Anti-tumor application of R406 and derivatives thereof in cooperation with TRAIL

Abstract

The invention provides a series of compounds with a general formula as shown in a formula (I), pharmaceutically acceptable salts, hydrates or solvates of the compounds, and application of the compounds and TRAIL combined medication in tumor treatment or tumor cell inhibition. The pharmaceutical composition disclosed by the invention is used for resisting tumors, has the characteristics of good safety, strong tolerance, high sensitivity and the like, and is very suitable for the development of antitumor drugs. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of medicines, and in particular, provides the anti-tumor use of the drug R406 and its derivatives in coordination with TRAIL. Background Art

[0002] In recent years, the incidence and mortality of tumors have increased day by day, which has brought new challenges to tumor treatment. At present, many studies have shown that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has the effect of selectively inducing apoptosis of tumor cells and has almost no obvious toxic side effects on normal tissue cells. In human cells, there are four types of TRAIL membrane surface receptors, namely TRAIL-R1, TRAIL-R2, TRAIL-R3 and TRAIL-R4. Among them, TRAIL-R1 and TRAIL-R2 contain death domains (DD) in cells. When stimulated by TRAIL, TRAIL-R1 and TRAIL-R2 can initiate cell apoptosis by recruiting the protein FADD containing the death domain. TRAIL-R3 and TRAIL-R4 are decoy receptors, which do not contain death domains in cells, so they will not induce apoptosis when stimulated by TRAIL. Different TRAIL receptors have different expressions in normal cells and tumor cells. TRAIL-R1 and TRAIL-R2 are highly expressed in tumor cells, while TRAIL-R3 and TRAIL-R4 are highly expressed in normal cells. Based on the above characteristics, TRAIL can specifically induce the death of tumor cells, so the research on the anti-tumor effect of TRAIL is very popular. At present, the treatment of TRAIL has been applied to many clinical trials, including clinical studies of TRAIL in non-small cell lung cancer (NSCLC), B-cell Non-Hodgkin's Lymphoma (NHL) and colorectal cancer. Although TRAIL showed high tolerance and safety in clinical phase I, many clinical results showed that the effect of using TRAIL alone for tumor treatment is very limited. Many preclinical studies have shown that most tumor cells are not sensitive to single TRAIL treatment, so more and more studies have begun to try the combination of TRAIL and other small molecules to increase the sensitivity of tumor cells to TRAIL. At present, the research on TRAIL combined with drugs has entered the clinical stage, and clinical results have shown that TRAIL combined with drugs has a good effect on tumor inhibition. Therefore, the development of new small molecule drugs that can cooperate with TRAIL to exert better anti-tumor function can provide a good reference and hope for subsequent targeted tumor treatment.

[0003] R406 is a reported inhibitor of tyrosine kinase Syk, and R788, also known as Fostamatinib, is the phosphate form of R406. As a prodrug molecule of R406, R788 can be rapidly converted into R406 in vivo. Currently, R788 has been approved by the FDA for the treatment of idiopathic thrombocytopenic purpura (ITP), but there is no research report on the combination of R788 and TRAIL in the treatment of tumors. Therefore, there is no application of R406 or its phosphate R788 combined with TRAIL in the treatment of tumors in the art.

[0004] Therefore, there is an urgent need to develop small molecule drugs that can synergize with TRAIL to exert better anti-tumor function. Summary of the invention

[0005] The object of the present invention is to provide a new use of drug R406 and its derivatives, or pharmaceutically acceptable salts, hydrates or solvates thereof in tumor treatment.

[0006] In the first aspect of the present invention, there is provided a compound as shown in formula (I), or a pharmaceutically acceptable salt, hydrate or solvate thereof,

[0007]

[0008] in,

[0009] n is 0, 1, 2, 3, 4 or 5;

[0010] Ring A is selected from the following group: saturated or partially unsaturated C 3-8 Carbocyclic group, saturated or partially unsaturated 5-10 membered heterocyclic group, C 6-10 Aryl, 5-12 membered heteroaryl;

[0011] R1 is selected from the following group: H, D, C 1-6 Alkyl, C 2-6 Acyl, C 2-6 Ester group, C 1-6 Halogenated alkyl, saturated or partially unsaturated C 3-8 Carbocyclic group, saturated or partially unsaturated 5-10 membered heterocyclic group, C 6-10 Aryl, 5-12 membered heteroaryl;

[0012] R2 and R3 are each independently selected from the following group: 1-6 Alkyl, C 2-6 Acyl, C 1-6 Haloalkyl;

[0013] Alternatively, R2 and R3 and the atoms therebetween together form a substituted or unsubstituted 5-10 membered saturated or partially unsaturated heterocyclic group, or a substituted or unsubstituted 5-10 membered heteroaryl group;

[0014] X1 is selected from the group consisting of O, NH, NR4, CH2, CHR4, C(R4)2;

[0015] Each R4 is independently selected from the following groups: D, C 1-6 Alkyl, C 2-6 Acyl, C 1-6 Haloalkyl, -C 1-3 Alkyl-OP(O)(OH)2, -C 1-3 Alkyl-OS(O)2OH;

[0016] Each L1 is independently selected from the following group: none, -O-, -NH-, -C 1-6 Alkyl-, -OC 1-6 Alkyl-, -NH-C 1-6 alkyl-;

[0017] Each R5 is independently selected from the group consisting of hydrogen, CN, C 1-6 Alkyl-C(O), C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylamino, saturated or partially unsaturated C 3-8 Carbocyclic group, saturated or partially unsaturated 5-10 membered heterocyclic group, C 6-10 Aryl, 5-12 membered heteroaryl;

[0018] R 11 Selected from the group consisting of halogen, CN, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkenyl, C 1-6 Alkynyl;

[0019] Among them, ring A, R1, R2, R3, R4, R5, R 11 Each independently is one or more (e.g., 2, 3, 4, etc.) R e substituted, and the R e Selected from the group consisting of D, hydroxy, halogen, cyano, amino, oxo (=O), C 1-6 Alkyl, C 1-6 Alkoxy, NHR f 、C(O)NHR f 、C(O)R f 、NHC(O)R f 、C(O)OR f 、OC(O)R f 、-S(O)2NHRf 、-S(O)2R f , saturated or partially unsaturated C 3-8 Carbocyclic group, saturated or partially unsaturated 5-10 membered heterocyclic group, C 6-10 Aryl, 5-12 membered heteroaryl; and the Re may be substituted by one or more (e.g., 2, 3, 4, etc.) unsubstituted or halogenated Rf;

[0020] R f Selected from the group consisting of halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, methylsulfone, -S(=O)2NH2, -S(=O)2NHCH3, oxo (=O), -CN, hydroxyl, -NH2, C 1-6 Alkyl-NH-, carboxyl, C1-C6 amide (-C(=O)-N(Rc)2 or -NH-C(=O)(Rc, Rc is H or C1-C5 alkyl), C 6-10 Aryl, 5-10 membered heteroaryl, 5-10 membered heterocyclic group, -(CH2)-C 6-10 Aryl, -(CH2)-(5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O); wherein the carbocyclic group, heterocyclic group, aryl group, heteroaryl group may be a monocyclic, spirocyclic, bridged, fused or condensed ring structure; the heterocyclic group or heteroaryl group may have 1-3 heteroatoms selected from N, S and O on the ring skeleton;

[0021] Furthermore, the compound is not a structure selected from the following group:

[0022]

[0023] In some embodiments, the ring A is selected from the following group:

[0024]

[0025] In some embodiments, the R5 is selected from the group consisting of saturated or partially unsaturated C 3-6 Carbocyclic group, saturated or partially unsaturated 5-8 membered heterocyclic group, C 6-10 Aryl, 5-10 membered heteroaryl; and said R5 is substituted or unsubstituted.

[0026] In another preferred embodiment, R5 is selected from the following group:

[0027] In some embodiments, the R1 is selected from the following group: H, C 1-6 Alkyl, C 2-6 Acyl, C 2-6 Ester group, C 1-6Halogenated alkyl.

[0028] In another preferred embodiment, the compound of formula I has a structure as shown below:

[0029]

[0030] in,

[0031] L1 is selected from the following group: none, -O-, -NH-, -C 1-6 Alkyl-, -OC 1-6 Alkyl-, -NH-C 1-6 alkyl-;

[0032] R5 is selected from the following group: CN, C 1-6 Alkyl-C(O), C 1-6 Haloalkyl, C 1-6 Alkylamino, saturated or partially unsaturated C 3-8 Carbocyclic group, saturated or partially unsaturated 5-10 membered heterocyclic group, C 6-10 Aryl, 5-12 membered heteroaryl; and said R5 may be substituted or unsubstituted.

[0033] In another preferred embodiment, R5 is a 5-7 membered saturated carbocyclic or heterocyclic ring, and said R5 may be substituted or unsubstituted.

[0034] In another preferred embodiment, R5 is a benzene ring or a 5-7 membered heteroaromatic ring, and the R5 may be substituted or unsubstituted.

[0035] In another preferred embodiment, the ring A, R1, R2, R3, R4, R5, R 11 Each is independently substituted by one or more substituents selected from the group consisting of D, hydroxy, halogen, cyano, amino, oxo (=O), C 1-4 Alkyl, C 1-4 Halogenated alkyl, C(O)NH2, saturated or partially unsaturated C 3-8 Carbocyclic group, saturated or partially unsaturated 5-10 membered heterocyclic group, C 6-10 Aryl, 5-12 membered heteroaryl.

[0036] In another preferred embodiment, the A ring is a benzene ring or a pyridine ring.

[0037] In some embodiments, the compound is selected from the group consisting of:

[0038]

[0039]

[0040]

[0041] In a second aspect of the present invention, a pharmaceutical composition is provided, characterized in that the composition comprises:

[0042] (1) a therapeutically effective amount of a first active ingredient, wherein the first active ingredient is R406, R788 or the compound according to the first aspect of the present invention, or a pharmaceutically acceptable salt, hydrate or solvate thereof;

[0043]

[0044] (2) a therapeutically effective amount of a second active ingredient, wherein the second active ingredient is tumor necrosis factor-related apoptosis-inducing ligand (TRAIL);

[0045] Optional (3) a pharmaceutically acceptable carrier.

[0046] In some embodiments, the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is selected from the following group: membrane TRAIL (mTRAIL) and soluble TRAIL (sTRAIL).

[0047] In the third aspect of the present invention, there is provided a use of the compound according to the first aspect of the present invention, or a pharmaceutically acceptable salt, hydrate or solvate thereof, characterized in that the drug is used in combination with TRAIL to promote programmed cell death by continuously activating TAK1 kinase activity, for treating tumors and / or inhibiting tumor metastasis;

[0048] In another preferred embodiment, the compound directly binds to TAK1 and promotes the sustained activation of TAK1 kinase.

[0049] In another preferred embodiment, the tumor is associated with cell death induced by death receptor-related ligands such as tumor necrosis factor.

[0050] In some embodiments, the tumor is selected from the group consisting of lung cancer, liver cancer, colon cancer, gastric cancer, breast cancer, ovarian cancer, melanoma, lymphoma, blood tumors, pancreatic cancer, bone cancer, and endocrine system cancer.

[0051] In a fourth aspect of the present invention, there is provided an in vitro non-diagnostic non-therapeutic method for inhibiting tumor cells, comprising the steps of:

[0052] A therapeutically effective amount of the compound according to the first aspect of the present invention and a therapeutically effective amount of TRAIL are administered to tumor cells.

[0053] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Schematic representation of the chemical structures of R406 and its phosphate R788 is shown.

[0055] Figure 2 AUC analysis of the purity of sf9-expressing purified hTAK1-TAB1 is shown.

[0056] Figure 3 The effects of R406 and R788 on TAK1 thermostability are shown.

[0057] Figure 4 It was shown that R406 promoted the phosphorylation of TAK1 downstream proteins in an in vitro kinase reaction.

[0058] Figure 5 It was shown that R406 promoted the hyperactivation of TAK1 in an in vitro kinase reaction.

[0059] Figure 6 The synthesis and use of R406-derivative photosensitive affinity probes are shown.

[0060] Figure 7 The binding site of R406 to TAK1 was determined by photo-cross-linking mass spectrometry.

[0061] Figure 8 We show that R406 / R788 can promote TNFα-induced RIPK1-dependent programmed cell death in FADD-deficient Jurkat cells.

[0062] Fig. 9 It was shown that R406 could promote TNFα-induced RIPK1-dependent programmed cell necrosis in HT29 cells.

[0063] Fig.10 It was shown that R406 could promote TNFα-induced RIPK1-dependent programmed cell necrosis in NCI-H1975 cells.

[0064] Fig.11 It was shown that R406 could promote TNFα-induced RIPK1-dependent apoptosis in NCI-H1299 cells.

[0065] Fig.12 It was shown that R406 could promote TRAIL-induced apoptosis in E0771 cells.

[0066] Fig.13 It showed that the sensitivity of the modified ZsGreen-LUC-expressing E0771 cells to the compound remained unchanged.

[0067] Fig.14 It was shown that R406 could promote TRAIL-induced apoptosis in LLC cells.

[0068] Fig.15 Shown are images of ZsGreen-LUC-expressing E0771 tumor tissue visualization in C57BL / 6 mice.

[0069] Fig.16 Shown are bioluminescent imaging changes of ZsGreen-LUC-expressing E0771 in C57BL / 6 mice.

[0070] Fig.17 It was shown that R788 combined with TRAIL significantly inhibited tumor growth in the ZsGreen-LUC-expressing E0771 breast cancer model in C57BL / 6 mice.

[0071] Fig.18 Shown is the immunohistochemical staining of Cleaved Caspase-3 in ZsGreen-LUC-expressing E0771 tumor tissue sections.

[0072] Fig.19 Shown is immunohistochemical staining of Ki-67 in ZsGreen-LUC-expressing E0771 tumor tissue sections.

[0073] Fig. 20 Shown is immunohistochemical staining of CD31 in ZsGreen-LUC-expressing E0771 tumor tissue sections.

[0074] Fig.21 Shown are images of ex vivo visualization of LLC tumor tissue in C57BL / 6 mice.

[0075] Fig. 22 Shown are the effects of R788 combined with TRAIL on tumor volume and tumor weight in LLC tumor-bearing mice.

[0076] Fig.23 The effects of R788 combined with TRAIL on HE staining of tumor tissue sections, immunohistochemistry of Ki-67, Cleaved caspase-3 and CD31 in LLC tumor-bearing mice are shown.

[0077] Fig.24 The effects of R788 combined with TRAIL on PARP1 cleavage and RIPK1 activation in tumor tissues of LLC tumor-bearing mice are shown. DETAILED DESCRIPTION

[0078] The inventors unexpectedly discovered that the small molecule R406 and its phosphate R788 can promote TRAIL-induced tumor cell death through large-scale screening of a known active compound library. As a prodrug of R406, R788 has been approved by the FDA for the treatment of idiopathic thrombocytopenic purpura, indicating that it has good safety and tolerability in the human body. On this basis, the present invention was completed.

[0079] the term

[0080] In the present invention, unless otherwise specified, the terms used have the general meanings well known to those skilled in the art.

[0081] In the present invention, the term "halogen" refers to F, Cl, Br or I.

[0082] In the present invention, “C 1-6 "Alkyl" refers to a straight or branched chain alkyl group containing 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, neopentyl, tert-pentyl, or the like. The term "C 1-4 Alkyl", "C 1-3 "Alkyl" has a similar meaning.

[0083] In the present invention, the term "C 2-6 "Alkenyl" refers to a straight or branched alkenyl group having 2 to 6 carbon atoms and containing one double bond, including but not limited to ethenyl, propenyl, butenyl, isobutenyl, pentenyl and hexenyl. The term "C 2-4 "Alkenyl" has a similar meaning.

[0084] In the present invention, the term "C 2-6 "Alkynyl" refers to a straight or branched alkynyl group having 2 to 6 carbon atoms and containing one triple bond, including but not limited to ethynyl, propynyl, butynyl, isobutynyl, pentynyl and hexynyl. The term "C 2-4 "Alkynyl" has a similar meaning.

[0085] In the present invention, the term "C 3-8 "Cycloalkyl" refers to a cyclic alkyl group having 3 to 8 carbon atoms in the ring, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc. The term "Cycloalkyl" refers to a cyclic alkyl group having 3 to 8 carbon atoms in the ring, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl 3-6 "Cycloalkyl" has a similar meaning.

[0086] In the present invention, the term "C 1-6 "Alkoxy" refers to a straight or branched alkoxy group having 1 to 6 carbon atoms, including but not limited to methoxy, ethoxy, propoxy, isopropoxy and butoxy. Preferably, C 1-4 Alkoxy, more preferably C 1-3 Alkoxy.

[0087] In the present invention, the term "heterocyclyl" or "heterocycloalkyl" refers to a 4-8 membered heterocyclyl containing 1, 2 or 3 heteroatoms selected from N, O, S, including (but not limited to) the following groups:

[0088] In the present invention, the term "aromatic ring" or "aryl group" has the same meaning, preferably "C 6-10 The term "C 6-10 The term "aryl" refers to an aromatic ring group having 6 to 10 carbon atoms and containing no heteroatoms in the ring, such as phenyl, naphthyl, etc.

[0089] In the present invention, the term "aromatic heterocycle" or "heteroaryl" has the same meaning, referring to a heteroaromatic group containing one to multiple heteroatoms. 3-10 "Heteroaryl" refers to an aromatic heterocycle containing 1 to 4 heteroatoms selected from oxygen, sulfur and nitrogen and 3 to 10 carbon atoms. Non-limiting examples include: furanyl, thienyl, pyridyl, pyrazolyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring. The heteroaryl group may be optionally substituted or unsubstituted.

[0090] In the present invention, the term "halo" means substituted with halogen.

[0091] In the present invention, the term "deuterated" means substituted with deuterium.

[0092] In the present invention, the term "substituted" means that one or more hydrogen atoms on a specific group are replaced by a specific substituent. The specific substituent is the substituent described above or the substituent appearing in the embodiments. Unless otherwise specified, a substituted group may have a substituent selected from a specific group at any substitutable position of the group, and the substituent may be the same or different at each position. It should be understood by those skilled in the art that the combination of substituents contemplated by the present invention is those that are stable or chemically feasible. The substituents include, but are not limited to, halogen, hydroxyl, carboxyl (-COOH), C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-8 Cycloalkyl, 3- to 12-membered heterocyclyl, aryl, heteroaryl, C 1-8 Aldehyde, C 2-10 Acyl, C 2-10 Ester group, amino group, C 1-6 Alkoxy, C 1-10 Sulfonyl, etc.

[0093] In the present invention, the term 1-6 refers to 1, 2, 3, 4, 5 or 6. Other similar terms independently have similar meanings. The term "plurality" refers to 2-6, such as 2, 3, 4, 5 or 6.

[0094] It should be understood that when a group is present in multiple different positions of a compound at the same time, its definition at each position is independent of each other and may be the same or different. That is, the term "selected from the following group:" has the same meaning as the term "each independently selected from the following group:".

[0095] Compound

[0096] The present invention provides a compound that can be used in combination with TRAIL to enhance its inhibitory effect on tumor cells, and a pharmaceutically acceptable salt, hydrate or solvate thereof. The compound structure is shown in formula (I):

[0097]

[0098] wherein each group is as defined above.

[0099] As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by a compound of the present invention and an acid or base that is suitable for use as a drug. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts is a salt formed by a compound of the present invention and an acid. Suitable acids for forming salts include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, and naphthalenesulfonic acid; and amino acids such as proline, phenylalanine, aspartic acid, and glutamic acid.

[0100] Another preferred salt is a salt of the compound of the present invention and a base, such as an alkali metal salt (e.g., sodium salt or potassium salt), an alkaline earth metal salt (e.g., magnesium salt or calcium salt), an ammonium salt (e.g., lower alkanolammonium salt and other pharmaceutically acceptable amine salts), for example, methylamine salt, ethylamine salt, propylamine salt, dimethylamine salt, trimethylamine salt, diethylamine salt, triethylamine salt, tert-butylamine salt, ethylenediamine salt, hydroxyethylamine salt, dihydroxyethylamine salt, trihydroxyethylamine salt, and amine salts formed from morpholine, piperazine, and lysine, respectively.

[0101] The present invention's various embodiments specifically describe the preparation methods of the compounds of formula I of the present invention, but these specific methods do not constitute any limitation to the present invention. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, and such combinations can be easily performed by those skilled in the art to which the present invention belongs.

[0102] Typically, the raw materials and reagents used in the preparation process of the compounds of the present invention can be purchased through commercial channels unless otherwise specified.

[0103] Pharmaceutical compositions and methods of administration

[0104] The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more safe and effective amounts of the compounds.

[0105] Since the compounds of the present invention have excellent anti-tumor activity, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat, prevent and alleviate tumor-related diseases.

[0106] The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, and more preferably, contains 10-1000 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.

[0107] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0108] The pharmaceutical composition is in the form of injection, capsule, tablet, pill, powder or granule.

[0109] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administrations include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0110] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0111] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0112] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0113] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0114] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.

[0115] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0116] Dosage forms for topical administration of the compounds of the invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0117] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds (such as anti-tumor drugs).

[0118] The treatment method of the present invention can be used alone or in combination with other treatment methods or therapeutic drugs.

[0119] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg, preferably 50 to 1000 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.

[0120] The main advantages of the present invention are:

[0121] 1. The present invention found that compounds that activate TAK1 kinase activity can promote TRAIL-induced tumor cell apoptosis and necrosis.

[0122] 2. The TAK1 kinase activator and TRAIL combination composition of the present invention can significantly inhibit the growth of tumor cells.

[0123] Preparation Example

[0124] General synthetic route of compounds:

[0125]

[0126] The synthesis of the compounds of the present invention all uses the corresponding aminopyridine derivatives as raw materials, and the reaction process is as follows:

[0127] Add the corresponding aminopyridine derivative (2.59mmol), 5-fluoro-2,4-dichloropyrimidine (650mg, 3.9mmol), DIPEA (850μL, 5.18mmol) and DMF (5mL) into a 15mL reaction bottle. Heat to 90℃ and stir overnight. After the reaction is complete, dilute with ice water, filter, wash the filter cake with water and organic mixed solvent (PE / DCM=10 / 1), and dry to obtain the pyrimidine-substituted intermediate.

[0128] The intermediate (0.31 mmol) was dissolved in NMP (2 mL) with the corresponding primary amine (0.46 mmol) and 2M dioxane hydrochloride solution (15 μL, 0.03 mmol) and subjected to microwave reaction at 120°C for 1 h. After the reaction was complete, the reaction solution was purified by reverse phase (H2O / MeCN=1:1) and HPLC to obtain the target compound.

[0129] Among them, the preparation routes of various aminopyridine derivatives are as follows:

[0130] 1. Preparation of compound TXY-4-44

[0131]

[0132] (1) Preparation of compound TXY-3-21:

[0133]

[0134] 6-Bromo-2-nitropyridine-3-ol (1.5 g, 6.85 mmol), potassium hydroxide (1.5 g, 27 mmol) and DMSO (20 mL) were added to a round-bottom flask. The reaction was pre-stirred for 5 min at room temperature, and iodomethane (850 μL, 13.7 mmol) was added and stirred for 30 min. After the reaction was complete, it was diluted with water, extracted with DCM, and the organic phase was washed with potassium hydroxide aqueous solution and saturated brine in sequence, concentrated, and purified by silica gel column chromatography, eluted with PE / EA (2:1) to obtain the target compound with a yield of 63%. MS-ESI: m / z calculated for C6H5BrN2O3, Exact Mass 231.95, found 233.0 [M+H] + .

[0135] (2) Preparation of compound TXY-3-22:

[0136]

[0137] Add compound TXY-3-21 (1 g, 4.3 mmol), iron powder (670 mg, 12.9 mmol), ammonium chloride (240 mg, 4.7 mmol), 10 mL ethanol and 10 mL water to the reaction bottle. Heat to 70 ° C for 30 min, filter while hot, wash with DCM, combine the filtrate, wash with saturated sodium bicarbonate solution and saturated brine in turn, and concentrate to obtain 670 mg of crude product with a yield of 77%. MS-ESI: m / z calculated for C6H7BrN2O, Exact Mass 201.97, found 202.9 [M+H] + .

[0138] (3) Preparation of compound TXT-3-40:

[0139]

[0140] Compound TXY-3-22 (300 mg, 1.48 mmol), paraformaldehyde (1.08 g, 4.4 mmol), 20 μL acetic acid and 1,2-dichloroethane (3 mL) were added to the reaction flask. After reacting at room temperature for 1 hour, the mixture was heated to 50°C and sodium triacetoxyborohydride was added in batches within 6 hours, totaling 800 mg (3.77 mmol). After the reaction was complete, the reaction solution was concentrated and purified by silica gel column chromatography, eluted with PE / DCM (1:1), and 200 mg of solid product was obtained, with a yield of 59%. MS-ESI: m / z calculated for C8H 11 BrN2O,ExactMass 230.01,found 231.1[M+H] + .

[0141] (4) Preparation of compound TXY-4-44:

[0142]

[0143] Add compound TXY-3-40 (100 mg, 0.43 mmol), 2,4-dimethoxybenzylamine (80 μL, 0.47 mmol), cuprous iodide (4 mg, 0.02 mmol), compound Ligand (8 mg, 0.02 mmol), potassium hydroxide (50 mg, 0.86 mmol) and DMSO (2 mL) to the reaction flask. Heat to 90 ° C under nitrogen protection and react for 3 h. After the reaction is complete, the reaction solution is diluted with water, extracted with DCM, and the organic phase is washed with water and saturated brine in turn, concentrated, and purified by silica gel column chromatography, eluted with PE / EA (2:1) to obtain a crude product of 2,4-dimethoxybenzylamine substituted bromine. Dissolve the crude product in DCM (1 mL), add 200 μL trifluoroacetic acid, and stir overnight at room temperature. After the reaction was complete, the reaction solution was concentrated and purified by silica gel column chromatography, eluted with DCM / MeOH (2MNH3) (4:1) to obtain 50 mg of solid with a yield of 70%. MS-ESI: m / z calculated for C8H 13 N3O,Exact Mass167.11,found 168.2[M+H] + .

[0144] 2. Preparation of compound TXY-3-60

[0145]

[0146] (1) Preparation of compound TXY-3-26:

[0147]

[0148] The same preparation process as compound TXY-3-22, yield 58%. MS-ESI: m / z calculated for C5H5BrN2O, Exact Mass 187.96, found 188.9 [M + H] + .

[0149] (2) Preparation of compound TXY-3-58:

[0150]

[0151] Compound TXY-3-26 (300 mg, 1.59 mmol) and THF (9 mL) were added to the reaction flask, and chloroacetyl chloride (150 μL in 1 mL THF, 1.9 mmol) was added dropwise at 0°C. The mixture was gradually restored to room temperature, and after reacting for 2 h, triethylamine (660 μL, 4.77 mmol) was added, and the temperature was raised to 50°C and stirred overnight. After the reaction was complete, the reaction solution was concentrated and purified by silica gel column chromatography, eluted with PE / EA (3:1), and 248 mg of solid was obtained, with a yield of 68%. MS-ESI: m / z calculated for C7H5BrN2O2, Exact Mass 227.95, found 229.1 [M+H] + .

[0152] (3) Preparation of compound TXY-3-60:

[0153]

[0154] The same preparation process as compound TXY-4-44, yield 38%. MS-ESI: m / z calculated for C7H7N3O2, Exact Mass 165.05, found 166.0[M+H] + .

[0155] 3. Preparation of compound TXY-4-34

[0156]

[0157] The same preparation process as TXY-4-44, after the coupling product is obtained and purified in the first step, 100 mg of the intermediate (0.32 mmol) is dissolved in DMF (1 mL), potassium carbonate (88 mg, 0.64 mmol) and iodomethane (30 μL, 0.48 mmol) are added, and the reaction is carried out at room temperature for 2 hours, then diluted with water, extracted with EA, and the organic phase is washed with water and saturated brine in turn, dried over anhydrous sodium sulfate, and concentrated to obtain a methylated crude product. The crude product is dissolved in DCM (1 mL), 150 μL (1.9 mmol) of trifluoroacetic acid is added, and the reaction is carried out at room temperature for 3 hours, then concentrated, and purified by silica gel column chromatography, eluted with PE / EA (1:1), and about 40 mg of solid is obtained, with a yield of 70%. MS-ESI: m / z calculated for C8H9N3O2, Exact Mass 179.07, found 180.0 [M+H] + .

[0158] 4. Preparation of compound TXY-4-41

[0159]

[0160] (1) Preparation of compound TXY-4-40:

[0161]

[0162] The preparation process was the same as that of TXY-4-44. The product was purified by reverse phase, H2O / MeCN (1:1) elution, with a yield of 55%. MS-ESI: m / z calculated for C8H 11 N3O4,Exact Mass 213.07,found 214.1[M+H] + .

[0163] (2) Preparation of compound TXY-4-41:

[0164]

[0165] Compound TXY-4-40 (100 mg, 0.47 mmol), Pd / C (15 mg) and 5 mL methanol were added to a round-bottom flask, and hydrogen was introduced to react for 3 h. After the reaction was complete, the mixture was filtered and the filtrate was concentrated to obtain 80 mg of black liquid, which was directly used for the next step, with a yield of 93%. MS-ESI: m / z calculated for C8H 13 N3O2,Exact Mass183.10,found 184.0[M+H] + .

[0166] 5. Preparation of compound TXY-3-34

[0167]

[0168] The compound 2,6-dichloro-3-nitropyridine (500 mg, 2.59 mmol) was dissolved in 7M ammonia methanol solution (10 mL), heated to 100°C and microwaved for 30 min. After the reaction was complete, the reaction solution was concentrated to obtain a light yellow solid, which was washed with a small amount of DCM and dried to obtain a 350 mg crude product, which was directly used in the next step, with a yield of 88%. MS-ESI: m / z calculated for C5H6N4O2, Exact Mass 154.05, found 155.1 [M+H] + .

[0169] 6. Preparation of compound TXY-4-59

[0170]

[0171] Compound TXY-3-34 (150 mg, 1 mmol), Pd / C (15 mg) and 5 mL methanol were added to a round-bottom flask, filled with hydrogen, and stirred at room temperature overnight. The intermediate can be detected by LCMS (125 [M+H] + ). The reaction solution was filtered and concentrated to obtain a blue solid. DMF (5 mL) was added to dissolve, and N, N'-disuccinimidyl carbonate (DSC) (250 mg, 1 mmol) was slowly added dropwise at 0 ° C and under nitrogen protection. After the addition was completed, the temperature was gradually restored to room temperature and the reaction was continued for 3 h. After the reaction was complete, an appropriate amount of silica gel was added to the reaction solution, concentrated, and purified by silica gel column chromatography, eluted with DCM / MeOH (2M NH3) (5:1), and 50 mg of the product was obtained with a yield of 33%. 1 H NMR (400MHz, DMSO-d6) δ10.72(s,1H),10.14(s,1H),6.93(d,J=8.2Hz,1H),6.02(d,J=8.2Hz,1H),5.37(s,2H).MS-ESI:m / z calculated for C6H6N4O,Exact Mass 150.05,found151.0[M+H] + .

[0172] 7. Preparation of compound TXY-4-70

[0173]

[0174] Add compound 2,2-dimethyl-6-nitro-2H-pyrido[3,2-B][1,4]oxazine-3(4H)-one (100 mg, 0.45 mmol), 2-bromoethanol (62 mg, 0.5 mmol), potassium carbonate (125 mg, 0.9 mmol) and DMF (2 mL) to the reaction bottle. Heat to 60 ° C for 1 h. After the reaction is complete, concentrate and purify by silica gel column chromatography, eluting with PE / EA (3:1). The obtained product is directly added to MeOH (5 mL) with Pd / C (15 mg), and hydrogen is charged for catalytic hydrogenation. The reaction is allowed to proceed overnight, filtered, and concentrated to obtain 100 mg of the target product with a total yield of 90%. MS-ESI: m / z calculated for C 11 H 15 N3O3,Exact Mass237.11,found 238.2[M+H] + .

[0175] The structures of the compounds obtained by the general method are shown in Table 1, and the NMR data are shown in Table 3.

[0176] Biological Test Examples

[0177] Example 1 R406 and its phosphate R788 directly bind to and activate TAK1

[0178] 1 Experimental Materials and Methods

[0179] 1.1 Drugs and reagents

[0180] R406 and its phosphate R788 were purchased from Selleck, dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution and stored at -20°C. DMEM culture medium and fetal bovine serum were purchased from Gibco (Life Technologies, Grand Island, NY, USA).

[0181] The antibodies used are as follows: p-S321RIPK1, RIPK1, p-TAK1, TAK1, p-IKKα / β, IKKβ, FADD, His were all purchased from Cell signaling technology, β-actin was purchased from TransGen Biotech, GST was purchased from SantaCruz, and mouse anti-Tubulin was purchased from Sigma-Aldrich.

[0182] 1.2 Western blot analysis

[0183] 1) Vertical protein electrophoresis separation: 8%-10% SDS-polyacrylamide (SDS-PAGE) gel electrophoresis separation, 5% stacking gel. The stacking gel separation voltage is 80V, the separation gel separation voltage is 120V, and the bromophenol blue indicator stops when it runs to the bottom.

[0184] 2) Transfer: Semi-dry transfer, transfer current is 0.2-0.4V, adjust the time according to the size of the protein.

[0185] 3) Pre-stain with Ponceau red and observe the transfer effect.

[0186] 4) Blocking: Block with TBST containing 5% skim milk at room temperature on a decolorizing shaker for 1 hour.

[0187] 5) Binding to primary antibody: dilute the antibody at 1:2000-1:10000 in TBST containing 5% BSA, place on a decolorizing shaker at room temperature for 1 hour, and incubate at 4°C overnight. Wash three times with TBST, 10 minutes each time.

[0188] 6) Binding secondary antibody: dilute the antibody in TBST containing 5% BSA at 1:5000-1:10000, place on a decolorizing shaker at room temperature for 1 hour, and wash three times with TBST, 10 minutes each time.

[0189] 7) Develop the ECL kit substrate for 2-3 minutes, detect by chemiluminescence, and expose to X-ray film.

[0190] 1.3 Cell viability assay

[0191] Cells in the logarithmic growth phase were inoculated in a 384-well culture plate at an appropriate density, with 40 μL per well. After overnight culture, different concentrations of drugs were added for different time points, with triplicate wells for each concentration. After the end of the treatment, 15 μL / well of ATP was added to each well for 15 minutes, and the chemiluminescence value was detected with an enzyme-labeled instrument.

[0192] 1.4 In vitro kinase reaction

[0193] 1) Overexpress WT-RIPK1 and IKKβ-K44A in HEK293T cells, and add 5Z7 (500 nM) and Nec-1s (10 μM) during transient transfection;

[0194] 2) 24 hours after transfection, cells were lysed with NP-40 lysis buffer and centrifuged at 12000 rpm for 20 minutes;

[0195] 3) After centrifugation, RIPK1 and IKKβ in the supernatant were enriched and purified using beads with Flag, and the beads were washed 6-8 times with high-salt NP-40 buffer to remove as much impurity as possible, and then equilibrated with kinase buffer 3-5 times;

[0196] 4) The Flag beads were evenly divided into several equal parts, incubated with 5mM R406 and 1μM 5Z7 for 30 minutes respectively, and reacted at a kinase reaction temperature of 30°C for 60 minutes;

[0197] 5) Add 2* loading buffer to boil the sample and terminate the reaction.

[0198] 1.5 Protein thermal stability test

[0199] 1) The concentration of hTAK1-TAB1 fusion recombinant protein expressed and purified by sf 9 was adjusted to 3 μM, and the recombinant protein was mixed evenly with 10 μM R406 and R788 respectively, and placed at room temperature for 2 hours, and then 5X SYRO orange Protein gelstain dye was added and incubated in the dark for 30 minutes;

[0200] 2) Seal the q-PCR plate with a transparent sealing film;

[0201] 3) Set the Q7 thermoshift program, the temperature rises from room temperature to 105°C, the program takes about 1.5 hours to run;

[0202] 4) After the program is finished, Protein Thermal Shift Microsoft (PTSM) software is used to process and analyze the data.

[0203] 1.6 Photocrosslinking mass spectrometry

[0204] 1) Incubate the photoaffinity probe T-5-27 and its control compound S-8-56 with cell lysate at 4°C for 2 h. The protein concentration of the cell lysate is approximately 2.5 mg / ml (0.6 mg / ml).

[0205] 2) Alternatively, the photoaffinity probe and its control compound were incubated with the protein for 2 h, the concentration of the compound was 10 μM, the concentration of the protein was 2 mg / ml, and a total of 300 μl of the system solution;

[0206] 3) The mixed solution of the small molecule compound and the protein incubation was divided into several equal parts, added to a 96-well plate, 30 μl of the reaction solution was added to each well, and then photocrosslinked under ultraviolet light. After 30 minutes, the mixed solution divided into several equal parts was remixed and the SDS concentration was increased to 1%;

[0207] 4) Then conduct click chemistry reaction. TCEP should be prepared and used immediately. React at 25℃, 700-800rpm for 40min-1h.

[0208] 5) Then add 4 times the volume of the reaction solution of methanol, 1 times the volume of the reaction solution of chloroform and 3 times the volume of the reaction solution of distilled water, mix all the reactions evenly, place on ice for 40 minutes, centrifuge at 12000 rpm for 20 minutes, wash twice with 1 ml of 4°C pre-cooled methanol, and then spin dry with a desiccator and redissolve;

[0209] 6) Enrich the target protein with beads containing Biotin and bind overnight at 4°C;

[0210] 7) Centrifuge at 5000 rpm for 15 min and discard the supernatant. Repeat three times;

[0211] 8) Wash with ddH2O 7-10 times;

[0212] 9) After processing, the samples are sent to the mass spectrometry platform for analysis.

[0213] 1.7 Experimental Results

[0214] 1.7.1 R406 directly binds to TAK1

[0215] Currently, insect cells are used to express the recombinant protein of TAK1 kinase domain. Since TAK1 has kinase activity only after fusion with TAB1, insect cell expression system is used to express TAK1-TAB1, such as Figure 2 As shown, it is used to study the binding of R406, R788 and TAK1-TAB1. The thermal stability of proteins can be used to study the stability changes of proteins and small molecules or other proteins. When small molecules bind to proteins, they can stabilize the globular structure of proteins and change the Tm value of proteins in thermal stability experiments. Therefore, the change of Tm can reflect the binding status of proteins.

[0216] R406 and R788 were incubated with hTAK1-TAB1 protein for 60 minutes, and then the thermal stability experiment was started. The results are as follows Figure 3 As shown, the Tm value and thermal stability fluorescence change spectrum of TAK1 show that R406 and R788 can increase the Tm value of the thermal stability of the hTAK1-TAB1 reaction by 4-6°C, which has a significant effect on thermal stability, indicating that R406 can directly bind to TAK1.

[0217] 1.7.2R406 directly activates TAK1

[0218] Plasmids of Flag-RIPK1 and Flag-IKKβK44A were overexpressed in HEK293T cells, respectively. To minimize the background of RIPK1 autophosphorylation, 10 μM Nec-1s was added during overexpression. After 24 h, RIPK1 and IKKβ were immunoprecipitated with Flag beads, respectively. Then, in vitro kinase reactions were performed with recombinant His-tagged TAK1-TAB1 proteins. At the same time, 20 μM R406 and 1 μM 5Z7 were added, respectively. After reacting at 30°C for 60 min, the samples were boiled for Western detection. Figure 4 shown.

[0219] The results showed that R406 could promote the phosphorylation of IKKβ and RIPK1-S321 in vitro, while 5Z7 as a control could inhibit their phosphorylation. Figure 5 The results showed that R406 can also promote its own phosphorylation level in vitro and the phosphorylation level when TAK1 small peptide is used as a substrate. These data indicate that R406 can directly activate TAK1 in an in vitro system.

[0220] 1.7.3 Design and use of R406 photosensitive affinity probe

[0221] Based on the structure of R406, small molecules were modified, as shown in Table 1, and their structure-activity relationship for TAK1 was analyzed. Then, through cell survival assay, it was detected whether the modified small molecules still had the activity of R406 in promoting TNFα-induced cell death, as shown in Table 2. Then, suitable structural sites were found to introduce photo-crosslinking tags without affecting the activity of R406, and photo-crosslinking probes for TAK1 were synthesized, as shown in Table 1, to assist in finding the interaction sites between R406 and TAK1, as shown in Table 2.

[0222] Table 1 R406 derivatives

[0223]

[0224]

[0225]

[0226]

[0227] Table 2 Activity data of R406 derivatives ("+" represents that the compound promotes the death of MEFs cells by ≤10%, "++" represents that the compound promotes the death of MEFs cells by ≤20%, but >10%, "+++" represents that the compound promotes the death of MEFs cells by ≥30%)

[0228]

[0229] Table 3 Characterization data of some compounds of the present invention

[0230]

[0231]

[0232]

[0233]

[0234]

[0235] The results showed that the small molecules based on the structure modification of R406 were sequentially tested for cell survival assays, and it was finally found that S-8-56 was the most active small molecule. T-5-27 was a photo-crosslinking probe derived from S-8-56. Cells were treated with small molecules, or small molecules and proteins were co-incubated, such as Figure 6 After photocrosslinking and click chemistry reactions, the biotin-labeled target protein can be co-precipitated and detected through the biotin-avidin system.

[0236] The diaziridine group of T-5-27 can undergo a photochemical reaction under ultraviolet light of a certain wavelength, so that this part of the group can be covalently cross-linked to the vicinity of the target protein binding site, and the alkynyl group on the left can undergo a click reaction with the azide group of the biotin probe to enrich the protein labeled with the photocross-linking probe. The enriched protein can be subjected to mass spectrometry analysis after trypsin digestion, attempting to find the approximate binding site of T-5-27 on TAK1 by increasing the molecular weight peptide information shown in the figure.

[0237] like Figure 6 As shown in the figure, it can be found that the photosensitive affinity probe T-5-27 can indeed enrich and separate TAK1 from cell lysate, while S-8-56, a derivative of the light-stable control small molecule R406 of T-5-27, cannot co-precipitate TAK1. In addition, under the competition condition of S-8-56, the cross-linking effect of the probe will be weakened, which proves the effectiveness of the photo-cross-linking probe.

[0238] Next, the probe and TAK1 protein were photocrosslinked in vitro. The probe was linked to Biotin through a click chemistry reaction. Then, streptavidin agarose beads were used to precipitate the TAK1 protein crosslinked with the probe. The TAK1 protein was then digested with trypsin and the peptides with the probe and biotin were eluted for protein spectrum analysis in order to identify the interaction site between R406 and TAK1. Figure 7 If the peptide is linked to the probe and biotin, the peptide will increase the mass migration of 1000.4865Da. The amino acids in the peptide that are linked to the probe and biotin can be identified through the secondary mass spectrometry. Finally, through mass spectrometry analysis, it was identified that the peptide "35-EIEVEEVGR-44" has a mass increase of 1000.4865Da. The E6 in the peptide, that is, the glutamic acid at position 140 of TAK, is covalently coupled to the probe.

[0239] Example 2 Effects of R406 and its phosphate R788 combined with TNFα / TRAIL on tumor cells

[0240] 1 Experimental Materials and Methods

[0241] 1.1 Drugs and reagents

[0242] R406 and its phosphate R788 were purchased from Selleck, dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution and stored at -20°C. Recombinant human TRAIL protein was purchased from R&D. DMEM, RMI1640, McCoy's 5A medium and fetal bovine serum were purchased from Gibco (Life Technologies, Grand Island, NY, USA).

[0243] The antibodies used in the study are as follows: rabbit anti-PARP, p-S166RIPK1, RIPK1, p-S227RIPK3, RIPK3, p-S358MLKL, Cleavedcaspase-3, caspase 3, TAK1, p-IKKα / β, IKKα / β, FADD were all purchased from Cellsignaling technology, and mouse anti-Tubulin was purchased from Sigma-Aldrich.

[0244] 1.2 Cell lines and cell culture

[0245] Table 3A. Sources of human tumor tissue cell lines

[0246]

[0247]

[0248] Table 4. Sources of mouse tumor tissue cell lines

[0249] Cell lines source Organization Source Culture medium LLC ATCC Mouse lung cancer cells DMEM + 10% FBS E0771 ATCC Mouse breast cancer cells DMEM + 10% FBS

[0250] The above cell lines were all cultured in a cell culture incubator at 37°C containing .5% CO2, and the experiments were performed when the cells were in the logarithmic growth phase.

[0251] 1.3 Detection of cell death using SYTOX Green

[0252] 1) Inoculate relevant cells into appropriate well plates and add drugs according to experimental needs until the corresponding time point;

[0253] 2) Stain the cells with SYTOX Green at a final concentration of 200 nM;

[0254] 3) The number of SYTOXGreen positive cells is the number of cell death, and the ratio is the final result.

[0255] 1.4 Experimental Results

[0256] 1.4.1 R406 / R788 promotes TNFα-induced RIPK1-dependent programmed cell death in tumor cells

[0257] In human leukemia FADD deficient Jurkat cells, TNFα (20 ng / ml) was added to induce cell necrosis after pretreatment with R406 (3 μM), R788 (3 μM) or Nec-1s (10 μM) for 30 minutes.

[0258] like Figure 8 As shown in the results, R406 and R788 can significantly promote TNFα-induced cell necrosis as the TNFα treatment time increases, and this promotion can be protected by Nec-1s. In addition, as the TNFα treatment time increases, R406 and R788 significantly enhance the phosphorylation of RIPK1 and MLKL, and this phosphorylation can be protected by Nec-1s. This indicates that R406 can promote TNFα-induced RIPK1-dependent programmed necrosis.

[0259] Human colon cancer cells HT-29 and human lung adenocarcinoma cells NCI-H1975 were pretreated with R406 (3 μM) and Nec-1s (10 μM) for 30 minutes, and then TNFα (20 ng / ml) and caspase inhibitor zVAD.fmk (20 μM) were added to induce cell necrosis. The cell SYTOX Green level was measured every 7 h to evaluate cell death.

[0260] like Figure 9-12 As shown in the results, R406 significantly promoted cell necrosis induced by TNFα / zVAD.fmk as the treatment time of TNFα and zVAD.fmk was prolonged, and this promotion effect could be protected by Nec-1s. In addition, as the treatment time of TNFα / zVAD.fmk increased, R406 significantly enhanced the phosphorylation of RIPK1 and MLKL, and this phosphorylation could be protected by Nec-1s. This indicates that R406 can promote TNFα-induced RIPK1-dependent programmed necrosis.

[0261] Subsequently, human lung cancer cells NCI-H1299 were pretreated with R406 (3 μM) and Nec-1s (10 μM) for 30 minutes, and then TNFα (20 ng / ml) was added. Similarly, the cell SYTOX Green level was measured every 7 hours to evaluate cell death.

[0262] like Fig.11As shown in the results, as the TNFα treatment time increases, R406 can significantly promote cell death induced by TNFα, and this promotion can be protected by Nec-1s. In addition, as the TNFα treatment time increases, R406 significantly enhances the phosphorylation of RIPK1 and the cleavage of PARP1, and this phosphorylation and cleavage can be protected by Nec-1s. This indicates that R406 can promote TNFα-induced RIPK1-dependent cell apoptosis.

[0263] 1.4.2 R406 / R788 promotes TRAIL-induced apoptosis

[0264] Different concentrations of TRAIL were used to induce cell apoptosis in E0771 cells, and 1μM, 3μM and 5μM R406 were added to treat the cells. The cellular ATP level was detected after 24 hours, and the cell survival rate was evaluated by CellTiter-Glo assay.

[0265] like Fig.12 As shown, when TRAIL alone treated E0771 cells, its IC 50 is 34.21nM. With the addition of R406, the IC 50 It showed a gradually decreasing trend, and the higher the concentration of R406, the higher the IC of TRAIL 50 The lower.

[0266] In order to establish an animal model with ZsGreen-LUC-expressing E0771, it is first necessary to construct a stable cell line with a luciferase gene, and this cell line has unchanged sensitivity to the compound. Fig.13 The experiment shown.

[0267] The experimental results showed that the cells with the strongest luciferase activity of the modified ZsGreen-LUC-expressing E0771 were not sensitive to the compound compared to the E0771-BLANK cells. The compound alone or TRAIL alone could promote 20%-30% cell death (12h). When the compound and TRAIL were used in combination, they could promote 70%-80% cell death (12h) in both cells. This promotion effect was significantly different. In addition, the cell death promoted by compound R788 under TRAIL-induced conditions could be inhibited by Nec-1s, but the cell death promoted by TRAIL itself could not be inhibited by Nec-1s.

[0268] LLC cells were treated with TRAIL at different concentrations to induce apoptosis, and 1 μM, 3 μM and 5 μM R406 were used, respectively. Cell ATP levels were detected after 24 h, and cell viability was evaluated by CellTiter-Glo assay.

[0269] like Fig.14 As shown, when TRAIL alone treated E0771 cells, its IC 50 is 37.77nM. With the addition of R406, the IC 50 It showed a gradually decreasing trend, and the higher the concentration of R406, the higher the IC of TRAIL 50 The lower.

[0270] like Fig.12 and 16 Western Blot results showed that in LLC and E0771 cells, R406 could promote TRAIL-induced PARP1 cleavage and RIPK1-S166 phosphorylation, thereby promoting RIPK1 activation.

[0271] This indicates that R406 / R788 can promote TRAIL-induced apoptosis in E0771 and LLC cells.

[0272] Example 3 Inhibitory effect of R788 combined with TRAIL on LLC and E0771 transplanted tumors in mice

[0273] 1 Materials and methods

[0274] 1.1 Test Articles

[0275] Name:R788

[0276] Item No.: S2625

[0277] Appearance: White powder

[0278] Purity: 98%

[0279] Source: Selleck

[0280] Storage: Store at -20℃ before preparation

[0281] 1.2 Combined drug protein

[0282] mTRAIL: Purified endotoxin-free mTRAIL was prepared in endotoxin-free PBS at a concentration of 10 mg / kg.

[0283] 1.3 Experimental animals

[0284] C57BL / 6 mice, male and female, weighing 18-22 g, were purchased from Shanghai Lingchang Biotechnology Co., Ltd. The breeding environment was: SPF animal room, free food intake, 12 h light / 12 h dark.

[0285] 1.4 Small animal in vivo imaging experiment (bioluminescence imaging BLI)

[0286] 1) First, 100 μL of D-Luciferin substrate was injected into the mouse at a dose of 150 mg / kg;

[0287] 2) Place the mouse in an anesthesia box, and then place it in the operating platform of the small animal in vivo imaging instrument after isoflurane anesthesia for no more than 5 minutes;

[0288] 3) When the internal control platform of the small animal live imaging instrument rises to a suitable position, manually turn on the background plate lighting, that is, the bright field, and take the first white light background image;

[0289] 4) Then turn off the light manually, and then switch to the bioluminescence channel, i.e., the luciferase channel, to take a picture of the bioluminescence emitted from the mouse;

[0290] 5) The superposition of the bright field and dark field background images can intuitively display the location and intensity of specific photons in the animal body, completing the imaging operation.

[0291] 1.5 Hematoxylin-eosin staining (HE staining)

[0292] 1) The tumor tissue was embedded in paraffin, sliced ​​into 4 μm thick sections, spread in 42°C water and placed in a 37°C oven. The dried tissue sections were dewaxed the next day using xylene for 5-10 minutes.

[0293] Replace with fresh xylene and dewax for another 5-10 minutes.

[0294] 2) Dehydrate with ethanol, first dehydrate with anhydrous ethanol for 5 minutes, then dehydrate with 90% ethanol for 2 minutes, 80% ethanol for 2 minutes, and 70% ethanol for 2 minutes, and finally soak in distilled water for 5-10 minutes.

[0295] 3) For the above treated samples, stain them with hematoxylin staining solution for 5-10 minutes.

[0296] 4) Soak in tap water to rinse off excess staining solution for about 10 minutes.

[0297] 5) Rinse again with distilled water (for a few seconds).

[0298] 6) Stain with eosin solution for 30 seconds to 2 minutes.

[0299] 7) Dehydrate with ethanol, using 70% ethanol for 10 seconds, 80% ethanol for 10 seconds, 90% ethanol for 10 seconds, and anhydrous ethanol for 10 seconds.

[0300] 8) Then use xylene to make it clear for 5 minutes, then use fresh xylene to make it clear for another 5 minutes.

[0301] 9) Use neutral gum or other mounting media to seal the slides.

[0302] 1.6 Immunohistochemical staining (IHC staining)

[0303] 1) Incubate the sections in xylene solution three times, 5 min each time.

[0304] 2) Place the slices in 100% ethanol and incubate the slices twice, each time for 10 minutes.

[0305] 3) Place the slices in 95% ethanol and incubate the slices twice, each time for 10 minutes.

[0306] 4) Wash the sections twice with ddH2O, 5 min each time.

[0307] 5) Immerse the sections in 1* citrate repair solution and heat in a microwave until boiling.

[0308] 6) Continue to maintain the sub-boiling temperature for 10 minutes and cool the slices on the laboratory bench for 30 minutes.

[0309] 7) Wash the sections 3 times with ddH2O, 5 min each time.

[0310] 8) Place the slices in a 3% hydrogen peroxide solution and incubate for 10 minutes.

[0311] 9) Wash the sections twice with ddH2O, 5 min each time.

[0312] 10) Wash the sections with wash buffer for 5 min.

[0313] 11) Use 100 μL-400 μL of blocking solution to block each section at room temperature for 1 hour.

[0314] 12) Remove the blocking solution and add 100 μL-400 μL of Incubate with primary antibody diluted in Antibody Diluent at 4°C overnight.

[0315] 13) Use Equilibrate Boost Detection Reagent to room temperature.

[0316] 14) Remove the antibody solution and wash the antibody three times with washing buffer, 5 min each time.

[0317] 15) Apply 1-3 drops on the slices as needed Boost Detection Reagent and incubate at room temperature in a humidified chamber for 30 min.

[0318] 16) Wash the sections three times with washing buffer, 5 min each time.

[0319] 17) Add 1 drop (30 μL) DAB Chromogen Concentrate into 1 ml of SignalStainDAB Diluent and mix well before use.

[0320] 18) Apply 100μL-400μL to each slice DAB and observe closely. Usually, appropriate staining intensity can be obtained within 1-10 minutes.

[0321] 19) Immerse the sections in ddH2O.

[0322] 20) Wash the sections twice with ddH2O, 5 min each time.

[0323] 21) Dehydrate the sections by incubating them in 95% ethanol twice for 10 s each time, then repeating the incubation in 100% ethanol twice for 10 s each time, and finally repeating the incubation in xylene twice for 10 s each time.

[0324] 22) Mount the sections using coverslips and mounting media.

[0325] 1.7 Experimental procedures

[0326] 1) Six-week-old C57BL / 6 male and female mice were placed in a sterile environment with artificially controlled light, humidity and temperature for at least one week (light-dark cycle with light from 7:00 to 17:00, with a gradual change from light to dark, temperature 21-25℃ and humidity 45-65%), and the experiment was performed after one week of stabilization;

[0327] 2) Before the experiment, weigh the mice to ensure that the weight fluctuation of all mice does not exceed 1g;

[0328] 3) LLC cells and ZsGreen-LUC-expressing E0771 cells were cultured in 15 cm dishes, digested with trypsin and centrifuged, the culture medium and trypsin were washed off with PBS, and the cells were resuspended in serum-free DMEM (1,000,000 cells were resuspended in 100 μl DMEM);

[0329] 4) The LLC cell suspension was subcutaneously injected into the armpit of male mice at a cell concentration of 1.0*10 6 -1.5*10 6 between;

[0330] 5) The resuspended solution of ZsGreen-LUC-expressing E0771 cells was subcutaneously injected near the mammary gland of female mice at a cell concentration of 1.0*10 6 -1.5*10 6 between;

[0331] 6) When the tumor of the mouse is visible to the naked eye (tumor volume is 60mm 3 The mice were divided into groups, and the mice that were not successfully vaccinated were eliminated. The successfully vaccinated mice were evenly divided into 4 groups, namely, the control group, the R788 group, the TRAIL group, and the TRAIL+R788 group;

[0332] 7) After grouping, mice were gavaged with R788 (200 μl R788 dissolved in CMC-Na solution) every day according to the treatment group, and mTRAIL was injected intraperitoneally once a day;

[0333] 8) Use a vernier caliper to measure the long and short diameters of the subcutaneous tumor every day and record the tumor volume of the mouse (V = a*b 2 / 2,a:the length of the tumor; b:the width of the tumor) and the weight of the mice.

[0334] 9) LLC tumor inoculation lasted for 17 days and drug treatment lasted for 14 days;

[0335] 10) On the 11th and 15th days after inoculation of ZsGreen-LUC-expressing E0771 tumors, mice were injected orthotopically with D-Luciferin (150 mg / kg), and the changes in light intensity were detected by a small animal in vivo imaging device to monitor the drug effect;

[0336] 11) ZsGreen-LUC-expressing E0771 tumors were inoculated for 16 days and drug treatment lasted for 11 days.

[0337] 1.8 Experimental Results

[0338] 1.8.1 R788 enhances the anti-tumor effect of TRAIL in the mouse E0771 model (statistical analysis)

[0339] The constructed ZsGreen-LUC-expressing E0771 tumor cells were implanted into C57BL / 6 female mice. On the 11th and 15th days, the mice were injected with D-Luciferin (150 mg / kg) in situ. The light intensity changes were detected by a small animal in vivo imager to monitor the effect of the drug.

[0340] like Fig.15 As shown, the tumor size of the mice in the R788 alone treatment group and the TRAIL alone treatment group was reduced compared with the control group. After the combination of TRAIL and R788, the tumors of the mice were not only significantly reduced compared with the control group, but also significantly reduced compared with the tumors of the mice in the R788 alone and TRAIL alone groups.

[0341] The growth curve of the tumor and the size of the tumor were statistically analyzed, such as Fig.16 As shown, a total of 32 6-8 week old C57BL / 6 female mice successfully inoculated with ZsGreen-LUC-expressing E0771 tumor cells were used for the experiment, 8 mice were used for each experiment, and there were 4 experimental groups in total. Starting from the first day of the treatment, the volume of the mouse tumor and the weight of the mouse were recorded every day. After 11 days of treatment, the tumor of the mouse was weighed in vitro, and the statistical results were compared and analyzed.

[0342] Compared with the control group, the tumor growth rate of the two groups of mice treated with R788 alone and TRAIL alone was slowed down. When the drug was stopped, the tumor volume was about 1500mm 3 When the drug was stopped in the control group, the tumor volume was about 2300mm 3 The tumor volume of the mice treated with R788 and TRAIL was significantly slower than that of the control group and the groups treated with R788 or TRAIL alone. When the drug was stopped, the tumor volume of the mice treated with the combination drug was about 700 mm 3 .

[0343] The statistical results of the in vitro weighing of ZsGreen-LUC-expressing E0771 tumors are shown in Fig.17 As shown, the single administration of R788 and TRAIL had a certain anti-tumor effect, but the combined use of R788 and TRAIL had a better effect.

[0344] 1.8.2 R788 enhances the anti-tumor effect of TRAIL in the mouse E0771 model (tissue section analysis)

[0345] The shearing of Caspase-3 represents the occurrence of cell apoptosis. The larger the shearing area, the more intense the apoptosis. Fig.18 As shown, after the combination of R788 and TRAIL, the immunohistochemical staining area of ​​cleaved Caspase-3 in the mouse ZsGreen-LUC-expressing E0771 tumor tissue sections was the largest, proving that cell apoptosis occurred most obviously at this time.

[0346] This proves that R788 has an anti-tumor effect by promoting TRAIL-induced cell apoptosis, thereby slowing down the growth rate of tumors.

[0347] Ki-67 is a marker of cell proliferation. The deeper the Ki-67, the more vigorous the cell proliferation. Fig.19 As shown, Vehicle has the darkest color and the ZsGreen-LUC-expressing E0771 tumor cells proliferate most vigorously. Compared with the control group, Ki-67 is weaker in TRAIL alone and R788 alone, and the tumor cell proliferation ability is reduced. The contrast is more obvious in the TRAIL+R788 group. Compared with the control group, Ki-67 has a significant change and has become significantly weaker, and the tumor cell proliferation ability is the worst.

[0348] CD31 is a marker for endothelial cells. The darker the CD31 immunohistochemical staining, the faster the endothelial cells grow, the greater the density of microvessels, and the richer the nutrition supplied to tumor cells, which will accelerate the growth of tumor cells.

[0349] like Fig. 20 As shown, Vehicle had the darkest immunohistochemical staining and the fastest endothelial cell proliferation rate, while CD31 was weaker in R788 alone and TRAIL alone compared with the control group, and the endothelial cell proliferation ability decreased. The contrast was more obvious in the TRAIL+R788 group. Compared with the control group, CD31 had a significant change and became significantly weaker. The endothelial cell proliferation ability was the worst, the blood vessels were the least, the nutrition supplied to the tumor cells was the poorest, and the tumor cells grew the slowest.

[0350] 1.8.3 R788 enhances the anti-tumor effect of TRAIL in the LLC mouse model (statistical analysis)

[0351] like Fig.21As shown, 17 days after LLC was subcutaneously inoculated in C57BL / 6 mice, the tumors of the mice treated with drugs in the four groups were taken out for comparison as shown in the figure. The tumor size of the R788-treated group was not significantly different from that of the control group, and the tumor size of the mice in the TRAIL-treated group was smaller than that of the control group. After TRAIL and R788 were combined, the tumor size of the mice was not only significantly reduced compared with the control group, but also significantly reduced compared with the tumor size of the mice in the TRAIL-treated group. The growth curve of the tumor and the tumor size were statistically analyzed.

[0352] like Fig. 22 As shown, a total of 24 6-8 week old C57BL / 6 male mice successfully inoculated with LLC tumor cells were used for the experiment, and 6 mice were used in each experiment, with a total of 4 experimental groups. Starting from the first day of the drug treatment, the volume of the mouse tumor and the weight of the mouse were recorded every day. After 14 days of drug treatment, the tumor of the mouse was weighed in vitro, and the statistical results were compared and analyzed.

[0353] There was no significant difference in tumor growth rate between the R788 alone group and the control group. After 13 days of inoculation, both groups showed exponential growth rate. When the administration was stopped, the tumor volume was nearly 1000mm 3 In the group of mice that were intraperitoneally injected with TRAIL alone, the growth rate of tumors slowed down compared with the control group 14 days after inoculation. When the drug was stopped, the tumor volume was about 600mm 3 The tumor volume of the mice treated with R788 and TRAIL was significantly slower than that of the control group and the group treated with TRAIL alone. When the drug was stopped, the tumor volume of the combined drug group was about 300mm 3 .

[0354] The statistical results of LLC tumor ex vivo weighing are as follows Fig. 22 As shown, R788 alone had no effect, TRAIL had a certain anti-tumor effect, but the combination of R788 and TRAIL had a better effect.

[0355] 1.8.4 R788 enhances the anti-tumor effect of TRAIL in the mouse LLC model (tissue section analysis)

[0356] like Fig.23As shown in the figure, the tumor tissue sections of mice treated with R788 alone showed basically no damage and necrosis compared with the control group. Some damage and necrosis areas can be clearly found in the tissue sections of mice after intraperitoneal injection of TRAIL, but the area of ​​cell death is small. After the combination of R788 and TRAIL, the area of ​​tissue damage and cell necrosis is significantly enlarged, accompanied by a large area of ​​bleeding spots. It is speculated that R788 can promote TRAIL-induced cell death in mouse LLC tumor tissue. The immunohistochemical staining results of Ki-67, CD31 and cleaved Caspase-3 show that R788 does indeed promote TRAIL-induced cell apoptosis, thereby slowing down the growth rate of tumors and playing an anti-tumor effect.

[0357] 1.8.5R788 promotes TRAIL-induced activation of RIPK1 and caspase (in vivo)

[0358] like Fig.24 As shown, 1 / 2 / 3 represent the numbers of mice treated with drugs in each group. There are 6 mice in each group, and tumor tissue samples of 3 mice were randomly selected for Western Blot detection. The results showed that neither R788 alone nor the control group could activate RIPK1 and caspase; while the phosphorylation of RIPK1-S166 site in tumor tissue of mice injected intraperitoneally with TRAIL alone was slightly activated, and caspase-3 and PARP1 cleavage occurred at the same time; however, when R788 and TRAIL were used in combination, the phosphorylation of RIPK1-S166 site was significantly enhanced, and the cleavage of caspase-3 and PARP1 was also more obvious.

[0359] This indicates that R788 can be well absorbed in vivo and can promote TRAIL-induced activation of RIPK1 and caspase in LLC and E0771 tumor tissues.

[0360] 2 Discussions

[0361] 2.1 The anti-tumor effect of R788, a phosphate of R406, was evaluated by evaluating its ability to promote apoptosis of tumor cells mediated by other death receptor family members such as TRAIL in mice. Existing experimental data have shown that the compound has the most obvious effect in the mouse lung cancer LLC and breast cancer E0771 cells. In addition, in the mouse tumor models of these two cells, whether through biological characteristics observation, HE staining, or immunohistochemistry, it has been proven that R788 can promote the anti-tumor effect of TRAIL.

[0362] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A compound as represented by formula (I), or a pharmaceutically acceptable salt, hydrate or solvate thereof, in, n is 0, 1, 2, 3, 4 or 5; Ring A is selected from the group consisting of: saturated or partially unsaturated C 3-8 Carbocyclic group, saturated or partially unsaturated 5-10 membered heterocyclic group, C 6-10 Aryl, 5-12 membered heteroaryl; R1 is selected from the following group: H, D, C 1-6 Alkyl, C 2-6 Acyl, C 2-6 Ester group, C 1-6 Halogenated alkyl, saturated or partially unsaturated C 3-8 Carbocyclic group, saturated or partially unsaturated 5-10 membered heterocyclic group, C 6-10 Aryl, 5-12 membered heteroaryl; R2 and R3 are each independently selected from the following group: 1-6 Alkyl, C 2-6 Acyl, C 1-6 Haloalkyl; Alternatively, R2 and R3 and the atoms therebetween together form a substituted or unsubstituted 5-10 membered saturated or partially unsaturated heterocyclic group, or a substituted or unsubstituted 5-10 membered heteroaryl group; X1 is selected from the group consisting of O, NH, NR4, CH2, CHR4, C(R4)2; Each R4 is independently selected from the following groups: D, C 1-6 Alkyl, C 2-6 Acyl, C 1-6 Haloalkyl, -C 1-3 Alkyl-OP(O)(OH)2, -C 1-3 Alkyl-OS(O)2OH; Each L1 is independently selected from the following group: none, -O-, -NH-, -C 1-6 Alkyl-, -OC 1-6 Alkyl-, -NH-C 1-6 alkyl-; Each R5 is independently selected from the group consisting of hydrogen, CN, C 1-6 Alkyl-C(O), C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkylamino, saturated or partially unsaturated C 3-8 Carbocyclic group, saturated or partially unsaturated 5-10 membered heterocyclic group, C 6-10 Aryl, 5-12 membered heteroaryl; R 11 Selected from the group consisting of halogen, CN, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Alkenyl, C 1-6 Alkynyl; Among them, ring A, R1, R2, R3, R4, R5, R 11 Each independently is one or more (e.g., 2, 3, 4, etc.) R e substituted, and the R e Selected from the group consisting of D, hydroxy, halogen, cyano, amino, oxo (=O), C 1-6 Alkyl, C 1-6 Alkoxy, NHR f 、C(O)NHR f 、C(O)R f 、NHC(O)R f 、C(O)OR f 、OC(O)R f 、-S(O)2NHR f 、-S(O)2R f , saturated or partially unsaturated C 3-8 Carbocyclic group, saturated or partially unsaturated 5-10 membered heterocyclic group, C 6-10 Aryl, 5-12 membered heteroaryl; and the Re may be substituted by one or more (e.g., 2, 3, 4, etc.) unsubstituted or halogenated Rf; R f Selected from the group consisting of halogen, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, methylsulfone, -S(=O)2NH2, -S(=O)2NHCH3, oxo (=O), -CN, hydroxyl, -NH2, C 1-6 Alkyl-NH-, carboxyl, C1-C6 amide (-C(=O)-N(Rc)2 or -NH-C(=O)(Rc, Rc is H or C1-C5 alkyl), C 6-10 Aryl, 5-10 membered heteroaryl, 5-10 membered heterocyclic group, -(CH2)-C 6-10 Aryl, -(CH2)-(5-10 membered heteroaryl having 1-3 heteroatoms selected from N, S and O); wherein the carbocyclic group, heterocyclic group, aryl group, heteroaryl group may be a monocyclic, spirocyclic, bridged, fused or condensed ring structure; the heterocyclic group or heteroaryl group may have 1-3 heteroatoms selected from N, S and O on the ring skeleton; Furthermore, the compound is not a structure selected from the following group:

2. The compound according to claim 1, or a pharmaceutically acceptable salt, hydrate or solvate thereof, characterized in that: The ring A is selected from the following group:

3. The compound according to claim 1, or a pharmaceutically acceptable salt, hydrate or solvate thereof, characterized in that: The R5 is selected from the following group: saturated or partially unsaturated C 3-6 Carbocyclic group, saturated or partially unsaturated 5-8 membered heterocyclic group, C 6-10 Aryl, 5-10 membered heteroaryl; and said R5 is substituted or unsubstituted. In another preferred embodiment, R5 is selected from the following group:

4. The compound according to claim 1, or a pharmaceutically acceptable salt, hydrate or solvate thereof, characterized in that: The R1 is selected from the following group: H, C 1-6 Alkyl, C 2-6 Acyl, C 2-6 Ester group, C 1-6 Halogenated alkyl. In another preferred embodiment, the compound of formula I has a structure as shown below: in, L1 is selected from the following group: none, -O-, -NH-, -C 1-6 Alkyl-, -OC 1-6 Alkyl-, -NH-C 1-6 alkyl-; R5 is selected from the following group: CN, C 1-6 Alkyl-C(O), C 1-6 Haloalkyl, C 1-6 Alkylamino, saturated or partially unsaturated C 3-8 Carbocyclic group, saturated or partially unsaturated 5-10 membered heterocyclic group, C 6-10 Aryl, 5-12 membered heteroaryl; and said R5 may be substituted or unsubstituted. In another preferred embodiment, R5 is a 5-7 membered saturated carbocyclic or heterocyclic ring, and said R5 may be substituted or unsubstituted. In another preferred embodiment, R5 is a benzene ring or a 5-7 membered heteroaromatic ring, and the R5 may be substituted or unsubstituted. In another preferred embodiment, the ring A, R1, R2, R3, R4, R5, R 11 Each is independently substituted by one or more substituents selected from the group consisting of D, hydroxy, halogen, cyano, amino, oxo (=O), C 1-4 Alkyl, C 1-4 Halogenated alkyl, C(O)NH2, saturated or partially unsaturated C 3-8 Carbocyclic group, saturated or partially unsaturated 5-10 membered heterocyclic group, C 6-10 Aryl, 5-12 membered heteroaryl. In another preferred embodiment, the A ring is a benzene ring or a pyridine ring.

5. The compound according to claim 1, or a pharmaceutically acceptable salt, hydrate or solvate thereof, characterized in that: The compound is selected from the following group:

6. A pharmaceutical composition, characterized in that The composition comprises: (1) a therapeutically effective amount of a first active ingredient, wherein the first active ingredient is R406 or a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, hydrate or solvate thereof; (2) a therapeutically effective amount of a second active ingredient, wherein the second active ingredient is tumor necrosis factor-related apoptosis-inducing ligand (TRAIL); Optional (3) a pharmaceutically acceptable carrier; Preferably, the first active ingredient and the second active ingredient act in combination to promote the sustained activation of TAK1 kinase.

7. The composition according to claim 6, characterized in that The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is selected from the following group: membrane TRAIL (mTRAIL) and soluble TRAIL (sTRAIL).

8. Use of the compound according to any one of claims 1 to 5, its pharmaceutically acceptable salt, hydrate or solvate, or the pharmaceutical composition according to claim 6, characterized in that: The drug is used in combination with TRAIL to promote programmed cell death of tumor cells by continuously activating TAK1 kinase, and is used to treat tumors and / or inhibit tumor metastasis.

9. The use according to claim 8, characterized in that The tumor is selected from the group consisting of lung cancer, liver cancer, colon cancer, stomach cancer, breast cancer, ovarian cancer, melanoma, lymphoma, blood tumor, pancreatic cancer, bone cancer and endocrine system cancer.

10. An in vitro non-diagnostic non-therapeutic method for inhibiting tumor cells, comprising the steps of: A therapeutically effective amount of a compound according to any one of claims 1 to 5 and a therapeutically effective amount of TRAIL are administered to tumor cells.

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