Quinazolinone-based LSD1 inhibitors

Abstract

The invention provides an LSD1 inhibitor based on quinazolinone, the LSD1 inhibitor comprises a compound with the following structural formula (I) or (II) or pharmaceutically acceptable salt of the compound, # imgabs0 #, the LSD1 inhibitor has excellent LSD1 inhibition activity, a new structure and thought are provided for research and development of LSD1-targeted drugs, and meanwhile, a new choice is provided for treatment of LSD1-related diseases.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, in particular to a quinazolinone-based LSD1 inhibitor. Background Art

[0002] Lysine specific demthylase 1 (LSD1), also known as KIAA0601, KDM1, AOF2 or BHC110, is a flavin adenine dinucleotide (FAD)-dependent monoamine oxidase that can specifically remove mono- and di-methyl groups from histone H3 lysine 4 / 9 (H3K4 / 9). Studies have confirmed that abnormal expression of LSD1 is closely related to tumor metastasis and proliferation, and is currently one of the important targets for targeted tumor therapy. In addition, LSD1 is also involved in the occurrence and development of a variety of diseases such as neurodegenerative diseases, cardiovascular diseases, and inflammatory responses. Currently, several inhibitors have entered the clinical research stage.

[0003] According to the different binding modes and mechanisms of action of inhibitors with LSD1, the existing LSD1 inhibitors can be divided into two types: irreversible LSD1 inhibitors and reversible LSD1 inhibitors. Among them, irreversible LSD1 inhibitors may covalently bind to non-target proteins containing FAD structures, thereby increasing the risk of unexpected toxic reactions. Reversible LSD1 inhibitors bind to LSD1 in the form of hydrogen bonds, van der Waals forces, and hydrophobic forces. Reversible inhibitors are relatively more conducive to avoiding adverse reactions. Therefore, the development of new and highly efficient reversible LSD1 inhibitors is the research direction of LSD1 inhibitors. Summary of the invention

[0004] The invention provides a quinazolinone-based LSD1 inhibitor having excellent LSD1 inhibitory activity.

[0005] The technical solution of the present invention is achieved as follows: a quinazolinone-based LSD1 inhibitor, wherein the LSD1 inhibitor has a compound of the following structural formula (I) or (II) or a pharmaceutically acceptable salt thereof,

[0006]

[0007] in:

[0008] R 1 and R 2Independently selected from hydrogen, -OH, substituted or unsubstituted 5-10 membered heterocyclic group or -N(R), the heterocyclic group contains 1-3 heteroatoms of N, O or S, substituted or unsubstituted means that the corresponding functional group has or does not have a substituent, the substituent is C1-C6 alkyl, C1-C6 alkoxy, amino, C1-C6 alkylamino; in -N(R), each R is independently selected from hydrogen, C1-C7 fatty primary amine, C1-C7 fatty secondary amine, C1-C7 fatty tertiary amine;

[0009] W 1 and W 2 Independently selected from N, CH, CF, C-NH2, C-CH3;

[0010] R 3 Selected from substituted or unsubstituted 5-10 membered aryl, 5-10 membered heteroaryl, C3-C6 cycloalkyl, C1-C6 alkyl, 5-10 membered heterocyclic group, substituted or unsubstituted means that the corresponding functional group has or does not have a substituent, and the substituent is C1-C6 alkyl, C1-C6 alkoxy, halogen, halogenated C1-C6 alkyl, nitro, amino, 5-10 membered aryl.

[0011] Furthermore, R 1 and R 2 independently selected from substituted or unsubstituted 5-6 membered heterocyclic group or -N(R 4 )2, the heterocyclic group contains 1-2 N heteroatoms, and the substituent is C1-C6 alkyl, C1-C6 alkoxy, amino, C1-C6 alkylamino; -N(R 4 )2, each R 4 are independently selected from propylamine, N-methylpropylamine, N,N-dimethylpropylamine;

[0012] R 3 Selected from substituted or unsubstituted 5-10 membered aryl, 5-10 membered heteroaryl, C3-C6 cycloalkyl, C1-C6 alkyl, 5-10 membered heterocyclic group, the substituent is C1-C3 alkyl, C1-C3 alkoxy, halogen, halogenated C1-C3 alkyl, amino.

[0013] Furthermore, the R 1 and R 2 Independently selected from hydrogen, methyl, methoxy, hydroxyl, substituted or unsubstituted groups:

[0014]

[0015] W 1 and W 2 Independently selected from N, CH, CF, C-CH3;

[0016] Furthermore, R 3 is optionally substituted or unsubstituted 5-10 membered aryl or 5-10 membered heteroaryl, and the substituent is C1-C3 alkyl, C1-C3 alkoxy, halogen, halogenated C1-C3 alkyl, or amino.

[0017] Furthermore, R 3 is optionally substituted or unsubstituted as follows:

[0018]

[0019] The substituents are C1-C3 alkyl, C1-C3 alkoxy, halogen, halogenated C1-C3 alkyl, and amino.

[0020] The structure of the compound is selected from:

[0021] 4-(7-((S-3-aminopiperidin-1-yl)-3-(4-methoxyphenyl)-4-oxo-1,2,3,4-tetrahydroquinazolin-2-yl)benzonitrile (I-1)

[0022] 4-(7-((S)-3-aminopiperidin-1-yl)-4-oxo-3-(p-tolyl)-1,2,3,4-tetrahydroquinazolin-2-yl)benzonitrile (I-2)

[0023] (S)-4-(7-(3-aminopiperidin-1-yl)-3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)benzonitrile (II-3)

[0024] (S)-4-(7-(3-aminopiperidin-1-yl)-3-(4-methylphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)benzonitrile (II-4)

[0025] (S)-4-(7-(3-aminopiperidin-1-yl)-3-(4-fluorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)benzonitrile (II-5)

[0026] (S)-4-(7-(3-aminopyrrol-1-yl)-3-(4-fluorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)benzonitrile (II-6)

[0027] 4-(3-(4-methoxyphenyl)-4-oxo-7-(piperazin-1-yl)-1,2,3,4-tetrahydroquinazolin-2-yl)benzonitrile (I-7)

[0028] 4-(3-(4-Fluorophenyl)-4-oxo-7-(piperazin-1-yl)-1,2,3,4-tetrahydroquinazolin-2-yl)benzonitrile (I-8)

[0029] 4-(3-(4-methoxyphenyl)-4-oxo-7-(piperazin-1-yl)-3,4-dihydroquinazolin-2-yl)benzonitrile (II-9)

[0030] 4-(3-(4-Fluorophenyl)-4-oxo-7-(piperazin-1-yl)-3,4-dihydroquinazolin-2-yl)benzonitrile (II-10).

[0031] The molecular structural formulas of the above compounds are as follows:

[0032]

[0033]

[0034] Furthermore, the pharmaceutically acceptable salt of the compound is an acid addition salt formed by formula (I) or formula (II) and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

[0035] The preparation methods of some compounds of the present invention are as follows:

[0036]

[0037]

[0038] Beneficial effects of the present invention:

[0039] The present invention develops a novel LSD1 inhibitor, which provides a new structure and idea for the development of drugs targeting LSD1, and also provides a new option for the treatment of diseases related to LSD1. The results of pharmacological tests show that the compounds of the present invention have strong inhibitory activity against LSD1, and exhibit certain inhibitory activity against other kinases, and can be used to prevent or treat clinical diseases related to LSD1, which can be: leukemia, lymphoma (non-Hodgkin's lymphoma), Hodgkin's disease (also known as Hodgkin's lymphoma) and myeloma, for example, acute lymphocytic leukemia (ALL), acute granulocytic leukemia or acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic neutrophilic leukemia (CNL), acute undifferentiated cell leukemia (AUL), running developmental large cell lymphoma (ALCL), adult T cell ALL, with blue trilineage AML with myelodysplasia (AML / TMDS), mixed trilineage leukemia (MLL), myelodysplastic syndromes (MDSs), myelodysplasia (MPD), multiple myeloma (MM) and spinal cord sarcoma, lung cancer, melanoma, liver cancer, kidney cancer, leukemia, non-small cell lung cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, testicular cancer, breast cancer, bladder cancer, gallbladder cancer, myelodysplastic syndrome, lymphoma, esophageal cancer, thyroid follicular cancer, gastrointestinal cancer, tumors of the central or peripheral nervous system (such as astrocytoma, neuroblastoma, glioma or schwannoma), mesothelioma, type II or non-insulin-dependent diabetes mellitus, autoimmune diseases, psoriasis, etc. DETAILED DESCRIPTION

[0040] The specific implementation modes of the present invention are described in detail below in combination with embodiments and specific situations.

[0041] The present invention can be specifically implemented by the following examples.

[0042] Methyl 4-fluoro-2-nitrobenzoate (1a)

[0043] 4-Fluoro-2-nitrobenzoic acid (2 g, 10.80 mmol) was added to a 150 ml single-necked bottle, dissolved in anhydrous methanol (30 ml), and thionyl chloride (8 ml, 54.02 mmol) was slowly added dropwise under an ice bath. After the addition was completed, the temperature was raised to 70°C and heated in an oil bath for 3 h. The raw material disappeared after TLC detection (ethyl acetate: petroleum ether = 1:3). After the reaction liquid was cooled to room temperature, the solvent was evaporated under reduced pressure, an excess of saturated NaCl solution was added, and EA was used for extraction. The organic layer was collected, dried over anhydrous Na2SO4, filtered, and the solvent was evaporated under reduced pressure. Purification was performed by column chromatography, ethyl acetate: petroleum ether = 1:20 as the eluent, to obtain a colorless oily liquid 1a (2.12 g), with a yield of 98.5%, MS [M+H] + :200.02.

[0044] (S)-4-(3-((tert-Butyloxycarbonyl)amino)piperidin-1-yl)-2-nitrobenzoic acid methyl ester (2a)

[0045] 1a (2 g, 10.04 mmol) was added to a 150 ml single-necked flask, dissolved with DMF (30 ml), and (S)-piperidine-3-carbamic acid tert-butyl ester (4 g, 19.97 mmol) was added in batches. The reaction was allowed to react at room temperature for 8 h. The starting material disappeared after TLC detection (ethyl acetate: petroleum ether = 1:2). Water was added to the reaction solution until no more precipitation occurred. The filter cake was filtered to obtain a filter cake. The filter cake was purified by column chromatography using ethyl acetate: petroleum ether = 1:9 as the eluent to obtain solid 2a (3.60 g). The yield was 94.5%. MS [M+H] + :380.16.

[0046] (S)-4-(3-((tert-Butyloxycarbonyl)amino)pyrrol-1-yl)-2-nitrobenzoic acid methyl ester (2b)

[0047] Using 1a (2 g, 10.04 mmol) and (S)-pyrrole-3-carbamic acid tert-butyl ester (3.6 g, 19.32 mmol) as raw materials, the preparation method was the same as 2a to obtain solid 2b (3.55 g) with a yield of 95.2%. MS [M+H] + :366.15.

[0048] tert-Butyl 4-(4-(methoxycarbonyl)-3-nitrophenyl)piperazine-1-carboxylate (2c)

[0049] Using 1a (2 g, 10.04 mmol) and 1-Boc-piperazine (4.73 g, 25.11 mmol) as raw materials, the preparation method was the same as 2a to obtain solid 2c (3.46 g), with a yield of 94.2%, MS [M+H] + :366.15.

[0050] (S)-4-(3-((tert-Butyloxycarbonyl)amino)piperidin-1-yl)-2-nitrobenzoic acid (3a)

[0051] 2a (3 g, 7.91 mmol) was added to a 150 ml single-necked bottle, followed by THF (20 ml), methanol (10 ml), distilled water (10 ml), and KOH (2.2 g, 39.21 mmol). The reaction was heated in an oil bath at 70 °C for 0.5 h. The starting material disappeared after TLC detection (ethyl acetate: petroleum ether = 1:2). After the reaction solution was cooled to room temperature, the solvent was evaporated under reduced pressure. The residue was acidified with 2N HCl and filtered to obtain a filter cake. The product was used directly in the next step without further purification to obtain solid 3a (2.84 g) with a yield of 98.5%. MS [M+H] + :366.15.

[0052] (S)-4-(3-((tert-Butyloxycarbonyl)amino)pyrrol-1-yl)-2-nitrobenzoic acid (3b)

[0053] Using 2b (3 g, 8.21 mmol) as raw material, the preparation method was the same as 3a to obtain yellow solid 3b (2.87 g), with a yield of 99.5%, MS [M+H] + :352.13.

[0054] 4-(4-(tert-Butyloxycarbonyl)piperazin-1-yl)-2-nitrobenzoic acid (3c)

[0055] Using 2c (3 g, 8.21 mmol) as raw material, the preparation method was the same as 3a to obtain solid 3c (2.78 g), with a yield of 96.4%, MS [M+H] + :352.13.

[0056] (S)-tert-Butyl (1-(4-((4-methoxyphenyl)carbamoyl)-3-nitrophenyl)piperidin-3-yl)carbamate (4a)

[0057] 3a (1 g, 2.74 mmol) was added to a 50 ml single-necked flask and dissolved with DCE (10 ml). DMF (0.2 ml, 2.73 mmol) and (CClO)2 (0.46 ml, 5.47 mmol) were added under ice bath. The temperature was raised to room temperature for reaction for 5 min. The starting material disappeared by TLC (ethyl acetate: petroleum ether = 1:6). The reaction solution was spin-dried and redissolved with DCE (10 ml). A solution of p-methoxyaniline (402 mg, 3.26 mmol) and pyridine (0.6 ml, 6.84 mmol) dissolved in DCE (5 ml) was added under ice bath. The reaction was reacted at room temperature for 10 min. The starting material disappeared by TLC (ethyl acetate: petroleum ether = 1:3). EA (20 ml) was added for extraction. The organic layer was collected, dried over anhydrous Na2SO4, filtered, and the solvent was evaporated under reduced pressure. The solid 4a (839 mg) was obtained with a yield of 65.2%. MS [M+H] + :471.21.

[0058] (S)-tert-Butyl (1-(4-((4-methylphenyl)carbamoyl)-3-nitrophenyl)piperidin-3-yl)carbamate (4b)

[0059] Using 3b (1 g, 2.74 mmol) and p-methylaniline (880 mg, 8.22 mmol) as raw materials, the preparation method was the same as 4a to obtain solid 5b (651 mg) with a yield of 52.3%. MS [M+H] + :455.21.

[0060] (S)-tert-Butyl (1-(4-((4-fluorophenyl)carbamoyl)-3-nitrophenyl)piperidin-3-yl)carbamate (4c)

[0061] Using 3c (1 g, 2.74 mmol) and p-fluoroaniline (0.3 ml, 3.13 mmol) as raw materials, the preparation method was the same as 4a to obtain solid 4c (654 mg) with a yield of 51.7%. MS [M+H] + :459.19.

[0062] (S)-tert-Butyl (1-(3-amino-4-((4-methoxyphenyl)carbamoyl)phenyl)piperidin-3-yl)carbamate (5a)

[0063] 4a (800 mg, 1.70 mmol) was added to a 150 ml single-necked bottle and dissolved in methanol (50 ml). Then 10% Pd / C (80 mg) was added. Hydrogen was evacuated three times and the reaction was carried out at room temperature for 3 h. The starting material disappeared after TLC detection (ethyl acetate: petroleum ether = 1:2). The filtrate was filtered and the solvent was evaporated under reduced pressure. The product was purified by column chromatography using ethyl acetate: petroleum ether = 1:4 as the eluent to obtain a solid (564 mg). The yield was 75.4%. MS [M+H] + :441.23.

[0064] (S)-tert-Butyl (1-(3-amino-4-((4-methylphenyl)carbamoyl)phenyl)piperidin-3-yl)carbamate (5b)

[0065] Using 4b (600 mg, 1.32 mmol) as raw material, the preparation method was the same as 5a to obtain solid 5b (450 mg), with a yield of 80.1%, MS [M+H] + :425.24.

[0066] (S)-tert-Butyl (1-(3-amino-4-((4-fluorophenyl)carbamoyl)phenyl)piperidin-3-yl)carbamate (5c)

[0067] Using 4c (600 mg, 1.35 mmol) as raw material, the preparation method was the same as 5a to obtain solid 5c (462 mg), with a yield of 82.5%, MS [M+H] + :429.21.

[0068] Tert-butyl (3S)-1-(2-(4-cyanophenyl)-3-(4-methoxyphenyl)-4-oxo-1,2,3,4-tetrahydroquinazolin-7-yl)piperidin-3-yl)carbamate (6a)

[0069] 5a (500 mg, 1.14 mmol) was added to a 50 ml single-necked bottle and dissolved in methanol (20 ml). Then p-cyanobenzaldehyde (223 mg, 1.70 mmol) was added and the mixture was heated in an oil bath at 70°C for 1.5 h. The starting material disappeared after TLC detection (ethyl acetate: petroleum ether = 1:2). The reaction solution was poured into water and a precipitate was precipitated. The solid was obtained by suction filtration and purified by column chromatography using ethyl acetate: petroleum ether = 1:3 as the eluent to obtain a solid (529 mg). The yield was 84.2%. MS [M+H] + :554.26.

[0070] Tert-butyl (3S)-1-(2-(4-cyanophenyl)-3-(4-methylphenyl)-4-oxo-1,2,3,4-tetrahydroquinazolin-7-yl)piperidin-3-yl)carbamate (6b)

[0071] Using 5b (400 mg, 0.94 mmol) as raw material, the preparation method was the same as 6a to obtain solid 6b (405 mg), with a yield of 80.2%, MS [M+H] + :538.26.

[0072] Tert-butyl (3S)-1-(2-(4-cyanophenyl)-3-(4-fluorophenyl)-4-oxo-1,2,3,4-tetrahydroquinazolin-7-yl)piperidin-3-yl)carbamate (6c)

[0073] Using 5c ​​(400 mg, 0.96 mmol) as raw material, the preparation method was the same as 6a to obtain solid 6c (333 mg), with a yield of 65.4%, MS [M+H] + :542.24.

[0074] (S)-tert-Butyl (1-(2-(4-cyanophenyl)-3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-7-yl)piperidin-3-yl)carbamate (7a)

[0075] 6a (500 mg, 0.90 mmol) was added to a 50 ml single-necked bottle and dissolved in DCM (15 ml). DDQ (225 mg, 0.99 mmol) was then added and reacted at room temperature for 5 min. The starting material disappeared after TLC detection (ethyl acetate: petroleum ether = 1:1). The solvent was evaporated under reduced pressure, an excess of saturated NaCl solution was added, and the mixture was extracted with EA. The organic layer was collected, dried over anhydrous Na2SO4, and filtered. The solvent was evaporated from the filtrate under reduced pressure and purified by column chromatography using ethyl acetate: petroleum ether = 1:2 as the eluent to obtain solid 7a (424 mg). The yield was 85.2%. MS [M+H] + :552.24.

[0076] (S)-tert-Butyl (1-(2-(4-cyanophenyl)-3-(4-methylphenyl)-4-oxo-3,4-dihydroquinazolin-7-yl)piperidin-3-yl)carbamate (7b)

[0077] Using 6b (400 mg, 0.74 mmol) as raw material, the preparation method was the same as 7a to obtain solid 7b (234 mg), with a yield of 58.9%, MS [M+H] + :536.25.

[0078] (S)-tert-Butyl (1-(2-(4-cyanophenyl)-3-(4-fluorophenyl)-4-oxo-3,4-dihydroquinazolin-7-yl)piperidin-3-yl)carbamate (7c)

[0079] Using 6c (300 mg, 0.57 mmol) as raw material, the preparation method was the same as 7a to obtain solid 7c (183 mg), with a yield of 61.2%, MS [M+H] + :540.22.

[0080] Example 3: (S)-4-(7-(3-aminopiperidin-1-yl)-3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)benzonitrile (II-3)

[0081] 7a (400 mg, 0.72 mmol) was added to a 25 ml single-necked bottle, dissolved in anhydrous dichloromethane (15 ml), and CF3COOH (3.8 ml) was added dropwise. The reaction was allowed to react at room temperature for 1 h. The starting material disappeared after TLC detection (ethyl acetate: petroleum ether = 1:1). The pH was adjusted to 9 with saturated NaHCO3 solution, and the product was extracted with dichloromethane. The organic layer was collected and dried over anhydrous Na2SO4, filtered, and the solvent was evaporated from the filtrate under reduced pressure. The product was purified by column chromatography using methanol: dichloromethane = 1:40 as the eluent to obtain solid II-3 (172 mg). The yield was 52.6%, and the melting point was 145.5°C-148.3°C. 1 H NMR (500MHz, DMSO-d6): δ7.95(d,J=8.9Hz,1H,ArH),7.74(d,J=8.0Hz,2H,ArH),7.57( d,J=8.0Hz,2H,ArH),7.22(d,J=8.0Hz,3H,ArH),7.04(d,J=2.5Hz,1H,ArH),6.84(d,J =8.5Hz,2H,ArH),3.97–3.86(m,2H,-CH2-),3.71(s,3H,-OCH3),2.92(t,J=10.9Hz,1H ,-CH-),2.79–2.67(m,2H,-CH2-),1.92–1.70(m,2H,-CH2-),1.52–1.25(m,2H,-CH2-); 13 C NMR (126MHz, DMSO-d6): δ161.16,159.10,155.19,154.63,149.54,140.96,132.05,131.13,130.57,130.23,128. 20,118.80,115.61,114.25,111.72,110.72,108.86,55.89,55.73,47.68,47.43,33.66,23.55; HRMS(ESI)calcd for C 27 H 25 N5O2[M+H] + :452.2087.Found:452.2038.

[0082] Example 4: (S)-4-(7-(3-aminopiperidin-1-yl)-3-(4-methylphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)benzonitrile (II-4)

[0083] Using 7b (200 mg, 0.37 mmol) as raw material, the preparation method was the same as II-3 to obtain solid II-4 (73 mg) with a yield of 45.1% and a melting point of 160.1°C-163.7°C. 1 H NMR (500MHz, DMSO-d6): δ7.96 (d, J=9.0Hz, 1H, ArH), 7.73 (d, J=8.4Hz, 2H, ArH), 7.57 (d, J=8.4Hz, 2H,ArH),7.23(dd,J=9.1,2.6Hz,1H,ArH),7.18(d,J=8.4Hz,2H,ArH),7.11(d,J=8.1Hz,2H,ArH),7 .05(d,J=2.5Hz,1H,ArH),3.92(dd,J=19.4,3.4Hz,2H,-CH3),2.93(ddd,J=13.6,11.5,2.9Hz,1H, -CH-),2.82–2.69(m,2H,-CH2-),2.25(s,3H,-CH3),1.88(s,2H,-CH2-),1.74–1.47(m,2H,-CH2-); 13 C NMR (126MHz, DMSO-d6): δ161.01,155.19,154.42,149.52,140.86,138.08,135.41,128.21,118. 77,115.68,111.78,110.75,108.93,55.47,47.68,47.30,33.26,23.47,21.10; HRMS(ESI)calcd for C 27 H 25 N5O[M+H] + :436.2137.Found:436.2103.

[0084] Example 5: (S)-4-(7-(3-aminopiperidin-1-yl)-3-(4-fluorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)benzonitrile (II-5)

[0085] Using 7c (100 mg, 0.19 mmol) as raw material, the preparation method was the same as II-3 to obtain solid II-5 (40 mg) with a yield of 50% and a melting point of 198.7°C-200.2°C. 1H NMR (500MHz, Methanol-d4): δ8.06(d,J=9.1Hz,1H,ArH),7.64(d,J=8.3Hz,2H,ArH),7.57(d,J=8.4Hz,2H,ArH),7.27(dt,J=9.4,4.0Hz,3H,ArH),7.0 9–7.04(m,3H,ArH),3.98–3.84(m,2H,-CH2-),3.06–3.01(m,1H,-CH-),2.9 2–2.81(m,2H,-CH2-),2.05–1.85(m,2H,-CH2-),1.68–1.41(m,2H,-CH2-); 13 C NMR (126MHz, DMSO-d6): δ151.39,149.05,147.32,144.84,143.03,138.07,136.39,135.05,132.41,131.00,1 30.04,129.85,129.18,121.25,119.12,111.34,103.35,56.49,51.36,46.71,34.52,21.27; HRMS(ESI)calcd for C 26 H 22 FN5O[M+H] + :440.1887.Found:440.1831.

[0086] (S)-tert-Butyl (1-(4-((4-fluorophenyl)carbamoyl)-3-nitrophenyl)pyrrol-3-yl)carbamate (8a)

[0087] Using 3b (1 g, 2.85 mmol) and p-fluoroaniline (348 mg, 3.13 mmol) as raw materials, the preparation method was the same as 4a to obtain white solid 8a (816 mg) with a yield of 64.5%, MS [M+H] + :445.17.

[0088] Example 81: (S)-tert-butyl (1-(3-amino-4-((4-fluorophenyl)carbamoyl)phenyl)pyrrol-3-yl)carbamate (9a)

[0089] Using 8a (800 mg, 1.80 mmol) as raw material, the preparation method was the same as 5a to obtain yellow solid 9a (615 mg) with a yield of 82.5%, MS [M+H] + :415.20.

[0090] Tert-butyl (3S)-1-(2-(4-cyanophenyl)-3-(4-fluorophenyl)-4-oxo-1,2,3,4-tetrahydroquinazolin-7-yl)pyrrol-3-yl)carbamate (10a)

[0091] Using 9a (600 mg, 1.45 mmol) as raw material, the preparation method was the same as 6a to obtain solid 10a (569 mg) with a yield of 74.5%, MS [M+H] + :528.22.

[0092] (S)-tert-Butyl (1-(2-(4-cyanophenyl)-3-(4-fluorophenyl)-4-oxo-3,4-dihydroquinazolin-7-yl)pyrrol-3-yl)carbamate (11a)

[0093] Using 10a (500 mg, 0.95 mmol) as raw material, the preparation method was the same as 7a to obtain solid 11a (305 mg) with a yield of 61.4%, MS [M+H] + :526.21.

[0094] Example 6: (S)-4-(7-(3-aminopyrrol-1-yl)-3-(4-fluorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)benzonitrile (II-6)

[0095] Using 11a (300 mg, 0.57 mmol) as raw material, the preparation method was the same as II-1 to obtain solid II-6 (151 mg) with a yield of 62.4% and a melting point of 217.9°C-220.1°C. 1 H NMR (500MHz, DMSO-d6): δ7.96 (d, J=8.9Hz, 1H, ArH), 7.76 (d, J=7.8Hz, 2H, ArH), 7. 59(d,J=7.9Hz,2H,ArH),7.40(dd,J=8.6,4.8Hz,2H,ArH),7.15(t,J=8.5Hz,2H,ArH ),6.85(dd,J=8.9,2.5Hz,1H,ArH),6.63(d,J=2.4Hz,1H,ArH),3.65–3.38(m,4H,- CH2-×2),3.06(dd,J=10.1,4.6Hz,1H,-CH-),1.95(dd,J=153.2,6.2Hz,2H,-CH2-); 13C NMR (126MHz, DMSO-d6): δ162.70,161.16,160.75,154.14,152.56,149.23,140.75,134.45,134.42,132.34,132.27,132.1 1,130.30,128.25,118.75,116.02,115.84,113.75,111.84,109.10,106.10,56.33,51.22,46.58,34.39; HRMS(ESI)calcd forC 25 H 20 FN5O[M+H] + :426.1730.Found:426.1738.

[0096] tert-Butyl 4-(4-(4-methoxyphenyl)carbamoyl)-3-nitrophenyl)piperazine-1-carboxylate (12a)

[0097] Using 3c (1 g, 2.85 mmol) and p-methoxyaniline (421 mg, 3.42 mmol) as raw materials, the preparation method was the same as 4a to obtain white solid 12a (850 mg) with a yield of 65.4%, MS [M+H] + :457.19.

[0098] tert-Butyl 4-(4-(4-fluorophenyl)carbamoyl)-3-nitrophenyl)piperazine-1-carboxylate (12b)

[0099] Using 3c (1 g, 2.85 mmol) and p-fluoroaniline (348 mg, 3.13 mmol) as raw materials, the preparation method was the same as 4a to obtain white solid 12b (832 mg) with a yield of 65.8%, MS [M+H] + :445.17.

[0100] tert-Butyl 4-(3-amino-4-(4-methoxyphenyl)carbamoyl)phenyl)piperazine-1-carboxylate (13a)

[0101] 12a (800 mg, 1.75 mmol) was added to a 150 ml single-necked bottle, dissolved in 95% ethanol (40 ml), and then hydrazine hydrate (1.62 ml, 33.33 mmol) and FeO(OH) / C (285 mg) were added. The mixture was heated to reflux at 85°C for 8 h, and the raw material disappeared after TLC monitoring (methanol: dichloromethane = 1:10). FeO(OH) / C was removed by suction filtration, and the filtrate was dried by spin drying. The mixture was purified by column chromatography using methanol: dichloromethane = 1:30 as the eluent to obtain a white solid 13a (720 mg). The yield was 93.1%, and the MS [M+H] + :441.28.

[0102] tert-Butyl 4-(3-amino-4-(4-fluorophenyl)carbamoyl)phenyl)piperazine-1-carboxylate (13b)

[0103] Using 12b (800 mg, 1.80 mmol) as raw material, the preparation method was the same as 13a to obtain white solid 13b (622 mg) with a yield of 83.5%, MS [M+H] + :415.20.

[0104] 4-(2-(4-cyanophenyl)-3-(4-methoxyphenyl)-4-oxo-1,2,3,4-tetrahydroquinazolin-7-yl)piperazine-1-carboxylic acid tert-butyl ester (14a)

[0105] Using 13a (600 mg, 1.41 mmol) as starting material, the preparation method was the same as 6a to obtain white solid 14a (572 mg) with a yield of 75.4%, MS [M+H] + :540.24.

[0106] 4-(2-(4-cyanophenyl)-3-(4-fluorophenyl)-4-oxo-1,2,3,4-tetrahydroquinazolin-7-yl)piperazine-1-carboxylic acid tert-butyl ester (14b)

[0107] Using 13b (600 mg, 1.45 mmol) as raw material, the preparation method was the same as 6a to obtain white solid 14b (536 mg) with a yield of 70.2%. MS [M+H] + :528.22.

[0108] Example 7: 4-(3-(4-methoxyphenyl)-4-oxo-7-(piperazin-1-yl)-1,2,3,4-tetrahydroquinazolin-2-yl)benzonitrile (I-7)

[0109] Using 14a (300 mg, 0.55 mmol) as raw material, the preparation method was the same as II-1 to obtain white solid II-7 (132 mg) with a yield of 54.1% and a melting point of 189.5°C-192.1°C. 1H NMR (500MHz, DMSO-d6): δ7.80 (d, J=8.4Hz, 2H, ArH), 7.54 (dd, J=8.6, 5.4Hz, 3H, ArH), 7.4 9(d,J=3.0Hz,1H,ArH),7.15(d,J=9.0Hz,2H,ArH),6.88(d,J=9.0Hz,2H,ArH),6.39(dd,J =8.9,2.3Hz,1H,ArH),6.24(d,J=2.9Hz,1H,ArH),6.15(d,J=2.4Hz,1H,ArH),3.72(s,3H, -CH3),3.22(d,J=4.4Hz,4H,-CH2-×2),2.94(t,J=5.2Hz,4H,-CH2-×2),1.24(s,1H,-NH); 13 C NMR (126MHz, DMSO-d6): δ162.42,157.53,155.13,147.95,147.15,134.11,132.90,129.80,128.01,1 27.94,119.00,114.32,111.38,106.95,106.34,98.38,72.79,55.69,46.65,44.56; HRMS(ESI)calcd for C 26 H 25 N5O2[M+H] + :440.2087.Found:440.2039.

[0110] Example 8: 4-(3-(4-fluorophenyl)-4-oxo-7-(piperazin-1-yl)-1,2,3,4-tetrahydroquinazolin-2-yl)benzonitrile (I-8)

[0111] Using 14b (300 mg, 0.57 mmol) as raw material, the preparation method was the same as II-1 to obtain white solid II-7 (143 mg) with a yield of 58.7% and a melting point of 184.2°C-187.5°C. 1H NMR (500MHz, DMSO-d6): δ7.81(d,J=8.3Hz,2H,ArH),7.58–7.51(m,3H,ArH),7.49(d ,J=3.0Hz,1H,–NH-),7.27(dd,J=9.0,5.0Hz,2H,ArH),7.16(t,J=8.9Hz,2H,ArH),6. 39(dd,J=8.9,2.4Hz,1H,ArH),6.31(d,J=2.9Hz,1H,ArH),6.14(d,J=2.4Hz,1H,-CH -),3.16(q,J=4.5Hz,4H,-CH2-×2),2.85(t,J=5.1Hz,4H,-CH2-×2),1.24(s,1H-NH); 13 C NMR (126MHz, DMSO-d6): δ162.53,161.09,159.15,155.53,148.08,146.86,137.56,137.54,132.94,129.84,128.6 1,128.54,127.99,118.96,115.91,115.73,111.47,106.45,106.33,98.11,72.51,47.47,45.24; HRMS(ESI)calcd forC 25 H 22 FN5O[M+H] + :428.1887.Found:428.1829.

[0112] tert-Butyl 4-(2-(4-cyanophenyl)-3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-7-yl)piperazine-1-carboxylate (15a)

[0113] Using 14a (500 mg, 0.93 mmol) as starting material, the preparation method was the same as 7a to obtain white solid 15a (327 mg) with a yield of 65.7%, MS [M+H] + :538.23.

[0114] 4-(2-(4-cyanophenyl)-3-(4-fluorophenyl)-4-oxo-3,4-dihydroquinazolin-7-yl)piperazine-1-carboxylic acid tert-butyl ester (15b)

[0115] Using 14b (500 mg, 0.95 mmol) as raw material, the preparation method was the same as 7a to obtain white solid 15b (278 mg) with a yield of 55.8%, MS [M+H] + :526.21.

[0116] Example 9: 4-(3-(4-methoxyphenyl)-4-oxo-7-(piperazin-1-yl)-3,4-dihydroquinazolin-2-yl)benzonitrile (II-9)

[0117] Using 15a (300 mg, 0.56 mmol) as raw material, the preparation method was the same as II-1 to obtain solid II-9 (138 mg) with a yield of 56% and a melting point of 166.0°C-168.3°C. 1 H NMR (500MHz, DMSO-d6): δ7.98 (d, J=9.0Hz, 1H, ArH), 7.74 (d, J=8.4Hz, 2H, ArH), 7.57(d,J=8.4Hz,2H,ArH),7.27(dd,J=9.0,2.5Hz,1H,ArH),7.22(d,J=8.9Hz,2 H,ArH),7.07(d,J=2.5Hz,1H,ArH),6.84(d,J=8.9Hz,2H,ArH),3.71(s,3H,-OCH 3),3.43–3.39(m,4H,-CH2-×2)),3.00–2.89(m,4H,-CH2-×2)),1.23(s,1H,-NH); 13 C NMR (126MHz, DMSO-d6): δ161.23,159.13,155.75,154.70,149.34,140.89,132.06,131.12,130.51,13 0.23,128.06,118.78,115.91,114.27,111.76,111.74,109.48,55.73,47.36,44.90; HRMS(ESI)calcd for C 26 H 23 N5O2[M+H] + :438.1930.Found:438.1927.

[0118] Example 10: 4-(3-(4-fluorophenyl)-4-oxo-7-(piperazin-1-yl)-3,4-dihydroquinazolin-2-yl)benzonitrile (II-10)

[0119] Using 15b (200 mg, 0.38 mmol) as raw material, the preparation method is the same as II-1 to obtain solid II-10 (87 mg), with a yield of 53.7%, melting point: 189.3℃-192.1℃. Melting point: 189.3℃-192.1℃. 1H NMR (500MHz, DMSO-d6): δ7.98(d,J=9.0Hz,1H,ArH),7.76(d,J=8.1Hz,2H,ArH),7.58(d,J=8.0Hz,2H,ArH),7.40(dd,J=8.7,5.0Hz,2H,ArH),7.2 6(dd,J=9.1,2.5Hz,1H,ArH),7.16(t,J=8.8Hz,2H,ArH),7.05(d,J=2.5Hz,1H,ArH),3.33–3.18(m,4H,-CH2-×2),2.85(t,J=5.0Hz,4H,-CH2-×2); 13 CNMR (126MHz, DMSO-d6): δ162.75,161.06,160.80,156.19,154.23,149.34,140.63,134.34,132.29,132.21,13 2.13,130.28,128.00,118.73,116.09,115.90,115.80,111.90,111.28,109.20,48.38,45.71; HRMS(ESI)calcd for C 25 H 20 FN5O[M+H] + :426.1730.Found:426.1729.

[0120] For the structure determination of the above examples, the melting point was determined using a b-type melting point tube, the medium was methyl silicone oil, and the thermometer was not calibrated; 1 HNMR was measured using a JEOL FX90Q Fourier transform nuclear magnetic resonance instrument and a BRUKER nuclear magnetic resonance instrument (TMS internal standard); MS was measured using a Nicolet 2000 Fourier transform mass spectrometer and a MAT-212 mass spectrometer.

[0121] Pharmacological activity test

[0122] 1. Determination of the inhibitory activity of LSD1

[0123] The compound was prepared into a DMSO solution with a stock concentration of 20 mmol / L and stored in the dark for future use.

[0124] (1) First prepare 1× buffer;

[0125] (2) Compound concentration preparation: Compound test IC 50The final concentration was 10 μmol / L, 3-fold dilution, 8 concentrations, and each concentration was tested in a single well. The inhibition rate test concentration was 1 μmol / L, and the test was repeated. The positive control compound ORY-1001 test final concentration was 100 nmol / L, 3-fold dilution, 10 concentrations, and each concentration was tested in repeated wells. Diluted to the corresponding 1000-fold final concentration solution in the 384-well Source plate, and then transferred 10nl to the 384-well reaction plate for testing using Echo550. 10 nmol / L of 100% DMSO was transferred to the Min and Max wells.

[0126] (3) Prepare 2× enzyme solution using 1× reaction solution.

[0127] (4) Use 1× reaction solution to prepare 2× substrate mixed solution.

[0128] (5) Add 5 μl of 2× enzyme solution to each well; add 5 μl of 1× reaction solution to the Min well, centrifuge at 1000 rpm for 1 min, and incubate at room temperature for 15 min.

[0129] (6) Add 5 μl of 2× substrate mixture solution to each well of the reaction plate to start the reaction, centrifuge at 1000 rpm for 1 min, and incubate at room temperature for 60 min.

[0130] (7) Add 10 μl of detection solution to each well, centrifuge at 1000 rpm for 1 min, and incubate at room temperature for 60 min.

[0131] (8) Use EnVision to read the signal Intensity (665nm) / Intensity (615nm).

[0132] 2. Data Analysis

[0133] Calculation formula: Inhibition percentage (%) = (maximum value - signal value) / (maximum value - minimum value) × 100

[0134] Fitting the dose-effect curve: With the logarithm of the concentration as the X-axis and the percentage inhibition rate as the Y-axis, the log(inhibitor) vs.response-Variable slope formula of the analysis software GraphPad Prism 5 was used to fit the dose-effect curve, thereby obtaining the IC value of each compound on the enzyme activity. 50 value.

[0135] 3. MTS assay to detect cell activity

[0136] TS is a new MTT analogue, the full name is 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyph enyl)-2-(4-sulfopheny)-2H-tetrazolium, a yellow dye. Succinate dehydrogenase in the mitochondria of living cells can metabolize and reduce MTS to generate soluble formazan compounds, and the content of formazan can be measured at 490nm using an enzyme marker. Under normal circumstances, the amount of formazan generated is proportional to the number of living cells, so the number of living cells can be inferred based on the optical density OD value.

[0137] 4. Experimental Methods

[0138] (1) Inoculation of cells: Prepare a single cell suspension in a culture medium (DMEM or RMPI1640) containing 10% fetal bovine serum, and inoculate 3,000 to 15,000 cells per well into a 96-well plate. The volume of each well is 100 μl. The cells are inoculated and cultured 12 to 24 hours in advance. (2) Add the solution of the compound to be tested: Dissolve the compound in DMSO, and screen the compound at a concentration of 40 μmol / L. The final volume of each well is 200 μl. Three replicate wells are set for each treatment. The compound is screened at a concentration of 40, 8, 1.6, 0.32, 0.064, 0.0128, 0.00256, and 0.000512 μmol / L. The specific screening concentrations of each compound are detailed in the rescreening data. The final volume of each well is 200 μl. Three replicate wells are set for each treatment.

[0139] (3) Color development: After culturing at 37°C for 48 hours, discard the culture medium in the wells for adherent cells and add 20 μl of MTS solution and 100 μl of culture medium to each well; discard 100 μl of culture supernatant for suspended cells and add 20 μl of MTS solution to each well; set up 3 blank replicate wells (a mixture of 20 μl MTS solution and 100 μl culture medium) and continue incubating for 2 to 4 hours to allow the reaction to proceed fully before measuring the absorbance value.

[0140] (4) Colorimetry: Select 492 nm wavelength and use a multifunctional microplate reader (MULTISKAN FC) to read the light absorption value of each well and record the results. After data processing, draw a cell growth curve with concentration as the horizontal axis and cell survival rate as the vertical axis. Calculate the IC of the compound using the two-point method (Reed and Muench method) 50 value.

[0141] (5) Positive control compounds: Two positive control compounds, cisplatin (DDP) and paclitaxel (Taxol), were set in each experiment. The cell growth curve was drawn with the concentration as the horizontal axis and the cell survival rate as the vertical axis. The IC of the compound was calculated using the two-point method (Reed and Muench method). 50 value.

[0142] 5. Target compound activity test results

[0143] Based on the designed synthetic route, we synthesized 10 target compounds, using ORY-1001() as the positive control compound to test the inhibition rate of the target compounds on LSD1, and the IC values ​​of the compounds with better activity on LSD1 were measured. 50 The test results are as follows:

[0144] Table 1 Activity test results of target compounds against LSD1

[0145]

[0146]

[0147] a Inhibition rate at a compound concentration of 1 μmol / L; b A: inhibition rate>80%, B: 79%>inhibition rate>50%, C: 49%>inhibition rate; c The highest concentration of the compound was 10 μmol / L; d D:IC 50 <1μmol / L, E: 5μmol / L>IC 50 >1μmol / L, F:IC 50 >5μmol / L; ND not tested.

Claims

1. A quinazolinone-based LSD1 inhibitor characterized in that: The LSD1 inhibitor includes a compound having the following structural formula (I) or (II) or a pharmaceutically acceptable salt thereof, in: R 1 and R 2 Independently selected from hydrogen, -OH, substituted or unsubstituted 5-10 membered heterocyclic group or -N(R), the heterocyclic group contains 1-3 heteroatoms of N, O or S, substituted or unsubstituted means that the corresponding functional group has or does not have a substituent, the substituent is C1-C6 alkyl, C1-C6 alkoxy, amino, C1-C6 alkylamino; in -N(R), each R is independently selected from hydrogen, C1-C7 fatty primary amine, C1-C7 fatty secondary amine, C1-C7 fatty tertiary amine; W 1 and W 2 Independently selected from N, CH, CF, C-NH2, C-CH3; R 3 Selected from substituted or unsubstituted 5-10 membered aryl, 5-10 membered heteroaryl, C3-C6 cycloalkyl, C1-C6 alkyl, 5-10 membered heterocyclic group, substituted or unsubstituted means that the corresponding functional group has or does not have a substituent, and the substituent is C1-C6 alkyl, C1-C6 alkoxy, halogen, halogenated C1-C6 alkyl, nitro, amino, 5-10 membered aryl.

2. The quinazolinone-based LSD1 inhibitor according to claim 1, characterized in that: R 1 and R 2 independently selected from substituted or unsubstituted 5-6 membered heterocyclic group or -N(R 4 )2, the heterocyclic group contains 1-2 N heteroatoms, and the substituent is C1-C6 alkyl, C1-C6 alkoxy, amino, C1-C6 alkylamino; -N(R 4 )2, each R 4 are independently selected from propylamine, N-methylpropylamine, N,N-dimethylpropylamine; R 3 Selected from substituted or unsubstituted 5-10 membered aryl, 5-10 membered heteroaryl, C3-C6 cycloalkyl, C1-C6 alkyl, 5-10 membered heterocyclic group, the substituent is C1-C3 alkyl, C1-C3 alkoxy, halogen, halogenated C1-C3 alkyl, amino.

3. The quinazolinone-based LSD1 inhibitor according to claim 1, characterized in that: The R 1 and R 2 Independently selected from hydrogen, methyl, methoxy, hydroxyl, substituted or unsubstituted groups: W 1 and W 2 Independently selected from N, CH, CF, C-CH 3。 4. The quinazolinone-based LSD1 inhibitor according to claim 3, characterized in that: R 3 is optionally substituted or unsubstituted 5-10 membered aryl or 5-10 membered heteroaryl, and the substituent is C1-C3 alkyl, C1-C3 alkoxy, halogen, halogenated C1-C3 alkyl, or amino.

5. The quinazolinone-based LSD1 inhibitor according to claim 3, characterized in that: R 3 is optionally substituted or unsubstituted as follows: The substituents are C1-C3 alkyl, C1-C3 alkoxy, halogen, halogenated C1-C3 alkyl, and amino.

6. The quinazolinone-based LSD1 inhibitor according to claim 1, characterized in that The compound is selected from: 4-(7-((S-3-aminopiperidin-1-yl)-3-(4-methoxyphenyl)-4-oxo-1,2,3,4-tetrahydroquinazolin-2-yl)benzonitrile (I-1) 4-(7-((S)-3-aminopiperidin-1-yl)-4-oxo-3-(p-tolyl)-1,2,3,4-tetrahydroquinazolin-2-yl)benzonitrile (I-2) (S)-4-(7-(3-aminopiperidin-1-yl)-3-(4-methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)benzonitrile (II-3) (S)-4-(7-(3-aminopiperidin-1-yl)-3-(4-methylphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)benzonitrile (II-4) (S)-4-(7-(3-aminopiperidin-1-yl)-3-(4-fluorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)benzonitrile (II-5) (S)-4-(7-(3-aminopyrrol-1-yl)-3-(4-fluorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)benzonitrile (II-6) 4-(3-(4-methoxyphenyl)-4-oxo-7-(piperazin-1-yl)-1,2,3,4-tetrahydroquinazolin-2-yl)benzonitrile (I-7) 4-(3-(4-Fluorophenyl)-4-oxo-7-(piperazin-1-yl)-1,2,3,4-tetrahydroquinazolin-2-yl)benzonitrile (I-8) 4-(3-(4-methoxyphenyl)-4-oxo-7-(piperazin-1-yl)-3,4-dihydroquinazolin-2-yl)benzonitrile (II-9) 4-(3-(4-Fluorophenyl)-4-oxo-7-(piperazin-1-yl)-3,4-dihydroquinazolin-2-yl)benzonitrile (II-10).

7. The quinazolinone-based LSD1 inhibitor according to claim 1, characterized in that: The pharmaceutically acceptable salt of the compound is an acid addition salt formed by formula (I) or formula (II) and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

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