Bifunctional compounds that inhibit mTOR while degrading gspt1 protein

Abstract

The present disclosure relates to a bifunctional compound for inhibiting mTOR while degrading GSPT1 protein, a preparation method and application thereof, the compound having a structure shown in formula (I), wherein each group is defined in detail in the specification, and the present disclosure also relates to a tautomer, an enantiomer, a diastereomer, a mixture of enantiomers and diastereomers, a racemate, an endo-racemate, a mixture of exo-racemate and endo-racemate, a pharmaceutically acceptable hydrate, a pharmaceutically acceptable salt, a solvate, a polymorph or a nitrogen oxide of the compound, the compound being a bifunctional inhibitor of mTor and GSPT1 protein, and having high activity and good selectivity.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of medicinal chemistry, in particular to a bifunctional compound for inhibiting mTOR while degrading GSPT1 protein, and a preparation method and application thereof. BACKGROUND

[0002] Tumor-targeted drug therapy has the great advantage of "precise treatment, high efficiency, and few side effects", which promotes the development of tumor treatment. The emerging drug resistance of tumor-targeted drugs is a major problem faced by tumor research. Traditional small molecule kinase inhibitors inhibit enzyme activity by occupying the active pocket of the target enzyme. Due to the high concentration and long time occupation of the active site, it is easy to cause the target to be resistant to traditional small molecule inhibitors, and thus cause the disease to relapse.

[0003] Small molecule-induced protein degradation technology is a research field that has been booming in recent years and has attracted much attention from the pharmaceutical industry. Small molecule-induced degradation of the entire protein is a repeated process with catalytic properties, as it triggers ubiquitination of the target protein and then dissociates into the next catalytic cycle. Therefore, the degrader can function at very low doses and show better tolerance in the case of target protein mutation or overexpression. At the same time, this mechanism of action does not require high binding force, and even in the case of target mutation, it can still degrade the target protein to overcome drug resistance. As a targeted protein degradation technology, degraders have a broader prospect in drug development, as they can overcome drug resistance, target difficult-to-drug targets, eliminate protein non-scaffold functions, and other advantages. The process of degraders inducing the formation of a ternary complex between the target protein and E3 ubiquitin ligase and mediating the interaction between the two proteins is quite important, so when designing degrader drugs, it is necessary to first achieve effective interaction between the target protein and E3 ubiquitin ligase, while ensuring the high drugability of the degrader molecule, thus increasing the difficulty of degrader design.

[0004] To solve the problems of inhibitor resistance and degrader design difficulty, the inventors propose to combine the two, design a bifunctional molecule that retains the activity of the inhibitor and functions as a degrader, to overcome clinical inhibitor resistance and provide a new direction for the treatment of malignant tumors.

[0005] The mammalian target of rapamycin (mTOR) regulates several fundamental biological functions, including cell growth, metabolism, survival, and immune responses, by forming two important complexes, mTOR complex 1 (mTORC1) and complex 2 (mTORC2). mTOR signaling is frequently dysregulated in cancer and is considered an attractive target for cancer therapy. Pharmaceutical companies have made great efforts to develop potent mTOR inhibitors, particularly kinase inhibitors that inhibit both mTORC1 and mTORC2; however, no kinase inhibitor targeting mTOR has been approved for marketing. The main reason for the lack of sensitivity or response of clinical patients to cancer treatment targeting mTOR is the emergence of mTOR inhibitor resistance, such as the induced activation of PI3K / Akt and MEK / ERK survival signaling pathways to compensate for mTOR inhibition caused by rapamycin. In addition, mTOR is also involved in the regulation of neurodegenerative diseases, metabolic diseases, and aging.

[0006] G1 to S phase transition 1 (GSPT1) was originally identified as a gene essential for the transition from G1 to S phase in the cell cycle, and was later renamed eukaryotic releasing factor 3a (eRF3a). eRF3a, as a key member of the peptide release factor, is involved in various biological processes, including terminating protein translation, regulating intracellular mRNA degradation, regulating cell growth cycle and apoptosis, and participating in cytoskeleton formation. Abnormal function of GSPT1 is closely related to the occurrence and evolution of various common malignant tumors, such as breast cancer, gastric cancer, colorectal cancer, etc. However, as a GTPase, it is very difficult to develop competitive compounds targeting GSPT1 due to the high concentration of GTP in cells and the strong binding of the GTP binding pocket to GTPase. Therefore, GSPT1 has been considered as an undruggable target.

[0007] Currently, there is no compound that can simultaneously inhibit mTOR and degrade GSPT1, making it difficult to achieve both. It is of great significance to develop a compound that can selectively inhibit mTOR and degrade GSPT1. SUMMARY

[0008] The inventors of the present patent chose mTOR inhibition and GSPT1 degradation as a solution to overcome the problem of clinical resistance to mTOR inhibitors. The purpose of the present disclosure is to provide a bifunctional compound for inhibiting mTor and degrading GSPT1 protein, as well as a preparation method and application thereof. The compound as a bifunctional inhibitor of mTor and GSPT1 protein has the advantages of high activity, good selectivity, and low toxicity and side effects.

[0009] Specifically, this disclosure relates to compounds represented by formula (I) or their tautomers, enantiomers, diastereomers, mixtures of enantiomers and diastereomers, racemates, mesomates, mixtures of racemates and mesomates, pharmaceutically acceptable hydrates, pharmaceutically acceptable salts, solvates, polymorphs, or nitrogen oxides:

[0010]

[0011] in,

[0012] G is connected to L via Z1, and G has the following structure:

[0013]

[0014] X1, X2, X 10 X 11 X 12 Each is independently selected from CH, C, or N.

[0015] R1 is selected from hydrogen, amino, or...

[0016] R6, R7, R8, and R9 are each independently selected from hydrogen and C. 1-6 Alkyl, the C 1-6 The alkyl group may optionally be replaced by one or more halogens.

[0017] Preferably, R6, R7, R8, and R9 are each independently selected from hydrogen, methyl, and ethyl, wherein the methyl and ethyl groups are optionally replaced by one or more fluorine molecules.

[0018] Preferably, R6, R7, R8, and R9 are each independently selected from hydrogen, methyl, ethyl, trifluoromethyl, -CF2CH3, -CF2CF3, and -CH2CF3.

[0019] Preferably, R1 is selected from hydrogen, amino,

[0020] R2 does not exist or is

[0021] R3 is absent or is selected from hydrogen, halogens, Where n7 is selected from integers from 1 to 5.

[0022] R 10 Selected from hydroxyl, amino, halogen, C 1-5 Alkoxy, C 1-5 alkylamine group, the C 1-5 Alkoxy, C 1-5 The alkylamine group may optionally be replaced by one or more deuterium or halogens.

[0023] Preferably, R 10 Selected from hydroxyl, deuterated methylamino, deuterated methoxy, trifluoromethoxy, and difluoromethoxy.

[0024] A1 is selected from hydrogen, or C with any substitution. 5-18 aryl, 5 to 18-membered heteroaryl, 5 to 18-membered heterocyclic, or -NH-R5, wherein R5 is selected from any substituted C 5-18 Aryl, 5 to 18 heteroaryl compounds.

[0025] Preferably, A1 is selected from hydrogen, C 5-18 aryl, 5 to 18-membered heteroaryl, 5 to 18-membered heterocyclic, or -NH-R5, wherein R5 is selected from C 5-18 Aryl, 5- to 18-membered heteroaryl, said C 5-18 The aryl, 5- to 18-membered heteroaryl, and 5- to 18-membered heterocyclic groups are optionally replaced by amino, hydroxyl, nitro, cyano, methyl, or trifluoromethyl groups.

[0026] Preferably, A1 is selected from hydrogen, phenyl, pyrrolyl, pyrazolyl, imidazoleyl, pyridyl, pyrimidinyl, benzopyrazolyl, morpholinyl, piperazineyl, -NH-phenyl, -NH-pyridyl, -NH-pyrimidinyl, wherein the phenyl, pyrrolyl, pyrazolyl, imidazoleyl, pyridyl, pyrimidinyl, benzopyrazolyl, morpholinyl, and piperazineyl are optionally substituted with amino, hydroxyl, nitro, cyano, methyl, or trifluoromethyl.

[0027] Preferably, A1 is selected from hydrogen, phenyl,

[0028]

[0029] Preferably, G is selected from:

[0030]

[0031] Preferably, G is selected from Preferably, G is selected from Z1 is selected from direct bond, -CH2-,

[0032]

[0033] Preferably, Z1 is selected from direct bonds, -CH2-,

[0034] L has the following structure:

[0035] Where n1 and n3 are each independently selected from 0 or 1; n2 and n4 are each independently selected from integers from 0 to 4;

[0036] M is selected from O or H; J is selected from -O- or -NH-;

[0037] n2 R 11 Whether they are the same or different, n4 R 12 Whether they are the same or different, R 11 and R 12 Each is independently selected from -O-, amino, -C(O)-, C 1-6 Alkyl, C 2-6 alkynyl group, C 3-9 cycloalkyl, C 5-12 aryl, 4-12-membered heterocyclic alkyl, 5-9-membered heteroaryl, wherein C 3-9 cycloalkyl, C 5-12 Aryl, 4-12-membered heterocyclic alkyl, 5-9-membered heteroaryl groups are optionally coated with halogens, hydroxyl groups, amino groups, C 1-6 Alkyl group, -(CH2) n5 OH or -(CH2) n5 The form is replaced by COOH, where n5 is an integer selected from 0 to 3.

[0038] Preferably, R 11 and R 12 Each of the following groups is independently selected from -O-, amino, -C(O)-, -CH2-, -CH2CH2-, divalent tert-butyl, ethynyl, cyclopentyl, cyclohexyl, bicyclo[1.1.1]pentyl, phenyl, naphthyl, pyridyl, pyrazinyl, pyrimidinyl, thiophene, azircyclic butyl, pyrrolyl, piperidinyl, piperazinyl, diazaspiroheptyl, diazaspironyl, dioxane, wherein the cyclopentyl, cyclohexyl, bicyclo[1.1.1]pentyl, phenyl, naphthyl, pyridyl, pyrazinyl, pyrimidinyl, thiophene, azircyclic butyl, pyrrolyl, piperidinyl, piperazinyl, diazaspiroheptyl, diazaspironyl, dioxane, and dioxane are optionally halogenated, hydroxyl, amino, methyl, ethyl, or -(CH2). n5 OH or -(CH2) n5 The form is replaced by COOH, where n5 is an integer selected from 0 to 3.

[0039] Preferably, L is selected from single bonds,

[0040] Preferably, L is selected from single bonds.

[0041] Y is selected from

[0042] K1 is selected from hydrogen, halogens, and C.1-5 Alkoxy group, K2 is selected from O or H.

[0043] Preferably, Y is selected from

[0044] This disclosure relates to a series of compounds or their tautomers, enantiomers, diastereomers, mixtures of enantiomers and diastereomers, racemates, mesomates, mixtures of racemates and mesomates, pharmaceutically acceptable hydrates, pharmaceutically acceptable salts, solvates, polymorphs or nitrides, comprising compounds represented by formula (IIa) or (IIb):

[0045]

[0046]

[0047] Among them, X1, X2, R1, R7, R 7a The definitions of A1, Z1, and L are consistent with those in the previous text.

[0048] This disclosure relates to a compound or its tautomers, enantiomers, diastereomers, mixtures of enantiomers and diastereomers, racemates, mesomates, mixtures of racemates and mesomates, pharmaceutically acceptable hydrates, pharmaceutically acceptable salts, solvates, polymorphs, or nitrides, wherein the compound comprises the compound represented by formula (IIIa1), (IIIa2), or (IIIb1):

[0049]

[0050] The definitions of A1, n5, and L are consistent with those in the previous text.

[0051] This disclosure relates to the following compounds or their tautomers, enantiomers, diastereomers, mixtures of enantiomers and diastereomers, racemates, mesomates, mixtures of racemates and mesomates, pharmaceutically acceptable hydrates, pharmaceutically acceptable salts, solvates, polymorphs, or nitrogen oxides:

[0052]

[0053]

[0054] This disclosure also relates to an intermediate compound having the structure of formula (IVa),

[0055]

[0056] The definitions of L and Y are consistent with those in the previous text, while U is either absent or selected from halogens, hydroxyl groups, or hydrogen.

[0057] Any of the compounds disclosed herein has mTOR inhibition or GSPT1 degradation activity.

[0058] Any of the compounds disclosed herein possesses both mTOR inhibition and GSPT1 degradation activity.

[0059] This disclosure also relates to a pharmaceutical composition comprising, in a physiologically acceptable medium, the aforementioned compound or its tautomers, enantiomers, diastereomers, mixtures of enantiomers and diastereomers, racemates, mesomates, mixtures of racemates and mesomates, pharmaceutically acceptable hydrates, pharmaceutically acceptable salts, solvates, polymorphs, or nitrogen oxides.

[0060] This disclosure also relates to the use of the compounds described above or their tautomers, enantiomers, diastereomers, mixtures of enantiomers and diastereomers, racemates, mesomates, mixtures of racemates and mesomates, pharmaceutically acceptable hydrates, pharmaceutically acceptable salts, solvates, polymorphs or nitrogen oxides, and the use of the pharmaceutical compositions described above in the preparation of medicaments for mTOR inhibition or GSPT1 degradation.

[0061] This disclosure also relates to the use of the compounds described above or their tautomers, enantiomers, diastereomers, mixtures of enantiomers and diastereomers, racemates, mesomates, mixtures of racemates and mesomates, pharmaceutically acceptable hydrates, pharmaceutically acceptable salts, solvates, polymorphs or nitrogen oxides, and the use of the pharmaceutical compositions described above in the preparation of medicaments for mTOR inhibition and GSPT1 degradation.

[0062] This disclosure also relates to a method of inhibiting mTOR or GSPT1 in a patient in need, comprising administering to the patient the compound described above or a tautomer thereof, enantiomer, diastereomer, mixture of enantiomers and diastereomers, racemic, meso, mixture of racemic and meso, pharmaceutically acceptable hydrate, pharmaceutically acceptable salt, solvate, polymorph or nitride, or the pharmaceutical composition described above.

[0063] This disclosure also relates to a method for simultaneously inhibiting mTOR and degrading GSPT1 in a patient in need, comprising administering to the patient the compound described above or a tautomer thereof, enantiomer, diastereomer, mixture of enantiomers and diastereomers, racemic, meso, mixture of racemic and meso, pharmaceutically acceptable hydrate, pharmaceutically acceptable salt, solvate, polymorph or nitride, or the pharmaceutical composition described above.

[0064] This disclosure relates to a method for inhibiting mTOR or GSPT1 protein in a biological sample, comprising contacting the biological sample with the aforementioned compound or its tautomers, enantiomers, diastereomers, mixtures of enantiomers and diastereomers, racemates, mesomates, mixtures of racemates and mesomates, pharmaceutically acceptable hydrates, pharmaceutically acceptable salts, solvates, polymorphs or nitrides, or the aforementioned pharmaceutical compositions.

[0065] This disclosure relates to a method for simultaneously inhibiting mTOR and degrading GSPT1 protein in a biological sample, comprising contacting the biological sample with the aforementioned compound or its tautomers, enantiomers, diastereomers, mixtures of enantiomers and diastereomers, racemic mixtures, mesomixes, mixtures of racemic mixtures and mesomixes, pharmaceutically acceptable hydrates, salts, solvates, polymorphs or nitrides, or the aforementioned pharmaceutical compositions.

[0066] This disclosure relates to a method of treating malignant diseases by inhibiting the mTOR signaling pathway and / or degrading GSPT1 protein, comprising administering to the patient the aforementioned compound or its tautomers, enantiomers, diastereomers, mixtures of enantiomers and diastereomers, racemic mixtures, mesomixes, mixtures of racemic mixtures and mesomixes, pharmaceutically acceptable hydrates, pharmaceutically acceptable salts, solvates, polymorphs or nitrides, or the pharmaceutical composition thereof.

[0067] This disclosure relates to the use of the compounds described above or their tautomers, enantiomers, diastereomers, mixtures of enantiomers and diastereomers, racemates, mesosomes, mixtures of racemates and mesosomes, pharmaceutically acceptable hydrates, pharmaceutically acceptable salts, solvates, polymorphs or nitrides, and the use of the pharmaceutical compositions described above in the preparation of medicaments for treating diseases abnormally mediated by the mTOR signaling pathway and GSPT1 protein, respectively or simultaneously.

[0068] The malignant diseases mentioned include, but are not limited to, tumors and leukemia.

[0069] This disclosure relates to conditions treated by inhibiting the mTOR signaling pathway and / or degrading the GSPT1 protein, including but not limited to cancer and leukemia.

[0070] Preferably, the tumor includes malignant tumors such as colorectal cancer, pancreatic cancer, liver cancer, breast cancer, ovarian cancer, endometrial cancer, lung cancer, prostate cancer, and lymphoma; preferably, the leukemia includes one or more of chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, and acute myeloid leukemia.

[0071] In the structure of the compound disclosed herein, X serves as an mTOR-binding group, L as a linking group, and Y as a ubiquitin ligase-binding group.

[0072] The inventors of this disclosure have experimentally verified the independence between the mTOR inhibition and GSPT1 degradation of the above-mentioned compounds. That is, the bifunctional compounds of this disclosure combine inhibitor technology and degrader technology.

[0073] The disclosed compound can simultaneously inhibit mTOR and degrade GSPT1 in various solid tumor cells such as breast cancer, lung cancer, colorectal cancer, pancreatic cancer, liver cancer, glioma, and hematologic malignancies, and can effectively inhibit the proliferation of tumor cells. Attached Figure Description

[0074] Figure 1 The image shows the Western blot plot of compound 2 in Example 1, which inhibits mTOR protein kinase and degrades GSPT1 protein.

[0075] Figure 2 The Western blot plots show the effects of compounds 2 and 7 in Example 1 on the inhibition of mTOR protein kinase and the degradation of GSPT1 protein.

[0076] Figure 3 Western blot plots showing the inhibition of mTOR protein kinase and degradation of GSPT1 protein by compounds 1 and 9 in Effect Example 1.

[0077] Figure 4 The image shows the Western blot plot of compound 5 in Example 1, which inhibits mTOR protein kinase and degrades GSPT1 protein.

[0078] Figure 5 This is the proteomics detection result in Example 1. Detailed Implementation

[0079] I. Definition

[0080] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0081] The compounds disclosed herein may be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers are included, such as enantiomers and diastereomers. The compounds containing asymmetric carbon atoms of this disclosure can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents. Racemic, diastereomer, and enantiomers are all included within the scope of this disclosure.

[0082] In this disclosure, This refers to the position where the substituents are bonded. It should be noted that the structural fragments described in this disclosure (e.g., L or Z1) do not necessarily represent a left-to-right sequence of the corresponding groups. For example, when L is... When, it can represent It can also mean

[0083] In this disclosure, a range of numbers refers to individual integers within a given range. For example, "C 1-6 "" means that the group can have 1, 2, 3, 4, 5, or 6 carbon atoms; "C 1-3 "" means that the group can have 1 carbon atom, 2 carbon atoms or 3 carbon atoms.

[0084] The term “optional” or “optionally” means that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0085] The terms “substituted” or “substituted” refer to the substitution of one or more hydrogen atoms on a particular atom or group by a substituent, provided that the valence state of the particular atom or group is normal and the substituted compound is stable.

[0086] When any variable (e.g., R) n When a substituent appears more than once in the composition or structure of a compound, its definition is independent in each case. Therefore, for example, if a group is substituted by one to three Rs, the group can optionally be substituted by up to three Rs, and the Rs in each case have independent options. Furthermore, combinations of substituents and / or their variants are only permitted if such combinations produce a stable compound.

[0087] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight-chain or branched saturated hydrocarbon groups, having the indicated number of carbon atoms. For example, the term "C..." 1-6 "Alkyl" includes C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl, examples of which include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, n-hexyl, 2-hexyl, 3-hexyl, etc. It can be divalent, such as methylene and ethylene.

[0088] The term "alkoxy" can be linear, branched, or cyclic. Specific examples include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, i-propyloxy, n-butoxy, isobutoxy, tert-butoxy, sec-butoxy, n-pentoxy, neopentoxy, and isopentoxy.

[0089] In this disclosure, the term "deuterated" means that one or more hydrogen atoms of a compound are replaced by deuterium atoms, and deuteration can be monosubstituted, disubstituted, polysubstituted, or total substituted; the term "deuterated compound" means a compound containing deuterium atoms.

[0090] In this disclosure, examples of halogen groups may include fluorine, chlorine, bromine, or iodine.

[0091] In this disclosure, the term "cycloalkyl" refers to a monocyclic saturated hydrocarbon system without heteroatoms and double bonds. For example, the term "C 3-9 Examples of "cycloalkyl" include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and cyclononyl.

[0092] In this disclosure, the term "aryl" refers to an aromatic ring group consisting of an all-carbon monocyclic or fused polycyclic ring having a conjugated π-electron system, obtained by removing a hydrogen atom from a single carbon atom of the parent aromatic ring system. It includes bicyclic groups comprising an aromatic ring fused with a saturated, partially unsaturated, or aromatic carbon ring. Specific examples include, but are not limited to, phenyl or naphthyl groups.

[0093] In this disclosure, the term "heterocyclic alkyl" refers to a 5-12 member saturated non-aromatic system having a ring carbon atom and one to two ring heteroatoms. Specific examples of heterocyclic groups include, but are not limited to, piperidinyl or tetrahydropyrroleyl.

[0094] In this disclosure, the term "heteroaryl" refers to a monovalent aryl group comprising at least one heteroatom independently selected from nitrogen, oxygen, and sulfur. The heteroaryl group can be monocyclic or polycyclic, such as bicyclic, wherein two or more rings exist in the form of fused rings, bridged rings, or spirocyclic rings, and at least one ring contains one or more heteroatoms. Specific examples of heteroaryl groups include, but are not limited to, pyridyl, thiophene, imidazolyl, pyrimidinyl, furanyl, pyrazinyl, thiazolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, imidazopyridyl, benzofuranyl, pyridazinyl, isoyindolyl, and pyridinoneyl.

[0095] The term "heterocycle" refers to a 5-12 member saturated non-aromatic system having a ring carbon atom and one to two ring heteroatoms, wherein the heteroatoms are independently selected from nitrogen, sulfur, or oxygen atoms. In heterocyclic groups containing one or more nitrogen atoms, the connecting point can be a carbon or nitrogen atom, provided the valence allows. Heterocycles can be monocyclic or polycyclic systems, such as bicyclic systems, wherein two or more rings exist in the form of fused, bridged, or spirocyclic rings, wherein at least one ring contains one or more heteroatoms. In spirocyclic heterocyclic groups, two different rings share a common atom; an example of a spirocyclic heterocyclic group is an azeropeptyl group, but it is not limited thereto.

[0096] The term "nitrogen oxide" refers to the oxidation of one or more nitrogen atoms to form N-oxides when a compound contains several amine functional groups. Specific examples of N-oxides are N-oxides of tertiary amines or N-oxides of nitrogen-containing heterocyclic nitrogen atoms. The corresponding amines can be treated with oxidizing agents such as hydrogen peroxide or peracids (e.g., peroxycarboxylic acids) to form N-oxides.

[0097] Drug or pharmaceutical composition

[0098] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that are suitable for use in human and animal tissues to the extent of reasonable medical judgment without excessive toxicity, irritation, allergic reactions, or other problems or complications in proportion to a reasonable benefit / risk ratio.

[0099] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological potency of a particular compound as a free acid or base without any adverse biological effects. Examples include acid (including organic and inorganic acids) addition salts or base addition salts (including organic and inorganic bases).

[0100] The pharmaceutically acceptable salts disclosed herein can be synthesized from parent compounds containing an acid radical or a base using conventional chemical methods. Generally, such salts are prepared by reacting these compounds, in their free acid or base form, with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture thereof.

[0101] The pharmaceutical or pharmaceutical composition disclosed herein can be administered orally, topically, parenterally, or via mucosal routes (e.g., sublingually, by inhalation, or rectally) in dosage units comprising a conventional, non-toxic, pharmaceutically acceptable carrier.

[0102] For oral administration in tablet or capsule form, the active pharmaceutical ingredient may be combined with non-toxic, pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, sucrose, glucose, mannitol, sorbitol, and other reducing and non-reducing sugars, microcrystalline cellulose, calcium sulfate, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica, stearic acid, sodium stearyl fumarate, glyceryl docosanoate, calcium stearate, etc.); disintegrants (e.g., potato starch or sodium hydroxyacetic acid starch); or wetting agents (e.g., sodium lauryl sulfate), colorants and flavorings, gelatin, sweeteners, natural and synthetic gums (e.g., gum arabic, tragacanth, or alginate), buffer salts, carboxymethyl cellulose, polyethylene glycol, waxes, etc. For oral administration in liquid form, the pharmaceutical component may be combined with a non-toxic, pharmaceutically acceptable inert carrier (e.g., ethanol, glycerol, water), an anti-settling agent (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), an emulsifier (e.g., lecithin or gum arabic), a non-aqueous carrier (e.g., almond oil, esters, ethanol, or fractionated vegetable oils), and a preservative (e.g., methylparaben, propylparaben, or sorbic acid). Stabilizers such as antioxidants (BHA, BHT, propyl iodide, sodium ascorbate, citric acid) may also be added to stabilize the dosage form.

[0103] Tablets containing the active compound can be coated using methods well known in the art. The compositions of this disclosure containing a compound of formula I as the active compound can also incorporate beads, microspheres, or microcapsules, for example, constructed from polyglycolic acid / lactic acid (PGLA). Liquid formulations for oral administration can take the form of, for example, solutions, syrups, emulsions, or suspensions, or they can be presented as dry products reconstituted with water or other suitable excipients prior to use. Formulations for oral administration can be suitably formulated to allow for controlled or delayed release of the active compound.

[0104] The pharmaceutical products or pharmaceutical compositions disclosed herein can be delivered parenterally, i.e., administered intravenously (IV), intraventricularly (ICV), subcutaneously (SC), intraperitoneally (IP), intramuscularly (IM), subcutaneously (SD), or intradermally (ID), by direct injection, such as rapid concentration or continuous infusion. Formulations for injection may be presented in unit dosage forms, such as in ampoules or multi-dose containers with added preservatives. The compositions may be in the form of excipients, suspensions, solutions, or emulsions in oil or aqueous carriers, and may contain formulation agents such as anti-settling agents, stabilizers, and / or dispersants. Alternatively, the active ingredient may be reconstituted in powder form with a suitable carrier (e.g., sterile, pyrogen-free water) prior to use.

[0105] The pharmaceutical or pharmaceutical composition disclosed herein can also be formulated for rectal administration, for example as a suppository or retention enema (e.g., containing a conventional suppository base such as cocoa butter or other glycerides).

[0106] The term "treatment" includes the suppression, relief, prevention, or elimination of one or more symptoms or side effects associated with the disease, condition, or disorder being treated. The term "effective dose" or "therapeutic effective dose" refers to a dose sufficient to treat, suppress, or reduce one or more symptoms of the treated disease state or otherwise provide the desired pharmacological and / or physiological effect. Precise dosages will vary depending on a variety of factors, such as subject-dependent variables (e.g., age, immune system health, etc.), the disease or disorder, and the treatment administered. The effect of an effective dose can be relative to a control. These controls are known in the art and discussed herein, and can be, for example, the condition of the subject before or without administration of the drug or combination of drugs, or, in the case of a combination of drugs, the combined effect can be compared to the effect of administration of only one drug.

[0107] The term "pharmaceutical composition" means a composition comprising the compounds described in this disclosure or their pharmaceutically acceptable salts, and at least one pharmaceutically acceptable ingredient selected from the following, depending on the manner of administration and the nature of the dosage form: carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, thermosensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc.

[0108] II. Examples

[0109] The present disclosure is further illustrated below with reference to embodiments. The description of specific exemplary embodiments of the present disclosure is for illustrative and explanatory purposes. These descriptions are not intended to limit the present disclosure to the precise form disclosed, and it will be apparent that many changes and variations can be made in accordance with the teachings of this specification. The exemplary embodiments were chosen and described in order to explain the specific principles of the present disclosure and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the present disclosure, as well as various different choices and variations.

[0110] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0111] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0112]

[0113]

[0114] Preparation Example 1: Preparation of Intermediate 1:

[0115]

[0116] Step 1: Intermediate 1-1

[0117] tert-butyl-4-formylcyclohexane-1-carboxylic acid ester (311 mg, 1.46 mmol) was dissolved in N,N-dimethylformamide (6 mL), followed by the addition of acetic acid (0.9 mL), 5-amino-2-(2,6-dicarbonylpiperidin-3-yl)isodihydroindole-1,3-dione (200 mg, 0.732 mol), and the mixture was stirred at room temperature for 1 hour. The mixture was then placed in an ice bath at 0 °C, and sodium cyanoborohydride (931 mg, 4.39 mmol) was added. The mixture was then stirred at room temperature for 16 hours. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated sodium chloride aqueous solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 0-100%) to give 300 mg of intermediate 1-1, a yellow oil, in 82.9% yield.

[0118] Step 2: Intermediate 1

[0119] Intermediate 1-1 (300 mg, 0.639 mmol) was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (728 mg, 6.39 mmol) was added. The mixture was stirred overnight at 25 °C. The mixture was concentrated under vacuum, and the residue was purified by reversed-phase preparative liquid chromatography (LCMS) (column: Gemini-C18 150 x 21.2 mm, 5 μm; mobile phase: acetonitrile / water, 0.1% FA; gradient: 20%–50%) to give 101.8 mg of intermediate 1 as a white solid, in approximately 38.4% yield. LCMS (HCOOH), [M+H) + =413.9. 1 H NMR (400MHz, DMSO-d6) δ12.06(s,1H),11.06(s,1H),7.55(dd,J=8.4,1.2Hz,1H),7.16(dd,J=9.2,5.4Hz ,1H),6.95(d,J=1.6Hz,1H),6.86(dd,J=8.4,2.0Hz,1H),5.03(dd,J=12.8,5.4Hz,1H),3.04(dt,J=12.1, 6.2Hz,2H),2.87(ddd,J=17.4,14.2,5.4Hz,1H),2.57(dd,J=17.0,3.2Hz,1H),2.48–2.45(m,0.6H),2.1 5(tt,J=12.1,3.4Hz,0.4H),2.02–1.82(m,4H),1.66–1.44(m,3H),1.32–1.21(m,2H),1.08–0.94(m,1H).

[0120] Preparation Example 2: Preparation of Intermediate 2

[0121]

[0122] 2-(2,6-dicarbonylpiperidin-3-yl)-5-fluoroisodihydroindole-1,3-dione (3.0 g, 10.86 mmol) and trans-4-(aminomethyl)cyclohexanoic acid (3.4 g, 21.62 mmol) were dissolved in DMSO (90 mL), and DIEA (6.9 g, 53.39 mmol) was added. The mixture was heated to 95 °C for 3–4 hours under nitrogen protection, cooled to room temperature in an ice-water bath, and dichloromethane (100 mL) was added. The pH was adjusted to 3–4 with 2N hydrochloric acid. The organic phase was washed once with saturated brine (70 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 5–0:1) to give 1.31 g of yellow solid intermediate 2, yield 29.2%.

[0123] LCMS(HCOOH),[M+H]+ =413.9.

[0124] Preparation Example 3: Preparation of Intermediate 3

[0125]

[0126] Step 1: Intermediate 3-1

[0127] A mixed solution of methyl 2-methyl-4-nitrobenzene (5.00 g, 0.0256 mol), N-bromosuccinimide (4.78 g, 0.0268 mol), azobisisobutyronitrile (0.250 g, 0.00153 mol), and carbon tetrachloride (30 mL) was stirred at 70 °C for 16 hours. The reaction solution was filtered, and the filtrate was evaporated to dryness and purified by rapid chromatography (ethyl acetate to petroleum ether = 0.5%) to give 6.50 g of pale yellow solid intermediate 3-1, yield 74%. 1 H NMR (400MHz, CDCl3) δ8.34 (d, J = 2.2 Hz, 1H), 8.22-8.19 (m, 1H), 8.12 (d, J = 8.6 Hz, 1H), 4.98 (s, 2H), 4.01 (s, 3H).

[0128] Step 2: Intermediate 3-2

[0129] A mixture of intermediate 3-1 (6.50 g, 0.0237 mol), 3-aminopiperidine-2,6-dione hydrochloride (5.82 g, 0.0355 mol), N,N-diisopropylethylamine (9.19 g, 0.0711 mol), and acetonitrile (40 mL) was stirred at 80 °C for 16 hours. The reaction mixture was filtered, and the filter cake was washed with acetonitrile to give 4.00 g of black solid intermediate 3-2, yield 50%. LC-MS (HCOOH), [M+H) + =290.9. 1 H NMR (400MHz, DMSO-d6) δ11.06(s,1H),8.54(d,J=1.6Hz,1H),8.36(dd,J=8.2,2.0Hz,1H),7.98(d,J=8.4Hz,1H),5.17(dd,J=13.2,5.0Hz,1 H), 4.61 (d, J = 18.2Hz, 2H), 4.49 (d, J = 18.2Hz, 1H), 2.97-2.86 (m, 1H), 2.68-2.57 (m, 1H), 2.43 (dd, J = 13.0, 4.4Hz, 1H), 2.06-2.03 (m, 1H).

[0130] Step 3: Intermediate 3-3

[0131] A mixture of intermediate 3-2 (4.00 g, 13.8 mmol), palladium on carbon (0.730 g, 6.90 mmol), and methanol (20 mL) was stirred at room temperature for 16 hours under a hydrogen atmosphere. The reaction solution was filtered, and the filter cake was washed with methanol to give 1.60 g of a blue-black solid intermediate 3-3, in 36% yield. LC-MS (HCOOH), [M+H) + =259.9. 1 H NMR(400MHz,DMSO-d6)δ10.90(s,1H),7.31(d,J=8.8Hz,1H),6.59-6.57(m,2H),5.79(s,2H),4.97(dd,J=13.2,5.0Hz,1H), 4.21(d,J=16.6Hz,1H), 4.07(d,J=16.6Hz,1H), 2.9-2.81(m,1H), 2.59-2.49(m,1H), 2.35-2.23(m,1H), 1.93-1.86(m,1H).

[0132] Step 4: Intermediate 3-4

[0133] 4-Formylcyclohexane-1-carboxylic acid tert-butyl ester (328 mg, 1.543 mmol) was dissolved in N,N-dimethylformamide (4 mL), followed by the addition of acetic acid (0.62 mL), intermediate 3-3 (200 mg, 0.771 mol), and the mixture was stirred at room temperature for 1 hour. The mixture was then placed in an ice bath at 0 °C, and sodium triacetoxyborohydride (981 mg, 4.63 mmol) was added. The mixture was then stirred at room temperature for 16 hours. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with saturated sodium chloride aqueous solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum. The residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 0-100%) to give 220 mg of colorless oily intermediate 3-4, in 59.5% yield. LCMS(HCOOH), [M+H] + =456.0. 1H NMR(400MHz,DMSO-d6)δ10.93(s,1H),7.40–7.33(m,1H),6.69–6.58(m,2H),6.42( t,J=5.5Hz,1H),5.01(dd,J=13.2,5.0Hz,1H),4.26(d,J=16.6Hz,1H),4.12(d,J=1 6.6Hz,1H),2.98–2.84(m,3H),2.63–2.54(m,2H),2.45–2.05(m,1H),1.96–1.81(m ,4H),1.69–1.48(m,3H),1.41–1.38(m,9H),1.29–1.21(m,2H),1.03–0.90(m,1H).

[0134] Step 5: Intermediate 3

[0135] Intermediate 3-4 (220 mg, 0.483 mmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (551 mg, 4.83 mmol) was added. The mixture was stirred overnight at 25 °C. The mixture was concentrated under vacuum, and the residue was separated by preparative liquid chromatography (column: Gemini-C18 150 x 21.2 mm, 5 μm; mobile phase: acetonitrile / water, 0.1% FA; gradient: 20%-50%) to give 56.8 mg of white solid intermediate 5, in approximately 29.5% yield. LCMS (HCOOH), [M+H) + =400.0. 1 H NMR (400MHz, DMSO-d6) δ12.05(s,1H),10.90(d,J=8.6Hz,1H),7.34(t,J=5.8Hz,1H),6.68 –6.50(m,2H),6.38(q,J=5.4Hz,1H),4.97(dd,J=13.2,5.0Hz,1H),4.22(d,J=16.8Hz,1H), 4.09(d,J=16.8Hz,1H),2.96–2.80(m,3H),2.65–2.50(m,2H),2.35–2.06(m,1H),1.87(ddd d,J=13.8,12.8,4.2,1.8Hz,4H),1.71–1.37(m,3H),1.30–1.17(m,2H),1.02–0.87(m,1H).

[0136] Preparation Example 4: Preparation of Intermediate 4

[0137]

[0138] Step 1: Intermediate 4-1

[0139] 5-Bromobenzyl[D]oxazol-2-amine (500 mg, 2.35 mmol) and pinacol diborate (715 mg, 2.82 mmol) were dissolved in 1,4-dioxane, followed by the addition of potassium acetate (690 mg, 7.05 mmol) under argon protection. The reaction mixture was refluxed for 5 hours. After the reaction was completed and cooled to room temperature, the reaction mixture was filtered through diatomaceous earth, concentrated under reduced pressure, and purified by thin-layer chromatography (dichloromethane:methanol = 30:1) to obtain intermediate 4-1.

[0140] Step 2: Intermediate 4-2

[0141] Intermediate 4-aminopyrazolo[3,4-d]pyrimidine (1.35 g, 0.01 mol) was dissolved in DMF, followed by the addition of N-bromosuccinimide (NBS) (1.87 g, 0.0105 mol). The reaction mixture was stirred at 60°C for 3 hours. After the reaction was complete, the mixture was cooled to room temperature, and diethyl ether was added to the reaction mixture. The product precipitated out, and the reaction mixture was filtered to obtain the target intermediate 4-2, which could be used directly in the next reaction without purification.

[0142] Step 3: Intermediate 4-3

[0143] Intermediate 4-2 (1.8 g, 8.4 mmol) was dissolved in DMF, followed by the addition of tert-butyl 4-bromopiperidine-1-carboxylate (2.83 g, 12.6 mmol) and potassium carbonate (3.5 g, 16.8 mmol). The reaction mixture was reacted at 80 °C for 12 hours. After the reaction was completed, an aqueous sodium chloride solution was added, and the mixture was extracted with ethyl acetate. The extract was concentrated, and the concentrate was purified by thin-layer chromatography (dichloromethane:methanol = 60:1) to obtain intermediate 4-3.

[0144] Step 4: Intermediate 4

[0145] Intermediate 4-3 (210 mg, 0.6 mmol) and intermediate 4-1 (200 mg, 0.78 mmol) were dissolved in 1,4-dioxane, followed by the addition of sodium carbonate (324 mg, 3.0 mmol) and water. Tetraphenylphosphine palladium (55 mg, 0.048 mmol) was then added with stirring under argon protection. The reaction mixture was incubated at 110 °C for 3 hours. After the reaction, the mixture was filtered, concentrated under reduced pressure, and purified by thin-layer chromatography to obtain the target compound. A trifluoroacetic acid / dichloromethane (1 / 9) mixture was then added, and the mixture was stirred at room temperature for 1 hour. After the reaction, the mixture was concentrated to obtain intermediate 4, which could be directly proceeded to the next step without further purification. LCMS(HCOOH), [M+H) + =451.30. 1H NMR (400MHz, DMSO) δ8.23(s,1H),7.54(s,2H),7.46(d,J=7.2Hz,1H),7.40(s,1H),7.23(d,J=8.0 Hz,1H),4.89(s,1H),4.10(s,2H),3.17(s,1H),2.99(s,2H),2.02(d,J=10.5Hz,2H),1.94(s,2H).

[0146] Preparation Example 5: Preparation of Intermediate 5

[0147]

[0148] Step 1: Intermediate 5-1

[0149] 4,6-Dichloro-1H-pyrazolo[3,4-d]pyrimidine (2080 mg, 11.0 mmol), (S)-3-methylmorpholine (1100 mg, 11.0 mmol), and triethylamine (2230 mg, 22.0 mmol) were dissolved in dichloromethane solution (30 mL). The mixture was reacted in an ice bath for 30 minutes, and then reacted at room temperature for 16 hours. After the reaction was complete, the reaction solution was filtered, washed with water, and the filtrate was evaporated to dryness to give 2400 mg of a yellow solid intermediate 5-1, with a yield of 85.7%. LC-MS (HCOOH), [M+H) + =254.1.

[0150] Step 2: Intermediate 5-2

[0151] Intermediate 5-1 (1500 mg, 5.90 mmol), tert-butyl-4-((methanesulfonyl)oxo)piperidine-1-carboxylic acid ester (1651.7 mg, 5.90 mmol), and potassium carbonate (1634.4 mg, 11.8 mmol) were dissolved in N,N-dimethylformamide solution (15 mL). The mixture was reacted at 80 °C for 16 hours under nitrogen protection. After the reaction was complete, a suitable amount of water was added to the mixture, and the solution was extracted with ethyl acetate. The resulting organic phase was evaporated to dryness and purified by column chromatography (petroleum ether:ethyl acetate = 5:1) to give 786 mg of a yellow, transparent, oily intermediate 5-2, with a yield of 28.9%. LC-MS (HCOOH), [M+H) + =436.

[0152] Step 3: Intermediate 5-3

[0153] Intermediate 5-2 (436 mg, 1.00 mmol), 4-fluorophenylboronic acid (279.3 mg, 2.00 mmol), and potassium carbonate (275.8 mg, 2.00 mmol) were dissolved in 1,4-dioxane (12 mL). Under nitrogen protection, [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex (121.3 mg, 0.150 mmol) was added, and the mixture was reacted at 110 °C for 6 hours. After the reaction was complete, the reaction solution was evaporated to dryness and purified by column chromatography (dichloromethane:methanol = 6:1) to give 220 mg of pale yellow solid intermediate 5-3, with a yield of 42.1%. LC-MS (HCOOH), [M+H) + =497.1.

[0154] Step 4: Intermediate 5

[0155] To tert-butyl intermediate 5-3 (220 mg, 0.400 mmol), 1,4-dioxane hydrochloride solution (1.1 mL, 4 M, 4.40 mmol) and dichloromethane solution (5 mL) were added, and the reaction was carried out at room temperature for 16 hours. After the reaction was complete, the reaction solution was evaporated to dryness, yielding 164.7 mg of white solid intermediate 5, with a yield of 85.5%. LC-MS (HCOOH), [M+H) + =397.1. 1 H NMR (400MHz, CD3OD) δ8.47(dd,J=8.8,5.6Hz,2H),8.24(s,1H),7.20(t,J=8.6Hz,2H),5.32–5.19(m,1H),4.62(s,1H),4.09(d,J= 7.8Hz,1H),3.89–3.78(m,2H),3.70–3.57(m,4H),3.34–3.31(m,3H),2.54–2.44(m,2H),2.32–2.24(m,2H),1.45(d,J=6.8Hz,3H).

[0156] Preparation Example 6: Preparation of Intermediate 6

[0157]

[0158] Step 1: Intermediate 6-1

[0159] Intermediate 6-1 (4.5 g, 17.74 mmol), 4-fluorophenylboronic acid (2.98 g, 21.29 mmol), [1,1-Pd(dppf)Cl2CH2Cl2 (4.35 g, 5.32 mmol), and sodium carbonate (3.76 g, 35.48 mmol) were dissolved in a mixed solvent of 1,4-dioxane (40 mL) and water (20 mL). The mixture was stirred at 100 °C for 12 h under nitrogen protection. LCMS analysis confirmed the reaction was complete. The reaction solution was evaporated to dryness, and the crude product was separated by prep-HPLC to obtain 1.64 g of a yellow oily intermediate 6-1, with a yield of 29.51%. LCMS (HCOOH): m / z = 314.2 (M+H).

[0160] Step 2: Intermediate 6-2

[0161] Intermediate 6-1 (1 g, 3.19 mmol) was dissolved in DMF (10 mL), and tert-butyl-3-iodoaceridine-1-carboxylic acid ester (1.99 g, 7.02 mmol) and potassium carbonate (882.18 mg, 6.38 mmol) were added. The mixture was stirred at 80 °C for 12 h. LCMS analysis confirmed the reaction was complete. The reaction solution was diluted with water (10 mL), extracted with ethyl acetate (3 x 10 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 0–100%) to give 1.18 g of white solid intermediate 6-2, yield 78.91%. LCMS (HCOOH): m / z = 469.2 (M+H).

[0162] Step 3: Intermediate 6-3

[0163] Intermediate 6-2 (1.18 g, 2.52 mmol) was dissolved in DCM (10 mL), and TFA (3.08 g, 27.01 mmol, 2 mL) was added. The reaction mixture was stirred at 30 °C for 24 h. LCMS analysis showed that the reaction was complete. The reaction mixture was evaporated to dryness to obtain 1.22 g of colorless oily intermediate 6-3 (TFA salt). LCMS (HCOOH): m / z = 369.1 (M+H).

[0164] Step 4: Intermediate 6-4

[0165] Intermediate 6-3 (610 mg, 1.26 mmol, TFA salt) was dissolved in DCE (5 mL), and TEA (127.95 mg, 1.26 mmol, 175.99 μL) was added. The mixture was stirred at 20 °C for 0.5 hours. Then, tert-butyl-3-oxoylidene acridine-1-carboxylic acid ester (259.75 mg, 1.52 mmol) and HOAc (75.93 mg, 1.26 mmol) were added. After stirring at 20 °C for 0.5 hours, NaBH(OAc)3 (401.98 mg, 1.90 mmol) was added, and the reaction mixture was stirred at 30 °C for 11 hours. LCMS analysis confirmed the reaction was complete. The reaction solution was evaporated to dryness, and the crude product was separated by prep-HPLC (column: Phenomenex Luna C18 250*50mm*10um; mobile phase: [water(TFA)-ACN]; B%: 30%-70%, 10 min) to obtain 570 mg of white solid intermediate 6-4, yield 86.10%. LCMS (HCOOH): m / z = 524.2 (M+H).

[0166] Step 5: Intermediate 6

[0167] Intermediate 6-4 (570 mg, 1.09 mmol) was dissolved in DCE (10 mL), and TFA (3.08 g, 27.01 mmol, 2 mL) was added. The reaction mixture was stirred at 30 °C for 2 hours. LCMS analysis showed that the reaction was complete. The reaction mixture was evaporated to dryness to give 570 mg of white solid intermediate 6, with a yield of 97.41%. LCMS (HCOOH): m / z = 424.1 (M+H). 1 H NMR(DMSO-d6,400MHz)δ9.0–9.2(m,2H),8.4–8.5(m,3H),7.33(t,2H,J=8.8Hz),5.89(quin,1H,J=7.3Hz),4.3-5.0(m,7H),4.22(br s,4H),4.05(br d,1H,J=8.0Hz),3.8–3.9(m,1H),3.7–3.8(m,1H),3.4–3.7(m,2H),1.34(br d,3H,J=6.3Hz)

[0168] Preparation Example 7: Preparation of Intermediate 7

[0169]

[0170] Step 1: Intermediate 7-1

[0171] Intermediate 6-3 (610 mg, 1.26 mmol) was dissolved in DCE (5 mL), and triethylamine (127.95 mg, 1.26 mmol) was added. The reaction mixture was stirred at 20 °C for 0.5 h. N-tert-butyloxycarbonyl-4-oxoperididine (302.32 mg, 1.52 mmol) and HOAc (75.93 mg, 1.26 mmol) were added, and the mixture was stirred at 20 °C for 0.5 h. Then, NaBH(OAc)3 (401.98 mg, 1.90 mmol) was added, and the mixture was stirred at 30 °C for 11 h. The reaction mixture was analyzed by TLC to confirm the complete reaction of the starting material. The reaction mixture was evaporated to dryness, and the residue was separated by prep-HPLC to obtain 450 mg of white solid intermediate 7-1, with a yield of 64.51%.

[0172] Step 2: Intermediate 7

[0173] Intermediate 7-1 (450 mg, 815.73 μmol) was dissolved in DCE (10 mL), and TFA (3.47 g, 30.39 mmol) was added. The reaction mixture was stirred at 30 °C for 12 hours. LCMS analysis confirmed the reaction was complete. The reaction mixture was evaporated to dryness to obtain 432.6 mg of white solid intermediate 7, yield 93.77%. LCMS (HCOOH): m / z = 452.1 (M+H). 1 H NMR(DMSO-d6,400MHz)δ8.8–8.9(m,1H),8.5–8.7(m,1H),8.49(br dd,3H,J=5.8,8.6Hz),7.34(t,2H,J=8.8Hz),5.8–6.0(m,1H),4.7–4.8(m,2H),4.66(br d,2H,J=7.0Hz),4.05(br d,1H,J=8.0Hz),3.7–3.9(m,2H),3.6–3.7(m,2H),3.43(br s,3H),2.94(br d,2H,J=9.5Hz),2.5–2.6(m,2H),2.2–2.3(m,2H),1.6–1.7(m,2H),1.34(br d,3H,J=6.3Hz).

[0174] Preparation Example 8: Preparation of Intermediate 8

[0175]

[0176] Step 1: Intermediate 8-1

[0177] Intermediate 5 (600 mg, 1.18 mmol, TFA salt) was dissolved in DCE (2 mL), and triethylamine (118.93 mg, 1.18 mmol) was added. The mixture was stirred at 20 °C for 0.5 h. N-tert-butoxycarbonyl-3-azacyclobutanone (301.82 mg, 1.76 mmol) and HOAc (70.58 mg, 1.18 mmol) were added, and the reaction mixture was stirred at 30 °C for 0.5 h. NaBH(OAc)3 (373.66 mg, 1.76 mmol) was added, and the mixture was stirred at 30 °C for 11 h. TLC analysis showed the reaction was complete. The reaction mixture was evaporated to dryness, and the residue was separated by prep-HPLC to give 375 mg of white solid intermediate 8-1, yield 57.84%. LCMS (HCOOH): m / z = 552.4 (M+H).

[0178] Step 2: Intermediate 8

[0179] Intermediate 8-1 (375 mg, 679.77 μmol) was dissolved in DCM (5 mL), and TFA (1.54 g, 13.51 mmol) was added. The reaction mixture was stirred at 30 °C for 2 hours, and the reaction was confirmed by LCMS. The reaction mixture was evaporated to dryness to obtain 306.6 mg of brown oily intermediate 8. LCMS (HCOOH): m / z = 452.3 (M+H). 1 H NMR (400MHz, DMSO-d6) δppm 9.17–9.47(m,2H),8.43–8.52(m,2H),8.33(s,1H),7.32(t,J=8.88Hz,2H),5.05–5.19(m,1H),4.37(br s,2H),4.20(br d,J=7.88Hz,3H),4.03(br d,J=8.13Hz,1H),3.68–3.86(m,2H),3.39-3.64(m,4H),3.05-3.26(m,2H),2.49-2.52(m,2H),2.31-2.46(m,2H),2.20(br d, J=13.01Hz, 2H), 1.34 (br d, J=6.50Hz, 3H).

[0180] Preparation Example 9: Preparation of Intermediate 9

[0181]

[0182] Step 1: Intermediate 9-1

[0183] Intermediate 4-3 (4.00 g, 9.01 mmol) was dissolved in dioxane (40 mL), and tert-butyl nitrite (890 mg, 9.01 mmol) was added. The mixture was then heated to 100 °C and stirred for 16 h. The reaction was monitored by LCMS until completion. The reaction solution was concentrated and water (50 mL) was added. The solution was then extracted with ethyl acetate (80 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 4:1) to give 1.20 g of intermediate 9-1, yield 31.0%. LCMS (HCOOH): m / z = 381.9 (M+H).

[0184] Step 2: Intermediate 9-2

[0185] At 0 °C, intermediates 9-1 (600 mg, 1.40 mmol), 4-1 (434 mg, 1.67 mmol), tetrakis(triphenylphosphine)palladium (323 mg, 0.280 mmol), and potassium carbonate (580 mg, 4.20 mmol) were dissolved in toluene / ethanol / water (18 mL, 5:5:2). The mixture was heated to 90 °C and stirred for 1 h. The reaction was monitored by LCMS to be essentially complete. The reaction solution was concentrated and diluted with water (30 mL), then extracted with ethyl acetate (50 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated. The residue was separated by prep-HPLC to give 35 mg of intermediate 9-2, with a yield of 5.74%. LCMS (HCOOH): m / z = 436.1 (M+H).

[0186] Step 3: Intermediate 9

[0187] Intermediate 9-2 (35 mg, 0.08 mmol) was dissolved in dichloromethane (0.5 ml), and a dioxane solution of hydrochloric acid (0.1 ml) was added dropwise. The mixture was stirred for 1 h, and the reaction was monitored by LCMS until complete. The reaction solution was concentrated, and the residue was separated by prep-HPLC to give 17 mg of white solid intermediate 9, with a yield of 63.4%. LCMS (HCOOH): m / z = 336.1 (M+H). 1 H NMR (400MHz, DMSO-d6) δ9.71 (s, 1H), 9.15-0.19 (m, 1H), 9.10 (s, 1H), 8.83 (br, 1H), 8.12 (br, 2H), 7.89-7.90 (d, J=1.6Hz, 1H), 7.79-7.809 (dd, J=8.0, 2.0Hz, 1H), 7.56-7.58 (d, J=8.4Hz, 1H), 5.22-5.27 (m, 1H), 3.34 -3, 49(m, 2H), 3.20-3.28(m, 2H), 2.43-2.47(m, 2H), 2.21-2.23(m, 2H).

[0188] Preparation Example 10: Preparation of Intermediate 10

[0189]

[0190] Step 1: Intermediate 10-1

[0191] Intermediate 5-2 (500 mg, 1.14 mmol), p-nitrophenylboronic acid (210.1 mg, 1.26 mmol), Pd(dppf)Cl2·CH2Cl2 (93.4 mg, 114.43 μmol), and sodium carbonate (242.6 mg, 2.29 mmol) were added to a mixed solvent of 1,4-dioxane (10 mL) and water (1 mL). The mixture was stirred at 100 °C for 12 hours under nitrogen protection, and the reaction was confirmed to be complete by LCMS. The reaction solution was added to water (20 mL), extracted with dichloromethane (10 mL * 3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 0–50%) to give 0.48 g of yellow oily intermediate 10-1, yield 80.11%. LCMS (HCOOH): m / z = 468.3 (M + H - 56).

[0192] Step 2: Intermediate 10

[0193] Intermediate 10-1 (0.48 g, 916.76 μmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (1.05 g, 9.17 mmol, 2 mL) was slowly added. The mixture was stirred at 20 °C for 12 hours, and the reaction was monitored by LCMS until complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 0–50%) to give 183.6 mg of white solid intermediate 10, with a yield of 46.82%. LCMS (NH4HCO3): m / z = 424.2 (M+H). 1 HNMR (400MHz, Methanol-d4) δ8.71 (d, J=8.88Hz, 2H), 8.31 (d, J=8.88Hz, 2H), 8.24 (s, 1H), 5.23-5.35 (m, 1H), 4.12 (br d, J=7.88Hz, 1H), 3.78-3.97(m, 2H), 3.52-3.75(m, 4H), 3.32-3.43(m, 3H), 3.31(br s, 1H), 2.46-2.59(m, 2H), 2.31(br dd, J=14.01, 3.13Hz, 2H), 1.46 (d, J=6.88Hz, 3H).

[0194] Example 1: Preparation of 2-(2,6-dicarbonylpiperidin-3-yl)-5-(((trans-4-(4-(6-(4-fluorophenyl)-4-((S)-3-methylmorpholino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-carbonyl)cyclohexyl)methyl)amino)isodihydroindole-1,3-dione (compound 1)

[0195]

[0196] Intermediate 5 (1 equiv), intermediate 2 (1.05 equiv), DIPEA (5 equiv), and HATU (20 mg, 0.0508 mmol, 1.3 equiv.) were dissolved in DMF. The reaction mixture was stirred at room temperature for 4 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic phase was concentrated and further purified by silica gel column chromatography (DCM:MeOH = 20:1) to give compound 1. 1 H NMR (400MHz, Methano1-d4) δ8.46-8.41 (m, 2H), 7.92 (s, 1H), 7.56 (d, J=8.3Hz, 1H), 7.11 (d, J=8.7Hz, 2 H), 6.94 (d, J=2.2Hz, 1H), 6.77-6.73 (m, 1H), 5.14-5.05 (m, 1H), 4.93-4.88 (m, 1H), 4.78-4.71 (m, 1H), 3 .91-3.79 (m, 3H), 3.71-3.65 (m, 1H), 3.06 (d, J=6.7Hz, 2H), 2.87-2.72 (m, 5H), 2.62-2.54 (m, 1H), 2.35- 2.09(m, 6H), 1.99-1.82(m, 6H), 1.69-1.54(m, 4H), 1.46(d, J=6.8Hz, 3H), 1.11-1.05(m, 2H).LCMS[M+H] + =792.5.

[0197] Example 2: Preparation of 5-(((4-(4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)cyclohexyl)methyl)amino)-2-(2,6-dioxadipinidin-3-yl)isodihydroindole-1,3-dione (compound 2)

[0198]

[0199] Intermediate 4 (1 equiv), intermediate 1 (1.05 equiv), DIPEA (5 equiv), and HATU (20 mg, 0.0508 mmol, 1.3 equiv) were dissolved in DMF. The reaction mixture was stirred at room temperature for 4 hours. Water was added to the reaction mixture and extracted with ethyl acetate. The organic phase was concentrated and further purified by silica gel column chromatography (DCM:MeOH = 20:1) to obtain compound 2. 1 H NMR (400MHz, DMSO) δ11.06 (s, 1H), 8.24 (s, 1H), 7.55 (d, J=6.1Hz, 3H), 7.46 (d, J=8.1Hz, 1H), 7.40 (s, 1H), 7.23 (d, J=8.1Hz, 1H), 7.17 (s, 1H), 6.96 (s, 1H), 6.86 (d, J=8.5Hz, 1H), 5.08-4.91 (m, 2H), 4.54 (d, J=12.6Hz, 1H), 4.11 (d, J=13.7Hz, 1H), 3.68-3.52(m, 1H), 3.13(dd, J=7.2, 4.2Hz, 1H), 3.03(s, 2H), 2.93-2.74(m, 3H), 2.01(m, 3H), 1.86( d, J=11.8Hz, 2H), 1.74 (d, J=10.3Hz, 2H), 1.57 (m, 2H), 1.38 (dd, J=24.2, 11.4Hz, 3H), 1.17-1.00 (m, 2H).LCMS[M+H] + =746.50.

[0200] Example 3: Preparation of 3-(5-(((4-(4-(4-(4-amino-3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)cyclohexyl)methyl)amino)-1-carbonylisodihydroindole-2-yl)piperidine-2,6-dione (compound 3)

[0201]

[0202] Intermediate 4 (1 equiv.), intermediate 3 (1.05 equiv.), DIPEA (5 equiv.), and HATU (20 mg, 0.0508 mmol, 1.3 equiv.) were dissolved in DMF. The reaction mixture was stirred at room temperature for 4 hours. Water was added to the reaction mixture and extracted with ethyl acetate. The organic phase was concentrated and further purified by silica gel column chromatography (DCM:MeOH = 20:1) to obtain compound 3. 1H NMR (400MHz, DMSO) δ10.90 (s, 1H), 8.25 (s, 1H), 7.53 (s, 2H), 7.46 (d, J = 8.1Hz, 1H), 7.40 (s, 1H), 7.36 (d, J = 8.3Hz, 1H), 7.23 (d , J=8.1Hz, 1H), 6.69-6.60 (m, 2H), 6.41 (s, 1H), 5.00 (dd, J=13.0, 4.7Hz, 2H), 4.55 (m, 1H), 4.26 (d, J=16.1Hz, 1H), 4.13 (d, J=1 6.5Hz, 1H), 3.60 (m, 1H), 3.15 (d, J=13.2Hz, 1H), 3.07 (m, 1H), 2.87 (m, 3H), 2.61 (dd, J=30.7, 15.0Hz, 2H), 2.33 (d, J=8.1Hz, 1H ), 2.14-1.82(m, 2H), 1.73(m, 1H), 1.60(m, 3H), 1.51-1.31(m, 2H), 1.26(dd, J=13.7, 6.2Hz, 4H), 1.15-0.99(m, 2H).LCMS[M+H] + =732.62.

[0203] Example 4: Preparation of 2-(2,6-dicarbonylpiperidin-3-yl)-5-(((trans-4-(3-(3-(6-(4-fluorophenyl)-4-((S)-3-methylmorpholino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)azacyclobutane-1-yl)azacyclobutane-1-carbonyl-cyclohexyl)methyl)amino)isodihydroindole-1,3-dione (compound 4)

[0204]

[0205] Intermediate 6 (1 equiv.), intermediate 2 (1.05 equiv.), DIPEA (5 equiv.), and HATU (20 mg, 0.0508 mmol, 1.3 equiv.) were dissolved in DMF. The reaction mixture was stirred at room temperature for 4 hours. Water was added to the reaction mixture and the mixture was extracted with ethyl acetate. The organic phase was concentrated and further purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to give compound 4. 1H NMR (400MHz, CDCl3) δ8.46 (dd, J=8.6, 5.7Hz, 2H), 7.99 (s, 1H), 7.61 (d, J=8.2Hz, 1H), 7.15 (t, J=8.5Hz, 2H), 6. 97 (s, 1H), 6.74 (dd, J=8.6, 2.1Hz, 1H), 4.92 (dd, J=12.1, 5.3Hz, 1H), 4.60 (s, 1H), 4.14 (d, J=9.6Hz, 2H), 3.94-3 .80 (m, 2H), 3.75-3.63 (m, 2H), 3.09 (t, J=5.9Hz, 2H), 2.93-2.65 (m, 3H), 2.21-2.07 (m, 2H), 2.00-1.76 (m, 4H), 1 .73-1.51 (m, 8H), 1.48 (d, J=6.8Hz, 3H), 1.29 (d, J=3.6Hz, 1H), 1.14-0.96 (m, 3H), 0.93-0.75 (m, 3H).LCMS[M+H] + =819.4.

[0206] Example 5: Preparation of 2-(2,6-dicarbonylpiperidin-3-yl)-5-(((trans-4-(4-(3-(6-(4-fluorophenyl)-4-((S)-3-methylmorpholino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)azacyclobutane-1-yl)piperidin-1-carbonyl)cyclohexyl)methyl)amino)isodihydroindole-1,3-dione (compound 5)

[0207]

[0208] Intermediate 7 (1 equiv.), intermediate 2 (1.05 equiv.), DIPEA (5 equiv.), and HATU (20 mg, 0.0508 mmol, 1.3 equiv.) were dissolved in DMF. The reaction mixture was stirred at room temperature for 4 hours. Water was added to the reaction mixture and the mixture was extracted with ethyl acetate. The organic phase was concentrated and further purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to give compound 5. 1H NMR (400MHz, CDCl3) δ8.46 (dd, J=8.6, 5.6Hz, 2H), 8.03-7.96 (m, 2H), 7.61 (d, J=8.3Hz, 1H), 7.14 (t, J=8.6Hz, 2H), 6.99-6.94 (m, 1H ), 6.75 (d, J = 9.0Hz, 1H), 4.93 (dd, J = 12.1, 5.3Hz, 1H), 4.62 (s, 1H), 4.12 (q, J = 7.2Hz, 3H), 3.93-3.87 (m, 1H), 3.83 (d, J = 12.7Hz, 1H) , 3.73-3.63 (m, 2H), 3.11 (t, J=6.1Hz, 3H), 2.94-2.67 (m, 4H), 2.50 (d, J=10.9Hz, 1H), 2.11 (d, J=11.4Hz, 2H), 1.95 (d, J=12.4Hz, 3H) , 1.82 (s, 3H), 1.55-1.50 (m, 3H), 1.50-1.45 (m, 4H), 1.43 (d, J = 9.0Hz, 2H), 1.29 (d, J = 3.5Hz, 3H), 1.08 (q, J = 11.8Hz, 4H). LCMS [M+H] + =847.3.

[0209] Example 6: Preparation of 2-(2,6-dicarbonylpiperidin-3-yl)-5-(((trans-4-(3-(4-(6-(4-fluorophenyl)-4-((s)-3-methylmorpholino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)azacyclobutane-1-carbonyl)cyclohexyl)methyl)amino)isodihydroindole-1,3-dione (compound 6)

[0210]

[0211] Intermediate 8 (1 equiv.), intermediate 2 (1.05 equiv.), DIPEA (5 equiv.), and HATU (20 mg, 0.0508 mmol, 1.3 equiv.) were dissolved in DMF. The reaction mixture was stirred at room temperature for 4 hours. Water was added to the reaction mixture and the mixture was extracted with ethyl acetate. The organic phase was concentrated and further purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to give compound 6. 1H NMR (400MHz, CD3OD) δ8.46 (dd, J=8.6, 5.6Hz, 2H), 8.06 (d, J=12.5Hz, 1H), 7.53 (d, J=8.4Hz, 1H), 7.12 (t, J=8.6Hz, 2H), 6.95 (d, J=2. 2Hz, 1H), 6.79 (dd, J=8.5, 2.2Hz, 1H), 4.97 (dd, J=12.2, 5.4Hz, 1H), 4.93-4.84 (m, 1H), 4.34-4.25 (m, 1H), 4.16-4.02 (m, 3H), 3.92-3 .79 (m, 3H), 3.68 (t, J = 11.8Hz, 2H), 3.61-3.50 (m, 1H), 3.12-3.01 (m, 4H), 2.83-2.67 (m, 3H), 2.39 (d, J = 11.9Hz, 2H), 2.32-2.19 (m, 3 H), 2.11-2.03(m, 3H), 1.99-1.92(m, 2H), 1.87-1.75(m, 3H), 1.72-1.60(m, 2H), 1.44(d, J=6.8Hz, 3H), 1.14-1.02(m, 4H).LCMS[M+H] + =847.3.

[0212] Example 7: Preparation of 5-(((4-(3-(2-aminobenzo[d]oxazol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)cyclohexyl)methyl)amino)-2-(2,6-dioxadipinidin-3-yl)isodihydroindole-1,3-dione (compound 7)

[0213]

[0214] Intermediate 9 (1 equiv.), intermediate 2 (1.05 equiv.), DIPEA (5 equiv.), and HATU (20 mg, 0.0508 mmol, 1.3 equiv.) were dissolved in DMF. The reaction mixture was stirred at room temperature for 4 hours. Water was added to the reaction mixture and the mixture was extracted with ethyl acetate. The organic phase was concentrated and further purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to give compound 7. 1H NMR (400MHz, DMSO) δ11.04 (s, 1H), 9.65 (s, 1H), 9.05 (s, 1H), 8.68 (s, 1H), 7.85 (s, 1H), 7.71 (d, J=8.1Hz, 1H), 7.55 (d, J =6.0Hz, 2H), 7.47 (d, J = 8.2Hz, 1H), 7.17 (s, 1H), 6.96 (s, 1H), 6.87 (d, J = 8.2Hz, 1H), 5.14 (s, 1H), 5.02 (dd, J = 12.8, 5.4H z, 1H), 4.58 (s, 1H), 4.13 (s, 1H), 3.60 (s, 2H), 3.13 (s, 2H), 3.04 (s, 2H), 2.86 (d, J=12.5Hz, 2H), 2.67 (s, 1H), 2.13 (d, J =18.8Hz, 2H), 2.03 (s, 2H), 1.88 (d, J = 10.5Hz, 2H), 1.77 (s, 2H), 1.58 (s, 1H), 1.38 (s, 2H), 1.17-1.01 (m, 3H).LCMS[M+H] + =731.5.

[0215] Example 8: Preparation of 2-(2,6-dicarbonylpiperidin-3-yl)-5-(((trans-4-(4-(6-(4-nitrophenyl)-4-((S)-3-methylmorpholino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-carbonyl)cyclohexyl)methyl)amino)isodihydroindole-1,3-dione (compound 8)

[0216]

[0217] Intermediate 10 (20.0 mg, 0.97 equiv.), intermediate 2 (15.4 mg, 1.00 equiv.), DIPEA (25.8 mg, 5 equiv.), and HATU (20 mg, 0.0508 mmol, 1.32 equiv.) were dissolved in DMF, and the reaction mixture was stirred overnight at room temperature. Water was added to the reaction system and the mixture was extracted with ethyl acetate. The organic phase was concentrated and further purified by PTLC (DCM:MeOH = 15:1) to give compound 8. 1H NMR (400MHz, DMSO-d6) δ11.04 (s, 1H), 8.67 (d, J=8.6Hz, 2H), 8.39-8.29 (m, 3H), 7.55 (d, J=8.4Hz, 1H), 7.16 (t, J=5.7Hz, 1H), 6.97 (s, 1H ), 6.87 (d, J=8.5Hz, 1H), 5.19-5.08 (m, 1H), 5.02 (dd, J=12.9, 5.4Hz, 1H), 4.56 (d, J=12.8Hz, 1H), 4.13 (d, J=12.7Hz, 1H), 4.05 (d, J=9.6H z, 1H), 3.82 (d, J = 11.5Hz, 1H), 3.73 (d, J = 11.7Hz, 1H), 3.59 (t, J = 8.9Hz, 1H), 3.04 (t, J = 5.8Hz, 2H), 2.86 (d, J = 13.7Hz, 2H), 2.73-2.56 (m , 2H), 2.13-1.94(m, 5H), 1.88(d, J=12.9Hz, 3H), 1.82-1.71(m, 2H), 1.68-1.52(m, 2H), 1.50-1.31(m, 7H), 1.19-1.07(m, 3H)..LCMS[M+H] + =819.2.

[0218] Example 9: Preparation of Compound 9

[0219]

[0220] Step 1: Compound 9-1

[0221] 4-Chloro-1H-pyrazolo[3,4-d]pyrimidine (2000 mg, 13.0 mmol), tert-butyl-4-hydroxypiperidine-1-carboxylic acid ester (2600 mg, 13.0 mmol), and triphenylphosphine (4020 mg, 15.6 mmol) were dissolved in tetrahydrofuran solution (30 mL). Under ice bath and argon protection, diisopropyl azodicarbonate (2680 mg, 15.6 mmol) was added. The mixture was reacted at room temperature for 16 hours. After the reaction was complete, the reaction solution was filtered, the filtrate was evaporated to dryness, and purified by column chromatography (petroleum ether:ethyl acetate = 1:1) to give a white solid compound 9-1 (2500 mg), in 58% yield. LC-MS (HCOOH): m / z = 338.0 (M+H).

[0222] Step 2: Compound 9-2

[0223] tert-butyl-4-(4-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylic acid ester (370 mg, 1.00 mmol), (S)-3-methylmorpholine (111.8 mg, 1.10 mmol), and triethylamine (201.9 mg, 2.00 mmol) were dissolved in tetrahydrofuran solution (15 mL). The mixture was stirred at 0 °C for half an hour, and then reacted at 60 °C for 16 hours. After the reaction was complete, the mixture was purified by column chromatography (petroleum ether:ethyl acetate = 1:1) to give a yellow, transparent oily compound 9-2 (402 mg), with a yield of 99%. LC-MS (HCOOH): m / z = 403.0 (M+H).

[0224] Step 3: Compound 9-3

[0225] A solution of tert-butyl(S)-4-(4-(3-methylmorpholino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylic acid ester (402 mg, 1.00 mmol) in dichloromethane (5 mL) was added to a solution of hydrogen chloride / 1,4-dioxane (1.30 mL, 4 M, 5.20 mmol), and the reaction was carried out at room temperature for 16 hours. After the reaction was complete, the reaction solution was evaporated to dryness to give a white solid compound 9-3 (130.3 mg), with a yield of 43%. 1 H NMR (400MHz, Methanol-d4) δ8.58 (s, 1H), 8.47 (s, 1H), 5.24 (ddd, J=14.8, 10. 8, 4.2Hz, 1H), 4.67 (d, J = 6.2Hz, 1H), 4.32 (d, J = 11.4Hz, 1H), 4.17 (d, J = 7.8Hz, 1H), 3.96-3.71(m, 4H), 3.68-3.60(m, 2H), 3.39-3.34(m, 2H), 2.50(td, J=14. 6, 4.0Hz, 2H), 2.32 (dd, J=14.0, 3.0Hz, 2H), 1.57 (d, J=6.8Hz, 3H). LC-MS [M+H] + =303.2.

[0226] Step 4: Compound 9-4

[0227] A mixture of 4-formylbenzoic acid (164 mg, 1.10 mmol) and N,N-dimethylformamide (4 mL) was added to glacial acetic acid (1 mL). After stirring the mixture at room temperature for 10 min, 5-amino-2-(2,6-dicarbonylpiperidin-3-yl)isodihydroindole-1,3-dione (150 mg, 0.549 mmol) was added. The mixture was stirred at room temperature for 1 h. Sodium triacetoxyborohydride (698 mg, 3.29 mmol) was added at 0 °C, and the mixture was stirred at room temperature overnight. The reaction mixture was evaporated to dryness and purified by reverse-phase preparative liquid chromatography (Gemini-C18 150 x 21.2 mm, 5 μm, ACN--H2O (0.1% FA30-50) to give 125.2 mg of the title intermediate as a yellow solid, yield 55.1%. LCMS tR = 1.09 min, [M+H]+ = 407.9.1 H⁺. NMR (400MHz, DMSO-d6) δ12.90 (s, 1H), 11.06 (s, 1H), 7.93-7.90 (m, 2H), 7.78 (t, J=6.0Hz, 1H), 7.57 (d, J=8.4Hz, 1H), 7.47-7.45 (m, 2H), 6.96 (s, 1H), 6.89 (d, J=8.4Hz, 1H), 5.02 (dd, J=12.8, 5.4Hz, 1H), 4.55 (d, J=5.8Hz, 2H), 2.93-2.80 (m, 1H), 2.69-2.32 (m, 2H), 2.02-1.93 (m, 1H).

[0228] Step 5: Compound 9

[0229] Intermediate 7 (1 equiv.), intermediate 4 (1.05 equiv.), DIPEA (5 equiv.), and HATU (20 mg, 0.0508 mmol, 1.3 equiv.) were dissolved in DMF. The reaction mixture was stirred at room temperature for 4 hours. Water was added to the reaction mixture and the mixture was extracted with ethyl acetate. The organic phase was concentrated and further purified by silica gel column chromatography (DCM:MeOH = 20:1) to give compound 9. 1H NMR (400MHz, Methanol-d4) δ8.30 (s, 1H), 8.00 (s, 1H), 7.62-7.34 (m, 5H), 6.7 0 (d, J=8.2Hz, 1H), 6.56 (s, 1H), 5.12-4.94 (m, 2H), 4.88-4.61 (m, 3H), 4.32-4 .21(m, 2H), 4.14-3.98(m, 3H), 3.92-3.75(m, 2H), 3.72-3.41(m, 3H), 3.17-3. 02(m, 1H), 2.90-2.75(m, 2H), 2.39-1.90(m, 7H), 1.51-1.35(m, 3H).LCMS[M+H] + =692.6.

[0230] Example 1: Inhibition of mToR and degradation of GsPT1 target (Western blot method)

[0231] Cells (MDA-MB-468, U87, and A549 cells) were suspended in 1.5 mL of culture medium and placed in 12-well cell culture plates (5 × 10⁻⁶ cells per well). 5 ~1×10 6 Cells were incubated in wells (cells / well) with different concentrations of the compound described in this application and the mTOR inhibitor MLN0128 (Biode Pharmaceuticals, BD305515). After incubation for the specified time, cells were collected, centrifuged to remove the supernatant, and then washed twice with PBS. Cell samples were lysed on ice using RIPA for 20 minutes, and then 2× protein loading buffer (50mM Tris-HCl (pH 6.8), 2% (w / v) sodium dodecyl sulfate, 0.1% (w / v) bromophenol blue, 10% (v / v) glycerol, and 10% (v / v) β-mercaptoethanol in an ultrapure aqueous solution) was added. The mixture was heated in a metal bath at 100°C for 20 minutes and then cooled to obtain protein samples. 10 μL of protein sample was then taken and separated by 10% SDS-PAGE gel electrophoresis. After electrophoresis, the samples were wet-transferred to a PVDF membrane at 4°C (100V, 1.5h). After transfer, the membrane was blocked with 5% milk for one hour. After blocking, wash three times with PBST for 5 minutes each time, then incubate with primary antibody overnight at 4°C. After incubation, recover the antibody and wash three more times with PBST for 5 minutes each time. Then incubate with the corresponding rabbit / mouse secondary antibody at room temperature for 1 hour. Recover the secondary antibody and wash the membrane three times with PBST for 10 minutes each time. Develop using a chemiluminescent solution on a Tianneng imaging instrument, such as... Figures 1-4 As shown in Table 1, the development results were analyzed using ImageJ for grayscale, and the degradation ratio was obtained by normalization.

[0232] U87 cells were treated with 300 nM compound 2 for 4 h, and quantitative proteomics analysis was performed to analyze degradation selectivity. Figure 5 Compound 2 selectively degrades GSPT1.

[0233] Table 1: Inhibitory effects of the disclosed compounds on mTOR protein kinase and degradation effects on GSPT1 protein.

[0234] Compound mTOR inhibition GSPT1 degradation 1 ++++ ++++ 2 ++++ ++++ 3 ++++ +++ 4 ++ ++ 5 ++ +++ 6 ++ ++ 7 +++ +++ 8 +++ +++ 9 No inhibition +++ MLN0128 ++++ No degradation

[0235] The structural formula of MLN0128 is:

[0236] In the table above, for mTor inhibition, "++++" indicates that at 100 nM or less, the pS6K band in the immunoblotting drug group is almost invisible; "+++" indicates that at 100 nM or less than or equal to 300 nM, the gray level of the pS6K band in the immunoblotting drug group is weaker than that in the DMSO group; "++" indicates that at 300 nM or less than or equal to 500 nM, the gray level of the pS6K band in the immunoblotting drug group is weaker than that in the DMSO group; "+" indicates that at 500 nM or less than or equal to 1000 nM, the gray level of the pS6K band in the immunoblotting drug group is weaker than that in the DMSO group; "no inhibition" indicates that at 1000 nM, the gray level of the pS6K band in the immunoblotting drug group is consistent with that in the DMSO group. Regarding the degradation of GSPT1, "++++" indicates that when the concentration is less than or equal to 100 nM, the GSPT1 band in the immunoblotting drug group is almost invisible; "+++" indicates that when the concentration is greater than 100 nM and less than or equal to 300 nM, the gray level of the GSPT1 band in the immunoblotting drug group is weaker than that in the DMSO group; "++" indicates that when the concentration is greater than 300 nM and less than or equal to 500 nM, the gray level of the GSPT1 band in the immunoblotting drug group is weaker than that in the DMSO group; "+" indicates that when the concentration is greater than 500 nM and less than or equal to 1000 nM, the gray level of the GSPT1 band in the immunoblotting drug group is weaker than that in the DMSO group; "no inhibition" indicates that when the concentration is greater than 1000 nM, the gray level of the pS6K band in the immunoblotting drug group is basically the same as that in the DMSO group.

[0237] Example of effect 2: Inhibitory activity of the disclosed compound against tumor cell proliferation

[0238] The compounds disclosed herein exhibit significant inhibitory effects on the proliferation of various solid tumor cells.

[0239] In the cell proliferation inhibition assay, MDA-MB-468 and U87 cells were suspended in 50 μL of culture medium and placed in 96-well cell culture plates (5000 cells / well). The DMSO stock solution of the specific compound disclosed herein was diluted with 50 μL of culture medium and added to the 96-well cell culture plates for culture. Cells were cultured at 37°C in a 5% CO2 incubator. After 72 h, 10 μL of CCK-8 reagent was added to each well of the 96-well cell culture plate. After incubation for 1–4 hours, the absorbance was read at 450 nm using a multi-mode microplate reader. Cells treated with DMSO were considered to have 100% viability. The IC50 was calculated using GraphPad Prism 8 software through nonlinear regression analysis. 50 The formula for calculating cell viability is:

[0240] Survival rate = [(dose - blank) / (control - blank)] × 100%.

[0241] Table 2: IC50 values ​​of the compounds in each example. 50 value

[0242]

[0243] The foregoing description of specific exemplary embodiments of this disclosure is for illustrative and explanatory purposes. These descriptions are not intended to limit this disclosure to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of this disclosure and their practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of this disclosure, as well as various different choices and variations. The scope of this disclosure is intended to be defined by the claims and their equivalents.

Claims

1. The compound represented by formula (I) or its tautomers, enantiomers, diastereomers, mixtures of enantiomers and diastereomers, racemates, mesosomes, mixtures of racemates and mesosomes, or pharmaceutically acceptable salts: in, G is connected to L via Z1, and G has the following structure: ； X1 and X2 are each independently N; R1 is selected from hydrogen, amino, ; R2 does not exist or is ; A1 is selected from hydrogen, , ; Z1 is selected from , , , ; L is selected from , , The left side of L connects to the right side of Z1; Y is selected from , .

2. The compound according to claim 1, or its tautomers, enantiomers, diastereomers, mixtures of enantiomers and diastereomers, racemates, mesosomes, mixtures of racemates and mesosomes, or pharmaceutically acceptable salts, characterized in that, Compounds containing formula (IIa) or (IIb): The definitions of X1, X2, R1, A1, Z1, and L are as described in claim 1; R7 is a methyl group.

3. The compound according to claim 1 or 2, or its tautomers, enantiomers, diastereomers, mixtures of enantiomers and diastereomers, racemic mixtures, meso mixtures, mixtures of racemic mixtures and meso mixtures, or pharmaceutically acceptable salts, wherein, Compounds comprising formula (IIIa1), formula (IIIa2), or formula (IIIb1): ， Wherein, A1 and L are defined as described in claim 1; n5 is 1.

4. The following compounds or their tautomers, enantiomers, diastereomers, mixtures of enantiomers and diastereomers, racemates, mesosomes, mixtures of racemates and mesosomes, and pharmaceutically acceptable salts: 。 5. A pharmaceutical composition comprising, in a physiologically acceptable medium, the compound according to any one of claims 1-4 or a tautomer, enantiomer, diastereomer, mixture of enantiomers and diastereomers, racemic, meso, mixture of racemic and meso, or a pharmaceutically acceptable salt.

6. The use of the compound of any one of claims 1-4 or its tautomers, enantiomers, diastereomers, mixtures of enantiomers and diastereomers, racemic mixtures, meso mixtures, mixtures of racemic mixtures and meso mixtures, pharmaceutically acceptable salts, or the pharmaceutical composition of claim 5 in the preparation of a medicament for the inhibition of mTOR and degradation of GSPT1.

7. The use of the compound of any one of claims 1-4 or its tautomers, enantiomers, diastereomers, mixtures of enantiomers and diastereomers, racemic mixtures, meso mixtures, mixtures of racemic mixtures and meso mixtures, pharmaceutically acceptable salts, or the pharmaceutical composition of claim 5 in the preparation of a medicament for treating diseases abnormally mediated by the mTOR signaling pathway and GSPT1 protein, respectively or simultaneously.

8. In the application according to claim 7, the disease abnormally mediated by the mTOR signaling pathway and GSPT1 protein, either individually or simultaneously, is a malignant disease, including tumors and leukemia.

9. The application according to claim 8, wherein the tumor disease includes colorectal cancer, pancreatic cancer, liver cancer, breast cancer, ovarian cancer, endometrial cancer, lung cancer, prostate cancer, and lymphoma.

10. The application according to claim 8, wherein the leukemia includes one or more of chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, and acute myeloid leukemia.

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