A USP14 covalent inhibitor and its preparation method and application

Abstract

The present invention discloses a USP14 covalent inhibitor, its preparation method, and application, belonging to the field of pharmaceutical technology. The present invention provides a compound containing a novel heterocyclic structure that has significant inhibitory activity against USP14 and can effectively inhibit the growth of human colon cancer cells. The compound can be used for the development of covalent USP14 inhibitors and tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a heterocyclic compound and a drug with USP14 covalent inhibition effect, and further relates to a preparation method and pharmaceutical use thereof. Background Art

[0002] Ubiquitin is a small protein consisting of 76 amino acids, which is important for the regulation of protein function in cells. Ubiquitination and deubiquitination are enzymatically mediated processes by which ubiquitin is covalently bound to a target protein or cleaved from the target protein by a deubiquitinating enzyme (DUB). There are approximately 95 DUBs in human cells, which are divided into subfamilies based on sequence homology. The USP family is characterized by their common Cys and His boxes, which contain Cys and His residues that are crucial for their DUB activity. Ubiquitination and deubiquitination processes are involved in the regulation of many cellular functions, including cell cycle progression, apoptosis, modification of cell surface receptors, regulation of DNA transcription, and DNA repair. Therefore, the ubiquitin system is involved in the pathogenesis of many disease states, including inflammation, viral infection, metabolic dysfunction, CNS disorders, and tumorigenesis.

[0003] The proteasome is a large protein complex composed of 33 different subunits. The proteasome complex functions as a protease, partially degrading unwanted or misfolded proteins. The proteasome regulates numerous aspects of cellular physiology, and its malfunction contributes to a variety of diseases, including cancer and neurodegenerative disorders. Most, but not exclusively, proteasome substrates are targeted for degradation through covalent attachment of multimeric chains of ubiquitin, a small, highly conserved protein. Because longer ubiquitin chains interact more strongly with the proteasome than shorter ones, processes that frequently alter the length of ubiquitin chains can also affect the rate of substrate degradation. Certain proteasome-associated deubiquitinating enzymes and ubiquitin ligases regulate the length of ubiquitin chains attached to substrates marked for proteasomal degradation. These deubiquitinating enzymes and ligases appear to regulate proteasome activity by cleaving and extending ubiquitin chains bound to the proteasome.

[0004] The mammalian proteasome contains three major deubiquitinating enzymes: Rpn11, Uch37, and Usp14. Rpn11 removes ubiquitin from labeled substrates by severing the binding site between the ubiquitin chain and the substrate. Because Rpn11-mediated cleavage occurs after the substrate has been committed to proteolysis but before degradation, Rpn11 helps prevent ubiquitin from being degraded along with the substrate, thereby minimizing fluctuations in cellular ubiquitin levels. Furthermore, because proteasomal substrates must pass through a narrow translocation channel before accessing the proteasome's cryptic proteolytic sites, removal of large ubiquitin chains may also facilitate substrate translocation. Thus, Rpn11 promotes substrate degradation by removing ubiquitin chains en bloc at a relatively late stage in the proteasomal pathway. In contrast, Uch37 acts before the substrate has been committed to proteasomal degradation. Uch37 cleaves the terminal ubiquitin chains of substrates, and its enzymatic activity shortens the chains rather than completely removing them. It has been proposed that chain trimming by Uch37 enhances the ability of the proteasome to discriminate between polyubiquitin chain lengths.

[0005] USP14 is one of three deubiquitinating enzymes (USP14, Uch37, and Rpn11) associated with the proteasome. It can be recruited to the 19S granule and cleaves ubiquitin chains on protein substrates to avoid proteasome recognition and degradation. Inhibition of USP14 leads to the rapid accumulation of polyubiquitin-linked proteins, endoplasmic reticulum stress, and the generation of reactive oxygen species (ROS), which impairs organelle trafficking, inhibits microtubule transport of toxic protein aggregates, and induces apoptosis. However, it does not affect the proteolytic activity of the 20S proteasome. Following the approval of the proteasome inhibitor bortezomib for the treatment of multiple myeloma, the ubiquitin-proteasome system has garnered attention as a target for cancer therapy. Extended treatment with bortezomib is limited by its associated toxicity and drug resistance. However, therapeutic strategies targeting specific aspects of the ubiquitin-proteasome pathway upstream of the proteasome, such as DUBs, are expected to be better tolerated. Therefore, there is a need for compounds and pharmaceutical compositions that inhibit DUBs, such as USP14, for the treatment of indications where DUB activity has been observed, including, but not limited to, cancer. Summary of the Invention

[0006] The purpose of the present invention is to provide a USP14 covalent inhibitor that can be used to treat various diseases related to USP14 deubiquitinase, such as tumors.

[0007] In order to achieve the above-mentioned purpose of the invention, the technical solution of the present invention is as follows:

[0008] In one aspect, the present invention provides a compound represented by formula (I) or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, pharmaceutically acceptable salts or prodrugs;

[0009]

[0010] in,

[0011] X is independently selected from

[0012] R1 is selected from hydrogen, hydroxy, amino, halogen, unsubstituted or substituted alkyl, cycloalkyl or heterocycloalkyl, wherein the substituent is one or more of alkyl, halogen, hydroxy or amino;

[0013] R2 is a group containing at least one of an alkenyl group, an alkynyl group, a cyano group, an acylamino group, an oxirane group, and a sulfonyl fluoride group;

[0014] R3 is selected from hydrogen, hydroxy, amino, dialkylamino, halogen, C 1 4 Alkoxy, substituted or unsubstituted alkyl, cycloalkyl, phenyl, wherein the substituent is one or more of alkyl, halogen, hydroxy, alkoxy, amino, and nitrile, and the value of n is an integer between 1 and 6;

[0015] The substituent is selected from unsubstituted or substituted aromatic heterocycles, and the substituent is selected from one or more of alkyl, halogen, hydroxyl, and amino.

[0016] Preferably, R1 is selected from hydrogen, hydroxy, amino, halogen, unsubstituted or substituted with 1-3 substituents C 1 4 Alkyl, C 3 6 Cycloalkyl or C 3 6 Heterocycloalkyl, wherein the substituent is one or more of methyl, ethyl, fluorine, hydroxyl or amino;

[0017] R2 is selected from

[0018] R3 is selected from hydrogen, hydroxy, amino, dimethylamino, halogen, C 1 4 Alkoxy, substituted or unsubstituted C 1 4 Alkyl, C 3 6 Cycloalkyl, phenyl, the substituent is one or more of methyl, ethyl, fluorine, chlorine, hydroxyl, methoxy, amino, and nitrile, and the value of n is an integer between 1 and 4;

[0019] Selected from unsubstituted or 1-3 substituent-substituted C 4-9 Aromatic heterocycle, wherein the substituent is selected from one or more of methyl, fluorine, hydroxyl and amino.

[0020] As some examples of the present invention, preferably, X is R1 is selected from hydrogen, unsubstituted or substituted with 1 to 3 substituents C1 4 Alkyl, C 3 6 Cycloalkyl or C 3 6 Heterocycloalkyl, wherein the substituent is one or more of methyl, ethyl, fluorine, hydroxyl or amino.

[0021] As some examples of the present invention, more preferably, X is R1 is selected from hydrogen, unsubstituted or substituted with 1 to 3 substituents C 3 6 Cycloalkyl or C 3 6 Heterocycloalkyl, wherein the substituent is one or more of methyl, ethyl, fluorine, hydroxyl or amino.

[0022] As some examples of the present invention, more preferably, X is R1 is selected from hydrogen, unsubstituted or substituted with 1 to 3 substituents C 3 6 Heterocycloalkyl, wherein the substituent is one or more of methyl, ethyl, fluorine, hydroxyl or amino.

[0023] As some examples of the present invention, preferably, X is R2 is selected from

[0024] As some examples of the present invention, preferably, X is R3 is selected from C 1 4 Alkoxy, substituted or unsubstituted C 1 4 Alkyl, C 36 Cycloalkyl, phenyl, the substituent is one or more of methyl, ethyl, fluorine, chlorine, hydroxyl, methoxy, amino, and nitrile, and the value of n is an integer between 1 and 4.

[0025] As some examples of the present invention, more preferably, X is R3 is selected from C 1 4 Alkoxy, substituted or unsubstituted C 3 6 Cycloalkyl, phenyl, the substituent is one or more of methyl, ethyl, fluorine, chlorine, hydroxyl, methoxy, amino, and nitrile, and the value of n is an integer between 1 and 4.

[0026] As some examples of the present invention, more preferably, X is R3 is selected from C 1 4 Alkoxy, substituted or unsubstituted C 3 6 Cycloalkyl, phenyl, the substituent is one or more of chlorine, methoxy, and nitrile, and the value of n is an integer between 1 and 4.

[0027] As some examples of the present invention, preferably, X is Selected from substituted or unsubstituted pyridine, pyrrole, pyrazine, pyrimidine, pyridazine, thiophene, thiazole, furan, imidazole, pyrazole, triazole, tetrazole, oxazole, isoxazole, diazole, isothiazole, thiadiazole, benzotriazole, indole, isoindole, dihydroindole, isoindole, quinoxaline, quinazoline, cinnoline, chroman, isochroman, tetrahydroquinoline, quinoline, tetrahydroisoquinoline, isoquinoline, 2,3-dihydrobenzofuran, 2,3-dihydrobenzo-1,4-diene, imidazo(2,1-b)(1,3)thiazole, benzo-1,3-dioxole, and the substituent is selected from one or more of methyl, fluorine, hydroxyl, and amino.

[0028] As some examples of the present invention, more preferably, X is Selected from substituted or unsubstituted pyridine, pyrrole, pyrazine, pyrimidine, pyridazine, thiophene, thiazole, furan, imidazole, pyrazole, triazole, tetrazole, oxazole, isoxazole, diazole, isothiazole, thiadiazole, the substituent is selected from one or more of methyl, fluorine, hydroxyl, amino, the substituent is selected from one or more of methyl, fluorine, hydroxyl, amino.

[0029] As some examples of the present invention, more preferably, X is Selected from substituted or unsubstituted pyrrole, thiazole, imidazole, pyrazole, triazole, tetrazole, oxazole, isoxazole, diazole, isothiazole, thiadiazole, the substituent is selected from one or more of methyl, fluorine, hydroxyl, and amino.

[0030] As some examples of the present invention, preferably, X is R1 is selected from hydrogen, hydroxy, amino, and halogen.

[0031] As some examples of the present invention, more preferably, X is R1 is selected from hydrogen, hydroxy, amino, fluorine, and chlorine.

[0032] As some examples of the present invention, more preferably, X is R1 is hydrogen.

[0033] As some examples of the present invention, preferably, X is R2 is selected from

[0034]

[0035] As some examples of the present invention, preferably, X is R3 is selected from hydrogen, hydroxy, amino, dimethylamino, halogen, C 1 4 In the alkoxy group, the value of n is an integer between 1 and 4.

[0036] As some examples of the present invention, more preferably, X is R3 is selected from methoxy or n=1.

[0037] As some examples of the present invention, preferably, X is is selected from substituted or unsubstituted benzotriazole, indole, isoindole, dihydroindole, isoindole, quinoxaline, quinazoline, cinnoline, chroman, isochroman, tetrahydroquinoline, quinoline, tetrahydroisoquinoline, isoquinoline, 2,3-dihydrobenzofuran, 2,3-dihydrobenzo-1,4-diene, imidazo(2,1-b)(1,3)thiazole, benzo-1,3-dioxole, wherein the substituent is selected from one or more of methyl, fluorine, hydroxyl and amino. As some examples of the present invention, more preferably, X is The substituent is selected from substituted or unsubstituted indole, isoindole, dihydroindole, isoindole, quinoline and isoquinoline, wherein the substituent is selected from one or more of methyl, fluorine, hydroxyl and amino.

[0038] As some examples of the present invention, more preferably, X is Selected from substituted or unsubstituted indoles, wherein the substituent is selected from one or more of methyl, fluorine, hydroxyl and amino.

[0039] Preferably, R1 is selected from hydrogen,

[0040] R2 is selected from

[0041] R3 is selected from

[0042] Selected from

[0043] Preferably, the compound has a structure represented by formula (II) or formula (III):

[0044]

[0045]

[0046] Wherein, X is selected from R1 is selected from hydrogen,

[0047] R2 is selected from

[0048]

[0049] R3 is selected from

[0050] More preferably, and as a specific example of the present invention, the compound is selected from Table 1.

[0051] Table 1

[0052]

[0053]

[0054] More preferably, and as a specific example of the present invention, the compound is selected from Table 2.

[0055] Table 2

[0056]

[0057]

[0058] Further preferably, and as a specific example of the present invention, the compound is selected from Table 3.

[0059] Table 3

[0060]

[0061] Unless otherwise specified, the term "alkyl" as used herein includes branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms, including all isomers. Common abbreviations for alkyl groups include "Me" or CH3 for methyl, "Et" or CH2CH3 for ethyl, "Pr" or CH2CH2CH3 for propyl, and "Bu" or CH2CH2CH2CH3 for butyl. For example, "C 1-4 "C1-C4 alkyl" refers to a straight or branched chain alkyl group having the specified number of carbon atoms, including all isomers. 1-4 Alkyl includes n-, iso-, sec- and t-butyl, n- and iso-propyl, ethyl and methyl. The term "C 1-10 "Alkyl" and the like have similar meanings.

[0062] The term "alkoxy" represents straight and branched chain alkyl groups having the indicated number of carbon atoms attached through an oxygen bridge.

[0063] The term "halogen" (or "halo") refers to fluorine, chlorine, bromine, and iodine (alternatively referred to as fluoro (F), chloro (Cl), bromo (Br), and iodo (I)).

[0064] The term "aryl" refers to aromatic mono- and polycyclic carbocyclic ring systems wherein the individual carbon rings in the polycyclic ring system are fused or linked to each other by single bonds. Typical aryl groups include phenyl, naphthyl and biphenylene.

[0065] The term "heterocycle" refers to a cyclic structure composed of carbon atoms and non-carbon atoms such as nitrogen, oxygen, and sulfur. Typical heterocyclic groups include pyridine, quinoline, tropane, phenothiazine, benzodiazepine, furan, pyrazolone, and pyrimidine.

[0066] The term "aromatic heterocycle" refers to a 5- or 6-membered monocyclic aromatic ring or a 7- to 12-membered bicyclic ring composed of carbon atoms and one or more heteroatoms selected from N, O and S. Examples of aromatic heterocycles include pyridyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, thienyl (or thiophenyl), thiazolyl, furanyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, diazolyl, thiazolyl, isothiazolyl and thiadiazolyl, benzotriazolyl, indolyl, isoindolyl, indazolyl, indolinyl, isoindolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, chromanyl, isochromanyl, tetrahydroquinolinyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzo-1,4-dienyl, imidazo(2,1-b)(1,3)thiazole and benzo-1,3-dioxolyl.

[0067] The aryl group in the term "substituted aryl" is as defined above. When the substituent of the substituted aryl group is not specified, the substituent group may be selected from the following groups, including but not limited to: halogen, C1-C 20 Alkyl, CF3, NH2, N(C1-C6 alkyl)2, NO2, oxo, CN, N3, -OH, -O(C1-C6 alkyl), C3-C 10 Cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, (C0-C6 alkyl)S(O) 0-2 -, aryl-S(O) 0-2 -, (C0-C6 alkyl)S(O) 0-2 (C0-C6 alkyl)-, (C0-C6 alkyl)C(O)NH-, H2N-C(NH)-, -O(C1-C6 alkyl)CF3, (C0-C6 alkyl)C(O)-, (C0-C6 alkyl)OC(O)-, (C0-C6 alkyl)2NC(O)-(C0-C6 alkyl)O(C1-C6 alkyl)-, (C0-C6 alkyl)C(O) 1-2 (C0-C6 alkyl)-, (C0-C6 alkyl)OC(O)NH-, aryl, aralkyl, heteroaryl, heterocyclylalkyl, halogen-aryl, halogen-aralkyl, halogen-heterocycle, halogen-heterocyclylalkyl, cyano-aryl, cyano-aralkyl, cyano-heterocycle and cyano-heterocyclylalkyl. The term "substituted phenyl" has a similar definition.

[0068] Unless otherwise specified, all ranges listed herein are inclusive. For example, "n is an integer between 0 and 2" means that n can be 0, 1 or 2.

[0069] The term "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic base or acid. When the compound of the present invention is acidic, its corresponding salt can be readily prepared from an inorganic or organic base. Salts derived from such inorganic bases include salts of aluminum, ammonium, calcium, copper (copper and cuprous), iron, ferrous, lithium, magnesium, manganese (manganese and manganous), potassium, sodium, zinc, and the like. Preferred are salts of ammonium, calcium, magnesium, potassium, and sodium. Salts prepared from organic bases include primary, secondary, and tertiary amines derived from natural and synthetic sources. Pharmaceutically acceptable organic non-toxic bases that can form salts include arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydrabamine, isopropylamine, dicyclohexylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc. When the compound of the present invention is basic, its corresponding salt can be easily prepared from inorganic or organic acids. Such acids include, for example, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, and the like.

[0070] The term "solvate" refers to a complex of variable stoichiometry formed by a solute (i.e., a compound of Formula I) or a pharmaceutically acceptable salt thereof and a solvent that does not interfere with the biological activity of the solute. Examples of solvents include, but are not limited to, water, ethanol, and acetic acid. When the solvent is water, the solvate is referred to as a hydrate. Hydrates include, but are not limited to, hemihydrates, monohydrates, monosesquihydrates, dihydrates, and trihydrates.

[0071] The term "prodrug" is a functional derivative of the compounds of the invention which is readily convertible in vivo into the required compound.

[0072] In another aspect, the present invention provides a method for preparing the above-mentioned compound, comprising at least one of the following synthetic routes:

[0073] Synthesis Route 1:

[0074]

[0075] Synthesis route 2:

[0076]

[0077] Synthesis route 3:

[0078]

[0079] Synthesis Route 4:

[0080]

[0081] wherein X, R1, and R2 have the same definitions as those of the corresponding products.

[0082] In another aspect, the present invention provides use of the above-mentioned compound or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, pharmaceutically acceptable salts or prodrugs in the preparation of USP14 covalent inhibitors.

[0083] In another aspect, the present invention provides the use of the above-mentioned compound or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, pharmaceutically acceptable salts or prodrugs in the preparation of anti-tumor drugs.

[0084] Preferably, the tumor is selected from one or more of cervical squamous cell carcinoma, bile duct carcinoma, hepatocellular carcinoma, gastric adenocarcinoma, sarcomatoid lung cancer, thymic carcinoma, pancreatic cancer, colorectal cancer, neuroblastoma, and multiple myeloma.

[0085] In another aspect, the present invention provides a drug comprising a compound represented by formula (I) or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, pharmaceutically acceptable salts or prodrugs, and one or more pharmaceutically acceptable carriers, diluents, and excipients.

[0086] The drug can be a raw material drug, a pharmaceutical composition, or a pharmaceutical preparation.

[0087] The medicaments include products comprising an active ingredient and an inert ingredient (pharmaceutically acceptable excipient) constituting a carrier, as well as any product obtained directly or indirectly by the combination, complexation or aggregation of two or more ingredients, or the decomposition of one or more ingredients, or other types of reactions or interactions of one or more ingredients. Therefore, the medicaments of the present invention include any composition prepared by mixing a compound of formula I, other active ingredients, and a pharmaceutically acceptable excipient.

[0088] Medicine of the present invention comprises the compound (or its pharmaceutically acceptable salt or solvate) shown in the formula I as active ingredient, pharmaceutically acceptable carrier and optional other therapeutic ingredients or adjuvant.Pharmaceutical composition comprises the composition that is applicable to oral, rectal, local and parenteral (including subcutaneous, intramuscular and intravenous) administration, although the most suitable approach depends on specific main body, the character and the severity of the disease of active ingredient to it in any particular case.Pharmaceutical composition can be prepared by any method known to field of pharmacy.

[0089] The active ingredient can be administered orally in solid dosage forms such as capsules, tablets, lozenges, troches, granules, and powders, or in liquid dosage forms such as elixirs, syrups, emulsions, dispersions, and suspensions. The active ingredient can also be administered parenterally in sterile liquid dosage forms such as dispersions, suspensions, or solutions. Other dosage forms that can be used to administer the active ingredient include ointments, creams, drops, transdermal patches, or powders for topical administration; ophthalmic solutions or suspensions, i.e., eye drops, for administration to the eye; sprays or powder compositions for inhalation or intranasal administration, or creams, ointments, sprays, or suppositories for rectal or vaginal administration. Gelatin capsules contain the active ingredient and a powdered carrier such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Similar diluents can be used to prepare compressed tablets. Both tablets and capsules can be formulated as sustained-release products to provide sustained release of the drug over several hours. Compressed tablets can be coated with sugar or film to cover any unpleasant taste and protect the tablet from air, or can be enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can include coloring agents and flavoring agents to increase patient acceptance. Generally speaking, water, suitable oil, saline, dextrose (glucose) aqueous solution and related sugar solutions and glycols such as propylene glycol or polyethylene glycol are suitable carriers of parenteral solutions. The solution for parenteral administration preferably includes a water-soluble salt of the active ingredient, a suitable stabilizer and the buffer substance used as needed. Antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid, alone or in combination, are suitable stabilizers. Citric acid and its salts and sodium EDTA can also be used. In addition, parenteral solutions can also include preservatives, such as benzalkonium chloride, methylparaben or propylparaben and chlorobutanol. For inhalation administration, the compound of the present invention can be easily delivered in a spray form from a pressurized package or a sprayer. This compound can also be sent in the powder form that prepares, and this powder composition can be sucked with the help of being blown into powder inhaler device.The preferred delivery system for sucking is metered dose inhalation (MDI) aerosol, and it can be mixed with suspension or the solution of the compound of formula I in suitable propellant, and described propellant is fluorocarbon or hydrocarbon for example.For eye administration, ophthalmic preparation can be prepared with the solution or the suspension of the suitable weight percentage of compound of formula I in suitable eye carrier, thereby keeps compound and eye surface contact enough time so that compound is penetrated into cornea and the inner area of ​​eyes.

[0090] Useful pharmaceutical dosage forms for administering the compounds of the present invention include, but are not limited to, hard and soft gelatin capsules, tablets, parenteral injection solutions, and oral suspensions.

[0091] When the compounds of the present invention are administered stepwise or in combination with other therapeutic agents, the same dosage forms as described above can be used. When the drugs are administered in a physical combination, the dosage form and route of administration should be selected based on the compatibility of the combined drugs. The compounds of the present invention can be administered as the sole active ingredient or in combination with a second active ingredient, including those known to be useful as adjunctive inhibitors of USP proteins and / or anti-tumor agents.

[0092] The beneficial effects of the present invention are as follows:

[0093] The present invention proposes a new compound that can serve as an inhibitor of the deubiquitinating enzyme USP14 protein, effectively inhibits the activity of USP14 protein, can effectively treat and prevent USP14 protein-related and / or tumor diseases, and provides new ideas for tumor treatment. DETAILED DESCRIPTION

[0094] The following non-limiting examples are provided to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way. The following is merely an illustrative description of the scope of protection claimed in this application. Those skilled in the art may make various changes and modifications to the invention of this application based on the disclosed content, and such changes and modifications should also fall within the scope of protection claimed in this application.

[0095] The present invention is further described below by way of specific examples. Unless otherwise specified, the various chemical reagents used in the examples of the present invention were obtained through conventional commercial channels.

[0096] In the following examples, the English abbreviations involved and their corresponding Chinese meanings are shown in Table 4.

[0097] Table 4

[0098]

[0099] The compounds of the present invention can be synthesized using the following four general synthetic schemes.

[0100] General Solution 1

[0101]

[0102] Reagents and conditions: a) NIS, acetonitrile, rt, 4h, b) tetrahydropyrrole, K2CO3, DMF, 85℃, 6h, c) n-butyllithium, THF, -78℃, 10h, d) H2, palladium on carbon, methanol, rt, 12h, e) R1-N-BOC, HATU, DIPEA, DCM, rt, 6h, f) hydrochloric acid ethyl acetate solution, rt, 1h, g) R2-Cl, TEA, DCM, rt, 1h.

[0103] General Solution 2

[0104]

[0105] Reagents and conditions: a) thionyl chloride, DMF, DCM, rt, 1h, b) 1-(4-chlorophenyl)-2,5-dimethylpyrrole, AlCl3, DCM, rt, 8h, c) H2, palladium on carbon, methanol, rt, 12h, d) R1-N-BOC, HATU, DIPEA, DCM, rt, 6h, e) hydrochloric acid ethyl acetate solution, rt, 1h, f) R2-Cl, TEA, DCM, rt, 1h.

[0106] General Solution 3

[0107]

[0108] Reagents and conditions: a) thionyl chloride, DMF, DCM, rt, 1h, b) 5-methoxyindole, AlCl3, DCM, rt, 8h, c) borane dimethyl sulfide, rt, 8h, d) boron tribromide, DCM, rt, 3h, e) (2-bromomethyl)dimethylamine, KOH, ethanol, 60°C, 6h, f) H2, palladium on carbon, methanol, rt, 12h, g) acryloyl chloride, TEA, DCM, rt, 1h.

[0109] General Solution 4

[0110]

[0111] Reagents and conditions: a) thionyl chloride, DMF, DCM, rt, 1 h, b) 5-methoxyindole, AlCl3, DCM, rt, 8 h, c) H2, Raney nickel, methanol, rt, 12 h, d) 4-(dimethylamino)but-2-enoic acid hydrochloride, HATU, DIPEA, DCM, rt, 6 h.

[0112] The present invention is further described below with reference to the embodiments.

[0113] Example 1 N-(2-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-5-(pyrrolidin-1-yl)phenyl)acrylamide

[0114] Synthesized according to Scheme 1 steps a, b, c, d, g.

[0115]

[0116] Step 1. Methyl 4-chloro-2-nitrobenzoate (108 mg, 0.5 mmol) was dissolved in N,N-dimethylformamide (2 ml). Potassium carbonate (212 mg, 1.5 mmol) and tetrahydropyrrole (42 mg, 0.6 mmol) were added sequentially and stirred at 85°C for 2 h. After the reaction, the reaction solution was quenched with water and extracted with ethyl acetate five times. The mixture was backwashed twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate and purified by column chromatography (PE:EA = 7:1) to obtain 127 mg of methyl 2-nitro-4-(pyrrolidin-1-yl)benzoate. ESI-MS: m / z = 251 [M+H] + . Step 2. Weigh 205 mg (1 mmol) of 1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole and add it to 8 ml of acetonitrile, then add 248 mg (1.1 mmol) of N-iodosuccinimide and stir at room temperature for 3 hours. TLC detection shows that the reaction is complete. Use DCM to extract and wash the organic phase with water several times, dry it with MgSO4, and separate the crude product by flash column chromatography with an elution gradient of pure DCM. The products are combined, concentrated, and dried to obtain 272 mg of 1-(4-chlorophenyl)-3-iodo-2,5-dimethyl-1H-pyrrole. ESI-MS: m / z=332[M+H] + .

[0117] Step 3. Weigh 331 mg (1 mmol) of 1-(4-chlorophenyl)-3-iodo-2,5-dimethyl-1H-pyrrole into anhydrous tetrahydrofuran. Cool to -78°C under nitrogen, then add 77 mg (1.2 mmol) of n-butyl lithium. After stirring at -78°C under nitrogen for 1 hour, add 250 mg (1 mmol) of methyl 2-nitro-4-(pyrrolidin-1-yl)benzoate, and then stir at room temperature overnight. TLC analysis showed no change in the reaction. The organic phase was extracted with DCM several times and washed with water, dried over MgSO4, and the crude product was separated by flash column chromatography with a pure DCM gradient. The products were combined, concentrated, and dried to yield 101 mg of (1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)(2-nitro-4-(pyrrolidin-1-yl)phenyl)methanone. ESI-MS: m / z = 424 [M+H] + .

[0118] Step 4. Weigh 42.3 mg (0.1 mmol) of (1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)(2-nitro-4-(pyrrolidin-1-yl)phenyl)methanone and dissolve it in 4 ml of methanol. Then add 4.3 mg (1%) of palladium on carbon and stir at room temperature overnight. TLC confirmed the reaction was complete. After filtration, the mixture was concentrated to dryness to yield 37.2 mg of the product (2-amino-4-(pyrrolidin-1-yl)phenyl)(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methanone. ESI-MS: m / z = 393 [M+H] + ;

[0119] Step 5. 39 mg (0.1 mmol) of (2-amino-4-(pyrrolidin-1-yl)phenyl)(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methanone was dissolved in 4 ml of anhydrous dichloromethane mixed with 30.3 mg (0.3 mmol) of triethylamine. 8 mg (0.09 mmol) of acryloyl chloride was then added and stirred at room temperature for 1 hour. TLC confirmed the reaction was complete. The organic phase was extracted with DCM several times and washed with water, dried over MgSO₄, and the crude product was separated by flash column chromatography with an elution gradient of MeOH:DCM = 1:10. The combined products were concentrated to dryness to yield 21 mg of N-(2-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-5-(pyrrolidin-1-yl)phenyl)acrylamide. ESI-MS: m / z = 448 [M+H] + ; 1 H NMR(400MHz,Chloroform-d)δ12.27(s,1H),8.07(d,J=2.4Hz,1H),7.79(d,J=8.9Hz,1H),7.51-7.44(m,2H),7.21-7.12(m,2H),6.42 -6.30(m,2H),6.21(dd,J=8.9,2.5Hz,1H),6.14(s,1H),5.72(dd,J=9.7,1.8Hz,1H),3.41(d,J=6.2Hz,4H),2.17(s,3H),1.98(s,3H).

[0120] Example 2 N-(5-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-2-(pyrrolidin-1-yl)phenyl)acrylamide

[0121] Synthesized according to Scheme 2 steps a, b, c, f.

[0122]

[0123] Step 1. Weigh 236 mg (1 mmol) of 3-nitro-4-(pyrrolidin-1-yl)benzoic acid and add it to a solution of 10 ml of dry dichloromethane mixed with three drops of N,N-dimethylformamide. Then add 476 mg (4 mmol) of thionyl chloride and stir at room temperature for 1 hour. The reaction is complete by TLC. Concentrate and dry to obtain 3-nitro-4-(pyrrolidin-1-yl)benzoyl chloride, which is then used directly in the next step.

[0124] Step 2. Aluminum chloride (27 mg, 0.2 mmol) was added to anhydrous DCM (1 ml), stirred at 0°C, and 3-nitro-4-(pyrrolidin-1-yl)benzoyl chloride (51 mg, 0.2 mmol) was slowly added. The mixture was stirred at 0°C for half an hour. Subsequently, 1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole (20 mg, 0.1 mmol) was dissolved in anhydrous DCM (1 ml) and slowly added dropwise to the aluminum chloride DCM solution of chloroacetyl chloride at 0°C. The mixture was stirred at room temperature overnight. After the reaction was complete, the reaction mixture was quenched with water and extracted with DCM (dichloromethane, 3 × 20 ml). The organic phase was dried over anhydrous sodium sulfate and purified by column chromatography (DCM:CH3OH = 40:1) to obtain 22 mg of the product (1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)(3-nitro-4-(pyrrolidin-1-yl)phenyl)methanone. ESI-MS: m / z = 424 [M+H] + .

[0125] Step 3. Weigh 42.3 mg (0.1 mmol) of (1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)(3-nitro-4-(pyrrolidin-1-yl)phenyl)methanone and dissolve it in 4 ml of methanol. Then add an appropriate amount of Raney nickel and stir at room temperature overnight. TLC confirmed the reaction was complete. After filtration, the mixture was concentrated to dryness to obtain 37.2 mg of the product (3-amino-4-(pyrrolidin-1-yl)phenyl)(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methanone. ESI-MS: m / z = 393 [M+H] + ;

[0126] Step 4. 39 mg (0.1 mmol) of (3-amino-4-(pyrrolidin-1-yl)phenyl)(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methanone was weighed and dissolved in 4 ml of anhydrous dichloromethane mixed with 30.3 mg (0.3 mmol) of triethylamine. 8 mg (0.09 mmol) of acryloyl chloride was then added and stirred at room temperature for 1 hour. The reaction was determined to be complete by TLC. The organic phase was extracted with DCM several times and washed with water, dried over MgSO₄, and the crude product was separated by flash column chromatography with an elution gradient of MeOH:DCM = 1:10. The products were combined, concentrated, and dried to obtain 15 mg of N-(5-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-2-(pyrrolidin-1-yl)phenyl)acrylamide. ESI-MS: m / z = 448 [M+H] + ;1H NMR(400MHz,Chloroform-d)δ8.30(s,1H),7.88(s,1H),7.68-7.59(m,1H),7.48-7.46(m,2H),7.17-7.15(m,2H),6.95(d,J=8.6Hz,1 H),6.44-6.33(m,2H),6.29(s,1H),5.78-5.70(m,1H),3.21(d,J=6.4Hz,4H),2.26(s,3H),1.98(d,J=1.0Hz,3H),1.94-1.91(m,4H).

[0127] Example 3 N-(2-((5-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-2-(pyrrolidin-1-yl)phenyl)amino)-2-oxoethyl)acrylamide

[0128] Synthesized according to Scheme 2 steps a, b, c, d, e, f.

[0129]

[0130] Step 1. Weigh 236 mg (1 mmol) of 3-nitro-4-(pyrrolidin-1-yl)benzoic acid and add it to a solution of 10 ml of dry dichloromethane mixed with three drops of N,N-dimethylformamide. Then add 476 mg (4 mmol) of thionyl chloride and stir at room temperature for 1 hour. The reaction is complete by TLC. Concentrate and dry to obtain 3-nitro-4-(pyrrolidin-1-yl)benzoyl chloride, which is then used directly in the next step.

[0131] Step 2. Aluminum chloride (27 mg, 0.2 mmol) was added to anhydrous DCM (1 ml), stirred at 0°C, and 3-nitro-4-(pyrrolidin-1-yl)benzoyl chloride (51 mg, 0.2 mmol) was slowly added. The mixture was stirred at 0°C for half an hour. Subsequently, 1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole (20 mg, 0.1 mmol) was dissolved in anhydrous DCM (1 ml) and slowly added dropwise to the aluminum chloride DCM solution of chloroacetyl chloride at 0°C. The mixture was stirred at room temperature overnight. After the reaction was complete, the reaction mixture was quenched with water and extracted with DCM (dichloromethane, 3 × 20 ml). The organic phase was dried over anhydrous sodium sulfate and purified by column chromatography (DCM:CH3OH = 40:1) to obtain 22 mg of the product (1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)(3-nitro-4-(pyrrolidin-1-yl)phenyl)methanone. ESI-MS: m / z = 424 [M+H] + .

[0132] Step 3. Weigh 42.3 mg (0.1 mmol) of (1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)(3-nitro-4-(pyrrolidin-1-yl)phenyl)methanone and dissolve it in 4 ml of methanol. Then add an appropriate amount of Raney nickel and stir at room temperature overnight. TLC confirmed the reaction was complete. After filtration, the mixture was concentrated to dryness to obtain 37.2 mg of the product (3-amino-4-(pyrrolidin-1-yl)phenyl)(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methanone. ESI-MS: m / z = 393 [M+H] + ;

[0133] Step 4. 17.5 mg (0.1 mmol) of (tert-butyloxycarbonyl)glycine was weighed and added to 2 ml of dichloromethane, followed by the addition of 57 mg (0.15 mmol) of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and 39 mg (0.3 mmol) of N,N-diisopropylethylamine. The mixture was stirred at room temperature for 1 hour. Then, 39 mg (0.1 mmol) of (3-amino-4-(pyrrolidin-1-yl)phenyl)(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methanone was added and stirred at room temperature for 4 hours. The reaction was complete by TLC. The organic phase was extracted with DCM several times and washed with water, dried over MgSO4, and the crude product was separated by flash column chromatography with an elution gradient of EA:PE = 1:5. The products were combined, concentrated, and dried to obtain 22 mg of tert-butyl (2-((5-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-5-(pyrrolidin-1-yl)phenyl)amino)-2-oxoethyl)carbamate. ESI-MS: m / z = 551 [M+H] + ;

[0134] Step 5. 55 mg (0.1 mmol) of tert-butyl (2-((5-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-5-(pyrrolidin-1-yl)phenyl)amino)-2-oxoethyl)carbamate was added to 0.5 ml (1 mmol, 2 M) hydrochloric acid and ethyl acetate solution and stirred at room temperature for 0.5 hour. TLC indicated that the reaction was complete. The reaction was quenched with saturated aqueous sodium bicarbonate solution, extracted with EA several times, and the organic phase was washed with water and dried over MgSO4. The products were combined and concentrated to dryness to obtain 42 mg of 2-amino-N-(5-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-2-(pyrrolidin-1-yl)phenyl)acetamide. ESI-MS: m / z = 451 [M+H] + ;

[0135] Step 6. 45 mg (0.1 mmol) of 2-amino-N-(5-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-2-(pyrrolidin-1-yl)phenyl)acetamide was dissolved in 4 ml of anhydrous dichloromethane mixed with 30.3 mg (0.3 mmol) of triethylamine. 8 mg (0.09 mmol) of acryloyl chloride was then added and stirred at room temperature for 1 hour. The reaction was determined to be complete by TLC. The organic phase was extracted with DCM several times and washed with water, dried over MgSO₄, and the crude product was separated by flash column chromatography using a gradient of MeOH:DCM = 1:10. The combined products were concentrated to dryness to yield 19 mg of N-(2-((5-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-5-(pyrrolidin-1-yl)phenyl)amino)-2-oxoethyl)acrylamide. ESI-MS: m / z = 505 [M+H] + ; 1 HNMR(400MHz,Chloroform-d)δ8.27(s,1H),8.19(d,J=2.0Hz,1H),7.64(dd,J=8.5,2 .1Hz,1H),7.49-7.46(m,2H),7.17(d,J=6.6Hz,2H),6.93(d,J=8.5Hz,1H),6.36(dd,J =16.9,1.6Hz,1H),6.28-6.24(m,2H),5.71(dd,J=10.1,1.6Hz,1H),4.18(d,J=5.4Hz, 2H), 3.20 (t, J = 5.7Hz, 4H), 2.27 (s, 3H), 1.98 (d, J = 0.9Hz, 3H), 1.92 (q, J = 3.3Hz, 4H).

[0136] Example 4 N-(3-((5-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-2-(pyrrolidin-1-yl)phenyl)amino)-3-oxopropyl)acrylamide

[0137]

[0138] Synthesized using a method similar to that described in Example 3, using BOC-β-alanine in step d.

[0139] ESI-MS: m / z = 519 [M+H] + ; 1 H NMR(400MHz,Chloroform-d)δ8.14(d,J=2.2Hz,1H),7.85(s,1H),7.64(dd,J=8.5,2.1Hz,1H ),7.50-7.46(m,2H),7.18-7.15(m,2H),6.91(d,J=8.5Hz,1H),6.28-6.25(m,1H),6.23(d,J= 1.6Hz,1H),6.07(dd,J=17.0,10.2Hz,1H),5.60(dd,J=10.2,1.6Hz,1H),3.68(q,J=5.9Hz,2H ), 3.26-3.19 (m, 4H), 2.68 (t, J = 5.8Hz, 2H), 2.26 (s, 3H), 1.98 (s, 3H), 1.92 (q, J = 3.4Hz, 4H).

[0140] Example 5 N-(5-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-2-(pyrrolidin-1-yl)phenyl)-3-propionamidopropionamide

[0141]

[0142] Synthesized using a similar method as described in Example 4, using propionyl chloride in step f.

[0143] ESI-MS: m / z = 521 [M+H] +;1H NMR(400MHz,Chloroform-d)δ8.15(d,J=2.1Hz,1H),7.83(s,1H),7.64(dd,J=8.5 ,2.1Hz,1H),7.49-7.46(m,2H),7.18-7.15(m,2H),6.92(d,J=8.5Hz,1H),6.25(s, 1H),3.60(q,J=5.9Hz,2H),3.26-3.19(m,4H),2.64(t,J=5.8Hz,2H),2.26(s,3H) ,2.17(q,J=7.6Hz,2H),1.98(s,3H),1.93(q,J=3.3Hz,4H),1.11(t,J=7.6Hz,3H).

[0144] Example 6 N-(2-((2-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-5-(pyrrolidin-1-yl)phenyl)amino)-2-oxoethyl)acrylamide

[0145] Synthesized according to Scheme 1 steps a, b, c, d, e, f, g.

[0146]

[0147] Step 1. Methyl 4-chloro-2-nitrobenzoate (108 mg, 0.5 mmol) was dissolved in N,N-dimethylformamide (2 ml). Potassium carbonate (212 mg, 1.5 mmol) and tetrahydropyrrole (42 mg, 0.6 mmol) were added sequentially and stirred at 85°C for 2 h. After the reaction, the reaction solution was quenched with water and extracted with ethyl acetate five times. The mixture was backwashed twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate and purified by column chromatography (PE:EA = 7:1) to obtain 127 mg of methyl 2-nitro-4-(pyrrolidin-1-yl)benzoate. ESI-MS: m / z = 251 [M+H] + . Step 2. Weigh 205 mg (1 mmol) of 1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole and add it to 8 ml of acetonitrile, then add 248 mg (1.1 mmol) of N-iodosuccinimide and stir at room temperature for 3 hours. TLC detection shows that the reaction is complete. Use DCM to extract and wash the organic phase with water several times, dry it with MgSO4, and separate the crude product by flash column chromatography with an elution gradient of pure DCM. The products are combined, concentrated, and dried to obtain 272 mg of 1-(4-chlorophenyl)-3-iodo-2,5-dimethyl-1H-pyrrole. ESI-MS: m / z=332[M+H] + .

[0148] Step 3. Weigh 331 mg (1 mmol) of 1-(4-chlorophenyl)-3-iodo-2,5-dimethyl-1H-pyrrole into anhydrous tetrahydrofuran. Cool to -78°C under nitrogen, then add 77 mg (1.2 mmol) of n-butyl lithium. After stirring at -78°C under nitrogen for 1 hour, add 250 mg (1 mmol) of methyl 2-nitro-4-(pyrrolidin-1-yl)benzoate, and then stir at room temperature overnight. TLC analysis showed no change in the reaction. The organic phase was extracted with DCM several times and washed with water, dried over MgSO4, and the crude product was separated by flash column chromatography with a pure DCM gradient. The products were combined, concentrated, and dried to yield 101 mg of (1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)(2-nitro-4-(pyrrolidin-1-yl)phenyl)methanone. ESI-MS: m / z = 424 [M+H] + .

[0149] Step 4. Weigh 42.3 mg (0.1 mmol) of (1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)(2-nitro-4-(pyrrolidin-1-yl)phenyl)methanone and dissolve it in 4 ml of methanol. Then add 4.3 mg (1%) of palladium on carbon and stir at room temperature overnight. TLC confirmed the reaction was complete. After filtration, the mixture was concentrated to dryness to yield 37.2 mg of the product (2-amino-4-(pyrrolidin-1-yl)phenyl)(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methanone. ESI-MS: m / z = 393 [M+H] + ;

[0150] Step 5. 17.5 mg (0.1 mmol) of (tert-butyloxycarbonyl)glycine was weighed and added to 2 ml of dichloromethane, followed by the addition of 57 mg (0.15 mmol) of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and 39 mg (0.3 mmol) of N,N-diisopropylethylamine. The mixture was stirred at room temperature for 1 hour. Then, 39 mg (0.1 mmol) of (2-amino-4-(pyrrolidin-1-yl)phenyl)(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methanone was added and stirred at room temperature for 4 hours. The reaction was complete by TLC. The organic phase was extracted with DCM several times and washed with water, dried over MgSO4, and the crude product was separated by flash column chromatography with an elution gradient of EA:PE = 1:5. The products were combined, concentrated, and dried to obtain 22 mg of tert-butyl (2-((2-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-5-(pyrrolidin-1-yl)phenyl)amino)-2-oxoethyl)carbamate. ESI-MS: m / z = 551 [M+H] + ;

[0151] Step 6. 55 mg (0.1 mmol) of tert-butyl (2-((2-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-5-(pyrrolidin-1-yl)phenyl)amino)-2-oxoethyl)carbamate was added to 0.5 ml (1 mmol, 2 M) hydrochloric acid and ethyl acetate solution and stirred at room temperature for 0.5 hour. TLC indicated that the reaction was complete. The reaction was quenched with saturated aqueous sodium bicarbonate solution, extracted with EA several times, and the organic phase was washed with water and dried over MgSO4. The products were combined and concentrated to dryness to obtain 42 mg of 2-amino-N-(2-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-2-(pyrrolidin-1-yl)phenyl)acetamide. ESI-MS: m / z = 451 [M+H]. + ;

[0152] Step 7. 45 mg (0.1 mmol) of 2-amino-N-(2-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-2-(pyrrolidin-1-yl)phenyl)acetamide was dissolved in 4 ml of anhydrous dichloromethane mixed with 30.3 mg (0.3 mmol) of triethylamine. 8 mg (0.09 mmol) of acryloyl chloride was then added and stirred at room temperature for 1 hour. The reaction was determined to be complete by TLC. The organic phase was extracted with DCM several times and washed with water, dried over MgSO₄, and the crude product was separated by flash column chromatography using a gradient of MeOH:DCM = 1:10. The combined products were concentrated to dryness to yield 17 mg of N-(2-((2-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-5-(pyrrolidin-1-yl)phenyl)amino)-2-oxoethyl)acrylamide. ESI-MS: m / z = 506 [M+H] + ; 1 HNMR(400MHz,Chloroform-d)δ7.86(d,J=2.4Hz,1H),7.79(d,J=8.9Hz,1H),7.52-7.4 5(m,2H),7.21-7.15(m,2H),6.70(s,1H),6.34(d,J=1.7Hz,1H),6.28(d,J=10.0Hz,1H ),6.25-6.21(m,1H),6.13(d,J=1.2Hz,1H),5.68(ddd,J=10.1,1.7,0.8Hz,1H),4.28- 4.22(m,2H),3.44-3.36(m,4H),2.17(s,3H),2.06-2.01(m,4H),1.99(d,J=0.9Hz,3H).

[0153] Example 7 2-Chloro-N-(2-((2-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-5-(pyrrolidin-1-yl)phenyl)amino)-2-oxoethyl)acetamide

[0154]

[0155] Synthesized using a similar method as described in Example 6, using chloroacetyl chloride in step g.

[0156] ESI-MS: m / z = 527 [M+H] + ; 1 H NMR(400MHz,Chloroform-d)δ7.87(d,J=2.4Hz,1H),7.80(d,J=8.9Hz,1H),7.51-7.45(m,2H),7.36(t,J=5.3Hz,1H),7.20-7.15(m,2H), 6.13 (d, J = 1.0Hz, 1H), 4.19 (d, J = 5.9Hz, 4H), 3.40 (td, J = 6.7, 5.4, 3.2Hz, 4H), 2.18 (s, 3H), 2.01 (d, J = 3.3Hz, 4H), 1.98 (d, J = 1.0Hz, 3H).

[0157] Example 8 N-(2-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-5-(pyrrolidin-1-yl)phenyl)-2-cyanaminoacetamide

[0158]

[0159] Synthesized using a method similar to that described in Example 6, using cyanogen bromide in step g.

[0160] ESI-MS: m / z = 476 [M+H] + ; 1 H NMR(400MHz,Chloroform-d)δ7.83-7.78(m,2H),7.50(d,J=2.8Hz,1H),7.49-7.48(m,1H),7.47(d,J=2.7Hz,1H),7.18(d,J=2.1Hz,1H),7.16 (d,J=2.0Hz,1H),6.12(d,J=1.1Hz,1H),3.98(d,J=5.1Hz,2H),3.40(d,J=4.6Hz,4H),2.17(s,3H),2.03-2.01(m,4H),1.98(d,J=0.9Hz,3H).

[0161] Example 9 N-(2-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-5-(pyrrolidin-1-yl)phenyl)-2-(prop-2-yn-1-ylamino)acetamide

[0162]

[0163] Synthesized using a similar method as described in Example 6, using 3-bromopropyne in step g.

[0164] ESI-MS: m / z = 489 [M+H] + ; 1 H NMR(400MHz,Chloroform-d)δ7.94(d,J=2.4Hz,1H),7.75(d,J=8.8Hz,1H),7.50-7.44(m,2H),7.19-7.14(m,2H),6.21(dd,J=8.9,2.5Hz, 1H), 6.14 (d, J = 1.1Hz, 1H), 3.65 (s, 2H), 3.62 (d, J = 2.5Hz, 2H), 3.44-3.37 (m, 4H), 2.26 (t, J = 2.4Hz, 1H), 2.04-2.00 (m, 5H), 1.98 (s, 3H).

[0165] Example 10 N-(2-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-5-(pyrrolidin-1-yl)phenyl)-2-(di(prop-2-yn-1-yl)amino)acetamide

[0166]

[0167] Synthesized using a method similar to that described in Example 9.

[0168] ESI-MS: m / z = 527 [M+H] + ;1H NMR(400MHz,Chloroform-d)δ7.94(d,J=2.4Hz,1H),7.74(d,J=8.9Hz,1H),7. 52-7.41(m,2H),7.22-7.13(m,2H),6.20(dd,J=8.9,2.5Hz,1H),6.15(t,J=1. 0Hz,1H),3.62(d,J=2.4Hz,4H),3.45(s,2H),3.39(td,J=5.4,4.2,2.5Hz,4H) ,2.23(t,J=2.4Hz,2H),2.16(s,3H),2.05-1.99(m,4H),1.98(d,J=1.0Hz,3H).

[0169] Example 11 N-(3-(5-(2-(dimethylamino)ethoxy)-1H-indole-3-carbonyl)phenyl)acrylamide was synthesized according to Scheme 3, steps a, b, c, e, f, g.

[0170]

[0171] Step 1. Dissolve 167 mg (1 mmol) of 3-nitrobenzoic acid in 3 ml of anhydrous dichloromethane and add 3 drops of N,N-dimethylformamide. Then, add 357 mg (3 mmol) of thionyl chloride and stir at room temperature for 30 minutes. TLC confirms the reaction is complete. Concentrate under reduced pressure to obtain a pale yellow solid, 3-nitrobenzoyl chloride, which is then used for further use.

[0172] Step 2. Dissolve 3-nitrobenzoyl chloride in 3 ml of anhydrous dichloromethane, then add 133 mg (1 mmol) of aluminum chloride and stir at 0°C under nitrogen for 30 minutes. After the aluminum chloride solid dissolves, add 147 mg (1 mmol) of 5-methoxyindole at 0°C, then return to room temperature and stir for 6 hours under nitrogen. TLC detection shows that the reaction is complete. Use DCM to extract and wash the organic phase with water several times, dry it with MgSO4, and separate the crude product by flash column chromatography with an elution gradient of EA:PE=1:2. The products are combined, concentrated, and dried to obtain 212 mg of (5-methoxyindol-3-yl)(3-nitrophenyl)methanone. ESI-MS: m / z=297[M+H] + Step 3. Weigh 296 mg (1 mmol) of (5-methoxyindol-3-yl)(3-nitrophenyl)methanone and dissolve it in 4 ml of anhydrous dichloromethane. Then add 250 mg (1 mmol) of boron tribromide and stir at room temperature for 2 hours. TLC indicates that the reaction is complete. The organic phase is extracted with DCM several times and washed with water, then dried over MgSO4. The crude product is separated by flash column chromatography with an elution gradient of MeOH:DCM = 1:20. The products are combined, concentrated, and dried to obtain 101 mg of (5-hydroxy-1H-indol-3-yl)(3-nitrophenyl)methanone. ESI-MS: m / z = 283 [M+H] + ;

[0173] Step 4. Weigh 282 mg (1 mmol) of (5-hydroxy-1H-indol-3-yl)(3-nitrobenzene)methanone and add it to 112 mg (2 mmol) of potassium hydroxide dissolved in 6 ml of ethanol. Heat to 60°C and stir for 5 minutes. Then add 233 mg (1 mmol) of N,N-dimethylaminoethyl bromide hydrobromide and stir at 60°C for 4 hours. TLC indicates that the reaction is complete. The organic phase is extracted with DCM several times and washed with water, dried over MgSO4, and the crude product is separated by flash column chromatography with an elution gradient of MeOH:DCM = 1:20. The products are combined, concentrated, and dried to obtain 177 mg of (5-(2-(dimethylamino)ethoxy)-1H-indol-3-yl)(3-nitrophenyl)methanone. ESI-MS: m / z = 354 [M+H] + ;

[0174] Step 5. 353 mg (1 mmol) of (5-(2-(dimethylamino)ethoxy)-1H-indol-3-yl)(3-nitrophenyl)methanone was dissolved in 4 ml of methanol, followed by the addition of 3.4 mg (1%) of palladium on carbon, and stirred at room temperature overnight. The reaction was determined to be complete by TLC. The mixture was filtered and concentrated to dryness to afford 344 mg of (3-aminophenyl)(5-(2-(dimethylamino)ethoxy)-1H-indol-3-yl)methanone. ESI-MS: m / z = 324 [M+H] + ;

[0175] Step 6. 323 mg (1 mmol) of (3-aminophenyl)(5-(2-(dimethylamino)ethoxy)-1H-indol-3-yl)methanone was dissolved in 4 ml of anhydrous dichloromethane mixed with 303 mg (3 mmol) of triethylamine. 81 mg (0.9 mmol) of acryloyl chloride was then added and stirred at room temperature for 1 hour. The reaction was complete by TLC. The organic phase was extracted multiple times with DCM and washed with water, dried over MgSO₄, and the crude product was separated by flash column chromatography with an elution gradient of MeOH:DCM = 1:10. The products were combined, concentrated, and dried to yield 67 mg of N-(3-(5-(2-(dimethylamino)ethoxy)-1H-indole-3-formyl)phenyl)acrylamide.

[0176] ESI-MS: m / z = 378 [M+H] +;1H NMR (400MHz, DMSO-d6) δ10.35(s,1H),7.87(s,1H),7.85(s,1H),7.83(s,1H),7.74(d,J=2.5Hz,2H),7.45(s,1H),7.43(s,1H),7 .39(s,1H),7.37(s,1H),6.86(dd,J=8.8,2.5Hz,1H),6.27(d,J=2.1Hz,1H),5.73(s,1H),4.05(s,2H),3.19(s,2H),2.22(s,6H).

[0177] Example 12 N-(2-(1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonyl)-5-(pyrrolidin-1-yl)phenyl)-2-(di(prop-2-yn-1-yl)amino)acetamide

[0178]

[0179] The compound was synthesized by a method similar to that described in Example 11, with the addition of step d.

[0180] ESI-MS: m / z = 364 [M+H] + ;1H NMR (400MHz, DMSO-d6) δ10.65(s,-1H),10.03(s,-1H),7.47(d,J=8.2Hz,1H),7.18(s, 1H),7.16(d,J=1.9Hz,1H),7.06(d,J=2.5Hz,1H),6.97(d,J=8.0Hz,1H),6.85(d,J=2. 4Hz,1H),6.65(dd,J=8.7,2.5Hz,1H),6.36(dd,J=17.1,10.1Hz,1H),6.19(d,J=2.2Hz ,1H),5.70-5.63(m,2H),3.94(d,J=5.8Hz,2H),3.92(s,2H),3.13(s,2H),2.17(s,6H).

[0181] Example 13 4-(Dimethylamino)-N-(3-(6-methoxy-1H-indole-3-carbonyl)phenyl)but-2-enamide was synthesized according to Scheme 3, steps a, b, and d.

[0182]

[0183] Step 1. Dissolve 167 mg (1 mmol) of 3-nitrobenzoic acid in 3 ml of anhydrous dichloromethane, add 3 drops of N,N-dimethylformamide, then add 357 mg (3 mmol) of thionyl chloride and stir at room temperature for 30 minutes. TLC confirms the reaction is complete. Concentrate under reduced pressure to obtain a pale yellow solid, 3-nitrobenzoyl chloride, for later use. Dissolve 3-nitrobenzoyl chloride in 3 ml of anhydrous dichloromethane, then add 133 mg (1 mmol) of aluminum chloride. Stir at 0°C under nitrogen for 30 minutes. Once the aluminum chloride solid has dissolved, add 147 mg (1 mmol) of 5-methoxyindole at 0°C. Return the mixture to room temperature and stir under nitrogen for 6 hours. TLC confirms the reaction is complete. Extract the organic phase several times with DCM, wash with water, and dry it over MgSO4. The crude product is separated by flash column chromatography using an elution gradient of EA:PE = 1:2. The products were combined, concentrated, and dried to obtain 221 mg of (5-methoxyindol-3-yl)(3-nitrophenyl)methanone. ESI-MS: m / z = 297 [M+H] + ;

[0184] Step 2. Weigh 296 mg (1 mmol) of (5-methoxyindol-3-yl)(3-nitrophenyl)methanone and dissolve it in 4 ml of methanol. Then add an appropriate amount of Raney nickel and stir at room temperature overnight. TLC confirms the reaction is complete. After filtration, the mixture is concentrated to dryness to obtain 233 mg of the product (3-aminophenyl)(6-methoxy-1H-indol-2-yl)methanone. ESI-MS: m / z = 267 [M+H] + ;

[0185] Step 3. 12.9 mg (0.1 mmol) of 4-(dimethylamino)but-2-enoic acid hydrochloride was weighed and added to 2 ml of dichloromethane. 57 mg (0.15 mmol) of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and 39 mg (0.3 mmol) of N,N-diisopropylethylamine were then added. The mixture was stirred at room temperature for 1 hour. Then, 26 mg (0.1 mmol) of (3-aminophenyl)(6-methoxy-1H-indol-2-yl)methanone was added and stirred at room temperature for 4 hours. The reaction was determined to be complete by TLC. The organic phase was extracted with DCM several times and washed with water, dried over MgSO₄, and the crude product was separated by flash column chromatography using an elution gradient of EA:PE = 1:5. The combined products were concentrated to dryness to yield 10 mg of (E)-4-(dimethylamino)-N-(3-(6-methoxy-1H-indole-2-carbonyl)phenyl)but-2-enoic acid amide. ESI-MS: m / z = 378 [M+H] + ; 1H NMR(400MHz,Methanol-d4)δ8.01(t,J=1.9Hz,1H),7.84(d,J=2.5Hz,1H),7.8 1(ddd,J=8.1,2.3,1.2Hz,1H),7.73(s,1H),7.50(d,J=6.7Hz,2H),7.41(t,J= 7.8Hz,1H),7.32(d,J=8.8Hz,1H),6.88(dd,J=8.8,2.5Hz,1H),6.85-6.77(m, 1H), 6.42-6.32 (m, 1H), 3.85 (s, 4H), 3.50 (dd, J = 6.8, 1.4Hz, 2H), 2.55 (s, 7H).

[0186] Example 14 4-(Dimethylamino)-N-(3-((6-methoxy-1H-indol-3-yl)methyl)phenyl)but-2-enamide

[0187]

[0188] The product was synthesized by a method similar to that described in Example 13, with the addition of step c.

[0189] ESI-MS: m / z = 364 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ8.00(s,1H),7.73(t,J=6.4Hz,2H),7.61(d,J=2.6Hz,1H),7.29(q,J=9.0,8.2Hz,4H),6.72(dd,J =8.8, 2.6Hz, 1H), 6.60 (dt, J = 15.4, 6.2Hz, 1H), 6.26 (d, J = 15.4Hz, 1H), 3.06 (d, J = 6.2Hz, 2H), 2.11 (s, 6H), 1.59 (s, 2H).

[0190] Example 15 Determination of the inhibitory activity of compounds on USP14 protein

[0191] Compounds were added to a 384-well plate (containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mg / mL ovalbumin, 5 mM ATP / MgCl2, 1 mM DTT (dithiothreitol), 1 nM Ptsm-VS, and 15 nM USP14) at gradient concentrations (ranging from 0.1 μM to 50 μM) and pre-incubated for 30 minutes. To start the reaction, 1 μM Ub-AMC was added to the system. Fluorescence was measured by real-time monitoring of the reaction at room temperature for 45 minutes using an Envision plate reader. Each experiment was repeated 3 times, and the average value was calculated. The IC values ​​of the compounds for USP14 were 0.05, 0.1, and 0.1, respectively. 50 The data are shown in Table 5.

[0192] Table 5

[0193] Compound number <![CDATA[IC 50 (μM)]]> Compound number <![CDATA[IC 50 (μM)]]> A1 25.12 A8 22.69 A2 29.51 A9 26.77 A3 3.87 A10 >50 A4 8.54 A11 >50 A5 13.28 A12 >50 A6 5.01 A13 >50 A7 >30 A14 >50

[0194] It can be seen from the above table that the compounds of the present invention can inhibit the activity of USP14 protein.

[0195] Example 16 Determination of IC of Compounds against HCT116 Cell Line 50

[0196] HCT116 cells (Institute of Cell Collection, Chinese Academy of Sciences) were seeded in 96-well microplates with 5000 cells per well. The compound was dissolved in DMSO and diluted to a test gradient concentration (concentration range 0.1μM-50μM). After adding all reagents, the 96-well plate was placed in an incubator for 24 hours, and then 10μl of MTT solution was added to each well. The incubation continued for 4h, the supernatant was removed, and 150μl of DMSO was added to each well. The plate was placed on a shaker and shaken at low speed for 10min to fully dissolve the crystals. The absorbance of each well was measured at OD490nm in an enzyme-linked immunosorbent assay. IC inhibition of cell proliferation of the compound on the HCT116 cell line 50 As shown in Table 6.

[0197] Table 6

[0198] Compound number <![CDATA[IC 50 (μM)]]> Compound number <![CDATA[IC 50 (μM)]]> A1 >50 A8 >50 A2 >50 A9 >50 A3 4.61 A10 >50 A4 9.41 A11 >50 A5 37.62 A12 >50 A6 7.83 A13 >50 A7 >50 A14 >50

[0199] It can be seen from the above table that the compounds of the present invention can inhibit the proliferation of HCT116 cell line.

[0200] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A compound or its stereoisomers, geometric isomers, tautomers, or pharmaceutically acceptable salts, characterized in that: The compound is selected from: 。 2. A method for preparing the compound according to claim 1 or its stereoisomers, geometric isomers, tautomers, or pharmaceutically acceptable salts, characterized in that: Includes at least one of the following synthetic routes: Synthesis Route 1: ； Synthesis route 2: ； Among them, R1 is or , R2 is or .

3. Use of the compound according to claim 1 or its stereoisomers, geometric isomers, tautomers, and pharmaceutically acceptable salts in the preparation of USP14 covalent inhibitors.

4. Use of the compound according to claim 1 or its stereoisomers, geometric isomers, tautomers, and pharmaceutically acceptable salts as a USP14 covalent inhibitor in the preparation of antitumor drugs.

5. The use according to claim 4, characterized in that The tumor is selected from one or more of cervical squamous cell carcinoma, bile duct carcinoma, hepatocellular carcinoma, gastric adenocarcinoma, sarcomatoid lung cancer, thymic carcinoma, pancreatic cancer, colorectal cancer, neuroblastoma, and multiple myeloma.

6. A drug, characterized in that The invention comprises the compound according to claim 1 or its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts and one or more pharmaceutically acceptable carriers, diluents and excipients.

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