Anti-tumor use of r406 and derivative thereof in synergy with trail

Abstract

Provided are a series of compounds having the general formula as shown in formula (I), pharmaceutically acceptable salts, hydrates or solvates thereof, and a use of the compounds in combination with TRAIL in the treatment of tumors or inhibition of tumor cells. The pharmaceutical composition is used for resisting tumors, has the characteristics of good safety, strong tolerance, high sensitivity, etc., and is suitable for development of anti-tumor drugs.

Description

Anti-tumor application of R406 and its derivatives in synergy with TRAIL Technical Field

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

[0002] In recent years, the incidence and mortality of tumors have been increasing, posing new challenges to tumor treatment. Numerous studies have shown that TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in tumor cells, with virtually no significant toxic side effects on normal tissue cells. In human cells, there are four types of TRAIL surface receptors: TRAIL-R1, TRAIL-R2, TRAIL-R3, and TRAIL-R4. TRAIL-R1 and TRAIL-R2 contain a death domain (DD) within their cells. Upon TRAIL stimulation, TRAIL-R1 and TRAIL-R2 can initiate apoptosis by recruiting the death domain-containing protein FADD. TRAIL-R3 and TRAIL-R4 are decoy receptors that do not contain a death domain within their cells, and therefore do not induce apoptosis upon TRAIL stimulation. Different TRAIL receptors are differentially expressed in normal 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. These characteristics allow TRAIL to specifically induce tumor cell death, making research on its anti-tumor properties highly sought after. Currently, TRAIL-based treatments have been used in numerous clinical trials, including studies investigating TRAIL in non-small cell lung cancer (NSCLC), B-cell non-Hodgkin's lymphoma (NHL), and colorectal cancer. Although TRAIL demonstrated high tolerability and safety in Phase I clinical trials, multiple clinical trials have shown that the efficacy of TRAIL alone for tumor treatment is very limited. Numerous preclinical studies have demonstrated that most tumor cells are insensitive to TRAIL alone, leading to a growing number of studies exploring the combination of TRAIL with other small molecules to enhance tumor cell sensitivity to TRAIL. Currently, research on TRAIL combination therapy has entered the clinical stage, and clinical results have shown that TRAIL combination therapy has a good effect on tumor suppression. Therefore, the development of new small molecule drugs that can synergize with TRAIL to achieve better anti-tumor effects can provide a good reference and hope for subsequent targeted tumor treatment.

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

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

[0005] Summary of the Invention

[0006] 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.

[0007] In the first aspect of the present invention, there is provided a compound as represented by formula (I), or a pharmaceutically acceptable salt, hydrate or solvate thereof,

[0008] in,

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

[0010] 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;

[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 alkyl halide;

[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 (eg 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, methylsulfonyl, -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 fused 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] In some embodiments, the ring A is selected from the following group:

[0023] 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.

[0024] In another preferred embodiment, R5 is selected from the following group:

[0025] 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 1-6 Halogenated alkyl.

[0026] In another preferred embodiment, the compound of formula I has the structure shown below:

[0027] in,

[0028] L1 is selected from the following group: none, -O-, -NH-, -C 1-6 Alkyl-, -OC 1-6 Alkyl-, -NH-C 1-6 alkyl-;

[0029] R5 is selected from the following group: CN, C 1-6 Alkyl-C(O), C 1-6 Halogenated alkyl, C 1-6 Alkylamino, saturated or partially unsaturated C 3- 8-carbon ring 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.

[0030] In another preferred embodiment, R5 is a 5-7 membered saturated carbocyclic or heterocyclic ring, and said R5 may be substituted or unsubstituted.

[0031] In another preferred embodiment, R5 is a benzene ring or a 5-7 membered heteroaromatic ring, and the R5 may be substituted or unsubstituted.

[0032] 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.

[0033] In another preferred embodiment, the ring A is a benzene ring or a pyridine ring.

[0034] In some embodiments, the compound is selected from the group consisting of:

[0035] In a second aspect of the present invention, a pharmaceutical composition is provided, characterized in that the composition comprises:

[0036] (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;

[0037] (2) a therapeutically effective amount of a second active ingredient, wherein the second active ingredient is tumor necrosis factor-related apoptosis-inducing ligand (TRAIL);

[0038] Optional (3) a pharmaceutically acceptable carrier.

[0039] In some embodiments, the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is selected from the group consisting of membrane-bound TRAIL (mTRAIL) and soluble TRAIL (sTRAIL).

[0040] In a third aspect, the present invention provides a use of the compound according to the first aspect of the present invention, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein 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;

[0041] In another preferred embodiment, the compound directly binds to TAK1 and promotes the sustained activation of TAK1 kinase.

[0042] In another preferred embodiment, the compound is covalently coupled to the glutamic acid at position 40 of TAK1.

[0043] In another preferred embodiment, the tumor is associated with cell death induced by death receptor-related ligands such as tumor necrosis factor.

[0044] 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 cancer, pancreatic cancer, bone cancer, and endocrine system cancer.

[0045] 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:

[0046] 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.

[0047] 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 shows a schematic diagram of the chemical structures of R406 and its phosphate R788.

[0049] FIG2 shows the purity of hTAK1-TAB1 purified by sf9 expression analyzed by AUC.

[0050] Figure 3 shows the effects of R406 and R788 on the thermal stability of TAK1.

[0051] FIG4 shows that R406 promotes the phosphorylation of TAK1 downstream proteins in an in vitro kinase reaction.

[0052] FIG5 shows that R406 promotes TAK1 hyperactivation in an in vitro kinase reaction.

[0053] FIG6 shows the synthesis and use of R406 derivative photosensitive affinity probes.

[0054] FIG7 shows the binding site of R406 to TAK1 determined by photocross-linking mass spectrometry.

[0055] FIG8 shows that R406 / R788 can promote TNFα-induced RIPK1-dependent programmed cell death in FADD-deficient Jurkat cells.

[0056] FIG9 shows that R406 can promote TNFα-induced RIPK1-dependent programmed cell necrosis in HT29 cells.

[0057] FIG10 shows that R406 can promote TNFα-induced RIPK1-dependent programmed cell necrosis in NCI-H1975 cells.

[0058] FIG11 shows that R406 can promote TNFα-induced RIPK1-dependent apoptosis in NCI-H1299 cells.

[0059] FIG12 shows that R406 can promote TRAIL-induced apoptosis in E0771 cells.

[0060] FIG13 shows that the sensitivity of the modified ZsGreen-LUC-expressing E0771 cells to the compounds remains unchanged.

[0061] FIG14 shows that R406 can promote TRAIL-induced apoptosis in LLC cells.

[0062] FIG15 shows images of ZsGreen-LUC-expressing E0771 tumor tissues in C57BL / 6 mice.

[0063] FIG16 shows the changes in bioluminescence imaging of ZsGreen-LUC-expressing E0771 in C57BL / 6 mice.

[0064] FIG17 shows that R788 combined with TRAIL significantly inhibited tumor growth in the ZsGreen-LUC-expressing E0771 breast cancer model in C57BL / 6 mice.

[0065] FIG18 shows immunohistochemical staining of ZsGreen-LUC-expressing E0771 tumor tissue sections for Cleaved Caspase-3.

[0066] FIG19 shows immunohistochemical staining of ZsGreen-LUC-expressing E0771 tumor tissue sections for Ki-67.

[0067] FIG20 shows immunohistochemical staining of CD31 in ZsGreen-LUC-expressing E0771 tumor tissue sections.

[0068] FIG21 shows in vitro visualization images of LLC tumor tissues in C57BL / 6 mice.

[0069] FIG22 shows the effects of R788 combined with TRAIL on tumor volume and tumor weight in LLC tumor-bearing mice.

[0070] FIG23 shows 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.

[0071] FIG24 shows the effects of R788 combined with TRAIL on PARP1 cleavage and RIPK1 activation in tumor tissues of LLC tumor-bearing mice. DETAILED DESCRIPTION

[0072] Through extensive screening of a library of known active compounds, the inventors unexpectedly discovered that the small molecule R406 and its phosphate R788 can promote TRAIL-induced tumor cell death. R788, a prodrug of R406, has been approved by the FDA for the treatment of idiopathic thrombocytopenic purpura, demonstrating its good safety and tolerability in humans. This work led to the present invention.

[0073] the term

[0074] In the present invention, unless otherwise specified, the terms used have the general meanings commonly known to those skilled in the art.

[0075] In the present invention, the term "halogen" refers to F, Cl, Br or I.

[0076] 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.

[0077] In the present invention, the term "C 2-6 "Alkenyl" refers to a straight chain or branched alkenyl group having 2 to 6 carbon atoms and containing one double bond, including but not limited to vinyl, propenyl, butenyl, isobutenyl, pentenyl and hexenyl. The term "C 2-4 "Alkenyl" has a similar meaning.

[0078] In the present invention, the term "C 2-6 "Alkynyl" refers to a straight or branched chain 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.

[0079] 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 "C 3-6 "Cycloalkyl" has a similar meaning.

[0080] In the present invention, the term "C 1-6 "Alkoxy" refers to a straight or branched chain 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.

[0081] In the present invention, the term "heterocyclyl" or "heterocycloalkyl" refers to a 4-8 membered heterocyclic group containing 1, 2 or 3 heteroatoms selected from N, O and S, including (but not limited to) the following groups:

[0082] In the present invention, the term "aromatic ring" or "aryl group" has the same meaning, preferably "C 6-10 Aryl". The term "C 6- 10 The term "aryl" refers to an aromatic ring group having 6 to 10 carbon atoms and containing no heteroatoms, such as phenyl and naphthyl.

[0083] In the present invention, the term "aromatic heterocycle" or "heteroaryl" has the same meaning, referring to a heteroaromatic group containing one to multiple heteroatoms. For example, "C 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 furyl, thienyl, pyridyl, pyrazolyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is the heteroaryl ring. The heteroaryl group may be optionally substituted or unsubstituted.

[0084] In the present invention, the term "halo" means substituted with halogen.

[0085] In the present invention, the term "deuterated" means substituted with deuterium.

[0086] 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 site 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 a combination that is 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-membered cycloalkyl, 3- to 12-membered heterocyclic group, 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.

[0087] 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.

[0088] It should be understood that when a group is present at multiple different positions in a compound, its definition at each position is independent of each other and may be the same or different. That is, the term "selected from the group:" has the same meaning as the term "each independently selected from the group:".

[0089] Compound

[0090] 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 structure of the compound is shown in formula (I):

[0091] wherein each group is as defined above.

[0092] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention formed with an acid or base that is suitable for pharmaceutical use. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts are salts formed with 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.

[0093] Another preferred salt is a salt of the compound of the present invention formed with 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.

[0094] The present invention's various embodiments specifically describe methods for preparing compounds of formula I of the present invention, but these specific methods do not limit the present invention in any way. 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. Such combinations can be easily performed by those skilled in the art.

[0095] Typically, the raw materials and reagents used in the preparation of the compounds of the present invention can be purchased through commercial channels unless otherwise specified.

[0096] Pharmaceutical compositions and methods of administration

[0097] The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a safe and effective amount of one or more of the compounds.

[0098] 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.

[0099] The pharmaceutical composition of the present invention comprises a safe and effective amount of a compound of the present invention or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable excipient or carrier. "Safe and effective amount" means an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably 10-1000 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0100] "Pharmaceutically acceptable carriers" refer 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, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0101] The pharmaceutical composition is in the form of injection, capsule, tablet, pill, powder or granule.

[0102] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administration routes include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0103] 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 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.

[0104] Solid dosage forms such as tablets, dragees, 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 opacifying agents, and the release of the active compound or compounds in such compositions can be delayed in a certain portion of the digestive tract. Examples of useful encapsulating components are polymeric substances and waxes. If desired, the active compound can also be microencapsulated with one or more of the above-mentioned excipients.

[0105] 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, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, or mixtures thereof.

[0106] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0107] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0108] Compositions for parenteral injection may comprise 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.

[0109] Dosage forms for topical administration of the compounds of this 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.

[0110] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds (such as anti-tumor drugs).

[0111] The treatment method of the present invention can be used alone or in combination with other treatment methods or therapeutic drugs.

[0112] When using a pharmaceutical composition, a safe and effective amount of the compound of the present invention is administered to a mammal (e.g., a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage. For a 60 kg human, the daily dosage is generally 1 to 2000 mg, preferably 50 to 1000 mg. Of course, the specific dosage will also take into account factors such as the route of administration and the patient's health condition, all of which are within the skill of a skilled physician.

[0113] The main advantages of the present invention are:

[0114] 1. The present invention discovered that compounds that activate TAK1 kinase activity can promote TRAIL-induced tumor cell apoptosis and necrosis.

[0115] 2. The TAK1 kinase activator and TRAIL combination composition of the present invention can significantly inhibit the growth of tumor cells.

[0116] Preparation Example

[0117] General synthetic route of compounds:

[0118] 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:

[0119] To a 15 mL reaction flask, add the corresponding aminopyridine derivative (2.59 mmol), 5-fluoro-2,4-dichloropyrimidine (650 mg, 3.9 mmol), DIPEA (850 μL, 5.18 mmol), and DMF (5 mL). Heat to 90°C and stir overnight. After the reaction is complete, dilute with ice water, filter, and wash the filter cake with water and an organic mixed solvent (PE / DCM = 10 / 1) before drying to obtain the pyrimidine-substituted intermediate.

[0120] 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). The mixture was microwaved at 120°C for 1 h. After completion, the reaction solution was purified by reverse phase chromatography (H₂O / MeCN = 1:1) and HPLC to yield the title compound.

[0121] Among them, the preparation routes of various aminopyridine derivatives are as follows:

[0122] 1. Preparation of compound TXY-4-44

[0123] (1) Preparation of compound TXY-3-21:

[0124] To a round-bottom flask, 6-bromo-2-nitropyridin-3-ol (1.5 g, 6.85 mmol), potassium hydroxide (1.5 g, 27 mmol), and DMSO (20 mL) were added. The reaction was pre-stirred at room temperature for 5 min, followed by the addition of iodomethane (850 μL, 13.7 mmol) and continued stirring for 30 min. After completion of the reaction, the mixture was diluted with water and extracted with dichloromethane. The organic phase was washed sequentially with aqueous potassium hydroxide and saturated brine, concentrated, and purified by silica gel column chromatography using PE / EA (2:1) as the eluent to obtain the title compound in 63% yield. MS-ESI: m / z calculated for C6H5BrN2O3, Exact Mass 231.95, found 233.0 [M+H] + .

[0125] (2) Preparation of compound TXY-3-22:

[0126] 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 of ethanol, and 10 mL of water were added to a reaction flask. The mixture was heated to 70°C for 30 min, filtered while hot, washed with DCM, and the combined filtrates were washed sequentially with saturated sodium bicarbonate solution and saturated brine. The product was concentrated to give 670 mg of crude product in a 77% yield. MS-ESI: m / z calculated for C6H7BrN2O, Exact Mass 201.97, found 202.9 [M+H] + .

[0127] (3) Preparation of compound TXT-3-40:

[0128] Compound TXY-3-22 (300 mg, 1.48 mmol), paraformaldehyde (1.08 g, 4.4 mmol), 20 μL of acetic acid, and 1,2-dichloroethane (3 mL) were added to a reaction flask. The mixture was allowed to react at room temperature for 1 hour, then heated to 50°C. Sodium triacetoxyborohydride (800 mg, 3.77 mmol) was added in batches over 6 hours. After completion of the reaction, the solution was concentrated and purified by silica gel column chromatography using PE / DCM (1:1) as the eluent to afford 200 mg of a solid product in a 59% yield. MS-ESI: m / z calculated for C8H 11 BrN2O,Exact Mass 230.01,found 231.1[M+H] + .

[0129] (4) Preparation of compound TXY-4-44:

[0130] To a reaction flask, 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). Under nitrogen, heat to 90°C and react for 3 h. After completion, the reaction solution is diluted with water and extracted with DCM. The organic phase is washed sequentially with water and saturated brine, concentrated, and purified by silica gel column chromatography with PE / EA (2:1) as the eluent to obtain the crude product substituted with bromine from 2,4-dimethoxybenzylamine. The crude product is dissolved in DCM (1 mL), and 200 μL of trifluoroacetic acid is added. Stir at room temperature overnight. After the reaction was complete, the reaction solution was concentrated and purified by silica gel column chromatography using DCM / MeOH (2M NH3) (4:1) as eluent to obtain 50 mg of solid with a yield of 70%. MS-ESI: m / z calculated for C8H 13 N3O,Exact Mass 167.11,found 168.2[M+H] + .

[0131] 2. Preparation of compound TXY-3-60

[0132] (1) Preparation of compound TXY-3-26:

[0133] The preparation process of compound TXY-3-22 was the same, with a yield of 58%. MS-ESI: m / z calculated for C5H5BrN2O, Exact Mass 187.96, found 188.9[M+H] + .

[0134] (2) Preparation of compound TXY-3-58:

[0135] Compound TXY-3-26 (300 mg, 1.59 mmol) and THF (9 mL) were added to a reaction flask. Chloroacetyl chloride (150 μL in 1 mL THF, 1.9 mmol) was added dropwise at 0°C. The mixture was allowed to gradually return to room temperature and react for 2 hours. Triethylamine (660 μL, 4.77 mmol) was then 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 using PE / EA (3:1) as the eluent to obtain 248 mg of a solid in a 68% yield. MS-ESI: m / z calculated for C7H5BrN2O2, Exact Mass 227.95, found 229.1 [M+H] + .

[0136] (3) Preparation of compound TXY-3-60:

[0137] The preparation process of compound TXY-4-44 was the same, with a yield of 38%. MS-ESI: m / z calculated for C7H7N3O2, Exact Mass 165.05, found 166.0[M+H] + .

[0138] 3. Preparation of compound TXY-4-34

[0139] Similar to the preparation of TXY-4-44, after the coupling product was obtained in the first step and purified, 100 mg of the intermediate (0.32 mmol) was dissolved in DMF (1 mL), potassium carbonate (88 mg, 0.64 mmol) and iodomethane (30 μL, 0.48 mmol) were added, and the mixture was allowed to react at room temperature for 2 h. The mixture was then diluted with water and extracted with EA. The organic phase was washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the crude methylated product. The crude product was redissolved in DCM (1 mL), and 150 μL (1.9 mmol) of trifluoroacetic acid was added. The reaction was allowed to react at room temperature for 3 h, then concentrated and purified by silica gel column chromatography with PE / EA (1:1) as the eluent to obtain approximately 40 mg of a solid in a 70% yield. MS-ESI: m / z calculated for C8H9N3O2, Exact Mass 179.07, found 180.0 [M+H] + .

[0140] 4. Preparation of compound TXY-4-41

[0141] (1) Preparation of compound TXY-4-40:

[0142] The preparation process was the same as that of TXY-4-44. This product was purified using reverse phase chromatography, eluting with H2O / MeCN (1:1), with a yield of 55%. MS-ESI: m / z calculated for C8H 11 N3O4,Exact Mass 213.07,found 214.1[M+H] + .

[0143] (2) Preparation of compound TXY-4-41:

[0144] Compound TXY-4-40 (100 mg, 0.47 mmol), Pd / C (15 mg), and 5 mL of methanol were added to a round-bottom flask and filled with hydrogen gas. The reaction was allowed to proceed for 3 h. After the reaction was complete, the mixture was filtered and the filtrate was concentrated to yield 80 mg of a black liquid, which was used directly in the next reaction with a yield of 93%. MS-ESI: m / z calculated for C8H 13 N3O2,Exact Mass 183.10,found 184.0[M+H] + .

[0145] 5. Preparation of compound TXY-3-34

[0146] 2,6-Dichloro-3-nitropyridine (500 mg, 2.59 mmol) was dissolved in 7 M methanolic ammonia (10 mL) and heated to 100°C for 30 min in a microwave oven. After completion of the reaction, the reaction solution was concentrated to yield a pale yellow solid, which was washed with a small amount of DCM and dried to obtain 350 mg of crude product, which was used directly in the next reaction with an 88% yield. MS-ESI: m / z calculated for C5H6N4O2, Exact Mass 154.05, found 155.1 [M+H] + .

[0147] 6. Preparation of compound TXY-4-59

[0148] Compound TXY-3-34 (150 mg, 1 mmol), Pd / C (15 mg) and 5 mL of methanol were added to a round-bottom flask, filled with hydrogen, and stirred at room temperature overnight. The intermediate was detected by LCMS (125 [M+H] +The reaction mixture was filtered and concentrated to obtain a blue solid. DMF (5 mL) was added to dissolve the mixture. N,N'-disuccinimidyl carbonate (DSC) (250 mg, 1 mmol) was slowly added dropwise at 0°C under nitrogen protection. After the addition was complete, the temperature was gradually returned to room temperature and the reaction was allowed to react for 3 h. After the reaction was complete, an appropriate amount of silica gel was added to the reaction mixture, which was concentrated and purified by silica gel column chromatography using DCM / MeOH (2M NH3) (5:1) as the eluent to obtain 50 mg of the product in a 33% yield. 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,found 151.0[M+H] + .

[0149] 7. Preparation of compound TXY-4-70

[0150] To a reaction flask were added 2,2-dimethyl-6-nitro-2H-pyrido[3,2-B][1,4]oxazin-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). The reaction was heated to 60°C for 1 h. After completion of the reaction, the mixture was concentrated and purified by silica gel column chromatography using PE / EA (3:1) as the eluent. The resulting product was directly added to MeOH (5 mL) with Pd / C (15 mg) and catalytic hydrogenation was performed by introducing hydrogen. The reaction was allowed to proceed overnight, filtered, and concentrated to afford 100 mg of the desired product in a 90% overall yield. MS-ESI: m / z calculated for C 11 H 15 N3O3,Exact Mass 237.11,found 238.2[M+H] + .

[0151] The structures of the compounds obtained by the general method are shown in Table 1, and the NMR data are shown in Table 3.

[0152] Biological Test Examples

[0153] Example 1 R406 and its phosphate R788 directly bind to and activate TAK1

[0154] 1 Experimental Materials and Methods

[0155] 1.1 Drugs and Reagents

[0156] 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 medium and fetal bovine serum were purchased from Gibco (Life Technologies, Grand Island, NY, USA).

[0157] The antibodies used are as follows: p-S321 RIPK1, 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 Santa Cruz, and mouse anti-Tubulin was purchased from Sigma-Aldrich.

[0158] 1.2 Western blot analysis

[0159] 1) Vertical protein electrophoresis separation: 8%-10% SDS-polyacrylamide (SDS-PAGE) gel electrophoresis separation, 5% stacking gel. Separation voltage of the stacking gel is 80V, and separation voltage of the separating gel is 120V. The bromophenol blue indicator stops when it reaches the bottom.

[0160] 2) Transfer: Semi-dry transfer method, transfer current is 0.2-0.4V, and the time is adjusted according to the size of the protein.

[0161] 3) Pre-stain with Ponceau red and observe the transfer effect.

[0162] 4) Blocking: Block with TBST containing 5% skim milk at room temperature on a decolorizing shaker for 1 hour.

[0163] 5) Binding of primary antibody: Dilute the antibody 1:2000-1:10000 in TBST containing 5% BSA. Allow to bind on a shaker at room temperature for 1 hour, then overnight at 4°C. Wash three times with TBST for 10 minutes each.

[0164] 6) Binding secondary antibody: Dilute the antibody at 1:5000-1:10000 in TBST containing 5% BSA and shake on a decolorizing shaker at room temperature for 1 hour. Wash three times with TBST, 10 minutes each time.

[0165] 7) Develop the ECL kit substrate for 2-3 minutes, detect by chemiluminescence, and expose to X-ray film.

[0166] 1.3 Cell viability assay

[0167] Cells in the logarithmic growth phase were seeded at an appropriate density in 384-well culture plates, with 40 μL per well. After overnight culture, different drug concentrations were added for different time points, with triplicate wells set for each concentration. After the treatment period, 15 μL / well of ATP was added to each well for 15 minutes, and chemiluminescence was measured using a microplate reader.

[0168] 1.4 In vitro kinase reaction

[0169] 1) WT-RIPK1 and IKKβ-K44A were overexpressed in HEK293T cells, and 5Z7 (500 nM) and Nec-1s (10 μM) were added during transient transfection.

[0170] 2) 24 hours after transfection, cells were lysed with NP-40 lysis buffer and centrifuged at 12000 rpm for 20 minutes;

[0171] 3) After centrifugation, RIPK1 and IKKβ in the supernatant were enriched and purified using Flag-labeled beads. The beads were washed 6-8 times with high-salt NP-40 buffer to remove as much contaminants as possible, and then equilibrated with kinase buffer 3-5 times.

[0172] 4) Divide the Flag beads into several equal portions and incubate them with 5 mM R406 and 1 μM 5Z7 for 30 minutes respectively. Incubate the beads at 30°C for 60 minutes.

[0173] 5) Add 2* loading buffer to boil the sample and terminate the reaction.

[0174] 1.5 Protein thermal stability test

[0175] 1) Adjust the concentration of hTAK1-TAB1 fusion recombinant protein expressed and purified by sf9 to 3 μM. Mix the recombinant protein with 10 μM R406 and R788, respectively, and let it stand at room temperature for 2 hours. Then, add 5X SYRO orange protein gel stain and incubate in the dark for 30 minutes.

[0176] 2) Seal the q-PCR plate with a transparent sealing film;

[0177] 3) Set the Q7 thermoshift program to increase the temperature from room temperature to 105°C. The program should run for approximately 1.5 hours.

[0178] 4) After the program is completed, Protein Thermal Shift Microsoft (PTSM) software is used to process and analyze the data.

[0179] 1.6 Photocrosslinking mass spectrometry

[0180] 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 used is approximately 2.5 mg / ml (0.6 mg / ml).

[0181] 2) Alternatively, the photoaffinity probe and its control compound were incubated with the protein for 2 h, with the compound concentration being 10 μM and the protein concentration being 2 mg / ml, for a total of 300 μl of the system solution;

[0182] 3) The mixture of the small molecule compound and protein incubation solution was divided into several equal portions and added to a 96-well plate, with 30 μl of the reaction solution added to each well. The mixture was then photocrosslinked under UV light. After 30 minutes, the equal portions were remixed and the SDS concentration was increased to 1%.

[0183] 4) Then, perform the click chemistry reaction. TCEP should be prepared immediately and react at 25°C, 700-800 rpm, for 40 min-1 h.

[0184] 5) Then, add 4 times the reaction volume of methanol, 1 times the reaction volume of chloroform, and 3 times the reaction volume of distilled water. Mix all the reaction mixtures evenly, place on ice for 40 minutes, centrifuge at 12,000 rpm for 20 minutes, wash twice with 1 ml of 4°C pre-cooled methanol, spin dry in a desiccator, and redissolve.

[0185] 6) Enrich the target protein using beads containing Biotin and bind overnight at 4°C;

[0186] 7) Centrifuge at 5000 rpm for 15 min, discard the supernatant, and repeat three times;

[0187] 8) Wash with ddH2O 7-10 times;

[0188] 9) After processing, the sample is sent to the mass spectrometry platform for analysis.

[0189] 1.7 Experimental Results

[0190] 1.7.1 R406 directly binds to TAK1

[0191] Currently, recombinant protein expression of the TAK1 kinase domain is typically performed using insect cells. Since TAK1 only possesses kinase activity when fused to TAB1, TAK1-TAB1 was expressed using an insect cell expression system, as shown in Figure 2. This system was then used to study the binding of R406 and R788 to TAK1-TAB1. Protein thermal stability can be used to investigate changes in protein stability when binding to small molecules or other proteins. When small molecules bind to a protein, they stabilize its globular structure, causing changes in the protein's Tm value during thermal stability experiments. Therefore, changes in Tm can reflect the binding activity.

[0192] R406 and R788 were incubated with hTAK1-TAB1 protein for 60 minutes respectively, and then the thermal stability experiment was started. The results are shown in Figure 3. From the Tm value and thermal stability fluorescence change spectrum of TAK1, it can be seen 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.

[0193] 1.7.2 R406 directly activates TAK1

[0194] Flag-RIPK1 and Flag-IKKβK44A plasmids were overexpressed in HEK293T cells, respectively. To minimize the background autophosphorylation of RIPK1, 10 μM Nec-1s was added during overexpression. 24 h later, RIPK1 and IKKβ were immunoprecipitated using Flag beads, respectively. Then, in vitro kinase reactions were performed with recombinant His-tagged TAK1-TAB1 protein. At the same time, 20 μM R406 and 1 μM 5Z7 were added, respectively. After incubation at 30°C for 60 min, the samples were boiled for Western detection, as shown in Figure 4.

[0195] The results showed that R406 promoted the phosphorylation of IKKβ and RIPK1-S321 in vitro, while 5Z7, as a control, inhibited their phosphorylation. As shown in Figure 5, R406 also promoted its own phosphorylation in vitro, as well as the phosphorylation of TAK1 peptide substrates. These data indicate that R406 can directly activate TAK1 in vitro.

[0196] 1.7.3 Design and Utilization of the R406 Photosensitive Affinity Probe

[0197] 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, a cell survival assay was performed to detect 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 targeting TAK1 were synthesized, as shown in Table 1, to assist in finding the interaction sites between R406 and TAK1, as shown in Table 2.

[0198] Table 1 R406 derivatives

[0199] Table 2 Activity data of various 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%)

[0200] Table 3 Characterization data of some compounds of the present invention

[0201] The results showed that the small molecules based on the structural modification of R406 were sequentially subjected to cell survival assays, and it was finally found that S-8-56 was the most active small molecule. T-5-27 was a photocrosslinking probe derived from S-8-56. Cells were treated with small molecules, or small molecules and proteins were co-incubated, as shown in Figure 6. After photocrosslinking and click chemistry reactions, the biotin-labeled target protein could be co-precipitated and detected through the biotin-avidin system.

[0202] The diazirine group of T-5-27 can undergo a photochemical reaction under ultraviolet light of a certain wavelength, allowing this part of the group to be covalently cross-linked to the vicinity of the target protein binding site, while the alkyne 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. An attempt is made to find the approximate binding site of T-5-27 on TAK1 by increasing the molecular weight peptide information shown in the figure.

[0203] As shown in Figure 6, the photosensitized affinity probe T-5-27 can indeed enrich and isolate TAK1 from cell lysates, whereas S-8-56, a derivative of the R406 photostable control small molecule, fails to coprecipitate TAK1. Furthermore, in the presence of S-8-56, the cross-linking effect of the probe is weakened, demonstrating the effectiveness of the photocross-linking probe.

[0204] Next, the probe was photocrosslinked to TAK1 protein in vitro. The probe was linked to the biotin via a click chemistry reaction. The probe-crosslinked TAK1 protein was then precipitated using streptavidin agarose beads. The TAK1 protein was then digested with trypsin, and peptides containing the probe and biotin were eluted for protein mass spectrometry analysis to identify the interaction sites between R406 or its derivatives and TAK1. As shown in Figure 7, if the peptide is linked to the probe and biotin, it will have an increased mass shift of 1000.4865 Da. The amino acids in the peptide that are linked to the probe and biotin can be identified through the secondary mass spectrometry spectrum. Finally, mass spectrometry analysis identified the peptide "35-EIEVEEVGR-44" as having a mass increase of 1000.4865 Da. This indicates that E6 in the peptide, or glutamic acid at position 40 of TAK1, is covalently coupled to the probe.

[0205] Example 2 Effects of R406 and its phosphate R788 combined with TNFα / TRAIL on tumor cells

[0206] 1 Experimental Materials and Methods

[0207] 1.1 Drugs and Reagents

[0208] R406 and its phosphate, R788, were purchased from Selleck and dissolved in dimethyl sulfoxide (DMSO) to a 10 mM stock solution, which was 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).

[0209] The antibodies used in the study are as follows: rabbit anti-PARP, p-S166 RIPK1, RIPK1, p-S227 RIPK3, RIPK3, p-S358 MLKL, Cleaved caspase-3, caspase 3, TAK1, p-IKKα / β, IKKα / β, FADD were all purchased from Cell signaling technology, and mouse anti-Tubulin was purchased from Sigma-Aldrich.

[0210] 1.2 Cell lines and cell culture

[0211] Table 3. Sources of human tumor tissue cell lines

[0212] Table 4. Sources of mouse tumor tissue cell lines

[0213] The above cell lines were all cultured in a cell culture incubator at 37°C containing 0.5% CO2, and experiments were performed when the cells were in the logarithmic growth phase.

[0214] 1.3 Detection of cell death using SYTOX Green

[0215] 1) Inoculate relevant cells into appropriate well plates and treat with drugs as needed until the corresponding time point;

[0216] 2) Stain the cells with SYTOX Green at a final concentration of 200 nM;

[0217] 3) The number of SYTOX Green-positive cells is the number of cell death, and the ratio is the final result.

[0218] 1.4 Experimental Results

[0219] 1.4.1 R406 / R788 promotes TNFα-induced RIPK1-dependent programmed cell death in tumor cells

[0220] In human leukemia FADD-deficient Jurkat cells, after pretreatment with R406 (3 μM), R788 (3 μM) or Nec-1s (10 μM) for 30 minutes, TNFα (20 ng / ml) was added to induce cell necrosis.

[0221] As shown in Figure 8, R406 and R788 significantly promoted TNFα-induced cell necrosis with prolonged TNFα treatment, and this promotion was protected by Nec-1s. Furthermore, R406 and R788 significantly enhanced the phosphorylation of RIPK1 and MLKL with increasing TNFα treatment time, and this phosphorylation was protected by Nec-1s. This suggests that R406 can promote TNFα-induced RIPK1-dependent programmed necrosis.

[0222] 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 SYTOX Green level of the cells was measured every 7 hours to assess cell death.

[0223] As shown in Figures 9-12, R406 significantly promoted TNFα / zVAD.fmk-induced cell necrosis with prolonged TNFα and zVAD.fmk treatment, and this promotion was protected by Nec-1s. Furthermore, R406 significantly enhanced the phosphorylation of RIPK1 and MLKL with increasing TNFα / zVAD.fmk treatment time, and this phosphorylation was protected by Nec-1s. This suggests that R406 can promote TNFα-induced RIPK1-dependent programmed necrosis.

[0224] 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 assess cell death.

[0225] As shown in Figure 11, R406 significantly promoted TNFα-induced cell death with prolonged TNFα treatment, and this promotion was protected by Nec-1s. Furthermore, R406 significantly enhanced RIPK1 phosphorylation and PARP1 cleavage with increasing TNFα treatment time, and these phosphorylation and cleavage were protected by Nec-1s. This suggests that R406 can promote TNFα-induced RIPK1-dependent cell apoptosis.

[0226] 1.4.2 R406 / R788 promotes TRAIL-induced apoptosis

[0227] E0771 cells were treated with different concentrations of TRAIL to induce apoptosis and 1 μM, 3 μM, and 5 μM R406, respectively. Cellular ATP levels were detected 24 hours later, and cell viability was evaluated using the CellTiter-Glo assay.

[0228] As shown in Figure 12, when TRAIL alone treated E0771 cells, its IC 50 was 34.21 nM. 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.

[0229] In order to establish an animal model with ZsGreen-LUC-expressing E0771, it is first necessary to construct a stable cell line with the luciferase gene, and this cell line has unchanged sensitivity to the compound. Therefore, the experiment shown in Figure 13 was performed.

[0230] The experimental results showed that the modified ZsGreen-LUC-expressing E0771 cells with the strongest luciferase activity showed no difference in sensitivity to the compound compared to E0771-BLANK cells. Treatment with either the compound or TRAIL alone promoted 20%-30% cell death (12 hours). The combination of the compound and TRAIL promoted 70%-80% cell death (12 hours) in both cell types. This promotion effect was significantly different. In addition, the cell death promoted by compound R788 under TRAIL-induced conditions was inhibited by Nec-1s, but the cell death promoted by TRAIL itself was not inhibited by Nec-1s.

[0231] LLC cells were treated with different concentrations of TRAIL to induce apoptosis and 1 μM, 3 μM, and 5 μM R406, respectively. Cell ATP levels were detected 24 h later, and cell viability was evaluated using the CellTiter-Glo assay.

[0232] As shown in Figure 14, 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.

[0233] As shown in the Western Blot results of Figures 12 and 16 , in LLC and E0771 cells, R406 can promote TRAIL-induced PARP1 cleavage and RIPK1-S166 phosphorylation, thereby promoting RIPK1 activation.

[0234] These results indicate that R406 / R788 can promote TRAIL-induced apoptosis in E0771 and LLC cells.

[0235] Example 3 Inhibitory Effects of R788 Combined with TRAIL on LLC and E0771 Transplanted Tumors in Mice

[0236] 1. Materials and Methods

[0237] 1.1 Test sample

[0238] Name:R788

[0239] Item No.: S2625

[0240] Appearance: white powder

[0241] Purity: 98%

[0242] Source: Selleck

[0243] Storage: Store at -20℃ before preparation

[0244] 1.2 Combined drug protein

[0245] mTRAIL: Purified endotoxin-free mTRAIL was prepared in endotoxin-free PBS at a concentration of 10 mg / kg.

[0246] 1.3 Experimental animals

[0247] C57BL / 6 mice, male and female, weighing 18-22 g, were purchased from Shanghai Lingchang Biotechnology Co., Ltd. and housed in an SPF-grade animal room with free access to food and a 12-h light / 12-h dark cycle.

[0248] 1.4 In vivo imaging of small animals (bioluminescence imaging BLI)

[0249] 1) First, 100 μL of D-Luciferin substrate was injected into the mouse at a dose of 150 mg / kg.

[0250] 2) Place the mouse in an anesthesia box and, after isoflurane anesthesia for no more than 5 minutes, place it in the operating platform of the small animal in vivo imaging system;

[0251] 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;

[0252] 4) Then manually turn off the light and switch to the bioluminescence channel, i.e., the luciferase fluorescence channel, to capture the bioluminescence image emitted by the mouse;

[0253] 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.

[0254] 1.5 Hematoxylin-eosin staining (HE staining)

[0255] 1) The tumor tissue was embedded in paraffin, sliced ​​serially at 4 μm thickness, spread in 42°C water, and then placed in a 37°C oven. The next day, the dried tissue sections were dewaxed with xylene for 5-10 minutes.

[0256] Replace with fresh xylene and dewax for another 5-10 minutes.

[0257] 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.

[0258] 3) For the above-treated samples, stain them with hematoxylin staining solution for 5-10 minutes.

[0259] 4) Soak in tap water to rinse off excess staining solution for about 10 minutes.

[0260] 5) Rinse again with distilled water (for a few seconds).

[0261] 6) Stain with eosin solution for 30 seconds to 2 minutes.

[0262] 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.

[0263] 8) Then use xylene to make it clear for 5 minutes, and then use fresh xylene to make it clear for another 5 minutes.

[0264] 9) Seal the slides with neutral gum or other mounting media.

[0265] 1.6 Immunohistochemical staining (IHC staining)

[0266] 1) Incubate the sections in xylene solution three times for 5 minutes each time.

[0267] 2) Place the slices in 100% ethanol and incubate the slices twice, each time for 10 minutes.

[0268] 3) Place the slices in 95% ethanol and incubate the slices twice, each time for 10 minutes.

[0269] 4) Wash the sections twice with ddH2O, 5 min each time.

[0270] 5) Immerse the sections in 1* citrate repair solution and heat in a microwave until boiling.

[0271] 6) Maintain the sub-boiling temperature for another 10 minutes and cool the slices on the bench for 30 minutes.

[0272] 7) Wash the sections with ddH2O three times, 5 min each time.

[0273] 8) Place the slices in a 3% hydrogen peroxide solution and incubate for 10 minutes.

[0274] 9) Wash the sections twice with ddH2O, 5 min each time.

[0275] 10) Wash sections with wash buffer for 5 min.

[0276] 11) Block each section with 100 μL-400 μL of blocking solution at room temperature for 1 hour.

[0277] 12) Remove the blocking solution and add 100 μL-400 μL of Incubate with primary antibody diluted in Antibody Diluent overnight at 4°C.

[0278] 13) Use Equilibrate Boost Detection Reagent to room temperature.

[0279] 14) Remove the antibody solution and wash the antibody three times with washing buffer, each time for 5 minutes.

[0280] 15) Apply 1-3 drops to the slices as needed Boost Detection Reagent and incubate at room temperature in a humidified chamber for 30 min.

[0281] 16) Wash the sections three times with wash buffer, 5 min each time.

[0282] 17) Add 1 drop (30 μL) Dissolve DAB Chromogen Concentrate in 1 ml of SignalStain DAB Diluent and mix thoroughly before use.

[0283] 18) Apply 100 μL-400 μL to each slice DAB and observe closely. Usually, appropriate staining intensity can be obtained within 1-10 minutes.

[0284] 19) Immerse the sections in ddH2O.

[0285] 20) Wash the sections twice with ddH2O, 5 min each time.

[0286] 21) Dehydrate the sections by incubating them in 95% ethanol twice for 10 seconds each, then in 100% ethanol twice for 10 seconds each, and finally in xylene twice for 10 seconds each.

[0287] 22) Mount the sections using coverslips and mounting medium.

[0288] 1.7 Experimental Procedure

[0289] 1) Six-week-old C57BL / 6 male and female mice were housed in a sterile environment with 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°C and humidity 45-65%). Experiments were performed after one week of stabilization.

[0290] 2) Before the experiment, weigh the mice to ensure that the weight of all mice does not fluctuate by more than 1g;

[0291] LLC cells and ZsGreen-LUC-expressing E0771 cells were cultured in 15 cm dishes, trypsinized, 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).

[0292] 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;

[0293] 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;

[0294] 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;

[0295] 7) After grouping, mice were gavaged with R788 (200 μl R788 dissolved in CMC-Na solution) daily according to the treatment group, and mTRAIL was injected intraperitoneally once a day.

[0296] 8) Measure the length and short diameter of the subcutaneous tumor with a vernier caliper 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.

[0297] 9) LLC tumor inoculation lasted for 17 days and drug treatment lasted for 14 days;

[0298] 10) On days 11 and 15 after inoculation of ZsGreen-LUC-expressing E0771 tumors, mice were orthotopically injected with D-Luciferin (150 mg / kg), and the drug effect was monitored by measuring changes in light intensity using a small animal in vivo imaging system.

[0299] 11) ZsGreen-LUC-expressing E0771 tumors were inoculated for 16 days and drug treatment lasted for 11 days.

[0300] 1.8 Experimental Results

[0301] 1.8.1 R788 enhances the anti-tumor effect of TRAIL in the mouse E0771 model (statistical analysis)

[0302] The constructed ZsGreen-LUC-expressing E0771 tumor cells were implanted into C57BL / 6 female mice. On days 11 and 15, respectively, the mice were injected orally with D-Luciferin (150 mg / kg). The light intensity changes were detected by a small animal in vivo imaging device to monitor the drug effect.

[0303] As shown in Figure 15, 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.

[0304] Tumor growth curves and tumor size were statistically analyzed, as shown in Figure 16. A total of 32 6-8 week old C57BL / 6 female mice successfully inoculated with ZsGreen-LUC-expressing E0771 tumor cells were used in the experiment. Eight mice were used in each experiment, for a total of four experimental groups. Tumor volume and body weight were recorded daily, starting from the first day of treatment. Eleven days after treatment, tumors were weighed ex vivo, and the statistical results were compared and analyzed.

[0305] 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 control group stopped taking the drug, the tumor volume was about 2300mm 3 The tumor volume of mice treated with R788 and TRAIL combined was significantly slower than that of the control group and the R788 and TRAIL alone groups. When the drug was stopped, the tumor volume of the mice treated with the combined drug was approximately 700 mm 3 .

[0306] The statistical results of in vitro weighing of ZsGreen-LUC-expressing E0771 tumors are shown in FIG17 . R788 and TRAIL administered alone had certain anti-tumor effects, but the combined use of R788 and TRAIL had a better effect.

[0307] 1.8.2 R788 Enhances the Antitumor Effect of TRAIL in the E0771 Mouse Model (Tissue Section Analysis)

[0308] The cleavage of Caspase-3 represents the occurrence of cell apoptosis. The larger the cleavage area, the more intense the apoptosis. As shown in Figure 18, after the combination of R788 and TRAIL, the immunohistochemical staining area of ​​cleaved Caspase-3 in mouse ZsGreen-LUC-expressing E0771 tumor tissue sections was the largest, indicating that cell apoptosis is most obvious at this time.

[0309] This proves that R788 has an anti-tumor effect by promoting TRAIL-induced cell apoptosis, thereby slowing down tumor growth.

[0310] Ki-67 is a marker of cell proliferation. The darker the Ki-67, the more vigorous the cell proliferation. As shown in Figure 19, Vehicle has the darkest color, and the ZsGreen-LUC-expressing E0771 tumor cells proliferate the most vigorously. Compared with the control group, Ki-67 levels in the TRAIL alone and R788 alone treatments were weaker, and the tumor cell proliferation ability decreased. The contrast was more obvious in the TRAIL+R788 group. Compared with the control group, Ki-67 showed significant changes and became significantly weaker, and the tumor cell proliferation ability was the worst.

[0311] CD31 is a marker for endothelial cells. The darker the CD31 immunohistochemical staining, the faster the endothelial cells grow, the greater the microvessel density, and the richer the nutrition supplied to tumor cells, which will accelerate the growth of tumor cells.

[0312] As shown in Figure 20, Vehicle had the darkest immunohistochemical staining and the fastest endothelial cell proliferation rate, while CD31 was weaker in the R788 alone and TRAIL alone treatment groups 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 showed significant changes 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.

[0313] 1.8.3 R788 enhances the anti-tumor effect of TRAIL in the mouse LLC model (statistical analysis)

[0314] As shown in Figure 21, 17 days after LLC was subcutaneously inoculated into C57BL / 6 mice, tumors were removed from the four drug-treated groups and compared as shown in the figure. Tumor size in the R788-only group was not significantly different from that in the control group, while tumors in the TRAIL-treated group were slightly smaller than those in the control group. Combination therapy with TRAIL and R788 resulted in significantly smaller tumors not only compared to the control group but also compared to those in the TRAIL-only group. Tumor growth curves and tumor sizes were statistically analyzed.

[0315] As shown in Figure 22, a total of 24 6-8 week old C57BL / 6 male mice successfully inoculated with LLC tumor cells were used in the experiment. Six mice were used in each experiment, resulting in four experimental groups. Tumor volume and body weight were recorded daily starting from the first day of treatment. After 14 days of treatment, the tumors were weighed ex vivo, and the statistical results were compared and analyzed.

[0316] 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, and when the drug was stopped, the tumor volume was nearly 1000 mm 3 Fourteen days after the injection, the group of mice that received intraperitoneal injection of TRAIL alone showed a slower tumor growth rate compared to the control group. When the drug was stopped, the tumor volume was approximately 600 mm. 3 The tumor volume of the mice treated with R788 and TRAIL was significantly slower than that of the control group and the TRAIL-only group. When the drug was stopped, the tumor volume of the combined treatment group was approximately 300 mm 3 .

[0317] The statistical results of LLC tumor in vitro weighing are shown in Figure 22. R788 alone had no effect, TRAIL had a certain anti-tumor effect, but the combination of R788 and TRAIL had a better effect.

[0318] 1.8.4 R788 enhances the antitumor effect of TRAIL in the mouse LLC model (tissue section analysis)

[0319] As shown in Figure 23, tumor tissue sections from mice treated with R788 alone showed virtually no damage or necrosis compared to the control group. Tissue sections from mice treated with intraperitoneal TRAIL showed some areas of damage and necrosis, but the areas of cell death were smaller. However, combined administration of R788 and TRAIL significantly expanded the areas of tissue damage and cell necrosis, accompanied by extensive hemorrhages. This suggests that R788 can promote TRAIL-induced cell death in LLC tumor tissues in mice. Immunohistochemical staining for Ki-67, CD31, and cleaved Caspase-3 demonstrates that R788 indeed exerts its anti-tumor effect by promoting TRAIL-induced apoptosis, thereby slowing tumor growth.

[0320] 1.8.5 R788 promotes TRAIL-induced activation of RIPK1 and caspase (in vivo)

[0321] As shown in Figure 24, 1 / 2 / 3 represent the numbers of mice treated with drugs in each group. Each group consisted of 6 mice, and tumor tissue samples from 3 of these mice were randomly selected for Western Blot analysis. The results showed that neither R788 alone nor the control group activated RIPK1 or caspases. However, in mice injected intraperitoneally with TRAIL alone, phosphorylation of RIPK1 at S166 was slightly activated, accompanied by caspase-3 and PARP1 cleavage. However, when R788 and TRAIL were combined, phosphorylation of RIPK1 at S166 was significantly enhanced, and caspase-3 and PARP1 cleavage was even more pronounced.

[0322] This indicates that R788 can be well absorbed in vivo and can promote TRAIL-induced RIPK1 and caspase activation in LLC and E0771 tumor tissues.

[0323] 2 Discussions

[0324] 2.1 The anti-tumor effect of R406 phosphate R788 was evaluated by using it in mice to determine whether it could promote the apoptosis of tumor cells mediated by other death receptor family members, such as TRAIL. Existing experimental data showed that the compound had the most significant effect in two mouse lung cancer cells, LLC and breast cancer E0771. In addition, in the mouse tumor models of these two cells, whether through biological characteristics observation, HE staining, or immunohistochemistry, it was demonstrated that R788 could promote the anti-tumor effect of TRAIL.

[0325] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

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 the R5 may be substituted or unsubstituted. replaced. 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, R788 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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