PRMT5 inhibitors and uses thereof

Abstract

The present invention provides a class of compounds having methyltransferase inhibitory activity, specifically, the present invention provides a novel PRMT5 inhibitor represented by formula (I) and its use. The compound can be used to prepare a pharmaceutical composition for treating diseases related to PRMT5 activity.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical compounds. Specifically, the present invention provides a class of compounds for inhibiting PRMT5, and use thereof in pharmaceutical compositions. Background Art

[0002] Epigenetic regulation of gene expression is an important biological determinant of protein production and cellular differentiation and plays a major pathogenic role in many human diseases.

[0003] Epigenetic regulation involves heritable modifications of genetic material without changing its nucleotide sequence. Typically, epigenetic regulation is mediated by selective and reversible modifications (e.g., methylation) of DNA and proteins (e.g., histones) that control conformational transitions between transcriptionally active and inactive states of chromatin. These covalent modifications can be controlled by enzymes such as methyltransferases (e.g., PRMT5), many of which are associated with specific genetic alterations that may cause human disease. PRMT5 plays a role in diseases such as proliferative, metabolic, and hematological disorders.

[0004] PRMT5 is a known cellular essential gene, and conditional PRMT5 knockout and siRNA knockdown studies have shown that PRMT5 inhibition in normal tissues is associated with a range of diseases (e.g., pancytopenia, infertility, skeletal muscle loss, cardiac hypertrophy). Therefore, new strategies are needed to exploit this metabolic vulnerability and preferentially target PRMT5 in MTAP-deficient tumors while sparing PRMT5 in normal tissues (MTAPWT). Targeting PRMT5 with MTA-synergistic small molecule inhibitors can preferentially target the MTA-bound state of PRMT5, which is enriched in MTAP-deficient tumor cells, while providing a therapeutic index superior to normal cells with intact MTAP and low MTA levels.

[0005] Therefore, there is a need in the art to provide new small molecule compounds that target PRMT5 in MTAP-deficient tumors. Summary of the invention

[0006] The purpose of the present invention is to provide a new class of small molecule compounds targeting PRMT5 in MTAP-deficient tumors.

[0007] The first aspect of the present invention provides a compound as shown in the following formula I, or a pharmaceutically acceptable salt thereof:

[0008]

[0009] in,

[0010] R1 is selected from the following group: -X-(CH2) n R4; wherein X is NH or O; n is 1, 2, 3 or 4;

[0011] R2 and R3 are each independently selected from the following group: hydrogen, substituted or unsubstituted C1-C6 alkyl; or R2 and R3 and the carbon atom to which they are connected together form a 5-7 membered carbocyclic ring;

[0012] R4 is selected from the group consisting of a substituted or unsubstituted 5-6 membered heteroaromatic ring;

[0013] Unless otherwise specified, in the above formulae, the substitution refers to the replacement of the hydrogen atoms on the corresponding groups by one or more substituents selected from the group consisting of halogen, unsubstituted or halogenated C1-C6 alkyl.

[0014] In another preferred embodiment, the R2 is a substituted or unsubstituted C1-C6 alkyl group.

[0015] In another preferred embodiment, the R3 is H.

[0016] In another preferred embodiment, R2 and R3 together form -(CH2)3-.

[0017] In another preferred embodiment, the R4 is unsubstituted or substituted by C1-C6 alkyl. .

[0018] In another preferred embodiment, n is 2.

[0019] In another preferred embodiment, the compound is selected from Figure 1 The compounds shown in .

[0020] In the second aspect of the present invention, a pharmaceutical composition is provided, which contains a therapeutically effective amount of the compound as described in the first aspect, one or more of its pharmaceutically acceptable salts, racemates, optical isomers, stereoisomers or tautomers, and one or more pharmaceutically acceptable carriers, excipients, adjuvants, auxiliary materials and / or diluents.

[0021] In the third aspect of the present invention, there is provided a use of the compound as described in the first aspect, its racemate, optical isomer or pharmaceutically acceptable salt in the preparation of a drug for treating or preventing diseases associated with abnormal gene levels or abnormal expression of PRMT5 (such as corresponding nucleic acid mutations, deletions, or ectopic or fused or overexpressed methyltransferase).

[0022] In another preferred embodiment, the disease is a malignant tumor or cancer, which can be selected from the following group: ovarian cancer, lung cancer, lymphoma, glioblastoma, colon cancer, melanoma, malignant peripheral nerve sheath tumor (MPNST), esophageal cancer (e.g., esophageal squamous cell carcinoma or esophageal adenocarcinoma), bladder cancer (e.g., bladder cancer, urothelial carcinoma), mesothelioma, non-small cell lung cancer (NSCLC; such as lung squamous cell carcinoma or lung adenocarcinoma), astrocytoma, undifferentiated pleomorphic sarcoma, diffuse large B-cell lymphoma (DLBCL), leukemia, head and neck cancer, gastric adenocarcinoma, myxofibrosarcoma, bile duct sarcoma, as well as brain cancer, gastric cancer, kidney cancer, breast cancer, endometrial cancer, urinary tract cancer, liver cancer, soft tissue cancer, pleural cancer and colorectal cancer or sarcoma.

[0023] The fourth aspect of the present invention provides a pharmaceutical composition, which contains a therapeutically effective amount of a compound as described in any of the preceding aspects, one or more of its pharmaceutically acceptable salts, racemates, optical isomers, stereoisomers or tautomers, and one or more pharmaceutically acceptable carriers, excipients, adjuvants, auxiliary materials and / or diluents.

[0024] The fifth aspect of the present invention provides a compound as described in any of the preceding aspects, its racemate, optical isomer or pharmaceutically acceptable salt for use in the preparation of a drug for treating or preventing diseases associated with abnormal gene levels or abnormal expression of PRMT5 (such as corresponding nucleic acid mutations, deletions, or ectopic or fused or overexpressed methyltransferase).

[0025] In another preferred embodiment, the disease is selected from the following group: the disease or condition is ovarian cancer, lung cancer, lymphoma, glioblastoma, colon cancer, melanoma, gastric cancer, pancreatic cancer or bladder cancer.

[0026] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is the structural formula of the compound claimed in the present invention.

[0028] Figure 2 These are the corresponding meanings of various abbreviations in the present invention. DETAILED DESCRIPTION

[0029] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that when a specific substituent (R1) is introduced into a PRMT5 modulator compound, the cell proliferation inhibition effect of the compound is significantly enhanced and the selectivity is improved. On this basis, the inventors completed the present invention.

[0030] the term

[0031] In the present invention, the halogen is F, Cl, Br or I.

[0032] In the present invention, unless otherwise specified, the terms used have the general meanings known to those skilled in the art. In the present invention, if not otherwise specified, all chemical formulae are intended to cover any possible optical or geometric isomers (e.g., R-type, S-type or racemate, or cis-trans isomers of olefins, etc.).

[0033] In the present invention, the term "C1-C6 alkyl" refers to a straight or branched alkyl group having 1 to 6 carbon atoms, including but not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl and hexyl, etc.; preferably ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl and tert-butyl.

[0034] In the present invention, the term "aromatic heterocycle" or "heteroaryl" has the same meaning and refers to a heteroaromatic group containing one to multiple heteroatoms. The heteroatoms referred to here include oxygen, sulfur and nitrogen. For example, furanyl, thienyl, pyridyl, pyrazolyl, pyrrolyl, N-alkylpyrrolyl, pyrimidyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring can be fused to an aryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring. The heteroaryl group can be optionally substituted or unsubstituted.

[0035] Pharmaceutical compositions and methods of administration

[0036] Since the compounds of the present invention have excellent methyltransferase inhibitory activity, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat, prevent and alleviate related diseases caused by abnormal activity or expression of methyltransferases (such as PRMT5).

[0037] The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, and more preferably, contains 5-200 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.

[0038] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween®), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0039] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administrations include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0040] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0041] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0042] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0043] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0044] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.

[0045] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0046] Dosage forms for topical administration of the compounds of the invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0047] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds. In some preferred embodiments, the compounds of the present invention can be administered together with other small molecule compounds to form PROTAC, or together with other macromolecular compounds such as monoclonal antibodies to form ADC.

[0048] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg, preferably 5 to 500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill range of a skilled physician.

[0049] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0050] The definitions of each abbreviation are as follows Figure 2 shown.

[0051] The raw materials can be obtained from commercial sources or prepared by methods known or disclosed in the art.

[0052] Purification of intermediates and compounds is carried out by normal or reverse phase chromatography or by recrystallization and other conventional laboratory chemical procedures.

[0053] Normal phase chromatography is a pre-packed silica gel column or preparative thin layer chromatography. The silica gel column is mainly a glass column or a rapid preparative chromatograph. The mobile phase of normal phase chromatography is selected and proportioned from petroleum ether / ethyl acetate, dichloromethane / methanol or other suitable solvents for elution. Reverse phase preparative liquid chromatography uses a C18 column, and is performed using a preparative liquid chromatograph or a rapid preparative chromatograph, 214nM and 254nM or a preparative liquid chromatography-mass spectrometry instrument for detection, and uses water / acetonitrile containing 0.1% hydrochloric acid, water / acetonitrile, water / acetonitrile containing 0.1% ammonium bicarbonate, water / acetonitrile containing 0.1% formic acid, water / acetonitrile containing 0.1% ammonia water / acetonitrile, water / acetonitrile containing 0.1% trifluoroacetic acid or other suitable solvent systems as mobile phases for gradient elution.

[0054] The structures of intermediates and compounds were characterized by nuclear magnetic resonance (NMR) and mass spectrometry (LCMS). The NMR spectrometer used for NMR was Bruker Ascend 400 or Varian 400 or ZKNJ BIXI-1 300MHz or Bruker Avance III 400 MHz or Bruker AVANCE Neo 400 MHz. The solvents used were deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol or other deuterated solvents as noted. Spectral data were reported in the mode: chemical shift δ (peak splitting number, coupling constant J (Hz), number of hydrogens). Tetramethylsilane was used as an internal standard for chemical shift and its chemical shift was set to zero (δ, 0 ppm). The meanings of some abbreviations are: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad peak).

[0055] LCMS spectra were acquired on an Agilent 1260–6120 system equipped with an SQ mass spectrometer detector using a Waters CORTEC S-18, 2.7 μm, 4.6*30 mm column (solvent A: 0.05% formic acid (FA) in water; solvent B: 0.05% FA in acetonitrile (ACN); change from 5% ACN to 95% ACN within 1.0 min, hold for 1.0 min, total 2.5 min; flow rate: 1.8 mL / min; column temperature 40°C) or an XSelect CSH C18, 3.5 μm, 4.6*50 mm column (solvent A: 0.05% NH3 in water; solvent B: 0.05% NH3 in acetonitrile; change from 5% ACN to 95% ACN within 1.0 min, hold for 1.0 min, total 2.5 min; flow rate: 1.8 mL / min; column temperature 40°C). HPLC analysis was performed on a Waters Acquity UPLCH-Class instrument using an Acquity BEHC18, 1.7 μm, 50*2.1 mm column (solvent A: 0.05% trifluoroacetic acid (TFA) in water; solvent B: 0.05% TFA in acetonitrile; 5% acetonitrile to 95% acetonitrile took 2.0 min, held for 0.5 min, and re-equilibrated to 5% ACN took 2.7 min; flow rate: 0.5 mL / min; column temperature 45 °C).

[0056] Embodiment Synthesis general method:

[0057]

[0058] General Method: Synthesis of Intermediate B1

[0059]

[0060] Step 1: Preparation of 5-(2-((tert-butyldimethylsilyl)oxy)ethyl)-1-methyl-1H-pyrazole (Compound B1-3)

[0061] To a solution of 1-methyl-1H-pyrazole (5.0 g, 0.06 mol) in THF (50 mL) was added n-BuLi (2.4 M hexane solution, 30 mL, 0.07 mol) at -78 °C. The mixture was stirred at -78 °C for 1 hour. Then (2-bromoethoxy)(tert-butyl)dimethylsilane (18 g, 0.07 mol) was added dropwise. The mixture was stirred at -78 °C for 1 hour. The temperature of the reaction mixture was then raised to 25 °C. The reaction was stirred for 18 hours. The mixture was poured into ice water (50 mL) and EtOAc (50 mL × 3) was added. The organic layer was separated, dried (Na2SO4), filtered and the solvent was evaporated. The residue was purified by silica gel chromatography eluting with EA / PE from 0 to 30% in 20 min to give 5-(2-((tert-butyldimethylsilyl)oxy)ethyl)-1-methyl-1H-pyrazole (3.0 g, 18%) as a yellow oil.

[0062] LC-MS: Rt =1.401 min, (ESI) m / z . [M+H] + 241.2.

[0063] Step 2: Preparation of 2-(1-methyl-1H-pyrazol-5-yl)ethan-1-ol (Compound B1-4)

[0064] 5-(2-((tert-butyldimethylsilyl)oxy)ethyl)-1-methyl-1H-pyrazole (3.0 g, 13 mmol) was dissolved in HCl / MeOH (4 mol / L, 10 mL) and stirred at 25°C for 2 hours. The mixture was concentrated under reduced pressure and then dried in vacuo to give 2-(1-methyl-1H-pyrazol-5-yl)ethan-1-ol (1.5 g, 76%) as a yellow oil.

[0065] LC-MS: Rt =0.390 min, (ESI) m / z . [M+H] + 127.1.

[0066] Step 3: Preparation of 2-(1-methyl-1H-pyrazol-5-yl)ethyl methanesulfonate (Compound B1-5)

[0067] To a mixture of 2-(2-methylpyrazol-3-yl)ethanol (550 mg, 4.4 mmol) and triethylamine (1.3 g, 13 mmol) in DCM (20 mL) was added MsCl (1.50 g, 13 mmol) at 0 ° C. The mixture was stirred at 25 ° C for 14 hours. Water (20 mL) was added to the mixture. The mixture was extracted with DCM (20 mL×3). The combined organic layers were dried over Na2SO4 and concentrated to dryness to give 2-(1-methyl-1H-pyrazol-5-yl)ethyl methanesulfonate (632 mg, crude product) as a yellow oil.

[0068] LC-MS: Rt = 0.924 min, (ESI) m / z . [M+H] + 205.1.

[0069] Step 4: Preparation of 5-(2-(1-methyl-1H-pyrazol-5-yl)ethoxy)pyridinecarboxaldehyde (Compound B1-7)

[0070] 5-hydroxypyridinecarboxaldehyde (0.64g, 5.1mmol) and cesium carbonate (2.2g, 6.9mmol) were added to the stirred 2-(1-methyl-1H-pyrazole-5-yl)ethyl methanesulfonate (0.7g, 3.4mmol) solution. The reaction mixture was stirred for 2 hours at 25 ° C. The reaction mixture was filtered through diatomaceous earth and then washed with MeOH (20mL). The filtrate was concentrated under reduced pressure to obtain 5-(2-(1-methyl-1H-pyrazole-5-yl)ethoxy)pyridinecarboxaldehyde (800mg, crude product), which was a yellow oil.

[0071] LC-MS: Rt =1.090 min, (ESI) m / z . [M+H] + 232.2.

[0072] Step 5: Preparation of N-((5-(2-(1-methyl-1H-pyrazol-5-yl)ethoxy)pyridin-2-yl)methyl)-1-(pyrimidin-2-yl)ethane-1-amine (Compound B1-9)

[0073] To a solution of 5-[2-(2-methylpyrazol-3-yl)ethoxy]pyridine-2-carboxaldehyde (100 mg, 0.43 mmol) in DCM (5 mL) were added 1-(pyrimidin-2-yl)ethylamine (59 mg, 0.48 mmol), sodium triacetoxyborohydride (275 mg, 1.30 mmol) and HOAc (3 mg, 0.04 mmol). The reaction mixture was stirred at 25 °C for 1 hour. The reaction mixture was quenched with H2O (10 mL) and then extracted with DCM (10 mL×3). The combined organic phases were dried over sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography eluting with EA in PE from 0 to 20% in 10 min to give N-((5-(2-(1-methyl-1H-pyrazol-5-yl)ethoxy)pyridin-2-yl)methyl)-1-(pyrimidin-2-yl)ethan-1-amine (50 mg, 34%) as a yellow oil.

[0074] LC-MS: Rt =0.878 min, (ESI) m / z . [M+H] + 339.2.

[0075] Example 1 Synthesis of Compound 1

[0076]

[0077] 2-Amino-3-methylquinoline-6-carboxylic acid (72 mg, 0.35 mmol) was dissolved in SOCl2 (5 mL) and stirred at 80°C for 1 hour. The mixture was concentrated to dryness. The residue was added to a solution of 2-amino-3-methyl-N-((5-(2-(1-methyl-1H-pyrazol-5-yl)ethoxy)pyridin-2-yl)methyl)-N-(1-(pyrimidin-2-yl)ethyl)quinoline-6-carboxamide (120 mg, 0.35 mmol) and Et3N (72 mg, 0.71 mmol) dissolved in THF (10 mL). The mixture was stirred at 25°C for 13 hours. The residue was purified by preparative HPLC (column: XbridgeprepC18 10umOBD19×150mm / WELCHXtimateC18 21.2×250mm10um conditions: A water (0.1% FA) B (acetonitrile) 10-20% B in 8 minutes, hold at 100% B for 2 minutes, return to 5% B in 0.5 minutes, stop at 13 minutes) to give 2-amino-3-methyl-N-((5-(2-(1-methyl-1H-pyrazol-5-yl)ethoxy)pyridin-2-yl)methyl)-N-(1-(pyrimidin-2-yl)ethyl)quinoline-6-carboxamide (12 mg, 6.5%) as a white solid.

[0078] 1 H NMR (400 MHz, DMSO- d 6) δ 8.78 (d, J = 4.8 Hz, 2H), 8.32 (s, 1H), 8.16(s, 1H), 7.78 (s, 1H), 7.60 – 7.45 (m, 1H), 7.41 – 7.36 (m, 1H), 7.35 – 7.29(m, 1H), 7.28 – 7.24 (m, 1H), 6.46 (brs, 2H), 6.14 (d, J = 2.0 Hz, 1H), 5.39(s, 1H), 4.80 (s, 1H), 4.28 – 4.23 (m, 2H), 3.79 (s, 3H), 3.35 (s, 2H), 3.14- 3.07 (m, 2H), 2.20 (s, 2H), 1.55(d, J = 6.8 Hz, 2H). LC-MS: Rt =1.002 min, (ESI) m / z. 523.3 [M+H] + . HPLC purity: 99.60% (214 nm), 99.66% (254 nm).

[0079] Example 2 Synthesis of Compound 2

[0080]

[0081] 4-amino-2,3-dihydro-1H-cyclopenta[c]quinoline-8-carbonyl chloride (36 mg, 0.15 mmol) and Et3N (45 mg, 0.44 mmol) were added to a solution of N-((5-(2-(1-methyl-1H-pyrazol-5-yl)ethoxy)pyridin-2-yl)methyl)-1-(pyrimidin-2-yl)ethane-1-amine (50 mg, 0.15 mmol) in THF (5 mL). The reaction mixture was stirred at 25 ° C for 1 hour. The reaction mixture was quenched with H2O (10 mL) and then extracted with DCM (10 mL×3). The combined organic phases were dried over sodium sulfate and filtered, the filtrate was concentrated under reduced pressure and the residue was purified by silica gel chromatography eluting with EA in PE from 0% to 20% in 10 min to give 4-amino-N-((5-(2-(1-methyl-1H-pyrazol-5-yl)ethoxy)pyridin-2-yl)methyl)-N-(1-(pyrimidin-2-yl)ethyl)-2,3-dihydro-1H-cyclopenta[c]quinoline-8-carboxamide (2 mg, 2%) as a yellow solid.

[0082] 1 H NMR (400 MHz, MeOD) δ 9.05 (d, J = 3.6 Hz, 2H), 8.48 – 7.87 (m, 4H), 7.70 – 7.58 (m, 4H), 6.50 (d, J = 1.6 Hz, 1H), 4.60 (t, J = 6.4 Hz, 2H), 4.17 (s,3H), 3.48 (t, J = 6.4 Hz, 2H), 3.25 (s, 2H), 2.63 (s, 2H), 2.26 (s, 5H), 1.95(s, 3H), 1.59 (s, 2H), 1.20 (t, J = 6.4 Hz, 1H). LC-MS: Rt =1.009 min, (ESI) m / z . [M+H] + 549.4; HPLC purity: 94.31% (214 nm), 91.33% (254 nm).

[0083] General Method: Synthesis of Intermediate B2

[0084]

[0085] Step 1: 5-(2-azidoethyl)-1-methyl-1H-pyrazole (Compound B2-2)

[0086] NaN3 (477 mg, 7.4 mmol) was stirred and added to a DMF (50 mL) solution of 2-(1-methyl-1H-pyrazol-5-yl)ethyl methanesulfonate (0.50 g, 2.50 mmol). The reaction mixture was stirred at 80 ° C for 12 hours under N2 atmosphere. The reaction mixture was quenched with water (10 mL) and then extracted with ethyl acetate (20 mL×3). The combined organic phases were dried over sodium sulfate and then filtered. The filtrate was concentrated under reduced pressure to give 5-(2-azidoethyl)-1-methyl-1H-pyrazole (300 mg crude product), which can be used directly in the next step.

[0087] LC-MS: Rt =1.120 min, (ESI) m / z. [M+H]+ 152.2.

[0088] Step 2: 2-(1-methyl-1H-pyrazol-5-yl)ethan-1-amine (Compound B2-3)

[0089] A mixture of 5-(2-azidoethyl)-1-methyl-1H-pyrazole (300 mg, 2.0 mmol) and Pd / C (22 mg) in MeOH (30 mL) was stirred under H2 at 25°C for 12 h. The mixture was filtered, concentrated under reduced pressure, and then dried in vacuo to give 2-(1-methyl-1H-pyrazol-5-yl)ethan-1-amine (200 mg crude) as a yellow oil, which was used directly in the next step without further purification.

[0090] LC-MS: Rt = 0.190 min, (ESI) m / z. [M+H] + 126.2.

[0091] Example 3 Synthesis of Compound 3

[0092]

[0093] 2-amino-N-((5-bromopyridin-2-yl)methyl)-3-methyl-N-(1-(pyrimidin-2-yl)ethyl)quinoline-6-carboxamide (30 mg, 0.063 mmol), tBuXPhos Pd G3 (5.0 mg, 0.0062 mmol), tBuXphos (5.3 mg, 0.013 mmol), Cs2CO3 (62 mg, 0.19 mmol), dioxane (10 mL) and 2-(1-methyl-1H-pyrazol-5-yl)ethane-1-amine (16 mg, 0.13 mmol) were added to a microwave tube, stirred at 25°C for 10 seconds, and then heated to 100°C under microwave conditions and maintained for 60 minutes. The reaction mixture was filtered through celite and then washed with EA (50 mL). The filtrate was concentrated under reduced pressure and the residue was purified by pre-HPLC, eluting with acetonitrile aqueous solution (0.1% NH3) from 5% to 40% within 8.0 minutes (instrument: Waters MS-triggered Prep-LC with QDA detector; chromatographic column: Xbridge 5uC18 150×19 mm; flow rate: 20 ml / min) to obtain 2-amino-3-methyl-N-((5-((2-(1-methyl-1H-pyrazol-5-yl)ethyl)amino)pyridin-2-yl)methyl)-N-(1-(pyrimidin-2-yl)ethyl)quinoline-6-carboxamide (10 mg, 30%) as a white solid.

[0094] 1 H NMR (400 MHz, DMSO- d6) δ 8.79 – 8.78 (m, 2H), 7.85 (s, 1H), 7.76 (s, 2H), 7.54 (s, 1H), 7.46 (s, 1H), 7.39 – 7.37 (m, 1H), 7.29 (s, 1H), 7.08– 7.06 (m, 1H), 6.91 – 6.89 (m, 1H), 6.44 (s, 2H), 6.13 (s, 1H), 5.85 (s,1H), 5.34 (s, 2H), 4.77 (s, 1H), 3.73 (s, 3H), 3.27 (s, 2H), 2.88 – 2.84 (m,2H), 2.20 (s, 3H), 1.55 – 1.53 (m, 3H). LC-MS: Rt =1.168 min, (ESI) m / z.445.2 [M+H] + . HPLC purity: 91.02% (214 nm), 91.40% (254 nm).

[0095] Biological test example 1 PRMT5 in vitro inhibitory activity experiment

[0096] Experimental Materials:

[0097] PRMT5 (Active Motif, catalog number 31921), [ 3 H]-SAM (PerkinElmer, catalog number NET155V001MC), SAM (Sigma, catalog number A7007), MTA (Sigma, catalog number D5011), SAH (Sigma, catalog number A9384), 384-well plate (Perkin Elmer, catalog number 6007299), Echo 550 (manufacturer: Labcyte, model: Echo 550), 384-well Flashplate (manufacturer: Perkin Elmer, model: SMP410A001PK).

[0098] Experimental methods

[0099] 1. Enzyme reaction process

[0100] (1) Configure 1x assay buffer (modified Tris Buffer).

[0101] (2) Compound dilution: The compound was dissolved in 100% DMSO and the compound solution was added to a 384-well plate using an Echo 550.

[0102] (3) Prepare enzyme solution: Add PRMT5 to 1x assay buffer to prepare enzyme solution 1; add PRMT5 and MTA to 1x assay buffer to prepare enzyme solution 2.

[0103] (4) Prepare substrate solution: Add peptide and [3H]-SAM to 1x assay buffer.

[0104] (5) Add 15 μL of enzyme solution to a 384-well plate and 15 μL of 1x assay buffer to the negative control wells. Incubate at room temperature for 30 min.

[0105] (6) Add 15 μL of substrate solution to each well and incubate at room temperature for 90 min.

[0106] (7) Prepare the termination reaction solution: add pre-cooled SAM to 1x assay buffer.

[0107] (8) Add 10 µL of stop reaction solution to each well to terminate the reaction.

[0108] (9) Transfer 25 µL / well of the mixed solution to the Flashplate and incubate at room temperature for 1 hour.

[0109] (10) Wash the Flashplate three times with dH2O + 0.1% Tween-20 solution.

[0110] (11) Read the radioactivity value using Microbeta.

[0111] 2. Data Analysis

[0112] (1) Convert the original data into % inhibition according to formula 1:

[0113] Formula 1: % inhibition = (Max-Signal) / (Max-Min) *100

[0114] (2) Substitute the % inhibition data into XL-Fit formula 2 to obtain IC 50 value:

[0115] Formula 2: Y=Bottom + (Top-Bottom) / (1+(IC 50 / X)*HillSlope)

[0116] Where Y is % inhibition and X is the compound concentration.

[0117] The biological activities of some compounds were measured by experimental methods, see Table 1

[0118] Table 1

[0119]

[0120] Biological test example: HCT116 and HCT116-MTAP-KO cell proliferation inhibition experiment in vitro

[0121] Experimental Materials

[0122] The HCT116 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences. The MTAP gene was knocked out using CRISPR / Cas9 technology to obtain the HCT116-MTAP-KO cell line.

[0123] McCoy's 5A medium (Gibco, catalog number 16600082), fetal bovine serum (Gibco, catalog number 10099141C), penicillin-streptomycin double antibody (Gibco, catalog number 15140122), trypsin (Gibco, catalog number 25200056), CellTiter-Glo detection kit (Promega, catalog number G7572), 384-well clear flat-bottom black-walled cell culture plates (Corning, catalog number 3764), ultra-micropipette instrument (Tecan, catalog number D300e), multi-function microplate reader (Biotek, catalog number SynergyHTX).

[0124] Experimental methods

[0125] 1. Cell culture: HCT116 and HCT116-MTAP-KO cells were cultured in McCoy's 5A medium + 10% fetal bovine serum + 1% penicillin-streptomycin; they were kept in the logarithmic growth phase and the cell viability was greater than 95%.

[0126] 2. Preparation of compound concentration gradient: The test compound was added to a 384-well plate using an ultra-micropipette, starting from 30 μM (HCT116 cells) or 3 μM (HCT116-MTAP-KO cells), and diluted 3-fold with DMSO, for a total of 9 concentrations, with triplicate wells.

[0127] 3. Compound-treated cells: Add the trypsin-digested HCT116 or HCT116-MTAP-KO cell suspension to the 384-well plate with the test compound, 40 μL per well, i.e., 100 cells per well, and the final DMSO concentration is 0.4%. The cell culture plate is placed in a 37°C, 5% carbon dioxide incubator for 6 days.

[0128] 4. Detection: Add 20 μL of CellTiter-Glo reagent to each well of the cell culture plate and incubate at room temperature for 30 minutes with shaking. Use a multi-function microplate reader to detect the luminescent signal at 578 nm.

[0129] 5. Data Analysis:

[0130] Among them, the first column is the cell proliferation inhibition rate HCT116 MTAP WT IC 50 (nm), the second column is the cell proliferation inhibition rate HCT116-MTAP null IC 50 (nm).

[0131] Structure of reference compound: Compound 4.

[0132] The examples and compound activity test results are as follows:

[0133] Table 2

[0134] .

[0135] The results showed that when the specific substituent (R1) of the present invention was not present, the cell proliferation inhibition effect of the compound was significantly deteriorated. Therefore, the introduction of the substituent achieved unexpected technical effects.

[0136] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A compound represented by the following formula, or a pharmaceutically acceptable salt thereof: 。 2. A pharmaceutical composition, characterized in that The pharmaceutical composition contains a therapeutically effective amount of the compound according to claim 1 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

3. Use of the compound or pharmaceutically acceptable salt according to claim 1 in the preparation of a medicament for treating or preventing a disease associated with abnormal gene level or expression of PRMT5.

4. The use according to claim 3, characterized in that The disease is a malignant tumor, and the malignant tumor is selected from the following group: ovarian cancer, lung cancer, colon cancer, melanoma, esophageal cancer, leukemia, head and neck cancer, brain cancer, gastric cancer, breast cancer, endometrial cancer, urinary tract cancer, liver cancer, pleural cancer or sarcoma.

5. The use according to claim 3, characterized in that The disease is a malignant tumor, and the malignant tumor is selected from the following group: colorectal cancer, myxofibrosarcoma, cholangiosarcoma, and malignant peripheral nerve sheath tumor (MPNST).

6. The use according to claim 3, characterized in that The disease is a malignant tumor, and the malignant tumor is selected from the following group: lymphoma, non-small cell lung cancer, glioblastoma, astrocytoma, gastric adenocarcinoma, kidney cancer, bladder cancer.

7. The use according to claim 3, characterized in that The disease is a malignant tumor, and the malignant tumor is selected from the following group: diffuse large B-cell lymphoma (DLBCL) and mesothelioma.

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