Heterocyclic immunomodulatory substance

Abstract

The present invention relates to heterocyclic compounds or pharmaceutically acceptable salts thereof as immunomodulators. Specifically, the present invention relates to compounds represented by general formula (I) and their pharmaceutically acceptable salts. The present invention also relates to a method for preparing the compounds or their pharmaceutically acceptable salts. The compounds can be used for the treatment and / or prevention of tumors, inflammation, or immune diseases.

Description

Technical Field

[0001] The present invention relates to a heterocyclic compound as an immunomodulatory substance, or a pharmaceutically acceptable salt thereof. The present invention also relates to a method for preparing the compound or a pharmaceutically acceptable salt thereof. The present invention further relates to the use of the compound or a pharmaceutically acceptable salt thereof in immunomodulation, cancer resistance, anti-inflammation, etc., and a method of using the same.

Background Art

[0002] "Lidmid" compounds such as lenalidomide are immunomodulatory substances (immunomodulatory drugs; IMIDs) and have multiple mechanisms of action. At the molecular level, this type of immunomodulatory substance binds to an E3 ubiquitin ligase to regulate the function of the CRL4 CRBN -E3 ubiquitin ligase complex, causing ubiquitination of Ikaros (IKZF1), Aiolos (IKZF3), protein kinase CK1α (CK1α), translational termination factor GSPT1, etc., and being degraded by the 26S proteasome, thereby modifying the secretion of various cytokines (such as IL-2, IL-6, IL-10, TNFα, and IL-1β), and affecting the activity of immune cells. Different "lidmid" compounds have CRL4 CRBN-When combined with the E3 ubiquitin ligase complex, it exhibits diverse substrate protein degradation specificities and thus provides different signals. Both lenalidomide and pomalidomide can induce cereblon (CRBN) to degrade the transcription factors Ikaros and Aiolos, which play important roles in blood generation and differentiation, and are used in the treatment of multiple myeloma; however, only lenalidomide can cause the degradation of CK1α and is used in the treatment of myelodysplastic syndrome associated with 5q deletion. In addition, CC-885 promotes the degradation of GSPT1, while neither lenalidomide nor pomalidomide has such an effect. In non-immunomodulation, "thalidomide" compounds can inhibit tumor cell angiogenesis by inhibiting vascular endothelial growth factor (VEGF), directly inhibit tumor cell proliferation, and also induce the degradation of abnormal cells.

[0003] Thalidomide compounds have attracted wide attention in the treatment of many types of malignant tumors and immune diseases, such as multiple myeloma, myelodysplastic syndrome, hematological tumors / solid tumors (e.g., lymphoma, non-small cell lung cancer, pancreas, prostate, brain, kidney, ovary, etc.), and systemic lupus erythematosus, through immune and non-immune regulation. In addition, "thalidomide" compounds can also be combined with drugs for treating other diseases, such as small molecule target drugs, chemotherapeutic drugs, and macromolecular pharmaceuticals (PD-1 antibody, CD20 antibody, CD19 antibody, etc.). Moreover, with the further development of the research and development of such compounds in aspects such as immunomodulation, cancer resistance, and anti-inflammation, the clinical applications of such compounds are also continuously increasing.

[0004] The chemical structures of "thalidomide" compounds are similar, but their mechanisms of action, clinical treatment effects, and toxicities and side effects are different. Clinically common side effects of thalidomide such as neurological disorders, constipation, and drowsiness are rare in lenalidomide, and pomalidomide does not have side effects such as rash like lenalidomide. Therefore, in order to meet different clinical needs, the development of new immunomodulatory substances and the expansion of indications are required. The present invention relates to a compound having a structure represented by the general formula (I), which is used as an immunomodulatory substance and, when combined with an E3 ubiquitin ligase, effectively regulates the expression and / or biological functions of proteins such as IL-2, IL-6, IL-10, TNFα, and VEGF.

Summary of the Invention

[0005] Definition The following terms used in the present specification and claims have the following meanings unless otherwise specified.

[0006] "C x-y " indicates the range of carbon atoms, where x and y are integers. For example, C 3-8 Cycloalkyl refers to cycloalkyl having 3 to 8 carbon atoms, that is, cycloalkyl having 3, 4, 5, 6, 7, or 8 carbon atoms. Also, "C 3-8 " includes any sub-range therein, for example, C 3-7 , C 3-6 , C 4-7 , C 4-6 , C 5-6 etc. should be understood to be included.

[0007] "Alkyl" means a saturated straight-chain or branched-chain hydrocarbyl group containing 1 to 20 carbon atoms, such as 1 to 8 carbon atoms, 1 to 6 carbon atoms or 1 to 4 carbon atoms. Non-limiting examples of alkyl include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, and the like.

[0008] "Alkylene" means a saturated straight-chain or branched-chain hydrocarbon divalent group containing 1 to 20 carbon atoms, such as 1 to 6 carbon atoms or 1 to 4 carbon atoms. Non-limiting examples of alkylene include -CH2-, -CH(CH3)-, -CH2CH2-, -CH2CH2CH2-, -(CH3)C(CH3)-, -CH2CH2CH2CH2-, -CH2CH(CH3)CH2-, and the like.

[0009] "Cycloalkyl" means a saturated cyclic hydrocarbyl substituent containing 3 to 14 carbon ring atoms. Cycloalkyl may typically be a monocyclic carbon ring containing 3 to 8, 3 to 7, or 3 to 6 carbon ring atoms. Non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like. Cycloalkyl may also be bicyclic or tricyclic which are fused, bridged or spiro, such as decahydronaphthalenyl, bicyclo[2.2.2]octane, spiro[3.3]heptane, and the like.

[0010] "Heterocyclyl or heterocycle" means a saturated or partially unsaturated monocyclic or polycyclic cyclic group containing 3 to 20 ring atoms, such as 3 to 14, 3 to 12, 3 to 10, 3 to 8, 3 to 6 or 5 to 6 ring atoms, one or more of which is nitrogen, oxygen or S(O)m (Here, m is an integer from 0 to 2) (provided that it excludes parts other than -O-O-, -O-S-, or -S-S- ring moieties), and the remaining ring atoms are carbon atoms. Preferably, it contains 3 to 12 ring atoms, more preferably 3 to 10 ring atoms, more preferably 4 to 7 ring atoms, more preferably 4 to 6 ring atoms, and most preferably 5 or 6 ring atoms, among which 1 to 4 are heteroatoms, more preferably 1 to 3 are heteroatoms, and most preferably 1 to 2 are heteroatoms. Non-limiting examples of monocyclic heterocyclyl include pyrrolidinyl, oxetanyl, piperidinyl, piperazinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, homopiperazinyl, azetidinyl, etc. Non-limiting examples of polycyclic heterocyclyl include fused, bridged or spiro polycyclic heterocyclic groups, such as octahydrocyclopenta[c]pyrrole, octahydropyrrolo[1,2-a]pyrazine, 3,8-diazabicyclo[3.2.1]octane, 5-azaspiro[2.4]heptane, 2-oxa-7-azaspiro[3.5]nonane, etc.

[0011] "Aryl or aromatic ring" means an aromatic monocyclic or fused polycyclic group containing 6 to 14 carbon atoms, preferably 6 to 10 members, such as phenyl and naphthyl, and most preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring. Here, the ring bonded to the parent structure is the aryl ring, and non-limiting examples thereof include

Chemical Structure

[0012] "Heteroaryl or heteroaromatic ring" means a heteroaromatic system containing 5 to 14 ring atoms, of which 1 to 4 ring atoms are selected from heteroatoms including oxygen, sulfur, and nitrogen. Heteroaryl is preferably 5 to 10 membered, more preferably 5 membered or 6 membered, and examples thereof include furyl, thienyl, pyridinyl, pyrrolyl, pyrimidinyl, pyrazinyl, pyrazolyl, imidazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl, and the like. The heteroaryl ring may be condensed with an aryl, heterocyclyl or cycloalkyl ring, where the ring attached to the parent structure is the heteroaryl ring, and non-limiting examples thereof include [Chemical formula] and the like.

[0013] "Halogen" means fluorine, chlorine, bromine or iodine.

[0014] "Cyano" means -CN.

[0015] "Oxo" means =O.

[0016] "Carbonyl" means -C(O)- group.

[0017] "Sulfonyl" means -S(O)2- group.

[0018] "Sulfinyl" means -S(O)- group.

[0019] "Optionally" means that the event or situation described thereafter may occur but need not occur, and the expression means that it includes both the case where the event or situation occurs and the case where it does not occur. For example, "a heterocyclic group optionally substituted by an alkyl group" means that the alkyl group may or may not be present, and the expression includes the case where the heterocyclic group is substituted by an alkyl group and the case where the heterocyclic group is not substituted by an alkyl group. "Substituted" means that one or more hydrogen atoms in the group, preferably 5, more preferably 1 to 3 hydrogen atoms, are independently substituted by the corresponding number of substituents. The substituents are only at their possible chemical positions, and it goes without saying that a person skilled in the art can determine (by experiment or theory) possible substitutions or impossible substitutions without undue effort. For example, an amino group or a hydroxyl group having a free hydrogen may become unstable when bonded to a carbon atom having an unsaturated (e.g., ethylenic) bond. Examples of substituents include halogen, cyano, nitro, oxo, -SF5, C 1-4 alkyl, C 3-7 cycloalkyl, 4- to 7-membered heterocyclyl, phenyl, 5- to 6-membered heteroaryl, etc., but are not limited thereto.

[0020] "Isomer" means a compound having the same molecular formula but different in the nature or order of atomic bonds or the spatial arrangement of atoms. Isomers with different spatial arrangements of atoms are called "stereoisomers". Stereoisomers include optical isomers, geometric isomers, conformational isomers, and the like.

[0021] The compounds of the present invention may exist in the form of optical isomers. Optical isomers include enantiomers and diastereoisomers. Enantiomers are two stereoisomers that are mirror images of each other but cannot be superimposed. A racemic mixture or racemate means a mixture of equal amounts of the left- and right-handed enantiomers of a chiral molecule. Diastereoisomers mean two stereoisomers that are not mirror images of each other and cannot be superimposed. When the optical isomer is a single isomer and its absolute configuration is determined, it has an absolute configuration of "R" or "S" according to the configuration of the substituents on the chiral carbon atom, and when the absolute configuration of the optical isomer is not determined, it is (+) or (-) according to the measured optical rotation. Methods for preparing and separating optical isomers are known in the art.

[0022] Furthermore, the compounds of the present invention may exist as geometric isomers. The present invention contemplates various geometric isomers and mixtures thereof resulting from the distribution of substituents around carbon-carbon double bonds, carbon-nitrogen double bonds, cycloalkyl groups or heterocyclic groups. Substituents around a carbon-carbon double bond or a carbon-nitrogen bond are assigned the Z or E configuration, and substituents around a cycloalkyl or heterocycle are assigned the cis or trans configuration.

[0023] In addition, the compounds of the present invention may exhibit tautomerism, such as keto-enol tautomerism.

[0024] It should be understood that the present invention includes any tautomeric or stereoisomeric forms and mixtures thereof, and is not limited to any one tautomeric or stereoisomeric form used in the nomenclature or chemical structural formula of the compound.

[0025] "Isotope" means all isotopes of the atoms present in the compounds of the present invention. Isotopes include atoms having the same atomic number but different mass numbers. Examples of isotopes suitable for incorporation into the compounds of the present invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, for example, 2 H(D), 3 H, 13C, 14 C, 15 N, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F and 36 Cl are present, but are not limited thereto. The isotopically labeled compounds of the present invention can generally be prepared by methods similar to those of the prior art known to those skilled in the art or described in the attached examples, using appropriate isotopically labeled reagents in place of non-isotopically labeled reagents. Such compounds have various potential uses, for example, as standards and reagents in assays of biological activity. In the case of stable isotopes, such compounds have the potential to advantageously alter biological, pharmacological or pharmacokinetic properties. Deuterium (D) is a preferred isotope of the present invention, and for example, the hydrogen in methyl, methylene or methine may be replaced by deuterium.

[0026] The compounds of the present invention can be administered in the form of prodrugs. A "prodrug" means a derivative that is converted into the bioactive compound of the present invention under physiological conditions in vivo by oxidation, reduction, hydrolysis, etc. (performed either with or without the involvement of enzymes). Examples of prodrugs include the following compounds: those in which the amino group in the compound of the present invention is acylated, alkylated or phosphorylated, such as eicosanoylamino, alanyl amino, pivaloyloxymethylamino, or those in which the hydroxyl group is converted to acylation, alkylation, phosphorylation or borate, such as acetoxy, palmitoyloxy, pivaloyloxy, succinyloxy, fumaryloxy, alanyloxy, or those in which the carboxyl group is esterified or amidated, or those in which the sulfhydryl group forms a disulfide bridge with a carrier molecule such as a peptide that selectively delivers the drug to the target and / or the cytosol of the cell. These compounds can be prepared from the compounds of the present invention according to known methods.

[0027] "Pharmaceutically acceptable salts" mean salts prepared from pharmaceutically acceptable bases or acids including inorganic bases or acids and organic bases or acids. When the compounds of the present invention contain one or more acidic or basic groups, the present invention also includes their corresponding pharmaceutically acceptable salts. Compounds according to the present invention containing acidic groups can thus exist in the form of salts and can be used according to the present invention, for example, as alkali metal salts, alkaline earth metal salts or ammonium salts. More specific examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or organic amines such as ethylamine, ethanolamine, triethanolamine or amino acids. Compounds according to the present invention containing basic groups can exist in the form of salts and can be used according to the present invention in the form of addition salts with inorganic or organic acids. Examples of suitable acids include hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and other acids known to those skilled in the art. When the compounds according to the present invention contain both acidic and basic groups in the molecule, the present invention includes inner salts or betaines in addition to the salt forms mentioned. Individual salts can be obtained by conventional methods known to those skilled in the art, for example, by contacting them with organic or inorganic acids or bases in a solvent or dispersion medium, or by anion exchange or cation exchange with other salts.

[0028] "Pharmaceutical composition" means a composition containing one or more compounds of the present invention or their pharmaceutically acceptable salts, stable isotope derivatives, isomers, prodrugs or mixtures thereof, and other components such as pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to a living body, facilitate absorption of the active ingredient, and as a result, exert biological activity.

[0029] Accordingly, when referring to "compound", "compound of the present invention" or "compounds of the present invention" in the present application, all forms of said compound are included, such as pharmaceutically acceptable salts, stable isotope derivatives, isomers, prodrugs, or mixtures thereof.

[0030] "Cancer / tumor" includes, but is not limited to, gastrointestinal / gastrointestinal cancer, colon cancer, liver cancer, skin cancer (including mastocytoma and squamous cell carcinoma), breast cancer, ovarian cancer, prostate cancer, lymphoma, leukemia (including acute myeloid leukemia and chronic myeloid leukemia), kidney cancer, lung cancer, muscle cancer, bone cancer, bladder cancer, brain cancer, melanoma (oral and metastatic melanoma), Kaposi's sarcoma (myeloma of multiple myeloma), myeloproliferative diseases, proliferative diabetic retinopathy, angiogenesis-related diseases / tumors, etc.

[0031] "Inflammatory disease" includes, but is not limited to, arthritis, Hashimoto's thyroiditis, autoimmune hemolytic anemia, autoimmune atrophic gastritis of pernicious anemia, autoimmune encephalomyelitis, autoimmune orchitis, Goodpasture's disease, autoimmune thrombocytopenia, sympathetic ophthalmia, myasthenia gravis, Graves' disease, primary biliary cirrhosis, hepatitis, primary sclerosing cholangitis, chronic aggressive hepatitis, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, ulcerative colitis, membranous glomerulopathy, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, Sjogren's syndrome, Reiter's syndrome, polymyositis, dermatomyositis, type I interferon diseases (including Eckardt-Gutier syndrome and other systemic sclerosis (overexpressing type I interferon, Mendelian diseases, polyarteritis nodosa, multiple sclerosis, relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis, pemphigoid)); additional O-cell (humoral) or T-cell-based autoimmune diseases (including Cogan's syndrome, ankylosing spondylitis, Wegener's granulomatosis, alopecia areata universalis, type I or juvenile diabetes, thyroiditis).

[0032] "Therapeutically effective amount" means an amount of the compound of the present invention effective to treat or prevent a disease.

[0033] "Patient" means a mammal, particularly a human being.

[0034] The present invention relates to a compound of general formula (I):

Chemical formula

[0035] In one embodiment, ring A is a 4- to 10-membered monocyclic heterocycle containing 1 N atom, or a fused-, bridged- or spiro-bicyclic heterocycle (e.g., morpholine, piperidine, thiomorpholine-1,1-dioxide, 2-oxa-5-azabicyclo[2.2.1]heptane, etc.); R 4 is D, halogen, oxo, -CF3 or C 1-6 alkyl.

[0036] In one embodiment, ring A is a 6- to 10-membered monocyclic heterocycle containing 2 N atoms, or a fused-, bridged- or spiro-bicyclic heterocycle (e.g., piperazine, 3,6-diazabicyclo[3.1.1]heptane, 2,6-diazaspiro[3.3]heptane, etc.); R 4 is bonded to the second N atom and is C 1-6 alkyl, C 3-6 cycloalkyl, -C(O)C 1-6 alkyl, -C(O)C 3-6 cycloalkyl, -S(O)2C 1-6 alkyl, -S(O)2C 3-6 cycloalkyl, phenyl, or a 5- to 6-membered heteroaryl containing N, O and / or S, wherein one or more hydrogens of alkyl, cycloalkyl, phenyl and heteroaryl are optionally substituted by D, halogen, cyano, C 1-2 alkyl or fluoro C 1-2 alkyl.

[0037] In a preferred embodiment, ring A is piperazine; R 4 is bonded to the second N atom and is C 1-6 alkyl, C 3-6 cycloalkyl, -C(O)C 1-6 alkyl, -C(O)C 3-6 cycloalkyl, -S(O)2C 1-6 alkyl, phenyl, pyridinyl or pyrimidinyl, wherein one or more hydrogens of alkyl, cycloalkyl, phenyl, pyridinyl or pyrimidinyl are optionally substituted by D, halogen, cyano or C 1-2It may be substituted by alkyl; n is 0 or 1.

[0038] In one embodiment, R 2 is H; R 3 is cyano, C 1-6 alkyl or C 3-6 cycloalkyl.

[0039] In one embodiment, R 2 and R 3 are both methyl.

[0040] In one embodiment, R 2 and R 3 together with the carbon atom to which they are attached form a C 3-6 cycloalkane.

[0041] In a preferred embodiment, m is 0.

[0042] In some embodiments, the compound represented by general formula (I) is the following general formula (II):

Chemical formula

[0043] In a preferred embodiment, R 5is phenyl, pyridinyl or pyrimidinyl, wherein one or two hydrogens of phenyl, pyridinyl and pyrimidinyl may optionally be substituted by F or cyano.

[0044] The present invention further relates to the following compounds 1-22, or pharmaceutically acceptable salts, stable isotope derivatives, isomers, prodrugs thereof, or mixtures thereof: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0045] The compounds of the present invention can effectively inhibit the proliferation of NCI-H929 cells, preferably, the IC 50 is less than 50 nM, more preferably the IC 50 is less than 10 nM. The compounds of the present invention have the effect of significantly inhibiting the secretion of TNFa in human PBMC cells, preferably the IC 50 is less than 20 nM, more preferably the IC 50 is less than 10 nM. In addition, the compounds of the present invention have a significant stimulating effect on the secretion of IL-2 in human PBMC cells, preferably the EC 50 is less than 50 nM, more preferably the EC 50 is less than 10 nM.

[0046] The present invention relates to a pharmaceutical composition comprising a compound represented by general formula (I) or a pharmaceutically acceptable salt, stable isotope derivative, isomer, prodrug thereof, or a mixture thereof, and one or more pharmaceutically acceptable carriers or excipients.

[0047] One aspect of the present invention is to provide a compound represented by general formula (I) or a pharmaceutically acceptable salt, stable isotope derivative, isomer, prodrug thereof, or a mixture thereof, or a pharmaceutical composition, for regulating or inhibiting the activity of E3 ubiquitin ligase to affect the expression and / or biological function of proteins (including but not limited to aiolos, ikaros, helios, CK1α, GSPT1, IL-2, IL-6, TNFα, IFNγ, VEGF, etc.).

[0048] Another aspect of the present invention is a method for treating or preventing related diseases mediated by aiolos, ikaros, helios, CK1α, GSPT1, IL-2, IL-6, TNFα, IFNγ, VEGF, etc., which comprises administering to a subject in need thereof a therapeutically effective amount of a compound represented by general formula (I) or a pharmaceutically acceptable salt, stable isotope derivative, isomer, prodrug thereof, or a mixture thereof, or a pharmaceutical composition comprising the compound. The diseases include hematological malignancies (such as multiple myeloma, lymphoma, and leukemia), solid tumors (such as lung cancer, prostate cancer, head and neck cancer, breast cancer, ovarian cancer, uterine cancer, pancreatic cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, brain cancer, liver cancer, kidney cancer, skin cancer, epithelial cancer, bladder cancer, and neuroblastoma), autoimmune diseases (such as systemic lupus erythematosus, psoriasis, and inflammatory bowel disease), inflammation (such as rheumatoid arthritis), neurodegenerative diseases (such as multiple sclerosis, Alzheimer's disease, and Parkinson's disease), fibrosis (such as pulmonary fibrosis), skin diseases (such as melanoma), eye diseases, chronic obstructive pulmonary disease, etc., and particularly include but are not limited to multiple myeloma, lymphoma, myelodysplastic syndrome, systemic lupus erythematosus, solid tumors, etc.

[0049] The present invention also relates to a pharmaceutical composition comprising a compound represented by general formula (I) or a pharmaceutically acceptable salt, stable isotope derivative, isomer, prodrug, or a mixture thereof, and at least one additional drug, wherein the at least one additional drug can be a small molecule chemotherapeutic agent (e.g., NSAIDs, steroid anti-inflammatory drugs, kinase target drugs, cytotoxic drugs, and DNA damage-related drugs) or a macromolecular immune and / or inflammatory regulator (e.g., PD-1 antibody, CD20 antibody, CD19 antibody, TNFα antibody, and IL-6 antibody).

[0050] According to the present invention, it can be a pharmaceutical dosage form including, but not limited to, tablets, capsules, solutions, lyophilized preparations, and injections.

[0051] The pharmaceutical preparation of the present invention can be administered in unit dosage form containing a predetermined amount of the active ingredient per dosage unit. Such units can contain, for example, 0.1 mg to 500 mg, preferably 0.5 mg to 100 mg of the compound of the present invention, depending on the condition to be treated, the method of administration, and the age, weight, and condition of the patient. Furthermore, this type of pharmaceutical preparation can be prepared using methods known in the pharmaceutical art, such as mixing the active ingredient with one or more excipients and / or adjuvants.

[0052] The pharmaceutical preparation of the present invention can be adapted for administration by any desired suitable method, such as oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual, or transdermal), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, or intradermal) administration.

[0053] The present invention also provides a method for preparing the above-mentioned compound. The preparation of the compound represented by the general formula (I) of the present invention can be achieved by the following exemplary methods and examples, but these methods and examples should not be considered as limiting the scope of the present invention in any way. The compounds described in the present invention can also be synthesized by synthetic techniques known to those skilled in the art, or the methods known to those skilled in the art can be combined with the methods described in the present invention for use. The products obtained in each reaction step can be obtained by separation techniques known to those skilled in the art, including but not limited to extraction, filtration, distillation, crystallization, chromatographic separation, etc. The starting materials and chemical reagents required for the synthesis can be routinely synthesized according to the literature (available on SciFinder) or can also be purchased.

[0054] Synthesis method The heterocyclic compound represented by the general formula (I) of the present invention can be synthesized according to the following route: 1) Subject the "ridomide" compound A1 and the intermediate B having a leaving group X (such as halogen) to a substitution reaction under heating under the catalysis of a base (such as potassium carbonate) to produce the target compound A4 (where the "ridomide" compound A1 may be purchased or can be synthesized according to the methods in the literature (such as CN107739389, etc.). Also, the target compound A4 can be obtained by first subjecting the compound A2 having the intermediate B to a substitution reaction to generate A3, and then subjecting A3 to thermal cyclization under the catalytic reaction with an acid (such as TsOH, etc.). Here, the compound A2 can be purchased or synthesized according to the methods in the literature (such as WO2014025978, etc.).

[0055]

Chemical formula

[0056] Intermediate B can be synthesized according to the following route: 1) Subjecting starting material B1 and a raw material or reagent having two leaving groups X (such as halogen) to cyclization under alkaline conditions (for example, organic base DIPEA in DMF or inorganic base potassium carbonate) and heating to produce intermediate B2; 2) Subjecting a solution of B2 in methanol to a carbonyl insertion reaction under a carbon monoxide atmosphere using a palladium catalyst (such as PdCl2(dppf)) to produce intermediate ester B3 (B3 can also be produced by subjecting B4 and a raw material or reagent of NH-containing heterocyclic ring A to a substitution reaction under the catalysis of a base (such as potassium carbonate)); 3) Reducing the ester group of B3 with LAH to produce intermediate alcohol B5; 4) Further halogenating B5 (for example, with CBr4 / PPh3 or SOCl2) to produce important intermediate B.

[0057]

Chemical Structure

Examples

[0058] The starting materials of the present invention can be synthesized according to methods known in the art or can be purchased from chemical companies such as ABCR GmbH&Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., and Beijing OUHE technology Co. Ltd.

[0059] The structure of the compounds of the present invention was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The determination by NMR was performed using a Bruker ASCEND-400 nuclear magnetic analyzer with solvents such as deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), or deuterated methanol (CD3OD), and using tetramethylsilane (TMS) as an internal standard, at 10 -6The chemical shift was obtained in units of (ppm). MS measurement was performed using an Agilent SQD (ESI) mass spectrometer (Agilent 6120).

[0060] Determination by HPLC was carried out using an Agilent 1260 DAD high-pressure liquid chromatograph (Poroshell 120 EC-C18, 50×3.0 mm, 2.7 μm chromatographic column) or a Waters Arc high-pressure liquid chromatograph (Sunfirc C18, 150×4.6 mm, 5 μm chromatographic column).

[0061] Unless otherwise specified in the examples, the reaction temperature was room temperature (20 °C to 30 °C).

[0062] Unless otherwise specified in the examples, the reaction was carried out under an argon or nitrogen atmosphere. An argon or nitrogen atmosphere means that the reaction flask was connected to an argon balloon or nitrogen balloon with a volume of about 1 L.

[0063] A hydrogen atmosphere means that after evacuating the reaction flask, it was connected to a hydrogen balloon with a volume of about 1 L and filled with hydrogen (repeated 3 times).

[0064] For microwave reactions, a CEM Discover-SP type microwave reactor was used. The reaction process in the examples was monitored by Agilent's LC / MS chromatography (1260 / 6120) or thin-layer chromatography (TLC) using a silica gel plate (Qingdao Haiyang GF254) with a thickness of 0.15 - 0.2 mm.

[0065] Purification of the compound was carried out by column chromatography using 200 - 300 mesh silica gel from Qingdao Haiyang or thin-layer chromatography using a GF254 silica gel plate with a thickness of 0.4 - 0.5 mm from Qingdao Haiyang.

[0066] The developing solvent systems for column chromatography or thin-layer chromatography usually included a) dichloromethane and methanol systems, b) petroleum ether and ethyl acetate systems, or those shown in the examples. The volume ratio of the solvents was adjusted according to the polarity of the compound, and could be further adjusted by adding a small amount of triethylamine or other acidic or basic reagents.

[0067] The purification of the compound was also carried out using Waters' Mass Spectrometer-Guided Automatic Preparation System (Mass Spectrometer Detector: SQD2). According to the polarity of the compound, a reverse-phase high-pressure column (XBridge-C18, 19×150mm, 5μm) was eluted at a flow rate of 20 mL / min with an appropriate acetonitrile / water (containing 0.1% trifluoroacetic acid or formic acid, or 0.05% aqueous ammonia) gradient. In some of the examples, 1N dilute hydrochloric acid was added after purification by the automatic preparation system, and the solvent was removed under reduced pressure to produce the hydrochloride.

[0068] The abbreviation PdCl2(dppf) means [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride.

[0069] The abbreviation LAH means lithium aluminum hydride.

[0070] The abbreviation DIPEA means N,N-diisopropylethylamine.

[0071] The abbreviation TsOH·H2O means p-toluenesulfonic acid monohydrate.

[0072] The abbreviation THF means tetrahydrofuran.

[0073] The abbreviation DMF means N,N-dimethylformamide.

[0074] Example 1 3-(4-((4-(1-Morpholinoethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (Compound 1)

[0075] [Chemical formula]

[0076] Step 1 3-(1-Oxo-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoindolin-2-yl)piperidine-2,6-dione (1b) 3-(7-Amino-3-oxo-1H-isoindolin-2-yl)piperidine-2,6-dione 1a (5g, 19.3 mmol), bis(pinacolato)diboron (7.35g, 29.0 mmol) and acetonitrile (100 mL) were mixed at room temperature, and then tert-butyl nitrite (2.19g, 21.3 mmol) was added. The mixture was stirred at room temperature for 18 hours, and then the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 1) to give the target product 1b (3.9g, 55%). MS m / z (ESI): 371 [M+1]

[0077] Step 2 (2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)boronic acid (1c) 1b (3.9g, 10.5 mmol), THF (100 mL) and water (20 mL) were mixed at room temperature, and then sodium periodate (6.7g, 31.6 mmol) was slowly added. The mixture was stirred at room temperature for 2 hours, and then dilute hydrochloric acid (1N, 7.3 mL, 7.3 mmol) was added. The mixture was stirred at room temperature for 18 hours, and the solvent was removed under reduced pressure. The residue was dissolved in dichloromethane (20 mL) and water (20 mL). The mixture was stirred at room temperature for 18 hours and filtered to give the target product 1c (2.3g, 76%). MS m / z (ESI): 289 [M+1]

[0078] Step 3 3-(4-Hydroxy-1-oxoisoindolin-2-yl)piperidine-2,6-dione (1d) A solution of 1c (1 g, 3.5 mmol) in dimethyl sulfoxide (20 mL) was added with aqueous hydrogen peroxide solution (2.76 mL, 27 mmol) under a nitrogen gas atmosphere. The mixture was stirred at room temperature for 18 h, and then water (30 mL) was added. The resulting mixture was extracted with ethyl acetate (3 × 50 mL). The organic phases were combined, washed with saturated aqueous sodium chloride solution (3 × 50 mL), and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 1 - 85 / 15) to give the target product 1d (110 mg, 12%). MS m / z (ESI): 261 [M+1]

[0079] Step 4 Methyl 4-(1-morpholinoethyl)benzoate (1f) Methyl 4-(1-bromoethyl)benzoate 1e (242 mg, 1 mmol), morpholine (87 mg, 1 mmol), potassium carbonate (151.8 mg, 1.1 mmol) and acetonitrile (1 mL) were mixed at room temperature and stirred for 18 h, and then water (3 mL) was added. The mixture was extracted with ethyl acetate (3 × 3 mL). The organic phases were combined, washed with water (3 × 2 mL), and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure to give the target product 1f (280 mg, crude product). The product was used directly in the reaction of the next step without further purification.

[0080] Step 5 (4-(1-Morpholinoethyl)phenyl)methanol (1g) 1f (280 mg, crude product) was dissolved in THF (2 mL) and cooled to 0 °C. Next, under a nitrogen gas atmosphere, LAH (128 mg, 3.37 mmol) was added. The mixture was stirred at 0 °C for 1 hour and then at room temperature for 3 hours. Water (2 mL) was added to the mixture, and then the resulting mixture was extracted with ethyl acetate (3 × 3 mL). The organic phases were combined, washed with water (3 × 2 mL), and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 1 / 1) to give 1 g (160 mg, 73%) of the target product. MS m / z (ESI): 222 [M+1]

[0081] Step 6 4-(1-(4-(Bromomethyl)phenyl)ethyl)morpholine (1h) 1g (80 mg, 0.36 mmol), carbon tetrabromide (143 mg, 0.43 mmol) and dichloromethane (5 mL) were mixed, and then triphenylphosphine (114 mg, 0.43 mmol) was added under a nitrogen gas atmosphere. The mixture was stirred at room temperature for 48 hours, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 7 / 3) to give 1h (20 mg, 20%) of the target product. MS m / z (ESI): 284 [M+1]

[0082] Step 7 3-(4-((4-(1-Morpholinoethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (1) 1h (20 mg, 0.07 mmol), 1d (18.4 mg, 0.07 mmol), potassium iodide (17.6 mg, 0.11 mmol), potassium carbonate (14.6 mg, 0.11 mmol) and acetonitrile (1 mL) were mixed at room temperature. The mixture was heated to 80 °C, stirred for 18 h, and cooled to room temperature. The solvent was removed under reduced pressure. The residue was purified by reverse-phase preparative high performance liquid chromatography to give the target product 1 (6.1 mg, solid, 19%). MS m / z (ESI): 464 [M+1] TIFF0007712703000013.tif27156

[0083] Example 2 3-(4-((4-(1-Morpholinocyclopropyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (Compound 2)

[0084]

Chemical Structure

[0085] Step 1 Methyl 4-(1-morpholinocyclopropyl)benzoate (2b) Methyl 4-(1-aminocyclopropyl)benzoate 2a (191 mg, 1 mmol), 1-bromo-2-(2-bromoethoxy)ethane (696 mg, 3 mmol), DIPEA (645 mg, 5 mmol) and DMF (3 mL) were mixed at room temperature. The mixture was heated to 110 °C, stirred for 18 h, and cooled to room temperature. Water (10 mL) was added. Then, the resulting mixture was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, washed with water (3 × 5 mL), and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 1 - 19 / 1) to give the target product 2b (150 mg, 57%). MS m / z (ESI): 262 [M+1]

[0086] Step 2 (4-(1-Morpholinocyclopropyl)phenyl)methanol (2c) At 0 °C, LAH (43.6 mg, 1.15 mmol) was added to a solution of 2b (150 mg, 0.57 mmol) in THF (5 mL). The mixture was stirred at room temperature for 2 hours, and water (2 mL) was added. Next, the resulting mixture was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, washed with water (3 × 5 mL), and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure to give the target product 2c (110 mg, 84%). The product was used directly in the reaction of the next step without further purification. MS m / z (ESI): 234 [M+1]

[0087] Step 3 4-(1-(4-(Chloromethyl)phenyl)cyclopropyl)morpholine (2d) At room temperature, thionyl chloride (84 mg, 0.71 mmol) was added to a solution of 2c (110 mg, 0.47 mmol) in dichloromethane (5 mL). The mixture was stirred at room temperature for 2 hours, and the solvent was removed under reduced pressure to give the target product 2d (100 mg, 20%). The product was used directly in the reaction of the next step without further purification. MS m / z (ESI): 252 [M+1]

[0088] Step 4 3-(4-((4-(1-Morpholinocyclopropyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (2) 2d (100 mg, 0.4 mmol), 1d (52 mg, 0.2 mmol), potassium iodide (66 mg, 0.4 mmol), potassium carbonate (55 mg, 0.4 mmol) and acetonitrile (5 mL) were mixed at room temperature. The mixture was heated to 100 °C, stirred for 18 hours, cooled to room temperature, and filtered. The filtrate was concentrated to dryness under reduced pressure. The residue was purified by reverse-phase preparative high performance liquid chromatography to give the target product 2 (5.2 mg, solid, 5.5%). MS m / z (ESI): 476 [M+1] TIFF0007712703000015.tif32156

[0089] Example 3 3-(4-((4-((R)-1-Morpholinoethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (Compound 3)

[0090] [Chemical Structure]

[0091] Step 1 (R)-4-(1-(4-Bromophenyl)ethyl)morpholine (3b) (R)-1-(4-Bromophenyl)ethylamine 3a (2 g, 10 mmol) was dissolved in DMF (40 mL), and then potassium carbonate (4.15 g, 30 mmol) and 1-bromo-2-(2-bromoethoxy)ethane (2.58 g, 11.1 mmol) were added. The mixture was stirred at room temperature for 3 hours, then heated to 80 °C and stirred for 3 hours. The mixture was cooled to room temperature, and 1-bromo-2-(2-bromoethoxy)ethane (258 mg, 1.11 mmol) was added. The mixture was heated to 80 °C and stirred for 2 hours. The mixture was cooled to room temperature, diluted with water (250 mL), and extracted with ethyl acetate (2 × 150 mL). The organic phases were combined, successively washed with saturated sodium bicarbonate solution (2 × 150 mL) and saturated brine (150 mL), and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 0 - 96 / 4) to give the target product 3b (2.14 g, 79%). MS m / z (ESI): 270 [M+1] TIFF0007712703000017.tif22156

[0092] Step 2 (R)-Methyl 4-(1-morpholinoethyl)benzoate (3c) 3b (2.14 g, 7.92 mmol) was dissolved in methanol (80 mL), and triethylamine (4 g, 39.6 mmol) was added. The mixture was cooled to -40 °C and bubbled with carbon monoxide gas for 30 minutes. Next, PdCl2(dppf) (1.16 g, 1.58 mmol) was added. The mixture was heated to 100 °C in a sealed tube and stirred for 20 hours. The mixture was cooled to room temperature and filtered. The filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 0 to 3 / 7) to give the target product 3c (1.7 g, 86%). MS m / z (ESI): 250 [M+1] TIFF0007712703000018.tif16156

[0093] Step 3 (R)-(4-(1-Morpholinoethyl)phenyl)methanol (3d) 3c (1.7 g, 6.82 mmol) was dissolved in anhydrous THF (40 mL) and cooled to 0 °C. Next, LAH (600 mg, 15.8 mmol) was added. The mixture was stirred at room temperature for 16 hours, cooled to 0 °C, and then quenched with NaOH solution (1 N, 8 mL). After filtration, the filtrate was dried over anhydrous sodium sulfate and then filtered again. The obtained filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 0 to 98 / 2) to give the target product 3d (1.45 g, 96%). MS m / z (ESI): 222 [M+1] TIFF0007712703000019.tif16156

[0094] Step 4 (R)-4-(1-(4-(Chloromethyl)phenyl)ethyl)morpholine (3e) 3d (98 mg, 0.44 mmol) was dissolved in dichloromethane (10 mL). Next, thionyl chloride (1 mL) was added. The mixture was stirred at room temperature for 2 hours and concentrated to dryness to produce the target product 3e (120 mg, crude product). Without further purification, the product was directly used in the next step reaction. MS m / z (ESI): 240 [M+1]

[0095] Step 5 3-(4-((4-((R)-1-Morpholinoethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione formate (3) 3e (120 mg, crude product), potassium carbonate (182 mg, 1.32 mmol), 1d (92 mg, 0.35 mmol), potassium iodide (73 mg, 0.44 mmol) and acetonitrile (10 mL) were mixed, heated to 80 °C and stirred for 3 hours. The mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was diluted with water (20 mL) and then extracted with ethyl acetate (2 × 20 mL). The organic phases were combined, dried over anhydrous sodium sulfate and filtered. The resulting filtrate was concentrated to dryness under reduced pressure. The residue was purified by reverse phase preparative high performance liquid chromatography to produce the target product 3 (34.5 mg, solid, 19%). MS m / z (ESI): 464 [M+1] TIFF0007712703000020.tif32156

[0096] Compound 4 was prepared according to the experimental procedures of Steps 1 to 5 of Compound 3, but in Step 1, (S)-1-(4-bromophenyl)ethylamine was used instead of 3a. [Table 2]

[0097] The NMR data of Compound 4 are as follows: [Table 3]

[0098] Example 4 (S)-3-(4-((4-(2-Morpholinopropan-2-yl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (Compound 5)

[0099] [Chemical formula]

[0100] Step 1 Methyl 3-hydroxy-2-methylbenzoate (5b) 3-Hydroxy-2-methylbenzoic acid 5a (5g, 33 mmol) was dissolved in methanol (50 mL). Next, concentrated sulfuric acid (980 mg, 9.9 mmol) was added. The mixture was heated to 70 °C, stirred for 20 hours, then cooled to room temperature and concentrated to about 10 mL. Next, the residue was slowly added to cold water (100 mL). Next, the resulting mixture was adjusted to pH 4 with saturated sodium bicarbonate solution, stirred for 20 minutes, and filtered to collect the precipitate to produce the target product 5b (4.48 g, 82%). MS m / z (ESI): 167 [M+1] TIFF0007712703000024.tif16156

[0101] Step 2 Methyl 3-((tert-butyldimethylsilyl)oxy)-2-methylbenzoate (5c) 5b (4.48 g, 27 mmol) was dissolved in DMF (25 mL). The mixture was cooled to 0 °C, and imidazole (4.59 g, 67 mmol) and tert-butyldimethylchlorosilane (4.47 g, 30 mmol) were added successively. The mixture was warmed to room temperature, stirred for 16 h, and then diluted with water (125 mL). The resulting mixture was extracted with ethyl acetate (2 × 100 mL). The organic phases were combined, washed with water (3 × 100 mL), and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 0 to 93 / 7) to give the target product 5c (7.57 g, 100%). MS m / z (ESI): 281[M+1] TIFF0007712703000025.tif22156

[0102] Step 3 Methyl 2-(bromomethyl)-3-((tert-butyldimethylsilyl)oxy)benzoate (5d) 5c (561 mg, 2 mmol) was dissolved in carbon tetrachloride (30 mL). N-Bromosuccinimide (374 mg, 2.1 mmol) and dibenzoyl peroxide (41 mg, 0.2 mmol) were added. The mixture was heated to 80 °C, stirred for 3 h, then cooled to room temperature and filtered. The filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 0 to 97 / 3) to give the target product 5d (630 mg, 88%). TIFF0007712703000026.tif22156

[0103] Step 4 Tert-butyl (S)-5-amino-4-(4-((tert-butyldimethylsilyl)oxy)-1-oxoisoindolin-2-yl)-5-oxopentanoate (5e) 5d (630 mg, 1.75 mmol) and tert-butyl (S)-4,5-diamino-5-oxopentanoate hydrochloride (460 mg, 1.93 mmol) were dissolved in acetonitrile (5 mL). Next, DIPEA (566 mg, 4.38 mmol) was added. The mixture was heated to 50 °C, stirred for 18 h, and cooled to room temperature. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 44 / 56 - 0 / 100) to give the target product 5e (545 mg, 69%). MS m / z (ESI): 449[M+1] TIFF0007712703000027.tif27156

[0104] Step 5 tert-butyl (S)-5-amino-4-(4-hydroxy-1-oxoisoindolin-2-yl)-5-oxopentanoate (5f) At room temperature, 5e (545 mg, 1.21 mmol) was dissolved in THF (3 mL), and a solution of tetrabutylammonium fluoride in THF (1 M, 1.5 mL, 1.5 mmol) was added. The mixture was stirred for 30 min, diluted with water (20 mL), and then extracted with ethyl acetate (3 × 20 mL). The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1 - 0 / 100) to give the target product 5f (270 mg, 67%). MS m / z (ESI): 335[M+1]

[0105] Step 6 4-(2-(4-bromophenyl)prop-2-yl)morpholine (5h) Dissolve 5g (214mg, 1mmol) of 2-(4-bromophenyl)propyl-2-amine in DMF (4mL), and add potassium carbonate (415mg, 3mmol) and 1-bromo-2-(2-bromoethoxy)ethane (278mg, 1.2mmol). Heat the mixture to 80 °C, stir for 20 hours, cool to room temperature, and dilute with water (80mL). Extract the mixture with ethyl acetate (3 × 40mL). Wash the organic phase successively with water (2 × 40mL) and brine (40mL), and dry over anhydrous sodium sulfate. After filtration, concentrate the filtrate to dryness under reduced pressure. Purify the residue by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 0 to 3 / 2) to give the target product 5h (207mg, 73%). MS m / z (ESI): 284 [M+1] TIFF0007712703000028.tif16156

[0106] Step 7 Methyl 4-(2-morpholinopropan-2-yl)benzoate (5i) Dissolve 5h (207mg, 0.73mmol) in methanol (20mL). Next, add triethylamine (369mg, 3.64mmol). Cool the mixture to -40 °C and bubble with carbon monoxide gas for 20 minutes. Then add PdCl2(dppf) (107mg, 0.15mmol). Heat the mixture in a sealed tube to 100 °C, stir for 20 hours, cool to room temperature, and filter through celite. Concentrate the filtrate to dryness under reduced pressure. Purify the residue by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 0 to 1 / 1) to give the target product 5i (189mg, 98%). MS m / z (ESI): 264 [M+1] TIFF0007712703000029.tif16156

[0107] Step 8 (4-(2-Morpholinopropan-2-yl)phenyl)methanol (5j) 5i (189 mg, 0.72 mmol) was dissolved in THF (20 mL) and cooled to 0 °C. Next, LAH (35 mg, 1.44 mmol) was added. The mixture was stirred at room temperature for 2 h, then quenched with NaOH solution (1 N, 0.2 mL) and filtered. The filtrate was dried over anhydrous sodium sulfate and filtered. The resulting filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1 - 3 / 7) to give the target product 5j (77 mg, 46%). MS m / z (ESI): 236 [M+1]

[0108] Step 9 4-(2-(4-(Chloromethyl)phenyl)prop-2-yl)morpholine (5k) 5j (77 mg, 0.33 mmol) was dissolved in dichloromethane (2 mL). Next, thionyl chloride (0.5 mL) was added. The mixture was stirred at room temperature for 16 h, then concentrated to dryness to give the target product 5k (solid, crude product). The product was used directly in the reaction of the next step without further purification. MS m / z (ESI): 254 [M+1]

[0109] Step 10 tert-Butyl (S)-5-amino-4-(4-((4-(2-morpholinopropan-2-yl)benzyl)oxy)-1-oxoisoindolin-2-yl)-5-oxopentanoate (5l) 5k (crude product, ca. 0.33 mmol) was dissolved in DMF (2 mL). Next, potassium carbonate (138 mg, 1 mmol) and 5f (110 mg, 0.33 mmol) were added. The mixture was heated to 50 °C and stirred for 24 h. The mixture was cooled to room temperature, then diluted with water (20 mL), and then extracted with ethyl acetate (3 × 20 mL). The organic phases were combined, washed with water (3 × 20 mL), and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 0 to 19 / 1) to give the target product 5l (163 mg, two steps: 90%). MS m / z (ESI): 552 [M+1]

[0110] Step 11 (S)-3-(4-((4-(2-Morpholinopropan-2-yl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione formate 5l (163 mg, 0.3 mmol) was dissolved in acetonitrile (2 mL), and TsOH·H2O (56 mg, 0.3 mmol) was added. The mixture was heated to 90 °C, stirred for 18 h, and cooled to room temperature. TsOH·H2O (56 mg, 0.3 mmol) was added again. Then, the mixture was heated to 90 °C and stirred for 2 h. After cooling the mixture at room temperature, saturated sodium bicarbonate solution (20 mL) was added. Then, the resulting mixture was extracted with ethyl acetate (3 × 20 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness under reduced pressure. The residue was purified by reversed-phase preparative high performance liquid chromatography to give the target product 5 (37 mg, solid, 26%). MS m / z (ESI): 478 [M+1] TIFF0007712703000030.tif32156

[0111] Example 5 4-(4-((R)-1-(4-(((2-((S)-2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)phenyl)ethyl)piperazin-1-yl)-3-fluorobenzonitrile (Compound 6)

[0112]

Chem.

[0113] Step 1 (R)-1-(1-(4-Bromophenyl)ethyl)-4-tosylpiperazine (6a) (R)-1-(4-Bromophenyl)ethylamine 3a (2 g, 10 mmol), N,N-bis(2-chloroethyl)-4-methylbenzenesulfonamide (3.11 g, 10.5 mmol) and DIPEA (2.58 g, 20 mmol) were placed in a 30 mL sealed tube, heated to 125 °C and stirred for 18 h. After cooling to room temperature, ethanol (80 mL) was added and water (120 mL) was slowly added dropwise while stirring the resulting mixture. The resulting mixture was stirred for 30 min, filtered and the precipitate was collected to produce the target product 6a (3.97 g, 94%). MS m / z (ESI): 423 [M+1] TIFF0007712703000032.tif22156

[0114] Step 2 (R)-1-(1-(4-Bromophenyl)ethyl)piperazine (6b) 6a (1 g, 2.36 mmol) was dissolved in trifluoroacetic acid (2.69 g, 23.6 mmol). Then concentrated sulfuric acid (1.62 g, 16.5 mmol) was added. The mixture was heated to 75 °C and stirred for 16 h. After cooling to room temperature, saturated sodium bicarbonate solution (100 mL) was slowly added and then the mixture was extracted with ethyl acetate (3 × 100 mL). The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure to produce the target product 6b (860 mg, crude product). The product was used directly in the reaction of the next step without further purification. MS m / z (ESI): 269 [M+1]

[0115] Step 3 (R)-4-(4-(1-(4-(Bromophenyl)ethyl)piperazin-1-yl)-3-fluorobenzonitrile (6c) 6b (860 mg, crude product, about 2.36 mmol) and 3,4-difluorobenzonitrile (328 mg, 2.36 mmol) were dissolved in DMF (15 mL). Next, potassium carbonate (978 mg, 7.08 mmol) was added. The mixture was heated to 10 °C and stirred for 16 h. After cooling to room temperature, water (150 mL) was added and the mixture was stirred for 1 h. The mixture was filtered to collect the precipitate, yielding the target product 6c (506 mg, 55%). MS m / z (ESI): 388 [M+1] TIFF0007712703000033.tif22156

[0116] Step 4 Methyl (R)-4-(1-(4-(4-cyano-2-fluorophenyl)piperazin-1-yl)ethyl)benzoate (6d) 6c (506 mg, 1.3 mmol) and methanol (40 mL) were mixed. Next, triethylamine (659 mg, 6.5 mmol) was added. The mixture was cooled to -40 °C and bubbled with carbon monoxide for 20 min. Then, PdCl2(dppf) (190 mg, 0.26 mmol) was added. The mixture was heated to 100 °C, stirred for 18 h, cooled to room temperature, and filtered through celite. The filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 0 to 1 / 1), yielding the target product 6d (360 mg, 75%). MS m / z (ESI): 368 [M+1] TIFF0007712703000034.tif27156

[0117] Step 5 (R)-3-Fluoro-4-(4-(1-(4-(hydroxymethyl)phenyl)ethyl)piperazin-1-yl)benzonitrile (6e) 6d (360 mg, 0.98 mmol) was dissolved in THF (20 mL), and the mixture was cooled to 0 °C. Next, LAH (45 mg, 1.18 mmol) was added. The mixture was stirred at room temperature for 2 hours, cooled to 0 °C, and quenched with NaOH solution (1 N, 1 mL). The reaction solution was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / ethyl acetate = 7 / 3 to 4 / 1) to give the target product 6e (100 mg, 30%). MS m / z (ESI): 340 [M+1]

[0118] Step 6 (R)-4-(4-(1-(4-(chloromethyl)phenyl)ethyl)piperazin-1-yl)-3-fluorobenzonitrile (6f) 6e (100 mg, 0.29 mmol) was dissolved in dichloromethane (5 mL). Next, thionyl chloride (69 mg, 0.58 mmol) was added. The mixture was stirred at room temperature for 16 hours and concentrated to dryness to give the target product 6f (an oily substance, crude product). The product was used directly in the next step reaction without further purification. MS m / z (ESI): 358 [M+1]

[0119] Step 7 Tert-butyl (S)-5-amino-4-(4-((4-((R)-1-(4-(4-cyano-2-fluorophenyl)piperazin-1-yl)ethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)-5-oxopentanoate (6g) 6f (crude product, ca. 0.29 mmol) was dissolved in DMF (5 mL). Next, 5f (97 mg, 0.29 mmol) and potassium carbonate (200 mg, 1.45 mmol) were added. The mixture was heated to 50 °C, stirred for 20 h, cooled to room temperature, and diluted with water (80 mL). The resulting mixture was filtered to collect the precipitate, yielding the target product 6g (197 mg, two steps: 104%). MS m / z (ESI): 656 [M+1]

[0120] Step 8 4-(4-((R)-1-(4-(((2-((S)-2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)phenyl)ethyl)piperazin-1-yl)-3-fluorobenzonitrile (6) 6g (197 mg, 0.29 mmol) was dissolved in acetonitrile (10 mL). Next, TsOH·H2O (110 mg, 0.58 mmol) was added. The mixture was heated to 90 °C, stirred for 3 h, cooled to room temperature, and diluted with saturated sodium bicarbonate solution (30 mL). Next, the resulting mixture was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by reverse-phase preparative high-performance liquid chromatography to yield the target product (21 mg, solid, 12%). MS m / z (ESI): 582 [M+1] TIFF0007712703000035.tif32156

[0121] Compounds 7, 8, and 9 were prepared according to the experimental procedures of Steps 1 to 8 for Compound 6, except that different compounds were used instead of (R)-1-(4-bromophenyl)ethylamine 3a in Step 1.

[0122]

Table 4

[0123] The NMR data of Compounds 7, 8 and 9 are as follows: [Table 5]

[0124] Example 6 2-(4-(((2-((S)-2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)phenyl)-2-morpholinoacetonitrile (Compound 10)

[0125] [Chemical formula]

[0126] Step 1 tert-Butyl (S)-5-amino-4-(4-((4-formylbenzyl)oxy)-1-oxoisoindolin-2-yl)-5-oxopentanoate (10a) 5f (167 mg, 0.5 mmol) and 4-(chloromethyl)benzaldehyde (77 mg, 0.5 mmol) were dissolved in DMF (3 mL). Next, potassium carbonate (207 mg, 1.5 mmol) was added. The mixture was heated to 50 °C, stirred for 18 hours, cooled to room temperature, and filtered. The filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 0 to 9 / 1) to give the target product 10a (164 mg, 72%). MS m / z (ESI): 453 [M+1]

[0127] Step 2 (S)-4-(((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)benzaldehyde (10b) 10a (164 mg, 0.36 mmol) was dissolved in acetonitrile (20 mL). Next, TsOH·H2O (138 mg, 0.72 mmol) was added. The mixture was heated to 90 °C, stirred for 18 h, and cooled to room temperature. The solvent was removed under reduced pressure. The residue was diluted with saturated sodium bicarbonate solution (20 mL) and then extracted with ethyl acetate (3 × 20 mL). The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 0 - 47 / 3) to give the target product 10b (48 mg, 35%). MS m / z (ESI): 379 [M+1]

[0128] Step 3 2-(4-(((2-((S)-2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)phenyl)-2-morpholinoacetonitrile (10) 10b (48 mg, 0.13 mmol) and dichloromethane (10 mL) were mixed. Next, morpholine (23 mg, 0.26 mmol), trimethylsilyl cyanide (26 mg, 0.26 mmol) and ytterbium(III) trifluoromethanesulfonate (8 mg, 0.013 mmol) were added. The mixture was stirred at room temperature for 61 h and then the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 0 - 91 / 9) and thin layer silica gel chromatography (dichloromethane / methanol = 8 / 1) to give the target product 10 (44.6 mg, solid, 72%). MS m / z (ESI): 475 [M+1] TIFF0007712703000039.tif32156

[0129] Example 7 (S)-3-(4-((4-((R)-1-(4-(Cyclopropanecarbonyl)piperazin-1-yl)ethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (Compound 11)

[0130]

Chem.

[0131] Project 1 (R)-(4-(1-(4-Bromophenyl)ethyl)piperazin-1-yl)(cyclopropyl)methanone (11a) 6b (200 mg, 0.75 mmol), cyclopropanecarbonyl chloride (93 mg, 0.89 mmol) and dichloromethane (5 mL) were mixed. Next, triethylamine (225 mg, 2.23 mmol) was added. The reaction mixture was stirred at room temperature overnight and then concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 19 / 1 - 4 / 1) to give the target product 11a (200 mg, 80%). MS m / z (ESI): 337 [M+1]

[0132] Project 2 Methyl (R)-4-(1-(4-(cyclopropylcarbonyl)piperazin-1-yl)ethyl)benzoate (11b) 11a (200 mg, 0.59 mmol), triethylamine (299 mg, 2.97 mmol) and methanol (20 mL) were mixed. The mixture was cooled to -40 °C and then bubbled with carbon monoxide for 30 minutes. Next, PdCl2(dppf) (43 mg, 0.06 mmol) was added. The mixture was heated to 100 °C in a sealed tube, stirred for 18 hours, and cooled to room temperature. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 9 / 1 - 3 / 2) to give the target product 11b (120 mg, 75%). MS m / z (ESI): 317 [M+1]

[0133] Project 3 (R)-Cyclopropyl(4-(1-(4-(hydroxymethyl)phenyl)ethyl)piperazin-1-yl)methanone (11c) A solution of 11b (120 mg, 0.38 mmol) in THF (5 mL) at 0 °C was added to LAH (22 mg, 0.57 mmol). The mixture was stirred for 1 hour, and then sodium sulfate decahydrate (100 mg) was added. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1 - 0 / 100) to give the target product 11c (100 mg, 59%). MS m / z (ESI): 289 [M+1]

[0134] Step 4 (R)-(4-(1-(4-(Chloromethyl)phenyl)ethyl)piperazin-1-yl)(cyclopropyl)methanone (11d) Thionyl chloride (0.5 mL) was added to a solution of 11c (100 mg, 0.35 mmol) in dichloromethane (5 mL). The reaction mixture was stirred overnight at room temperature, and then the solvent was removed under reduced pressure to give the target product 11d (100 mg, crude product). The product was used directly in the next step reaction without further purification. MS m / z (ESI): 307 [M+1]

[0135] Step 5 Tert-butyl (S)-5-amino-4-(4-((4-((R)-1-(4-(Cyclopropanecarbonyl)piperazin-1-yl)ethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)-5-oxopentanoate (11e) 11d (100 mg, crude product), 5f (101 mg, 0.30 mmol), potassium carbonate (83 mg, 0.60 mmol) and DMF (5 mL) were mixed. The mixture was heated to 50 °C, stirred overnight, cooled to room temperature, and diluted with water (10 mL). The resulting mixture was extracted with ethyl acetate (3 × 20 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The obtained filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 0 - 19 / 1) to give the target product 11e (100 mg, 53%). MS m / z (ESI): 605 [M+1]

[0136] Project 6 (S)-3-(4-((4-((R)-1-(4-(Cyclopropanecarbonyl)piperazin-1-yl)ethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (11) 11e (100 mg, 0.17 mmol), TsOH·H2O (66 mg, 0.35 mmol) and acetonitrile (2 mL) were mixed. The mixture was heated to 90 °C, stirred overnight and cooled to room temperature. The solvent was removed under reduced pressure. The residue was purified by reverse-phase preparative high-performance liquid chromatography to give the target product 11 (10 mg, solid, 22%). MS m / z (ESI): 531 [M+1] TIFF0007712703000041.tif32156

[0137] Example 8 (S)-3-(4-((4-((R)-1-(4-Cyclopropylpiperazin-1-yl)ethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (Compound 12)

[0138]

Chemical formula

[0139] Step 1 Tert-butyl (R)-4-(1-(4-bromophenyl)ethyl)piperazine-1-carboxylate (12a) 6b (p-toluenesulfonate, 4 g, 9 mmol) and THF (80 mL) were mixed. Next, triethylamine (3.7 g, 36 mmol) was added. The mixture was cooled to 0 °C, and di-tert-butyl dicarbonate (2.2 g, 9.9 mmol) was added dropwise. The mixture was stirred at room temperature for 17 hours, and water (160 mL) was added. Next, the resulting mixture was extracted with ethyl acetate (2 × 100 mL). The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was prepared by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 0 to 1 / 1) to produce the target product 12a (3.07 g, 92%). MS m / z (ESI): 369 [M+1]

[0140] Step 2 tert-Butyl (R)-4-(1-(4-(methoxycarbonyl)phenyl)ethyl)piperazine-1-carboxylate (12b) 12a (3.07 g, 8.3 mmol) was dissolved in methanol (120 mL) in a sealed tube, and triethylamine (4.21 g, 41.6 mmol) was added. The mixture was cooled to -40 °C and bubbled with carbon monoxide for 30 minutes. Next, PdCl2(dppf) (1.21 g, 1.66 mmol) was added. The mixture was heated to 100 °C, stirred for 18 hours, cooled to room temperature, and filtered through a celite pad. The filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 0 to 1 / 1) to produce the target product 12b (2.7 g, 93%). MS m / z (ESI): 349 [M+1]

[0141] Step 3 Methyl (R)-4-(1-(piperazin-1-yl)ethyl)benzoate (12c) To a solution of 12b (350 mg, 1.01 mmol) in dichloromethane (4 mL) was added trifluoroacetic acid (1 mL). The mixture was stirred at room temperature for 4 h, and the solvent was removed under reduced pressure to give the target product 12c (350 mg, crude product). The product was used directly in the next step reaction without further purification. MS m / z (ESI): 249 [M+1]

[0142] Step 4 Methyl (R)-4-(1-(4-(cyclopropylpiperazin-1-yl)ethyl)benzoate (12d) To a mixture of 12c (140 mg, 0.56 mmol), (1-ethoxycyclopropyloxy)trimethylsilane (295 mg, 1.69 mmol), acetic acid (100 mg, 1.69 mmol) and methanol (5 mL) was added sodium cyanoborohydride (106 mg, 1.69 mmol). The reaction mixture was stirred at room temperature overnight, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 9 / 1 - 7 / 3) to give the target product 12d (100 mg, 62%). MS m / z (ESI): 289 [M+1]

[0143] Step 5 (R)-(4-(1-(4-(Cyclopropylpiperazin-1-yl)ethyl)phenyl)methanol (12e) At 0 °C, to a solution of 12d (100 mg, 0.35 mmol) in THF (3 mL) was added LAH (20 mg, 0.52 mmol). The mixture was stirred for 1 h, and sodium sulfate decahydrate (100 mg) was added. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 99 / 1 - 19 / 1) to give the target product 12e (75 mg, 83%). MS m / z (ESI): 261 [M+1]

[0144] Step 6 (R)-1-(1-(4-(Chloromethyl)phenyl)ethyl)-4-cyclopropylpiperazine (12f) Thionyl chloride (1 mL) was added to a solution of 12e (80 mg, 0.31 mmol) in dichloromethane (5 mL). The mixture was stirred overnight and the solvent was removed under reduced pressure to give the title product 12f (80 mg, crude product). The product was used directly in the next step reaction without further purification. MS m / z (ESI): 279 [M+1]

[0145] Step 7 Tert-butyl (S)-5-amino-4-(4-((4-((R)-1-(4-cyclopropylpiperazin-1-yl)ethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)-5-oxopentanoate (12g) 12f (80 mg, crude product), 5f (101 mg, 0.30 mmol), potassium carbonate (83 mg, 0.60 mmol) and DMF (5 mL) were mixed. The mixture was heated to 50 °C, stirred overnight, cooled to room temperature and diluted with water (10 mL). Then the resulting mixture was extracted with ethyl acetate (3 × 20 mL). The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure to give the title product 12g (120 mg, crude product). The product was used directly in the next step reaction without further purification. MS m / z (ESI): 577 [M+1]

[0146] Step 8 (S)-3-(4-((4-((R)-1-(4-cyclopropylpiperazin-1-yl)ethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione formate (12) 12 g (120 mg, crude product), TsOH·H2O (95 mg, 0.50 mmol) and acetonitrile (2 mL) were mixed. The mixture was heated to 90 °C, stirred overnight, and cooled to room temperature. The solvent was removed under reduced pressure. The residue was purified by reverse-phase preparative high-performance liquid chromatography to give the target product 12 (10.0 mg, solid, 6.5%). MS m / z (ESI): 503 [M+1] TIFF0007712703000043.tif37156

[0147] Example 9 (S)-3-(4-((4-((R)-1-(4-(Methylsulfonyl)piperazin-1-yl)ethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (Compound 13)

[0148]

Chemical formula

[0149] Step 1 Methyl (R)-4-(1-(4-(Methylsulfonyl)piperazin-1-yl)ethyl)benzoate (13a) 12c (140 mg, 0.56 mmol), methanesulfonyl chloride (97 mg, 0.85 mmol), triethylamine (90 mg, 0.90 mmol) and dichloromethane (5 mL) were mixed. The mixture was stirred overnight and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 9 / 1 - 1 / 1) to give the target product 13a (100 mg, 54%). MS m / z (ESI): 327 [M+1]

[0150] Step 2 (R)-(4-(1-(4-(Methylsulfonyl)piperazin-1-yl)ethyl)phenyl)methanol (13b) A solution of 13a (100 mg, 0.31 mmol) in THF (3 mL) at 0 °C was added with LAH (18 mg, 0.46 mmol). The mixture was stirred for 1 hour, and sodium sulfate decahydrate (100 mg) was added. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 1% - 5%) to give the target product 13b (80 mg, 88%). MS m / z (ESI): 299 [M+1]

[0151] Step 3 (R)-1-(1-(4-(Chloromethyl)phenyl)ethyl)-4-(methylsulfonyl)piperazine (13c) Thionyl chloride (1 mL) was added to a solution of 13b (40 mg, 0.13 mmol) in dichloromethane (5 mL). The mixture was stirred overnight at room temperature, and the solvent was removed under reduced pressure to give the target product 13c (40 mg, crude product). The product was used directly in the reaction of the next step without further purification. MS m / z (ESI): 317 [M+1]

[0152] Step 4 Tert-butyl (S)-5-amino-4-(4-((4-((R)-1-(4-(methylsulfonyl)piperazine-1-yl)ethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)-5-oxopentanoate (13d) 13c (40 mg, crude product), 5f (101 mg, 0.30 mmol), potassium carbonate (83 mg, 0.60 mmol) and DMF (5 mL) were mixed. The mixture was heated to 50 °C, stirred overnight, cooled to room temperature and diluted with water (20 mL). Then, the mixture was extracted with ethyl acetate (3 × 20 mL). The organic phases were combined, dried over anhydrous sodium sulfate and filtered. The obtained filtrate was concentrated to dryness under reduced pressure to give the target product 13d (60 mg, crude product). The product was used directly in the reaction of the next step without further purification. MS m / z (ESI): 615 [M+1]

[0153] Project 5 (S)-3-(4-((4-((R)-1-(4-(Methylsulfonyl)piperazin-1-yl)ethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (13) 13d (60 mg, crude product), TsOH·H2O (95 mg, 0.50 mmol) and acetonitrile (2 mL) were mixed. The mixture was heated to 90 °C, stirred overnight, cooled to room temperature, and concentrated to dryness under reduced pressure. The residue was purified by reverse-phase preparative high-performance liquid chromatography to give the target product 13 (9.2 mg, solid, 13%). MS m / z (ESI): 541 [M+1] TIFF0007712703000045.tif32156

[0154] Example 10 (S)-3-(4-((4-((R)-1-(4-Methylpiperazin-1-yl)ethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (Compound 14)

[0155]

Chemical Structure

[0156] Step 1 (R)-1-(1-(4-(Bromophenyl)ethyl)-4-methylpiperazine (14a) 6b (200 mg, 0.75 mmol), aqueous formaldehyde solution (40%, 0.3 mL), acetic acid (122 mg, 2.0 mmol) and methanol (10 mL) were mixed. Next, sodium cyanoborohydride (127 mg, 2.0 mmol) was added. The mixture was stirred at room temperature overnight, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 3 / 2 - 1 / 4) to give the target product 14a (200 mg, 95%). MS m / z (ESI): 283 [M+1]

[0157] Steps 2 to 6 (S)-3-(4-((4-((R)-1-(4-Methylpiperazin-1-yl)ethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (14) Compound 14 was prepared according to the experimental procedures from Step 2 to Step 6 of Compound 11, but in Step 2, 14a was used instead of 11a. MS m / z (ESI): 283 [M+1] TIFF0007712703000047.tif32156

[0158] Example 11 (S)-3-(1-Oxo-4-((4-((R)-1-(4-(Pyrimidin-4-yl)piperazin-1-yl)ethyl)benzyl)oxy)isoindolin-2-yl)piperidine-2,6-dione (Compound 15)

[0159]

Chemical Structure

[0160] Step 1 Methyl (R)-4-(1-(4-(Pyrimidin-4-yl)piperazin-1-yl)ethyl)benzoate (15a) 12c (200 mg, 0.81 mmol), 4-Chloropyrimidine hydrochloride (243 mg, 1.61 mmol), potassium carbonate (334 mg, 2.42 mmol) and DMF (2 mL) were mixed. The mixture was heated to 110 °C, stirred overnight, cooled to room temperature, and diluted with water (10 mL). Next, the resulting mixture was extracted with ethyl acetate (3 × 20 mL). The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 9 / 1 to 1 / 1) to yield the target product 15a (100 mg, 38%). MS m / z (ESI): 327 [M+1]

[0161] Project 2 (R)-(4-(1-(4-(Pyrimidin-4-yl)piperazin-1-yl)ethyl)phenyl)methanol (15b) At 0 °C, LAH (18 mg, 0.46 mmol) was added to a solution of 15a (100 mg, 0.31 mmol) in THF (5 mL). The mixture was stirred for 1 hour, and then sodium sulfate decahydrate (100 mg) was added. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 99 / 1 to 19 / 1) to give the target product 15b (40 mg, 44%). MS m / z (ESI): 299 [M+1]

[0162] Project 3 (R)-4-(4-(1-(4-(Chloromethyl)phenyl)ethyl)piperazin-1-yl)pyrimidine (15c) Thionyl chloride (1 mL) was added to a solution of 15b (40 mg, 0.13 mmol) in dichloromethane (4 mL). The reaction mixture was stirred overnight and concentrated to dryness under reduced pressure to give the target product 15c (40 mg, crude product). The product was used directly in the next step reaction without further purification. MS m / z (ESI): 317 [M+1]

[0163] Project 4 Tert-butyl (S)-5-amino-5-oxo-4-(1-oxo-4-((4-((R)-1-(4-(pyrimidin-4-yl)piperazin-1-yl)ethyl)benzyl)oxy)isoindolin-2-yl)pentanoate (15d) 15c (40 mg, crude product), 5f (67 mg, 0.20 mmol), potassium carbonate (56 mg, 0.40 mmol) and DMF (1 mL) were mixed. The mixture was heated to 50 °C, stirred overnight, cooled to room temperature and diluted with water (10 mL). Next, the mixture was extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, dried over anhydrous sodium sulfate and filtered. The resulting filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 99 / 1 to 19 / 1) to give the target product 15d (40 mg, two steps: 49%). MS m / z (ESI): 615 [M+1]

[0164] Step 5 (S)-3-(1-Oxo-4-((4-((R)-1-(4-(pyrimidin-4-yl)piperazin-1-yl)ethyl)benzyl)oxy)isoindolin-2-yl)piperidine-2,6-dione (15) 15d (40 mg, 0.07 mmol), TsOH·H2O (37 mg, 0.20 mmol) and acetonitrile (1 mL) were mixed. The mixture was heated to 90 °C, stirred overnight, cooled to room temperature and concentrated to dryness under reduced pressure. The residue was purified by reverse-phase preparative high performance liquid chromatography to give the target product (20.0 mg, solid, 57%). MS m / z (ESI): 541 [M+1] TIFF0007712703000049.tif32156

[0165] Compounds 16 and 20 were prepared according to the experimental procedures of Steps 1 to 5 of Compound 15, but different compounds were used in Step 1 instead of 4-chloropyrimidine hydrochloride.

Table 6

[0166] The NMR data of Compounds 16 and 20 are as follows:

Table 7

[0167] Example 12 (S)-3-(4-((4-((R)-1-(4-(2,4-Difluorophenyl)piperazin-1-yl)ethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (Compound 17)

[0168] [Chemical formula]

[0169] Step 1 Methyl (R)-4-(1-(4-(2,4-difluorophenyl)piperazin-1-yl)ethyl)benzoate (17a) 12c (150 mg, 0.60 mmol), 1-bromo-2,4-difluorobenzene (232 mg, 1.20 mmol), potassium t-butoxide (202 mg, 1.80 mmol), tris(dibenzylideneacetone)dipalladium (35 mg, 0.06 mmol), XPhos (58 mg, 0.12 mmol) and toluene (2 mL) were mixed. Next, under a nitrogen gas atmosphere, the mixture was heated to 130 °C in a microwave reactor, stirred for 1 hour, and cooled to room temperature. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 9 / 1 to 1 / 1) to give the target product 17a (40 mg, 19%). MS m / z (ESI): 361 [M+1]

[0170] Steps 2 to 5 (S)-3-(4-((4-((R)-1-(4-(2,4-Difluorophenyl)piperazin-1-yl)ethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (17) Compound 17 was prepared according to the experimental procedures of Steps 2 to 5 of Compound 15, but in Step 2, 17a was used instead of 15a. MS m / z (ESI): 575 [M+1] TIFF0007712703000053.tif32156

[0171] Example 13 (3S)-3-(4-((4-(1-(2-Oxa-5-azabicyclo[2.2.1]heptan-5-yl)ethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (Compounds 18 and 19)

[0172] [Chemical Structure]

[0173] Step 1 Methyl 4-(1-(2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)ethyl)benzoate (18a) Methyl 4-(1-bromoethyl)benzoate 1e (243 mg, 1 mmol) and 2-oxa-5-azabicyclo[2.2.1]heptane hydrochloride (136 mg, 1 mmol) were dissolved in DMF (4 mL). Next, potassium carbonate (553 mg, 4 mmol) was added. The mixture was stirred at room temperature for 19 hours and diluted with water (50 mL). The resulting mixture was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, washed with water (3 × 30 mL), and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 0 to 4 / 1) to give the target product 18a (212 mg, 81%). MS m / z (ESI): 262 [M+1]

[0174] Step 2 (4-(1-(2-Oxa-5-azabicyclo[2.2.1]heptan-5-yl)ethyl)phenyl)methanol (18b) 18a (212 mg, 0.81 mmol) was dissolved in THF (5 mL). The mixture was cooled to 0 °C. Next, LAH (46 mg, 1.22 mmol) was added. The mixture was stirred at 0 °C for 30 minutes, then quenched with aqueous NaOH solution (2.5 N, 1 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 0 to 19 / 1) to give the target product 18b (158 mg, 84%). MS m / z (ESI): 234 [M+1]

[0175] Step 3 5-(1-(4-(Chloromethyl)phenyl)ethyl)-2-oxa-5-azabicyclo[2.2.1]heptane (18c) Thionyl chloride (161 mg, 1.35 mmol) was added to a solution of 18b (158 mg, 0.68 mmol) in dichloromethane (5 mL). The mixture was stirred at room temperature for 2 hours, then the solvent was removed under reduced pressure to give the target product 18c (crude product). The product was used directly in the next step reaction without further purification. MS m / z (ESI): 252 [M+1]

[0176] Step 4 Tert-butyl (4S)-4-(4-((4-(1-(2-oxa-5-azabicyclo[2.2.1]heptan-5-yl)ethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)-5-amino-5-oxopentanoate (18d) 18c (crude product, approximately 0.68 mmol), 5f (226 mg, 0.68 mmol), potassium carbonate (376 mg, 2.72 mmol) and DMF (5 mL) were mixed. The mixture was heated to 50 °C, stirred for 48 hours, cooled to room temperature and diluted with water (80 mL). Next, the mixture was filtered to collect the precipitate to give the target product 18d (280 mg, two steps: 75%). MS m / z (ESI): 550 [M+1]

[0177] Step 5 (3S)-3-(4-((4-(1-(2-Oxa-5-azabicyclo[2.2.1]heptan-5-yl)ethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (18 and 19) To a solution of 18d (280 mg, 0.51 mmol) in acetonitrile (30 mL) was added TsOH·H2O (194 mg, 1.02 mmol). The mixture was heated to 90 °C, stirred for 18 h, and cooled to room temperature. The solvent was removed under reduced pressure. Next, saturated sodium bicarbonate solution (50 mL) was added. The mixture was extracted with ethyl acetate (2 × 50 mL). The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by reverse-phase preparative high-performance liquid chromatography to give the title products 18 (isomer 1, 45 mg, solid, 19%) and 19 (isomer 2, 19 mg, solid, 8%).

[0178] The characteristic data of 18 were as follows: MS m / z (ESI): 476 [M+1] TIFF0007712703000055.tif37156

[0179] The characteristic data of 19 were as follows: MS m / z (ESI): 476 [M+1] TIFF0007712703000056.tif32156

[0180] Example 14 5-(4-((R)-1-(4-(((2-((S)-2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)phenyl)ethyl)piperazin-1-yl)-2-cyanopyridine (Compound 21)

[0181]

Chemical Structure

[0182] Step 1 tert-Butyl (R)-4-(1-(4-(hydroxymethyl)phenyl)ethyl)piperazine-1-carboxylate (21a) To a solution of 12b (2.7 g, 7.75 mmol) in THF (30 mL) at 0 °C was added LAH (353 mg, 9.30 mmol). The mixture was stirred at 0 °C for 1 h and quenched with water (0.5 mL). Next, NaOH solution (2.5 N, 4 mL) was added. The resulting mixture was diluted with THF (60 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure to give the title product 21a (2.75 g, crude product). 1.97 g of the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 0 to 1 / 4) to give the title product 21a (1.75 g). MS m / z (ESI): 321 [M+1]

[0183] Step 2 (R)-(4-(1-(Piperazin-1-yl)ethyl)phenyl)methanol dihydrochloride (21b) 21a (1.75 g, 5.46 mmol) was dissolved in 1,4-dioxane (20 mL). Next, hydrogen chloride in 1,4-dioxane (4 M, 20 mL) was added. The mixture was stirred at room temperature for 18 h and the solvent was removed under reduced pressure. The residue was purified by reverse-phase preparative high performance liquid chromatography to give the title product 21b (560 mg, 35%). MS m / z (ESI): 221 [M+1] TIFF0007712703000058.tif22156

[0184] Step 3 (R)-5-(4-(1-(4-(Hydroxymethyl)phenyl)ethyl)piperazin-1-yl)-2-cyanopyridine (21c) 21b (110 mg, 0.38 mmol), 2-cyano-5-fluoropyridine (51 mg, 0.42 mmol), cesium carbonate (371 mg, 1.14 mmol) and DMF (4 mL) were mixed. The mixture was heated to 60 °C, stirred for 2 h, and cooled to room temperature. The reaction mixture was filtered through celite. The filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 0 to 19 / 1) to give the target product 21c (24 mg, 52%). MS m / z (ESI): 323 [M+1]

[0185] Step 4 Tert-butyl (S)-5-amino-4-(4-((4-((R)-1-(4-(6-cyanopyridin-3-yl)piperazin-1-yl)ethyl)benzyl)oxy)-1-oxoisoindolin-2-yl)-5-oxopentanoate (21d) 21c (64 mg, 0.20 mmol) was dissolved in dichloromethane (10 mL). Then, thionyl chloride (48 mg, 0.40 mmol) was added. The mixture was stirred at room temperature for 2 h and concentrated to dryness under reduced pressure. The residue was dissolved in DMF (4 mL). Then, 5f (67 mg, 0.20 mmol) and potassium carbonate (138 mg, 1 mmol) were added. The mixture was heated to 50 °C, stirred for 20 h, cooled to room temperature, and filtered. The filtrate was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 100 / 0 to 19 / 1) to give the target product 21d (92 mg, 72%). MS m / z (ESI): 639 [M+1]

[0186] Step 5 5-(4-((R)-1-(4-(((2-((S)-2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)methyl)phenyl)ethyl)piperazin-1-yl)-2-cyanopyridine formate (21) 21d (156 mg, 0.29 mmol), TsOH·H2O (110 mg, 0.58 mmol) and acetonitrile (10 mL) were mixed. The mixture was heated to 90 °C, stirred for 20 hours, cooled to room temperature, and adjusted to pH = 8 with saturated sodium bicarbonate solution. Next, the resulting mixture was extracted with ethyl acetate (3 × 50 mL). The organic phases were combined, successively washed with saturated sodium bicarbonate solution (50 mL) and saturated brine (50 mL), and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to dryness under reduced pressure. The residue was purified by reverse-phase preparative high performance liquid chromatography to give the target product 22 (62 mg, solid, containing 0.65 equivalents of formic acid, 45%). MS m / z (ESI): 565 [M+1] TIFF0007712703000059.tif37156

[0187] Compound 22 was prepared according to the experimental procedures of steps 1 to 5 of compound 21, but in step 3, 6-chloro-5-fluoronicotinonitrile was used instead of 2-cyano-5-fluoropyridine.

[0188]

Table 8

[0189] The NMR data of compound 22 are as follows:

Table 9

[0190] Biological assay Example 15 Measurement of the inhibition of NCI-H929 cell proliferation The effect of the compounds of the present invention on the proliferation of NCI-H929 human myeloma cells was evaluated by a luminescence cell viability assay experiment. The experimental method was summarized as follows:

[0191] The compound was dissolved, diluted to 5 mM with DMSO, and then serially diluted 4-fold with DMSO to a minimum concentration of 0.31 μM. Each concentration was further diluted 50-fold with RPMI 1640 medium (Thermo Fisher, Cat. No. 72400-047). When the IC 50 value of the compound is relatively low, the initial concentration of the compound may be decreased.

[0192] NCI-H929 cells (Nanjing Cobioer, Cat. No. CBP60243) were cultured in RPMI 1640 complete medium [containing 10% FBS (GIBCO, Cat No. 10099-141) and 100 units / mL penicillin-streptomycin (Thermo Fisher, Cat No. 15140122)]. Cells (15000 cells / mL) were plated in 90 μL of complete medium in a 96-well plate and cultured overnight, and then 10 μL of the compound solution was added to each well. The cells were cultured in an incubator at 37 °C and 5% CO2 for 6 days. Next, according to the instructions of the CellTilter-Glo (CTG) kit (Promega, Cat.No.G7572), the cell culture plate was taken out and equilibrated to room temperature. 50 μL of the CTG reagent was added and dissolved completely, the plate was placed at room temperature for 10 minutes, and the luminescence signal was read with a microplate reader (EnVision, Perkin Elmer). The group containing 0.2% DMSO medium was set as 0% inhibition. Using XLfit software, the inhibition curve of the compound was plotted and the inhibitory IC 50 value was calculated. The experimental results are shown in Table 1.

[0193] Example 16 Measurement of the effects on the secretion of TNFα and IL-2 in human peripheral blood mononuclear cells The immunomodulatory function of the compound of the present invention was evaluated by detecting the effects on the secretion amounts of TNFα and IL-2 in human peripheral blood mononuclear cell (hPBMC) cells by enzyme-linked immunosorbent assay (ELISA).

[0194] The experimental method was summarized as follows: The peripheral blood of healthy volunteers was collected using EDTA anticoagulant tubes and diluted at an equal ratio with phosphate-buffered saline (PBS) containing 2% fetal bovine serum (Gibco, Cat No.10099-141). 30 mL of the obtained diluted sample was transferred to a Sepmate-50 centrifuge tube (Stemcell, Cat. No. 86450) to which 15 mL of a density gradient centrifugation solution (Sigma, Cat. No. 10771) had been previously added, and centrifuged at 1200×g for 10 minutes at room temperature. The liquid containing PBMC in the upper layer was transferred to a new 50 mL centrifuge tube and centrifuged at 300×g for 8 minutes at room temperature. The supernatant was discarded, and the obtained PBMC was resuspended at 5×10 6 / mL using RPMI1640 medium (Gibco, Cat. No. 72400-047). 80 μL of the obtained suspension was added to each well of a 96-well plate.

[0195] The compound was dissolved and diluted to 5 mM in DMSO (if the IC 50 value of the compound is relatively low, the initial concentration of the compound may be decreased), and then serially diluted 4-fold with DMSO to a minimum concentration of 0.31 μM. Each concentration was further diluted 50-fold with RPMI 1640 medium. 10 μL of each compound solution was added to the cells in the above 96-well plate. This 96-well plate was placed in an incubator and cultured at 37 °C and 5% CO2 for 1 hour. 10 μL of LPS (Sigma, Cat. No. L-2880) at a concentration of 100 ng / mL was added, and the cells were continuously cultured overnight in an incubator at 37 °C and 5% CO2. The supernatant was collected and TNFα was detected.

[0196] Also, the above 96-well plate was placed in an incubator and the cells were cultured at 37 °C and 5% CO2 for 1 hour. 10 μL of an anti-CD3 antibody (Thermo Fisher, Cat.No.14-0037-82) at a concentration of 500 ng / mL was added, and the cells were continuously cultured for 72 hours in an incubator at 37 °C and 5% CO2. The supernatant was collected and IL-2 was detected.

[0197] TNFα and IL-2 were detected according to the respective instructions of ELISA kits (R&D, Cat. No. DY210 and DY202 respectively), and the OD450 value of each well was obtained. Note that the group containing 0.2% DMSO medium was set as inhibition / stimulation 0%. Using XLfit software, the inhibition or stimulation curve of the compound was plotted, and the corresponding IC 50 or EC 50 was calculated. The experimental results are shown in Table 1.

[0198]

Table 10

[0199] Example 17 Measurement of hERG potassium ion channel blockade The effect of the compounds of the present invention on the potential for arrhythmia was evaluated by measuring the blockade of the hERG potassium ion channel.

[0200] The experimental method was summarized as follows.

[0201] Extracellular fluid: 140 mM NaCl, 3.5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM D-glucose, 10 mM HEPES, 1.25 mM NaH2PO4, pH = 7.4.

[0202] Electrode internal solution: 20 mM KCl, 115 mM K-aspartate, 1 mM MgCl2, 5 mM EGTA, 10 mM HEPES, 2 mM Na2-ATP, pH = 7.2.

[0203] Compound solution: The test compound was dissolved in DMSO to prepare a stock solution at a concentration of 10 mM, then diluted to a concentration of 3 mM with DMSO, and then diluted to a solution at a concentration of 3 μM with extracellular fluid and then used.

[0204] Cell culture: The HEK293 cell line (Creacell, Cat.No.A-0320) that stably overexpresses the hERG potassium channel was cultured in DMEM medium containing 10% fetal bovine serum (Gibco, Cat.No.1428478) and 0.8 mg / mL of G418 (Amresco, Cat.No.E859-5G) at a carbon dioxide concentration of 5% and a culture temperature of 37°C. The used medium was removed, and the cells were washed once with PBS (Gibco, Cat.No.1009-141). Next, 1 mL of TrypLE TM Express solution (Gibco, Cat.No.12604021) was added, and the cells were incubated at 37°C for 30 seconds. When the cells detached from the bottom of the dish, 5 mL of pre-warmed complete medium was added. The cell suspension was transferred to a sterile centrifuge tube, centrifuged at 1000 rpm for 5 minutes, and then the cells were collected. Cells were seeded in 6 cm cell culture dishes at a seeding density of 2.5*105 cells per dish (final volume: 5 mL). Before the patch clamp detection experiment, 3*10 3 cells were plated on cover slips, cultured in 24-well plates (final volume: 500 μL), and detected 18 hours later.

[0205] Voltage stimulation protocol for recording whole-cell hERG potassium current by whole-cell patch clamp: After forming a whole-cell seal, the cell membrane voltage was clamped at -80 mV. The clamp voltage was depolarized from -80 mV to -50 mV and maintained for 0.5 seconds (as a leak current detection), then stepped to 30 mV and maintained for 2.5 seconds, and then quickly returned to -50 mV and maintained for 4 seconds to excite the hERG channel tail current (peak tail current), and the hERG potassium current was recorded every 10 seconds. Experimental data were collected using an EPC-10 amplifier (HEKA) and saved in PatchMaster (HEKA v2x73) software.

[0206] Measurement: A capillary glass tube (Sutter Instruments) was drawn onto the recording electrode using a microelectrode puller (Sutter Instruments). The cover glass with cells was removed from a 24-well plate placed in an incubator and then placed under an inverted microscope. The recording electrode was filled with the intracellular solution, and then the microelectrode controller (Sutter Instruments) was operated to bring the recording electrode into contact with the cell surface. Negative pressure aspiration was performed to form a GΩ seal, followed by high-speed capacitance compensation. Then, negative pressure aspiration was continued until the cell membrane was ruptured by aspiration to complete the whole-cell recording mode. In the whole-cell recording mode, low-speed capacitance compensation was performed, and the membrane capacitance and series resistance were recorded, during which leak compensation was not applied. After the hERG tail current recorded in the whole cell was stable for 3 - 5 minutes, 8 mL of extracellular solution without compound (blank control) and 8 mL of a 3 μM solution of the test compound were sequentially flowed into the recording chamber by gravity perfusion and allowed to act on the cells for 5 minutes (or until the current was stable). The current detected in each cell with the extracellular solution without compound was used as its own control. Two to three cells were detected independently and repeatedly. All electrophysiological experiments were performed at room temperature.

[0207] Data analysis: First, the current with the test compound applied was normalized by the current of the blank control.

Number

Number

[0208]

Table 11

[0209] Example 18 In Vivo Pharmacokinetic Experiment in Rats The test substance was dissolved in a vehicle of 5% DMA + 20% Solutol + 75% saline to prepare a dosing solution of 0.5 mg / mL.

[0210] The dosing solution of 2 mL / kg was administered by intravenous injection at a dose of 1 mg / kg to each of three fed male Sprague-Dawley rats. Blood samples were collected at 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after administration. The dosing solution of 10 mL / kg was administered to another three fed male Sprague-Dawley rats by gavage (PO) at a dose of 5 mg / kg. Blood samples were collected at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after administration.

[0211] The concentration of the test compound in plasma was determined by LC-MS / MS quantitative analysis using an API-4500 mass spectrometer, and the limit of quantification (LOQ) in plasma was 1 ng / mL. Pharmacokinetic (PK) parameters were calculated using WinNonlin, and the results were summarized in Table 3.

[0212]

Table 12

Chemical

Chem.

Chem.

Chem.

Claims

1. General formula (I): 【Chemical 1】 [wherein, ring A is a nitrogen-containing 4- to 10-membered heterocycle; R 1 is D or halogen; R 2 and R 3 are each independently selected from H, D, halogen, cyano, C 1-6 alkyl, C 3-6 cycloalkyl or 3- to 8-membered heterocyclyl, provided that both are not simultaneously H and / or D, or R 2 and R 3 together with the carbon atom to which they are attached form a C 3-6 cycloalkane or 3- to 8-membered heterocycle; R 4 is D, halogen, cyano, oxo, C 1-6 alkyl, C 3-6 cycloalkyl, -OC 1-6 alkyl, -C(O)C 1-6 alkyl, -C(O)C 3-6 cycloalkyl, -S(O) 2 C 1-6 alkyl, -S(O) 2 C 3-6 cycloalkyl, aryl or heteroaryl, where one or more hydrogens of alkyl, cycloalkyl, aryl and heteroaryl may optionally be substituted by D, halogen, cyano, C 1-2 alkyl or fluoro C 1-2 alkyl; m is an integer from 0 to 4; and n is an integer from 0 to 2] A compound represented by the formula, or a pharmaceutically acceptable salt, stable isotope derivative, or stereoisomer thereof.

2. Ring A is a 4- to 10-membered monocyclic heterocycle containing 1 N atom, or a fused-, bridged- or spiro-bicyclic heterocycle; R 4 is D, halogen, oxo, -CF 3 or C 1-6 alkyl, a compound according to claim 1 or a pharmaceutically acceptable salt, stable isotope derivative, or stereoisomer thereof.

3. Ring A is a 6- to 10-membered monocyclic heterocycle containing two N atoms, or a fused-, bridged- or spiro-bicyclic heterocycle; R 4 is attached to the second N atom and is C 1-6 alkyl, C 3-6 cycloalkyl, -C(O)C 1-6 alkyl, -C(O)C 3-6 cycloalkyl, -S(O) 2 C 1-6 alkyl, -S(O) 2 C 3-6 cycloalkyl, phenyl, or a 5- or 6-membered heteroaryl containing N, O and / or S, where one or more hydrogens of alkyl, cycloalkyl, phenyl and heteroaryl may optionally be substituted by D, halogen, cyano, C 1-2 alkyl or fluoroC 1-2 alkyl; n is 0 or 1, the compound according to claim 1 or a pharmaceutically acceptable salt, stable isotope derivative, or stereoisomer thereof.

4. R 2 is H; R 3 is cyano, C 1-6 alkyl or C 3-6 cycloalkyl, a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt, stable isotope derivative, or stereoisomer thereof.

5. R 2 and R 3 wherein both are methyl, the compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, stable isotope derivative, or stereoisomer thereof.

6. R 2 and R 3 together with the carbon atom(s) to which they are attached form a C 3-6 cycloalkane, a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt, stable isotope derivative, or stereoisomer thereof.

7. The compound according to any one of Claims 1 to 6, or a pharmaceutically acceptable salt, stable isotope derivative, or stereoisomer thereof, wherein m is 0.

8. The compound according to any one of Claims 1 to 3, or a pharmaceutically acceptable salt, stable isotope derivative, or stereoisomer thereof, wherein ring A is morpholine, piperidine, thiomorpholine-1,1-dioxide or 2-oxa-5-azabicyclo[2.2.1]heptane.

9. The compound according to any one of Claims 1 to 3, or a pharmaceutically acceptable salt, stable isotope derivative, or stereoisomer thereof, wherein ring A is piperazine, 3,6-diazabicyclo[3.1.1]heptane or 2,6-diazaspiro[3.3]heptane.

10. General formula (II): 【Chemical 2】 [wherein, R 5 is H, C 1-6 alkyl, C 3-6 cycloalkyl, -C(O)C 1-6 alkyl, -C(O)C 3-6 cycloalkyl, -S(O) 2 C 1-6 alkyl, phenyl or 5- to 6-membered heteroaryl containing N, O and / or S, wherein one or more hydrogens of alkyl, cycloalkyl, phenyl and heteroaryl may optionally be substituted by D, halogen, cyano or C 1-2 alkyl which may be substituted] The compound according to Claim 1, or a pharmaceutically acceptable salt, stable isotope derivative, or stereoisomer thereof, represented by the formula.

11. The compound according to Claim 10, or a pharmaceutically acceptable salt, stable isotope derivative, or stereoisomer thereof, wherein R5 is phenyl, pyridinyl or pyrimidinyl, where one or two hydrogens of phenyl, pyridinyl and pyrimidinyl may optionally be substituted by F or cyano.

12. The compound according to Claim 1, or a pharmaceutically acceptable salt, stable isotope derivative, or stereoisomer thereof, wherein the compound is selected from 【Chemical Formula 3】 【Chemical Formula 4】

13. The compound according to Claim 12, wherein the compound is 【Chemical Formula 5】

14. A pharmaceutical composition comprising the compound according to any one of Claims 1 to 13, or a pharmaceutically acceptable salt, stable isotope derivative, or stereoisomer thereof, and a pharmaceutically acceptable carrier or adjuvant.

15. A pharmaceutical composition comprising a compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt, stable isotope derivative, or stereoisomer thereof, and at least one additional drug, wherein the at least one additional drug is selected from chemotherapeutic agents, immune and / or inflammation modulating substances, and substances modulating neurologically related diseases.

16. The pharmaceutical composition according to claim 14 or 15 for treating or preventing related diseases mediated by aiolos, ikaros, helios, CK1α, GSPT1, IL-2, IL-6, TNFα, IFNγ, VEGF, wherein the diseases are hematological tumors, solid tumors, autoimmune diseases, inflammation, neurodegenerative diseases, skin diseases.

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