Pyrazole-1(2H)-phthalazinone compounds and their uses

Abstract

Provided are pyrazole-1(2H)-phthalazinone compounds and their use as PRMT5·MTA complex inhibitors in the preparation of medicaments for treating related diseases. JPEG2025502791000092.jpg5465

Description

[Technical field]

[0001] This invention claims priority to the following: CN202111616977.2, the filing date is December 27, 2021; CN202210062133.6, the filing date is January 19, 2022; CN202210142771.9, the filing date is February 16, 2022; CN202211213451.4, application date is September 29, 2022.

[0002] The present invention relates to a series of pyrazole-1(2H)-phthalazinone compounds and uses thereof, in particular to the compounds represented by formula (III) and pharma- ceutically acceptable salts thereof: [Background technology]

[0003] Protein arginine methyltransferase 5 (PRMT5), an epigenetic target with clinical potential, is a type II arginine methyltransferase that post-translationally modifies histones and other proteins by symmetric dimethylation, and current studies have demonstrated that PRMT5 expression levels are elevated in a variety of tumors.

[0004] The gene for the metabolic enzyme 5'-methylthioadenosine phosphorylase (MTAP) is very close to the tumor inhibitor gene CDKN2A on human chromosome 9p21, so tumors with CDKN2A gene deletions often have MTAP deletions, and MTAP is one of the most frequently mutated genes in tumors. MTAP deletion leads to accumulation of its substrate, methylthioadenosine (MTA), which can selectively inhibit the activity of PRMT5 methyltransferase and increase sensitivity to further PRMT5 inhibition. PRMT5 inhibitors could be a highly selective treatment for patients with tumors that have MTAP deletion or low expression.

[0005] In recent years, PRMT5 has attracted special attention as a potential target for tumor therapy. At present, a large number of PRMT5 small molecule inhibitors have been reported, which can be classified into two categories, SAM non-competitive inhibitors and SAM competitive inhibitors, according to whether the compounds occupy the SAM binding site or not, among which SAM non-competitive inhibitors occupy the substrate binding site and compete with the substrate, while SAM competitive inhibitors occupy the SAM binding site.

[0006] Although SAM-noncompetitive inhibitors can effectively inhibit the proliferation of many cell lines in vitro, they lack selectivity for tumor cell lines and their ability to inhibit tumor cells is independent of the intracellular MTAP status, which may lead to the risk of potential clinical toxicity. In MTAP-deficient cells, MTA accumulates and partially inhibits intracellular PRMT5, making tumor cells sensitive to PRMT5 inhibitors. Therefore, designing small molecules that bind synergistically to MTA and PRMT5 and specifically to the PRMT5·MTA complex to inhibit tumor cell proliferation is an effective means to improve the selectivity of PRMT5 inhibitors. Summary of the Invention [Means for solving the problem]

[0007] The present invention provides a compound represented by formula (III) or a pharma- ceutically acceptable salt thereof. [ka]

[0008] however, R1 is C 2-5 Alkenyl, C 2-5 Alkynyl, -OC 3-5 Cycloalkyl, -OC 2-3 Alkynyl and C 3-5 cycloalkyl, 2-5 Alkenyl, C 2-5 Alkynyl, -OC 3-5 Cycloalkyl, -OC 2-3 Alkynyl and C 3-5 Cycloalkyl, each independently, optionally has 1, 2 or 3 R a is replaced by R2 is a halogen or C 2-3 alkynyl, 2-3 The alkynyl is optionally substituted by 1, 2 or 3 halogens; R3 is selected from CH3 and CD3; R4 is selected from H and halogen; Each R a each independently represents a halogen and C 1-3 alkyl, 1-3 The alkyl is optionally substituted with 1, 2 or 3 halogens; and, (1) When R2 is selected from halogen, R1 is C 2-5 Alkenyl, C 2-5 Alkynyl, -OC 3-5 Cycloalkyl, -OC 2-3 Alkynyl and C 3-5 cycloalkyl, 2-5 Alkenyl, C 2-5 Alkynyl and -OC 2-3 Alkynyl optionally has one, two or three R a and -OC3-5 Cycloalkyl and C 3-5 Cycloalkyl can have one, two or three R a is replaced by (2) when R2 is selected from halogen, R3 is selected from CD3; (3) At least one of the following conditions is satisfied: when R2 is selected from halogen, R4 is selected from halogen.

[0009] In some embodiments of the present invention, the above R a are each independently selected from F, Cl, CH3, CHF2, and CF3, and all other variables are as defined herein.

[0010] In some embodiments of the present invention, R1 is C 2-3 Alkenyl, C 2-3 Alkynyl, -O-cyclopropyl, -OC 2-3 alkynyl and cyclopropyl; 2-3 Alkenyl, C 2-3 Alkynyl, -O-cyclopropyl, -OC 2-3 Alkynyl and cyclopropyl each independently optionally have 1, 2 or 3 R a and other variables are as defined herein.

[0011] In some embodiments of the present invention, R1 above is [ka] and [ka] each independently optionally represents 1, 2 or 3 R a and other variables are as defined herein.

[0012] In some embodiments of the present invention, R1 above is [ka] and the other variables are as defined herein.

[0013] In some embodiments of the present invention, R2 above is selected from the group consisting of F, Cl and [ka] and [ka] is optionally substituted with 1, 2 or 3 halogens, and the other variables are as defined herein.

[0014] In some embodiments of the present invention, R2 above is Cl, [ka] and the other variables are as defined herein.

[0015] In some embodiments of the present invention, R2 above is selected from Cl and R1 is [ka] and the other variables are as defined herein.

[0016] In some embodiments of the invention, R2 above is selected from Cl and R3 is selected from CD3, with the other variables being as defined herein.

[0017] In some embodiments of the present invention, R3 above is selected from CD3, with other variables as defined herein.

[0018] In some embodiments of the present invention, R4 above is selected from H and F, with other variables as defined herein.

[0019] In some embodiments of the invention, the compound or a pharma- ceutically acceptable salt thereof is selected from the following formulas:

[0020] [ka]

[0021] However, R2, R3 and R a is as defined in the present invention.

[0022] In some embodiments of the invention, the compound or a pharma- ceutically acceptable salt thereof is selected from the following formulas:

[0023] [ka]

[0024] However, R3 and R a is as defined in the present invention.

[0025] In some embodiments of the invention, the compound or a pharma- ceutically acceptable salt thereof is selected from the following formulas:

[0026] [ka]

[0027] However, R a is as defined in the present invention.

[0028] In some embodiments of the invention, the compound or a pharma- ceutically acceptable salt thereof is selected from the following formulas:

[0029] [ka]

[0030] However, R a is as defined in the present invention.

[0031] The present invention further provides a compound represented by formula (III) or a pharma- ceutically acceptable salt thereof.

[0032] [ka]

[0033] however, R1 is C 2-5 Alkenyl, C 2-5 Alkynyl, -OC 3-5 Cycloalkyl, -OC 2-3 Alkynyl and C 3-5 cycloalkyl, 2-5 Alkenyl, C 2-5 Alkynyl, -OC 3-5 Cycloalkyl, -OC 2-3 Alkynyl and C 3-5 Cycloalkyl, each independently, optionally has 1, 2 or 3 R a is replaced by R2 is a halogen or C 2-3 alkynyl, 2-3 The alkynyl is optionally substituted by 1, 2 or 3 halogens; R3 is selected from CH3 and CD3; R4 is selected from H and halogen; Each R a each independently represents a halogen and C 1-3 alkyl, and, 1) When R2 is selected from halogen, R1 is C 2-5 Alkenyl, C 2-5 Alkynyl, -OC 3-5 Cycloalkyl, -OC 2-3 Alkynyl and C 3-5cycloalkyl, 2-5 Alkenyl, C 2-5 Alkynyl and -OC 2-3 Alkynyl optionally has one, two or three R a and -OC 3-5 Cycloalkyl and C 3-5 Cycloalkyl can have one, two or three R a or 2) when R2 is selected from halogen, R3 is selected from CD3; 3) When R2 is selected from halogen, R4 is selected from halogen. At least one of the following conditions is satisfied.

[0034] In some embodiments of the present invention, R a are each independently selected from F, Cl, CH3, CHF2, and CF3, and all other variables are as defined herein.

[0035] In some embodiments of the present invention, R is C 2-3 Alkenyl, C 2-3 Alkynyl, -O-cyclopropyl, -OC 2-3 alkynyl and cyclopropyl; 2-3 Alkenyl, C 2-3 Alkynyl, -O-cyclopropyl, -OC 2-3 Alkynyl and cyclopropyl each independently optionally have 1, 2 or 3 R a and other variables are as defined herein.

[0036] In some embodiments of the present invention, R1 is [ka] and [ka] each independently optionally represents 1, 2 or 3 Ra and other variables are as defined herein.

[0037] In some embodiments of the present invention, R1 is [ka] and the other variables are as defined herein.

[0038] In some embodiments of the present invention, R2 is selected from the group consisting of F, Cl and [ka] and [ka] is optionally substituted with 1, 2 or 3 halogens, and the other variables are as defined herein.

[0039] In some embodiments of the present invention, R2 is Cl, [ka] and the other variables are as defined herein.

[0040] In some embodiments of the invention, R3 is selected from CD3, and other variables are as defined herein.

[0041] In some embodiments of the present invention, R4 is selected from H and F, and other variables are as defined herein.

[0042] The present invention further provides a compound represented by formula (II) or a pharma- ceutically acceptable salt thereof.

[0043] [ka]

[0044] however, R1 is C 2-5 Alkenyl, C 2-5 Alkynyl, -OC 3-5 Cycloalkyl, -OC 2-3 Alkynyl and C 3-5 cycloalkyl, 2-5 Alkenyl, C 2-5 Alkynyl, -OC 3-5 Cycloalkyl, -OC 2-3 Alkynyl and C 3-5 Cycloalkyl, each independently, optionally has 1, 2 or 3 R a is replaced by R2 is a halogen or C 2-3 alkynyl, 2-3 The alkynyl is optionally substituted by 1, 2 or 3 halogens; R3 is selected from CH3 and CD3; Each R a each independently represents a halogen and C 1-3 alkyl, The conditions are: 1) When R2 is selected from halogen, R1 is C 2-5 Alkenyl, C 2-5 Alkynyl, -OC 3-5 Cycloalkyl, -OC 2-3 Alkynyl and C 3-5 cycloalkyl, 2-5 Alkenyl, C 2-5 Alkynyl and -OC 2-3 Alkynyl optionally has one, two or three R a and -OC 3-5 Cycloalkyl and C 3-5 Cycloalkyl can have one, two or three R a or 2) When R2 is selected from halogen, R3 is selected from CD3.

[0045] In some embodiments of the present invention, R a are each independently selected from F, Cl, CH3, CHF2, and CF3, and all other variables are as defined herein.

[0046] In some embodiments of the present invention, R is C 2-3 Alkenyl, C 2-3 Alkynyl, -O-cyclopropyl, -OC 2-3 alkynyl and cyclopropyl; 2-3 Alkenyl, C 2-3 Alkynyl, -O-cyclopropyl, -OC 2-3 Alkynyl and cyclopropyl each independently optionally have 1, 2 or 3 R a and other variables are as defined herein.

[0047] In some embodiments of the present invention, R1 is [ka] and [ka] each independently optionally represents 1, 2 or 3 R a and other variables are as defined herein.

[0048] In some embodiments of the present invention, R1 is [ka] and the other variables are as defined herein.

[0049] In some embodiments of the present invention, R2 is selected from the group consisting of F, Cl and [ka] and [ka] is optionally substituted with 1, 2 or 3 halogens, and the other variables are as defined herein.

[0050] In some embodiments of the present invention, R2 is Cl, [ka] and the other variables are as defined herein.

[0051] The present invention further provides a compound of formula (I) or a pharma- ceutically acceptable salt thereof.

[0052] [ka]

[0053] however, R1 is C 2-5 Alkenyl, C 2-5 Alkynyl, -OC 3-5 Cycloalkyl, -OC 2-3 Alkynyl and C 3-5 cycloalkyl, 2-5 Alkenyl, C 2-5 Alkynyl, -OC 3-5 Cycloalkyl, -OC 2-3 Alkynyl and C 3-5 Cycloalkyl, each independently, optionally has 1, 2 or 3 R a is replaced by R2 is a halogen or C 2-3 alkynyl, 2-3 The alkynyl is optionally substituted by 1, 2 or 3 halogens; Each R a each independently represents a halogen and C1-3 alkyl, The conditions are: When R2 is selected from halogen, R1 is selected from C 2-5 Alkenyl, C 2-5 Alkynyl, -OC 3-5 Cycloalkyl, -OC 2-3 Alkynyl and C 3-5 cycloalkyl, 2-5 Alkenyl, C 2-5 Alkynyl and -OC 2-3 Alkynyl optionally has one, two or three R a and -OC 3-5 Cycloalkyl and C 3-5 Cycloalkyl can have one, two or three R a is replaced by

[0054] In some embodiments of the present invention, R a are each independently selected from F, Cl, CH3, CHF2, and CF3, and all other variables are as defined herein.

[0055] In some embodiments of the present invention, R is C 2-3 Alkenyl, C 2-3 Alkynyl, -O-cyclopropyl, -OC 2-3 alkynyl and cyclopropyl; 2-3 Alkenyl, C 2-3 Alkynyl, -O-cyclopropyl, -OC 2-3 Alkynyl and cyclopropyl each independently optionally have 1, 2 or 3 R a and other variables are as defined herein.

[0056] In some embodiments of the present invention, R1 is [ka] and [ka] each independently optionally represents 1, 2 or 3 R a and other variables are as defined herein.

[0057] In some embodiments of the present invention, R1 is [ka] and the other variables are as defined herein.

[0058] In some embodiments of the present invention, R2 is selected from the group consisting of F, Cl and [ka] and [ka] is optionally substituted with 1, 2 or 3 halogens, and the other variables are as defined herein.

[0059] In some embodiments of the present invention, R2 is Cl, [ka] and the other variables are as defined herein.

[0060] Further embodiments of the present invention are formed by any combination of the above variables.

[0061] The present invention further provides a compound represented by the formula: or a pharma- ceutically acceptable salt thereof.

[0062] [ka]

[0063] In some embodiments of the invention, the compound or a pharma- ceutically acceptable salt thereof is selected from the following formulas:

[0064] [ka]

[0065] [ka]

[0066] [ka]

[0067] [ka]

[0068] The present invention further provides the use of the above compound or a pharma- ceutically acceptable salt thereof in medicine relating to a PRMT5 inhibitor.

[0069] The present invention further provides the use of the above compound or a pharma- ceutically acceptable salt thereof in a medicine related to a PRMT5·MTA complex inhibitor.

[0070] The present invention further provides the following synthetic methods.

[0071] Method 1 [ka]

[0072] Method 2 [ka]

[0073] Method 3 [ka]

[0074] Method 4 [ka]

[0075] Method 5 [ka]

[0076] Method 6 [ka]

[0077] The present invention further provides the following test methods.

[0078] Test method 1: PRMT5 enzyme inhibitory activity test Experimental objective: To test the inhibitory effect of compounds on PRMT5 enzyme activity. Experimental material: PRMT5 enzyme Experimental procedure: Using LABCYTE Echo 550, test compounds are added at specific concentrations to white clear bottom 384-well plates, PRMT5 is added, and a solvent control (DMSO added, no compound added) and a blank control (DMSO added, no PRMT5 added) are set up. After 30 minutes of incubation at 25°C, the substrate is added and reacted at 25°C for 90 minutes. After 90 minutes, detection reagent is added to the 384-well plate using PerkinElmer LANCE Ultra TR-FRET standard method to bind to the enzyme-substrate reaction product, and after 1 hour of reaction at 25°C, the signal is detected by PerkinElmer EnVision 2105 Multimode Plate Reader plate reader. The raw data is used to calculate the inhibition rate of the test compound using the following formula:

[0079]

number

[0080] The inhibition rates were respectively imported into Excel to calculate the inhibition rates of different concentrations of the compounds, and then the inhibition curves were generated using XLfit software to calculate the minimum inhibition rate, maximum inhibition rate, and IC 50 Calculate related parameters such as

[0081] Test method 2: Pharmacokinetic evaluation in mice Experimental Objective: Male C57BL / 6 mice were used as test animals, and LC / MS / MS method was used to measure the plasma drug concentration of the compound at different time points after intravenous and oral administration. The pharmacokinetic behavior of the compound in mice was studied, and its pharmacokinetic properties were evaluated. Experimental procedure: Weigh out an appropriate amount of sample and prepare a clear solution with sterile saline. Take two healthy male C57BL / 6 mice purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and feed them with normal diet. After the animals are administered, 25μL of blood is collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours, respectively, and placed into a commercial anticoagulant tube pre-added with EDTA-K2. Centrifuge the tube for 10 minutes to separate the plasma and store at -60℃. Use LC / MS / MS method to measure the content of the corresponding compound in the plasma sample.

[0082] Effect of the Invention The compounds of the present invention have excellent binding effect on PRMT5, can effectively inhibit PRMT5, and have significant inhibitory effect on PRMT5·MTA complex. They have significant inhibitory activity on MTAP-deficient tumor cells, weak inhibitory effect on wild-type tumor cells, and show good selectivity. They have good liver microsomal stability and good in vivo pharmacokinetic properties, with small inter-species differences. They have significant tumor inhibitory effect in subcutaneous xenograft tumor model of human large cell lung cancer LU99, and the body weight of mice after drug administration is well maintained.

[0083] Definitions and Explanations Unless otherwise explained, the following terms and phrases as used herein are intended to have the following meanings: Certain terms or phrases, unless specifically defined, should not be considered indefinite or unclear but should be understood according to their ordinary meaning. When trade names appear herein, it is intended to refer to the corresponding trade name or its active ingredients.

[0084] As used herein, the term "pharmacologically acceptable" refers to those compounds, materials, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals and are commensurate with a reasonable benefit / risk ratio without undue toxicity, irritation, allergic response or other problem or complication.

[0085] The term "pharmaceutical acceptable salt" refers to a salt of the compound of the present invention prepared from a compound having certain substituents and a relatively non-toxic acid or base. When the compound of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting such a compound with a sufficient amount of base in a pure solution or a suitable inert solvent. When the compound of the present invention contains a relatively basic functional group, an acid addition salt can be obtained by contacting such a compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Some specific compounds of the present invention contain basic and acidic functional groups and can therefore be converted into either a base addition salt or an acid addition salt.

[0086] The pharma- ceutically acceptable salts of the present invention can be synthesized from a parent compound that contains an acid or base group by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or free base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of both.

[0087] The compounds of the present invention can exist in specific geometric or stereoisomeric forms. All such compounds contemplated by the present invention include cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic and other mixtures thereof, such as mixtures enriched in enantiomers or diastereomers, all of which are within the scope of the present invention. Other asymmetric carbon atoms may be present in substituents such as alkyl. All of these isomers and mixtures thereof are both within the scope of the present invention.

[0088] [ka]

[0089] Certain compounds of the present invention may exist as atropisomers, which are conformational isomers that arise when rotation around a single bond in a molecule is hindered or significantly slowed down by spatial interactions with other parts of the molecule. The compounds disclosed in the present invention include all atropisomers, which may be pure individual atropisomers, or may be enriched in one of the atropisomers, or may be non-specific mixtures of each. If the rotational potential energy around a single bond is high enough and the interconversion between conformations is slow enough, separation of the isomers is possible. For example,

[0090] [ka] This indicates that the stereo orientation on this side is inward.

[0091] The terms "optionally" and "optionally" mean that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs as well as instances where the event or circumstance does not occur.

[0092] The term "optionally substituted" means that the group may be substituted or unsubstituted, and unless otherwise specified, the type and number of substituents are optional as long as they are chemically feasible.

[0093] When any variable (e.g., R) occurs more than one time in a composition or structure of a compound, its definition is independent at each occurrence. Thus, for example, if a group is substituted with 0-2 R, said group may be optionally substituted with up to 2 R, and each occurrence of R has independent options. Furthermore, combinations of substituents and / or variants thereof are permissible only if such combinations result in stable compounds.

[0094] Unless otherwise stated, when a group has one or more linkable sites, any one or more sites of the group can be linked to other groups via a chemical bond. When the linking method of the chemical bond is delocalized and there is an H atom at the linkable site, when the chemical bond is connected, the number of H atoms at the site is reduced to the corresponding valence of the group according to the number of connected chemical bonds. The chemical bond by which the site is linked to another group is: [ka] For example, the straight solid bond of -OCH3 represents a bond to another group via the oxygen atom in the group, [ka] The straight-dashed bond represents a bond between the nitrogen atom of the group and another group, [ka] The wavy lines in the formula (I) represent bonds to other groups via the 1st and 2nd carbon atoms of the phenyl group, [ka] represents that any available bonding site of the piperidinyl can be bonded to another group via one chemical bond, and at least [ka] Even if the H atom is drawn as -N-, [ka] for [ka] When one chemical bond is bonded, the H at that site decreases by one to the corresponding monovalent piperidinyl.

[0095] Unless otherwise stated, the terms "element halogen" or "halogen," by themselves or as part of another substituent, mean a fluorine, chlorine, bromine, or iodine atom.

[0096] Unless otherwise stated, "C 2-5 The term "alkenyl" is used to represent a straight or branched chain hydrocarbon group of 2 to 5 carbon atoms containing at least one carbon-carbon double bond, which may be located in any position of the group. 2-5 Alkenyl includes C 2-3 , C5, C4, C3 and C2 alkenyl, and the like. 2-5 Alkenyl may be monovalent, divalent or polyvalent. 2-4 Examples of alkenyl include, but are not limited to, vinyl, propenyl, butenyl, butadienyl, and the like.

[0097] Unless otherwise stated, "C 2-3The term "alkenyl" is used to represent a straight or branched chain hydrocarbon group of 2 to 3 carbon atoms containing at least one carbon-carbon double bond, which may be located in any position of the group. 2-3 Alkenyl includes C3 and C2 alkenyl, 2-3 Alkenyl may be monovalent, divalent or polyvalent. 2-3 Examples of alkenyl include, but are not limited to, vinyl, propenyl, and the like.

[0098] Unless otherwise stated, "C 2-5 The term "alkynyl" is used to represent a straight or branched chain hydrocarbon group of 2 to 5 carbon atoms containing at least one carbon-carbon triple bond, which may be located in any position of the group. 2-4 Alkynyl includes C 2-3 , C5, C4, C3 and C2 alkynyl, etc. It may be monovalent, divalent or polyvalent. 2-5 Examples of alkynyl include, but are not limited to, ethynyl, propynyl, butynyl, and the like.

[0099] Unless otherwise stated, "C 2-3 The term "alkynyl" is used to denote a straight or branched chain hydrocarbon group of 2 to 3 carbon atoms containing at least one carbon-carbon triple bond, which may be in any position of the group. It may be monovalent, divalent or polyvalent. 2-3 Alkynyl includes C3 and C2 alkynyl. 2-3 Examples of alkynyl include, but are not limited to, ethynyl, propynyl, and the like.

[0100] Unless otherwise stated, "C 3-5 "Cycloalkyl" means a saturated cyclic hydrocarbon group of 3 to 5 carbon atoms, which is a monocyclic ring system, 3-5 Cycloalkyl has C 3-4 and C 4-5Cycloalkyl and the like are included, which may be monovalent, divalent or polyvalent. C 3-5 Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and the like.

[0101] The compounds of the present invention can be prepared by various synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining with other chemical synthetic methods, and equivalent alternative methods known to those skilled in the art, and preferred embodiments include, but are not limited to, the examples of the present invention.

[0102] The structure of the compound of the present invention can be confirmed by conventional methods known to those skilled in the art, and when the present invention relates to the absolute configuration of a compound, the absolute configuration can be confirmed by conventional technical means in the art.For example, in single crystal X-ray diffraction (SXRD), the single crystal is grown and collected diffraction intensity data by Bruker D8 venture diffractometer, the light source is CuKα radiation, the scanning method is φ / ω scanning, and after collecting relevant data, the crystal structure can be further analyzed by direct method (Shelxs97) to confirm the absolute configuration.

[0103] In the present invention, the abbreviations are: solutol stands for polyethylene glycol-15 hydroxystearate, MC (4000 cps) stands for methylcellulose (viscosity 4000), tolbutamide stands for tolbutamide, and labetalol stands for labetalol.

[0104] The solvents used in the present invention can be obtained commercially.

[0105] Compounds are named according to conventional naming principles in the art or using ChemDraw® software; commercially available compounds are named by the supplier's catalogue name. [Brief description of the drawings]

[0106] [Figure 1]FIG. 1 is a diagram showing the predicted binding mode of compound A and PRMT5. [Diagram 2] FIG. 1 is a diagram showing the predicted binding mode of compound B (structure 1) with PRMT5. [Diagram 3] FIG. 1 is a diagram showing the predicted binding mode of compound B (structure 2) with PRMT5. [Figure 4] FIG. 1 is a diagram showing the predicted binding mode of compound B (structure 3) with PRMT5. [Diagram 5] FIG. 1 is a diagram showing the predicted binding mode of compound B (structure 4) with PRMT5. [Figure 6] FIG. 1 is a diagram showing the predicted binding mode of compound C (structure 1) with PRMT5. [Figure 7] FIG. 1 is a diagram showing the predicted binding mode of compound C (structure 2) with PRMT5. [Figure 8] FIG. 1 is a diagram showing the predicted binding mode of compound C (structure 3) with PRMT5. [Figure 9] FIG. 1 is a diagram showing the predicted binding mode of compound C (structure 4) with PRMT5. [Figure 10] FIG. 1 is a diagram showing the predicted binding mode of compound D and PRMT5. [Figure 11] FIG. 1 is a diagram showing the predicted binding mode of compound F and PRMT5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0107] The present invention will be described in detail below with reference to examples, but these are not intended to limit the present invention in any adverse manner. Although the present invention has been described in detail in the present specification, specific embodiments thereof have also been disclosed, and it is obvious to those skilled in the art that various changes and improvements can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0108] Calculation example 1 [ka]

[0109] The molecular docking process was performed using the Induced Fit Docking method in Maestro (Schrodinger version 2021-2). [1-2] The program was run using default options. The crystal structure of PRMT5 was selected in the PDB database (PDB ID: 5FA5) as the docking template. To prepare the protein, Maestro [2] Hydrogen atoms were added using the Protein Preparation Wizard module of the Sigma-Aldrich ... [3] The three-dimensional structure of the molecule was generated using FTIR and energy minimization was performed using OPLS4 force field. A box was automatically generated for placing the example compound during the molecular docking process, centered on Trp579 of 5FA5. The interaction between the protein and the example compound was analyzed, and a reasonable docking conformation was selected and saved based on the calculated IFD score and binding mode. The predicted binding mode diagram of compound A and PRMT5 is shown in Figure 1, the predicted binding mode diagram of compound B and PRMT5 is shown in Figures 2-5, the predicted binding mode diagram of compound C and PRMT5 is shown in Figures 6-9, the predicted binding mode diagram of compound D and PRMT5 is shown in Figure 10, and the predicted binding mode diagram of compound F and PRMT5 is shown in Figure 11.

[0110] [1] Induced Fit Docking protocol; Glide, Schrodinger, LLC, New York, NY, 2021. [2] Prime, Schrodinger, LLC, New York, NY, 2021. [3] Maestro, Schrodinger, LLC, New York, NY, 2021. [4] LigPrep, Schrodinger, LLC, New York, NY, 2021.

[0111] Conclusion: The compounds of the present invention had excellent binding effect on PRMT5.

[0112] Reference example 1: Compound M1 [ka]

[0113] Step 1 Compound M1-1 (4.2 g, 48.90 mmol) and compound M1-2 (9.99 g, 48.90 mmol) were dissolved in anhydrous dichloromethane (80 mL), 4.2 g of 3 Å molecular sieves were added, and the mixture was purged with nitrogen gas three times and stirred at 25° C. for 16 hours. The reaction solution was filtered and the cake was washed with 30 mL of dichloromethane. The filtrate was concentrated under reduced pressure to obtain compound M1-3, which was used directly in the next step without purification. 1 H NMR (400MHz, CDCl3) δ:6.62 - 6.78 (m, 1H), 5.57 (s, 2H), 5.41 - 5.50 (m, 1H), 3.21 (s, 6H), 2.98 (s, 6H), 1.81 - 1.89 (m, 3H).

[0114] Step 2 Anhydrous dichloromethane (120 mL) was added to a three-neck flask that had been dried in advance, and the flask was purged with nitrogen gas three times. Diethylzinc (1M, 140.50 mL) was added, cooled to -65°C, diiodomethane (56.45 g, 210.75 mmol) was added dropwise, and the mixture was stirred for 20 minutes. Anhydrous dichloromethane (120 mL) solution of compound M1-3 (11.9 g, 46.83 mmol) was added dropwise at -65°C. After the addition was completed, the mixture was stirred at -65°C for 2 hours. 150 mL of saturated aqueous ammonium chloride solution was slowly added dropwise to the reaction solution at -65°C, and the mixture was slowly warmed to room temperature. The solution was separated, and the aqueous phase was extracted with dichloromethane (30 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound M1-4, which was used directly in the next step without purification. 1H NMR (400MHz, CDCl3) δ: 5.49 (s, 2H), 3.17 (s, 6H), 2.97 (s, 6H), 0.95 - 1.12 (m, 4H), 0.68 - 0.78 (m, 1H), 0.38 - 0.46 (m, 1 H), 0.46 - 0.34 (m, 1H).

[0115] Step 3 Compound M1-4 (4.95 g, 18.46 mmol) was dissolved in methyl tert-butyl ether (75 mL) and water (75 mL), sodium perborate tetrahydrate (28.41 g, 184.62 mmol) was added, and the mixture was stirred at 25° C. for 12 hours. The solution was separated, and the aqueous phase was extracted with methyl tert-butyl ether (15 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give compound M1, which was used directly in the next step without purification. 1 H NMR (400 MHz, CDCl3) δ: 3.32 - 3.43 (m, 1H), 2.19 (br s, 1H), 1.08 - 1.19 (m, 4H), 0.83 - 0.96 (m, 1H), 0.48 (q, J=5.6 Hz, 1H).

[0116] Reference Example 2: Compounds M2A and M2B [ka]

[0117] Under nitrogen gas protection, compound M2-1 (30 g, 154.61 mmol) was dissolved in acetonitrile (300 mL), and then cesium carbonate (50.37 g, 154.61 mmol) and deuterated iodomethane (22.38 g, 157.70 mmol) were added and reacted for 24 hours at 20° C. The reaction system was filtered, the cake was washed with acetonitrile (300 mL), and the filtrate was concentrated under reduced pressure. Next, methyl tert-butyl ether (400 mL) was added, filtered, the filtrate was washed with methyl tert-butyl ether (300 mL), and the filtrate was concentrated under reduced pressure. The crude product was separated and purified by SFC (chromatography column: DAICEL CHIRALCEL OD (250 mm × 50 mm, 10 μm), mobile phase: phase A (CO2), phase B (isopropanol containing 0.1% aqueous ammonia), gradient: B% = 11% to 11%) to obtain compound M2A. 1 H NMR (400 MHz, CDCl3) δ:7.47 (d, J = 1.6 Hz, 1H), 6.70 (d, J = 1.6 Hz, 1H), 1.33 (s, 12H).

[0118] SFC analytical method: Chromatography column: Chiralcel OD-3, 150 × 4.6 mm ID, 3 μm, mobile phase: phase A (CO2), phase B (ethanol containing 0.1% isopropylamine), gradient: B% = 10% to 50%). The retention time of compound M2A was 0.895 min, and that of its isomer M2B was 1.398 min.

[0119] Reference example 3: Compound M3 [ka]

[0120] Step 1 Compound M3-1 (50 g, 234.71 mmol) was dissolved in N,N-dimethylformamide dimethyl acetal (180.12 g, 1.51 mol), potassium tert-butoxide (2.63 g, 23.47 mmol) was added, the mixture was purged with nitrogen gas three times, heated to 110° C., and stirred for 20 hours. The reaction solution was concentrated under reduced pressure. The crude product was added to 100 mL of petroleum ether and stirred at 16° C. for 0.5 hours. The mixture was filtered and the solid was collected, and 200 mL of ethyl acetate was added, heated to 80° C., and stirred for 5 hours. The mixture was filtered and the solid was collected to obtain compound M3-2. 1 H NMR (400 MHz, DMSO-d6) δ:7.98 (d, J = 0.8 Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.29 (dd, J = 8.4, 1.6 Hz, 1H), 7.01 (s, 1H), 3.11 (s, 6H).

[0121] Step 2 Compound M3-2 (29.1 g, 108.54 mmol) was dissolved in absolute ethanol (450 mL), 98% hydrazine hydrate (11.85 g, 236.71 mmol) was added, and the mixture was purged with nitrogen gas three times, stirred at 16° C. for 0.5 hours, then heated to 70° C. and stirred for 12 hours. The reaction solution was cooled to room temperature, filtered, and the cake was washed with 30 mL of ethanol. The cake was collected and concentrated under reduced pressure to remove residual solvent, obtaining compound M3-3. 1 H NMR (400 MHz, DMSO-d6) δ:12.66 (s, 1 H), 8.34 (d, J = 1.6 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.93 - 8.04 (m, 1H), 3.61 (s, 2H), 2.19 (s, 6H).

[0122] Step 3 Compound M3-3 (20.7 g, 73.37 mmol) was dissolved in anhydrous tetrahydrofuran (300 mL) and purged with nitrogen gas three times. The mixture was cooled to 0°C, and isobutyl chloroformate (12.02 g, 88.04 mmol) was added dropwise. After the addition was completed, the mixture was heated to 25°C and stirred for 12 hours. The reaction solution was cooled to 0°C, and 300 mL of 0.5 M aqueous hydrochloric acid was added dropwise, and the mixture was stirred for 0.5 hours. The reaction solution was filtered, and the cake was washed with tetrahydrofuran (30 mL x 3). The solid was collected and dried under reduced pressure to remove the solvent, to obtain compound M3-4. 1 H NMR (400 MHz, DMSO-d6) δ:12.92 (s, 1H), 7.90 - 8.52 (m, 3H), 5.07 (s, 2H).

[0123] Step 4 Compound M3-4 (15.6 g, 57.04 mmol) was dissolved in N,N-dimethylformamide (170 mL), phthalimide potassium salt (11.62 g, 62.74 mmol) was added, the mixture was purged with nitrogen gas three times, heated to 25°C, and stirred for 4 hours. 200 mL of 0.5 M dilute hydrochloric acid solution was added dropwise to the reaction solution, filtered, and the cake was washed with 100 mL of saturated sodium bicarbonate aqueous solution and 100 mL of water in sequence. 50 mL of ethanol was further added to the cake, and the mixture was stirred at 70°C for 1 hour. The mixture was filtered, the solid was collected, and the solid was dried under reduced pressure to obtain compound M3-5. 1 H NMR (400 MHz, DMSO-d6) δ:12.60 (s, 1H), 8.43 (d, J = 1.6 Hz, 1H), 8.03 - 8.23 ​​(m, 2H), 7.83 - 8.00 (m, 4H), 5.19 (s, 2H).

[0124] Step 5 Compound M3-5 (2.5 g, 6.51 mmol), bis(pinacolato)diboron (2.48 g, 9.76 mmol), potassium acetate (1.92 g, 19.52 mmol) were dissolved in anhydrous dioxane (25 mL), purged with nitrogen gas three times, [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (476.14 mg, 650.73 μmol) was added, purged with nitrogen gas three times, heated to 80 °C, and stirred for 2 hours. The reaction solution was concentrated under reduced pressure. The crude product was stirred with 20 mL of acetonitrile for 0.5 hours, filtered, and the cake was collected to obtain compound M3. 1 H NMR (400 MHz, DMSO-d6) δ:12.40 (s, 1H), 8.01 - 8.35 (m, 3H), 7.84 - 7.98 (m, 4H), 5.17 (s, 2H), 1.24 (s, 12H).

[0125] Reference example 4: Compound M4 [ka]

[0126] Compound 1-2 (20 g, 79.23 mmol), compound 1-8 (32.97 g, 158.45 mmol), sodium carbonate (25.19 g, 237.68 mmol) and [1,1'-bis(di-tert-butylphosphino)ferrocene]palladium(II) dichloride (2.58 g, 3.96 mmol) were dissolved in 600 ml of N,N-dimethylformamide, purged with nitrogen gas three times, and the mixture was stirred at 80 ° C for 48 hours. After the reaction was completed, the reaction solution was concentrated. The crude product was diluted with 300 ml of water and extracted with ethyl acetate (3 × 300 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the crude product was purified by flash chromatography (silica gel, eluent: ethyl acetate / petroleum ether, ethyl acetate ratio: 0-20%) to obtain compound M4. 1H NMR (400 MHz, CDCl3) δ = 7.67 (d, J=2.0 Hz, 1H), 7.47 (dd, J=5.6, 7.6 Hz, 1H), 6.57 (d, J=2.0 Hz, 1H), 3.86 (d, J=1.2 Hz, 3H).

[0127] Reference example 5: Compound M5 [ka]

[0128] Step 1 Compound M3-5 (6 g, 15.62 mmol) and hydrazine hydrate (3.68 g, 62.47 mmol, purity: 85%) were dissolved in 100 ml of ethanol, and the mixture was stirred at 80° C. for 15 hours. After the reaction was completed, the reaction solution was concentrated to obtain compound M5-1. MS-ESI calculated value [M+1] + 254.0, 256.0, actual values ​​253.8, 255.8.

[0129] Step 2 Compound M5-1 (3.9 g, 15.35 mmol), Boc anhydride (6.70 g, 30.70 mmol) and triethylamine (4.66 g, 46.05 mmol) were dissolved in 100 ml of dichloromethane, and the mixture was stirred at 25° C. for 24 hours. After the reaction was completed, it was filtered, and the cake was collected and dried to obtain compound M5-2. 1 H NMR (400 MHz, DMSO-d6) δ = 12.67 (br s, 1H), 8.27 (s, 1H), 8.17 (d, J=8.3 Hz, 1H), 8.02 (d, J=8.5 Hz, 1H), 4.41 (s, 2H), 1.41 (s, 9H).

[0130] Step 3 Compound M5-2 (4.1 g, 11.58 mmol), bis(pinacolato)diboron (4.41 g, 17.36 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (846.98 mg, 1.16 mmol) and potassium acetate (3.41 g, 34.73 mmol) were added to 100 ml of anhydrous 1,4-dioxane, purged with nitrogen gas three times, and the mixture was stirred at 100 ° C for 15 hours. After the reaction was completed, it was cooled to room temperature, left for 2 hours, filtered, and the filtrate was concentrated, and 20 ml of petroleum ether / ethyl acetate (volume ratio: 10:1) mixed solvent was added, slurried and stirred at room temperature for 1 hour, filtered, and the cake was collected to obtain compound M5. MS-ESI calculated value [M+1] + 402.2, measured value 402.2.

[0131] Example 1 [ka]

[0132] Step 1 Compound 1 (5 g, 28.81 mmol), N-bromosuccinimide (10.26 g, 57.62 mmol), p-toluenesulfonic acid (2.48 g, 14.41 mmol), 1,2-dichloroethane (50 mL), and palladium acetate (646.82 mg, 2.88 mmol) were added to a pre-dried one-neck flask, and the mixture was thoroughly purged with nitrogen gas, followed by stirring at 75°C for 20 hours. The reaction solution was filtered, the cake was washed with dichloromethane (20 mL x 2), the filtrate was concentrated under reduced pressure, and the crude product was separated and purified using a silica gel column (ethyl acetate / petroleum ether, ethyl acetate ratio: 0-10%) to obtain compound 1-2. 1 H NMR (400 MHz, CDCl3) δ ppm 7.28 (d, J=5.50 Hz, 1 H).

[0133] Step 2 Compound 1-7 (500 mg, 6.93 mmol) was dissolved in anhydrous tetrahydrofuran (50 mL), purged with nitrogen gas three times, cooled to 0 ° C, sodium hydrogen (416.06 mg, 10.40 mmol, purity: 60%) was added, and stirred for 10 minutes. Compound 1-2 (1.75 g, 6.93 mmol) was added at 0 ° C, slowly warmed to 16 ° C, and stirred for 2 hours. The reaction solution was poured into 100 mL of saturated ammonium chloride aqueous solution and extracted with ethyl acetate (10 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by a silica gel column (petroleum ether: ethyl acetate = 100: 1 to 10: 1) to obtain compound 1-3.

[0134] Step 3 Compound 1-3 (1.2 g, 3.94 mmol), compound 1-8 (2.46 g, 11.82 mmol) and sodium carbonate (1.67 g, 15.76 mmol) were dissolved in dioxane (20 mL) and water (5 mL), purged with nitrogen gas three times, [1,1'-bis(di-tert-butylphosphino)ferrocene]palladium(II) dichloride (256.81 mg, 394.03 μmol) was added, the temperature was raised to 80 ° C, and the mixture was stirred for 16 hours. The reaction system was poured into 100 mL of saturated saline and extracted with ethyl acetate (30 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by a silica gel column (petroleum ether: ethyl acetate = 10: 1 to 3: 1) to obtain compound 1-4. 1 H NMR (400 MHz, CDCl3) δ ppm 7.61 (d, J = 2.0 Hz, 1H), 7.42 (d, J = 6.0 Hz, 1H), 6.50 (d, J = 2.0 Hz, 1H), 3.81 (d, J =1.2 Hz, 3H), 1.63 (s, 3H), 1.09 - 1.16 (m, 2H), 0.82 - 0.90 (m, 2H).

[0135] Step 4 Compound 1-4 (760 mg, 2.49 mmol) was dissolved in acetonitrile (40 mL), purged with nitrogen gas three times, N-bromosuccinimide (884.87 mg, 4.97 mmol) was added, the temperature was raised to 40°C, and the mixture was stirred for 2 hours. The mixture was concentrated under reduced pressure. The crude product was purified using a silica gel column (petroleum ether: ethyl acetate = 10: 1 to 3: 1) to obtain compound 1-5. 1 H NMR (400 MHz, CDCl3) δ ppm 7.61 (s, 1H), 7.48 (d, J = 6.0 Hz, 1H), 3.80 (s, 3H), 1.65 (s, 3H), 1.10 - 1.19 (m, 2H), 0.83 - 0.94 (m, 2H).

[0136] Step 5 Compound 1-5 (400 mg, 1.04 mmol), compound M3 (538.18 mg, 1.25 mmol) and anhydrous potassium phosphate (662.24 mg, 3.12 mmol, 3 eq) were dissolved in dioxane (6 mL) and water (1.5 mL), purged with nitrogen gas three times, [1,1'-bis(di-tert-butylphosphino)ferrocene]palladium(II) dichloride (67.78 mg, 104.00 μmol) was added, the temperature was raised to 80 ° C, and the mixture was stirred for 12 hours. The reaction product was poured into 30 mL of saturated saline and extracted with ethyl acetate (10 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by a silica gel column (dichloromethane:methanol = 100: 1 to 30: 1) to obtain compound 1-6.

[0137] Step 6 Compound 1-6 (330 mg, 541.87 μmol) was dissolved in anhydrous ethanol (7 mL), 98% hydrazine hydrate (310 mg, 6.19 mmol) was added, and the mixture was heated to 80 ° C. and stirred for 2 hours. The reaction solution was concentrated under reduced pressure, and 4 mL of DMF was added to the crude product, filtered, and the filtrate was collected. The crude product was separated by prep-HPLC (chromatography column: Phenomenex C18 75 × 30 mm × 3 μm, mobile phase A: water (0.1% HCl), mobile phase B: acetonitrile, running gradient: B%: 10% to 45%) to obtain compound 1.

[0138] Step 7 Compound 1 was further separated by SFC (chromatography column: DAICEL CHIRAL PAKIC (250 mm × 30 mm, 10 μm), mobile phase: phase A (CO2), phase B (ethanol containing 0.1% aqueous ammonia), gradient (B%): 50% to 50%) to obtain compounds 1A and 1B.

[0139] Compound 1A: SFC analytical method: Chromatographic column: Chiralpak IC-3, 150 × 4.6 mm ID, 3 μm, mobile phase: [A (CO2) and B (isopropanol containing 0.1% diethylamine)], gradient: B% = 50%, retention time was 4.221 min, ee = 98.74%; 1 HNMR (400 MHz, CD3OD) δ ppm 8.27 (d, J=8.53 Hz, 1H), 7.76-7.82 (m, 2H), 7.61 (d, J=8.03 Hz, 1H), 7.25 (s, 1H), 4.05-4.18 (m, 2H), 3.85 (s, 3H), 1.64 (s, 3H), 1.07-1.13 (m, 2H), 0.89-0.94 (m, 2H). MS m / z: 479.0[M+H] + .

[0140] Compound 1B: SFC analytical method: Chromatographic column: Chiralpak IC-3, 150 × 4.6 mm ID, 3 μm, mobile phase: [A (CO2) and B (isopropanol containing 0.1% diethylamine)], gradient: B% = 50%, retention time was 6.128 min, ee = 99.01%; 1 HNMR (400 MHz, CD3OD) δ ppm 8.24-8.31 (m, 1H), 7.74-7.84 (m, 2H), 7.61 (br d, J=9.79 Hz, 1H), 7.17-7.29 (m, 1H), 4.03-4.17 (m, 2H), 3.85 (s, 3H), 1.64 (s, 3H), 1.06-1.13 (m, 2H), 0.88-0.94 (m, 2H). MS m / z: 479.0[M+H] + .

[0141] Example 2 [ka]

[0142] Step 1 Compound M1 (270 mg, 3.74 mmol) and tetrahydrofuran (4 mL) were added to a three-neck flask that had been dried in advance, and sodium hydride (299.53 mg, 7.49 mmol, purity: 60%) was added at 0°C. The mixture was stirred at 0°C for 10 minutes, and a solution of compound 1-2 (945.28 mg, 3.74 mmol) in tetrahydrofuran (2 mL) was added and stirred at 20°C for 2 hours. The reaction solution (10 mL) was quenched, saturated saline (10 mL) was added, and the mixture was extracted with ethyl acetate (10 mL x 3). The combined organic phase was collected, anhydrous sodium sulfate was added, dried, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated and purified using a silica gel column (ethyl acetate: petroleum ether = 1:10) to obtain compound 2-1.

[0143] Step 2 Compound 2-1 (2.1 g, 6.90 mmol), compound 1-8 (2.87 g, 13.80 mmol), 1,4-dioxane (40 mL), water (10 mL), potassium phosphate (4.39 g, 20.70 mmol) were added to a pre-dried one-neck flask, and the flask was thoroughly purged with nitrogen gas. [1,1'-bis(di-tert-butylphosphino)ferrocene]palladium(II) dichloride (539.30 mg, 828.00 μmol) was added and stirred at 80 ° C for 20 hours. The reaction solution was concentrated under reduced pressure, saturated saline (15 mL) was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phase was collected, anhydrous sodium sulfate was added, dried, filtered, and the filtrate was concentrated under reduced pressure. The crude product was separated and purified using a silica gel column (ethyl acetate: petroleum ether = 1:10) to obtain compound 2-2. MS m / z: 305.9[M+H] + .

[0144] Step 3 Compound 2-2 (320 mg, 1.05 mmol), N-bromosuccinimide (372.58 mg, 2.09 mmol), and acetonitrile (5 mL) were added to a pre-dried one-neck flask and stirred at 40°C for 2 hours. The mixture was concentrated under reduced pressure. The crude product was separated and purified using a silica gel column (ethyl acetate:petroleum ether = 1:10) to obtain compound 2-3. MS m / z: 383.9 [M+H] + , 385.9[M+H] + .

[0145] Step 4 Compound 2-3 (500 mg, 1.30 mmol), compound M3 (689.54 mg, 1.60 mmol), potassium phosphate (551.87 mg, 2.60 mmol), 1,4-dioxane (10 mL), and water (2.5 mL) were added to a pre-dried one-neck flask, and the inside was thoroughly replaced with nitrogen gas. Then, [1,1'-bis(di-tert-butylphosphino)ferrocene]palladium(II) dichloride (101.67 mg, 155.99 μmol) was added, and the mixture was stirred at 85°C for 3 hours. Saturated saline (50 mL) was added, and the mixture was extracted with ethyl acetate (15 mL x 3). The combined organic phase was collected and concentrated under reduced pressure. The crude product was separated and purified using a silica gel column (ethyl acetate: petroleum ether = 1:10) to obtain compound 2-4. MS m / z: 609.1 [M + H] + .

[0146] Step 5 Compound 2-4 (100 mg, 164.20 μmol), ethanol (2 mL), and 98% hydrazine hydrate (16.78 mg, 328.40 μmol) were added to a pre-dried one-neck flask and stirred at 80 ° C for 2 hours. The reaction solution was concentrated under reduced pressure and first separated by prep-HPLC (chromatography column: Phenomenex luna C18 80 × 40 mm × 3 μm, mobile phase: [water (hydrochloric acid) - acetonitrile], gradient (acetonitrile %): 1% to 35%) to obtain compound 2.

[0147] Step 6 Compound 2 was further separated by SFC (chromatography column: DAICEL CHIRALPAK IC (250 mm × 30 mm, 10 μm), mobile phase: [A: CO2, B: isopropanol containing 0.1% aqueous ammonia], B%: 65% to 65%) to obtain compounds 2A and 2B.

[0148] Compound 2A: SFC analysis: Chromatography column: Chiralpak IC-3, 50 x 4.6 mm ID, 3 μm, mobile phase: [A: CO2, B: isopropanol with 0.1 isopropylamine)], A:B = 50:50, retention time was 1.352 min, ee = 96.56%.1 H NMR (400 MHz, CDCl3) δ ppm 9.50 - 10.07 (m, 1 H) 8.24 (br dd, J=3.94, 2.31 Hz, 1 H) 7.83 (s, 1 H) 7.56 (s, 1 H) 7.40 - 7.46 (m, 1 H) 7.38 (br d, J=5.75 Hz, 1 H) 3.96 (br d, J=1.88 Hz, 2 H) 3.76 (s, 3 H) 3.40 - 3.49 (d, 1 H) 1.14 - 1.21 (m, 1 H) 1.04 - 1.12 (m, 3 H) 0.90 - 1.02 (m, 1 H) 0.71 - 0.73 (m, 1H). MS m / z: 479.2[M+H] + .

[0149] Compound 2B was purified by SFC (chromatographic column: Chiralpak IC-3, 50 × 4.6 mm ID, 3 μm, mobile phase: [A: CO, B: isopropanol containing 0.1% isopropylamine, A:B = 50:50, retention time was 2.269 min, ee = 97.23%. 1 H NMR (400 MHz, CDCl3) δ ppm 10.07 - 10.67 (s, 1 H) 8.26 ( d, J=7.75 Hz, 1 H) 7.84 (s, 1 H) 7.58 (s, 1 H) 7.33 - 7.49 (m, 2 H) 3.98 (d, 2 H) 3.76 (s, 3 H) 3.45 (d, J=3.00 Hz, 1 H) 1.18 (m, J=1.75 Hz, 1 H) 1.05 - 1.14 (m, 3 H) 0.85 - 0.99 (m, 1 H) 0.52 - 0.72 (m, 1 H). MS m / z: 479.2[M+H] + .

[0150] Example 3 [ka]

[0151] Step 1 Compound 1-3 (1g, 3.28mmol), compound M2A (1.73g, 8.21mmol), anhydrous potassium phosphate (2.79g, 13.13mmol) were dissolved in dioxane (16mL) and water (4mL), purged with nitrogen gas three times, [1,1'-bis(di-tert-butylphosphino)ferrocene]palladium(II) dichloride (214.01mg, 328.36μmol) was added, purged with nitrogen gas three times, heated to 80℃, and stirred for 2 hours. The reaction solution was concentrated under reduced pressure. The crude product was purified by a silica gel column (petroleum ether:ethyl acetate=10:1-3:1) to obtain compound 3-1. 1 H NMR (400 MHz, CDCl3) δ: 7.61 (d, J = 2.0 Hz, 1H), 7.42 (d, J = 6.0 Hz, 1H), 6.50 (d, J = 2.0 Hz, 1H), 1.63 (s, 3H), 1.07 - 1.19 (m, 2H), 0.81 - 0.89 (m, 2H).

[0152] Step 2 Compound 3-1 (480 mg, 1.55 mmol) and N-bromosuccinimide (553.40 mg, 3.11 mmol) were dissolved in acetonitrile (10 mL), purged with nitrogen gas three times, heated to 40°C, and stirred for 2 hours. The reaction solution was concentrated under reduced pressure. The crude product was purified using a silica gel column (petroleum ether:ethyl acetate=10:1 to 3:1) to obtain compound 3-2. 1 H NMR (400 MHz, CDCl3) δ: 7.61 (s, 1H), 7.48 (d, J = 6.0 Hz, 1H), 1.65 (s, 3H), 1.09 - 1.20 (m, 2H), 0.82 - 0.91 (m, 2H).

[0153] Step 3 Compound 3-2 (500 mg, 1.29 mmol), compound M3 (834.35 mg, 1.93 mmol) and anhydrous potassium phosphate (821.37 mg, 3.87 mmol) were dissolved in dioxane (8 mL) and water (2 mL), and the mixture was purged with nitrogen gas three times. [1,1'-bis(di-tert-butylphosphino)ferrocene]palladium(II) dichloride (84.06 mg, 128.98 μmol) was added, and the mixture was heated to 80°C and stirred for 2 hours. The reaction solution was concentrated under reduced pressure. The crude product was purified by a silica gel column (dichloromethane:methanol = 100:1 to 30:1) to obtain compound 3-3.

[0154] Step 4 Compound 3-3 (600 mg, 980.35 μmol) was dissolved in absolute ethanol (12 mL), 98% hydrazine hydrate (260 mg, 4.15 mmol) was added, and the mixture was heated to 80°C and stirred for 2 hours. The reaction solution was concentrated under reduced pressure. The crude product was first separated and purified by preparative high-performance liquid chromatography (chromatography column: Phenomenex Luna 75 × 30 mm × 3 μm, mobile phase A: water (0.1% HCl), mobile phase B: acetonitrile, running gradient: B%: 20% to 50%) to obtain compound 3.

[0155] Step 5 Compound 3 was separated and purified by SFC (column: DAICEL CHIRALPAK IC (250 mm × 30 mm, 10 μm), mobile phase: A (CO2) and B (acetonitrile / isopropanol = 1:1, containing 0.1% ammonia water), gradient: B% = 60% ~ 60%), and the separated eluent containing the product was post-treated: concentrated under reduced pressure, and the residue was dissolved by adding acetonitrile / water, and then freeze-dried to obtain compound 3A and compound 3B.

[0156] Compound 3A: retention time under SFC analytical method (column: Chiralpak IC-3, 50 × 4.6 mm ID, 3 μm, mobile phase: A (CO2) and B (isopropanol containing 0.1% isopropylamine), gradient: B% = 50%) conditions was 2.221 min, ee = 96.70%;1 H NMR (400 MHz, CD3OD) δ ppm 8.24 (d, J = 8.4 Hz, 1H), 8.14 (s, 1H), 7.66 - 7.82 (m, 2H), 7.52 - 7.66 (m, 1H), 3.89 - 4.13 (m, 2H), 1.62 (s, 3H), 1.02 - 1.15 (m, 2H), 0.79 - 0.98 (m, 2H). MS m / z: 482.2[M+H] + .

[0157] Compound 3B: retention time under SFC analytical method (column: Chiralpak IC-3, 50 × 4.6 mm ID, 3 μm, mobile phase: A (CO2) and B (isopropanol containing 0.1% isopropylamine), gradient: B% = 50%) was 1.312 min, ee = 95.56%. 1 H NMR (400 MHz, CD3OD) δ : 8.22 (t, J = 9.2 Hz, 1H), 8.13 (d, J = 2.0 Hz, 1H), 7.77 (d, J = 6.0 Hz, 1H), 7.70 (d, J = 4.4 Hz, 1H), 7.59 (t, J = 7.0 Hz, 1H), 3.97 (s, 2H), 1.61 (s, 3H), 0.99 - 1.17 (m, 2H), 0.81 - 0.97 (m, 2H). MS m / z: 482.1[M+H] + .

[0158] Example 4 [ka]

[0159] Step 1 Compound 2-1 (1.04 g, 3.41 mmol), compound M2A (2.16 g, 10.24 mmol), anhydrous potassium phosphate (2.90 g, 13.66 mmol) were dissolved in dioxane (20 mL) and water (5 mL), purged with nitrogen gas three times, [1,1'-bis(di-tert-butylphosphino)ferrocene]palladium(II) dichloride (222.57 mg, 341.50 μmol) was added, purged with nitrogen gas three times, heated to 80 ° C., and stirred for 12 hours. The reaction solution was concentrated under reduced pressure, 20 mL of water and 10 mL of ethyl acetate were added, filtered, and the solution was separated. The aqueous phase was extracted with ethyl acetate (10 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column (petroleum ether:ethyl acetate=10:1 to 3:1) to obtain compound 4-1. 1 H NMR (400 MHz, CDCl3) δ: 7.61 (d, J = 2.0 Hz, 1H), 7.38 (d, J = 5.6 Hz, 1H), 6.50 (d, J = 2.0 Hz, 1H), 3.50 - 3.59 (m, 1H), 1.23 - 1.34 (m, 1H), 1.16 - 1.21 (m, 3H), 1.01 - 1.11 (m, 1H), 0.68 - 0.77 (m, 1H).

[0160] Step 2 Compound 4-1 (440.00 mg, 1.43 mmol) was dissolved in acetonitrile (2 mL), purged with nitrogen gas three times, N-bromosuccinimide (507.29 mg, 2.85 mmol) was added, heated to 40°C, and stirred for 8 hours. The reaction solution was concentrated under reduced pressure. The crude product was purified using a silica gel column (petroleum ether: ethyl acetate = 10: 1 to 3: 1) to obtain compound 4-2. 1H NMR (400 MHz, CDCl3) δ: 7.61 (s, 1 H), 7.45 (d, J = 6.0 Hz, 1 H), 3.50 - 3.60 (m, 1H), 1.25 - 1.38 (m, 1H), 1.20 (d, J = 6.4 Hz, 3 H), 1.02 - 1.14 (m, 1H), 0.68 - 0.80 (m, 1H).

[0161] Step 3 Compound 4-2 (500 mg, 1.29 mmol), compound M3 (834.35 mg, 1.93 mmol) and anhydrous potassium phosphate (821.37 mg, 3.87 mmol) were dissolved in dioxane (10 mL) and water (2 mL), purged with nitrogen gas three times, [1,1'-bis(di-tert-butylphosphino)ferrocene]palladium(II) dichloride (84.06 mg, 128.98 μmol) was added, heated to 80 ° C., and stirred for 12 hours. The reaction solution was concentrated under reduced pressure. The crude product was purified by a silica gel column (dichloromethane:methanol = 100:1, 50:1, 30:1) to obtain compound 4-3. 1 H NMR (400 MHz, DMSO-d6) δ: 12.44 (s, 1H), 8.33 (s, 1H), 8.12 - 8.20 (m, 1H), 7.87 - 8.01 (m, 5H), 7.82 - 7.87 (m, 1H), 7.52 - 7.65 (m, 1H), 5.00 - 5.15 (m, 2H), 3.89 - 3.98 (m, 1H), 1.14 - 1.23 (m, 1H), 1.04 - 1.12 (m, 3H), 0.89 - 0.96 (m, 1H), 0.68 - 0.76 (m, 1H).

[0162] Step 4 Compound 4-3 was dissolved in absolute ethanol (12 mL), hydrazine hydrate (420 mg, 8.22 mmol) was added, and the mixture was heated to 80°C and stirred for 2 hours. The reaction solution was concentrated under reduced pressure. The crude product was separated and purified by preparative high performance liquid chromatography (chromatography column: Phenomenex Luna 80 x 30 mm x 3 um, mobile phase A: water (0.1% HCl), mobile phase B: acetonitrile, running gradient: B%: 1% to 30%) to obtain compound 4.

[0163] Step 5 Compound 4 was separated and purified by SFC (column: DAICEL CHIRALPAK IC (250 mm × 30 mm, 10 μm), mobile phase: A (CO2) and B (ethanol containing 0.1% aqueous ammonia), gradient: B% = 60% to 60%) to obtain compounds 4A and 4B.

[0164] Compound 4A: Retention time in SFC analysis (column: Chiralpak IC-3, 50×4.6 mm ID, 3 um, mobile phase: A (CO2) and B (EtOH with 0.1% isopropylamine), gradient: B%=40%) was 2.056 min, ee=100%. 1 H NMR (400 MHz, CD3OD) δ : 8.21 (d, J=8.0 Hz, 1H), 8.13 (s, 1H), 7.66 - 7.79 (m, 2H), 7.59 (d, J=8.0 Hz, 1H), 3.98 (s, 2 H), 3.73 - 3.84 (m, 1H), 1.12 - 1.32 (m, 4H), 0.90 - 1.02 (m, 1H), 0.73 (J=6.0 Hz, 1H). MS m / z: 482.1[M+H] + .

[0165] Compound 4B: Retention time in SFC analysis (column: Chiralpak IC-3, 50×4.6 mm ID, 3 um, mobile phase: A (CO2) and B (EtOH with 0.1% isopropylamine), gradient: B%=40%) was 1.479 min, ee=98.83%. 1H NMR (400 MHz, CD3OD) δ : 8.23 ​​(d, J = 8.4 Hz, 1H), 8.13 (s, 1H), 7.76 (d, J = 6.0 Hz, 1H), 7.71 (s, 1H), 7.60 (dd, J = 8.4, 1.2 Hz, 1H), 3.97 (d, J = 3.2 Hz, 2H), 3.73 - 3.82 (m, 1H), 1.19 - 1.26 (m, 1H), 1.13 - 1.17 (m, 3H), 0.92 - 1.02 (m, 1 H), 0.73 (q, J=6.4 Hz, 1H). MS m / z: 482.1[M+H] + .

[0166] Example 5 [ka]

[0167] Step 1 2-(Methylsulfonyl)ethanol (587.42 mg) was added to N,N-dimethylformamide (6 mL) and the reaction system was protected with nitrogen gas. Sodium hydrogen (189.23 mg, purity: 60%) was added in batches at 0°C and stirred for 0.5 hours. A solution of compound M4 (600 mg) in N,N-dimethylformamide (6 mL) was added dropwise at 0°C and then stirred at 20°C for 1 hour. Water (30 mL) was added to dilute and extracted with ethyl acetate (10 mL x 2). The aqueous phase was adjusted to pH = 2-3 with 1 M hydrochloric acid, then extracted with ethyl acetate (15 mL x 3), the organic phase was combined, washed with saturated saline (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 5-1. MS m / z: 252.0 [M+H] + .

[0168] Step 2 Compound 5-1 (500 mg) was added to dichloromethane (10 mL), followed by the addition of pyridine (628.67 mg). Trifluoromethanesulfonic anhydride (840.89 mg) was added at 0°C, and the reaction solution was stirred at 20°C for 1 hour. Water (20 mL) was added, stirred for 5 minutes, and the solution was separated. The aqueous phase was extracted with dichloromethane (10 mL), and the organic phases were combined, washed with saturated saline (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by flash silica gel column (ethyl acetate / petroleum ether, ethyl acetate ratio: 0-20%) to obtain compound 5-2. MS m / z: 383.9[M+H] + .

[0169] Step 3 Compound 5-2 (600 mg) was added to N,N-dimethylformamide (9 mL), followed by the addition of trimethylsilylacetylene (168.94 mg), bis(triphenylphosphine)palladium(II) dichloride (109.76 mg), cuprous iodide (14.89 mg) and triethylamine (237.34 mg), and the reaction solution was stirred at 20°C for 3 hours. Water (30 mL) was added, followed by the addition of ethyl acetate (20 mL), and the mixture was stirred for 5 minutes and filtered. The filtrate was separated, and the aqueous phase was extracted with ethyl acetate (10 mL x 2). The organic phases were combined, washed with saturated saline, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by flash silica gel column (ethyl acetate / petroleum ether, ethyl acetate ratio: 0-20%) to obtain compound 5-3. MS m / z: 332.1 [M+H] + .

[0170] Step 4 Compound 5-3 (170 mg) was added to acetic acid (3 mL), then N-iodosuccinimide (230.51 mg) was added, and the reaction solution was stirred at 20° C. for 12 hours. The reaction solution was concentrated under reduced pressure, saturated sodium bicarbonate solution (10 mL) was added, ethyl acetate (10 mL) was added, and the mixture was stirred for 5 minutes, and the solution was separated. The aqueous phase was extracted with ethyl acetate (5 mL), and the organic phases were combined, washed with saturated saline (5 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by flash silica gel column (ethyl acetate / petroleum ether, ethyl acetate ratio: 0-20%) to obtain compound 5-4. MS m / z: 457.9 [M+H] + .

[0171] Step 5 Compound 5-4 (50 mg) was added to 1,4-dioxane (2 mL), water (0.2 mL) was added, and then compound M5 (65.75 mg), [1,1'-bis(di-tert-butylphosphino)ferrocene]palladium(II) dichloride (3.56 mg) and potassium phosphate (46.37 mg) were added, and the reaction solution was stirred at 80°C for 12 hours under nitrogen gas protection. The reaction solution was cooled to room temperature, water (5 mL) was added, ethyl acetate (5 mL) was added, stirred for 5 minutes, and filtered. The filtrate was separated, and the aqueous phase was extracted with ethyl acetate (5 mL). The organic phases were combined, washed with saturated saline (5 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by preparative thin layer chromatography (petroleum ether:ethyl acetate = 1:1) to obtain compound 5-5. MS m / z: 533.2 [M + H] + .

[0172] Step 6 Compound 5-5 (5 mg) was added to dichloromethane (0.6 mL), then trifluoroacetic acid (0.2 mL) was added, and the reaction solution was stirred at 20° C. for 0.5 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by preparative high performance liquid chromatography (column: Welch Xtimate C18 100×40 mm×3 μm, mobile phase: [water (trifluoroacetic acid)-acetonitrile], gradient (acetonitrile%): 10% to 40%) to obtain the trifluoroacetate salt of compound 5.

[0173] MS m / z: 433.1 [M+H] + . 1 H NMR (400 MHz, CD3OD) δ 8.28 (d, J=8.03 Hz, 1H), 8.18 (s, 1H), 8.14 (d, J=7.03 Hz, 1H), 7.85 (s, 1H), 7.50 (d, J=10.04 Hz, 1H), 4.51 (br d, J=5.02 Hz, 2H), 4.28 (s, 1H), 3.86 (s, 3H).

[0174] Biological Test Data Experimental Example 1: In vitro HCT116 WT / MTAP KO cell proliferation experiment Test materials: McCoy's 5A medium, penicillin / streptomycin antibiotics were purchased from Wisent, and fetal bovine serum was purchased from Biosera. 3D CellTiter-Glo (cell viability chemiluminescence detection reagent) reagent was purchased from Promega. HCT116 WT cell line was purchased from Nanjing Cobioer Biosciences Co., Ltd. HCT116 MTAP KO cells were purchased from Horizon. Envision multilabel analyzer (PerkinElmer).

[0175] Testing Method: HCT116 WT / MTAP KO cells were seeded into ultra-low attachment 96-well U-shaped plates with 80 μL / well of cell suspension containing 1000 HCT116 cells. The cell plates were cultured overnight in a carbon dioxide incubator.

[0176] The test compounds were diluted 5-fold with a pipette into eight concentrations, i.e., from 2 mM to 25.6 nM, and a double-well experiment was set up. 78 μL of medium was added to the middle plate, and then the gradient-diluted compounds were transferred to the middle plate at 2 μL / well according to the corresponding positions, mixed uniformly, and then transferred to the cell plate at 20 μL / well. The concentration range of the compounds transferred to the cell plate was 10 μM to 0.128 nM. The cell plate was cultured in a carbon dioxide incubator for 10 days. Another cell plate was prepared, and the signal value on the day of drug administration was read as the maximum value (Max value in the formula below) and participated in the data analysis.

[0177] 100 μL / well of cell viability chemiluminescence detection reagent was added to the cell plate and incubated at room temperature for 10 minutes to stabilize the luminescence signal. The measurements were read using a multilabel analyzer.

[0178] Data Analysis: Convert raw data into percentage inhibition using the equation (Sample-Min) / (Max-Min) × 100% to IC 50 The values ​​of were obtained from curve fitting with four parameters (obtained in the "log(inhibitor) vs. response--Variable slope" mode of GraphPad Prism). Table 1 shows the inhibitory activity of the compounds of the present invention against the proliferation of HCT116WT / MTAP KO cells.

[0179] Table 1: In vitro screening test results for compounds of the present invention [Table 1] Conclusion: The compounds of the present invention had significant inhibitory activity against MTAP-deficient HCT116 cells and showed weak inhibitory activity against wild-type tumor cells, demonstrating good selectivity.

[0180] Experimental Example 2: Enzyme Inhibitory Activity Test Experimental objective: To test the inhibitory effect of the compounds of the present invention on the PRMT5·MTA complex. Experimental materials: PRMT5 / MEP50, Polypeptide H4 (1-21), LANCE® Ultra Europium Anti-Methyl Histone H4 Arginine 3 (H4R3me) Antibody, Ulight Streptavidin, LANCE Detection Buffer, SAM (S-Adenosylmethionine), Pig Skin Collagen, MTA (Methylthioadenosine)

[0181] Testing Method: (1) Preparation of buffer solution: Take 10 ml as an example. 10 mM MTA: 10 mg of MTA was added to 3296 μl of DMSO, dissolved, dispensed, and stored at -80° C. It was prepared on the day of the experiment when used, and the preparation scheme is as shown in Tables 2 and 3.

[0182] Table 2. Scheme for preparing buffer solutions when MTA is added [Table 2]

[0183] Table 3. Scheme for preparing buffer solutions without adding MTA [Table 3]

[0184] (2) Preparation of Compounds Compounds were dissolved in DMSO to obtain a compound mother solution with a concentration of 10 mM. A gradient dilution was performed in the compound dilution plate to obtain four compound wells with sequential concentrations of 1 mM, 37.037 μM, 1.3717 μM, and 0.0508 μM. These four concentrations of compounds were transferred to the compound transfer plate with a transfer volume of 8 μL per concentration. DMSO was added to the empty wells of the compound transfer plate for later use. The serial dilution function of the Echo550 liquid handler was used to transfer the liquid to the experimental plate, and the experimental plate was obtained once the liquid transfer was completed.

[0185] (3) Reaction A mixed solution of the enzyme solution and the substrate was prepared using a buffer solution, in which the concentration of PRMT5 was 7.6 nM, the concentration of polypeptide H4(1-21) was 0.32 μM, and the concentration of SAM was 2.6 μM.

[0186] Using an electronic multichannel pipette, PRMT5 solution was added to the compound wells and negative control wells of the experimental plate at a volume of 5 μL / well, and the same volume of buffer was added to the positive control well. Using a centrifuge, the plates were centrifuged at 1000 rpm for 1 minute, and then the experimental plates were placed in a constant temperature incubator and incubated at 25°C for 30 minutes. Next, using the same method, the substrate mixture solution was added to the positive control, negative control, and compound wells of the experimental plate, centrifuged, and incubated at 25°C for 90 minutes.

[0187] (4) Detection and Calculation of Results Principle: In this experiment, detection was performed using PE's time-resolved fluorescence resonance energy transfer technology (LANCE® Ultra). During the reaction, after PRMT methylated the substrate polypeptide H4 (1-21), two antibodies were added, among which LANCE® Ultra Europium anti-methyl histone H4 arginine 3 (H4R3me) antibody specifically bound to the methylation site on polypeptide H4 (1-21) as the energy donor, and Ulight specifically bound to the biotin tag on polypeptide H4 (1-21) as the energy acceptor. When excited with a laser of a certain wavelength (the excitation light wavelength in this experiment is 340 nm), the energy donor can emit an emission light with a wavelength of 615 nm, and at the same time, if the spatial distance between the energy donor and the energy acceptor is close enough (i.e., the two antibodies are bound to the polypeptide H4 (1-21) at the same time), energy transfer can occur between the energy donor and the energy acceptor, so that the energy acceptor can emit an emission light with a wavelength of 665 nm. The two emitted lights are detected using a plate reader, and the ratio of the two signals at 665 nm and 615 nm is calculated, plotted, and calculated to determine the relevant parameters of the test sample.

[0188] The antibody mixture solution was prepared using LANCE buffer, the concentration of LANCE® Ultra Europium-anti-methyl-Histone H4 Arginine 3 (H4R3me) Antibody was 4 nM, and the concentration of Ulight was 53.3 nM. Using an electronic multichannel pipette, the detection solution was added to the positive control, negative control, and compound wells of the experimental plate in a volume of 10 μL / well, centrifuged, incubated at room temperature for 1 h, and read using a plate reader Envision 2104. The compound inhibition rate was calculated using the interpolation method, and the compound inhibition curve was drawn using 4-parameter logistic curve and XLfit software to calculate the minimum inhibition rate, maximum inhibition rate, and IC 50 The relevant parameters such as

[0189] Conclusion: The compounds of the present invention had significant inhibitory effects on the PRMT5·MTA complex.

[0190] Experimental Example 3: In vitro microsomal stability testing of compounds Experimental materials Liver microsomes: Human and animal microsomes were purchased from Corning or Xenotech and stored in a -80°C refrigerator. Reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH), Supplier: Chem-impex international, Catalogue Number: 00616. Control compounds: testosterone, diclofenac, propafenone.

[0191] Experimental procedure 2.1 Preparation of working solutions Stock solution: 10 mM in DMSO Preparation of working concentration: 100% acetonitrile diluted to 100 μM (content of organic phase: 99% acetonitrile, 1% DMSO).

[0192] 2.2 Experimental procedure Two 96-well culture plates, named T60 culture plate and NCF60 culture plate, were prepared.

[0193] 445 μL of the microsome working solution (liver microsomal protein concentration was 0.56 mg / mL) was added to each of the T60 culture plate and the NCF60 culture plate, and then the above culture plates were placed in a water bath at 37° C. and pre-incubated for about 10 minutes.

[0194] After the preliminary incubation was completed, 5 μL of the working solution of the test product or control compound was added to the T60 culture plate and the NCF60 culture plate, respectively, and mixed uniformly. 50 μL of potassium phosphate buffer was added to each well of the NCF60 culture plate to start the reaction, 180 μL of stop solution (acetonitrile solution containing 200 ng / mL tolbutamide and 200 ng / mL labetalol) and 6 μL of NADPH regenerating system working solution were added to the T0 stop plate, and 54 μL of the sample was transferred from the T60 culture plate to the T0 stop plate (generating the T0 sample). 44 μL of the NADPH regenerating system working solution was added to each well of the T60 culture plate to start the reaction. 54 μL of the microsome working solution, 6 μL of the NADPH regenerating system working solution, and only 180 μL of the stop solution were added to the blank plate. Thus, in the samples of test products or control compounds, the final reaction concentrations of the compounds, testosterone, diclofenac and propafenone were 1 μM, the concentration of liver microsomes was 0.5 mg / mL, and the final concentrations of DMSO and acetonitrile in the reaction system were 0.01% (v / v) and 0.99% (v / v), respectively.

[0195] After incubation for an appropriate time (e.g., 5, 15, 30, 45, and 60 min), 180 μL of stop solution (200 ng / mL tolbutamide and 200 ng / mL labetalol in acetonitrile) was added to the sample wells of each stop plate, and then 60 μL of sample was removed from the T60 incubation plate to stop the reaction.

[0196] All sample plates were shaken well and centrifuged at 3220×g for 20 min, then 80 μL of supernatant was taken from each well and diluted with 240 μL of pure water for liquid chromatography tandem mass spectrometry. The experimental results are shown in Table 4.

[0197] Table 4: Liver microsomal stability test results for compounds of the present invention [Table 4] Conclusion: The compounds of the present invention showed moderate metabolism in the liver microsomal assay.

[0198] Experimental Example 4: In vivo pharmacokinetics experiment in mice Experimental Objective: To study the in vivo pharmacokinetic properties of compounds of the present invention in CD-1 mice. Experimental materials: CD-1 mice (female, 15-30 g, 6-9 weeks, Beijing Vital River Laboratory Animal Technology Co., Ltd.)

[0199] Experimental Procedure: According to standard protocols, the pharmacokinetic properties of the compounds in rodents after intravenous injection and oral administration were tested. In the experiments, the candidate compounds were formulated into a clear solution and administered intravenously to mice in a single dose, and formulated into a homogeneous suspension and administered orally to mice in a single dose. The dose for intravenous injection was 3mg / kg, and the solvent was 5% DMSO / 10% Solutol / 85% water. The oral dose was 30mg / kg, and the solvent was 0.5% MC(4000cps) / 0.2% Tween80. Whole blood samples were collected within 24 hours and centrifuged at 3000g at 4℃ for 15 min, the supernatant was separated to obtain plasma samples, 20 volumes of acetonitrile solution containing internal standard was added to precipitate proteins, vortexed for 10 min, and then centrifuged at 3220g at 4℃ for 15 min, the supernatant was transferred to a 96-well plate and centrifuged at 3220g at 4℃ for 5 min, the supernatant was collected to perform analysis, and the blood drug concentration was quantitatively analyzed using LC-MS / MS analysis, and the pharmacokinetic parameters were calculated.

[0200] The experimental results are shown in Table 5.

[0201] Table 5: Pharmacokinetic study results in CD-1 mice [Table 5] Conclusion: The compounds of the present invention have good oral absorption bioavailability and plasma exposure in mice, and have good pharmacokinetic properties.

[0202] Experimental Example 5: In vivo pharmacokinetic study in beagle dogs Experimental Objective: To study the pharmacokinetic properties of compounds of the present invention in Beagle dogs. Experimental materials: Beagle dogs (male, ≥ 6 months, Beijing Marshall Biotechnology Co., Ltd.)

[0203] Experimental Procedure: According to the standard protocol, the pharmacokinetic properties of the compound in dogs after intravenous injection and oral administration were tested. In the experiment, the test compound was formulated into a clear solution and administered to dogs for a single intravenous injection and a single oral administration. The intravenous injection dose was 2mg / kg, the solvent was 5% DMSO / 10% Solutol / 85% water, and the oral administration dose was 10mg / kg, the solvent was 5% DMSO / 10% Solutol / 85% water. Whole blood samples were collected within 48 hours, centrifuged at 4℃ and 3000g for 15 minutes, and the supernatant was separated to obtain plasma samples, and 20 volumes of acetonitrile solution containing internal standard were added to precipitate proteins, vortexed for 10 minutes, and then centrifuged at 4℃ and 3220g for 15 minutes. The supernatant was transferred to a 96-well plate and centrifuged at 4℃ and 3220g for 5 minutes. The supernatant was collected to perform analysis, and the blood drug concentration was quantitatively analyzed using LC-MS / MS analysis, and the pharmacokinetic parameters were calculated.

[0204] Experimental results: The experimental results are shown in Table 6.

[0205] Table 6: Pharmacokinetic study results in beagle dogs [Table 6] Conclusion: The compounds of the present invention have good oral absorption bioavailability and plasma exposure in beagle dogs, and have good pharmacokinetic properties.

[0206] Experimental Example 6: In vivo efficacy test Purpose of the Test: The tumor inhibitory effects of the compounds of the present invention in a human large cell lung cancer LU99 subcutaneous xenograft tumor model are examined.

[0207] Testing Method: Female BALB / c nude mice were subcutaneously inoculated with human large cell lung cancer LU99 cell line, and after inoculation, they were randomly divided into groups of 6 mice each according to body weight and tumor volume, and drug administration was performed as described below.

[0208] Group 1 (solvent group): After inoculation, the tumor volume was 207±9 mm 3 When the rats reached this age, drug administration was started, and the vehicle (5% DMSO / 10% Solutol / 85% double-distilled water) was administered intragastrically at a dose of 0.1 mL / 10 g once a day.

[0209] Group 2 (treatment group): After inoculation, tumor volume was 207±10 mm 3 When the rats reached the age of 18, drug administration was started, and a test compound (dissolved in 5% DMSO / 10% Solutol / 85% double distilled water) was intragastrically administered at a dose of 12.5 mg / kg once a day.

[0210] Group 3 (treatment group): After inoculation, tumor volume was 207±10 mm 3 When the rats reached the age of 10, drug administration was started, and a test compound (dissolved in 5% DMSO / 10% Solutol / 85% double distilled water) was intragastrically administered at a dose of 25 mg / kg once a day.

[0211] Group 4 (treatment group): After inoculation, tumor volume was 207±14 mm 3 When the rats reached the age of 10, drug administration was started, and a test compound (dissolved in 5% DMSO / 10% Solutol / 85% double distilled water) was intragastrically administered at a dose of 50 mg / kg once a day.

[0212] After the experiment, the mice were divided into groups, and their body weights were measured twice a week. The diameters of the tumors were measured with calipers to calculate the tumor volumes, and the tumor growth inhibition rates (TGI) were calculated. On the 29th day after the experimental administration, the mice were euthanized and samples were collected. The calculation formula is as follows: Tumor volume = 0.5 major axis x width2 .

[0213] TGI(%)=[1-(T i -T0) / (C i -C0)] × 100%, where T i is the average tumor volume of a given treatment group on a given day, T0 is the average tumor volume of that treatment group at the start of treatment, and C i One day (T i C0 is the average tumor volume of the control group at the start of administration (the same day as the start of administration), and C0 is the average tumor volume of the control group at the start of administration.

[0214] Experimental results: The experimental results are shown in Table 7.

[0215] Table 7. Evaluation of the tumor-inhibitory effect of the compounds of the present invention on human large cell lung cancer LU99 subcutaneous xenograft tumor model. [Table 7] *: Calculated from tumor volume 29 days after administration

[0216] Conclusion: The compounds of the present invention showed significant tumor inhibition effects in human large cell lung cancer LU99 cell subcutaneous xenograft tumor models, and the body weight of mice was well maintained after administration.

Claims

1. A compound represented by formula (III) or a pharma- ceutically acceptable salt thereof. 【Chemistry 1】 (however, R １ is C ２－５ Alkenyl, C ２－５ Alkynyl, —O—C ３－５ Cycloalkyl, —O—C ２－３ Alkynyl and C ３－５ cycloalkyl, wherein C ２－５ Alkenyl, C ２－５ Alkynyl, —O—C ３－５ Cycloalkyl, —O—C ２－３ Alkynyl and C ３－５ Cycloalkyl, each independently, optionally has 1, 2 or 3 R ａ is replaced by R ２ is halogen and C ２－３ alkynyl, ２－３ The alkynyl is optionally substituted by 1, 2 or 3 halogens; R ３ is CH ３ and CDs ３ is selected from R ４ is selected from H and halogen; Each R ａ are each independently a halogen or C １－３ alkyl, １－３ The alkyl is optionally substituted with 1, 2 or 3 halogens; and, (1) R ２ When is selected from halogen, R １ is C ２－５ Alkenyl, C ２－５ Alkynyl, —O—C ３－５ Cycloalkyl, —O—C ２－３ Alkynyl and C ３－５ cycloalkyl, wherein C ２－５ Alkenyl, C ２－５ Alkynyl and —O—C ２－３ Alkynyl optionally has 1, 2 or 3 R ａ and is substituted by the -O-C ３－５ Cycloalkyl and C ３－５ Cycloalkyl is one, two or three R ａ is replaced by (2) R ２ When is selected from halogen, R ３ CD ３ is selected from (3) R ２ When is selected from halogen, R ４ is selected from halogen, and at least one of the following conditions is satisfied.

2. Each R ａ are each independently F, Cl, or CH ３ , C.H.F. ２ and C.F. ３ 2. The compound of claim 1, selected from:

3. R １ is C ２－３ Alkenyl, C ２－３ Alkynyl, —O-cyclopropyl, —O—C ２－３ alkynyl and cyclopropyl; ２－３ Alkenyl, C ２－３ Alkynyl, —O-cyclopropyl, —O—C ２－３ Alkynyl and cyclopropyl each independently optionally have 1, 2 or 3 R ａ or R １ teeth, 【Chemistry 2】 and 【Chemistry 3】 each independently optionally represents 1, 2 or 3 R ａ or a pharma- ceutically acceptable salt thereof.

4. R １ teeth, 【Chemistry 4】 4. The compound of claim 3, selected from:

5. R ２ is F, Cl and 【Chemistry 5】 and 【Chemistry 6】 is optionally substituted with 1, 2 or 3 halogens, or R ２ is Cl, 【Chemistry 7】 2. The compound of claim 1, selected from:

6. R ２ is selected from Cl; R １ teeth, 【Chemistry 8】 2. The compound of claim 1, selected from:

7. R ２ is selected from Cl; R ３ CD ３ 2. The compound of claim 1, selected from:

8. R ４ is selected from H and F, or a pharma- ceutically acceptable salt thereof.

9. 9. The compound according to any one of claims 1 to 8, selected from the following formulas: or a pharma- ceutically acceptable salt thereof. 【Chemistry 9】 (However, R ２ , R ３ and R ａ is as defined in any one of claims 1 to 8.

10. 10. The compound of claim 9 selected from the following formulas: or a pharma- ceutically acceptable salt thereof. 【Chemistry 10】 (However, R ３ and R ａ is as defined in claim 9.

11. 10. The compound of claim 9 selected from the following formulas: or a pharma- ceutically acceptable salt thereof. 【Chemistry 11】 (However, R ａ is as defined in claim 9.

12. 12. The compound of claim 11 selected from the following formulas: or a pharma- ceutically acceptable salt thereof. 【Chemistry 12】 (However, R ａ is as defined in claim 11.

13. A compound represented by the following formula or a pharma- ceutically acceptable salt thereof: 【Chemistry 13】 【Chemistry 14】

14. 14. The compound of claim 13 selected from the following formulas: or a pharma- ceutically acceptable salt thereof. 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 【Chemistry 18】 【Chemistry 19】

15. Use of a compound according to any one of claims 1 to 14 or a pharma- ceutically acceptable salt thereof in a medicine relating to a PRMT5·MTA complex inhibitor.

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