

Medium, m≥0 and m is an integer;

Where p ≥ 0 and p is an integer;

General formula

Where q ≥ 0 and q is an integer; W is CH or N.

Further, R ⁵ is one selected from the following structures:

Where k = 1, 2 or 3.

Further, R ⁶ is one selected from the following structures:

In the formula (VIII), n is a positive integer of 1 to 5; and in the formula (IX), X ⁴ to X ^{8 are} independently selected from CH ₂ , NH, O or S.

Further, the acyclic alkyl group is selected from one of the following functional groups:

Still further, the C3-C6 heterocyclic ring described in R ³ , R ⁴ , R ⁵ and R ⁶ is selected from one of the following functional groups:

Finally, the multi-target protein kinase inhibitor provided by the present invention is selected from the following compounds:

It will be appreciated by those skilled in the art that the corresponding salts of the above compounds also have the same effect, and the specific salt forms include, but are not limited to, maleate, hydrochloride, sulfate, phosphate, malate, tosylate , mesylate, acetate, tartrate, trifluoroacetate, and the like.

According to another aspect of the present invention, a pharmaceutical composition is prepared by using the compound of the formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable drug delivery system containing the compound as an active ingredient .

According to the last aspect of the present invention, the inhibitory activity of a compound against a plurality of protein kinases is investigated, and the compound of the present invention has excellent inhibitory activity against protein kinases in comparison with the effects of existing commercial preparations;

Preferably, the compounds of the invention are directed against a receptor tyrosine protein kinase, a non-receptor tyrosine protein kinase, and a serine/threonine protein kinase.

[Advantageous Effects] The protein kinase inhibition test indicates that the compound of the present invention has sufficient commercialization level of inhibition ability; inhibition tests of various cells indicate that the compound of the present invention has excellent selection specificity and can effectively prevent toxic side effects, which is complicated The treatment of heterogeneous diseases and the overcoming of drug resistance have great prospects.

detailed description

The technical solutions of the present invention will be further described below in conjunction with the embodiments, and the following examples are not intended to limit the invention.

The reagent materials of the present invention are all commercially available.

Example 1 [Synthesis of Compounds]

(1) Synthesis of compound td32-4

N-(3-Fluoro-4-((2-(1-(2-hydroxy)ethyl)-1H-pyrazol-4-yl)thieno[3,2-b]pyridin-7-yl)oxy The synthetic route of phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dimethylamide (compound td32-4) is as follows:

To a 100 mL single-necked flask, compound 5 (2.00 g, 5.18 mmol, 1.00 eq), 1A-4 (1.73 g, 6.22 mmol, 1.20 eq), Pd(dppf)Cl ₂ (379.03 mg, 518.00 μmol, 0.10 eq) was added. Cs ₂ CO ₃ (5.06 g, 15.54 mmol, 3.00 eq), the mixture was dissolved in 60 mL of THF / H ₂ O (5:1, v: v), and the resulting solution was stirred under nitrogen atmosphere and stirred at 50 ° C for 5 hours. The reaction was monitored by LC-MS until Compound 5 disappeared completely, and the objective product 1-4 was formed. After the reaction was completed, 50 mL of purified water was added, and ethyl acetate was extracted three times (50 mL each time), and the obtained organic layer was evaporated to give a solid, which was purified by column chromatography, toluene DCM/MeOH (70/1, 2L) 1-4 (4.14 mmoL, 1.70 g).

Yield: 79.96%.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.43 (d, J = 5.6 Hz, 1H), 7.99 (s, 1H), 7.78 (s, 1H), 7.48 (s, 1H), 6.97 (t, J = 8.8 Hz, 1H), 6.55 (dd, J = 11.8, 2.8 Hz, 1H), 6.48-6.46 (m, 2H), 5.47-5.43 (m, 1H), 4.18-4.09 (m, 1H), 3.77-3.71 (m, 1H), 2.18-2.13 (m, 2H), 1.75-1.65 (m, 4H), 1.22 (s, 2H).

To a 100 mL one-neck round bottom flask, compound 1-4 (1.50 g, 3.65 mmol, 1.00 eq), compound 6 (855.41 mg, 3.83 mmol, 1.05 eq), HATU (1.53 g, 4.02 mmol, 1.10 eq), DIEA (1.42 g, 10.95 mmol, 1.91 mL, 3.00 eq), dissolved in 20 mL of DMF, and the mixture was stirred at 50 ° C for 5 hours. The reaction was monitored by LC-MS. After completion of the reaction, 50 mL of purified water was added, and ethyl acetate was extracted four times (30 mL each time), and the organic layer was evaporated to give crude product 2-4 (1.60 g).

Yield: N/A.

¹ H NMR (400 MHz, CD ₃ OD) δ 8.41 (d, J = 5.6 Hz, 1H), 8.34 (s, 1H), 7.99 (s, 1H), 7.84 (dd, J = 12.8, 2.4 Hz, 1H) ), 7.59-7.55 (m, 3H), 7.42-7.37 (m, 1H), 7.37-7.35 (m, 1H), 7.11-7.06 (m, 2H), 6.59 (d, J = 5.6 Hz, 1H), 5.48 (dd, J=9.6, 2.0 Hz, 1H), 4.09-4.06 (m, 1H), 3.78-3.73 (m, 2H), 2.20-2.05 (m, 1H), 2.08-2.05 (m, 2H), 1.80-1.73 (m, 2H), 1.66 (s, 4H)

Compound 2-4 (1.40 g, 2.27 mmol, 1.00 eq) was dissolved in 20.00 mL HCl/MeOH solution. The solution was placed in a 100 mL single-neck round bottom flask and stirred at 50 ° C for 1 hour. LC-MS monitoring showed compound 2- 4 completely disappeared and the target product was obtained. The resulting solution was subjected to rotary distillation to give Compound 3-4 (1.10 g).

Yield: N/A.

¹ H NMR (400 MHz, CD ₃ OD) δ 8.68 (d, J = 6.4 Hz, 1H), 8.43 (s, 2H), 7.78 (s, 1H), 7.64-7.60 (m, 1H), 7.54 - 7.53 (m, 2H), 7.52-7.51 (m, 2H), 7.10 (s, 1H), 7.06 (d, J = 8.8 Hz, 2H), 1.62-1.57 (m, 4H).

To a 100 mL one-neck round bottom flask, compound 3-4 (1.00 g, 1.88 mmol, 1.00 eq), 2-bromoethanol (282.11 mg, 2.26 mmol, 160.29 uL, 1.20 eq) and Cs ₂ CO ₃ (1.84 g, 5.64 mmol, 3.00 eq), 20 mL of DMF was dissolved, and the resulting mixture was stirred at 90 ° C for 14 hours, and LC-MS monitoring showed that Compound 3-4 completely disappeared to give the desired product. After the completion of the reaction, 100 mL of purified water was added to the obtained mixed solution, and ethyl acetate was extracted 4 times (50 mL each time), and the organic layer was evaporated to give a crude product, which was purified by column chromatography, DCD/MeOH (40/1, 2L) The obtained compound was further purified using preparative chromatography to afford white crystals of product td32-4 (100.00mg, 172.00 umol, 9.15% yield, 99% purity).

Yield: 9.15%.

^{1 H NMR (400MHz, DMSO-} d 6) δ10.39 (s, 1H), 9.99 (s, 1H), 8.44 (d, J = 5.6Hz, 1H), 8.31 (s, 1H), 8.00 (s, 1H), 7.89 (dd, J = 13.2, 2.0 Hz, 1H), 7.69 (s, 1H), 7.64 (dd, J = 8.8, 4.0 Hz, 2H), 7.49-7.47 (m, 1H), 7.46-7.40 (m, 1H), 7.14 (t, J = 8.8 Hz, 2H), 6.54 (d, J = 5.2 Hz, 1H), 4.96 (t, J = 5.2 Hz, 1H), 4.18 (t, J = 5.2 Hz) , 2H), 3.77 (q, J = 5.2 Hz, 2H), 1.46 (d, J = 2.4 Hz, 4H).

LC-MS: 576.2 (100%), 598.2 (10%).

(2) Synthesis of compound td32-5

N-(3-Fluoro-4-((2-(pyrrolidin-1-carbonyl)thieno[3,2-b]pyridin-7-yl)oxo)phenyl)-N-(4-fluorobenzene) The synthetic route of cyclopropane-1,1-dimethylformamide (compound td32-5) is as follows:

To a 25 mL round bottom flask, compound 2 (184.16 mg, 2.59 mmol, 216.66 uL, 2.00 eq), compound 1 (500.00 mg, 1.29 mmol, 1.00 eq), Pd(OAc) ₂ (29.07 mg, 129.47 μmol, 0.10 eq) Xantphos (74.92 mg, 129.47 μmol, 0.10 eq) and Na ₂ CO ₃ (274.46 mg, 2.59 mmol, 2.00 eq) were added to 5 mL of toluene and stirred to dissolve. After the CO was ventilated, the pressure was maintained at 15 psi and heated to 80 ° C for 15 hours. . LC-MS monitoring showed complete disappearance of compound 1 to give the desired product. After completion of the reaction, the mixture was filtered, and then filtered, mjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj Compound 3 (300.00 mg, 839.40 μmol, 65.07% yield) was obtained. LC-MS: 358.0, 379.9.

To a 5 mL round bottom flask, compound 3 (250.00 mg, 699.50 μmol, 1.00 eq) and compound 4 (187.35 mg, 839.40 μmol, 1.20 eq) were added, dissolved in 2 mL DMF, and HATU (398.95 mg, 1.05 mmol, 1.50 eq) was added. And DIEA (271.21 mg, 2.10 mmol, 366.50 uL, 3.00 eq), the resulting mixed solution was reacted at 20 ° C for 15 hours, and LC-MS monitoring showed that Compound 3 completely disappeared to give the desired product. After completion of the reaction, the crude product was purified by preparative chromatography to afford pale white solid td32-5.

Yield: N/A

^{1 H NMR (400MHz, DMSO-} d6) δ10.42 (s, 1H), 10.02 (s, 1H), 8.61 (d, J = 5.6Hz, 1H), 8.05 (s, 1H), 7.94-7.88 (m , 1H), 7.64 (dd, J=8.8, 4.8 Hz, 2H), 7.52-7.49 (m, 2H), 7.16 (t, J = 8.8 Hz, 2H), 6.74 (d, J = 5.6 Hz, 1H) , 3.87 (t, J = 6.8 Hz, 2H), 3.55 (t, J = 6.8 Hz, 2H), 1.99-1.87 (m, 4H), 1.47 (s, 4H).

LC-MS: 563.1 (100%), 586.1 (30%).

(3) Synthesis of compound td32-6

N-(4-((2-(cyclopropanecarbamoyl) <ethylenediamino])thieno[3,2-b]pyridin-7-yl)oxo)-3-fluorophenyl)- The synthetic route of N-(4-fluorophenyl)cyclopropane-1,1-dimethylamide (compound td32-6) is as follows:

To a 100 mL one-neck round bottom flask, compound 1-6 (1.00 g, 1.69 mmol, 1.00 eq), compound 2A-6 (337.10 mg, 1.86 mmol, 312.13 uL, 1.10 eq), Pd(OAc) ₂ (75.93 mg) , 338.00umol, 0.20eq), Xantphos (195.69mg, 338.00umol, 0.20eq) and Cs ₂ CO ₃ (1.65g, 5.07mmol, 3.00eq), dissolved in 20mL DMF, the mixture was reacted under nitrogen for 10 hours at 100 ° C for 12 hours LC-MS monitoring showed that Compound 1-6 completely disappeared to give the desired product. After completion of the reaction, 100 mL of purified water was added, and ethyl acetate was extracted 6 times (100 mL each time), and the organic layer was evaporated to give a crude product which was purified by column chromatography (developing liquid PE/EA=3/2, 2L) to obtain compound 2 -6 (500.00 mg, 682.50 μmol, 88% purity).

Yield: 40.38%.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.84 (s, 1H), 8.26 (d, J = 5.6 Hz, 1H), 7.94 (s, 2H), 7.74 (d, J = 7.6 Hz, 2H), 7.47 -7.29 (m, 8H), 7.25 (d, J = 6.8 Hz, 2H), 7.16 (d, J = 10.0 Hz, 1H), 7.08 (s, 1H), 7.06-7.00 (m, 1H), 6.96 ( t, J = 8.6 Hz, 2H), 6.30 (d, J = 5.2 Hz, 1H), 1.72-1.69 (m, 2H), 1.60-1.55 (m, 2H).

LC-MS: 645.3.

To a 40 mL sealed tube, compound 2-6 (450.00 mg, 698.01 umol, 1.00 eq) was added, dissolved in 9 mL of THF, HCl _(aq) (2M, 9.00 mL, 25.79 eq) was added, and the mixture was reacted at 25 ° C for 0.5 hour, LC - MS monitoring showed that Compound 2-6 completely disappeared to give the desired product. After the reaction was completed, the mixture was added to 50 mL of saturated sodium hydrogencarbonate, and extracted with ethyl acetate three times (30 mL each time), and the organic layer was evaporated to give a crude product which was purified by column chromatography (eluent PE/EA = 1/1 to 1/1) Compound 2-6 (230.00 mg, 478.68 μmol) was obtained as 1,2L.

Yield: 68.58%.

_{^{1 H NMR (400MHz, CDCl 3}} ) δ10.01 (s, 1H), 8.48 (s, 1H), 8.24 (d, J = 5.6Hz, 1H), 7.74 (dd, J = 12.0,2.4Hz, 1H) , 7.49-7.41 (m, 2H), 7.24-7.13 (m, 2H), 7.08-7.04 (m, 2H), 6.44 (s, 1H), 6.31 (d, J = 5.6 Hz, 1H), 4.43 (s) , 2H), 1.80-1.77 (m, 2H), 1.64-1.61 (m, 2H).

LC-MS: 481.0.

To a 40 mL sealed tube, compound 3-6 (200.00 mg, 416.24 μmol, 1.00 eq) and pyridine (65.85 mg, 832.48 μmol, 67.19 uL, 2.00 eq) were added, dissolved in 10 mL of dichloromethane, and added dropwise at 0 °C. The cyclopropanecarbonyl chloride (56 mg) was reacted at 25 ° C for 1 hour. LC-MS monitoring showed the starting material was still present. therefore, cyclopropanecarbonyl chloride (56 mg) was again added dropwise at 0 ° C, and the mixture was reacted again at 25 ° C for 1 hour. LC-MS monitoring showed that most of the starting materials still existed, and the mixture was reacted at 25 ° C for 10 h. LC-MS monitoring showed that the starting materials were still present, and the temperature was raised to 25 ° C for 1 hour. LC-MS monitoring showed that the starting materials were completely reacted. , the target product is generated. After completion of the reaction, 30 mL of purified water was added, and the mixture was extracted with dichloromethane (3 mL), and the organic layer was evaporated. The obtained solid was purified by preparative chromatography to afford the desired product td32-6 (70.00 mg, 126.33 μmol).

Yield: 30.35%.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.03 (s, 1H), 10.39 (s, 1H), 10.02 (s, 1H), 8.39 (d, J = 5.2 Hz, 1H), 7.89 (d, J = 12.8 Hz, 1H), 7.64 (bs, 2H), 7.54 - 7.33 (m, 2H), 7.17 (d, J = 8.4 Hz, 2H), 7.00 (s, 1H), 6.46 (d, J = 4.8) Hz, 1H), 1.86 (s, 1H), 1.47 (bs, 4H), 0.92 (bs, 4H).

LC-MS: 549.3.

(4) Synthesis of Compound 51

N-(3-((2-(cyclopropanecarbamoyl) <oxalylamino>)thiazolo[5,4-b]pyridin-5-yl)amino)-4-fluorophenyl)-N The synthetic route of -(4-fluorophenyl)cyclopropane-1,1-dimethylamide (Compound 51) is as follows:

To a 250 mL one-neck round bottom flask, compound 3-51 (3.00 g, 10.06 mmol, 1.00 eq), compound 1D-53 (4.55 g, 20.12 mmol, 2.00 eq), Pd(OAc) ₂ (225.90 mg, 1.01 mmol) , 0.10 eq), Xantphos (582.19 mg, 1.01 mmol, 0.10 eq) and Cs ₂ CO ₃ (6.56 g, 20.12 mmol, 2.00 eq), dissolved in 100 mL of 1,4-dioxane, and the mixture was reacted at 110 ° C under nitrogen. After 3 hours, LC-MS monitoring showed that the starting material was reacted to produce the desired product. After completion of the reaction, the solution was filtered and the filtrate was evaporated to give a crude material. Purification by column chromatography (EtOAc EtOAcjjjjjjjj

Into a 100 mL one-neck round bottom flask, compound 4-51 (3.50 g, 7.89 mmol, 1.00 eq) was added, dissolved in DCM/TFA (4/1, 5 mL), and the mixture was reacted at 25 ° C for 2 hours, and LC-MS monitoring showed The starting material is reacted to produce the target product. After completion of the reaction, the pH of the solution was adjusted to 8 with saturated sodium bicarbonate to give a precipitate, which was filtered and dried to afford compound 5- 5- (850.00 mg, 2.07 mmol).

Yield: 26.23%.

^{1 H NMR (400MHz, DMSO-} d 6) δ8.49 (s, 1H), 7.77 (d, J = 8.8Hz, 1H), 7.34 (dd, J = 7.6,2.4Hz, 1H), 6.96 (d, J=8.8 Hz, 1H), 6.86-6.81 (m, 1H), 6.15-6.12 (m, 1H), 4.89 (s, 2H), 1.88 (bs, 1H), 0.87-0.83 (m, 4H).

LC-MS: 343.9.

To a 25 mL one-neck round bottom flask, compound 5-51 (800.00 mg, 2.33 mmol, 1.10 eq), compound 5B (472.73 mg, 2.12 mmol, 1.00 eq), DIEA (821.18 mg, 6.35 mmol, 3.00 eq) and HATU (885.94 mg, 2.33 mmol, 1.10 eq) was dissolved in 12 mL of DMF, and the mixture was reacted at 35 ° C for 12 hours. LC-MS monitoring showed that the starting material was reacted to give the desired product. After the completion of the reaction, the obtained mixture was concentrated to give a crude material (yield: EtOAc (EtOAc: EtOAc: EtOAc: EtOAc)

Yield: 17.71%.

^{1 H NMR (400MHz, DMSO-} d 6) δ12.50 (s, 1H), 10.13 (s, 1H), 10.02 (s, 1H), 8.93 (s, 1H), 8.49 (d, J = 1.6Hz, 1H), 7.93 (d, J = 8.8 Hz, 1H), 7.66 (dd, J = 8.8, 5.2 Hz, 2H), 7.26 (d, J = 8.0 Hz, 1H), 7.17-7.13 (m, 3H), 7.07 (d, J = 8.8 Hz, 1H).

LC-MS: 549.0.

(5) Synthesis of Compound 52

N-(4-Fluoro-3-((6-(pyridin-3-yl)pyrimidin-4-yl)oxo)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-di The synthetic route of formamide (Compound 52) is as follows:

To a 250 mL one-neck round bottom flask was added compound 7N-1 (5.00 g, 33.56 mmol, 1.00 eq), Compound 12A (4.13 g, 33.56 mmol, 1.00 eq), Na ₂ CO ₃ (10.67 g, 100.68 mmol, 3.00 Eq), and Pd(PPh ₃ ) ₂ Cl ₂ (1.41 g, 2.01 mmol, 0.06 eq), dissolved in 120 mL of THF/H ₂ O (5:1), and the mixture was reacted at 75 ° C for 3 hours, LC-MS monitoring showed The starting material is reacted to produce the desired product. After the completion of the reaction, the mixed solution was evaporated to give a crude product. To a crude product was added 300 mL of purified water, and ethyl acetate was extracted three times (200 mL each time), and the organic layer was dried over anhydrous sodium sulfate. The product was purified by EtOAc EtOAc EtOAc (EtOAc:EtOAc

Yield: 33.82%

¹ H NMR (DMSO, 400 MHz) δ 9.39 (d, J = 1.6 Hz, 1H), 9.15 (s, 1H), 8.77 (dd, J = 4.8, 2.0 Hz, 1H), 8.59-8.57 (m, 1H) ), 8.45 (s, 1H), 7.62 (dd, 8.0, 4.8 Hz, 1H).

To a 100 mL one-neck round bottom flask, compound 1-52 (796.08 mg, 6.26 mmol, 1.20 eq) was added, dissolved in 20 mL dry THF, and NaH (250.50 mg, 6.26 mmol, 1.20 eq) was added in portions at 0 ° C. After completion, the reaction was stirred at 0 ° C for 0.5 hr, and then compound 7N (1.00 g, 5.22 mmol, 1.00 eq) was added, and the reaction was stirred at 25 ° C for 12 hours. The TLC (PE/EA=1:3) monitoring showed that the starting material was completely reacted, and the mixed solution was subjected to rotary distillation to obtain a crude product, which was purified by column chromatography (developing liquid PE/EA=1/1, 4L) to obtain compound 2-52 ( 500 mg, 1.63 mmol, 92% purity). LC-MS: 283.0.

Yield: 31.22%

^{1 H NMR (400MHz, DMSO-} d6) δ9.39 (d, J = 2.0Hz, 1H), 8.62 (s, 1H), 8.75 (dd, J = 4.8,1.6Hz, 1H), 8.58-8.55 (m , 1H), 7.94 (s, 1H), 7.64 - 7.55 (m, 1H), 7.05 (dd, J = 10.4, 8.8 Hz, 1H), 6.51-6.45 (m, 2H), 5.187 (s, 2H) .

To a 40 mL sealed tube, compound 2-52 (440.00 mg, 1.56 mmol, 1.00 eq), compound 5B (347.92 mg, 1.56 mmol, 1.00 eq), HATU (651.97 mg, 1.71 mmol, 1.10 eq) and DIEA ( 604.37 mg, 4.68 mmol, 3.00 eq), dissolved in 10 mL of DMF, and the mixture was stirred at 30 ° C for 12 hours. LC-MS monitoring showed that the starting material was reacted to give the desired product. After completion of the reaction, 25 mL of purified water was added to precipitate a precipitate, which was filtered, and the filter cake was slurried in 10 mL of methanol to give Compound 52 (210.00 mg, 426.50 μmol, 99% purity).

Yield: 27.34%

^{1 H NMR (400MHz, DMSO-} d6) δ10.22 (s, 1H), 10.03 (s, 1H), 9.417 (s, 1H), 8.87 (s, 1H), 8.76 (d, J = 3.6Hz, 1H ), 8.59 (d, J = 8.0 Hz, 1H), 8.03 (s, 1H), 7.79 (d, J = 5.2 Hz, 1H), 7.64 - 7.58 (m, 3H), 7.53 - 7.51 (m, 1H) , 7.37 (t, J = 9.6 Hz, 1H), 7.13 (t, J = 8.8 Hz, 2H), 1.444 (s, 4H).

LC-MS: 488.2 (100%), 510.2 (60%).

(6) Synthesis of Compound 53

N-(4-Fluoro-3-((6-(pyridin-3-yl)pyrimidin-4-yl)amino)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dimethyl The synthetic route of the amide (Compound 53) is as follows:

To a 40 mL sealed tube, compound 7N (500.00 mg, 2.61 mmol, 1.00 eq), compound 1D-53 (936.98 mg, 2.87 mmol, 1.10 eq) and TsOH·H ₂ O (19.86 mg, 104.40 umol, 0.04 eq) After adding 20 mL of i-PrOH, the mixture was stirred and dissolved, and the mixture was stirred at 75 ° C for 12 hours. LC-MS monitoring showed that the starting material was reacted to give the desired product. After completion of the reaction, the mixture was evaporated to give purified crystals. mjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjj

To a 25 mL single-mouth round bottom flask, compound 1-53 (350.00 mg, 726.86 umol, 1.00 eq), 10 mL HCl/EA solution was added, and the mixture was stirred at 25 ° C for 1 hour. LC-MS monitoring showed that the starting material was completely reacted. , the target product is generated. After completion of the reaction, it was rotary evaporated to give the product 2-53.

¹ H NMR ES 2539-48-P1A (400 MHz, DMSO-d ₆ ) = 10.36 (br, 1H), 9.38 (s, 1H), 9.05-8.945 (q, J=16, 4.8 Hz, 2H), 8.84 (s) , 1H), 8.12-8.08 (m, 2H), 7.69 (s, 1H), 7.47 (t, J = 9.5 Hz, 1H), 7.29-7.26 (m, 1H)

To a 40 mL sealed tube was added compound 2-53 (300.00 mg, 1.07 mmol, 1.10 eq), compound 5B (217.11 mg, 972.73 umol, 1.00 eq), HATU (406.85 mg, 1.07 mmol, 1.10 eq) and DIEA ( 377.15 mg, 2.92 mmol, 3.00 eq), dissolved in 10 mL of DMF, and the mixture was stirred at 35 ° C for 12 hours. LC-MS monitoring showed that the starting material was reacted to give the desired product. After completion of the reaction, 20 mL of purified water was added, and ethyl acetate was extracted 4 times (30 mL each time), and the organic layer was evaporated to give a crude product which was purified to afford compound 53 as a white solid (100.00 mg, 205.56 μmol, 100%purity).

Yield: 21.13%

1H NMR (400MHz, DMSO-d6) δ 10.12 (s, 1H), 10.05 (s, 1H), 9.50 (s, 1H), 9.19 (d, J = 1.5 Hz, 1H), 8.70 (s, 2H) , 8.37 (d, J = 8.0 Hz, 1H), 8.19 (d, J = 5.2 Hz, 1H), 7.64-7.60 (dd, J = 8.8, 4.8 Hz, 2H), 7.57-7.54 (dd, J = 8.4 , 4.8 Hz, 1H), 7.41-7.39 (m, 2H), 7.24 (t, J = 9.6 Hz, 1H), 7.14 (t, J = 8.8 Hz, 2H), 1.45 (s, 4H).

LC-MS: 487.0 (100%), 509.0 (85%).

(7) Synthesis of Compound 29

1-(3-Fluoro-4-((2-(4-)4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)oxo)phenyl)-3-( The synthetic route of 4-fluorophenyl)urea (Compound 29) is as follows:

To a 250 mL one-neck round bottom flask, compound 8A-1 (5.00 g, 39.33 mmol, 1.00 eq) was added, 100 mL of DMF was dissolved, and compound 5A2 (5.39 g, 39.33 mmol, 1.00 eq) was added dropwise at 0 ° C, and added dropwise. After completion, the obtained solution was stirred at 15 ° C for 2 hours, TLC (PE / EA = 2 / 1) monitoring showed that the reaction was complete, 300 mL of purified water was added, the precipitate was precipitated, and the filter cake was washed three times with 20 mL each time, dried under reduced pressure. The crude product was about 7.0 g, and the crude product was slurried in a solution of PE/EA=4:1 at 15 ° C for 1 hour, filtered, and the filter cake was washed 3 times with petroleum ether for 5 mL each time, and the obtained cake was dried under reduced pressure. Compound 8c (6.40 g, 21.80 mmol, purity 90%) was obtained.

Yield: 55.43%

¹ H NMR (DMSO, 400 MHz), δ 9.44 (s, 1H), 8.65 (s, 1H), 8.54 (s, 1H), 7.46-7.42 (m, 3H), 7.13-7.09 (m, 2H), 6.91-6.68 (m, 2H).

To a 250 mL three-neck round bottom flask was added compound 8C (2.50 g, 9.46 mmol, 1.00 eq), dissolved in 30 mL DMF, and then compound 7D (1.41 g, 9.46 mmol, 1.00 eq), K ₂ CO ₃ (2.61 g, 18.92) Methylene, 2.00 eq), the resulting mixed solution was heated to 90 ° C for 16 hours, and the reaction was monitored by TLC (PE / EA = 1 / 1). After completion of the reaction, 150 mL of purified water was added thereto with stirring to precipitate a precipitate, which was filtered, and the residue was washed three times with 20 mL each time, and the obtained cake was dried under reduced pressure to give 2.8 g. Purification by column chromatography gave Compound 7D-1 (1.

Yield: 39.98%

¹ H NMR (DMSO, 400 MHz), δ 8.99 (s, 1H), 8.83 (s, 1H), 8.66 (d, J = 5.6 Hz, 1H), 7.69 (d, J = 12.8, 2.0 Hz, 1H) , 7.49-7.46 (m, 2H), 7.34-7.29 (m, 2H), 7.20 (d, J = 1.6 Hz, 1H), 7.13 (t, J = 8.8 Hz, 2H).

To a 25 mL one-neck round bottom flask, compound 7D-1 (458.37 mg, 1.22 mmol, 1.00 eq) was added, dissolved in 10 mL isopropanol, then compound 1-29 (280.00 mg, 1.46 mmol, 1.20 eq), TsOH (20.95) Mg, 121.67 μmol, 0.10 eq), the resulting mixed solution was heated to 85 ° C for 16.5 hours, LC-MS monitoring showed that the reaction of the starting material was complete, and the pH was adjusted to 8-9 with saturated sodium bicarbonate solution under stirring, and the product was obtained by rotary evaporation. 600mg. The crude product was purified using preparative chromatography to afford compound 29 (49.00mg, 87.

Yield: 7.20%; LC-MS: 531.6 (100), 554.0 (15%), 266.4 (40%), 286.9 (30%).

^{1 H NMR: (d-DMSO} , 400MHz) δ9.41 (s, 1H), 8.99 (s, 1H), 8.87 (s, 1H), 8.29 (d, J = 5.6Hz, 1H), 7.75 (d, J = 13.2 Hz, 1H), 7.53 (dd, J = 8.8, 4.8 Hz, 2H), 7.28-7.24 (m, 1H), 7.17-7.12 (m, 5H), 6.64 (d, J = 8.0 Hz, 2H) ), 6.47 (d, J = 5.6 Hz, 1H), 2.91 (bs, 4H), 2.19 (bs, 4H), 2.06 (s, 3H).

(8) Synthesis of Compound 30

1-{3-Fluoro-4-[(2-{[2-(4-methylpiperazin-1-yl)ethyl]amino}pyrimidin-4-yl)oxo]phenyl}-3-( The synthetic route of 4-fluorophenyl)urea (Compound 30) is as follows:

To a 25 mL single-neck round bottom flask, compound 7D-1 (300.00 mg, 796.31 umol, 1.00 eq) was added, dissolved in 10 mL isopropanol, and compound 1-30 (171.08 mg, 1.19 mmol, 1.50 eq), DIEA (205.83) was added. The mixture was reacted at 85 ° C for 16 hours, and LC-MS monitoring showed that the starting material was completely reacted and the desired product was formed. After completion of the reaction, the solution was evaporated to give a crude material which was purified to afford compound 30 (85.00 mg, 170.52.

Yield: 21.41%, LC-MS: 484.2.

¹ H NMR: (CD ₃ OD, 400 MHz) δ 8.11 (d, J = 5.2 Hz, 1H), 7.62 (d, J = 12.0 Hz, 1H), 7.47-7.44 (m, 2H), 7.16-7. m, 2H), 7.07-7.02 (m, 2H), 6.29 (d, J = 5.2 Hz, 1H), 3.06 (br, 5H), 2.67 (s, 3H), 2.67-2.55 (m, 7H).

^{1 H NMR: (d-DMSO} , 400MHz) δ9.66-9.54 (m, 1H), 9.41-9.36 (m, 1H), 8.15 (s, 1H), 7.62 (d, J = 12.8Hz, 1H), 7.47-7.45 (m, 2H), 7.24-7.20 (m, 1H), 7.14-7.11 (m, 3H), 6.97-6.95 (m, 1H), 6.24 (d, J = 5.2 Hz, 1H), 3.19- 2.57 (br, 12H), 2.51 (bs, 3H).

(9) Synthesis of Compound 31

1-(3-Fluoro-4-((2-(3-(4-methylpiperazin-1-yl)propyl)amino)pyrimidin-4-yl)oxo)phenyl)-3-( The synthetic route of 4-fluorophenyl)urea (Compound 31) is as follows:

To a 25 mL one-neck round bottom flask, compound 7D-1 (300.00 mg, 796.31 umol, 1.00 eq) was added, dissolved in 10 mL isopropanol, and then compound 1-31 (187.84 mg, 1.19 mmol, 1.50 eq) was added, DIEA (205.83 mg, 1.59 mmol, 2.00 eq), the mixture was heated to 85 ° C for 16 hr. After completion of the reaction, the mixture was evaporated to give 650 mg of crude material. After preparative chromatography, compound 31 (115.00 mg, 219.58.

Yield: 25.18%, LC-MS: 498.1 (100%), 249.6 (25%).

¹ H NMR: (CD ₃ OD, 400 MHz) δ 8.11 (d, J = 5.6 Hz, 1H), 7.63 (d, J = 12.4 Hz, 1H), 7.47 - 7.43 (m, 2H), 7.14 - 7.10 ( m, 2H), 7.08-7.04 (m, 2H), 6.26 (d, J = 5.6 Hz, 1H), 3.20 (bs, 2H), 2.87-2.55 (m, 13H), 1.69 (br, 2H).

¹ H NMR: (d-DMSO, 400 MHz) δ 9.18 (bs, 1H), 9.03 (s, 1H), 8.16 (s, 1H), 7.59 (d, J = 12.0 Hz, 1H), 7.47-7.44 ( m, 2H), 7.33 (bs, 1H), 7.22-7.19 (m, 1H), 7.15-7.11 (m, 3H), 6.21 (bs, 1H), 2.94-2.65 (m, 12H), 2.45-2.32 ( m, 3H), 1.63-1.49 (m, 2H).

(10) Synthesis of compound t-3

N-(4-((2-(2,7-diazaspiro[3.5]decan-2-yl)thieno[3,2-b]pyridin-7-yl)oxo)-3-fluoro The synthetic route of phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dimethylamide (compound t-3) is as follows:

Into a 100 mL three-neck round bottom flask, compound 5 (349.90 mg, 906.06 μmol, 1.00 eq) was added, dissolved in 20 mL of THF, and the compound Pd ₂ (dba) ₃ (82.97 mg, 90.61 μmol, 0.10 eq) was added under the protection of nitrogen, RuPhos (63.42 mg, 135.91 μmol, 0.15 eq), t-BuONa (261.22 mg, 2.72 mmol, 3.00 eq) and Compound 1A-3 (250.00 mg, 951.37 μmol, 1.05 eq). The reaction mixture was heated to 75 ° C for 12 hours under the protection of nitrogen. The LC-MS monitoring showed that the starting material was completely reacted and the target product was formed. After completion of the reaction, the solution was filtered, and the filtrate was evaporated to give a crude product (2 g). Purification by column chromatography gave Compound 1-3 (300.00 mg, 347.38.

To a 100 mL one-neck round bottom flask, compound 1-3 (300.00 mg, 619.08 umol, 1.00 eq) was added, 15 mL of THF was dissolved, and the compound triethylamine (375.87 mg, 3.71 mmol, 514.89 uL, 6.00 eq) was added at 0 ° C. The reaction was stirred for 0.5 hours, then compound 6A (0.3M, 5.17 mL of THF, 2.50 eq) was slowly added, and the mixture was stirred at 20 ° C for 17 hours, and LC-MS monitoring showed that the starting material was completely and the product was formed. After the reaction was completed, 50 mL of ethyl acetate was added, and the organic layer was washed three times with saturated sodium bicarbonate, 15 mL each time, and the mixture was washed three times with a saturated ammonium chloride solution, 15 mL each time, and washed with a saturated sodium chloride solution three times, each time 15 mL. The organic layer was dried over anhydrous sodium sulfate and evaporated. LC-MS: 690.0.

To a 50 mL one-neck round bottom flask, compound 2-3 (260.00 mg, 286.47 umol, 1.00 eq) was added, and 8 mL of dichloromethane was dissolved. The compound trifluoroacetic acid (132.39 mg, 1.16 mmol, 85.97 uL, 3.00 Eq), the resulting mixture was stirred at 20 ° C for 3 hours, TLC (EA / MeOH) monitoring showed that the starting material was completely converted, the resulting solution was washed twice with water, 5 mL each time, once with saturated sodium chloride solution, 5 mL, organic layer anhydrous sulfuric acid The sodium was dried, filtered, and evaporated to give a crystallite.

Yield: N/A, LC-MS: 590.3 (100%), 295.6 (80%).

^{1 H NMR (400MHz, DMSO-} d 6) δ10.36 (bs, 1H), 9.99 (bs, 1H), 8.19 (d, J = 5.2Hz, 1H), 7.86 (d, J = 10.6Hz, 1H) , 7.61 (t, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 1H), 7.37 (d, J = 8.8 Hz, 1H), 7.13 (t, J = 8.4 Hz, 2H), 6.25 (d, J = 5.6 Hz, 1H), 6.07 (s, 1H), 3.71 (bs, 4H), 2.63 (bs, 4H), 1.66 (bs, 4H), 1.45 (bs, 4H).

(11) Synthesis of compound t-11

N-(4-((2-(8-oxa-2-azaspiro[4.5]decan-2-yl)thieno[3,2-b]pyridin-7-yl)oxo)-3 The synthetic route of -fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dimethylamide (compound t-11) is as follows:

Into a 100 mL three-neck round bottom flask, compound 5 (651.14 mg, 1.69 mmol, 1.00 eq) was added, dissolved in 20 mL of THF, and the compound Pd ₂ (dba) ₃ (154.76 mg, 169.00 μmol, 0.10 eq) was added under the protection of nitrogen, RuPhos ( 118.29 mg, 253.50 μmol, 0.15 eq), t-BuONa (487.23 mg, 5.07 mmol, 3.00 eq) and Compound 1A-11 (250.58 mg, 1.77 mmol, 1.05 eq). The resulting mixture was stirred at 75 ° C for 12 hours under nitrogen. The LC-MS monitoring showed that the starting material was completely reacted and the target product was formed. After completion of the reaction, the obtained solution was filtered, and the filtrate was evaporated to dryness to give crystals of the crude product (2 g) and purified by column chromatography to give compound 1-11 (420.00 mg, 655.06 μmol, 62.31% purity). Yield: 38.76%, LC-MS: 400.0.

To a 100 mL one-neck round bottom flask, compound 1-11 (335.56 mg, 840.00 umol, 0.40 eq) was added, 15 mL of dry THF was dissolved, and triethylamine (531.25 mg, 5.25 mmol, 727.74) was added dropwise at 0-5 °C. uL, 2.50 eq) and Compound 6A (507.47 mg, 2.10 mmol, 1.00 eq) (7 mL, 0.3 mmol/mL in THF), and the mixture was stirred at 25 ° C for 16 hours, TLC (EA/MeOH = 10/1) The monitoring showed that the reaction material was completely converted, and 60 mL of ethyl acetate was added under stirring, and the organic layer was washed successively with saturated sodium hydrogen carbonate solution (15 mL×3), saturated ammonium chloride solution (15 mL) and sodium chloride solution (15 mL×2). The organic layer was dried over anhydrous sodium sulfate, filtered, and then evaporated to give the crude product (yield: 0.7 g,yield of white solid product 11 (40.00 mg, 65.32 μmol, 98.75% purity).

Yield: 3.11%, LC-MS: 605.1.

^{1 H NMR: (400MHz, DMSO} -d 6) 10.35 (s, 1H), 9.99 (s, 1H), 8.17 (d, J = 5.6Hz, 1H), 7.86 (d, J = 13.2Hz, 1H), 7.64 (dd, J = 8.8, 5.6 Hz, 2H), 7.46 (d, J = 8.8 Hz, 1H), 7.34 (d, J = 8.8 Hz, 1H), 7.13 (t, J = 8.8 Hz, 2H), 6.19 (d, J = 5.6 Hz, 1H), 6.03 (s, 1H), 3.64 - 3.58 (m, 4H), 3.44 - 3.33 (m, 2H), 3.33 - 3.28 (m, 2H), 1.98 - 1.94 ( m, 2H), 1.56 (bs, 4H), 1.45 (bs, 4H).

(12) Synthesis of compound td32-1

N-(4-((2-(4-(1H-pyrazol-1-yl)piperidin-1-yl)thieno[3,2-b]pyridin-7-yl)oxo)-3- The synthetic route of fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dimethylamide (compound td32-1) is as follows:

To a 100 mL one-neck round bottom flask, compound 5 (1.40 g, 3.63 mmol, 1.00 eq), compound 4-1 (1.64 g, 10.85 mmol, 2.99 eq), CuI (138.09 mg, 725.05 umol, 0.20 eq), -proline (166.95 mg, 1.45 mmol, 0.40 eq) and K ₂ CO ₃ (1.50 g, 10.88 mmol, 3.00 eq), 30 mL of DMSO as a reaction solvent, and the mixture was stirred at 100 ° C for 14 hours under nitrogen atmosphere, LC-MS monitoring showed The raw material is completely reacted and the target product is formed. After completion of the reaction, 50 mL of purified water was added, and ethyl acetate was extracted three times (30 mL each time), and the organic layer was evaporated to give a crude product which was purified to give compound 5-1 (450.00 mg, 1.10 mmol), yield 30.27%, LC -MS: 410.1.

^{1 H NMR: (400MHz, CDCl} 3) δ8.19 (d, J = 5.6Hz, 1H), 7.47 (d, J = 1.6Hz, 1H), 7.38 (d, J = 2.0Hz, 1H), 6.91 ( t, J = 8.8 Hz, 1H), 6.47 (d, J = 2.4 Hz, 1H), 6.43 (d, J = 8.8 Hz, 1H), 6.32 (s, 1H), 6.22 (dd, J = 4.0, 1.6 Hz, 2H), 4.34 - 4.27 (m, 1H), 3.81-3.75 (m, 4H), 3.16-3.09 (m, 2H), 2.232.11 (m, 4H).

To a 50 mL one-neck round bottom flask, compound 5-1 (400.00 mg, 976.85 umol, 1.00 eq), compound 6 (348.85 mg, 1.56 mmol, 1.60 eq), DIEA (378.74 mg, 2.93 mmol, 51.81 uL, 3.00 eq. And HATU (408.57 mg, 1.07 mmol, 1.10 eq), 6 mL of DMF as a reaction solvent, and the resulting mixture was stirred and reacted at 50 ° C for 3 hours, and LC-MS monitoring showed that the starting material was completely reacted and the target product was formed. After completion of the reaction, 20 mL of purified water was added, and ethyl acetate was extracted three times (20 mL each time), and the organic layer was evaporated to give a crude product which was purified to afford product td32-1 (60.00 mg, 96.64 μmol, 99% purity).

Yield: 9.89%, LC-MS: 615.3.

^{1 H NMR: (400MHz, DMSO} -d 6) δ10.38 (s, 1H), 1.01 (s, 1H), 8.25 (d, J = 6.0Hz, 1H), 7.82 (d, J = 2.0Hz, 1H ), 7.80 (s, 1H), 7.66-7.62 (m, 2H), 7.47-7.35 (m, 2H), 7.45-7.38 (m, 1H), 7.15 (t, J = 8.8 Hz, 2H), 6.50 ( s, 1H), 6.30 (d, J = 5.6 Hz, 1H), 6.25 (t, J = 2.0 Hz, 1H), 4.52-4.44 (m, 1H), 3.86-3.82 (m, 2H), 3.26-3.20 (m, 2H), 2.10-2.04 (m, 4H), 1.47-1.46 (m, 4H).

(13) Synthesis of compound t-27

N-(3-Fluoro-4-((2-morpholinothiazolo[4,5-d]pyrimidin-7-yl)amino)phenyl)-N-(4-fluorophenyl)cyclopropane-1 , 1-diformyl

The synthetic route of the amine (compound t-27) is as follows:

To a 25 mL one-neck round bottom flask, compound 8 (600.00 mg, 2.34 mmol, 1.00 eq) was added, dissolved in 2 mL isopropanol, and then compound 14 (634.57 mg, 2.80 mmol, 1.20 eq) and TsOH (80.50 mg, 467.45 μmol, 0.20 eq), the mixture was heated to 85 ° C for 16 hours. LC-MS monitoring showed complete reaction of the starting material and formation of the desired product. After the completion of the reaction, the solvent was evaporated to dryness, and the obtained solid was added to 15 mL of methylene chloride, and the mixture was washed twice with saturated sodium hydrogencarbonate (10 mL), dried over anhydrous sodium sulfate and filtered to give a crude product. 620 mg), purity 81%, LC-MS: 447.1.

To a 100 mL one-neck round bottom flask was added compound 9 (620.00 mg, 1.39 mmol, 1.00 eq), dissolved in 20 mL dichloromethane, then trifluoroacetic acid (7.70 g, 67.53 mmol, 5.00 mL, 48.58 eq). After reacting at 25 ° C for 1 hour, LC-MS monitoring showed that the starting material was completely reacted and the target product was formed. After the end of the reaction, the resulting solution was evaporated to give a crude product 10 which was used in the next step without purification. LC-MS: 346.9.

To a 25 mL one-neck round bottom flask was added compound 10 (950.00 mg, 2.06 mmol, 1.00 eq, TFA salt) and compound 11 (367.83 mg, 1.65 mmol, 0.80 eq), dissolved in 5 mL DMF, then HATU (1.17 g, 3.09 mmol, 1.50 eq) and DIPEA (1.60 g, 12.36 mmol, 2.16 mL, 6.00 eq), and the mixture was stirred and reacted at 25 ° C for 14 hours. LC-MS monitoring showed that the starting material was completely and the desired product was formed. After the reaction was completed, 20 mL of purified water was added, and ethyl acetate was extracted three times (15 mL each time), and the organic layer was washed twice with saturated sodium chloride (20 mL each time), dried over anhydrous sodium sulfate, filtered and evaporated. The product was purified to give 60 mg.

Yield: 5.17%, LC-MS: 552.1 (100%), 574.1 (15%).

^{1 H NMR: (400MHz, DMSO} -d 6) 10.30 (s, 1H), 10.01 (s, 1H), 9.22 (s, 1H), 8.27 (s, 1H), 7.70 (d, J = 19.2Hz, 1H ), 7.65-7.62 (m, 2H), 7.38-7.36 (m, 1H), 7.15 (t, J = 8.8 Hz, 2H), 3.73 (t, J = 4.8 Hz, 4H), 3.59 (d, J = 4.8 Hz, 4H), 1.48 (d, J = 2.4 Hz, 4H).

Example 2 [Screening of inhibitory activity of compounds against protein kinase]

Representative compounds (as shown in Table 2) were tested for inhibitory activity against the following kinases using the methods of Mobility Shift Assay and Lanthascreen Assay: VEGFR2, c-Met, c-kit, B-Raf, EGFR, RET.

Table 1 Purchasing Information for Kinases

Kinase name	source	Product number	lot number
BRAF	Invitrogen	PR 6995A	1258788L
CKIT	Millipore	14-559k	2060980
EGFR	Carna	08-115	13CBS-0005L
VEGFR2	Carna	08-191	07CBS-0540
CMET	Carna	08-151	10CBS-1118K
RET	Carna	08-159	06CBS-3284

The experimental method is as follows:

1) Prepare 1x kinase buffer and stop solution

1X kinase buffer: 50mM HEPES, pH 7.5, 0.0015% Brij-35, 2mM DTT;

Stop solution: 100 mM HEPES, pH 7.5, 0.015% Brij-35.

2) Compound preparation

A 50-fold compound was prepared: the final concentration of the compound was 2 μM, and the concentration was 50-fold, that is, 100 μM. 100 μL of 50-fold compound was added to the second well of a 96-well plate, and the other wells were added to 60 μL of 100% DMSO. 30 μL of the compound was taken from the second well and added to the third well, and 10 dilutions were sequentially performed, and a total of 10 concentrations were diluted.

Transfer 5 times of the compound to the reaction plate: 10 μL from each well of the above 96-well plate to another 96-well plate, and add 90 μL of 1X kinase buffer. 5 μL of the above 96-well plate was taken out to a 384-well reaction plate. Thus, in a 384-well reaction plate, there are 5 [mu]L of 10% DMSO dissolved 5 fold compound and 5 [mu]L of 10% DMSO. 5 μL of kinase buffer was added to the negative control wells.

3) Kinase reaction

Formulate 2.5 times the enzyme solution: Add the kinase to the 1X kinase buffer to form a 2.5-fold enzyme solution.

A 2.5-fold substrate solution was prepared: FAM-labeled polypeptide and ATP were added to 1X kinase buffer to form a 2.5-fold substrate solution.

The enzyme solution was added to the 384-well plate: 5 μL of 10% DMSO-dissolved 5-fold compound was present in the 384-well reaction plate. 10 μL of the 2.5-fold enzyme solution was added to the 384-well reaction plate, and the mixture was incubated at room temperature for 10 minutes. The substrate solution was added to the 384-well plate, and 10 μL of a 2.5-fold substrate solution was added to the 384-well reaction plate.

Kinase reaction and termination: reaction at 28 ° C; addition of 25 μL of stop solution to stop the reaction.

4) Read conversion rate data on Caliper

The conversion rate data was replicated from Caliper; the conversion rate was converted to inhibition rate data, where max is the conversion of the DMSO control and min is the conversion of the enzyme-free control.

Percent inhibition = (max-conversion) / (max-min) * 100.

The data was imported into MS Excel and curve fitted using XLFit excel add-in version 5.4.0.8.

Table 2: Inhibitory activity of compounds on related kinases

^* refers to the inhibition rate at 100 nM concentration

ND means not tested; NA means inhibition rate <10% at 100 nM.

It can be seen from Table 2 that the two compounds td32-4 and td32-5 have stronger inhibitory ability to VEGFR-2 and c-kit than the positive controls sorafenib and cabozantinib, and the inhibition ability against c-met is comparable to that of cabozantinib, t-3 The inhibitory effect on VEGFR-2 is stronger than that of sorafenib, which is slightly weaker than cabozantinib. The inhibitory ability against c-met is comparable to that of cabozantinib, and the inhibition ability of c-kit is stronger than that of sorafenib and cabozantinib. All three of the above compounds were inactive against Raf. Compound 51 has a stronger inhibitory effect on B-Raf than sorafenib and almost no activity on c-kit and RET. Representative compounds embody certain kinase inhibition selectivity.

Example 3 [Test for inhibiting tumor cell proliferation activity in vitro]

According to the results of the protein kinase inhibitory activity in Example 2, the selection effect was superior to or similar to that of the control products Sorafenib and Cabozantinib, that is, the selection compounds td32-4, td32-5, td32-6, t-3, 51, 29 were used. Inhibition of cell proliferation activity test.

Instruments and materials:

Cell Titer-Glo luminescent cell viability assay (Promega, Cat. No. G7573, Lot. No. 0000181739).

TT (ATCC, Cat. No. CRL-1803, Lot. No. 58785858)

SNU-5 (ATCC, Cat. No. CRL-5973, Lot. No. 58033358)

Hs746T (ATCC, Cat. No. HTB-135, Lot. No. 5006453)

U87MG (ATCC, Cat. No. HTB-14, Lot. No. 5018014)

HepG2 (ATCC, Cat. No. HB-8065, Lot. No. 7579337)

A673 (ATCC, Cat. No. CRL-1598, Lot. No. 58075870)

F-12K medium (Invitrogen, Cat. No. 21127-022, Lot. No. 1759876)

MEM medium (Invitrogen, Cat. No. 11095-098, Lot. No. 1798295)

IMDM medium (Invitrogen, Cat. No. 12440-061, Lot. No. 1806052)

DMEM medium (Invitrogen, Cat. No. 12430-062, Lot. No. 1810223)

MEM Non-Essential Amino Acids Solution, 100×(Invitrogen, Cat. No. 11140-050, Lot. No. 1806220)

FBS (Invitrogen, Cat. No. 10099141, Lot. No. 1652792)

DMSO (Sigma, Cat. No.276855-1L, Lot. No. STBD7938V)

experimental method:

Cell plating

1) Prepare complete medium and mix well.

2) Resuscitate the cells and transfer the cell lines with good growth conditions for about two generations.

3) Remove the cell culture flask from the incubator and check the cell name and medium type labeled on the culture flask.

4) Adherent cells: aspirate the medium, wash it with trypsin, discard the waste, and add 3 ml of fresh trypsin to the culture flask for digestion. When the cells are loose to leave the bottle wall, add 8 ml of complete medium to stop trypsin digestion and mix gently. The cell suspension was pipetted into a centrifuge tube and centrifuged at 800-1000 rpm for 3-5 minutes. Suspension of cells: Aspirate the cell suspension and transfer to a centrifuge tube and centrifuge at 800-1000 rpm for 3-5 minutes.

5) Discard the supernatant.

6) Add an appropriate volume of medium to the centrifuge tube and gently blister to resuspend the cells.

7) Count using a Vi-Cell XR cytometer.

8) Adjust the cell suspension to the appropriate concentration.

9) Add the cell suspension to a 96-well plate at 100 μL/well. Mark the cell name, plate density, date and other details and place the plate in a CO2 incubator overnight.

2. Preparation and addition of compound plates:

1) Test compound:

A solution of 20 mM or 10 mM was prepared in DMSO.

The test compound was diluted to 2 mM in DMSO, added to the compound plate, and subjected to a 3-fold gradient dilution with DMSO.

2) 0.4 mM staurosporin was prepared in DMSO, added to the compound plate, and diluted 3-fold in DMSO.

3) Compound addition: 0.5 μL of the compound plate to be tested and the compound in the Staurosporine compound plate were pipetted and added to the corresponding cell wells.

4) Incubate for 72 hours in a carbon dioxide incubator.

3. Reagent preparation and testing

1) Melt the CellTiter-Glo Buffer at room temperature. The lyophilized CellTiter Glo substrate was equilibrated to room temperature.

2) Add CellTiter-Glo Buffer to the CellTiter Glo substrate and mix well.

3) The cell plates were taken out to equilibration to room temperature.

4) Add 100 μl of the mixed CellTiter Glo reagent to each well, shake for 2 min in the dark, and incubate for 10 min.

5) Put the plate into the EnSpire reading plate and record the luminescence reading results;

Calculate the inhibition rate according to the following formula:

Inhibition rate (%) = (1-(RLU compound-RLU blank) / (RLU DMSO-RLU blank)) × 100%.

6) Use XLFit to plot the pharmacodynamic inhibition rate curve and calculate the IC ₅₀ value. The results of some data are shown in Table 3.

In vitro proliferation inhibitory activity of the compounds of Table 3 on some cell lines

ND: means not tested

As can be seen from Table 3, in the cell line expanded by c-met gene, the compounds t-3, td32-4, td32-5 and td32-6, which can significantly inhibit the activity of c-met protein kinase, have very strong cells. Proliferation inhibitory activity, while most of the cell lines amplified by non-c-met gene have moderate inhibitory activity, while compound 51 selectively inhibits HepG2 and A673 cell lines. The compound showed very good cell selective inhibition.

Example 4 [Evaluation of Compound Pharmacokinetics]

Single intravenous (IV) and oral (PO) administration of Sprague Dawley rats, blood samples were taken at different time points, and LC/MS/MS was used to determine the concentration of each substance in the plasma of rats after administration of the test substance and calculate relevant parameters. .

1) The time point for animal blood collection is:

Intravenous: 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after administration.

Oral: before administration, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after administration.

Each animal took about 0.2 mL of blood per jugular vein, and heparin sodium was anticoagulated. Blood samples were collected and placed on ice, and plasma was separated by centrifugation (centrifugation conditions: 8000 rpm, 6 minutes, 4 ° C). The collected plasma was stored at -70 °C prior to analysis.

2) Analytical instruments:

Liquid Chromatograph (Shimadzu LC), including solution transfer pump (LC-20AD), online degassing instrument (DGU-20A3), autosampler (SIL-20AHT), controller (CBM-20A), column Thermostat (CTO-20A).

Mass spectrometer (API 4000, Applied Biosystems, USA), electrospray ionization source (ESI), tandem quadrupole mass analyzer.

The data processing system is Analyst Software (Applied Biosystems, Inc., software version number 1.5.1).

3) Sample preparation

Take 50 μL of plasma sample into a 1.5 mL centrifuge tube, add 250 μL of the internal standard solution (with the addition of the same volume of methanol in the blank), vortex and mix, centrifuge at 14,000 rpm for 5 minutes, and add 200 μL of the supernatant to the solution. LC-MS/MS injection analysis in a 96-well sample plate.

The pharmacokinetic parameters of Compound 51 were determined as follows:

Table 4 pharmacokinetic parameters of the compounds

As can be seen from Table 4, Compound 51 has good pharmacokinetic parameters and the absolute bioavailability is about 79%. Similarly, the pharmacokinetic parameters of the compounds td32-4 and td32-5 were tested, respectively, and the absolute bioavailability was determined. It is 42% and 48%. It is apparent that representative compounds have better pharmacokinetic properties.

Claims (10)

1. A compound having multi-target protein kinase inhibitory activity, characterized in that the compound has the structural formula of formula (I):

   

   Wherein R ¹ is H, or a halogen atom in the ortho, meta or para position, an acyclic alkyl group, a C3-C6 unsubstituted or substituted heterocyclic ring, a C3-C7 cycloalkyl group;

   R ² is H, or a halogen atom or an acyclic alkyl group in the ortho or meta or para position of the linker;

   M ¹ is O or NH in the linker alignment or meta position;

   M ² is one selected from the group consisting of formula (II), (III) and (IV):

   

   Wherein, in the formula (II), R ³ is a spiro ring, or an unsubstituted or substituted C3-C6 heterocyclic ring; X ¹ is CH or N; Y ¹ is CH or N; Z ¹ is CH, S, NH, or O; Z ² is N, CH or NH;

   In the formula (III), X ² is CH or N; Z ³ is N or S; Z ⁴ is N or S; R ⁴ is an unsubstituted or substituted C3-C6 heterocyclic ring, or a group of the formula (V)

   

   In formula (V),

   X ⁴ is NH, S or O; M ³ is S or O; R ⁶ is a C3-C7 substituted or unsubstituted cycloalkane, or a substituted or unsubstituted C3-C6 heterocyclic ring;

   In the formula (IV), X ³ is CH or N; Y ² is CH or N; and R ⁵ is an unsubstituted or substituted C3-C6 heterocyclic ring;

   Linker is selected from one of formula (VI) and formula (VII):

   
2. The compound according to claim 1, wherein R ^{3 is} selected from one of the following structures,

   

   Or one selected from the following structures,

   

   Among them, the general formula

   

   Medium, m≥0 and m is an integer;

   

   Where p ≥ 0 and p is an integer;

   General formula

   

   Where q ≥ 0 and q is an integer; W is CH or N.
3. The compound according to claim 1, wherein R ⁵ is one selected from the group consisting of:

   

   Where k = 1, 2 or 3.
4. The compound of claim 1 wherein R ⁶ is one selected from the group consisting of:

   

   In the formula (VIII), n is a positive integer of 1 to 5; and in the formula (IX), X ⁴ to X ^{8 are} independently selected from CH ₂ , NH, O or S.
5. The compound of claim 1 wherein said acyclic alkyl group is selected from one of the group consisting of:

   
6. The compound according to claim 1, wherein the C3-C6 heterocyclic ring described in R ³ , R ⁴ , R ⁵ and R ⁶ is selected from one of the following functional groups:

   
7. The compound of claim 1 wherein said compound is selected from one of the following structures:

   1) N-(3-Fluoro-4-((2-(1-(2-hydroxyethyl))-1H-pyrazol-4-yl)thieno[3,2-b]pyridin-7-yl) Oxo)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dimethylamide

   Its structural formula is:

   

   2) N-(3-Fluoro-4-((2-(pyrrolidin-1-carbonyl)thieno[3,2-b]pyridin-7-yl)oxo)phenyl)-N-(4- Fluorophenyl)cyclopropane-1,1-dimethylformamide

   Its structural formula is:

   

   3) N-(4-((2-(cyclopropanecarbamoyl) <ethylenediamino])thieno[3,2-b]pyridin-7-yl)oxo)-3-fluorophenyl )-N-(4-fluorophenyl)cyclopropane-1,1-dimethylamide

   Its structural formula is:

   

   4) N-(3-((2-(cyclopropanecarbamoyl) <oxalylamino))thiazolo[5,4-b]pyridin-5-yl)amino)-4-fluorophenyl) -N-(4-fluorophenyl)cyclopropane-1,1-dimethylformamide

   Its structural formula is:

   

   5) N-(4-Fluoro-3-((6-(pyridin-3-yl)pyrimidin-4-yl)oxo)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1 -Dimethylamide has the structural formula:

   

   6) N-(4-Fluoro-3-((6-(pyridin-3-yl)pyrimidin-4-yl)amino)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1- The structure of dimethylformamide is:

   

   7) 1-(3-Fluoro-4-((2-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-3 -(4-Fluorophenyl)urea has the structural formula:

   

   8) 1-{3-Fluoro-4-[(2-{[2-(4-methylpiperazin-1-yl)ethyl]amino}pyrimidin-4-yl)oxo]phenyl}-3 -(4-Fluorophenyl)urea has the structural formula:

   

   9) 1-(3-Fluoro-4-((2-((3-(4-methylpiperazin-1-yl)propyl)amino)pyrimidin-4-yl)oxy)phenyl)-3 -(4-Fluorophenyl)urea has the structural formula:

   

   10) N-(4-((2-(2,7-diazaspiro[3.5]decan-2-yl)thieno[3,2-b]pyridin-7-yl)oxo)-3 -fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dimethylformamide

   Its structural formula is:

   

   11) N-(4-((2-(8-oxa-2-azaspiro[4.5]decane-2-yl)thieno[3,2-b]pyridin-7-yl)oxo) 3-fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dimethylformamide

   Its structural formula is:

   

   12) N-(4-((2-(4-(1H-pyrazol-1-yl)piperidin-1-yl)thieno[3,2-b]pyridin-7-yl)oxo)- 3-fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dimethylamide

   Its structural formula is:

   

   13) N-(3-Fluoro-4-((2-morpholinothiazolo[4,5-d]pyrimidin-7-yl)amino)phenyl)-N-(4-fluorophenyl)cyclopropane -1,1-dimethylformamide

   Its structural formula is:

   
8. The compound of claim 1 wherein said compound is selected from one of the following structures:

   (1) N-(3-Fluoro-4-((2-(1-(2-hydroxyethyl))-1H-pyrazol-4-yl)thieno[3,2-b]pyridine-7-yl Oxo)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dimethylamide

   Its structural formula is:

   

   (2) N-(3-Fluoro-4-((2-(pyrrolidin-1-carbonyl)thieno[3,2-b]pyridin-7-yl)oxo)phenyl)-N-(4) -fluorophenyl)cyclopropane-1,1-dimethylformamide

   Its structural formula is:

   

   (3) N-(4-((2-(cyclopropanecarbamoyl) <oxalylamino>)thieno[3,2-b]pyridin-7-yl)oxo)-3-fluorobenzene -N-(4-fluorophenyl)cyclopropane-1,1-dimethylformamide

   Its structural formula is:

   

   (4) N-(4-((2-(2,7-diazaspiro[3.5]decan-2-yl)thieno[3,2-b]pyridin-7-yl)oxo)- 3-fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dimethylamide

   Its structural formula is:

   

   (5) N-(3-((2-(cyclopropanecarbamoyl) <oxalylamino))thiazolo[5,4-b]pyridin-5-yl)amino)-4-fluorophenyl )-N-(4-fluorophenyl)cyclopropane-1,1-dimethylamide

   Its structural formula is:

   

   (6) 1-(3-Fluoro-4-((2-(4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)- 3-(4-fluorophenyl)urea has the structural formula:

   
9. A pharmaceutical composition comprising the compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof.
10. Use of a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a disease caused by abnormal protein kinase activity.