Kinase inhibitor activated in tumor microenvironment, composition and use

Abstract

A kinase inhibitor activated in a tumor microenvironment, a composition and a use. The kinase inhibitor activated in a tumor microenvironment has a structure shown as R-A-C-D. The present invention also relates to a pharmaceutical composition based on the kinase inhibitor and a use of the kinase inhibitor in preparing a drug for treatment of cancer. The provided kinase inhibitor activated in a tumor microenvironment has good water solubility, the toxic side effects of the kinase inhibitor on normal cells are reduced, and the present invention has great significance for cancer treatment.

Description

[Corrected 30.12.2024 according to Rule 26] Kinase inhibitors, compositions and applications based on tumor microenvironment activation Technical Field

[0001] The present invention relates to the field of biopharmaceutical synthesis technology, and in particular to a kinase inhibitor based on tumor microenvironment activation, a composition and an application thereof. Background Art

[0002] Kinases play a crucial role in regulating a wide range of cellular processes, paving the way for the development of kinase inhibitors as therapeutic agents for a wide range of conditions, including cancer. Cancer treatment is challenging because it is difficult to kill cancer cells while minimizing or eliminating normal cells. Killing or otherwise adversely affecting normal cells during cancer treatment can cause adverse side effects in patients. Therefore, improving drug targeting, minimizing drug toxicity, and avoiding effects on normal cells are key challenges in cancer drug development.

[0003] Protein kinase inhibitors are enzyme inhibitors that block the action of protein kinases. Protein kinases add a phosphate group to proteins in a process called phosphorylation, which can turn the protein on or off, affecting its activity and functional level. Protein kinase inhibitors are able to cross the blood-brain barrier (BBB) ​​and are used to treat brain metastases and / or leptomeningeal disease; chemotherapy drugs rarely cross the BBB, and the long-term side effects of radiation therapy can be devastating.

[0004] Furthermore, water solubility is a crucial physicochemical property of small organic molecule drugs and a key issue in their development. Good water solubility contributes to enhanced efficacy and pharmacokinetic properties. In medicinal chemistry, improving drug water solubility through chemical structure modification is a fundamental approach to addressing the fundamental nature of solubility.

[0005] Therefore, in the art, providing a kinase inhibitor with good water solubility and low toxic side effects is of great significance for improving the therapeutic effect of cancer. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a kinase inhibitor based on tumor microenvironment activation, wherein the kinase inhibitor is a compound having a structure represented by the following formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: RACD (I)

[0007] Where,

[0008] R is a modifying group used to improve the physicochemical properties of the drug;

[0009] A is an amino acid linker that can be cleaved by proteolytic enzymes;

[0010] C is a sterically hindered group or a self-removable group; and

[0011] D is the drug molecule.

[0012] Specifically, the R is a modifying group with an MI structure, such as the structure shown in the following formula (II):

[0013] Where,

[0014] MI is a structure with a conjugated system and can add an acceptor;

[0015] Rc is a structure that increases water solubility, compound rigidity, or expands spatial distance;

[0016] n1 is a positive integer between 1 and 12, i.e., n1 = any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; n2 is a positive integer between 1 and 12, i.e., n2 = any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12;

[0017] X1 is a trifunctional group;

[0018] The wavy line indicates where R and A are connected.

[0019] Specifically, the MI is selected from

[0020] wherein R1, R2 and R3 are CH or N;

[0021] or wherein the cyclic structure in the structure is a five-membered ring including any one of N, O or S, and R4, R5, R6 and R7 are one of CH, N, O or S;

[0022] or Among them, R8, R9, R 10 、R 11 、R 12 、R 13 、R 14 and R 15 is C, CH or N; Ra and Rb are

[0023] or The ring structure in the structure is a five-membered ring including any one of N, O or S, and R 16 、R 17 and R 18 is one of C, CH, N, O or S; R 19 、R 20 、R 21and R 22 is C, CH or N; Re and Rf are

[0024] Specifically, Rc is selected from C 1-12 Alkyl, C 1-12 Oxyalkyl-C 1-12 Alkyl, C 1-12 Alkyl-C 3-8 Cycloalkyl, (C 1-4 Alkyl-O)pC 1-12 Alkyl, phenyl-C 1-12 Alkyl, C 3-8 Cycloalkyl, C 1-12 Alkyl-C 3-8 Cycloalkyl-C 1-12 Alkyl, C 1-12 Alkyl-phenyl-C 1-12 Alkyl or C 1-12 Alkylcarbonylamino-(C 1-4 Alkyl-O)qC 1-12 alkyl;

[0025] Wherein, p is a positive integer from 1 to 12, i.e., p=any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; q is a positive integer from 1 to 12, i.e., q=any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; and phenyl is one or two substituted phenyl groups, and the substituent in the substituted phenyl group is a halogen atom.

[0026] Specifically, X1 is selected from lysine Lys or glutamic acid Glu;

[0027] When X1 is lysine, its structure is The carboxyl end of X1 is connected to A, and the two amino ends of X1 are connected to the side chain containing n1 or the side chain containing n2 in a one-to-one correspondence, that is, the side chain containing n1 can be connected to the amino group of the lysine main chain or the amino group of the lysine side chain; the side chain containing n2 can be connected to the amino group of the lysine main chain or the amino group of the lysine side chain;

[0028] When X1 is glutamic acid Glu, its structure is Among them, the amino end is connected to the side chain containing n1 or the side chain containing n2; the carboxyl end adjacent to the amino group on glutamic acid is connected to A, and the carboxyl end away from the amino group on glutamic acid is connected to the side chain containing n1 or the side chain containing n2.

[0029] Specifically, A is -Ala-Ala-Asn-, wherein the Ala end is connected to the lysine side and the Asn end is connected to C.

[0030] Specifically, C is Among them, R 23 、R 24 and R 25 is CH or N, the amino end of C is connected to Asn, and the carbonyl end of C is connected to D.

[0031] Specifically, the drug molecule D is one of the following chemical formulas:

[0032] The present invention also provides a pharmaceutical composition, which comprises the aforementioned kinase inhibitor based on tumor microenvironment activation and a pharmaceutically acceptable carrier or pharmaceutical composition thereof.

[0033] Pharmaceutically acceptable carriers are generally safe and non-toxic and, in a broad sense, can include any known substance used in the pharmaceutical industry to prepare pharmaceutical compositions, such as fillers, diluents, coagulants, binders, lubricants, glidants, stabilizers, colorants, wetting agents, disintegrants, and the like. Suitable pharmaceutically acceptable carriers include sugars, such as lactose or sucrose, mannitol, or sorbitol; cellulose preparations and / or calcium phosphates, such as tricalcium phosphate or dibasic calcium phosphate; starch pastes, including corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone; silica, talc, stearic acid or its salts, such as magnesium stearate or calcium stearate, and / or polyethylene glycol, among others. When selecting a pharmaceutically acceptable carrier, the primary consideration is the intended route of administration of the pharmaceutical composition.

[0034] The pharmaceutical composition provided by the present invention contains a therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0035] The dosage form of the pharmaceutical composition includes, but is not limited to, tablets, capsules, or injections. The routes of administration of the pharmaceutical composition include, but are not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, intrathecal, intracranial, nasal, or topical administration. The dosage of the drug can be determined based on the patient's age, health and weight, the type of concurrent treatment, and the frequency of treatment.

[0036] The subject of administration of the pharmaceutical composition is a mammal, preferably a human.

[0037] The compounds of formula (I) herein, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, are particularly useful for treating cancer or cancer cell metastasis. The cancers that can be treated with the compounds of formula (I) herein, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, can be determined based on the therapeutic activity of the drug molecule D itself.

[0038] The present invention also provides a use of the aforementioned tumor microenvironment-activated kinase inhibitor in the preparation of a drug for treating cancer, including colorectal cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, breast cancer, gastric cancer, liver cancer, head and neck cancer, prostate cancer and pancreatic cancer.

[0039] In certain aspects, the compound of formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutical composition provided by the present invention can also stimulate the proliferation of T cells and invasion of lesions, inhibit tumor-associated macrophages and / or promote the stimulation of immune responses.

[0040] Therefore, the present invention also provides a method for treating cancer or cancer cell metastasis, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I) disclosed herein, or a pharmaceutically acceptable salt or pharmaceutical composition thereof. The cancer or cancer cell metastasis is as described above.

[0041] In certain aspects, the present invention also provides a method for stimulating the proliferation and invasion of T cells into lesions, inhibiting tumor-associated macrophages and / or promoting the stimulation of an immune response, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I) disclosed herein or a pharmaceutically acceptable salt or pharmaceutical composition thereof.

[0042] The subject is a mammal, preferably a human.

[0043] The present invention also provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer or cancer cell metastasis, or in the preparation of an immunotherapy drug. The cancer or cancer cell metastasis is as described above; the immunotherapy drug can be used to stimulate T cell proliferation and invasion of lesions, inhibit tumor-associated macrophages, and / or promote immune responses.

[0044] The present invention also provides a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in treating cancer or cancer cell metastasis, or for stimulating T cell proliferation and invasion into lesions, inhibiting tumor-associated macrophages and / or promoting immune response.

[0045] The present invention also provides an application of administering the aforementioned compound of formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutical composition, which includes the use of the aforementioned tumor microenvironment-activated kinase inhibitor or a pharmaceutically acceptable salt thereof, or the aforementioned composition or a pharmaceutically acceptable salt thereof and an anti-PD-1 antibody in the preparation of a drug for combined treatment of tumors.

[0046] The present invention also provides a method for treating or preventing cancer, or inhibiting cancer cell proliferation, comprising: administering the aforementioned compound of formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof to a subject.

[0047] Specifically, it also includes: administering to the subject an immune checkpoint inhibitor in combination with the aforementioned compound of formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof.

[0048] Specifically, the immune checkpoint inhibitor is an anti-PD-1 antibody drug.

[0049] Compared with the prior art, the beneficial effects of the present invention include but are not limited to:

[0050] 1. The tumor-targeted kinase inhibitor provided by the present invention has improved water solubility and overall tumor targeting ability through structural modification of the R group. After the R group modification, the kinase inhibitor provided by the present invention releases the D drug molecule after entering the body, which directly enters the tumor, thereby improving the drug's tumor targeting ability and reducing the drug's toxic side effects on normal human cells.

[0051] 2. The tumor microenvironment-activated kinase inhibitors and pharmaceutical compositions provided by the present invention have been verified to be cleaved by the Legumain enzyme and release drug molecules. Compared with clinical drugs such as Frucitinib, they not only have better tumor targeting, but also significantly improve the killing effect on cancer cells and have good anti-tumor efficacy. At the same time, they also significantly improve the water solubility of kinase inhibitors and reduce the toxic side effects of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG1 is a chromatogram of the Legumain enzyme digestion of compound S1 in Example 5;

[0053] FIG2 is a graph showing the survival rate of B16F10 cells in the presence of Fletinib in Example 9;

[0054] FIG3 is a graph showing the survival rate of B16F10 cells in the presence of compound D1 in Example 9;

[0055] FIG4 is a graph showing the survival rate of B16F10 cells in the presence of compound S1 in Example 9;

[0056] FIG5 is a graph showing the survival rate of A549 cells in the presence of Fletinib in Example 10;

[0057] FIG6 is a graph showing the survival rate of A549 cells in the presence of compound D1 in Example 10;

[0058] FIG7 is a graph showing the survival rate of A549 cells in the presence of compound S1 in Example 10;

[0059] FIG8 is a graph showing the effect of the test substance on the growth of mouse transplanted tumor CT-26 in Example 11;

[0060] FIG9 is a graph showing the effect of the test substance on the growth of mouse transplanted tumor MKN45 in Example 12;

[0061] FIG10 is a graph showing the effects of the test substances on the growth of HUH7 transplanted tumors in mice in Example 13;

[0062] FIG11 is a graph showing the tumor volume results of the mouse colorectal cancer CT-26 model in Example 16;

[0063] FIG12 is a graph showing the tumor volume results of the mouse colorectal cancer CT-26 model in Example 17;

[0064] FIG13 is a graph showing the tumor volume results of the human gastric cancer MKN45 model in Example 18. DETAILED DESCRIPTION

[0065] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The materials, instruments and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. The technical means used in the embodiments, unless otherwise specified, are conventional means well known to those skilled in the art.

[0066] The kinase inhibitor based on tumor microenvironment activation provided by the present invention has a compound represented by the following formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof: RACD (I)

[0067] Where,

[0068] R is a modifying group used to improve the physicochemical properties of the drug;

[0069] A is an amino acid linker that can be cleaved by proteolytic enzymes;

[0070] C is a sterically hindered group or a self-removable group; and

[0071] D is the drug molecule.

[0072] Specifically, R is a modifying group with an MI structure, such as the structure shown in the following formula (II):

[0073] Where,

[0074] MI is a structure with a conjugated system and can add an acceptor;

[0075] Rc is a structure that increases water solubility, compound rigidity, or expands spatial distance;

[0076] n1 is a positive integer between 1 and 12, i.e., n1 = any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; n2 is a positive integer between 1 and 12, i.e., n2 = any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12;

[0077] X1 is a trifunctional group;

[0078] The wavy line indicates where R and A are connected.

[0079] Specifically, MI is selected from

[0080] wherein R1, R2 and R3 are CH or N;

[0081] or wherein the cyclic structure in the structure is a five-membered ring including any one of N, O or S, and R4, R5, R6 and R7 are one of CH, N, O or S;

[0082] or Among them, R8, R9, R 10 、R 11 、R 12 、R 13 、R 14 and R 15 is C or CH or N; Ra and Rb are

[0083] or The ring structure in the structure is a five-membered ring including any one of N, O or S, and R 16 、R 17 and R 18 is one of C, CH, N, O, or S; R 19 、R 20 、R 21 and R 22 is C or CH or N; Re and Rf are

[0084] Preferably, the MI structure includes but is not limited to the following structures:

[0085] since

[0086] Specifically, Rc is selected from C 1-12 Alkyl, C 1-12 Oxyalkyl-C 1-12 Alkyl, C 1-12 Alkyl-C 3-8Cycloalkyl, (C 1-4 Alkyl-O)pC 1-12 Alkyl, phenyl-C 1-12 Alkyl, C 3-8 Cycloalkyl, C 1-12 Alkyl-C 3-8 Cycloalkyl-C 1-12 Alkyl, C 1-12 Alkyl-phenyl-C 1-12 Alkyl or C 1-12 Alkylcarbonylamino-(C 1-4 Alkyl-O)qC 1-12 alkyl;

[0087] Wherein, p is a positive integer from 1 to 12, i.e., p=any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; q is a positive integer from 1 to 12, i.e., q=any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; and phenyl is one or two substituted phenyl groups, and the substituent in the substituted phenyl group is a halogen atom.

[0088] Preferably, Rc includes but is not limited to the following structures:

[0089] Specifically, X1 is selected from lysine Lys or glutamic acid Glu;

[0090] When X1 is lysine, its structure is The carboxyl end of X1 is connected to A, and the two amino ends of X1 are connected to the side chain containing n1 or the side chain containing n2 in a one-to-one correspondence, that is, the side chain containing n1 can be connected to the amino group of the lysine main chain or the amino group of the lysine side chain; the side chain containing n2 can be connected to the amino group of the lysine main chain or the amino group of the lysine side chain;

[0091] When X1 is glutamic acid Glu, its structure is Among them, the amino end is connected to the side chain containing n1 or the side chain containing n2; the carboxyl end adjacent to the amino group on glutamic acid is connected to A, and the carboxyl end away from the amino group on glutamic acid is connected to the side chain containing n1 or the side chain containing n2.

[0092] Specifically, A is -Ala-Ala-Asn-, wherein the Ala end is connected to the lysine side and the Asn end is connected to C.

[0093] Specifically, C is Among them, R 23 、R 24 and R 25 is CH or N, the amino end of C is connected to Asn, and the carbonyl end of C is connected to D.

[0094] Preferably, C includes but is not limited to the following structures:

[0095] Specifically, the drug molecule D is one of the following chemical formulas:

[0096] After the above drug molecules enter the human body and are cleaved by legumain, the resulting compound structure is as follows:

[0097] The chemical structures of representative drugs provided by the present invention are shown in Table 1:

[0098] Table 1 Structures of kinase inhibitor compounds based on tumor microenvironment

[0099] The above compounds are only a partial list of compound structures in the present invention. The compound structures involved in the invention content provided by the present invention are all within the protection scope of the present invention and will not be listed again here.

[0100] The present invention also provides comparative compounds as shown in the following table:

[0101] The comparative compound C1 is a compound without an MI group structure and will be compared with the compound provided by the present invention in subsequent experiments.

[0102] Example 1: Synthesis of Compound S1

[0103] 1. Pentaethylene glycol monomethyl ether acetic acid (compound S1-1) (10 g, 32.2 mmol), HBTU (12.2 g, 32.2 mmol), DIEA (12.5 g, 96.6 mmol), and (S)-tert-butyl 2-amino-6-((benzyloxy)carbonyl)amino)hexanoate (11.9 g, 35.4 mmol) were dissolved in DMF (200 mL). The mixture was stirred at room temperature for 2 h. The reaction was confirmed to be complete by TLC. 800 mL of water was added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic phase was washed with water and then with saturated brine, dried over anhydrous sodium sulfate, and the organic phase was spin-dried. The organic phase was separated on a silica gel column (DCM:MeOH = 20:1) to obtain compound S1-2 (18 g, 88.76%) as a colorless oily liquid. LCMS: 629.4 (M+H) +

[0104] 2. Compound S1-2 (18 g, 28.6 mmol) was dissolved in 300 ml of ethyl acetate, and 1.8 g of palladium on carbon was added. The mixture was replaced with hydrogen three times. The mixture was stirred at room temperature overnight, filtered through celite, and washed with ethyl acetate. The organic phase was dried to give S1-3 (14 g, 98.8%) as a colorless oil. This compound was used directly in the next reaction without purification. LCMS: 495.3 (M+H) +

[0105] 3. Compound S1-3 and DIEA (4.38 g, 34 mmol) were dissolved in DMF (200 mL), and 2,5-dioxopyrrolidin-1-yl 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22-hexaoxa-4-azapentacosane-25-oate (17 g, 28.3 mmol) was added. The mixture was stirred at room temperature for 4 h. 800 mL of water was added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic phase was washed with water and then with saturated brine, dried over anhydrous sodium sulfate, and the organic phase was spin-dried. The organic phase was separated on a silica gel column (DCM:MeOH = 10:1) to obtain S1-4 (22 g, 79.2%) as a colorless oily liquid. LCMS: 981.5 (M+H) +

[0106] 4. Compound S1-4 (22 g, 22.4 mmol) was dissolved in 150 mL of dichloromethane, and 50 mL of trifluoroacetic acid was added. The reaction mixture was stirred at room temperature for 2 h. The solvent was dried to give the crude product S1-5 (22 g) as a yellow oily liquid. LCMS: 925.5 (M+H) +

[0107] 5. S1-5 (22 g, 23.8 mmol), HBTU (9 g, 23.8 mmol), DIEA (9.2 g, 71.4 mmol), and H-Ala-Ala-Asn(Trt)-PABC-OH (14.7 g, 23.8 mmol) were dissolved in DMF (250 mL). The mixture was stirred at room temperature for 2 h. The solvent was evaporated using an oil pump and separated on a silica gel column (DCM:MeOH = 10:1) to give S1-6 (15 g, 41.2%) as a yellow solid. LCMS: 1528.4 (M+H) +

[0108] 6. S1-6 (15 g, 9.8 mmol) was dissolved in 95% trifluoroacetic acid in water and stirred at room temperature for 2 h. The solvent was evaporated by oil pump. The mixture was then dissolved in 80 mL of tert-butanol and DIEA (6.3 g, 49 mmol) was added. The mixture was stirred at room temperature overnight. The solvent was evaporated by oil pump. The mixture was separated on a silica gel column (DCM:MeOH = 4:1) to give S1-7 (5.5 g, 43.6%) as a pale yellow solid. LCMS: 1286.4 (M+H) +

[0109] 7. S1-7 (5.5 g, 4.3 mmol) and di(p-nitrobenzene) carbonate (3.9 g, 12.9 mmol) were dissolved in 70 mL of DMF, and DIEA (2.8 g, 21.5 mmol) was added. The mixture was allowed to react at room temperature overnight. Purification on a silica gel column with 15% methanol / dichloromethane afforded S1-8 (3.8 g, 60.9% yield) as a colorless oil. LCMS: 1451.4 (M+H) +

[0110] 8. S1-8 (500 mg, 0.35 mmol) and S1-9 (221 mg, 0.35 mmol) were dissolved in 8 mL of DMF, and DIEA (90 mg, 0.7 mmol) was added. The mixture was reacted at room temperature for 2 hours. Reverse liquid phase preparation and lyophilization gave compound S1 (350 mg, 51.5% yield) as a white solid. LCMS: 1945.1 (M+H) +

[0111] Example 2: Synthesis of Compound S2

[0112] Referring to Example 1, a white solid compound S2 (370 mg, yield 54.4%) was obtained. LCMS: 1945.1 (M+H) +

[0113] The synthesis routes and methods of compounds S3-S23 provided by the present invention are synthesized with reference to Examples 1 and 2. Compounds S3-S8 are products synthesized based on Examples 1 and 2 by adjusting the length of the PEG chain; compounds S9-S17 are products synthesized based on Examples 1 and 2 by selecting different groups for the MI structure; compounds S18-S20 are products produced by substitution of different drug molecules D; S21 is a PABC heteroatom substitution product; S22 and S23 are products produced based on Examples 1 and 2 by selecting the X1 group from glutamic acid; the synthesis methods of compounds S3-S23 are similar to those of Examples 1 and 2 and will not be repeated here.

[0114] Example 3: Synthesis of Compound S18

[0115] Compound S1-8 and niraparib (221 mg, 0.69 mmol) were dissolved in 15 mL of DMF, and DIEA (178 mg, 1.38 mmol) was added. The mixture was allowed to react at room temperature for 2 hours. Reverse liquid phase preparation and lyophilization afforded compound S18 (330 mg, 29.3% yield) as a white solid. LCMS: 1632.8 (M+H) +

[0116] Example 4: Synthesis of Compound S19

[0117] Compound S1-8 (1 g, 0.69 mmol) and rucaparib (223 mg, 0.69 mmol) were dissolved in 15 mL of DMF, and DIEA (178 mg, 1.38 mmol) was added. The mixture was allowed to react at room temperature for 2 hours. Reverse liquid phase preparation and lyophilization afforded compound S19 (300 mg, 26.6% yield) as a pale yellow solid. LCMS: 1635.8 (M+H) +

[0118] Example 5: Legumain cleavage assay of compounds

[0119] 1.1 Solution Preparation

[0120] Mobile phase A (0.1% TFA / H2O): Add 1 mL of TFA to 1000 mL of water, mix well, and ultrasonicate for 10 minutes before use.

[0121] Mobile phase B (ACN): 1000 mL acetonitrile, sonicated for 10 minutes.

[0122] Assay buffer: 50 mM MES, 250 mM sodium chloride, adjusted to pH 5.0.

[0123] Legumain: 1mg / mL

[0124] 1.2 Preparation of sample solution

[0125] Preparation of sample solution: Weigh appropriate amounts of 23 compound samples listed in Table 1 into sample tubes, add DMSO to dissolve the samples to 4 mM, and then add water to dilute the samples to 0.5 mM.

[0126] 1.3 Enzyme digestion test:

[0127] This experiment included a control group and a Legumain enzyme digestion group.

[0128] Add the sample solution and experimental buffer to the sample tube at a volume ratio of 1:3 (90 μL buffer to 30 μL sample solution) and mix thoroughly. HPLC analysis of the remaining sample volume was performed at various time points (0 h, 5 min, 1 h, 2 h, 4 h, 6 h, and 20 h). A 10 μL injection volume was used as a control.

[0129] Sample solution, legumain, and assay buffer were added to a sample tube at a volume ratio of 1:1:2 (30 μL of sample solution, 30 μL of legumain and 60 μL of assay buffer) and mixed thoroughly. The remaining sample was analyzed by HPLC at various time points (0 h, 5 min, 1 h, 2 h, 4 h, 6 h, and 20 h) and compared to the digestion products. A 10 μL injection volume was used.

[0130] 2.1 Enzyme digestion results

[0131] As shown in Figure 1, the enzymatic cleavage chromatogram of sample compound S1 by Legumain is shown. The results show that at 1 hour, compound S1 has been completely cleaved by Legumain enzyme to obtain compound D1. The subsequent HPLC charts at 2 hours, 4 hours, 6 hours, and 20 hours are the same as the result of 1 hour in Figure 1, and are not repeated here. Using the same method, the above enzymatic cleavage test was also performed on the compounds provided by the present invention. The results show that the other 22 compounds provided in Table 1 can be completely cleaved by Legumain under the above conditions within 1 hour, completely releasing the corresponding small molecule drugs. This indicates that the 23 compounds with similar structures to the 23 compounds provided by the present invention can be cleaved by Legumain in vivo and release the corresponding small molecule drugs, thereby targeting tumor cells for treatment.

[0132] The results of this enzymatic digestion experiment showed that the enzymatic digestion efficiency of compounds S1-S23 in Table 1 of the present invention reached 100% within 1 hour. This efficiency was significantly higher than that of compounds disclosed in the prior art (such as compound S1 disclosed in Chinese invention patent CN104262455B, which serves as the control compound S0 of the present invention and has a structure shown below). The enzymatic digestion efficiency of this compound was 80% within 2 hours, which is lower than that of compounds S1-S23 of the present invention.

[0133] Control compound S0

[0134] Example 6: Solubility experiment of compound:

[0135] 5.0 mg of each of the 23 compounds in Table 1 were accurately weighed and dissolved in 0.25 mL of sterile water for injection until the compound was completely dissolved into a clear solution. After standing at room temperature for 8 hours, there was still no change. The solubility of the 23 compounds was calculated and shown in Table 2 below.

[0136] Table 2: Solubility of Compound 1-Compound 23

[0137] Example 7: Compound Stability Test

[0138] As shown in the compound in Table 1, 23 groups of 2.0 mg of compound were accurately weighed, and 0.1 mol / L citric acid buffer (pH was 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.4) was added to dissolve it in 0.5 mmol / L clarified solution. The chemical stability of the sample at different pH values ​​was detected at 0 h, 4 h, and 8 h. After the sample was placed for 30 min under different pH conditions, HPLC and LCMS were used to detect the chemical stability. It was found that the sample was substantially unchanged when the sample was placed under neutral or weakly acidic conditions for 8 h, and when the pH was 7.4 and the sample was placed for 8 h, a small amount of impurities appeared in the sample, which was detected to be a ring-opening product of maleimide. The above stability test results indicate that the compound samples provided by the present invention with structures similar to 23 compounds in Table 1 are very stable under neutral or weakly acidic conditions.

[0139] Example 8: Analysis of the kinase inhibitory activity of compound D1, fretinib and staurosporine using the ADP-Glo ​​method

[0140] 1. Prepare 1× Kinase buffer. 1) Prepare compound concentration gradient: Start with a 10,000 nM concentration of the test compound and dilute 3-fold to 10 concentrations in a single well. Transfer 50 nl of the test compound to the compound well of a 384-well plate using an Echo. Add 50 nl of DMSO to each of the negative and positive control wells.

[0141] 2) Prepare a kinase solution with 1× Kinase buffer to a final concentration of 2x.

[0142] 3) Add 2.5 μL of kinase solution (2 times the final concentration) to the compound wells and positive control wells respectively; add 2.5 μL of 1× Kinase buffer to the negative control wells.

[0143] 4) Centrifuge at 1000 rpm for 30 seconds, shake to mix, and incubate at room temperature for 60 minutes.

[0144] 5) Prepare a mixed solution of ATP and substrate Fluorescein-MAP2K1 at 2 times the final concentration using 1× Kinase buffer.

[0145] 6) Add 2.5 μL of a mixed solution of ATP and substrate at 2 times the final concentration to start the reaction.

[0146] 7) Centrifuge the 384-well plate at 1000 rpm for 30 seconds, shake to mix, and incubate at room temperature for 120 minutes.

[0147] 8) Add 5 μL ADP-Glo ​​Reagent, centrifuge at 1000 rpm for 30 seconds, shake to mix, and incubate at room temperature for 180 minutes.

[0148] 9) Add 10 μL Kinase Detection Reagent, centrifuge at 1000 rpm for 30 seconds, shake to mix, and incubate at room temperature for 30 minutes.

[0149] 10) Read the luminescence value (RLU) using an Envision microplate reader.

[0150] 11) ADP-Glo ​​method calculation formula

[0151] (RLU: chemiluminescence value of the sample; Mean (NC): mean ratio of negative control wells; Mean (PC): mean ratio of positive control wells.)

[0152] 3. Experimental Results

[0153] The test results obtained through the above test process are shown in Table 3 below:

[0154] Table 3: IC values ​​of kinase inhibitory activity of compound D1, fretinib, and staurosporine detected by ADP-Glo ​​method 50 The analysis results

[0155] As shown in Table 3, the inhibitory activity of fractinib against AXL is within 1 nM, and the inhibitory activity of staurosporine against FLT3 is within 1 nM, which is consistent with the reported selectivity of fractinib and staurosporine for kinases. Compound D1 is significantly more active than fractinib against the kinases Met and VEGFR, with particularly high activity against the kinase AXL, significantly doubling the activity of compound D1 compared to fractinib.

[0156] Example 9: Cytotoxicity study of fretinib, compound D1 and compound S1 on B16F10 cells

[0157] 1. Experimental steps

[0158] 1) Collect logarithmic phase B16F10 cells (B16F10 mouse skin melanoma cells), adjust the cell suspension concentration, add 100 μL to each well, and adjust the density of the cells to be tested to 5000 cells / well when plating (fill the edge wells with sterile PBS).

[0159] 2) Incubate at 37°C with 5% CO2 until the cell monolayer fills the bottom of the wells (96-well flat-bottom plate). Add the drug in a concentration gradient. The final drug concentration ranges from 0 to 100 μM, with a total of 7 concentration points. Dilute the drug serially, adding 100 μL per well, with triplicate wells.

[0160] 3) Incubate at 37°C in 5% CO2 for 48 hours and observe under an inverted microscope.

[0161] 4) Add 20 μL MTT solution (5 mg / ml, i.e., 0.5% MTT) to each well and continue incubation for 4 hours. If the drug reacts with MTT, centrifuge and discard the culture medium. Carefully rinse with PBS 2-3 times before adding the MTT-containing culture medium.

[0162] 5) Terminate the culture and carefully remove the culture medium from the wells.

[0163] 6) Add 150 μL of dimethyl sulfoxide to each well and shake at low speed for 10 minutes to fully dissolve the crystals. Measure the absorbance of each well at OD490 nm using an enzyme-linked immunosorbent assay (ELISA).

[0164] 7) Set up zero-adjustment wells (culture medium, MTT, dimethyl sulfoxide) and control wells (cells, drug dissolution medium of the same concentration, culture medium, MTT, dimethyl sulfoxide) at the same time.

[0165] 8) Inhibition rate calculation formula: Inhibition rate = OD 样品 -OD 调零孔 / OD 对照孔 -OD 调零孔

[0166] 9) Using the logarithmic concentration as the X-axis and the percentage inhibition rate as the Y-axis, the dose-effect curve was fitted using the log (inhibitor) vs. response-variable slope analysis software GraphPad Prism 5 to obtain the IC50 value of each compound on the enzyme activity.

[0167] 2. Experimental Results

[0168] As shown in Figures 2-4, the inhibition curves of compound D1 and fractinib are consistent, and the toxicity of compound D1 to B16F10 cells is stronger than that of fractinib, and the toxicity of compound S1 is lower than that of compound D1 and fractinib. The above phenomenon shows that the therapeutic effect of compound D1 released after compound S1 targets and enters tumor cells is better than that of existing drugs.

[0169] Example 10: Cytotoxicity study of compound S1, fretinib and compound D1 on A549 cells

[0170] 1. Experimental steps

[0171] 1) Collect A549 cells in the logarithmic phase (A549 cells are human alveolar basal epithelial cells derived from lung cancer), adjust the concentration of the cell suspension, add 100 μL to each well, and adjust the density of the cells to be tested to 5000 cells / well when plating (fill the edge wells with sterile PBS).

[0172] 2) Incubate at 37°C with 5% CO2 until the cell monolayer fills the bottom of the wells (96-well flat-bottom plate). Add the drug in a concentration gradient. The final drug concentration ranges from 0 to 100 μM, with a total of 7 concentration points. Dilute the drug serially, adding 100 μL per well, with triplicate wells.

[0173] 3) Incubate at 37°C in 5% CO2 for 48 hours and observe under an inverted microscope.

[0174] 4) Add 20 μl of MTT solution (5 mg / ml, i.e., 0.5% MTT) to each well and continue incubation for 4 hours. If the drug reacts with MTT, centrifuge and discard the culture medium. Carefully rinse with PBS 2-3 times before adding the MTT-containing culture medium.

[0175] 5) Terminate the culture and carefully remove the culture medium from the wells.

[0176] 6) Add 150 μL of dimethyl sulfoxide to each well and shake at low speed for 10 minutes to fully dissolve the crystals. Measure the absorbance of each well at OD490 nm using an enzyme-linked immunosorbent assay (ELISA).

[0177] 7) Set up zero-adjustment wells (culture medium, MTT, dimethyl sulfoxide) and control wells (cells, drug dissolution medium of the same concentration, culture medium, MTT, dimethyl sulfoxide) at the same time.

[0178] 8) Inhibition rate calculation formula: Inhibition rate = OD 样品 -OD 调零孔 / OD 对照孔 -OD 调零孔

[0179] 9) Using the logarithmic concentration as the X-axis and the percentage inhibition rate as the Y-axis, the dose-effect curve was fitted using the log (inhibitor) vs. response-variable slope analysis software GraphPad Prism 5 to obtain the IC50 value of each compound on the enzyme activity.

[0180] 2. Experimental Results

[0181] As shown in Figures 5-7, compound D1 has stronger toxicity to A549 cells than fragrinib, indicating that compound S1 provided by the present invention has better effect on tumor cells than fragrinib, a drug used in the prior art, and has a higher targeting effect.

[0182] Examples 9 and 10 illustrate that compound D1 and its similar structures are more toxic than fretinib in B16F10 cell and A549 cell models, and compound S1 has a better effect on tumor cells in the above two cell models than fretinib used in the prior art, reduces damage to normal cells, and improves the targeted effect of treatment.

[0183] Example 11: Efficacy study of compound S1 and compound S1 combined with anti-mPD-1 in the treatment of CT-26 tumor model

[0184] 1. Experimental Materials

[0185] Experimental drugs: control compound C1, low dose 3.95 μmol / kg, medium dose 7.90 μmol / kg, high dose 47.42 μmol / kg; compound S1, low dose 3.95 μmol / kg, medium dose 7.90 μmol / kg, high dose 47.42 μmol / kg; Anti-mPD-1.

[0186] Experimental animals: Balb / C mice, 7-8 weeks old, female, purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[0187] 2. Preparation of tumor model:

[0188] 1) Cell culture: CT-26 cells were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Cells were passaged every three days, and cells within passage 15 were used.

[0189] 2) Cell inoculation: Use RPMI-1640 basal medium to adjust the cell density to 3.0×10 6 / mL, inoculated subcutaneously on the right side of the back of mice, 0.1mL / mouse,

[0190] 3) Treatment: When the tumor volume reaches 100mm 3 ~200mm 3 The mice were randomly divided into 11 groups and treatment began, with 6 mice in each group, namely, normal saline control group, fretinib control group, compound D1 control group, control compound C1 low-dose group, control compound C1 medium-dose group, control compound C1 high-dose group, compound S1 low-dose group, compound S1 medium-dose group, compound S1 high-dose group, Anti-mPD-1 alone group, and Compound S1 and Anti-mPD-1 combination group. The day of grouping was set as D0, and the experimental results were D20 (i.e., the results on the 20th day).

[0191] As shown in FIG8 and Table 4, the effects of the test substances on the growth of mouse transplanted tumor CT-26 are shown.

[0192] Table 4: Effects of corresponding compounds and control group on tumor inhibition

[0193] As shown in Figure 8 and Table 4, in the CT-26 homologous transplant tumor model, compared with the normal saline group, fretinib, control compound C1 and compound S1 all showed good tumor inhibitory effects and showed a dose-dependent effect, while compound D1 did not show a tumor inhibitory effect; compared with the positive control group fretinib, at equimolar doses, control compound C1 and compound S1 both showed certain pharmacodynamic advantages, and compound S1 was more effective than compound C1.

[0194] Example 12: Pharmacological efficacy study of compound S1, compound S2, compound S12 and compound S15 in the treatment of MKN45 tumor model

[0195] 1. Experimental Materials

[0196] Experimental drugs: control compound C1, low dose 7.90 μmol / kg, medium dose 39.5 μmol / kg, high dose 79 μmol / kg; compound S1, low dose 7.90 μmol / kg, medium dose 39.5 μmol / kg, high dose 79 μmol / kg; compound S2, low dose 7.90 μmol / kg, medium dose 39.5 μmol / kg, high dose 79 μmol / kg; compound S12, low dose 7.90 μmol / kg, medium dose 39.5 μmol / kg, high dose 79 μmol / kg; compound S15, low dose 7.90 μmol / kg, medium dose 39.5 μmol / kg, high dose 79 μmol / kg.

[0197] Experimental animals: NOD-SCID mice, 7-8 weeks old, female, purchased from Nanjing Jicui Biotechnology Co., Ltd.

[0198] 2. Preparation of tumor model:

[0199] 1) Cell culture: MKN45 cells were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in RPMI-1640 medium containing 20% ​​fetal bovine serum at 37°C in a 5% CO2 incubator. Cells were passaged every three days, and cells within passage 15 were used.

[0200] 2) Cell inoculation: Use RPMI-1640 basal medium to adjust the cell density to 5.0×10 7 / ml, inoculated subcutaneously on the right side of the back of mice, 0.1ml / mouse,

[0201] 3) Treatment: When the tumor volume reaches 100mm 3 ~200mm 3 The mice were randomly divided into groups and treatment began, with 6 mice in each group, namely: normal saline control group, fretinib control group, compound D1 control group, control compound C1 low-dose group, control compound C1 medium-dose group, control compound C1 high-dose group, compound S1 low-dose group, compound S1 medium-dose group, compound S1 high-dose group, compound S2 low-dose group, compound S2 medium-dose group, compound S2 high-dose group, compound S12 low-dose group, compound S12 medium-dose group, compound S12 high-dose group, compound S15 low-dose group, compound S15 medium-dose group, and compound S15 high-dose group. The day of grouping was set as D0, and the experimental results were D28 (i.e., the results on the 28th day).

[0202] The drug administration results are shown in Table 5.

[0203] Table 5: Effects of corresponding compounds and control group on tumor inhibition

[0204] As shown in Figure 9 and Table 5, the effects of the test substances on the growth of mouse transplanted tumor MKN45 are shown. In the MKN45 transplanted tumor model, compared with the normal saline group, fretinib, control compound C1 and compound S1, compound S2, compound S12 and compound S15 all showed good tumor inhibitory effects and presented a dose-dependent effect, while compound D1 did not show a tumor inhibitory effect; compared with the positive control group fretinib, at equimolar doses, the control compound C1 and compound S1, compound S2 and compound S12 all showed certain pharmacodynamic advantages, and the effects of compound S1, compound S2, compound S12 and compound S15 were all better than compound C1.

[0205] Example 13: Study on the efficacy of compound S1, compound S9 and compound S10 in the treatment of HUH7 tumor model

[0206] 1. Experimental Materials

[0207] Experimental drugs: control compound C1, low dose 3.95 μmol / kg, medium dose 7.90 μmol / kg, high dose 23.71 μmol / kg; compound S1, low dose 3.95 μmol / kg, medium dose 7.90 μmol / kg, high dose 23.71 μmol / kg; compound S9, low dose 3.95 μmol / kg, medium dose 7.90 μmol / kg, high dose 23.71 μmol / kg; compound S10, low dose 3.95 μmol / kg, medium dose 7.90 μmol / kg, high dose 23.71 μmol / kg.

[0208] Experimental animals: Balb / c Nude mice, 7-8 weeks old, female, purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[0209] 2. Preparation of tumor model:

[0210] 1) Cell culture: HUH7 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd. and cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Cells were passaged every three days, and cells within passage 15 were used.

[0211] 2) Cell inoculation: Use DMEM basal medium to adjust the cell density to 5.0×10 7 / ml, inoculated subcutaneously on the right side of the back of mice, 0.1ml / mouse,

[0212] 3) Treatment: When the tumor volume reaches 100mm 3 ~200mm 3 The mice were randomly divided into 9 groups and treatment began, with 6 mice in each group, namely, normal saline control group, fretinib control group, compound D1 control group, control compound C1 low-dose group, control compound C1 medium-dose group, control compound C1 high-dose group, compound S1 low-dose group, compound S1 medium-dose group, and compound S1 high-dose group. The day of grouping was set as D0, and the test results were D36 (i.e., the results on the 36th day).

[0213] The drug administration results are shown in Table 6.

[0214] Table 6: Effects of corresponding compounds and control group on tumor inhibition

[0215] As shown in Figure 10 and Table 6, in the HUH7 transplant tumor model, compared with normal saline, fretinib, control compound C1, compound S1, compound S9 and compound S10 all showed good tumor inhibitory effects and showed a dose-dependent effect, while compound D1 did not show a tumor inhibitory effect; compared with the positive control group fretinib, at equimolar doses, the control compound C1, compound S1, compound S9 and compound S10 all showed certain pharmacodynamic advantages, and the effects of compound S1 and compound S9 were better than those of compound C1.

[0216] Example 14: Pharmacological efficacy study of compound S19 and compound S21 in the treatment of MDA-MB-231 tumor model

[0217] 1. Experimental Materials

[0218] Experimental drugs: control compound C1, low dose 3.95 μmol / kg, medium dose 7.90 μmol / kg, high dose 23.71 μmol / kg; S21, low dose 3.95 μmol / kg, medium dose 7.90 μmol / kg, high dose 23.71 μmol / kg; S19, low dose 3.95 μmol / kg, medium dose 7.90 μmol / kg, high dose 23.71 μmol / kg.

[0219] Experimental animals: BALB / c Nude mice, 7-8 weeks old, female, purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[0220] 2. Preparation of tumor model:

[0221] 1) Cell culture: MDA-MB-231 cells were purchased from the Chinese Academy of Sciences Cell Bank and cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Cells were passaged every three days, and cells within passage 15 were used.

[0222] 2) Cell inoculation: Use RPMI-1640 basal medium to adjust the cell density to 2.0×10 7 / ml, inoculated subcutaneously on the right side of the back of mice, 0.1ml / mouse,

[0223] 3) Treatment: When the tumor volume reaches 80 mm 3 ~200mm 3 The mice were randomly divided into three groups and treatment was started, with 6 mice in each group: normal saline control group, niraparib group, and rucaparib group. The day of grouping was set as D0. The experimental results were D36 (i.e., the results on day 36).

[0224] The drug administration results are shown in Table 7.

[0225] Table 7: Effects of corresponding compounds and control group on tumor inhibition

[0226] As shown in Table 7, in the MDA-MB-231 tumor model, compared with normal saline, fretinib, control compound C1, compound S19 and compound S21 all showed good tumor inhibitory effects in a dose-dependent manner, while compound D1 did not show any tumor inhibitory effect; compared with the positive control group fretinib, at equimolar doses, control compound C1, compound S19 and compound S21 all showed certain pharmacodynamic advantages.

[0227] Example 15: Toxicity assays of Fractinib, Compound D1, Niraparib, Rucaparib, Palbociclib, S1, S18, S19, and S20

[0228] Experimental purpose: To understand the acute toxicity of the drug of the present invention by determining the MTD experiment of intravenous administration in mice.

[0229] Experimental drugs: Fractinib, compound D1, Niraparib group, Rucaparib group and Palbociclib, S1, S18, S19 and S20 were dissolved in DMSO and then diluted with normal saline to the corresponding dose during the experiment.

[0230] Animals: BALB / C mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.), weighing 19-21 g, all female.

[0231] Methods and Results: 84 female BALB / C mice, weighing 19-21 g, were randomly divided into 14 groups of 6 mice each. Each mouse received a single intravenous injection of 0.2 ml of saline, fractinib, compound D1, niraparib, rucaparib, palbociclib, S1, S18, S19, and S20, as indicated in Table 8. Each mouse received a high-dose dose, and the mice were observed for 17 days. Animals were observed daily for signs of erect piloerection, matted luster, lethargy, hunched back, or hyperreactivity. Body weight and mortality were recorded. Blood samples were collected for complete blood counts on days 3, 5, and 14. On day 14, the heart, liver, kidneys, lungs, spleen, and pancreas were dissected and analyzed for hematoxylin and eosin staining.

[0232] Table 8: Comparison of mortality rates of mice receiving different doses of compound injections and saline

[0233] In summary, it can be seen that when the compounds S1, S18, S19 and S20 provided by the present invention are in the same dose 50 μmol / kg group, compared with the Frozenib, Niraparib, Rucaparib and Palbociclib groups, the animals did not show erected hair, dullness, lethargy, hunchback, overreaction or death. In the high dose 200 μmol / kg group, no death occurred. This further shows that the toxicity of the albumin-conjugated drug is significantly reduced than that of the unconjugated drug, and the therapeutic window index is greatly improved.

[0234] Example 16: Study on the efficacy of compound S1 in the treatment of colorectal cancer CT-26 tumor model

[0235] Animals: 36 Balb / c female mice.

[0236] Experimental method: Mouse colorectal cancer CT-26 cells were subcutaneously inoculated to establish a mouse transplant tumor model. 3 They were randomly divided into 6 groups, including:

[0237] negative control group, given normal saline;

[0238] positive control group, 5 mg / kg (7.90 μmol / kg) of fractinib;

[0239] S1 low-dose group, 15.7 mg / kg (7.90 μmol / kg) of compound S1;

[0240] S1 medium-dose group, 47 mg / kg (12.71 μmol / kg) of compound S1;

[0241] S1 medium- and high-dose groups, 70.4 mg / kg (33.56 μmol / kg) of compound S1;

[0242] S1 high-dose group, 93.9 mg / kg (47.42 μmol / kg) of compound S1.

[0243] The drug was administered intravenously with a volume of 10 ml / kg, twice a week for four consecutive weeks.

[0244] Experimental results: The administration results are shown in Figure 11, which shows the tumor volume after different doses of S1 were administered to the mouse colorectal cancer CT-26 model. Compared with the saline group, compound S1 showed good tumor inhibition and a dose-dependent effect. At equimolar doses, compound S1 showed a certain pharmacodynamic advantage and was better than fretinib.

[0245] Example 17: Study on the efficacy of compound S1 combined with anti-PD-1 antibody in the treatment of colorectal cancer CT-26 tumor model

[0246] Animals: 24 female Balb / c mice.

[0247] Experimental method: Mouse colorectal cancer CT-26 cells were subcutaneously inoculated to establish a mouse transplant tumor model. 3 They were randomly divided into 4 groups, including:

[0248] negative control group, given normal saline;

[0249] S1 monotherapy group, 7.7 mg / kg (3.95 μmol / kg) of compound S1;

[0250] anti-PD-1 antibody treatment group, 5 mg / kg of Anti-mPD-1 antibody;

[0251] The combined treatment group received 7.7 mg / kg (3.95 μmol / kg) of compound S1 and 5 mg / kg of anti-mPD-1 antibody.

[0252] The drug was administered intravenously with a volume of 10 ml / kg, twice a week for three consecutive weeks.

[0253] Experimental results: The administration results are shown in Figure 12, which shows the tumor volume of the mouse colorectal cancer CT-26 model after different administration methods. Compared with the S1 monotherapy group and the anti-PD-1 treatment group, the combination treatment group showed better tumor inhibition, indicating that the combination of compound S1 and anti-PD-1 antibody has a synergistic effect, and compound S1 combined with anti-PD-1 antibody can cure tumors at a low dose.

[0254] Example 18: Study on the efficacy of compound S1 in the treatment of human gastric cancer MKN45 tumor model

[0255] Animals: 36 female nude mice.

[0256] Experimental method: Human gastric cancer MKN45 cells were subcutaneously inoculated to construct a nude mouse xenograft tumor model. 3 They were randomly divided into 6 groups, including:

[0257] negative control group, given normal saline;

[0258] positive control group, 5 mg / kg (7.90 μmol / kg) of compound D1;

[0259] S1 low-dose group, 15.8 mg / kg (7.90 μmol / kg) of compound S1;

[0260] S1 medium-dose group, 79.2 mg / kg (39.5 μmol / kg) of compound S1;

[0261] S1 medium- and high-dose groups, 158.3 mg / kg (79 μmol / kg) of compound S1;

[0262] S1 high-dose group, 200 mg / kg (99.8 μmol / kg) of compound S1.

[0263] The drug was administered intravenously with a volume of 10 ml / kg, twice a week for three consecutive weeks.

[0264] Experimental results: The administration results are shown in Figure 13, which shows the tumor volume after administration of different doses of S1 in the human gastric cancer MKN45 model. Compared with the normal saline group, compound S1 showed good tumor inhibition and a dose-dependent effect. At equimolar doses, compound S1 showed a certain pharmacodynamic advantage and was better than compound D1.

[0265] Example 19: Toxicity test of compound S1 on beagle dogs

[0266] Animals: Forty beagle dogs were randomly divided into four groups, with 10 dogs in each group, half male and half female.

[0267] Experimental Methods: Four groups were administered normal saline, 9, 18, and 36 mg / kg of Compound S1, respectively, once weekly for four weeks. This was followed by a four-week recovery period with no drug administration. The following examinations were performed: general observation, body weight, food intake, clinical pathology (including hematology, blood biochemistry, and coagulation), temperature, electrocardiogram, ophthalmological examination, urine, gross anatomy, organ weights, bone marrow smears, histopathological examination, and toxicokinetics.

[0268] The experimental results showed that animals tolerated all doses well, and the HNSTD dose was 36 mg / kg.

[0269] It should be noted that, as used herein, an "effective amount" refers to an amount of a component sufficient to produce the desired response. The specific effective amount depends on a variety of factors, such as the specific condition being treated, the patient's physical condition (e.g., weight, age, or gender), the duration of treatment, co-administered therapies (if any), and the specific formulation used. "Effective amount" also refers to an amount at which the kinase inhibitors described herein are not significantly outweighed by their toxic or adverse effects.

[0270] In the present disclosure, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof can be equivalent to the therapeutically effective amount of a conventional drug molecule D. In some cases, due to the coupling of the linker disclosed in the present disclosure (i.e., group A), it has a higher activation efficiency and anti-tumor effect. Therefore, the therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof is lower than the therapeutically effective amount of a conventional drug molecule D. In summary, the above embodiments are only preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A kinase inhibitor based on tumor microenvironment activation, characterized in that: The kinase inhibitor is a compound having a structure represented by the following formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof: RACD (I) In the formula, R is a modifying group used to improve the physicochemical properties of the drug; A is an amino acid linker that can be cleaved by proteolytic enzymes; C is a sterically hindered group or a self-removable group; and D is the drug molecule.

2. The kinase inhibitor based on tumor microenvironment activation according to claim 1, characterized in that The R is a modifying group with an MI structure, such as the structure shown in the following formula (II): In the formula, MI is a structure with a conjugated system and can add an acceptor; Rc is a structure that increases water solubility, compound rigidity, or expands spatial distance; n1 is a positive integer between 1 and 12, n2 is a positive integer between 1 and 12, and n3 is 1 or 2; X1 is a trifunctional group; The wavy line indicates where R joins A.

3. The kinase inhibitor based on tumor microenvironment activation according to claim 2, characterized in that: The MI is selected from Wherein, R1, R2 and R3 are CH or N; or Wherein, the cyclic structure in the structure is a five-membered ring including any one of N, O or S, and R4, R5, R6 and R7 are one of CH, N, O or S; or Among them, R8, R9, R 10 , R 11 , R 12 , R 13 , R 14 and R 15 is C, CH or N; Ra and Rb are or The cyclic structure in the structure is a five-membered ring including any one of N, O or S, and R 16 , R 17 and R 18 is one of C, CH, N, O or S; R 19 , R 20 , R 21 and R 22 is C, CH or N; Re and Rf are 4. The kinase inhibitor based on tumor microenvironment activation according to claim 2, characterized in that: Rc is selected from C 1-12 Alkyl, C 1-12 Oxyalkyl-C 1-12 Alkyl, C 1-12 Alkyl-C 3-8 Cycloalkyl, (C 1-4 Alkyl-O)pC 1-12 Alkyl, phenyl-C 1-12 Alkyl, C 3-8 Cycloalkyl, C 1-12 Alkyl-C 3-8 Cycloalkyl-C 1-12 Alkyl, C 1-12 Alkyl-phenyl-C 1-12 Alkyl or C 1-12 Alkylcarbonylamino-(C 1-4 Alkyl-O)qC 1-12 alkyl; Wherein, p is a positive integer between 1 and 12, q is a positive integer between 1 and 12, and the phenyl group is one or two substituted phenyl groups, wherein the substituent in the substituted phenyl group is a halogen atom.

5. The kinase inhibitor based on tumor microenvironment activation according to claim 2, characterized in that: X1 is selected from lysine Lys or glutamic acid Glu; When X1 is lysine, its structure is The carboxyl end of X1 is connected to A, and the two amino ends of X1 are connected to the side chain containing n1 or the side chain containing n2 in a one-to-one correspondence. When X1 is glutamic acid Glu, its structure is Among them, the amino terminal is connected to the side chain containing n1 or the side chain containing n2; the carboxyl terminal adjacent to the amino group on glutamic acid is connected to A, and the carboxyl terminal far from the amino group on glutamic acid is connected to the side chain containing n1 or the side chain containing n2.

6. The kinase inhibitor based on tumor microenvironment activation according to claim 1, characterized in that A is -Ala-Ala-Asn-.

7. The kinase inhibitor based on tumor microenvironment activation according to claim 1, characterized in that C is Among them, R 23 , R 24 and R 25 It is CH or N.

8. The kinase inhibitor based on tumor microenvironment activation according to claim 1, characterized in that The drug molecule D is one of the following chemical formulas:

9. The kinase inhibitor based on tumor microenvironment activation according to claim 1, characterized in that The structure of the kinase inhibitor is selected from:

10. A pharmaceutical composition, characterized in that The composition comprises the tumor microenvironment activated kinase inhibitor according to any one of claims 1 to 9, and a pharmaceutically acceptable carrier or pharmaceutical composition thereof.

11. A use of a tumor microenvironment activated kinase inhibitor as described in any one of claims 1 to 9 in the preparation of a drug for treating cancer, wherein the cancer includes colorectal cancer, ovarian cancer, fallopian tube cancer, peritoneal cancer, breast cancer, gastric cancer, liver cancer, head and neck cancer, prostate cancer and pancreatic cancer.

12. A use of a kinase inhibitor based on tumor microenvironment activation as claimed in any one of claims 1 to 9 in a drug for treating cancer, characterized in that: The invention relates to a method for preparing a drug for combined treatment of tumors comprising the use of a tumor microenvironment activated kinase inhibitor or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, or a composition or a pharmaceutically acceptable salt thereof according to claim 10 and an anti-PD-1 antibody.

13. A method for treating or preventing cancer, or inhibiting the proliferation of cancer cells, characterized in that: include: The kinase inhibitor based on tumor microenvironment activation according to any one of claims 1 to 9 is administered to the subject.

14. The method according to claim 13, characterized in that Also includes: An immune checkpoint inhibitor and a kinase inhibitor based on tumor microenvironment activation as described in any one of claims 1 to 9 are administered to the subject.

15. The method according to claim 14, characterized in that The immune checkpoint inhibitor is an anti-PD-1 antibody drug.

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