Compound for inhibiting PRMT5 and pharmaceutical composition thereof

Abstract

The invention provides a compound with methyltransferase inhibitory activity, and particularly provides a compound with PRMT5 inhibitory activity as shown in a formula I which is described in the specification. The compound can be used for preparing a pharmaceutical composition for treating diseases related to PRMT5 activity. # imgabs0 # I

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] in, Ring A is a substituted or unsubstituted 5-membered heterocyclic ring or a 5-membered heteroaromatic ring; R1 is selected from the following groups: unsubstituted or halogenated C1-C6 alkyl, substituted or unsubstituted -(CH2) m-Ar; wherein Ar is selected from the following group: a benzene ring, a 5-6 membered heteroaromatic ring; m is 0, 1 or 2; R2 is selected from the following group: substituted or unsubstituted benzene ring, substituted or unsubstituted 5-6 membered heteroaromatic ring; R3 is selected from the group consisting of H, unsubstituted or halogenated C1-C6 alkyl; R4 is selected from the group consisting of H, halogen; 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.

[0009] In another preferred embodiment, the is a structure selected from the group consisting of: , .

[0010] In another preferred embodiment, the Ar is selected from the following group: .

[0011] In another preferred embodiment, the R2 is selected from the following groups: substituted or unsubstituted benzene ring, substituted or unsubstituted , substituted or unsubstituted .

[0012] In another preferred embodiment, the R4 is H or F.

[0013] In another preferred embodiment, m is 1.

[0014] In another preferred embodiment, the compound is selected from the following group: , , , , , , .

[0015] 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 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, excipients, adjuvants, auxiliary materials and / or diluents.

[0016] In the third aspect of the present invention, there is provided a use of a compound as described in the first aspect or a pharmaceutically acceptable salt thereof 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 methyltransferases).

[0017] 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.

[0018] 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 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, excipients, adjuvants, auxiliary materials and / or diluents.

[0019] The fifth aspect of the present invention provides a use of a compound as described in any of the preceding aspects or a pharmaceutically acceptable salt thereof 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 methyltransferases).

[0020] 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.

[0021] 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. DETAILED DESCRIPTION

[0022] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that when a specific substituent (biaryl substituent) 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.

[0023] the term In the present invention, the halogen is F, Cl, Br or I.

[0024] 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.).

[0025] 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.

[0026] 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, furyl, 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.

[0027] Pharmaceutical compositions and methods of administration 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).

[0028] 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.

[0029] "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.

[0030] The administration method of the compound or pharmaceutical composition of the present invention is not particularly limited, and representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] The definitions of various abbreviations are shown in Table 1 below: Table 1

[0042] The raw materials can be obtained from commercial sources or prepared by methods known or disclosed in the art.

[0043] The purification of intermediates and compounds is carried out by normal phase or reverse phase chromatography or recrystallization and other conventional chemical experimental operations. Normal phase chromatography is pre-packed silica gel chromatography column or preparative thin layer chromatography. Silica gel chromatography column is mainly glass column or 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 C18 column, preparative liquid chromatograph or rapid preparative chromatograph, 214nM and 254nM or preparative liquid chromatography-mass spectrometry instrument detection, with water / acetonitrile containing 0.1% hydrochloric acid, water / acetonitrile, water / acetonitrile containing 0.1% ammonium bicarbonate, water / acetonitrile containing 0.1% formic acid, 0.1% ammonia water / acetonitrile, 0.1% trifluoroacetic acid water / acetonitrile or other suitable solvent system as mobile phase for gradient elution.

[0044] 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 Ascend400 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 the 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).

[0045] 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); changing from 5% ACN to 95% ACN within 1.0 min, holding for 1.0 min, and a total of 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; changing from 5% ACN to 95% ACN within 1.0 min, holding for 1.0 min, and a total of 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).

[0046] Embodiment Synthesis general method:

[0047] Example 1 Synthesis of Compound 6

[0048] Step 1: Preparation of 4-[1-(trifluoromethyl)pyrazol-4-yl]thiazole-2-carbaldehyde 4-Bromothiazole-2-carboxaldehyde (a) (135 mg, 703 umol, 1 eq), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-(trifluoromethyl)pyrazole (202 mg, 773 umol, 1.1 eq) were dissolved in THF (2 mL) and H2O (0.4 mL), then K3PO4 (448 mg, 2.11 mmol, 3 eq) was added, degassed and purged with N2 for 3 times, then XPhosPdG3 (59.5 mg, 70.3 umol, 0.1 eq) was added, and then the mixture was stirred at 80 °C under N2 atmosphere for 16 hours. LC-MS showed that the reaction mixture was complete. The residue was diluted with 20 mL of H2O and extracted with ethyl acetate (10 mL*3). The combined organic layer was washed with 15 mL of brine, dried over MgSO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® silica gel flash chromatography column, 0-7.5% ethyl acetate / petroleum ether gradient elution, flow rate 30 mL / min) to obtain 4-[1-(trifluoromethyl)pyrazol-4-yl]thiazole-2-carbaldehyde (b) (75 mg, 300 umol, yield 42.73%, purity 99%) as a white solid.

[0049] LC-MS: Rt = 0.840 min, (ESI) m / z. [M+H] + 247.9.

[0050] Step 2: Preparation of 2-methyl-N-[[4-[1-(trifluoromethyl)pyrazol-4-yl]thiazol-2-yl]methyl]propan-1-amine 4-[1-(Trifluoromethyl)pyrazol-4-yl]thiazole-2-carbaldehyde (b) (75 mg, 303.40 μmol, 1 eq) was dissolved in MeOH (2 mL) and a solution of 2-methylpropan-1-amine (44.4 mg, 606 μmol, 60.3 μL, 2eq) was added at 25°C. After addition, the mixture was stirred at 25°C for 16 hours. NaBH4 (11.5 mg, 303 μmol, 1 eq) was added portionwise at 5-20°C over 1 minute and the mixture was stirred at 25°C for another 2 hours. LC-MS showed that the reaction mixture was complete. The reaction mixture was terminated by the addition of 1 mL of MeOH at 20°C and the reaction was concentrated in vacuo to obtain a residue. The residue was dissolved in DCM (15 mL), washed with NaHCO3 (10 mL), then washed with brine (10 mL), then dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® silica gel flash chromatography column, elution with a gradient of 0-20% ethyl acetate / petroleum ether @ 35 mL / min) to afford 2-methyl-N-[[4-[1-(trifluoromethyl)pyrazol-4-yl]thiazol-2-yl]methyl]propan-1-amine (c) (60 mg, 197 umol, 64.98% yield) as a yellow solid.

[0051] LC-MS: Rt = 0.721 min, (ESI) m / z. [M+H] + 305.1.

[0052] Step 3: Preparation of 4-amino-7-fluoro-N-isobutyl-1-methyl-N-[[4-[1-(trifluoromethyl)pyrazol-4-yl]thiazol-2-yl]methyl]pyrazolo[4,3-c]quinoline-8-carboxamide (Compound 6) To a solution of 2-methyl-N-[[4-[1-(trifluoromethyl)pyrazol-4-yl]thiazol-2-yl]methyl]propan-1-amine (c) (60 mg, 197 μmol, 1.08 eq) 4-amino-7-fluoro-1-methyl-pyrazolo[4,3-c]quinoline-8-carbonyl chloride (64.3 mg, 183 μmol, 1 eq, 2 HCl) in THF (2 mL) was added DIEA (118 mg, 915 μmol, 159 μL, 5 eq). The mixture was stirred at 25 °C for 3 hours. LC-MS showed that the reaction mixture was complete. The reaction mixture was concentrated under reduced pressure to remove the solvent. The crude product was purified by reverse phase HPLC (column: Boston Prime C18150*30mm*5um; mobile phase: [water (ammonia v / v)-ACN]; B%: 37%-67%, 8min) to obtain 4-amino-7-fluoro-N-isobutyl-1-methyl-N-[[4-[1-(trifluoromethyl)pyrazol-4-yl]thiazol-2-yl]methyl]pyrazolo[4,3-c]quinoline-8-carboxamide (Compound 6) (21.9 mg, 40.0 umol, yield 21.88%, purity 100%) as a white solid.

[0053] LC-MS: Rt = 2.147 min, (ESI) m / z. [M+H] + 547.2 1 H NMR (400 MHz, DMSO-d6) δ (ppm) 8.61-8.96 (m, 1 H), 8.18-8.47 (m, 2H), 7.88-8.18 (m, 2 H), 7.09-7.43 (m, 3 H), 4.71-5.23 (m, 2 H), 3.99-4.50 (m, 3 H), 3.15-3.29 (m, 2 H), 1.79-2.28 (m, 1 H), 0.57-1.05 (m, 6 H).

[0054] Example 2 Synthesis of Compound 7

[0055] Step 1: Preparation of 4-[6-(trifluoromethyl)-3-pyridyl]thiazole-2-carbaldehyde 4-Bromothiazole-2-carboxaldehyde (a) (1 g, 5.21 mmol, 1 eq), [6-(trifluoromethyl)-3-pyridyl]boronic acid (1.49 g, 7.81 mmol, 1.5 eq) were dissolved in THF (20 mL) and H2O (4 mL), then K3PO4 (3.32 g, 15.6 mmol, 3 eq) was added, degassed and purged with N2 for 3 times, then XPhos Pd G3 (440 mg, 520 umol, 0.1 eq) was added, and then the mixture was stirred at 80 ° C for 16 hours under N2 atmosphere. LC-MS showed that the reaction mixture was complete. The residue was diluted with 30 mL of H2O and extracted with ethyl acetate (20 mL*3). The combined organic layer was washed with brine 30 mL, dried over MgSO4, filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by flash silica gel chromatography (ISCO®; 12+40 g SepaFlash® silica gel flash chromatography column, elution with 0-10.2% ethyl acetate / petroleum ether gradient, flow rate 40 mL / min) to obtain 4-[6-(trifluoromethyl)-3-pyridyl]thiazole-2-carbaldehyde (b) (1.12 g, 4.35 mmol, yield 83.46%) as a yellow solid.

[0056] LC-MS: Rt = 0.865 min, (ESI) m / z. [M+H] + 258.9; 1 H NMR (400 MHz, CDCl3) δ (ppm) 10.06-10.13 (m, 1 H), 9.24-9.33 (m, 1H), 8.42-8.51 (m, 1 H), 8.11 (d, J = 1.00 Hz, 1 H), 7.82 (d, J = 8.25 Hz, 1H).

[0057] Step 2: Preparation of 2-methyl-N-[[4-[6-(trifluoromethyl)-3-pyridinyl]thiazol-2-yl]methyl]propan-1-amine 4-[6-(Trifluoromethyl)-3-pyridyl]thiazole-2-carboxaldehyde (b) (200 mg, 774 umol, 1 eq) was dissolved in MeOH (4 mL) and stirred at 25°C. A solution of 2-methylpropan-1-amine (113 mg, 1.55 mmol, 153 uL, 2 eq) was added. After addition, the mixture was stirred at 25°C for 16 hours. NaBH4 (29.3 mg, 774 umol, 1 eq) was added in portions over 2 minutes at 5-20°C, and the mixture was stirred at 25°C for 2 hours. LC-MS showed that the reaction mixture was complete. The reaction mixture was quenched by adding 2 mL of MeOH at 20°C, and the reaction was concentrated in vacuo to obtain a residue. The residue was dissolved in DCM (30 mL), washed with NaHCO3 (20 mL), then washed with brine (20 mL), then dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® silica gel flash chromatography column, elution with a gradient of 0-20% ethyl acetate / petroleum ether @ 35 mL / min) to afford 2-methyl-N-[[4-[6-(trifluoromethyl)-3-pyridinyl]thiazol-2-yl]methyl]propan-1-amine (c) (158 mg, 475 umol, 61.45% yield, 95% purity) as a yellow solid.

[0058] LC-MS: Rt = 0.734 min, (ESI) m / z. [M+H] + 316.1

[0059] Step 3: Preparation of 4-amino-7-fluoro-N-isobutyl-1-methyl-N-[[4-[6-(trifluoromethyl)-3-pyridinyl]thiazol-2-yl]methyl]pyrazolo[4,3-c]quinoline-8-carboxamide (Compound 7) To a solution of 2-methyl-N-[[4-[6-(trifluoromethyl)-3-pyridinyl]thiazol-2-yl]methyl]propan-1-amine (c) (53.9 mg, 171 umol, 1.2 eq), 4-amino-7-fluoro-1-methyl-pyrazolo[4,3-c]quinoline-8-carbonyl chloride (50.1 mg, 142 umol, 1 eq, 2HCl) in THF (2 mL) was added DIEA (92.1 mg, 713 umol, 124 uL, 5 eq). The mixture was stirred at 25 °C for 3 hours. LC-MS showed that the reaction mixture was complete. The reaction mixture was concentrated under reduced pressure to remove THF. The crude product was purified by reverse phase HPLC (column: Boston Prime C18 150*30mm*5um; mobile phase: [water (ammonia v / v)-ACN]; B%: 17%-47%, 8min) to obtain 4-amino-7-fluoro-N-isobutyl-1-methyl-N-[[4-[6-(trifluoromethyl)-3-pyridinyl]thiazol-2-yl]methyl]pyrazolo[4,3-c]quinoline-8-carboxamide (Compound 7) (30 mg, 53.3 umol, yield 37.35%, purity 99%) as a white solid.

[0060] LC-MS: Rt = 2.00 min, (ESI) m / z. [M+H] + 558.2.

[0061] 1 H NMR (400 MHz, DMSO-d6) δ (ppm) 9.12-9.46 (m, 1 H), 8.48-8.64 (m, 1H), 8.33-8.46 (m, 1 H), 7.84-8.30 (m, 3 H), 7.17-7.44 (m, 3 H), 4.77-5.26 (m, 2 H), 3.98-4.46 (m, 3 H), 3.41-3.52 (m, 2 H), 1.86-2.24 (m, 1 H), 0.55-1.11 (m, 6 H).

[0062] 19 F NMR (376 MHz, DMSO-d6) δ (ppm) -116.65 - -115.72 (m, 1 F) -66.27 (br d, J = 13.7 Hz, 3 F).

[0063] Example 3: Synthesis of Compound 5

[0064] Step 1: Preparation of 2-methyl-N-[[4-(6-methyl-3-pyridyl)thiazol-2-yl]methyl]propan-1-amine 4-(6-Methyl-3-pyridyl)thiazole-2-carboxaldehyde (a) (200 mg, 979 umol, 1 eq) and 2-methylpropan-1-amine (145 mg, 1.98 mmol, 197 uL, 2.02 eq) were dissolved in MeOH (5 mL) and stirred at 25-30°C for 12 hours. NaBH4 (52.2 mg, 1.38 mmol, 1.41 eq) was added and stirred at 25-30°C for 4 hours. LC-MS indicated that the starting material had been consumed and the desired compound was detected. The reaction was quenched with AcOH (60 uL) and concentrated under reduced pressure. The residue was partitioned between 10% Na2CO3 aqueous solution (25 mL) and CH2Cl2 / EtOH (10:1, 25 mL). The layers were separated and the aqueous phase was extracted with CH2Cl2 / EtOH (10:1, 10 mL*3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give a yellow solid P1. The crude 2-methyl-N-[[4-(6-methyl-3-pyridyl)thiazol-2-yl]methyl]propan-1-amine (b) (276 mg, crude) was obtained as a yellow solid and used in the next step without further purification.

[0065] LC-MS: Rt = 1.796 min, (ESI) m / z . [M+H] + 262.2;

[0066] Step 2: Preparation of 4-amino-7-fluoro-N-isobutyl-1-methyl-N-[[4-(6-methyl-3-pyridyl)thiazol-2-yl]methyl]pyrazolo[4,3-c]quinoline-8-carboxamide (Compound 5) To a solution of 2-methyl-N-[[4-(6-methyl-3-pyridyl)thiazol-2-yl]methyl]propan-1-amine (b) (50 mg, 191umol, 1 eq) and DIPEA (123 mg, 956 umol, 166 uL, 5 eq) in THF (5 mL) was added 4-amino-7-fluoro-1-methyl-pyrazolo[4,3-c]quinoline-8-carbonyl chloride (65 mg, 184 umol, 0.97 eq, 2HCl). The reaction mixture was stirred at 25-30°C for 16 hours. LC-MS indicated that the starting material was consumed and the desired compound was detected. The reaction mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC (basic conditions; chromatographic column: Boston Prime C18150*30mm*5um; mobile phase: [water (ammonia v / v)-ACN]; B%: 38%-58%, 9min). The fractions of the target compound were combined and lyophilized to obtain a white solid P1. 4-Amino-7-fluoro-N-isobutyl-1-methyl-N-[[4-(6-methyl-3-pyridyl)thiazol-2-yl]methyl]pyrazolo[4,3-c]quinoline-8-carboxamide (Compound 5) (52 mg, 103.26umol, yield 53.98%, purity 100%) was obtained as a white solid.

[0067] LC-MS: Rt = 1.712 min, (ESI) m / z . [M+H] + 504.2; 1 H NMR (400 MHz, DMSO- d 6) δ (ppm) 8.79-9.15 (m, 1H), 7.88-8.39 (m, 4H),7.04-7.47 (m, 4H), 4.60-5.25 (m, 2H), 3.82-4.49 (m, 3H), 3.15-3.34 (m, 2H),2.48 (s, 3H), 1.83-2.22 (m, 1H), 0.41-1.06 (m, 6H) 19 F NMR (376.5 MHz, DMSO- d 6) δ (ppm) -115.68, -116.40

[0068] Example 4 Synthesis of Compound 3

[0069] Step 1: Preparation of 1,3-dimethyl-N-((4-(6-methylpyridin-3-yl)thiazol-2-yl)methyl)-1H-pyrazol-4-amine A mixture of 4-(6-methylpyridin-3-yl)thiazole-2-carboxaldehyde (a) (100 mg, 490 μmol, 1.0 eq.), 1,3-dimethyl-1H-pyrazol-4-amine (b) (63 mg, 567 μmol, 1.2 eq.) and HOAc (35 mg, 583 μmol, 1.19 eq.) was dissolved in DCM (6 mL) and stirred at 15-20 °C for 4 hours. NaBH(OAc)3 (312 mg, 1.47 mmol, 3.01 eq.) was added and stirred at 15-20 °C for 12 hours. The reaction mixture was heated at 40 °C for another 16 hours. LCMS indicated that the starting material had been consumed and the desired compound was detected. The reaction mixture was diluted with H2O and CH2Cl2 and basified to pH = 12 with 2M K2CO3 aqueous solution while stirring. The layers were separated and the aqueous phase was extracted with CH2Cl2 (10 mL*3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO®; 12 g SepaFlash® silica gel flash chromatography column, eluent 0-30% (EtOAc: EtOH = 3: 1) / 5% Et3N in petroleum ether gradient @ 30 mL / min; petroleum ether: (ethyl acetate: EtOH = 3: 1), Rf = 0.43). The components of the desired compound were combined and concentrated under reduced pressure to give 1,3-dimethyl-N-((4-(6-methylpyridin-3-yl)thiazol-2-yl)methyl)-1H-pyrazole-4-amine (c) (117 mg, yield 79.82%) as a brown solid.

[0070] LC-MS: Rt = 0.49 min, (ESI) m / z. [M+H] + 300.1.

[0071] Step 2: Preparation of 4-amino-N-(1,3-dimethyl-1H-pyrazol-4-yl)-N-((4-(6-methylpyridin-3-yl)thiazol-2-yl)methyl)-1,3-dihydrofuro[3,4-c]quinoline-8-carboxamide (Compound 3) 1,3-Dimethyl-N-((4-(6-methylpyridin-3-yl)thiazol-2-yl)methyl)-1H-pyrazol-4-amine (c) (80 mg, 267 μmol, 1.0 eq.), 4-amino-1,3-dihydrofuro[3,4-c]quinoline-8-carboxylic acid (INT14) (62 mg, 269 μmol, 1.0 eq.) and NMI (67 mg, 814 μmol, 3.0 eq.) were dissolved in NMP (10 mL) and TCFH (91 mg, 325 μmol, 1.2 eq.) was added. The reaction mixture was stirred at 10-15 °C for 16 hours. The reaction was heated at 40 °C for another 16 hours. LC-MS indicated that the starting material was consumed and the desired compound was detected. The reaction mixture was purified by preparative high performance liquid chromatography (column: Boston Prime C18 150*30mm*5um; mobile phase: [water (ammonia v / v)-ACN]; gradient: 23%-43% B, 16 minutes). The fractions of the desired compound were combined and lyophilized to obtain 4-amino-N-(1,3-dimethyl-1H-pyrazol-4-yl)-N-((4-(6-methylpyridin-3-yl)thiazol-2-yl)methyl)-1,3-dihydrofurano[3,4-c]quinoline-8-carboxamide (Compound 3) (23 mg, yield 16.75%, purity 99.53%) as a white solid.

[0072] LC-MS: Rt = 1.270 min, (ESI) m / z. [M+H] + 512.1.

[0073] 1 H NMR (400 MHz, DMSO-d6) δ (ppm) 8.98 (d, J = 2.0 Hz, 1H), 8.20 (s,1H), 8.14 (dd, J = 2.2, 7.9 Hz, 1H), 7.73 (s, 1H), 7.44-7.53 (m, 2H), 7.35-7.42 (m, 1H), 7.32 (d, J = 8.1 Hz, 1H), 6.75 (s, 2H), 5.19 (s, 4H), 4.97 (s,2H), 3.63 (s, 3H), 2.49 (s, 3H), 1.65 (s, 3H).

[0074] The following compounds were synthesized using a similar general method to that in the previous examples, and the characterization results are as follows: Table 2 .

[0075] Biological test example 1 PRMT5 in vitro inhibitory activity experiment

[0076] Experimental Materials

[0077] 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)

[0078] Experimental methods 1. Enzyme reaction process (1) Configure 1x assay buffer (modified Tris Buffer).

[0079] (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.

[0080] (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.

[0081] (4) Prepare substrate solution: Add peptide and [3H]-SAM to 1x assay buffer.

[0082] (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.

[0083] (6) Add 15 μL of substrate solution to each well and incubate at room temperature for 90 min.

[0084] (7) Prepare the termination reaction solution: add pre-cooled SAM to 1x assay buffer.

[0085] (8) Add 10 µL of stop reaction solution to each well to terminate the reaction.

[0086] (9) Transfer 25 µL / well of the mixed solution to the Flashplate and incubate at room temperature for 1 hour.

[0087] (10) Wash the Flashplate three times with dH2O + 0.1% Tween-20 solution.

[0088] (11) Read the radioactivity value using Microbeta.

[0089] 2. Data Analysis (1) Convert the original data into % inhibition according to formula 1: Formula 1: % inhibition = (Max-Signal) / (Max-Min) *100 (2) Substitute the % inhibition data into XL-Fit formula 2 to obtain the IC50 value: Formula 2: Y=Bottom + (Top-Bottom) / (1+(IC50 / X)*HillSlope) Where, Y is % inhibition and X is the concentration of the compound. The biological activities of some compounds were measured by the above experimental method.

[0090] Biological test example: HCT116 and HCT116-MTAP-KO cell proliferation inhibition experiment in vitro

[0091] Experimental Materials 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.

[0092] 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)

[0093] Experimental methods 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%.

[0094] 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.

[0095] 3. Compound-treated cells: Add the trypsin-digested HCT116 or HCT116-MTAP-KO cell suspension to a 384-well plate with the test compound, 40 μL per well, i.e., 100 cells per well, with a final DMSO concentration of 0.4%. The cell culture plate was placed in a 37°C, 5% carbon dioxide incubator for 6 days.

[0096] 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.

[0097] 5. Data Analysis: 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) The examples and compound activity test results are shown in Table 3 below: Table 3

[0098] 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 I or a pharmaceutically acceptable salt thereof: ； in, Ring A is a substituted or unsubstituted 5-membered heterocyclic ring or a 5-membered heteroaromatic ring; R1 is selected from the following groups: unsubstituted or halogenated C1-C6 alkyl, substituted or unsubstituted -(CH2) m -Ar; wherein Ar is selected from the following group: a benzene ring, a 5-6 membered heteroaromatic ring; m is 0, 1 or 2; R2 is selected from the following group: substituted or unsubstituted benzene ring, substituted or unsubstituted 5-6 membered heteroaromatic ring; R3 is selected from the group consisting of H, unsubstituted or halogenated C1-C6 alkyl; R4 is selected from the group consisting of H, halogen; 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.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that The is a structure selected from the group consisting of: .

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The Ar is selected from the following group: .

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The R2 is selected from the following groups: substituted or unsubstituted benzene ring, substituted or unsubstituted , substituted or unsubstituted .

5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The R4 is H or F.

6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: m is 1.

7. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The compound is selected from the following group: 、 、 、 、 、 、 。 8. A pharmaceutical composition, characterized in that The pharmaceutical composition contains a therapeutically effective amount of a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, excipients, adjuvants, auxiliary materials and / or diluents.

9. Use of the compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing a disease associated with abnormal gene level or expression of PRMT5.

10. The use according to claim 9, characterized in that 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, bladder cancer, mesothelioma, non-small cell lung cancer, astrocytoma, undifferentiated pleomorphic sarcoma, diffuse large B-cell lymphoma (DLBCL), leukemia, head and neck cancer, gastric adenocarcinoma, myxofibrosarcoma, cholangiosarcoma, 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.

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